U.S. patent application number 10/789400 was filed with the patent office on 2004-12-02 for recombinant human metapneumovirus and its use.
This patent application is currently assigned to The Government of the U.S.A. as represented by the Secretary of the Dept. of Health & Human Services. Invention is credited to Biacchesi, Stephane, Buchholz, Ursula, Collins, Peter L., Murphy, Brian R., Skiadopoulos, Mario H..
Application Number | 20040241188 10/789400 |
Document ID | / |
Family ID | 34138424 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040241188 |
Kind Code |
A1 |
Collins, Peter L. ; et
al. |
December 2, 2004 |
Recombinant human metapneumovirus and its use
Abstract
Recombinant HMPV (rHMPV) and related immunogenic compositions
and methods are provided. The rHMPVs, including chimeric and
chimeric HMPV vectors viruses, provided according to the current
disclosure are infectious and attenuated in permissive mammalian
subjects, including humans. The rHMPVs are useful in immunogenic
compositions for eliciting an immune response against HPIV, against
one or more non-HMPV pathogens, or against a HMPV and a non-HMPV
pathogen. Also provided are isolated polynucleotide molecules and
vectors incorporating a recombinant HMPV genome of antigenome.
Inventors: |
Collins, Peter L.;
(Kensington, MD) ; Biacchesi, Stephane;
(Washington, DC) ; Buchholz, Ursula; (Bethesda,
MD) ; Skiadopoulos, Mario H.; (Potomac, MD) ;
Murphy, Brian R.; (Bethesda, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE #1600
ONE WORLD TRADE CENTER
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Government of the U.S.A. as
represented by the Secretary of the Dept. of Health & Human
Services
|
Family ID: |
34138424 |
Appl. No.: |
10/789400 |
Filed: |
February 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60451119 |
Feb 28, 2003 |
|
|
|
60478667 |
Jun 13, 2003 |
|
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Current U.S.
Class: |
424/199.1 ;
435/235.1; 435/5 |
Current CPC
Class: |
C12N 2760/18351
20130101; C12N 2760/18362 20130101; C12N 15/86 20130101; A61K
39/155 20130101; C12N 2760/18361 20130101; A61K 2039/70 20130101;
A61K 2039/5254 20130101; C12N 2760/18643 20130101; C12N 2760/18634
20130101; C12N 7/00 20130101; A61P 37/02 20180101; A61K 2039/5256
20130101; A61K 39/12 20130101; C12N 2760/18343 20130101; C12N
2760/18334 20130101 |
Class at
Publication: |
424/199.1 ;
435/005; 435/235.1 |
International
Class: |
C12Q 001/70; A61K
039/12; C12N 007/00 |
Claims
1. An isolated recombinant human metapneumovirus (rHMPV),
comprising a partial or complete, recombinant HMPV genome or
antigenome comprising one or more attenuating nucleotide
modifications, and a major nucleocapsid (N) protein, a nucleocapsid
phosphoprotein (P), and a large polymerase protein (L).
2. The rHMPV of claim 1, wherein the rHMPV is replication
competent.
3. The rHMPV of claim 1, wherein the recombinant HMPV genome or
antigenome further comprises a detectable heterologous sequence
encoding a polypeptide.
4. The rHMPV of claim 3, wherein the detectable heterologous
sequence encodes a reporter.
5. The rHMPV of claim 4, wherein the reporter comprises green
fluorescent protein (GFP).
6. The rHMPV of claim 3, wherein the detectable heterologous
sequence is operably linked to HMPV transcription gene start and
gene end signals.
7. The rHMPV of claim 1, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of one or more rHMPV SH, G, M2-1, M2-2, or M2 ORFs or one or more
nucleotide substitutions that reduces or ablates expression of the
one or more rHMPV SH, G, M2-1, M2-2, or M2 ORFs.
8. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of one or more of the rHMPV SH, G, M2-1, M2-2, or M2 ORFs, such
that a functional protein is not produced.
9. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a rHMPV SH ORF, such that a wild type SH protein is not
produced.
10. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a SH ORF of SEQ ID NO: 1, such that a SH protein comprising a
sequence set forth as SEQ ID NO: 5 is not produced.
11. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a rHMPV G ORF, such that a wild type G protein is not
produced.
12. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a G ORF of SEQ ID NO: 1, such that a G protein comprising a
sequence set forth as SEQ ID NO: 6 is not produced.
13. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a rHMPV SH and G ORFs, such that a wild type SH protein and a
wild type G protein are not produced.
14. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a SH ORF and a G ORF of SEQ ID NO: 1.
15. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises one or more nucleotide
substitutions that reduces or ablates expression of a rHMPV M2-2
ORF.
16. The rHMPV of claim 15, wherein the one or more nucleotide
substitutions that reduces or ablates expression of the rHMPV M2-2
ORF comprises one or more nucleotide substitutions that ablates one
or more potential translation initiation codons of the rHMPV M2-2
ORF or introduces one or more in-frame stop codons into the r HMPV
M2-2 ORF.
17. The rHMPV of claim 16, wherein the one or more nucleotide
substitutions comprises substitutions of one or more nucleic acids
of a rHMPV sequence set forth as SEQ ID NO: 1.
18. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a rHMPV M2-2 ORF, such that a wild type M2-2 protein is not
produced.
19. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a M2-2 ORF of SEQ ID NO: 1.
20. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises one or more nucleotide
substitutions that reduces or ablates expression of a rHMPV M2-1
ORF, such that a wild type M2-1 protein is not produced.
21. The rHMPV of claim 20, wherein the one or more nucleotide
substitutions that reduces or ablates expression of the rHMPV M2-1
ORF comprises one or more nucleotide substitutions that ablates the
translation initiation codon of the rHMPV M2-1 ORF and further
ablates additional ATG triplets in each reading frame of the rHMPV
M2-1 ORF.
22. The rHMPV of claim 21, wherein the one or more nucleotide
substitutions comprises substitutions at one or more positions of
SEQ ID NO: 1.
23. The rHMPV of claim 7, wherein the one or more attenuating
nucleotide modifications comprises a partial or complete deletion
of a rHMPV M2 ORF.
24. The rHMPV of claim 23, wherein the partial or complete deletion
comprises a partial or complete deletion of the M2 ORF of SEQ ID
NO: 1.
25. The rHMPV of claim 1, wherein the one or more attenuating
nucleotide modifications produces at least one desired phenotypic
change in the rHMPV, wherein the phenotypic change comprises at
least one of a change in growth properties in cell culture, a
change in growth properties or virulence in the upper or lower
respiratory tract of a mammalian host, a change in viral plaque
size, a change in sensitivity or adaptation to temperature, a
change in cytopathic effect, a change in the efficiency of
transcription or genome replication, a change in sensitivity to
interferon, a change in the efficiency of expression of one or more
genes, or a change in immunogenicity.
26. The rHMPV of claim 25, wherein the one or more attenuating
nucleotide modifications produces a change in viral growth in the
upper respiratory tract, lower respiratory tract, or both, such
that viral growth is attenuated by about 50-100 fold or greater,
compared to growth of the corresponding wild type HMPV strain.
27. The rHMPV of claim 1, wherein the one or more attenuating
nucleotide modifications comprises one or more nucleotide
substitutions that produce one or more amino acid substitutions in
a M2-1 or a L protein in the rHMPV.
28. An isolated, replication competent recombinant human
metapneumovirus (rHMPV), comprising a partial or complete,
recombinant HMPV genome or antigenome, a major nucleocapsid (N)
protein, a nucleocapsid phosphoprotein (P), and a large polymerase
protein (L), wherein the genome or antigenome of the rHMPV is
rearranged such that an order of one or more genes or genome
segments in the recombinant HMPV genome or antigenome is altered as
compared to a wild type HMPV.
29. The rHMPV of claim 28, wherein the order of a SH, G, or F gene
or genome segment is altered in the rHMPV genome or antigenome.
30. The rHMPV of claim 28, wherein the rHMPV genome or antigenome
comprises a SH gene or genome segment and a G gene or genome
segment inserted after a M gene and before a F gene of the rHMPV
genome or antigenome.
31. The rHMPV of claim 28, wherein the rHMPV genome or antigenome
comprises at least two copies of a SH gene or genome segment and at
least two copies of a G gene or genome segment inserted after a M
gene and before a F gene of the rHMPV genome or antigenome.
32. The rHMPV of claim 28, wherein the rHMPV genome or antigenome
comprises a F gene or genome segment inserted after a 3' leader
sequence and before a N gene of the rHMPV genome or antigenome.
33. The rHMPV of claim 28, wherein the rHMPV genome or antigenome
comprises a G gene or genome segment inserted after a 3' leader
sequence and before a N gene of the rHMPV genome or antigenome.
34. The rHMPV of claim 28, wherein the rHMPV genome or antigenome
comprises a F gene or genome segment and a G gene or genome segment
inserted after a 3' leader sequence and before a N gene of the
rHMPV genome or antigenome.
35. The rHMPV of claim 28, wherein the rHMPV genome or antigenome
comprises a G gene or genome segment and a F gene or genome segment
inserted after a 3' leader sequence and before a N gene of the
rHMPV genome or antigenome.
36. The rHMPV of claim 1, wherein the one or more attenuating
nucleotide modifications comprises inserting one or more additional
copies of one or more rHMPV G or F genes or genome segments in the
rHMPV genome or antigenome.
37. The rHMPV of claim 36, wherein the rHMPV genome or antigenome
comprises one or more additional copies of a rHMPV G gene or genome
segment, a F gene or genome segment, or both, inserted after a 3'
leader sequence and before a N gene of the rHMPV genome or
antigenome.
38. The rHMPV of claim 36, wherein the rHMPV genome or antigenome
comprises a single additional copy of a rHMPV G gene or genome
segment inserted after a 3' leader sequence and before a N gene of
the rHMPV genome or antigenome.
39. The rHMPV of claim 36, wherein the rHMPV genome or antigenome
comprises a single additional copy of a rHMPV F gene or genome
segment inserted after a 3' leader sequence and before a N gene of
the rHMPV genome or antigenome.
40. The rHMPV of claim 36, wherein the rHMPV genome or antigenome
comprises one additional copy of the rHMPV G gene and one
additional copy of the rHMPV F gene in the order G-F.
41. The rHMPV of claim 36, wherein the rHMPV genome or antigenome
comprises one additional copy of the recombinant HMPV G gene and
one additional copy of the recombinant HMPV F gene in the order
F-G.
42. The rHMPV of claim 36, wherein the rHMPV genome or antigenome
comprises one additional copy of a rHMPV G gene or genome segment
and two additional copies of the rHMPV F gene or genome segment in
the order G-F-F.
43. The rHMPV of claim 1, wherein the rHMPV genome or antigenome
further comprises one or more heterologous genes or genome segments
from a different paramyxovirus to form a chimeric recombinant HMPV
genome or antigenome.
44. The rHMPV of claim 43, wherein the rHMPV genome or antigenome
comprises one or more N, P, or M genes from a different
paramyxovirus.
45. The rHMPV of claim 44, wherein the paramyxovirus comprises
avian metapneumovirus.
46. The rHMPV of claim 1, wherein the rHMPV genome or antigenome
further comprises one or more rHMPV genes or genome segments from a
different subgroup of HMPV to form a chimeric recombinant HMPV
genome or antigenome.
47. An immunogenic composition comprising an immunogenically
effective amount of the isolated, replication competent recombinant
human metapneumovirus of claim 1 in a pharmaceutically acceptable
carrier.
48. A method for inducing an immune response in a subject against
human metapneumovirus, comprising administering to the subject a
therapeutically effective amount of the isolated, replication
competent recombinant human metapneumovirus of claim 1, thereby
inducing an immune response in the subject against human
metapneumovirus.
49. The method of claim 48, wherein the recombinant human
metapneumovirus is administered in a dose of 10.sup.3 to 10.sup.7
PFU.
50. The method of claim 49, wherein the recombinant human
metapneumovirus is administered to the upper respiratory tract.
51. The method of claim 48, wherein the recombinant human
metapneumovirus is administered by spray, droplet or aerosol.
52. An isolated, replication competent recombinant virus comprising
a paramyxovirus genome or antigenome and a major nucleocapsid (N)
protein, a nucleocapsid phosphoprotein (P), a large polymerase
protein (L), and one or more recombinant genes or genome segments
from human metapneumovirus.
53. The recombinant virus of claim 52, comprising a human
metapneumovirus F gene.
54. The recombinant virus of claim 52, wherein the virus is an
influenza virus or a parainfluenza virus.
55. An expression vector comprising an operably linked
transcriptional promoter, a partial or complete, recombinant human
metapneumovirus (rHMPV) genome or antigenome, and a transcriptional
terminator.
56. The expression vector of claim 55, wherein the rHMPV genome or
antigenome comprises one or more attenuating nucleotide
modifications.
57. A method of screening an antiviral compound for inhibition of a
biological activity of a human metapneumovirus, comprising
providing a recombinant human metapneumovirus (rHMPV) comprising a
major nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P),
a large polymerase protein (L), and a partial or complete,
recombinant HMPV genome or antigenome modified to incorporate a
detectable heterologous sequence encoding a polypeptide correlated
with the biological activity upon expression of the heterologous
sequence; exposing a test sample comprising the rHMPV or a host
cell amenable to infection by HMPV to a test compound or library of
test compounds that prospectively includes one or more antiviral
agents capable of inhibiting the biological activity of HMPV;
providing a control sample comprising the rHMPV or host cell under
suitable control conditions in the absence of the test compound or
library of test compounds; and detecting heterologous sequence in
the test and control samples to determine an increase or decrease
of the biological activity in the test sample compared to the
control sample to determine the presence or absence of the
antiviral compound in the test sample.
Description
PRIORITY CLAIM
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/451,119, filed Feb. 28, 2003, and U.S.
Provisional Patent Application No. 60/478,667, filed Jun. 13, 2003.
Both of the provisional applications are incorporated by reference
herein in their entirety.
FIELD OF THE DISCLOSURE
[0002] This invention relates to the field of virology and, more
specifically, to methods for producing recombinant human
metapneumovirus (rHMPV), for producing replication competent
derivatives with desirable properties such as attenuation and
protective efficacy, and to uses of rHMPV in immunoprophylaxis and
therapy.
BACKGROUND
[0003] Human metapneumovirus (HMPV) is a virus that was first
recovered in the Netherlands from infants and children experiencing
acute respiratory tract disease (van den Hoogen et al. Nat. Med.
7:719-724, 2001; De Jong et al. WO 02/057/302 A2).
[0004] HMPV is worldwide in prevalence and resembles human
respiratory syncytial virus (RSV or HRSV) with regard to disease
signs and the ability to infect and cause disease in the young
infant as well as individuals of all ages (for a review, see
Heikkinen and Jarvinen, Lancet 361:51-59, 2003).
[0005] HMPV is characterized as an enveloped virus with a genome
that is a single negative strand of RNA of approximately 13 kb. The
virus has been classified presumptively in the Metapneumovirus
genus, Pneumovirus subfamily, Paramyxovirus family of the Order
Mononegavirales, comprising the nonsegmented negative strand RNA
viruses or mononegaviruses. Mononegaviruses also are called
nonsegmented negative strand RNA viruses. The Paramyxovirus family
has two subfamilies, Paramyxovirinae and Pneumovirinae (also
referred to as paramyxoviruses and pneumoviruses,
respectively).
[0006] Several other mononegaviruses are important agents of
respiratory tract disease in pediatric and other populations, for
example, RSV and human parainfluenza virus types 1, 2 and 3 (HPIV1,
HPIV2 and HPIV3). Although these viruses have some similarities
with HMPV, their sequences are different from the HMPV viruses
disclosed herein.
[0007] Since HMPV was described only recently, there is little
documented experience with its propagation, manipulation and
stability, and there are no well established or widely available
reference strains, mutant strains, reported vaccine candidates, or
reference virus-specific antibodies or comparable reagents or
systems or experimental animals models to facilitate
characterization.
[0008] Because HMPV is associated with severe respiratory tract
disease, there is a need to develop methods to engineer sale and
effective vaccines to alleviate the serious health problems
attributable to this pathogen, particularly among young
infants.
BRIEF SUMMARY OF SPECIFIC EMBODIMENTS
[0009] HMPV is a significant agent of human respiratory tract
disease. Methods and compositions are provided herein for
recovering infectious, recombinant HMPV. Recombinant HMPV is
disclosed herein, including the complete nucleic acid sequence
encoding all of the protein products of HMPV. Compositions and
methods for introducing defined, predetermined structural and
phenotypic changes into an infectious HMPV are disclosed, as are
attenuated forms of HMPV as well as forms of HMPV that have been
modified to have improved qualities relevant to immunogenicity,
safety, protective efficacy, and breadth of vaccine coverage. Thus,
methods are disclosed for generating an immune response against
HMPV in a subject. The methods include administering to the subject
an attenuated HMPV, thereby producing the immune response. Also
described is the identification of a set of viral ORFs whose
expression is sufficient to direct viral transcription and RNA
replication, and to produce infectious HMPV entirely from cDNA.
[0010] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 is a diagrammatic representation of the negative
sense genomes of HMPV strain CAN97-83 (hereafter referred to as 83
or CAN-83) and RSV strain A2, showing a comparison of the two
genomes. Individual proposed genes are shown as shaded boxes
separated by proposed intergenic regions represented by open bars.
The 3' extragenic leader and 5' extragenic trailer regions also are
shown as open boxes. Tentative HMPV gene assignments are based on
ORFs whose potential protein exhibits partial amino acid sequence
identity or structural similarity with a known protein from RSV.
Note that HMPV lacks apparent counterparts to the NS1 and NS2 genes
present in RSV (boxed), and that the putative F and M2 gene pair
precedes the SH-G gene pair in HMPV but follows it in RSV (the
difference in the position of SH-G is indicated with dotted lines).
The M2 and L genes overlap in RSV but may not do so in HMPV. In
both viruses, the M2 gene contains two ORFs M2-1 and M2-2, depicted
as filled bars. Amino acid lengths are shown for the deduced
unmodified viral proteins. Each of the RSV proteins has been
directly identified; none of the proposed HMPV proteins had been
directly identified prior to the current disclosure. Abbreviations
(with HMPV assignments by analogy to RSV): N, major nucleocapsid
protein; P, nucleocapsid phosphoprotein; M, inner virion matrix
protein; M2-1 and M2-2, products of the first and second ORFs,
respectively, in the M2 mRNA; SH, small hydrophobic protein; G,
heavily glycosylated protein involved in attachment, F, fusion
protein involved in penetration; L, large polymerase protein: NS1
and NS2, nonstructural protein 1 and 2, respectively. This and all
subsequent diagrams are not to scale.
[0012] FIG. 2 is a diagram showing sequence alignments between the
3' leader (top) and 5' trailer (bottom) regions and flanking areas
of the genome of HMPV strain 83 (SEQ ID NO: 18 and SEQ ID NO: 22,
respectively), avian pneumovirus (APV or AMPV, SEQ ID NO: 19 and
SEQ ID NO: 23, respectively), and HRSV strain A2 (SEQ ID NO: 20 and
SEQ ID NO: 24, respectively). The sequences are in genome
(negative) sense. Nucleotide assignments that are the same in two
or more sequences between HMPV 83, APV and RSV are shaded; sequence
gaps introduced to make optimal alignments are indicated by dashes.
The bars over the left hand end of each sequence indicates regions
whose spacing and sequence had not been previously determined for
any HMPV. The partial sequences available for HMPV strain 00-1 3'
leader and 5' trailer regions (SEQ ID NO: 17 and SEQ ID NO: 21,
respectively) are indicated at the top: nucleotide assignments in
strain 00-1 that are identical to those of HMPV 83 are indicated by
asterisks while nucleotide differences are indicated. Sequences:
APV (Randhawa et al., J. Virol. 71:9849-54, 1997), RSV (Mink et
al., Virology 185:615-24, 1991), HMPV 00-1 (van den Hoogen et al.,
Nat. Med. 7:719-24,2001).
[0013] FIG. 3 is a diagram showing sequence alignments between the
3' and 5' ends of the genome of HMPV strain 83 (top; bases 1-41 of
SEQ ID NO: 18 and bases 1-40 of SEQ ID NO: 22, respectively) and
APV (bottom; bases 1-41 of SEQ ID NO: 19 and bases 1-42 of SEQ ID
NO: 23, respectively), with complementary nucleotides boxed. This
shows that the last 13 nucleotides of the two ends of APV are
perfect complements, whereas HMPV unexpectedly has two
noncomplementary nucleotides. Sequences for HMPV that were not
previously available are indicated with solid lines above (leader,
Le strand) or below (trailer, Tr strand) the sequence.
[0014] FIG. 4 is a diagram showing several examples of differences
in nucleotide and amino acid lengths between HMPV strains 83 and
00-1. The differences in G are illustrated by partial amino acid
sequences (upper alignment; amino acids 190-219 of SEQ ID NO: 6 and
amino acids 190-236 of SEQ ID NO: 11, respectively) and partial
nucleotide sequences (second alignment; bases 6871-6929 of SEQ ID
NO: 1 and bases 6886-6964 of SEQ ID NO: 36, respectively). The
sequences are numbered according to the amino acid sequence of HMPV
83 G (top alignment) or the complete nucleotide sequence of the
antigenome of HMPV 83 (second alignment). Examples of differences
include, (1) the SH protein for 83 is shorter by 4 aa due to an
earlier stop codon; (2) the G protein for 83 is shorter by 17 aa
due to an earlier stop codon, which is followed by a deletion in
the downstream noncoding region; and (3) the intergenic region
between the F and M2 genes of 83 is 13 nucleotides (nt) compared to
41 nt for 00-1.Taken together, these show that the G ORF and
encoded G protein of strain 83 are shorter by the equivalent of 17
amino acids from that of strain 00-1.
[0015] FIG. 5 is a chart showing the percent amino acid sequence
identity for the predicted, putative proteins of HMPV 83 compared
to those of two oilier strains of HMPV (00-(1), and 97-82), three
different antigenic types of APV (A, B and C), the two subgroups of
human RSV (A and B), bovine RSV (BRSV), and pneumonia virus of mice
(PVM, a murine counterpart of HRSV). Note that HMPV is most closely
related to APV C. ND indicates that a comparison was not done for
the indicated subject.
[0016] FIG. 6 is a taxonomy tree, illustrating putative
phylogenetic relationships between HMPV and other paramyxoviruses
based oil comparisons of the amino acid sequences of proposed
matrix M proteins. Pneumovirinae and Paramyxovirinae are the two
subfamilies of the Paramyxovirus family. The other taxonomic names
refer to genera. The numbers refer to the extent of amino acid
sequence divergence (percentage non-identity) divided by 100;
hence, 0.2 refers to 20% amino acid differences. Note that a system
for recovering and modifying infectious recombinant virus has not
been reported for any member of genus Metapneumovirus even though
the avian members of this genus have been known for almost 25
years.
[0017] FIG. 7 is a diagram illustrating the construction of a cDNA
encoding the complete antigenome of HMPV strain 83, designed from
the complete consensus genomic sequence. Three separate subgenomic
cloned fragments were created: fragment 1 contains the putative N,
P and M genes and is bordered on the upstream (left) side by an
added promoter for bacteriophage T7 RNA polymerase (T7p) and on the
right hand side by an NheI site that was added to the putative M-F
intergenic region as a marker. The sequence changes involved in
introducing the NheI site are shown. The T7 promoter was designed
to add three nonviral G residues to the 5' end of the antigenome, a
configuration chosen to improve the efficiency of the T7 promoter.
Fragment 2 included the putative F, M2, SH and G genes and is
bordered on the upstream side by the added NheI site and on the
downstream side by a naturally occurring Acc65I site. Fragment 3
consists of the L gene followed by part of the hepatitis delta
virus ribozyme (HDVribo) (Perrotta and Been, Nature 350:434-6,
1991) bordered by an RsrII site that occurs naturally within that
ribozyme. The vector for cloning and expressing the HMPV 83
antigenome cDNA was pBSKSII, which is a derivative of a modified
Bluescript II KS+(Stratagene) vector described previously (Durbin
et al., Virology 71:4272-7, 1997) and contains the hepatitis delta
virus ribozyme followed by a terminator for T7 RNA polymerase
(T7t). This vector was modified by the insertion of a polylinker
containing AvaI, AatII, NheI, and Acc65I sites, which in turn
served to accept the cloned fragments 1, 2 and 3.The final cDNA
encodes the complete 13335-nucleotide HMPV 83 antigenome containing
three added nonviral G residues at the 5' end. The complete
pHMPV-83 recombinant plasmid ("antigenome plasmid") contains 16333
bp.
[0018] FIG. 8 is a diagram showing putative transcription signals
for HMPV strain 83 based on the identification of semi-conserved
sequence motifs located between the major ORFs in the complete
sequence. Sequences are in positive (mRNA) sense. The
semi-conserved sequence motif that precedes most of the OFRs in the
complete sequence is shown is the putative gene start (GS) signal,
with some of the most highly conserved sequences shown in upper
case letters and with flanking sequence on either side in lower
case. The individual GS motifs are named according to the putative
ORF, that each precedes (SEQ ID NO: 1, bases 29-64 for N; bases
1237-1272 for P; bases 2154-2189 for M; bases 3041-3076 for F;
bases 4698-4733 for M2; bases 5455-5490 for SH; bases 6206-6241 for
G; and bases 7107-7142 for L). A consensus sequence is shown
underneath (SEQ ID NO: 25). For positions where alternative
assignments can occur, these are listed below the consensus (SEQ ID
NOs: 26 and 27). By this analysis, the major element of the GS
signal is contained within sixteen nucleotides, of which positions
1, 3, 4, 6, 7, 9, and 14-16 were exactly conserved, with 14-16 also
serving as potential translation start sites. Similarly, the
semi-conserved sequence motif that follows most of the ORFs in the
complete sequence is shown as the putative gene end (GE) signal,
with some of the most highly conserved sequences shown in upper
case and flanking sequence in lower case. The individual GE motifs
are named according to the ORF that each follows (SEQ ID NO: 1,
bases 1230-1249 for N; bases 2141-2166 for P; bases 3002-3032 for
M; bases 4680-4710 for F; bases 5440-5467 for M2; bases 6077-6107
for SH; bases 6912-6942 for G; and bases 13223-13253 for L). A
consensus sequence is shown underneath (SEQ ID NO: 28). For
positions where alternative assignments can occur, these are listed
below the consensus (SEQ ID NOs: 29 and 30). By this analysis, the
major element of the GE signal is contained within 12-13
nucleotides, of which positions 1-3 and 10-12 are exactly
conserved.
[0019] FIG. 9 is a diagram illustrating the construction of a
transcription cassette containing the ORF for jellyfish GFP under
the control of HMPV transcription GS and GE signals, and insertion
of this cassette into the HMPV strain 83 antigenomic cDNA clone
following antigenomic position 41. Sequence representing part of
the 3' leader region (bases 23-41 of SEQ ID NO: 1) and the putative
N GS signal and beginning of the putative N ORF (bases 42-66 of SEQ
ID NO: 1) is shown at the bottom. The GFP transcription cassette
consists of a 714-nucleotide cDNA containing the GFP ORF flanked on
the upstream side by 16 nucleotides (which include the initiating
ATG of the GFP ORF) representing the conserved putative N GS signal
(bases 42-57 of SEQ ID NO: 1) and flanked on the downstream side by
13 nucleotides representing the putative F GE signal (bases
46854697 of SEQ ID NO: 1; which include the termination codon of
the GFP ORF) followed by the trinucleotide AGT designed to function
as an intergenic (IG) region. The total length of the transcription
cassette including the trinucleotide intergenic region is 748
nucleotides. The F GE signal contains a naturally occurring PacI
site (italicized).
[0020] FIG. 10 is a diagram illustrating the construction of
expression vectors comprising support plasmid that contain ORFs
corresponding to the proposed nucleocapsid and polymerase proteins
of HMPV strain 83:N, P, M2-1 and L. These ORFs were clarified in
part based on sequence relatedness to the respective RSV ORF (see,
for example, FIG. 5). Each ORF was placed under the control of a T7
Transcription promoter (T7p) and terminator (T7t). The L cDNA was
assembled from two overlapping pieces. Each ORF conformed to the
consensus sequence of HMPV 83.
[0021] FIG. 11 is a diagram showing the strategy for recovery of a
recombinant version of HMPV strain 83, in this case expressing GFP
as an added marker gene. Plasmids encoding the HMPV-GFP antigenome
and the N, P, L and M2-1 support plasmids are transfected into BSR
T7/5 cells, which are a derivative of the baby hamster kidney-21
(1311K-21) cell line that was engineered to constitutively express
T7 RNA polymerase (Buchholz et al., J. Virol. 73:251-259. 1999).
The support plasmids were constricted to encode the proposed HMPV
nucleocapsid and polymerase proteins. By this strategy, the
expression of the antigenome together with HMPV
nucleocapsid/polymerase proteins provides the components for the
self-assembly of HMPV nucleocapsids functional for transcription
and RNA replication. These nucleocapsids then express all of the
HMPV RNAs and proteins, leading to a productive infection that
produces rHMPV.
[0022] FIG. 12A is a digitized image of an agarose gel showing that
the recovered recombinant HMPV-GFP (strain 83) contains the NheI
site that was engineered into the antigenome cDNA and is not
present in biologically-derived strain 83 parent. LLC-MK2 cells in
T25 flask were infected with an aliquot of the supernatant from the
transfected BSR T7/5 cells (first passage) and incubated at
32.degree. C. in the presence of 5 .mu.g/ml trypsin. Eleven days
post-infection, 1 ml of the supernatant was harvested, clarified by
centrifugation at 1500 rpm for 5 minutes and used to extract the
viral genomic RNA with the QIAamp viral RNA purification kit
(QIAGEN) according to the manufacturer's recommendations. The RNA
was subjected to reverse transcription (RT) using a positive sense
primer designed to hybridize within the M gene (nucleotides 2719 to
2738 in the HMPV antigenome exclusive of GFP). Polymerase chain
reaction (PCR) was performed using the RT primer and a reverse
primer designed to hybridize within the F gene (nucleotides 3894 to
3876 in the HMPV antigenome exclusive of GFP). An aliquot of each
RT-PCR reaction was subjected to digestion with NheI. The RT-PCR
products were analyzed by agarose gel electrophoresis and ethidium
bromide staining. Lanes: 1 and 7, molecular weight marker; 2, a
negative control in which RNA from the recombinant
HMPV-GFP-infected cell supernatant was assayed with the omission of
RT enzyme; lanes 3 and 4, RT-PCR products from RNA representing
recombinant HMPV-GFP and biologically-derived HMPV, respectively;
lanes 5 and 6, RT-PCR products from RNA representing recombinant
HMPV-GFP and biologically-derived HMPV, respectively, that had been
subjected to digestion with NheI.
[0023] FIG. 12B is a digitized image of a nylon membrane
illustrating Northern blot analysis of GFP mRNA expressed by the
rHMPV-GFP virus. LLC-MK2 cells were mock-infected (lanes 1 and 5)
or infected at an MOI of 3 PFU per cell with biologically-derived
HMPV83 (lanes 2 and 6), rHMPV (lanes 3 and 7), or rHMPV-GFP (lanes
4 and 8). Three days later, total intracellular RNA was isolated,
electrophoresed on 1% agarose-formaldehyde gels, transferred to
charged nylon and analyzed by hybridization to double-stranded
.sup.32P-labeled DNA probe specific to the GFP (lanes 1-4) or M
(lanes 5-8) gene. The identities and calculated sizes of individual
RNA species are indicated. This showed that GFP transcription
cassette was expressed predominantly as a single mRNA of the
appropriate size to be a monocistronic GFP mRNA.
[0024] FIG. 13 is a graph illustrating multi-step growth of the
recovered recombinant HMPV (strain 83) (rHMPV) compared with
biologically-derived HMPV 83. LLC-MK2 cells were inoculated with
0.01 plaque forming units (pfu) per cell and incubated at
32.degree. C. in the presence of 5 .mu.g/ml trypsin. Aliquots were
taken at 24 h intervals, flash-frozen, and viral liters were
measured by plaque assay and immunostaining with convalescent serum
from HMPV-infected hamsters. Each time point was represented by two
wells, and each virus titration was done in duplicate. Means are
shown.
[0025] FIG. 14 is a graph illustrating multi-step growth of
recombinant HMPV-GFP compared to recombinant HMPV in LLC-MK2 cells,
performed as described above for FIG. 13. Two separate infections
(a and b) were monitored for each virus, and each aliquot was
titrated in duplicate and the mean is shown.
[0026] FIG. 15 is a diagram illustrating deletion of the SH and G
genes singly and in combination from rHMPV and rHMPV-GFP (strain
83), each containing introduced BsiWI and BsrGI restriction sites.
The region of the antigenomic cDNA clone containing the putative SH
and G ORFs is illustrated, together with proposed GS and GE
transcription signals and proposed intergenic regions. BsiWI and
BsrGI sites were introduced into the putative M2-SH and SH-G,
respectively, intergenic regions of HMPV-GFP. In each case, the
mutant sequence (nucleotide substitutions in small case) is shown
below the wild type sequence. The restriction sites are numbered
according to the position of the first residue in the wild type
HMPV sequence (SEQ ID NO: 1) exclusive of GFP, a convention that
will be followed throughout this disclosure. The
naturally-occurring Acc65I sequence also is shown. Note that
cleavage of each of these restriction sites by its cognate enzyme
leaves a compatible overhang, namely GTAC, that is compatible with
each of the others. Thus, the genes can be readily deleted by
cutting with the appropriate pair of enzymes and religating. This
results in small changes in the length of the relevant intergenic
region, as noted. Each of these antigenomic cDNAs was successfully
used to recover infectious mutant virus.
[0027] FIG. 16 is a digitized image of an agarose gel showing that
the recovered recombinant HMPV-GFP.DELTA.SH, .DELTA.G and
.DELTA.SH/G viruses each contain a deletion of the appropriate size
in the region of the SH and G genes. Following the procedure
described above for FIG. 12, viral RNA was subjected to RT-PCR
using primers that span the SH and G genes; specifically a positive
sense primer designed to hybridize within the M2 and a negative
sense primer designed to hybridize within the L gene. The RT-PCR
products were analyzed by agarose gel electrophoresis and ethidium
bromide staining. Lanes: 1 and 8, molecular weight markers; lane 3,
a negative control in which RNA from recovered recombinant HMPV was
subjected to the RT-PCR procedure with the omission of RT enzyme;
lanes 2 and 4-7, RT-PCR products representing the indicated
viruses.
[0028] FIG. 17A is a graph illustrating multi-step growth of
rHMPV-GFP-.DELTA.SH, rHMPV-GFP-.DELTA.G, and rHMPV-GFP-.DELTA.SH/G
compared to rHMPV and rHMPV-GFP, performed in LLC-MK2 cells as
described above for FIG. 13. Since the deletions had been
introduced into rHMPV-GFP, rHMPV-GFP is the "wild type" equivalent
for comparison.
[0029] FIG. 17B is a graph illustrating the efficiency of
replication of biologically-derived HMPV83 and
recombinantly-derived rHMPV, rHMPV.DELTA.SH, rHMPV.DELTA.G, and
rHMPV.DELTA.SH/G in the upper (nasal turbinates) and lower (lungs)
respiratory tract of Golden Syrian hamsters. Animals in groups of
12 were infected intranasally with the following viruses at a dose
of 5.0.times.10.sup.5 TCID.sub.50 per animal in 0.1 ml:
biologically derived HMPV83, rHMPV, rHMPV.DELTA.SH, and
rHMPV.DELTA.SH/G, and the following virus at 1.6.times.10.sup.5
TCID.sub.50 per animal in 0.1 ml: rHMPV.DELTA.G. Six animals from
each group were sacrificed on days 3 and 5 and the nasal turbinates
and lungs were recovered and analyzed by limiting dilution to
determine the viral titer. This showed that the rHMPV replicated in
vivo with an efficiency similar to that of its biologically-derived
parent (HMPV83), that the replication of the rHMPV.DELTA.SH virus
was not significantly reduced, and that-the replication of the
rHMPV.DELTA.G and .DELTA.SH/G viruses were strongly reduced,
although virus replication was detectable in each case.
[0030] FIG. 17C is a graph illustrating the protective efficacy of
immunization with the indicated HMPV deletion mutant viruses.
Golden Syrian hamsters in groups of 6 were infected as described in
FIG. 17B and, 27 days later, serum samples were taken and analyzed
to determine the titers of HMPV-neutralizing serum antibodies.
These titers are shown at the bottom. The animals were then
challenged on day 28 by the intranasal instillation of 5.0
log.sub.10 TCID.sub.50 per animal of biologically-derived wild type
HMPV83.Three days later, the animals were sacrificed and the nasal
turbinates and lungs were harvested, homogenized, and analyzed by
limiting dilution to determine virus titers. This showed that there
was no detectable replication of the challenge virus in animals
that has received rHMPV or rHMPV.DELTA.SH, whereas animals that
initially had received rHMPV.DELTA.G or rHMPV.DELTA.SH/G had no
detectable challenge virus replication in the lungs and had reduced
replication in the nasal turbinates compared to the control that
had not been previously infected.
[0031] FIG. 18A is a diagram illustrating ablation of the putative
M2-2 ORF in infectious rHMPV and rHMPV-GFP (strain 83), each
containing an introduced BsiWI site. The region of the antigenomic
cDNA clone containing the putative M2-1 and M2-2 ORF is
illustrated, with each ORF depicted by a solid horizontal line. Two
nucleotide substitutions were made at positions 5236 and 5248 (SEQ
ID NO: 1; numbered according to the complete antigenomic cDNA
exclusive of GFP) that ablate two potential translation initiation
codons for the M2-2 ORF. A third substitution was made at position
5272 (SEQ ID NO: 1) that introduces an in-frame stop codon. In
addition, nucleotides 5289-5440 (SEQ ID NO: 1) were deleted,
removing most of the M2-2 ORF. The .DELTA.M2-2 mutant was
successfully recovered in both the rHMPV and rHMPV-GFP backbones
and designated rHMPV.DELTA.M2-2 and rHMPV-GFP.DELTA.M2-2,
respectively.
[0032] FIG. 18B is a graph illustrating evaluation of multi-step
growth kinetics of the recovered viruses in vitro. The
rHMPV.DELTA.M2-2 and rHMPV-GFP.DELTA.M2-2 viruses were compared
with rHMPV and rHMPV-GFP with regard to multi-step growth kinetics
and yield in LLC-MK2 cells as described above for FIG. 13.
rHMPV.DELTA.M2 clones #1 and #2 represent virus derived
independently from two sister cDNA clones.
[0033] FIG. 18C is a digitized image of a nylon membrane
illustrating Northern blot analysis of intracellular RNAs expressed
by rHMPV-GPV and rHMPV-GFP.DELTA.M2-2. Replicate monolayers of
LLC-MK2 cells were infected at an MOI of 3 PFU per cell with either
virus as indicated and incubated at 32.degree. C. Monolayers were
harvested at 9, 24, 36, 48, and 72 It post infection as indicated
and processed for purification of intracellular RNA. The RNA
preparations were analyzed by Northern blot hybridization with
strand-specific riboprobes representing the N gene: the upper
panels detect hybridization with the negative-sense probe and thus
represent antigenome and N-related mRNA, as indicated, and the
bottom panels detect hybridization of a replicate set of gel lanes
(that had been transferred in parallel onto the same membrane,
which was then cut and the replicate sets hybridized separately)
with positive-sense riboprobe and represent genome. Beneath the
Northern blot are calculations from four experiments involving
hybridization with a probe specific for N or for F, as indicated.
In each experiment, the amount of total N-related or F-related mRNA
for each time point was divided by the amount of antigenome from a
replicate gel lane (that had been transferred in parallel onto the
same membrane, which was then cut and the replicas hybridized
separately). Then, each time point for the rHMPV-GFP.DELTA.M2-2
virus was normalized relative to the corresponding time point for
rHMPV-GFP as 1.0.Thus, this provides a comparison of the efficiency
of mRNA expression for the .DELTA.M2-2 mutant compared to wild type
HMPV.
[0034] FIG. 19 is a diagram illustrating the introduction of single
amino acid substitutions into the Cys3-His1 motif of the M2-1
protein of rHMPV-GFP (strain 83). This motif consists of three
cysteine residues (C7, C15 and C21) and one histidine residue
(H25); it is also found in RSV and other pneumoviruses, and thus
represents a conserved pneumovirus motif. The amino acid sequence
shown is from methionine (M) 1 to asparagine (N) 26 of the deduced
complete M2-1 amino acid sequence (SEQ ID NO: 4). Each of these
mutants was successfully recovered as infectious recombinant
virus.
[0035] FIG. 20 is a graph illustrating multi-step growth of
derivatives of rHMPV-GFP containing mutations in the Cys3-His1
motif of the M2-1 ORF, compared to rHMPV-GFP and rHMPV, performed
in LLC-MK2 cells as described above for FIG. 13. The mutants
involve single amino acid point mutations in the Cys3-His1 motif:
C7S, Y9S, C15S, N16S and H25S. The mutations were introduced into
rHMPV-GFP, and thus rHMPV-GFP is the "wild type" or parental
equivalent for comparison.
[0036] FIG. 21 is a diagram illustrating silencing of the M2-1 ORF
in rHMPV-GFP (strain 83) by replacing the ATG translational start
site (shaded) with a TAG translational termination codon, and by
replacing additional ATG triplets (underlined) in each reading
frame with slop colons. The top line shows the nucleotide sequence
of the upstream end of the M2-1 mRNA (corresponding to nucleotides
4711 to 4775 in the complete HMPV antigenome sequence. SEQ ID NO:
1). The second line shows nucleotide substitutions (lower case
letters) introduced into the M2-1 sequence of rHMPV-GFP to yield
rHMPV-GFP-.DELTA.M2-1. The third line shows the first 17 amino
acids of the HMPV M2-1 protein (SEQ ID NO: 4), and the fourth line
shows coding changes introduced by the point mutations in
rHMPV-GFP-.DELTA.M2-1, with termination codons in the M2-1 ORF
indicated by asterisks. Note that the next in-frame ATG in the M2-1
ORF is at codon 134 out of the total of 187 codons in the ORF (SEQ
ID) NO: 4). This mutant was successfully recovered as infectious
recombinant virus.
[0037] FIG. 22A is a diagram illustrating deletion of the complete
M2 gene in infectious rHMPV-GFP (strain 83) containing an
introduced BsiWI site. The region of the antigenomic cDNA clone
containing the putative M2-1 and M2-2 ORF is illustrated, with each
ORF depicted by a solid horizontal line. Nucleotides 4701 to 5459
(SEQ ID NO: 1), representing a total of 759 nucleotides, were
deleted, resulting in the mutant rHMPV-GFP-.DELTA.M2(1+2). This
mutant was successfully recovered as infectious recombinant
virus.
[0038] FIG. 22B is a graph illustrating a comparison of the
efficiency of growth of viruses with alterations in the coding of
M2-1 and/or M2-2 proteins in LLC-MK2 cells (upper panel), which are
competent for expressing type I interferons versus Vero cells
(lower panel), which lack the interferon structural genes. Cells
were infected with 0.01 PFU per cell and incubated at 32.degree. C.
in the presence of 5 .mu.g/ml trypsin. Aliquots were taken at 24 h
intervals, flash-frozen, and viral titers were measured by plaque
assay and immunostaining with convalescent serum from hamsters that
had been infected with HMPV.
[0039] FIG. 22C is a chart demonstrating increased sensitivity of
HMPV .DELTA.M2(1+2) and .DELTA.M2-2 mutants to type I interferon.
Replicate cultures of Vero cells were treated overnight with the
indicated amount of interferon per 1.5.times.10.sup.6 cells. The
cells were infected with the indicated amount of wild type
rHMPV-GFP, rHMPV-GFP.DELTA.M2(1+2), rHMPV-GFP.DELTA.M2-2, or rgRSV,
the last being a recombinant RSV that expresses the GFP gene. The
GFP marker was used to visually monitor virus growth during the
experiment to judge the appropriate time of harvest, and is
otherwise irrelevant to this experiment. The cells were harvested
on day 4 and the yield of each virus was determined and compared
with additional replicate cultures that had been mock-interferon
treated, infected and harvested in parallel. The results are
expressed as the fold reduction of each interferon-treated culture
compared to its untreated counterpart.
[0040] FIG. 23A is a diagram illustrating the construction of two
rearrangements of the genes of rHMPV-GFP (strain 83). The wild type
gene order is shown on top for the M-L region of the rHMPV-GFP
antigenomic cDNA clone. The restriction sites shown are the ones
introduced as illustrated above in FIGS. 7 and 15 (the restriction
sites are numbered according to the position of the first residue
in the wild type HMPV sequence, SEQ ID NO: 1, exclusive of GFP),
and were used to rearrange the order if the putative SH and G gene
pair. Religations involving NheI were not compatible with the other
sites and necessitated fill-in of each end followed by blunt end
ligation. Two examples of gene rearrangements are shown: in the
mutant called "Order 1", the positions of the SH-G gene pair and
the F-M2 gene pair were swapped, resulting in a local gene order
that mimics that of RSV, namely M-SH-G-F-M2-L. This mutant was
successfully recovered as infectious virus. In a subsequent mutant,
"Order 2", the same RSV-like gene order was achieved, but the SH-G
gene pair was duplicated.
[0041] FIG. 23B is a graph illustrating the multi-step growth
kinetics of one of these mutants, rHMPV-GFP-Order #1 compared to
its direct parent rHMPV-GFP, as well as rHMPV, performed as
described above for FIG. 13. Clones #3 and #13 represent viruses
derived independently from two sister cDNA clones.
[0042] FIG. 24A is a diagram illustrating shifting of the F and/or
G genes of HMPV (strain 83) from their natural positions as the
fourth and seventh genes, respectively, to promoter-proximal
positions 1 or 2. As illustrated in the upper box, the putative F
and G ORFs were engineered to be flanked by putative GS (bases
3054-3069 and 6219-6234 of SEQ ID NO: 1, respectively) and GE
(bases 4685-4697 of SEQ ID NO: 1) signals and inserted individually
into the HMPV antigenomic cDNA clone. Sequence representing part of
the 3' leader region (bases 2341 of SEQ ID NO: 1) and the putative
N GS signal and beginning of the putative N ORF (bases 42-66 of SEQ
ID NO: 1) is shown. Note that the choice of transcription signals
and insertion site was the same as for the rHMPV-GFP construct
shown above in FIG. 9, and places the inserted gene as the first in
the gene order. As illustrated in the lower box, the putative F and
G ORFs were inserted as a pair, in the order G1-F2 or its converse
F1-G2, using the same transcription signals and insertion site as
in the single-gene rearrangements. Infectious virus was
successfully recovered from each of the constructs. These viruses
were designated rHMPV-F1, -G1, -F1G2, and G1F2, with the number
indicating the position of the gene in the gene order. In the virus
designation, the "-" symbol preceding the shifted gene indicates
that the shifted gene was removed from its normal position in the
gene order.
[0043] FIG. 24B is a graph illustrating multi-cycle growth kinetics
of the recovered viruses in vitro. The recovered rHMPV-G1, F1,
F1G2, and G1F2 viruses were compared to their rHMPV parent with
regard to multi-step growth in LLC-MK2 cells as described above for
FIG. 13.
[0044] FIG. 25A is a diagram showing representations of rHMPV in
which one or more extra copies of the F and/or G gene was placed in
promoter proximal positions 1, 2 or 3 in addition to the F and/or G
gene present in the normal genome position. The G or F genes were
inserted individually into promoter proximal position 1 (rHMPV+G1
or +F 1), or were inserted as a pair into positions 1 and 2 in each
of the two possible orders (rHMPV+G1F2 or +F1G2), or were inserted
as the pair G1F2 with an additional copy of F in the third position
(rHMPV+G1F2,3). The detailed structure of the gene insertions were
as shown in FIG. 24A. In the designations, the "+" symbol before
the named gene or genes indicates that it/they were additional to
the one or ones in the normal position. The added genes are
shaded.
[0045] FIG. 25B is a graph illustrating multi-cycle growth kinetics
in vitro. The recovered viruses were compared to their rHMPV parent
and to rHMPV-GFP with regard to multi-step growth in LLC-MK2 cells
as described above for FIG. 13.
[0046] FIG. 25C is a digitized image of a nylon membrane
illustrating Northern blot analysis of intracellular mRNAs
expressed by rHMPV+G1F23. LLC-MK2 cells were mock-infected (lanes
1, 5 and 9) or infected at an MOI of 3 PFU per cell with HMPV83
(lanes 2, 6 and 10), rHMPV (lanes 3, 7 and 11), or rHMPV+G1F23
(lanes 4, 8 and 12). Three days later, total intracellular RNA was
isolated, electrophoresed on 1% agarose-formaldehyde gels,
transferred to charged nylon and analyzed by hybridization to
double-stranded .sup.32P-labeled DNA probe specific to the F (lanes
1-4) G (lanes 5-8), or M (lanes 9-12) gene. The identities and
calculated sizes of individual RNA species are indicated.
[0047] FIGS. 26A-26F are diagrams illustrating amino acid locations
in the strain 83 HMPV L protein (SEQ ID NO: 7) that are targets for
mutagenesis based on mapping of attenuating mutations in a
heterologous virus (HRSV A2, SEQ ID NO: 13; HPIV3, SEQ ID NO: 14;
HPIV1, SEQ ID NO: 15; or BPIV3, SEQ ID NO: 16, as indicated). HMPV
strain 001, SEQ ID NO: 12, is included in the alignments for
comparison. Numbers at the left indicate the positional reference
of the first residue in the partial amino acid sequence shown, and
numbers to the right indicate the positional reference number of
the terminal residue in the partial sequence. The more highly
conserved or similar residues are denoted by shading. Dashes
indicate a gap in the particular sequence introduce to maximize the
alignment.
[0048] FIG. 27 is a graph illustrating multi-step growth of
rHMPV-GFP containing the F456L mutation (see FIG. 26A), compared to
rHMPV, performed in LLC-MK2 cells as described above for FIG. 13.
Clones #3 and #7 represent viruses derived independently from two
sister cDNA clones.
[0049] FIG. 28 is a diagram illustrating the GS signal of the M2
gene of wild type (WT) RSV (SEQ ID NO: 31), and a
highly-attenuating T to C nucleotide substitution at position nine
in the GS signal of the M2 gene of the attenuated cpts248/404 RSV
mutant (Whitehead et al., J. Virol. 72:4467-4471, 1998). There is a
corresponding T residue that is highly conserved among all of the
putative GS signals of HMPV. The individual GS motifs are named
according to the putative ORF that each precedes (SEQ ID NO: 1,
bases 41-57 for N; bases 1249-1265 for P; bases 2166-2182 for M;
bases 3053-3069 for F; bases 4710-4726 for M2;bases 5467-5483 for
SH; bases 6218-6234 for G; and bases 7119-7135 for L). The sequence
at the bottom (SEQ ID NO: 32) indicates in upper case letters those
positions in the putative HMPV GS signal that are exactly conserved
among all of the signal in strain 83, which includes the T at
position nine.
[0050] FIG. 29 is a diagrammatic representation of the genome
structure of biologically-derived HMPV strains 83 and CAN98-75
(hereafter referred to as 75 or CAN75), representing the two
proposed genetic HMPV subgroups and thus illustrating the diversity
that must be considered in designing an HMPV immunogenic
composition. Individual genes are indicated by boxes, with gene
lengths and boundaries within the complete genomic sequence given
in nucleotides together with the unmodified amino acid length
(italics) of the deduced protein (in the case of M2, two predicted
proteins, M2-1 and M2-2). The nucleotides lengths of the extragenic
3' leader, 5' trailer and intergenic regions are underlined.
[0051] FIG. 30 is a diagram illustrating the construction of
chimeric viruses constructed with genes of HMPV and APV, which
represent potential vaccine candidates. Part A illustrates the
genome of HMPV83 and derivatives in which the N, P or M ORF is
replaced individually by its APV counterpart (unshaded boxes).
Parts B, C and D illustrate the structures of cDNAs containing the
N, P and M genes, respectively of HMPV (upper line in each box)
compared to a version containing the indicated substituted APV ORF
(lower line in each box). Each ORF is shown as a shaded box and is
flanked by HMPV GS (SEQ ID NO: 1, bases 12-57 for the leader/N GS
signal; bases 1235-1265 for the N GE/P GS signal; and bases
2146-2182 for the P GE/M GS signal) and GE (SEQ ID NO: 1, bases
1235-1242 for the partial N GE signal; bases 2146-2151 for the
partial P GE signal; and bases 3007-3037 for the M GE signal)
transcription signals (boxed). In the case of the APV M gene, 64 nt
of noncoding sequence that separates the ORF from the GE signal
have been deleted -64 nts nc). The GE and GS signals in turn are
flanked by restriction sites selected from the following list, as
indicated: BbsI, BsmBI, BfuAI, BbsI. Each of these sites consists
of a recognition sequence (underlined) and has the property of
cutting in a sequence-independent fashion at a second site outside
of the recognition site (underlined tetranucleotide, bold) to leave
a 4-nt 5'-protruding overhang. Here, the overhangs were TAAT or
GTAG, as indicated, which matches the natural HMPV assignments at
these positions. Part E illustrates a novel assembly strategy that
utilizes the 4-nt overhangs, plus the flanking MluI and NheI sites,
for ligation of the N, P and M cDNAs simultaneously into the HMPV
backbone. By this strategy, this can be used to mix HMPV and APV N,
P and M genes in whatever combinations desired to make replacements
of single HMPV genes (as in part A) or to replace two or three
genes simultaneously.
[0052] FIG. 31 is a diagram showing the comparison of the predicted
gene boundaries, cis-acting GS and GE signals, and intergenic
regions of HMPV strains 83 (SEQ ID NO: 1) and 75 (SEQ ID NO: 2),
shown as positive-sense sequence. The top alignment shows the
leader/N gene boundary, specifically the last 16-18 nucleotides of
the leader region followed by the first 15 nucleotides of the N
gene. The second alignment shows the N/P gene boundary,
specifically the last 15 nucleotides of the N gene, followed by the
adjoining intergenic region, followed by the first 15 nucleotides
of the P gene. Subsequent alignments show the P/M, M/F, F/M2,
M2/SH, SH/G, G/L, and L/trailer boundaries. Conserved sequence
motifs at the end (gene end) and beginning (gene start) of each
gene are indicated in hold upper case, and a consensus is given
below (SEQ ID NOs: 34 and 35). Positions within these conserved
motifs are numbered. Translational stop and start codons are
underlined. Intergenic sequences are shown: in the case of the
longer intergenic regions, only the first 5 nucleotides on the
upstream and downstream ends are shown and the number of
nucleotides not shown is indicated. For each assignment in the
consensus sequence, no more than two of the sequence pairs (HMPV 83
versus 75) could have a heterologous assignment in both subgroup
sequences.
[0053] FIG. 32 is a chart illustrating the percent amino acid and
nucleotide sequence identity (the latter given in parentheses)
between the predicted proteins and ORFs of: HMPV 83 versus HMPV 75,
representing a comparison across subgroups; HMPV83 versus HMPV
00-1, representing a comparison within a subgroup; and RSV A2
versus RSV B1, representing a comparison across the two
well-characterized RSV antigenic subgroups A and B, which serve
here as a benchmark for assessing the magnitude and anticipated
significance of the differences in the HMPV subgroups. Amino acid
sequence identities were calculated based on the complete predicted
proteins; in the case of G and SH, overhangs on the
carboxy-terminal side of alignments due to length differences were
not included in the calculations. Nucleotide sequence identities
for the corresponding ORFs are shown in parentheses and are based
on the protein-coding sequence exclusive of flanking noncoding
sequence. For comparison, the HMPV intergenic regions were 48%
identical between subgroups and noncoding gene sequences exclusive
of conserved transcription signals were 54% identical, compared to
values of 42% and approximately 50% for RSV (Johnson et al., J.
Gen. Virol. 69:2901-2906, 1988).
[0054] FIGS. 33A-33B are diagrams showing the alignment of the
amino acid sequences of the SH (FIG. 33A) and G (FIG. 33B) proteins
of HMPV strains 75 (SEQ ID NOs: 8 and 9, respectively), 83 (SEQ ID
NOs: 5 and 6, respectively) and 00-1 (SEQ-ID NOs: 10 and 11,
respectively). This includes comparison between (75 versus 83) and
within (83 versus 00-1) the proposed HMPV genetic subgroups. For
strains 75 and 00-1, assignments that differ from that of 83 are
shown; dashes indicate gaps introduced to maximize the alignment or
to denote the absence of corresponding amino acids. Stars
underneath each alignment denote amino acid identity among all
three sequences; small dots indicate amino acid similarity among
all three. Proposed signal/transmembrane domains are boxed. Motifs
for N-linked carbohydrate are underlined (N-X-T/S, where X is not
proline). In panel A, cysteine residues conserved among all three
SH proteins are indicated with large dots, and potential sites for
O-linked glycosylation (Hansen et al., Glycoconj. J. 15:115-130,
1998) of the SH proteins are as follows: HMPV 83, 75, 77, 78, 81;
HMPV 75, 78, 79, 81; 00-1, 77, 78, 81.The ectodomain of the G
protein of each virus contained more than 40 potential acceptor
sites for O-linked carbohydrate.
[0055] FIG. 34 is a diagram illustrating the design of a chimeric
HMPV virus bearing antigens specific to HMPV strains that represent
two different antigenic subgroups of HMPV. The strain 83
antigenomic cDNA clone was used as the backbone (sequence
representing part of the 3' leader region (bases 23-41 of SEQ ID
NO: 1) and the putative N GS signal and beginning of the putative N
ORF (bases 42-66 of SEQ ID NO: 1) is shown), and a transcription
cassette was designed that consists of the ORF encoding the
putative G, F, or SH surface proteins of strain 75 under the
control of transcription signals from strain 83 (which including
bases 42-57 of SEQ ID NO: 1, representing the conserved putative N
GS signal, and bases 4685-4697 of SEQ ID NO: 1, representing the
putative F GE signal). This transcription cassette is then inserted
into the strain 83 backbone at a promoter-proximal site. This
strategy for constructing a bivalent immunogenic construct employs
the same strain 83 transcription signals and insertion site as
shown for the HMPV-GFP construct in FIG. 9.
[0056] FIG. 35 is a graph illustrating the daily level of
replication of HMPV strains 75 and 83 in the upper and lower
respiratory tract of African green monkeys. Mean daily virus titers
in the nasopharyngeal (upper panel) or tracheal lavage (lower
panel) specimens obtained on the indicated day post-inoculation
from animals infected simultaneously by the intranasal and
intratracheal routes with 105 TCID50 of the indicated virus: strain
75 (.circle-solid.), strain 83 (.box-solid.).
[0057] FIGS. 36A-36B are a diagram and digitized images showing the
construction and expression of a recombinant HPIV1 vector
expressing the F protein of HMPV CAN83 (rHPIV1-F.sub.83). The HPIV1
genome was modified by the creation of an MluI restriction site
(HPIV1 nt 113-118) one nucleotide prior to the translational start
codon of the N ORF (HPIV1 nt 119-121). The F ORF of HMPV strain
CAN83 (1620 nt in length and encoding a 539 aa polypeptide; bases
30544697 of SEQ ID NO: 1) was engineered by PCR to be followed by
the tetranucleotide TAAT and an HPIV1 gene junction consisting of a
GE signal, CTF intergenic region, and a GS signal. The length of
the entire cassette was 1656 nt and was designed, upon insertion
into the MluI site, to conform to the rule of six and to maintain
the HPIV1 GS signal sequence phasing. GS and GE signals are
indicated (FIG. 36A). A portion of the predicted viral promoter
that lies within the N gene is shown (FIG. 36A). LLC-MK2 cells were
infected with rHPIV-F83, HMPV CAN83, rHPIV1 or mock-infected, as
indicated, incubated for 72 hr, and analyzed by indirect
immunofluorescence using anti-HMPV polyclonal hamster serum or a
mixture of two mouse monoclonal antibodies to the HPIV1 HN protein,
as indicated (FIG. 36B).
[0058] FIGS. 37A-37D are the complete sequence of the genome of
biologically-derived strain 83, shown as positive sense DNA (SEQ ID
NO: 1). See also, GenBank Accession No. AY297749.
[0059] FIGS. 38A-38D are the complete sequence of the genome of
recombinant strain 83 bearing a transcription cassette expressing
GFP, which recombinant strain exemplifies rHMPV-GFP of the current
disclosure (SEQ ID NO: 3).
[0060] FIGS. 39A-39D are the complete sequence of the genome of
biologically-derived, biologically-cloned HMPV CAN75 (SEQ ID NO:
2). See also, GenBank Accession No. AY297748.
SEQUENCE LISTING
[0061] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand. In
the accompanying sequence listing:
[0062] SEQ ID NO: 1 shows the nucleic acid sequence of the genome
of biologically-derived HMPV (strain 83) as positive sense DNA.
[0063] SEQ ID NO: 2 shows the nucleic acid sequence of the genome
of biologically-derived HMPV (strain 75) as positive sense DNA.
[0064] SEQ ID NO: 3 shows the nucleic acid sequence of the genome
of recombinant HMPV (strain 83) with a transcription cassette
expressing GFP as positive sense DNA.
[0065] SEQ ID NO: 4 shows the amino acid sequence of the HMPV
(strain 83) M2-1 protein.
[0066] SEQ ID NO: 5 shows the amino acid sequence of the HMPV
(strain 83) SH protein.
[0067] SEQ ID NO: 6 shows the amino acid sequence of the HMPV
(strain 83) G protein.
[0068] SEQ ID NO: 7 shows the amino acid sequence of the HMPV
(strain 83) L protein.
[0069] SEQ ID NO: 8 shows the amino acid sequence of the HMPV
(strain 75) SH protein.
[0070] SEQ ID NO: 9 shows the amino acid sequence of the HMPV
(strain 75) G protein.
[0071] SEQ ID NO: 10 shows the amino acid sequence of the HMPV
(strain 00-1) SH protein.
[0072] SEQ ID NO: 11 shows the amino acid sequence of the HMPV
(strain 00-1) G protein.
[0073] SEQ ID NO: 12 shows the amino acid sequence of the HMPV
(strain 00-1) L protein.
[0074] SEQ ID NO: 13 shows the amino acid sequence of the HRSVA2 L
protein.
[0075] SEQ ID NO: 14 shows the amino acid sequence of the HPIV3 L
protein.
[0076] SEQ ID NO: 15 shows the amino acid sequence of the HPIV1 L
protein.
[0077] SEQ ID NO: 16 shows the amino acid sequence of the BPIV1 L
protein.
[0078] SEQ ID NO: 17 shows the nucleic acid sequence of the HMPV
(strain 00-1) 3' leader.
[0079] SEQ ID NO: 18 shows the nucleic acid sequence of the HMPV
(strain 83) 3' leader.
[0080] SEQ ID NO: 19 shows the nucleic acid sequence of the APV 3'
leader.
[0081] SEQ ID NO: 20 shows the nucleic acid sequence of the RSV 3'
leader.
[0082] SEQ ID NO: 21 shows the nucleic acid sequence of the HMPV
(strain 00-1) 5' trailer.
[0083] SEQ ID NO: 22 shows the nucleic acid sequence of the HMPV
(strain 83) 5' trailer.
[0084] SEQ ID NO: 23 shows the nucleic acid sequence of the APV 5'
trailer.
[0085] SEQ ID NO: 24 shows the nucleic acid sequence of the RSV 5'
trailer.
[0086] SEQ ID NO: 25 shows the nucleic acid sequence of the HMPV
(strain 83) GS consensus sequence.
[0087] SEQ ID NO: 26 shows the nucleic acid sequence of the HMPV
(strain 83) GS consensus sequence, with alternative nucleic acid
assignments.
[0088] SEQ ID NO: 27 shows the nucleic acid sequence of the HMPV
(strain 83) GS consensus sequence, with alternative nucleic acid
assignments.
[0089] SEQ ID NO: 28 shows the nucleic acid sequence of the HMPV
(strain 83) GE consensus sequence.
[0090] SEQ ID NO: 29 shows the nucleic acid sequence of the HMPV
(strain 83) GE consensus sequence, with alternative nucleic acid
assignments.
[0091] SEQ ID NO: 30 shows the nucleic acid sequence of the HMPV
(strain 83) GE consensus sequence, with alternative nucleic acid
assignments.
[0092] SEQ ID NO: 31 shows the nucleic acid sequence of the RSV M2
GS sequence.
[0093] SEQ ID NO: 32 shows the nucleic acid sequence of the HMPV
(strain 83) GS consensus sequence, with uniformly conserved
bases.
[0094] SEQ ID NO: 33 shows the nucleic acid sequence of a
polylinker used in the cloning and expression of rHMPV.
[0095] SEQ ID NO: 34 shows the nucleic acid sequence of the HMPV
(strain 83/75) GE consensus sequence.
[0096] SEQ ID NO: 35 shows the nucleic acid sequence of the HMPV
(strain 83/75) GS consensus sequence.
[0097] SEQ ID NO: 36 shows the nucleic acid sequence of the genome
of HMPV (strain 00-1) as positive sense DNA.
Detailed Description of Several Embodiments
[0098] In one embodiment, methods and compositions are provided for
producing an infectious, self-replicating, recombinant HMPV from
one or more isolated polynucleotide molecules encoding viral
sequences. The methods and compositions generally involve
coexpressing in a cell or cell-free system one or more expression
vectors comprising a polynucleotide molecule that encodes a partial
or complete, recombinant HMPV genome or antigenome and one or more
polynucleotide molecules encoding HMPV N, P, and L proteins, so as
to produce an infectious HMPV particle. In certain embodiments, the
methods and compositions for producing the recombinant HMPV further
include expression of the M2-1 gene.
[0099] Typically, the polynucleotide molecule that encodes the
recombinant HMPV genome or antigenome is a cDNA. Thus, disclosed
herein are polynucleotides such as cDNAs and their equivalents that
encode a recombinant HMPV. Expression vectors and constructs that
incorporate a polynucleotide molecule encoding a recombinant HMPV
genome or antigenome are also disclosed herein.
[0100] The HMPV genome or antigenome, and the N, P, M2-1 and L
proteins can all be produced from a single expression vector.
However, the genome or antigenome can be produced by a separate
expression vector, and the N, P, M2-1 and L proteins can be
produced by one, two, or more additional expression vectors. One or
more of the N, P, M2-1 and L proteins can be supplied by expression
of a recombinant HMPV genome or antigenome. These proteins can also
be supplied by coinfection with the same or different HMPV. Thus,
in several embodiments, one or more of the N, P, M2-1 and L
proteins are from a heterologous HMPV.
[0101] Infectious, recombinant, self-replicating viral particles
are disclosed that are produced according to the foregoing methods.
These particles include complete viruses as well as viruses that
lack one or more non-essential proteins or non-essential portions
(for example, a cytoplasmic, transmembrane or extracellular domain)
of a viral protein. Viruses of the current disclosure that lack one
or more such non-essential components (for example, a gene or
genome segment from the HMPV SH, G, M2-2 open reading frame (ORF)
or comparable accessory ORFs, or a segment of one or more other
ORFs, or an intergenic or other non-coding or non-essential genome
component) are referred to herein as incomplete viruses or
"subviral particles." Exemplary subviral particles can lack any
selected structural element, including a gene, gene segment,
protein, protein functional domain, etc. that is present in a
complete virus (that is, an assembled virion including a complete
genome or antigenome, nucleocapsid and envelope). For example, a
subviral particle of the current disclosure can include an
infectious nucleocapsid containing a genome or antigenome, and the
products of the N, P, and L genes. Other subviral particles are
produced by partial or complete deletions or substitutions of
non-essential genes and/or their products among other non-essential
structural elements.
[0102] Complete viruses and subviral particles produced by the
methods disclosed herein are typically infectious and
self-replicative through multiple rounds of replication in a
mammalian host amenable to infection by HMPV. These hosts include
various in vitro mammalian cell populations, in vivo animal models
widely known and accepted in the art as reasonably predictive of
HMPV activity in humans (including, mice, hamsters, cotton rats,
non-human primates including African green monkeys and
chimpanzees), and humans, including seronegative and seropositive
infants, children, juveniles, and adults. However, viruses and
subviral particles also can be produced that are highly defective
for replication in vivo.
[0103] In one embodiment, the polynucleotide molecule encodes a
sequence of a wild type HMPV (either the genome or the antigenome).
The term "wild type" refers to a gene or gene product which has the
characteristics of that gene or gene product when isolated from a
naturally occurring source. A wild-type gene is that which is most
frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene. In another
embodiment, the genome or antigenome can include one or more
mutations from a biologically derived mutant HMPV, or any
combination of recombinantly-introduced mutations; including one or
more polynucleotide insertions, deletions, substitutions, or
rearrangements that is/are selected to yield desired phenotypic
effects in the recombinant virus.
[0104] Thus, the recombinant HMPV genome or antigenome can be
engineered using the methods disclosed herein to incorporate a
recombinantly-introduced restriction site marker, or a
translationally silent point mutation for handling or marking
purposes. In other embodiments, the polynucleotide molecule
encoding the recombinant HMPV genome or antigenome can incorporate
one or more recombinantly-introduced attenuating mutations. In
specific examples, the recombinant HMPV genome or antigenome
incorporates one or more recombinantly-introduced, temperature
sensitive (ts) or host range (hr) attenuating (att) mutations.
[0105] The recombinant HMPV genome or antigenome can incorporate
one or more attenuating mutations identified in a biologically
derived mutant HMPV strain, or in another mutant nonsegmented
negative stranded RNA virus. For example, a mutation in a L, M, N,
13, M2-1, M2-2, SH, F, or G protein, or in an extragenic sequence
selected from a 3' leader or in a control signal such as the M2 GS
sequence. Where the mutation is of one or more particular amino
acid residues, the recombinant HMPV genome or antigenome can
incorporate multiple nucleotide changes in the codons specifying
the mutation to stabilize the modification against reversion.
[0106] The recombinant HMPV genome or antigenome can include an
additional nucleotide modification specifying a phenotypic change,
such as an alteration in growth characteristics, attenuation,
temperature-sensitivity, cold-adaptation, plaque size, host-range
restriction, or a change in immunogenicity. These additional
modifications can alter one or more of the HMPV N, P, M, SH, G, F,
M2-1, M2-2 and/or L genes and/or a 3' leader, 5' trailer, a
cis-acting sequence such as a GS or GE sequence, and/or intergenic
region within the HMPV genome or antigenome. For example, one or
more HMPV genes can be deleted in whole or in part, or expression
of the genes can be reduced or ablated by a frameshift mutation, by
a mutation that alters a translation start site, by introduction of
one or more stop codons in an ORF of the gene, or by a mutation in
a transcription signal. In several embodiments, the recombinant
HMPV genome or antigenome is modified by a partial or complete
deletion of the HMPV SH, G, or M2-2 gene or ORFs, or one or more
nucleotide changes that reduces or ablates expression of one or
more HMPV genes yet yields a viable, replication competent,
infectious viral construct. In other embodiments, the recombinant
HMPV genome or antigenome is modified to encode a non-HMPV molecule
such as a cytokine, a T-helper epitope, a restriction site marker,
or a protein of a microbial pathogen capable of eliciting a
protective immune response in a mammalian host.
[0107] A recombinant HMPV genome or antigenome can include a
partial or complete HMPV "vector" genome or antigenome combined
with one or more heterologous genes or genome segments (nucleic
acid sequences) encoding one or more antigenic determinants of one
or more heterologous pathogens to form a chimeric HMPV genome or
antigenome. The heterologous genes or genome segments encoding the
antigenic determinants can be added as supernumerary genes or
genome segments adjacent to or within a noncoding region of the
partial or complete HMPV vector genome or antigenome, or can be
substituted for one or more counterpart genes or genome segments in
a partial HMPV vector genome or antigenome. The heterologous genes
or genome segments can include one or more heterologous coding
sequences and/or one or more heterologous regulatory elements
comprising all extragenic 3' leader or 5' trailer region, a GS
signal, GE signal, editing region, translational start site,
intergenic region, or a 3' or 5' non-coding region.
[0108] In additional embodiments, the heterologous pathogen is one
or more heterologous pneumoviruses (for example, a heterologous
HMPV or RSV) and the heterologous genes or genome segments encodes
one or more HMPV or RSV N, P, M, SH, G, 1, M2-1, M2-2 and/or L
proteins or fragments thereof. Thus, the antigenic determinants can
be from a heterologous HMPV or RSV G, SH and F glycoproteins, and
antigenic domains, fragments and epitopes thereof, that is/are
added to or substituted within the partial or complete HMPV genome
or antigenome. In several examples, genes encoding G, SH and F
glycoproteins of a heterologous HMPV or RSV are substituted for
counterpart HMPV G, SH and F genes in a partial HMPV vector genome
or antigenome. In additional examples, genes encoding the G, SH,
and F glycoproteins of a heterologous HMPV or RSV are expressed in
addition to the HMPV sequences. In this manner, a plurality of
heterologous genes or genome segments encoding antigenic
determinants of multiple heterologous pneumoviruses can be added to
or incorporated within the partial or complete HMPV vector genome
or antigenome. In other embodiments, one or more genes from APV or
a related pneumovirus exhibiting a host range restriction in humans
is used to replace the corresponding genes in humans to achieve an
attenuated derivative and other improved properties.
[0109] The disclosed recombinant human metapneumoviruses (HMPVs)
can be used to generate a desired immune response against one or
more HMPVs, or against HMPV and one or more non-HMPV pathogens, in
a subject susceptible to infection. Recombinant HMPV as disclosed
herein are capable of eliciting a mono- or poly-specific immune
response in an infected mammalian host, yet are sufficiently
attenuated so as to not cause unacceptable symptoms of disease in
the immunized host. The attenuated viruses, including complete
viruses and subviral particles, can be present in a cell culture
supernatant, isolated from the culture, or partially or completely
purified. The virus can also be lyophilized, and can be combined
with a variety of other components for storage or delivery to a
host, as desired.
[0110] Immunogenic compositions are also disclosed herein that
include a physiologically acceptable carrier and/or adjuvant and an
isolated attenuated recombinant HMPV virus. In one embodiment, the
immunogenic composition includes a recombinant HMPV having at least
one, at least two, or more attenuating mutations or other
nucleotide modifications that specify a suitable balance of
attenuation and immunogenicity. In specific examples, the
immunogenic composition can be formulated in a dose of 10.sup.3 to
10.sup.7 PFU of attenuated virus. The immunogenic composition can
include an attenuated recombinant HMPV that elicits an immune
response against a single HMPV strain or against multiple HMPV
strains or serotypes or other pathogens such as RSV and/or HPIV. In
this regard, recombinant HMPV can be combined in formulations with
other HMPV strains, or with other candidate viruses such as a live
attenuated RSV. Methods are also disclosed herein for stimulating
the immune system of a mammalian subject to elicit an immune
response against one or more HMPVs, or against HMPV and a non-HMPV
pathogen. Thus, a method is provided herein for inducing an immune
response against a single HMPV, against multiple HMPVs, or against
one or more HMPVs and a non-HMPV pathogen such as RSV.
[0111] The disclosed recombinant HMPVs can be used to elicit a
monospecific immune response or a polyspecific immune response
against multiple HMPVs, or against one or more HMPVs and a non-HMPV
pathogen. Alternatively, recombinant HMPV having different
immunogenic characteristics can be combined in a mixture or
administered separately in a coordinated treatment protocol to
elicit an immune response against one HMPV, against multiple HMPVs,
or against one or more HMPVs and a non-HMPV pathogen such as RSV.
In one example, the immunogenic compositions are administered to
the upper respiratory tract, for example, by spray, droplet or
aerosol.
[0112] An operable set of viral open reading frames (ORFs)
sufficient to direct viral transcription and RNA replication are
disclosed herein. Diagnostic assays using this set of ORFs are
provided that determine viral activities based either on infectious
virus or on the expression of isolated polynucleotide molecules
encoding viral sequences. In one example, an infectious HMPV
recombinant virus is disclosed herein that expresses a detectable
marker or label, such as a recombinant HMPV expressing the
jellyfish green fluorescent protein (GFP). These and related tools
are effective, for example, to determine and characterize HMPV
infection in vitro and in vivo. In one exemplary embodiment,
recombinant HMPV expressing a detectable label can be utilized in
assays and related compositions for the detection of
HMPV-neutralizing antibodies in biological specimens (for example,
serum of patients at risk of HMPV infection or presenting with
respiratory symptoms). In additional embodiments the recombinant
HMPV expressing a detectable label can be used to screen compounds
for antiviral activity.
[0113] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes VII, published by
Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN
0471186341); and other similar references.
[0114] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. Also, as used
herein, the term "comprises" means "includes." Hence "comprising A
or B" means including A, B, or A and B. It is further to be
understood that all base sizes or amino acid sizes, and all
molecular weight or molecular mass values, given for nucleic acids
or polypeptide are approximate, and are provided for description.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
explanations of terms, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
[0115] As described above, methods and compositions for the
production and use of novel, recombinant HMPVs are provided herein.
The recombinant HMPVs are infectious and immunogenic in humans and
other mammals and are useful as immunogenic compositions to produce
immune responses against one or more HMPVs or other pneumoviruses.
Thus, the recombinant HMPVs can be used to produce an immune
response to, for example, RSV and/or one or more HMPV or other
pneumoviral strains, serotypes, or subgroups. Chimeric HMPVs are
provided herein that can be used to elicit an immune response
against a selected HMPV and one or more additional pathogens, for
example against multiple HMPVs or against a HMPV and a non-HMPV
virus such as RSV, or parainfluenza virus (PIV). The immune
response elicited can involve either or both humoral and/or cell
mediated responses. The HMPVs can be attenuated to yield a desired
balance of attenuation and immunogenicity.
[0116] Methods are provided herein for designing and producing
attenuated, HMPVs that are useful as agents for eliciting a desired
immune response against HMPV and other pathogens. Thus, using the
methods disclosed herein, recombinant HMPVs can be produced that
have a defined genome sequence and predictable characteristics.
[0117] An exemplary recombinant HMPV (rHMPV) includes a recombinant
HMPV genome or antigenome and encodes (or the antisense of which
encodes) HMPV major nucleocapsid (N) protein, nucleocapsid
phosphoprotein (P), and large polymerase protein (L) (see FIG. 1
and exemplary sequence information provided in FIG. 37). In
additional embodiments, the rHMPV can incorporate a recombinant
HMPV genome or antigenome, N, P, and L proteins, and a HMPV M2-1
protein. In further embodiments, one or more of the N, P, L, and/or
M2-1 proteins can be a mutant protein or partial protein, or
include a corresponding homologous protein or protein fragment of a
heterologous HMPV or non-HMPV virus such as APV. One or more
additional HMPV proteins can be coexpressed with the recombinant
HMPV genome or antigenome, in various combinations, to provide a
range of infectious viruses. As used herein the term "recombinant
HMPV" or "rHMPV" includes recombinantly produced subviral particles
lacking one or more non-essential viral components, complete
viruses having all native viral components, and viruses containing
supernumerary proteins, antigenic determinants or other additional
components. A recombinant virus is produced entirely from cloned
cDNA.
[0118] As set forth in the Examples below, a complete consensus
sequence was determined for the genomic RNA of an exemplary wild
type strain of HMPV. The sequence thus identified was used to
generate a full-length antigenomic cDNA and to recover a wild type
rHMPV. The biological properties of rHMPV in vitro and in vivo
demonstrates that the exemplary rHMPV sequence corresponds to a
wild type virus. This critical finding demonstrates that the
recombinant HMPV sequence disclosed herein is that of an authentic
wild type virus. This rHMPV serves as a novel substrate for
recombinant introduction of attenuating mutilations for the
generation of live-attenuated HMPV and HMPV-based chimeric and
vectors that can be used for immunogenic compositions.
[0119] As disclosed herein, infectious recombinant HMPVs can be
produced by a recombinant coexpression system that permits
introduction of defined changes into the recombinant HMPV and
provides for the generation, with high frequency and fidelity, of
HMPV having a defined genome sequence. These modifications are
useful in a wide-variety of applications, including the development
of live attenuated viral strains bearing predetermined, defined
attenuating mutations. Infectious HMPV can be produced by
intracellular or cell-free coexpression of one or more isolated
polynucleotide molecules that encode the HMPV genome or antigenome
RNA, together with one or more polynucleotides encoding viral
proteins to generate a transcribing, replicating nucleocapsid. An
"infectious virus" is a viral particle that has the ability to
deliver its genome to the cytoplasm of a host cell.
[0120] The cDNAs encoding a HMPV genome or antigenome are
constructed for intracellular or in vitro coexpression with the
selected viral proteins to form infectious HMPV. A "HMPV
antigenome" is an isolated positive-sense polynucleotide molecule
which serves as a template for synthesis of progeny HMPV genome. In
one embodiment a cDNA is constructed which is a positive-sense
version of the HMPV genome that corresponds to the replicative
intermediate RNA, or antigenome. This minimizes the possibility of
hybridizing with positive-sense transcripts of complementing
sequences encoding proteins necessary to generate a transcribing,
replicating nucleocapsid. A "replication competent virus" is a
viral particle capable of initiating an infection that produces
viral progeny.
[0121] In some embodiments the genome or antigenome of a
recombinant HMPV contains only those genes or portions thereof
necessary to render the viral or subviral particles encoded thereby
infectious. Further, the genes or portions thereof may be provided
by more than one polynucleotide molecule, that is, a gene may be
provided by complementation or the like from a separate nucleotide
molecule. In other embodiments, the HMPV genome or antigenome
encodes all functions necessary for viral growth, replication, and
infection without the participation of a helper virus or viral
function provided by a plasmid or helper cell line.
[0122] A "recombinant HMPV particle" is a HMPV or HMPV-like viral
or subviral particle derived directly or indirectly from a
recombinant expression system or propagated from virus or subviral
particles produced there from. The recombinant expression system
can employ a recombinant expression vector which includes an
operably linked transcriptional unit comprising an assembly of at
least a genetic element or elements having a regulatory role in
HMPV gene expression, for example, a promoter, a structural or
coding sequence which is transcribed into HMPV RNA, and appropriate
transcription initiation and termination sequences. A first nucleic
acid sequence is "operably linked" with a second nucleic acid
sequence when the first nucleic acid sequence is placed in a
functional relationship with the second nucleic acid sequence.
[0123] To produce infectious HMPV from a cDNA-expressed HMPV genome
or antigenome, the genomic or antigenome is coexpressed with those
HMPV or heterologous viral proteins necessary to produce a
nucleocapsid capable of RNA replication, and render progeny
nucleocapsids competent for both RNA replication and transcription.
Transcription by the genome nucleocapsid provides the other HMPV
proteins and initiates a productive infection. Alternatively,
additional HMPV proteins useful for a productive infection can be
supplied by coexpression.
[0124] In certain embodiments, complementing sequences encoding
proteins necessary to generate a transcribing, replicating HMPV
nucleocapsid are provided by one or more helper viruses. Such
helper viruses can be wild type or mutant. In one embodiment, the
helper virus can be distinguished phenotypically from the virus
encoded by the HMPV cDNA. For example, it may be desirable to
provide monoclonal antibodies that react immunologically with the
helper virus but not the virus encoded by the HMPV cDNA. Such
antibodies can be neutralizing antibodies. In some embodiments, the
antibodies can be used in affinity chromatography to separate the
helper virus from the recombinant virus. To aid the procurement of
such antibodies, mutations can be introduced into the HMPV cDNA to
provide antigenic diversity from the helper virus, such as in the
HMPV glycoprotein genes.
[0125] Expression of the HMPV genome or antigenome and proteins
from transfected plasmids can be achieved, for example, by each
cDNA being under the control of a selected promoter (for example,
for T7 RNA polymerase), which in turn is supplied by infection,
transfection or transduction with a suitable expression system (for
example, for the T7 RNA polymerase, such as a vaccinia virus MVA
strain recombinant which expresses the T7 RNA polymerase, as
described by Wyatt et al., Virology 210:202-205, 1995). The viral
proteins, and/or T7 RNA polymerase, can also be provided by
transformed mammalian cells (see Buchholz et al., J. Virol.
73:251-259. 1999) or by transfection of preformed mRNA or
protein.
[0126] A HMPV genome or antigenome can be constructed for use by,
for example, assembling cloned cDNA segments, representing in
aggregate the complete genome or antigenome, by PCR or the like
(described in, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202,
and PCR Protocols: A Guide to Methods and Applications, Innis et
al., eds., Academic Press, San Diego, 1990) of reverse-transcribed
copies HMPV mRNA or genome RNA. For example, a first construct can
be generated which includes cDNAs containing the left hand end of
the antigenomic, spanning from an appropriate promoter (for
example, 17 RNA polymerase promoter) and assembled in an
appropriate expression vector, such as a plasmid, cosmid, phage, or
DNA virus vector. The vector can be modified by mutagenesis and/or
insertion of a synthetic polylinker containing unique restriction
sites designed to facilitate assembly. For case of preparation the
N, P, L and other desired HMPV proteins can be assembled in one or
more separate vectors. The right hand end of the antigenome plasmid
may contain additional sequences as desired, such as a flanking
ribozyme and single or tandem T7 transcriptional terminators. The
ribozyme can be hammerhead type, which would yield a 3' end
containing a single nonviral nucleotide, or can be any of the other
suitable ribozymes such as that of hepatitis delta virus (Perrotta
et al., Nature 350:434-436, 1991) that would yield a 3' end free of
non-PIV nucleotides.
[0127] Alternative means to construct cDNA encoding the HMPV genome
or antigenome include RT-PCR using different l)CR conditions (for
example, as described in Cheng et al., Proc. Natl. Acad. Sci. USA
91:5695-5699, 1994) to reduce the number of subunit cDNA components
to as few as one or two pieces. In other embodiments different
promoters can be used (for example, T3, SPQ or different ribozymes,
such as that of a hammerhead variety. Different DNA vectors (for
example, cosmids) can be used for propagation to better accommodate
the larger size genome or antigenome.
[0128] By "infectious clone" or "infectious cDNA" of HMPV is meant
cDNA or its product, synthetic or otherwise, as well as RNA capable
of being directly incorporated into infectious virions which can be
transcribed into genomic or antigenomic HMPV RNA that can serve as
a template to produce the genome of infectious HMPV viral or
subviral particles. As noted above, defined mutations can be
introduced into an infectious HMPV clone by a variety of
conventional techniques (for example, site-directed mutagenesis)
into a cDNA copy of the genome or antigenome. The use of genomic or
antigenomic cDNA subfragments to assemble a complete genome or
antigenome cDNA as described herein has the advantage that each
region can be manipulated separately, where small cDNA constructs
provide for better ease of manipulation than large cDNA constructs,
and then readily assembled into a complete cDNA.
[0129] Isolated polynucleotides (for example, cDNA) encoding the
HMPV genome or antigenome can be introduced into cells that can
support a productive HMPV infection by transfection,
electroporation, mechanical insertion, transduction or the like.
Exemplary cells of use are HEp-2, FRhL-DBS2, LLC-MK2, MRC-5, baby
hamster kidney (BHK), and Vero cells. Isolated polynucleotide
sequences can be introduced into cultured cells by, for example,
calcium phosphate-mediated transfection (Wigler et al., Cell
14:725, 1978; Corsaro et al., Somatic Cell Genetics 7:603, 1981;
Graham et al., Virology 52:456, 1973, electroporation (Neumann et
al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection
(Ausubel et al., (ed.) Current Protocols in Molecular Biology, John
Wiley and Sons, Inc., NY, 1987), cationic lipid-mediated
transfection (Hawley-Nelson et al., Focus 15:73-79, 1993) or a
commercially available transfection regent, for example,
Lipofectamine-2000 (Invitrogen, Carlsbad, Calif.) or the like.
[0130] By providing infectious clones of HMPV, a wide range of
alterations can be recombinantly produced within the HMPV genome
(or antigenome), yielding defined mutations that specify desired
phenotypic changes. The compositions and methods disclosed herein
for producing recombinant HMPV permit ready detailed analysis and
manipulation of HMPV molecular biology and pathogenic mechanisms
using, for example, defined mutations to alter the function or
expression of selected HMPV proteins. Using these methods and
compositions, one can readily distinguish mutations responsible for
desired phenotypic changes from silent incidental mutations, and
select phenotype-specific mutations for incorporation into a
recombinant HMPV genome or antigenome. In this context, a variety
of nucleotide insertions, deletions, substitutions, and
rearrangements can be made in the HMPV genome or antigenome during
or after construction of the cDNA. For example, specific desired
nucleotide sequences can be synthesized and inserted at appropriate
regions in the cDNA using convenient restriction enzyme-sites.
Alternatively, such techniques as site-specific mutagenesis,
alanine scanning mutagenesis, PCR mutagenesis, or other such
techniques well known in the art can be used to introduce mutations
into the cDNA.
[0131] Recombinant modifications of HMPV can be directed toward the
production of improved candidate viruses, for example, to enhance
viral attenuation and immunogenicity, to ablate epitopes associated
with undesirable immunopathology, to accommodate antigenic drift,
etc. To achieve these and other objectives, the compositions and
methods disclosed herein allow for a wide variety of modifications
to be introduced into a HMPV genome or antigenome for incorporation
into infectious, recombinant HMPV. For example, foreign genes or
gene segments encoding antigenic determinants (for example,
protective antigens or immunogenic epitopes) can be added within a
HMPV clone to generate recombinant HMPVs capable of inducing
immunity to both HMPV and another virus or pathogenic agent from
which the antigenic determinants was/were derived. Alternatively,
foreign genes can be inserted, in whole or in part, encoding
modulators of the immune system, such as cytokines, to enhance
immunogenicity of a candidate virus. Other mutations that can be
included within HMPV clones are, for example, substitution of
heterologous genes or gene segments (for example, a gene segment
encoding a cytoplasmic tail of a glycoprotein gene) with a
counterpart gene or gene segment in a HMPV clone. Alternatively,
the relative order of genes within a HMPV clone can be changed, a
HMPV genome promoter or other regulatory element can be replaced
with its antigenome counterpart, or selected HMPV genes rendered
non-functional (for example, by functional ablation involving
introduction of a stop codon to prevent expression of the gene). In
addition, the codon selection of genes such as those encoding the
major protective antigens can be modified to improve the efficiency
of translation. Other modifications in a HMPV clone can be made to
facilitate manipulations, such as the insertion of unique
restriction sites in various non-coding or coding regions of the
HMPV genome or antigenome. In addition, nontranslated gene
sequences or intergenic regions can be shortened or removed to
increase capacity for inserting foreign sequences.
[0132] As noted above, it is often desirable to adjust the
phenotype of recombinant HMPVs for use by introducing additional
mutations that attenuate the virus, affect the virulence of the
virus, or otherwise alter the phenotype of the recombinant virus.
One of skill in the art can readily identify methods and procedures
for mutagenizing, isolating and characterizing HMPV to obtain
attenuated mutant strains (for example, temperature sensitive (ts),
cold passaged (c!)), cold-adapted (ca), small plaque (sp) and
host-range restricted (hr) mutant strains) and for identifying the
genetic changes that specify the attenuated phenotype (see, for
example, Durbin et al., Virology 235:323-332, 1997; U.S. patent
application Ser. No. 09/083,793, filed May 22, 1998; U.S. patent
application Ser. No. 09/458,813, filed Dec. 10, 1999; U.S. patent
application Ser. No. 09/459,062, filed Dec. 10, 1999; U.S.
Provisional Application No.60/047,575, filed May 23, 1997
(corresponding to International Publication No. WO 98/53078), and
U.S. Provisional Application No. 60/059,385, filed September 19,
1997). Thus, methods are provided herein for determining
replication, immunogenicity, genetic stability and immunogenic
efficacy of biologically derived and recombinantly produced
attenuated HMPVs in accepted model systems reasonably correlative
of human activity, including hamster or rodent and non-human
primate model systems.
[0133] In additional embodiments, rHMPV candidates are constructed
by introduction of nucleotide or amino acid point mutations that
confer attenuation or other desired phenotypes. Such mutations can
involve substitution of one or more nucleotides or amino acids at a
given locus, or can involve small deletions in which one or more
nucleotides or amino acids at a given locus are deleted. These
mutations are termed "point mutations" here to denote that each
particular mutation is circumscribed. For example, a typical
mutation can involve changing a single amino acid by substituting
1, 2 or 3 nucleotides in the corresponding codon. As another
example, the three nucleotides might be deleted altogether,
resulting in the deletion of a single amino acid in the encoded
protein. Point mutations can be identified empirically, such as by
systematically replacing charged amino acids in one or more of the
HMPV proteins with ones that are uncharged, for example, replacing
aspartate, glutamate, lysine or arginine with alanine.
[0134] Alternatively, attenuating amino acid substitutions and
other mutations can be devised using existing mononegavirus
mutations as a guide, for example mutations in a different
"biologically derived" HMPV, non-human pneumovirus, such as an APV,
or non-HMPV virus, such as a RSV or PIV. By "biologically derived"
is meant any virus not produced by recombinant DNA methods. Thus,
biologically derived HMPV include all naturally occurring HMPVs,
including, for example, naturally occurring HMPV having a wild type
genomic sequence and HMPV having allelic or mutant genomic
variations from a reference wild type HMPV sequence, for example,
HMPV having a mutation specifying an attenuated phenotype.
Likewise, biologically derived HMPV include HMPV mutants derived
from a parental HMPV by, inter alia, artificial mutagenesis and
selection procedures not involving direct recombinant DNA
manipulation. Attenuating mutations in biologically derived HMPV
and other nonsegmented negative stranded RNA viruses for
incorporation within recombinant HMPV can occur naturally or can be
introduced into wild type HMPV strains and thereafter identified
and characterized by well known mutagenesis and analytic
procedures. For example, incompletely attenuated parental HMPV or
other heterologous viral mutant strains can be produced by chemical
mutagenesis during virus growth in cell cultures to which a
chemical mutagen has been added, by selection of virus that has
been subjected to passage at suboptimal temperatures in order to
introduce growth restriction mutations, or by selection of a
mutagenized virus that produces small plaques (sp) in cell culture.
In addition, known mutations in existing mononegaviruses that have
been produced by recombinant methods also can serve as a guide for
devising desired mutations in HMPV. Such known mutations in
heterologous viruses can be ones that had been deliberately
introduced by directed mutagenesis, or can be ones that arose
spontaneously during the process of DNA manipulation and virus
recovery and propagation, or can ones that arose in recombinantly
derived viruses that were exposed subsequently to mutagenic agents
or passaged in a manner designed to favor phenotypic changes, such
as described above for generating mutations in biologically derived
viruses.
[0135] In certain embodiments, the HMPV genome or antigenome is
recombinantly modified to incorporate an attenuating mutation at an
amino acid position corresponding to an amino acid position of an
attenuating mutation identified in a heterologous, mutant
nonsegmented negative stranded RNA virus. The virus can be either
biologically derived or of recombinant origin. Based on routine
sequence alignments and other analyses, mutations previously
identified in a heterologous HMPV or non-HMPV virus are mapped to a
corresponding position in HMPV for "transfer" (that is,
introduction of an identical, conservative or non-conservative
mutation, potentially including a substitution, deletion or
insertion, at a homologous or corresponding position identified by
the alignment) into recombinant HMPV. In many cases, an alignment
of the two sequences of interest is sufficient to identify the
corresponding residue in HMPV, particularly if the percent amino
acid identity is substantial (for example, approximately 35-40%
identity or greater) or high (for example, 70% identity or greater,
such as at least about 80%, 90%, 95%, or 99% identity). In other
cases, particularly when the amino acid relatedness is not
substantial (for example, less than approximately 35% identity),
additional heterologous related viruses can be included in the
alignment in order to obtain reliably identify conserved residues
that serve as markers to identify corresponding positions. In one
embodiment, the percentage of sequence identity associated with the
terms "substantial" or "high" or "not high" are used herein in the
context of comparison of heterologous viruses from different
serotypes or taxonomic groups.
[0136] A large assemblage of such candidate mutations are available
for transfer into rHMPV of the current disclosure (see, for
example, (Durbin et al., Virology 235:323-332, 1997; Skiadopoulos
et al., J. Virol. 72:1762-1768, 1998; Skiadopoulos et al., J.
Virol. 73:1374-1381, 1999; Skiadopoulos et al., Virology
260:125-35, 1999; Durbin et al., Virology 261:319-30, 1999; Newman
et al., Virus Genes 24:77-92, 2002; Feller et al., Virology
276:190-201, 2000; U.S. patent applicalion Ser. No. 09/083,793,
filed May 22, 1998; U.S. patent application Ser. No. 09/458,813,
filed Dec. 10, 1999; U.S. patent application Ser. No. 09/459,062,
filed Dec. 10, 1999; U.S. Provisional Application No. 60/047,575,
filed May 23, 1997 (corresponding to International Publication No.
WO 98/53078); U.S. Provisional Application No. 60/059,385, filed
Sep. 19, 1997; U.S. patent application Ser. No. 10/302,547, filed
Nov. 21, 2002 and priority U.S. Provisional Application No.
60/331,961, filed Nov. 21, 2001; and U.S. Provisional Application
No. 60/412,053, filed Sep. 18, 2002).
[0137] Within these embodiments, it is often desired to modify the
recipient recombinant HMPV genome or antigenome to encode an
alteration at the subject site of mutation that corresponds
conservatively to the alteration identified in the heterologous
mutant virus. For example, if an amino acid substitution marks a
site of mutation in the mutant virus compared to the corresponding
wild type sequence, then a similar substitution can-be engineered
at the corresponding residues in the recombinant HMPV. In one
example, the substitution will specify an identical or conservative
amino acid to the substitute residue present in the mutant viral
protein (see below for a description of conservative substations).
However, it is also possible to alter the native amino acid residue
at the site of mutation non-conservatively with respect to the
substitute residue in the mutant protein (for example, by using any
other amino acid to disrupt or impair the function of the wild type
residue). Negative stranded RNA viruses from which exemplary
mutations are identified and transferred into a recombinant HMPV of
the current disclosure include heterologous strains of HMPV, other
non-HMPV pneumoviruses (for example, human RSV, bovine RSV, APV,
pneumonia virus of mice (PVM)), PIVs (for example, HPIV1, HPIV2,
HPIV3, BPIV3, and MPIV1), Newcastle disease virus (NDV), simian
virus 5 (SV5), measles virus (MeV), rinderpest virus, canine
distemper virus (CDV), rabies virus (RaV), and vesicular stomatitis
virus (VSV), among others.
[0138] As depicted in FIGS. 26A-26F, one or more corresponding
sites of mutation that specify a desired phenotypic change in a
heterologous virus (for example, when indicated wild type residues
at the designated positions is/are altered, for example, by
substitution) is identified by conventional sequence alignment. The
corresponding target sites for mutation (see, for example, boxed
sites in FIGS. 26A-26F) in HMPV is thereby mapped for transfer of
the mutation into a recombinant HMPV to yield attenuation or other
desired phenotypic changes. More specifically, corresponding amino
acids between the compared heterologous mutant and HMPV sequences
represent target sites for identical or conservative transfer (for
example, by recombinant engineering involving site-directed
mutagenesis of a HMPV antigenomic cDNA) of the subject mutation
into a recombinant HMPV. In some embodiments, attenuating and other
desired mutations identified in one negative stranded RNA virus are
thereby targeted for transfer (for example, to be copied
identically or conservatively by substitution mutagenesis) into a
corresponding position within the genome or antigenome of a
recombinant HMPV of the current disclosure. Related methods for
rational design of other recombinant mutant RNA viruses are
described, for example, in International Application No.
PCT/US00/09695, filed Apr. 12, 2000, published as WO 00/61737 on
Oct. 19, 2000 corresponding to U.S. National Phase application
09/958,292, filed on Jan. 08, 2002, and claiming priority to U.S.
Provisional Patent Application Serial No. 60/129,006, filed on Apr.
13, 1999. Additional description pertaining to this aspect of the
current disclosure is provided in Newman et al., Virus Genes
24:77-92,2002; Feller et al., Virology
10;276:190-201,2000;Skiadopoulos et al., Virology 260:125-35, 1999;
and Durbin et al., Virology 261:319-30, 1999.
[0139] In one exemplary embodiment, amino acid sequence alignments
are made between the L gene of HMPV and the L genes of other
mononegaviruses for which one or more attenuating substitutions in
the L protein have been identified. This embodiment of the current
disclosure is illustrated in FIGS. 26A-26F, which depict sequence
alignments of various segments of the putative L protein of HMPV
with corresponding segments of L proteins of a number of other
mononegaviruses in which attenuating mutations have been mapped. As
exemplified in FIGS. 26A-26F, various mutations identified in the L
gene of a heterologous negative stranded RNA virus can be
incorporated into recombinant HMPV to yield attenuation or other
desired phenotypic changes. These figures provide exemplary,
partial L gene sequence alignments between an HMPV wild type virus
and the indicated viruses (including as examples, a different HMPV,
RSV, HPIV1 HPIV3, and BPIV3). The examples of attenuating mutations
include ones identified in viruses of biological or recombinant
origin. The alignments are employed to identify regions containing
known attenuating mutations in the heterologous virus.
[0140] FIGS. 26A-26F illustrate the identification and mapping of
various exemplary attenuating mutations in the L gene for transfer
into rHMPV (note that the numbers at the left indicate the
positional reference number of the first residue in the partial
sequence shown, and the numbers at the right indicate the
positional reference number of the terminal residue in the partial
sequence). FIG. 26A shows a Phenylalanine-521 to Leucine (F521L)
substitution in the L protein specified by a mutation in the L gene
of an RSV cold-passage temperature-sensitive (cpts) mutant cpts530
(Juhasz et al., J. Virol. 71:5814-9, 1999). Also shown is an
attenuating double mutation of Arginine-588-Alanine (R588A) and
Aspartate-589-Alanine (D589A) that was originally developed in RSV
(Tang et al., Virology 302:207-16, 2002). FIG. 26B shows the
positions of attenuating Tyrosine-942-Histidine (Y942H) and
Leucine-992-Tyrosine (L992Y) mutations identified in the PIV3 cp45
vaccine candidate (Skiadopoulos et al. J. Virol. 72:31762-8,1998;
U.S. patent application Ser. No. 10/302,547, filed November 21,
2002 and priority U.S. Provisional Application No. 60/331,961,
filed Nov. 21, 2001; and U.S. Provisional Application No.
60/412,053, filed Sep. 18, 2002). FIG. 26C shows the positions of
the attenuating Isoleucine-I1103-Valine (I1103V) mutation
originally identified in BPIV3 (Haller et al., Virology 288:342-50,
2001), the attenuating Threonine-1558-Isoleucine (T1558I) mutation
identified in PIV3 cp45 (Skiadopoulos et al., J. Virol. 72:31762-8,
1998), and the Cysteine-319-Tyrosine (C)319Y) mutation identified
the attenuated cpRSV vaccine candidate (Connors et al., Virology
208:478-84, 1995). FIG. 26D shows the positions of the attenuating
Glutamine-831-Leucine (Q831L) mutation identified in cpts248 RSV
(Firestone et al., Virology 225:419-22, 1996), the attenuating
Methinone-1169-Valine (M1169V) mutation identified in the RSV
mutant cpts530/109 (Juhasz et al., J. Virol. 73:5176-80), the
Aspartate-1183-Glutamate (D1183) mutation identified in the RSV
mutant cpts248/404 (Firestone et al., Virology 225:419-22, 1996),
and the combination of six point mutations that constitute the
attenuating C9 cluster characterized in recombinant RSV (Tang et
al., Virology 302:207-16, 2002): Aspartate-1187-Alanine (D)1187A),
Lysine-1188-Alanine (K1188A), Arginine-1189-Alanine (R1189A),
Glutamate-1190-Alanine (E1190A), Glutamate-1208-Alanine (E1208A),
and Arginine-1209-Alanine (R1209A). FIG. 26E shows the position of
the attenuating Tyrosine-1321-Asparagine (Y1321N) mutation of RSV
cpts530/1030 (Whitehead et al., J. Virol. 73:871-7, 1999), and the
Histidine-1690-Tyrosine (H1690Y) mutation of the attenuated cpRSV
derivative (Connors et al., Virology 208, 478-84, 1995). FIG. 26F
shows the positions of an attenuating Asparagine-43-Isoleucine
(N431) mutation identified in the cpts RSV mutant 248/955, and the
attenuating Threonine-1711-Isoleucine (T1711I) mutation
characterized in the chimeric virus rHPIV3-L.sub.B, consisting of
HPIV3 in which the L gene was replaced by that of BPIV3
(Skiadopoulos et al., J. Virol. 77:1141-8, 2003).
[0141] The foregoing alignments show, for example, that the F521
residue in the RSV sequence is exactly conserved among most of the
other mononegaviruses examined (23 out of 24 paramyxoviruses and 1
out of 3 rhabdoviruses) and, in particular, is present in HMPV. It
should be noted that the amino acid position of the corresponding
residue is not the same in each L protein, reflecting differences
in length as well as small deletions and insertions elsewhere in
the various individual molecules. Thus, the corresponding residue
is F456 in HMPV. From this comparative mapping analysis, the F456
residue in HMPV is identified as a target for either an identical,
conservative, or even non-conservative amino acid substitution (for
example, substitution of the F456 residue by a leucine, or by a
conservative or non-conservative amino acid as compared to
leucine). The same alignment (FIG. 26A) shows that the D589
assignment is also exactly conserved in each of sequences. The R588
assignment is not conserved in each, but it is represented by the
homologous assignment of Lysine in HMPV. With respect to the
attenuating Tyrosine-942-Histidine (Y942H) and Leucine-992-Tyrosine
(L992Y) mutations of PIV3 cp45, in both of these instances the
exact amino acid assignment was not conserved, but the presence of
various highly-conserved residues throughout the alignment show
that these positions correspond (FIG. 26B). Positional
correspondence in this context indicates that the aligned,
corresponding target site for mutation (whether it is occupied by
an amino acid that is identical, conservative or divergent in
structural character compared the corresponding residue in the wild
type (for example, a non-attenuated parent) sequence of the
heterologous virus) will often yield an attenuation or other
desired phenotype in a rHMPV upon mutation from the HMPV wild type
sequence to a different residue (that is, to a residue identity
that is the same, conservative, or even distinct structurally in
comparison to the corresponding residue in the heterologous
mutant).
[0142] In addition to the foregoing L gene mutations, two
temperature sensitive (ts) mutations that were reported in the P
protein of recombinant RSV, namely Glycine-172-Serine and
Glutamate-176-Glycine (Lu et al., J. Virol. 76:2871-80, 2002), can
be transferred to yield a predicted attenuation phenotype in a
rHMPV P), by altering the corresponding one or both of the HMPV
residues Glycine-213 and Glutamate-217, respectively. Likewise, a
mutation in the N protein of the attenuated derivative cpRSV,
specifying a Valine-267-Isoleucine substitution (Connors et al.
Virology 208:478-84, 1996), maps to Serine-268 in HMPV, whereby an
Ile or other, conservative or non-conservative, substitution of
this residue in a rHMPV can yield useful candidates for
identification of additional attenuated derivatives. In the same
manner, two mutations in the F protein of cpRSV, namely
Glutamate-218-Alanine and Threonine-523-Isoleucine (Connors et al.
Virology 208:478-84, 1996), correspond to Lysine-188 and
Threonine-489, respectively, in the HMPV F protein, which therefore
represent target sites for introduction of an attenuating amino
acid substitution in rHMPV.
[0143] In other embodiments, mutations in a cis-acting regulatory
sequence or other non-coding sequence are identified in a
heterologous virus (including biologically or otherwise derived
mutant HMPV of the same or different strain, subtype, species, RSV,
PIV, APV, etc.) and incorporated in a rHMPV of the current
disclosure. For example, the identification of the GS and GE motifs
identifies these as targets for mutagenesis. The short,
circumscribed nature of these signals is amenable to a systematic
evaluation of the effects of mutations at each position. Mutations
in RNA signals such as these can be readily assayed in a
mini-replicon system as well as in complete virus.
[0144] FIG. 28 shows an example of transferring a mutation that
involves a cis-acting RNA signal. A potent attenuating point
mutation identified for the cpts 248/404 derivative of RSV involves
a point mutation in the GS signal of the M2 gene: specifically, the
T residue (positive sense) at position nine was changed to C (see,
for example, Whitehead et al., Virology 247:232-9, 1998). In the
proposed GS signal of HMPV, a T residue at position nine is
conserved in each of the proposed HMPV genes (FIG. 28). This
substitution can be made in any of the HMPV genes.
[0145] In accordance with the foregoing description, a large panel
of desired mutations identified in heterologous viruses can be
"transferred" into a rHMPV, even in cases of low sequence
relatedness. In these embodiments, there is not a strict
requirement that the corresponding wild type or final mutant amino
acid assignment be identical to that of the heterologous virus.
Also, rather than an amino acid substitution, it is possible to
delete one or several amino acids or, alternatively, insert one or
several amino acids. In designing substitution mutations for HMPV,
a useful strategy is to evaluate all possible alternative amino
acid assignments at this position. This is a useful method for
evaluating the full range of possible attenuation phenotypes
involving this position, and can produce mutants exhibiting a range
of temperature sensitivity and attenuation (see, for example, U.S.
patent application Ser. No. 10/302,547, filed Nov. 21, 2002 and
priority U.S. Provisional Application No. 60/331,961, filed Nov.
21, 2001; and U.S. Provisional Application No. 60/412,053, filed
Sep. 18, 2002). In addition, the choice of codon for substitution
can be made to involve the greatest number of nucleotide
differences relative to both the wild type and to any possible
alternative amino acid assignments that are found to not yield
attenuated phenotypes. This has the effect of reducing the
probability of reversion or mutation to lose the attenuated
phenotype. Specifically, it is estimated that the frequency of
reversion at any single nucleotide position is 10.sup.-4 to
10.sup.-5, and hence the frequency of reversion/mutation at two
nucleotide positions is 10.sup.-8 to 10.sup.-10, and the frequency
of reversion/mutation involving three nucleotides is 10.sup.-12 to
10.sup.-15. Thus, point mutations involving multiple nucleotide
substitutions can greatly increase genetic and phenotypic
stability. Also, the mutation can be designed to involve deletion
of a single amino acid or more than one amino acid, which can
reduce the possibility of reversion.
[0146] It should be noted that the mutations illustrated in FIGS.
26A-26F are only exemplary by virtue that the depicted mapping
exercise was limited to examples involving amino acid changes in
the L protein, whereas the same strategy can be applied in
accordance with the tools and description provided herein to
transfer attenuating mutations involving any viral protein. For
example, two ts mutations that were developed in the P protein of
recombinant RSV, namely Glycine-172-Serine and
Glutamate-176-Glycine (Lu et al., J. Virol. 76:2871-80, 2002),
correspond to HMPV P residues Glycine-213 and Glutamate-217,
respectively. A mutation in the N protein of the attenuated
derivative cpRSV, namely Valine-267-Isoleucine (Connors et al.
Virology 208:478-84, 1996), corresponds to Serine-268 in HMPV.
Also, two mutations in the F protein of cpRSV, namely
Glutamate-218-Alanine and Threonine-523-Isoleucine (Connors et al.
Virology 208:478-84, 1996), correspond to Lysine-188 and
Threonine489, respectively, in the HMPV F protein. Thus, any known
mutation having a desirable phenotypic property present in any
nucleotide or amino acid sequence is a useful candidate for
incorporation in rHMPV by mutation of corresponding residues in a
rHMPV RNA or protein employing the novel tools and methods of the
current disclosure.
[0147] Another method for devising attenuating or otherwise
desirable amino acid substitution mutations involve
"charge-to-alanine" or "alanine scanning" mutagenesis where, for
example, one or more charged amino acid residues are each changed
to alanine residues (Wertman et al., Genetics 132:337-50, 1992;
Hassett and Condit, Proc. Natl. Acad. Sci. USA 91:4554-58, 1994;
Tang et al., Virology 302:207-16, 2002.A typical target is a pair
of residues, but residues can be changed singly or in larger
combinations. It is often useful to systematically change ("scan")
residues across an encoded protein. Any protein can be a target,
although the L protein is the most common locus of mutations in
attenuated mononegaviruses. Alternatively, one advantage of this
strategy is that one can target proteins that are not represented
in available mutants. This strategy can be readily addressed with
the methods disclosed herein. In particular, the minireplicon
system offers a method for screening mutations in the N, P, L and
M2-1 proteins. In that strategy, mutations are introduced into the
appropriate support plasmid, such as the one encoding the L
protein, and evaluated for the ability to direct the replication
and transcription of a minigenome containing a reporter gene. For
example, the assay can be done in replica at 32.degree. C. and
37.degree. C. (or any temperature in the range of 35.degree. C.
-41.degree. C.) to identify ts mutations that operate at the lower,
permissive temperature and are inhibited at the higher temperature.
This offers a more rapid and technically simple method for
performing an initial screening, after which appropriate mutations
can be introduced into complete recombinant virus.
[0148] Thus, the methods disclosed herein can be used to develop
mutations that confer improved properties to HMPV for the purposes
of developing an immunogenic composition or for other purposes,
such as purifying viral proteins. Mutations can be optimized, such
as by evaluating all possible amino acid or nucleotide assignments
at a given position, or by making amino acid changes involve codon
choices that will be less likely to revert to wild type or to an
alternative assignment that yields a wild type phenotype. Other
changes can be made that fundamentally alter the viral backbone,
such as changes in gene order or movement of major protective
antigen genes to a promoter-proximal position. Importantly, these
various modifications can be introduced into rHMPV in combination
to develop viruses for use in immunogenic compositions or for other
purposes. The methods described herein allows for the systematic
modification of viruses as necessary, such as the introduction of
additional attenuating mutations into a recombinant virus in
response to clinical studies.
[0149] As noted above, production of a sufficiently attenuated
biologically derived HMPV or other viral mutant can be accomplished
by several known methods. One such procedure involves subjecting a
partially attenuated virus to passage in cell culture at
progressively lower, attenuating temperatures. For example,
partially attenuated mutants are produced by passage in cell
cultures at suboptimal temperatures. Thus, a cold-adapted (ca)
mutant or other partially attenuated HMPV strain is adapted to
efficient growth at a lower temperature by passage in culture. This
selection of mutant HMPV during cold-passage substantially reduces
any residual virulence in the derivative strains as compared to the
partially attenuated parent. Alternatively, specific mutations can
be introduced into biologically derived HMPV by subjecting a
partially attenuated parent virus to chemical mutagenesis, for
example, to introduce ts mutations or, in the case of viruses which
are already ts, additional ts mutations sufficient to confer
increased attenuation and/or stability of the ts phenotype of the
attenuated derivative. Means for the introduction of ts mutations
into HMPV include replication of the virus in the presence of a
mutagen such as 5-fluorouridine according to generally known
procedures. Other chemical mutagens can also be used. Attenuation
can result from a ts mutation in almost any HMPV gene, although a
particularly amenable target for this purpose has been found to be
the polymerase (L) gene. The level of temperature sensitivity of
replication in exemplary attenuated HMPV can be determined by
comparing its replication at a permissive temperature with that at
several restrictive temperatures. The lowest temperature at which
the replication of the virus is reduced 100-fold or more in
comparison with its replication at the permissive temperature is
termed the "shutoff temperature." In experimental animals and
humans, both the replication and virulence of HMPV correlate with
the mutant's shutoff temperature.
[0150] From biologically and recombinantly derived HMPV and other
nonsegmented negative stranded RNA viruses, a large "menu" of
attenutating mutations is identifiable by the teachings herein,
each of which can be combined with any other mutations for
adjusting the level of attenuation, immunogenicity and genetic
stability in recombinant HMPV. In this context, many recombinant
HMPV candidates will include one or more, and preferably two or
more, mutations from a biologically derived HMPV or other
heterologous viral mutant, for example, any one or combination of
mutations identified in APIV, HIPV, BPIV3, and/or RSV. Preferred
recombinant HMPVs can incorporate a plurality of mutations thus
identified. Often, these mutations are stabilized against reversion
in recombinant HMPV by multiple nucleotide substitutions in a codon
specifying each mutation.
[0151] Mutations compiled into a "menu" as described above are
introduced as desired, singly or in combination, to adjust
recombinant HMPV to an appropriate level of attenuation,
immunogenicity, genetic resistance to reversion from an attenuated
phenotype, etc. In accordance with the foregoing description, the
ability to produce infectious recombinant HMPV from cDNA permits
introduction of specific engineered changes within the recombinant
HMPV. In particular, infectious, recombinant HMPVs can be employed
for further identification of specific mutations in biologically
derived, attenuated HMPV strains, for example mutations that
specify ts, ca, att and other phenotypes. Desired mutations
identified by this and other methods are introduced into
recombinant HMPV candidate strains. The capability of producing
virus from cDNA allows for routine incorporation of these
mutations, individually or in various selected combinations, into a
full-length cDNA clone, where after the phenotypes of rescued
recombinant viruses containing the introduced mutations can be
readily determined.
[0152] By identifying and incorporating specific mutations
associated with desired phenotypes, for example, a ca or is
phenotype, into infectious recombinant HMPV, additional
site-specific modifications at, or within close proximity to, the
identified mutation are identified. Whereas most attenuating
mutations produced in biologically derived HMPVs are single
nucleotide changes, other "site specific" mutations can also be
incorporated by recombinant techniques into a recombinant HMPV. As
used herein, site-specific mutations include insertions,
substitutions, deletions or rearrangements of from about 1 to about
3, up to about 5-15 or more altered nucleotides (for example,
altered from a wild type HMPV sequence, from a sequence of a
selected mutant HMPV strain, or from a parent recombinant HMPV
clone subjected to mutagenesis). Such site-specific mutations can
be incorporated at, or within the region of, a selected,
biologically derived point mutation. Alternatively, the mutations
can be introduced in various other contexts within a recombinant
HMPV clone, for example at or near a cis-acting regulatory sequence
or nucleotide sequence encoding a protein active site, binding
site, immunogenic epitope, etc.
[0153] Site-specific recombinant HMPV mutants typically retain a
desired attenuating phenotype, but can additionally exhibit altered
phenotypic characteristics unrelated to attenuation, for example,
enhanced or broadened immunogenicity, and/or improved growth.
Further examples of desired, site-specific mutants include
recombinant HMPV mutants engineered to incorporate additional,
stabilizing nucleotide mutations in a codon specifying an
attenuating point mutation. Where possible, two or more nucleotide
substitutions are introduced at codons that specify attenuating
amino acid changes in a parent mutant or recombinant HMPV clone,
yielding a recombinant HMPV with greater genetic resistance to
reversion from an attenuated phenotype. In other embodiments,
site-specific nucleotide substitutions, additions, deletions or
rearrangements are introduced upstream or downstream, e. g., from
about 1 to about 3, about 5-10 and tip to 15 nucleotides or more 5'
or 3', relative to a targeted nucleotide position, for example, to
construct or ablate an existing cis-acting regulatory element.
[0154] In addition to single and multiple point mutations and
site-specific mutations, changes to the recombinant HMPV include
deletions, insertions, substitutions or rearrangements of one or
more genes or genome segments to increase, decrease, ablate or
otherwise alter gene expression. Expression of one or more
non-essential genes can be reduced or ablated by modifying the
recombinant HMPV genome or antigenome, for example, to incorporate
a mutation that alters the coding assignment of an initiation codon
or mutations that introduce one or one or more stop codons.
Alternatively, one or more non-essential genes or genome segments
can be deleted in whole or in part to render the corresponding
proteins partially or entirely non-functional or to disrupt protein
expression altogether. Exemplary recombinant HMPV within these
aspects of the current disclosure are provided herein that exhibit
partial or complete deletions of the G, SH and/or M2-2 ORFs,
including an exemplary mutant that has both the G and SH genes
deleted. These and other recombinants can be engineered, and
selected to possess highly desirable phenotypic characteristics for
development of immunogenic compositions. For example, these
modifications can specify one or more desired phenotypic changes
including (i) altered growth properties in cell culture, (ii)
attenuation in the upper and/or lower respiratory tract of mammals,
(iii) a change in viral plaque size, (iv) a change in cytopathic
effect, (v) a change in immunogenicity, and (vi) a change in RNA
replication and gene expression.
[0155] Thus a recombinant HMPV can incorporate one or more partial
or complete gene deletions, knock out mutations, or mutations that
simply reduce or increase expression of an HMPV gene. This can be
achieved, for example, by introducing a frame shift mutation or
termination codon within a selected coding sequence, altering
translational start sites, changing the position of a gene or
introducing an upstream start codon to alter its rate of
expression, changing GS and/or GE transcription signals to alter
phenotype, or modifying an RNA editing site (for example, growth,
temperature restrictions on transcription, etc.). In several
examples, recombinant HMPVs are provided in which expression of one
or more genes,-for example, a SH, G, or M2-2 ORF, is ablated at the
translational or transcriptional level without deletion of the gene
or of a segment thereof, by, for example, introducing multiple
translational termination codons into a translational ORF, altering
an initiation codon, or modifying an editing site. In one
embodiment, expression of the M2-2 ORF is ablated by removal of
both potential translational start codons, in combination with
partial deletion of the M2-2 ORF . These and other forms of
knock-out virus will often exhibit reduced growth rates and small
plaque sizes in tissue culture. This, additional novel types of
attenuating mutations are provided herein which ablate or alter
expression of a viral gene. In this context, knockout virus
phenotypes produced without (deletion of a gene or genome segment
can be alternatively produced by deletion mutagenesis, as
described, to effectively preclude correcting mutations that may
restore synthesis of a target protein. Other gene knock-outs can be
made using alternate designs and methods that are well known in the
art (as described, for example, in (Kretschmer et al., Virology
216:309-316, 1996; Radecke et al., Virology 217:418-421, 1996; Kato
et al., EMBO J. 16:578-587, 1987; and Schneider et al., Virology
277:314-322,1996).
[0156] Nucleotide modifications that may be introduced into
recombinant HMPV constructs can alter small numbers of bases (for
example, from 15-30 bases, up to 35-50 bases or more), large blocks
of nucleotides (for example, about 50-100, about 100-300, about
300-500, about 500-1,000 bases), or nearly complete or complete
genes (for example, about 1,000-1,500 nucleotides, about
1,500-2,500 nucleotides, about 2,500-5,000, nucleotides, about
5,000-6,0000 nucleotides or more) in the vector genome or
antigenome or heterologous, donor gene or genome segment, depending
upon the nature of the change (that is, a small number of bases may
be changed to insert or ablate an immunogenic epitope or change a
small genome segment, whereas large blocks of bases are involved
when genes or large genome segments are added, substituted, deleted
or rearranged).
[0157] In related aspects, the current disclosure provides for
supplementation of mutations adopted into a recombinant HMPV clone
from biologically derived HMPV, for example, ca and ts mutations,
with additional types of mutations involving the same or different
genes in a further modified recombinant HMPV. Each of the HMPV
genes can be selectively altered in terms of expression levels, or
can be added, deleted, substituted or rearranged, in whole or in
part, alone or in combination with other desired modifications, to
yield a recombinant HMPV exhibiting novel characteristics. Thus, in
addition to or in combination with attenuating mutations adopted
from biologically derived HMPV and/or non-HMPV mutants, the current
disclosure also provides a range of additional methods for
attenuating or otherwise modifying the phenotype of a recombinant
HMPV based on recombinant engineering of infectious HMPV clones. A
variety of alterations can be produced in an isolated
polynucleotide sequence encoding a targeted gene or genome segment,
including a donor or recipient gene or genome segment in a
recombinant HMPV genome or antigenome for incorporation into
partially attenuated, infectious clones. More specifically, to
achieve desired structural and phenotypic changes in recombinant
HMPV, the current disclosure allows for introduction of
modifications which delete, substitute, introduce, or rearrange a
selected nucleotide or nucleotide sequence from a parent genome or
antigenome, as well as mutations which delete, substitute,
introduce or rearrange whole genes or genome segments, within a
recombinant HMPV.
[0158] Thus provided are modifications in recombinant HMPV which
simply alter or ablate expression of a selected gene, for example,
by introducing a termination codon within a selected HMPV coding
sequence or altering its translational start site or RNA editing
site, changing the position of a HMPV gene relative to an operably
linked promoter, introducing an upstream start codon to alter rates
of expression, modifying (for example, by changing position,
altering an existing sequence, or substituting an existing sequence
with a heterologous sequence) GS and/or GE transcription signals to
alter phenotype (for example, growth, temperature restrictions on
transcription, etc.), and various other deletions, substitutions,
additions and rearrangements that specify quantitative or
qualitative changes in viral replication, transcription of selected
genes, or translation of selected proteins. In this context, any
HMPV gene or genome segment which is not essential for growth
can-be ablated or otherwise-modified in a recombinant HMPV to yield
desired effects on virulence, pathogenesis, immunogenicity and
other phenotypic characters. In addition to coding sequences,
noncoding, leader, trailer and intergenic regions can be similarly
deleted, substituted or modified and their phenotypic effects
readily analyzed, for example, by the use of minireplicons, and the
recombinant HMPV described herein.
[0159] In addition to these changes, the order of genes in a
recombinant HMPV construct can be changed, a HMPV genome promoter
replaced with its antigenome counterpart or vice versa, portions of
genes removed or substituted, and even entire genes deleted.
Different or additional modifications in the sequence can be made
to facilitate manipulations, such as the insertion of unique
restriction sites in various intergenic regions or elsewhere.
Nontranslated gene sequences can be removed to increase capacity
for inserting foreign sequences.
[0160] Other mutations for incorporation into recombinant HMPV
constructs of the current disclosure include mutations directed
toward cis-acting signals, which can be readily identified, for
example, by mutational analysis of HMPV minigenomes. For example,
insertional and deletional analysis of the leader, trailer and/or
flanking sequences identifies viral promoters and transcription
signals and provides a series of mutations associated with varying
degrees of reduction of RNA replication or transcription.
Saturation mutagenesis (whereby each position in turn is modified
to each of the nucleotide alternatives) of these cis-acting signals
also can be employed to identify many mutations that affect RNA
replication or transcription. Any of these mutations can be
inserted into a chimeric HMPV antigenome or genome as described
herein. Evaluation and manipulation of transacting proteins and
cis-acting RNA sequences using the complete antigenome cDNA is
assisted by the use of HMPV minigenomes as described in the
above-incorporated references.
[0161] Additional mutations within recombinant HMPVs can also
include replacement of the 3' end of genome with its counterpart
from antigenome or vice versa, which is associated with changes in
RNA replication and transcription. In one exemplary embodiment, the
level of expression of specific HMPV proteins, such as the
protective HN and/or F antigens, can be increased by substituting
the natural sequences with ones which have been made synthetically
and designed to be consistent with efficient translation. In this
context, it has been shown that codon usage can be a major factor
in the level of translation of mammalian viral proteins (Haas et
al., Current Biol. 6:315-324, 1996). Optimization by recombinant
methods of the codon usage of the mRNAs encoding one or more
immunogenic glycoproteins of recombinant HMPV will provide improved
expression for these genes.
[0162] In another exemplary embodiment, a sequence surrounding a
translational start site (preferably including a nucleotide in the
-3 position relative to the AUG start site) of a selected HMPV gene
or donor gene incorporated in an HMPV vector is modified, alone or
in combination with introduction of an upstream start codon, to
modulate gene expression by specifying up- or down-regulation of
translation. Alternatively, or in combination with other
recombinant modifications disclosed herein, gene expression of a
recombinant HMPV can be modulated by altering a transcriptional GS
or GE signal of any selected genes of the virus. In alternative
embodiments, levels of gene expression in a recombinant HMPV
candidate are modified at the level of transcription. In one
aspect, the position of a selected gene in the HMPV gene map can be
changed to a more promoter-proximal or promoter-distal position,
whereby the gene will be expressed more or less efficiently,
respectively. According to this aspect, modulation of expression
for specific genes can be achieved yielding reductions or increases
of gene expression from two-fold, more typically four-fold, up to
ten-fold or more compared to wild type levels often attended by a
commensurate decrease in expression levels for reciprocally,
positionally substituted genes. These and other transpositioning
changes yield novel recombinant HMPVs having attenuated phenotypes,
for example due to decreased expression of selected viral proteins
involved in RNA replication, or having other desirable properties
such as increased antigen expression.
[0163] In the latter context, any one or combination of antigenic
glycoproteins, prospectively including F, G, and SH, or antigenic
determinants thereof can be produced at elevated or decreased
levels (or with otherwise enhanced immunogenic activity) by, for
example, changing the promoter-relative position of the subject
gene or genome segment. For example, the wild type gene order of
the SH, G and F ORFs of HMPV are located at gene positions 6, 7 and
4, respectively. Recombinantly altering the promoter proximity of
one or more of these genes will modulated its expression, and/or
expression of normally "upstream" and "downstream" genes. The
change in gene expression due to the relocation of genes or to the
addition of a second copy can have other desirable effects, such as
increased or decreased growth in vitro or in vivo. In exemplary
embodiments, placement of putative protective antigen genes such as
SH, G, and F more proximal to the promoter, either singly, as a
pair, or as a triplet, will result in more efficient expression. As
described herein, exemplary embodiments of "promoter-shifted" HMPV
are provided wherein the position of the SH-G gene pair was
altered, for example by moving the pair from its wild type gene
order position following the M2 gene to a position preceding the F
gene. This resulted in a gene order in that region of the genome
that mimics that of RSV. In another exemplary embodiment, the SH-G
pair was moved to be upstream of the F gene, and a second copy of
the pair was inserted, resulting in multiple copies of the SH and G
prospective antigens. In yet additional embodiments, the
prospective antigens comprising one or more of the G and F proteins
are modified in their expression by promoter-relative shifting of
these genes (having wild type gene order positions at the 7th and
4th gene positions, respectively in the HMPV genome map) to more
promoter-proximal positions.
[0164] In other embodiments, recombinant HMPVs useful in
immunogenic compositions can be conveniently modified to
accommodate antigenic drift in circulating virus. In this context,
the Examples below provide the complete sequence for an HMPV
genome, and also provide a detailed comparison of complete genome
sequences for HMPV isolates representing two distinct antigenic
subgroups of HMPV, that are generally comparable in sequence
divergence to the antigenic subgroups of HRSV. Identification and
characterization of these distinct antigenic subgroups, including
detailed mapping of their prospective antigenic glycoprotein gene
structure, provides for construction of novel recombinant HMPVs for
use in immunogenic compositions to elicit an immune response
against one or more HMPV strains, including in bivalent immunogenic
composition to immunize against multiple HMPV strains and/or
accommodate antigenic drift. Typically the recombinant HMPV will
have a modification in one or more of the Sit, G, and/or F
glycoproteins. For example, an entire SH, G, and/or F gene, or a
genome segment encoding a particular antigenic determinant (for
example, immunogenic region or epitopes) thereof), from one HMPV
strain or group can be incorporated into a recombinant HMPV genome
or antigenome cDNA by replacement of a corresponding region in a
recipient clone of a different HMPV strain or group, or by adding
one or more copies of the gene, such that multiple antigenic forms
are represented in the recombinant virus. Progeny virus produced
from the modified recombinant HMPV can then be used in immunization
protocols against multiple, and emerging, HMPV strains.
[0165] In certain aspects of the current disclosure, replacement of
a HMPV coding sequence or non-coding sequence (for example, a
promoter, GE, GS, intergenic or other cis-acting element) with a
heterologous (for example, non-HMPV) counterpart yields chimeric
HMPV having a variety of possible attenuating and other phenotypic
effects. For example, host range and other desired effects can be
engineered by importing an APV or other non-HMPV virus (for
example, RSV, SV5, SV41, NDV, PIV) protein, protein domain, gene or
genome segment into a recombinant HMPV "background" genome or
antigenome, wherein the heterologous or "donor" gene or genome
segment does not function efficiently in a human cell, for example,
from incompatibility of the heterologous sequence or protein with a
biologically interactive HMPV sequence or protein (that is, a
sequence or protein that ordinarily cooperates with the substituted
sequence or protein for viral transcription, translation, assembly,
etc.) or, more typically in a host range restriction, with a
cellular protein or some other aspect other cellular milieu which
is different between the permissive and less permissive host. In
exemplary embodiments, APV, pneumovirus of mice, or RSV sequences
are selected for introduction into HMPV based on aspects of HMPV
structure and function described herein.
[0166] Methods are provided herein for attenuating recombinant HMPV
candidates based on the construction of chimeras between HMPV and a
non-human pneumovirus or other negative stranded RNA virus, for
example APIV, RSV, HPIV, MPIV1 (Sendai virus), BPIV3, SV5, SV4I,
and/or NDV (for example, as disclosed in U.S. patent application
Ser. No. 09/586,479, filed Jun. 1, 2000 by Schmidt et al.
(corresponding to PCT Publication WO 01/04320); Schmidt et al., J.
Virol. 74:8922-9, 2000). In exemplary embodiments, the recombinant
HMPV genome or antigenome is combined with a heterologous gene or
genome segment, such as an N, P, M, or L, ORF derived from a
non-HMPV paramyxovirus.
[0167] Paramyxoviruses are enveloped RNA viruses that include two
subfamilies (Paramyxovirinae and Pneumovirinae). Subfamily
Paramyxovirinae has five different genera, including Respirovirus
(containing parainfluenza viruses 1 and 3), Rubulavirus (containing
parainfluenza viruses 2 and 4 and mumps), and Morbillivirus
(containing measles and canine distemper viruses). Subfamily
Pneumovirinae has two genera, namely Pneumovirus (containing RSV)
and Metapneumovirus (containing HMPV). Paramyxoviruses encoded 6-11
proteins. Proteins common to all paramyxoviruses include the
nucleocapsid N protein that associates tightly with the RNA genome
to form the nucleocapsid, the phosphoprotein P and large L
polymerase protein that also associate with the nucleocapsid, the
matrix M protein that associates with the inner face of the viral
envelope, the transmembrane fusion F glycoprotein that mediates
penetration, and the attachment protein that, depending on the
virus, is called glycoprotein G, or the hemagglutinin HA or the
hemagglutinin-neuraminidase HN. Other proteins are found in some
viruses but not others, such as the nonstructural NS1 and NS2
proteins of RSV, the M2-1 and M2-2 proteins found in RSV and, as
disclosed here, in HMPV, the SH protein that is found in mumps,
simian virus 5, RSV and HMPV, and the C, D and V proteins found in
certain parainfluenza viruses. The sequences of non-HMPV HN, M, N,
P, L, F, G, and SH proteins, and nucleic acids encoding these
proteins are known in the art (see, Lamb, R A and Kolakofsky, D. pp
1305-1340 in Fields Virology, 4.sup.th addition, Knipe and Howley
(eds) Lippincott Williams and Wilkins, Philadelphia, 2001, Chanock,
Murphy and Collins, pp 1341-1379, ibid, Collins, Chanock and
Murphy, pp 1443-1485, ibid.).
[0168] Chimeric HMPV are therefore provided herein that include a
partial or complete "background" HMPV genome or antigenome derived
from or patterned after HMPV combined with one or more heterologous
genes or genome segments of a non-HMPV virus to form the chimeric
HMPV genome or antigenome. In one embodiment, chimeric HMPV of this
type incorporate a partial or complete HMPV background genome or
antigenome combined with one or more heterologous genes or genome
segments. The partial or complete background genome or antigenome
typically acts as a recipient backbone into which the heterologous
genes or genome segments of the counterpart, non-HMPV virus are
incorporated. Heterologous genes or genome segments from the
counterpart virus represent "donor" genes or polynucleotides that
are combined with, or substituted within, the background genome or
antigenome to yield a chimeric HMPV that exhibits novel phenotypic
characteristics compared to one or both of the contributing
viruses. For example, addition or substitution of heterologous
genes or genome segments within a selected recipient HMPV strain
may result in an increase or decrease in attenuation, growth
changes, altered immunogenicity, or other desired phenotypic
changes as compared with a corresponding phenotypes of the
unmodified recipient and/or donor (see, for example, U.S. patent
application Ser. No. 09/586,479, filed Jun. 1, 2000 by Schmidt et
al.; Schmidt et al., J. Virol. 74 8922-9, 2000).
[0169] Genes and genome segments that can be selected for use as
heterologous substitutions or additions within chimeric HMPV
include genes or genome segments encoding a HMPV N, P, M, F, M2,
SH, G and/or L proteins or portions thereof. In addition, genes and
genome segments encoding proteins found in other viruses, (for
example, an SH protein as found in mumps, RSV, and SV5 viruses),
may be incorporated within additional chimeric HMPV recombinants of
the current disclosure. Regulatory regions from heterologous
viruses, for example the extragenic 3' leader or 5' trailer
regions, and GS, GE, intergenic regions, or 3' or 5' non-coding
regions, are also useful as substitutions or additions within
recombinant HMPV. In exemplary aspects, chimeric HMPV bearing one
or more non-HMPV genes or genome segments exhibit a high degree of
host range restriction, for example, in the respiratory tract of
mammalian models of human HMPV infection such as hamsters and
non-human primates. In more detailed embodiments HMPV is attenuated
by the addition or substitution of one or more APV or RSV genes or
genome segments selected from N, P, M, F, M2, SH, G and/or L genes
and genome segments to a partial or complete HMPV background genome
or antigenome.
[0170] In one embodiment, the degree of host range restriction
exhibited by chimeric HMPV for use within immunogenic compositions
of the current disclosure is comparable to the degree of host range
restriction exhibited by the respective non-HMPV "donor" strain.
For example, the restriction should have a true host range
phenotype, that is, it should be specific to the host in question
and should not restrict replication in vitro in a suitable cell
line. In addition, chimeric HMPV bearing one or more heterologous
genes or genome segments elicit a desired immunogenic response in
hosts susceptible to HMPV infection. Thus, the current disclosure
provides a new basis for attenuating a live HMPV virus vector for
developing immunogenic compositions against HMPV and other
pathogens based on host range effects.
[0171] In combination with the host range phenotypic effects
provided in the chimeric HMPV, it is often desirable to adjust the
attenuation phenotype by introducing additional mutations that
increase or decrease attenuation of the chimeric virus. Thus, in
additional embodiments, attenuated, chimeric HMPV are produced in
which the chimeric genome or antigenome is further modified by
introducing one or more attenuating mutations specifying an
attenuating phenotype in the resultant virus or subviral particle.
These can include mutations generated de novo and tested for
attenuating effects according to a rational design mutagenesis
strategy. Alternatively, the attenuating mutations may be
identified in existing biologically derived mutant HMPV or
non-HMPVs and thereafter incorporated into a chimeric HMPV of the
current disclosure. Exemplary mutations specify lesions in RNA
regulatory sequences or in encoded proteins.
[0172] In certain chimeric HMPV, attenuation marked by replication
in the lower and/or upper respiratory tract in an accepted animal
model that is reasonably correlated with HMPV replication and
immunogenic activity in humans (for example, hamsters, rhesus
monkeys or chimpanzees), is reduced by at least about two-fold,
more often about 5-fold, 10-fold, or 20-fold, and preferably
50-100-fold and up to 1,000-fold or greater overall (for example,
as measured between 3-8 days following injection) compared to
growth of the corresponding wild type or mutant parental
strains.
[0173] Within the methods disclosed herein, additional genes or
genome segments can be inserted into or proximate to a recombinant
or chimeric HMPV genome or antigenome: For example, various
supernumerary heterologous genes or genome segments can be inserted
at any of a variety of sites within the recombinant genome or
antigenome, for example at a position 3' to N, between the N/P,
P/M, M/F, F/M2, M2/SH, SH/G, and/or G/L genes, or at another
non-coding region of the HMPV vector genome or antigenome (see FIG.
1). The inserted genes can be under common control with recipient
genes, or can be under the control of an independent set of
transcription signals. Genes of interest in this context include
genes encoding cytokines, for example, an interleukin (IL-2 through
IL-18, for example, interleukin 2 (IL-2), interleukin 4 (IL-4),
interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 18 (IL-18),
tumor necrosis factor alpha (TNF.alpha.), interferon gamma
(IFN.gamma.), or granulocyte-macrophage colony stimulating factor
(GM-CSF) (see, for example, U.S. application Ser. No. 09/614,285,
filed Jul. 12, 2000 and priority U.S. Provisional Application
Serial No. 60/143,425 filed Jul. 13, 1999). Coexpression of these
additional proteins provides the ability to modify and improve
immune responses against recombinant HMPV quantitatively and/or
qualitatively.
[0174] In additional embodiments, insertion of heterologous
nucleotide sequences into recombinant HMPV candidates are employed
separately to modulate the level of attenuation of candidate
recombinants, for example, for the upper respiratory tract. Thus,
it is possible to insert nucleotide sequences into a recombinant
HMPV that both direct the expression of a foreign protein and that
attenuate the virus in an animal host, or to use nucleotide
insertions separately to attenuate candidate viruses. To define
some of the rules that govern the effect of gene insertion on
attenuation, gene units of varying lengths are inserted into a wild
type HMPV backbone and the effects of gene unit length on
attenuation examined. Novel gene unit insertions are contemplated
in this regard that do not contain a significant ORF, permitting
identification of the effect of gene unit length independently of
an effect of the expressed protein of that gene. These heterologous
sequences may be inserted as an extra gene unit of various sizes,
for example, from about 150 or more nts in length up to 3,000 nts
or more in length. Gene unit (GU) insertions of about 1,000 or
2,000 nts in length will often substantially attenuate rHMPV
candidates for the upper respiratory tract of mammalian subjects.
In addition, gene unit insertions can have the dual effect of both
attenuating a candidate virus and inducing an immunogenic response
against a second virus. Alternately, gene extensions in the
3'-noncoding region (NCR) of a HMPV gene which cannot express
additional proteins, can also he attenuating in and of themselves.
Within these methods of the current disclosure, gene insertion
length is a determinant of attenuation (see, for example, U.S.
patent application Ser. No. 10/302,547, filed Nov. 21, 2002 and
priority U.S. Provisional Application No. 60/331,961, filed Nov.
21, 200 1; and U.S. Provisional Application No. 60/412,053, filed
Sep. 18, 2002).
[0175] Deletions, insertions, substitutions and other mutations
involving changes of whole viral genes or genome segments within
rHMPV yield highly stable candidates, which are particularly
important in the case of immunosuppressed individuals. Many of
these changes will result in attenuation of resultant strains,
whereas others will specify different types of desired phenotypic
changes. For example, accessory (that is, not essential for in
vitro growth) genes are excellent candidates to encode proteins
that specifically interfere with host immunity (see, for example,
Kato et al., EMBO. J. 16:578-87, 1997). Ablation of such genes in
candidate viruses can reduce virulence and pathogenesis and/or
improve immunogenicity.
[0176] In more detailed embodiments, chimeric HMPVs are constructed
using a HMPV "vector" genome or antigenome that is recombinantly
modified to incorporate one or more antigenic determinants of a
heterologous pathogen. The vector genome or antigenome includes a
partial or complete HMPV genome or antigenome, which may itself
incorporate nucleotide modifications such as attenuating mutations.
The vector genome or antigenome is modified to form a chimeric
structure though incorporation of a heterologous gene or genome
segment. More specifically, chimeric HMPVs of the current
disclosure are constructed through a cDNA-based virus recovery
system that yields recombinant viruses that incorporate a partial
or complete vector or "background" HMPV genome or antigenome
combined with one or more "donor" nucleotide sequences encoding the
heterologous antigenic determinants. In exemplary embodiments a
HMPV vector genome or antigenome is modified to incorporate one or
more genes or genome segments that encode antigenic determinants of
one or more heterologous negative stranded RNA viruses (for
example, another HMPV, RSV, PIV, or measles virus). Thus
constructed, chimeric HMPVs can elicit, for example, an immune
response against a specific HMPV or against a non-HMPV pathogen.
Alternatively, compositions and methods are provided employing a
HMPV-based chimeric virus to elicit a polyspecific immune response
against multiple HPIVs, against one or more HMPVs and a non-PIV
pathogen such as RSV, PIV, or measles virus.
[0177] A chimeric HMPV in this context can incorporate a partial or
complete human HMPV incorporating one or more heterologous
polynucleotides encoding one or more antigenic determinants of the
heterologous pathogen. These heterologous polynucleotides can be
added to or substituted within the HMPV vector genome or antigenome
to yield the chimeric HMPV recombinant. The chimeric HMPV virus
thus acquires the ability to elicit all immune response in a
selected host against the heterologous pathogen. In addition, the
chimeric virus can exhibit other novel phenotypic characteristics
compared to one or both of the vector HMPV and heterologous
pathogens. In addition to providing novel immunogenic
characteristics, the addition or substitution of heterologous genes
or genome segments within the vector HMPV strain may confer an
increase or decrease in attenuation, growth changes, or other
desired phenotypic changes as compared with the corresponding
phenotypes of the unmodified vector and donor viruses.
[0178] Heterologous genes or genome segments of a different HMPV or
non-HMPV virus can be added as a supernumerary genomic element to a
partial or complete genome or antigenome of a rHMPV. Alternatively,
one or more heterologous genes or genome segments may be
substituted into the rHMPV at a position corresponding to a wild
type gene order position of a counterpart genes or genome segments
that is deleted within the HMPV vector genome or antigenome. In yet
additional embodiments, the heterologous gene or genome segment is
added or substituted at a position that is more promoter-proximal
or promoter-distal compared to a wild type gene order position of
the counterpart gene or genome segment within the vector genome or
antigenome to enhance or reduce, respectively, expression of the
heterologous gene or genome segment. Additional methods and
compositions that are useful for engineering chimeric HMPV employ
certain known techniques, for example, as disclosed for PIV viruses
(Durbin et al., Virology 235:323-332, 1997; Skiadopoulos et al., J.
Virol. 72:1762-1768, 1998; Tao et al., J Virol 72:2955-2961, 1998;
Skiadopoulos et al., J. Virol. 73:1374-1381, 1999; Skiadopoulos et
al., Vaccine 18:503-510, 1999; Tao et al., Vaccine 17:1100-1108,
1999; Tao et al., Vaccine 18:1359-1366, 2000; U.S. patent
application Ser. No. 09/083,793, filed May 22, 1998; U.S. patent
application Ser. No. 09/458,813, filed Dec. 10, 1999; U.S. patent
application Ser. No. 09/459,062, filed Dec. 10, 1999; U.S.
Provisional Application No. 60/047,575, filed May 23, 1997
(corresponding to International Publication No. WO 98/53078), and
U.S. Provisional Application No. 60/059,385, filed Sep. 19,
1997).
[0179] Chimeric HMPV can also be constructed that express a
chimeric protein, for example an immunogenic glycoprotein having a
cytoplasmic tail and/or transmembrane domain specific to a HMPV
vector fused to a heterologous ectodomain (the ectodomain being
that part of a transmembrane surface protein that extends into the
luminal or extracellular space) of a different HMPV or non-HMPV
pathogen to provide a fusion protein that elicits an immune
response against the heterologous pathogen. For example, a
heterologous genome segment encoding a glycoprotein ectodomain from
a RSV F, SH, M2, or G glycoprotein can be joined with a genome
segment encoding the corresponding HMPV glycoprotein cytoplasmic
and transmembrane domains to form a chimeric glycoprotein that
elicits an immune response against RSV.
[0180] Briefly, HMPV expressing a chimeric glycoprotein includes a
major nucleocapsid protein, a nucleocapsid phosphoprotein, a large
polymerase protein, and a HMPV vector genome or antigenome that is
modified to encode a chimeric glycoprotein. The chimeric
glycoprotein incorporates one or more heterologous antigenic
domains, fragments, or epitopes of a second, antigenically distinct
HMPV or non-HMPV pathogen. In one embodiment, this is achieved by
substitution within the HMPV vector genome or antigenome of one or
more heterologous genome segments of the second virus that encodes
one or more antigenic domains, fragments, or epitopes, whereby the
recombinant genome or antigenome encodes the chimeric glycoprotein
that is antigenically distinct from the parent, vector virus. In
several examples, the heterologous genome segment or segments can
encode a glycoprotein ectodomain or immunogenic portion or epitope
thereof. The heterologous genome segment can also include other
portions of the heterologous or "donor" glycoprotein, for example
both an ectodomain and transmembrane region that are substituted
for counterpart glycoprotein ecto- and transmembrane domains in the
vector genome or antigenome.
[0181] As used herein, the term "gene" generally refers to a
portion of a subject genome, for example, a HMPV genome, encoding
an mRNA and typically begins at the upstream end with a GS signal
and ends at the downstream end with the GE signal. The term gene is
also interchangeable with the term "translational open reading
frame," or "ORF," particularly in the case where a protein, such as
the HPIV1 or HPIV3 C protein, is expressed from an additional ORF
rather than from a unique mRNA. The viral genome of all
mononegaviruses also contains extragenic leader and trailer
regions, possessing part of the promoters required for viral
replication and transcription, as well as non-coding and intergenic
regions. Transcription initiates at the 3' end and proceeds by a
sequential stop-start mechanism that is guided by short conserved
motifs found at the gene boundaries. The upstream end of each gene
contains a GS signal, that directs initiation of its respective
mRNA. The downstream terminus of each gene contains a GE motif that
directs polyadenylation and termination. The current disclosure
provides operative identification of transcription signals and
insertion sites, and provides the identification of the GS, GE and
potential insertion sites within the genome.
[0182] To construct-chimeric HMPVs, one or more HMPV genes or
genome segments can be deleted, inserted or substituted in whole or
in part. This means that partial or complete deletions, insertions
and substitutions may include ORFs and/or cis-acting regulatory
sequences of any one or more of the HMPV genes or genome segments.
By "genome segment" is meant any length of continuous nucleotides
from the HMPV genome, which might be part of an ORF, a gene, or an
extragenic region, or a combination thereof. In several examples,
at least about 10, at least about 20, at least about 30, at least
about 50, or at least about 100 continuous nucleotides are deleted,
inserted or substituted. When a subject genome segment encodes an
antigenic determinant, the genome segment encodes at least one
immunogenic epitope capable of eliciting a humoral or cell mediated
immune response in a mammalian host. The genome segment can also
encode an immunogenic fragment or protein domain. For example, the
donor genome segment can encode multiple immunogenic domains or
epitopes, including recombinantly synthesized sequences that
comprise multiple, repeating or different, immunogenic domains or
epitopes. Chimeric HMPV can be engineered to express one or more
major antigenic determinants of a wide range of non-HMPV pathogens.
The methods disclosed herein are generally adaptable for
incorporation of antigenic determinants from, for example, subgroup
A and subgroup B RSVs, HMPV, measles virus, mumps virus, human
papilloma viruses, type 1 and type 2 human immunodeficiency
viruses, herpes simplex viruses, cytomegalovirus, rabies virus,
Epstein Barr virus, filoviruses, buniyaviruses, flaviviruses,
alphaviruses and influenza viruses, among other pathogens.
Pathogens that can be targeted for development of immunogenic
compositions include viral and bacterial pathogens, as well as
protozoans and multicellular pathogens. Useful antigenic
determinants from many important human pathogens in this context
are known or readily identified for incorporation within chimeric
HMPV. Thus, major antigens have been identified for the foregoing
exemplary pathogens, including the measles virus HA and F proteins;
the F, G, SH and M2 proteins of RSV, mumps virus HN and F proteins,
human papilloma virus L1 protein, type 1 or type 2 human
immunodeficiency virus gp160 protein, herpes simplex virus and
cytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM
proteins, rabies virus G protein, Epstein Barr Virus gp350 protein;
filovirus G protein, bunyavirus G protein, flavivirus E and NS1
proteins, and alphavirus E protein. These major antigens, as well
as other antigens known in the art for the enumerated pathogens and
others, are well characterized to the extent that many of their
antigenic determinants, including the full length proteins and
their constituent antigenic domains, fragments and epitopes, are
identified, mapped and characterized for their respective
immunogenic activities.
[0183] Among the numerous, exemplary mapping studies that identify
and characterize major antigens of diverse pathogens for use in the
methods a chimeric HMPV disclosed herein are epitope mapping
studies directed, for example, to immunogenic glycoproteins of PIV.
Exemplifying the subject methods and tools, van Wyke Coelingh et
al. (J. Virol. 63:375-382, 1989) described twenty-six monoclonal
antibodies (MAbs) (14 neutralizing and 12 nonneutralizing) that
were used to examine the antigenic structure, biological
properties, and natural variation of the fusion (F) glycoprotein of
HPIV3. Analysis of laboratory-selected antigenic variants and of
PIV3 clinical isolates indicated that the panel of MAbs recognizes
at least 20 epitopes, 14 of which participate in neutralization.
Competitive binding assays confirmed that the 14 neutralization
epitopes are organized into three nonoverlapping principal
antigenic regions (A, B, and C) and one bridge site (AB), and that
the 6 nonneutralization epitopes form four sites (D, E, F, and G).
Most of the neutralizing MAbs were involved in nonreciprocal
competitive binding reactions, suggesting that they induce
conformational changes in other neutralization epitopes. These and
related methods will serve to readily determine candidate antigenic
determinants among heterologous viruses for expression by HMPV
vectors of the current disclosure.
[0184] Other antigenic determinants for use within the current
disclosure have been identified and characterized for RSV. For
example, Beeler et al., J. Virol. 63:2941-2950, 1989, employed
eighteen neutralizing monoclonal antibodies (MAbs) specific for the
fusion glycoprotein of the A2 strain of RSV to construct a detailed
topological and operational map of epitopes involved in RSV
neutralization and fusion. Competitive binding assays identified
three nonoverlapping antigenic regions (A, B, and C) and one bridge
site (AB). Thirteen MAb-resistant mutants (MARMs) were selected,
and the neutralization patterns of the MAbs with either MARMs or
RSV clinical strains identified a minimum of 16 epitopes. MARMs
selected with antibodies to six of the site A and AB epitopes
displayed a small-plaque phenotype, which is consistent with an
alteration in a biologically active region of the F molecule.
Analysis of MARMs also indicated that these neutralization epitopes
occupy topographically distinct but conformationally interdependent
regions with unique biological and immunological properties.
Antigenic variation in F epitopes was then examined by using 23
clinical isolates (18 subgroup A and 5 subgroup B) in
cross-neutralization assays with the 18 anti-F MAbs. This analysis
identified constant, variable, and hypervariable regions on the
molecule and indicated that antigenic variation in the
neutralization epitopes of the RSV F glycoprotein is the result of
a noncumulative genetic heterogeneity. Of the 16 epitopes, 8 were
conserved on all or all but 1 of 23 subgroup A or subgroup B
clinical isolates. These antigenic determinants, including the full
length proteins and their constituent antigenic domains, fragments
and epitopes, all represent useful candidates for integration
within chimeric HMPV of the current disclosure to elicit novel
immune responses as described above. (See also, Anderson et al., J.
Infect. Dis. 151:626-633, 1985; Coelingh et al., J. Virol.
63:375-382, 1989; Fenner et al., Scand. J. Immunol. 24:335-340,
1986; Fernie et al., Proc. Soc. Exp. Biol. Med. 171:266-271, 1982;
Sato et al., J. Gen. Virol. 66:1397-1409, 1985; Walsh et al., J.
Gen. Virol. 67:505-513, 1986, and Olmsted et al.; J. Virol.
63:411-420, 1989).
[0185] To express antigenic determinants of heterologous HMPVs and
non-HMPV pathogens, numerous methods and constructs are provided
herein. In certain detailed embodiments, a transcription unit
comprising an ORF of a gene encoding an antigenic protein (for
example, an RSV F or G gene) is added to a HMPV vector genome or
antigenome at various positions, yielding exemplary chimeric
"vector" candidates. In exemplary embodiments, chimeric HMPVs are
engineered that incorporate heterologous nucleotide sequences
encoding protective antigens from RSV to produce infectious,
attenuated candidates (see U.S. patent application Ser. No.
08/720,132, filed Sep. 27, 1996, corresponding to International
Publication WO 97/12032 published Apr. 03 1997, and U.S.
Provisional Patent Application No. 60/007,083, filed Sep. 27, 1995;
U.S. Provisional Patent Application No. 60/021,773, filed Jul. 15,
1996; U.S. Provisional Patent Application No. 60/046,141, filed May
9, 1997; U.S. Provisional Patent Application No. 60/047,634, filed
May 23, 1997; U.S. patent application Ser. No. 08/892,403, filed
Jul. 15, 1997 corresponding to International Publication No. WO
98/02530 published on Jan. 22, 1998; U.S. patent application Ser.
No. 09/291,894, filed on Apr. 13, 1999 corresponding to
International Publication No. WO 00/61611 published Oct. 19, 2000,
and priority U.S. Provisional Patent Application Serial No.
60/129,006, filed on Apr. 13, 1999; U.S. patent application Ser.
No. 09/602,212, filed Jun. 23, 2000 and corresponding International
Publication No. WO 01/04335 published on Jan. 18, 2001, and
priority U.S. Provisional Patent Application Nos. 60/129,006, filed
Apr. 13, 1999 60/143,097, filed Jul. 9, 1999, and 60/143,132, filed
Jul. 9, 1999; International Publication No. WO (0/61737 published
on Oct. 19. 2000; Collins et al., Proc Nat. Acad. Sci. U.S.A.
92:11563-11567, 1995; Bukreyev et al., J. Virol. 70:6634-41, 1996,
Juhasz et al., J. Virol. 71:5814-5819, 1997; Durbin et al.,
Virology 235:323-332, 1997; He et al. Virology 237:249-260, 1997;
Baroni et al. J. Virol. 71:1265-1271, 1997; Whitehead et al.,
Virology 247:232-9, 1998a; Whitehead et al., J. Virol.
72:4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998; and
Whitehead et al., J. Virol. 73:3438-3442,1999, and Bukreyev et al.,
Proc. Nat. Acad. Sci. U.S.A. 96:2367-72, 1999). Other reports and
discussion incorporated or set forth herein identify and
characterize RSV antigenome determinants that are useful within the
current disclosure.
[0186] HMPV chimeras incorporating one or more RSV antigenic
determinants, can include a HMPV vector genome or antigenome
combined with a heterologous gene or genome segment encoding an
antigenic RSV glycoprotein, protein domain (for example, a
glycoprotein ectodomain) or one or more immunogenic epitopes. In
one embodiment, one or more genes or genome segments from RSV SH,
M2, F, and/or G genes is/are combined with the HMPV vector genome
or antigenome to form the chimeric HMPV candidate. Certain of these
constructs will express chimeric proteins, for example fusion
proteins having a cytoplasmic tail and/or transmembrane domain of
HMPV fused to an ectodomain of a corresponding RSV glycoprotein to
yield a novel attenuated virus that optionally elicits a
multivalent immune response against both HMPV and RSV.
[0187] In certain embodiments, it is useful to administer
immunogenic compositions comprising a rHMPV in a predetermined
schedule with one or more additional immunogenic components, for
example a second immunogenic composition against RSV or PIV
administered before or after the anti-HMPV composition. RSV and
HPIV3 cause significant illness within the first four months of
life whereas most of the illness caused by HPIV1 and HPIV2 occur
after six months of age (Chanock et al., in Parainfluenza Viruses,
Knipe et al. (Eds.), pp. 1341-1379, Lippincott Williams &
Wilkins, Philadelphia, 2001; Collins et al., In Fields Virology,
Vol. 1, pp. 1205-1243, Lippincott-Raven Publishers, Philadelphia,
1996; Reed et al., J. Infect. Dis. 175:807-13, 1997). Accordingly,
certain sequential immunization protocols involve administration of
a immunogenic composition as described herein that elicits an
immune response against HMPV before, simultaneous with (for
example, as a combined immunogenic composition), or subsequent to,
administration of a second immunogenic composition directed toward
another virus. In one embodiment, an immunogenic composition that
elicits an immune response against HMPV, or against HMPV and RSV is
administered one, two or more times early in life, with the first
dose administered at or before one month of age, followed by an
immunogenic composition against HPIV I and/or HPIV2 at about four
and six months of age. Alternatively, it might be advantageous to
administer immunogenic components against HMPV, RSV, and one or
more PIVs at the same time, perhaps as a multi-component
immunogenic composition.
[0188] This, combinatorial immunogenic compositions and coordinate
immunization protocols are provided herein for multiple pathogenic
agents, including HMPV, RSV, and one or more PIVs. In exemplary
embodiments, these methods and formulations are temporally selected
to target early immunization against HMPV, RSV and/or PIV3. One
exemplary immunization sequence employs one or more live attenuated
immunogenic compositions against HMPV, RSV and/or PIV3 as early as
one month of age (for example, at one and two months of age)
followed by mono- or bivalent PIV1 and/or PIV2 immunogenic
composition at four and six months of age. The methods disclosed
herein can be used to administer multiple immunogenic compositions,
including one or more chimeric HMPV candidates, coordinately, for
example, simultaneously in a mixture or separately in a defined
temporal sequence (for example, in a daily or weekly sequence),
wherein each virus preferably expresses, for example, a different
heterologous protective antigen. Such a coordinate/sequential
immunization strategy, which is able to induce secondary antibody
responses to multiple viral respiratory pathogens, provides a
highly powerful and extremely flexible immunization regimen that is
driven by the need to immunize against multiple pathogens in early
infancy.
[0189] As noted above, the current disclosure permits a wide range
of alterations to be recombinantly produced within the HMPV genome
or antigenome, yielding defined mutations that specify desired
phenotypic changes. Defined mutations can be introduced by a
variety of conventional techniques (for example, site-directed
mutagenesis) into a cDNA copy of the genome or antigenome. The use
of genomic or antigenomic cDNA subfragments to assemble a complete
genome or antigenome cDNA as described herein has the advantage
that each region can be manipulated separately, where small cDNA
constructs provide for better ease of manipulation than large cDNA
constructs, and then readily assembled into a complete cDNA. Thus,
the complete antigenome or genome cDNA, or a selected subfragment
thereof, can be used as a template for oligonucleotide-directed
mutagenesis. This can be through the intermediate of a
single-stranded phagemid form, such as using the MUTA-gen.RTM. kit
of Bio-Rad Laboratories (Richmond, Calif.), or a method using the
double-stranded plasmid directly as a template such as the
Chameleon.RTM. mutagenesis kit of Strategene (La Jolla, Calif.), or
by the PCR employing either an oligonucleotide primer or a template
which contains the mutations of interest. A mutated subfragment can
then be assembled into the complete antigenome or genome cDNA. A
variety of other mutagenesis techniques are known and can be
routinely adapted for use in producing the mutations of interest in
a HMPV antigenome or genome cDNA of the current disclosure.
[0190] Thus, in one illustrative embodiment mutations are
introduced by using the MUTA-gene.RTM. phagemid in vitro
mutagenesis kit available from Bio-Rad Laboratories. In brief, cDNA
encoding a HMPV genome or antigenome is cloned into the plasmid
pTZ18U, and used to transform CJ236 cells (Life Technologies).
Phagemid preparations are prepared as recommended by the
manufacturer. Oligonucleotides are designed for mutagenesis by
introduction of an altered nucleotide at the desired position of
the genome or antigenome. The plasmid containing the genetically
altered genome or antigenome is then amplified.
[0191] Mutations can vary from single nucleotide changes to the
introduction, deletion or replacement of large cDNA segments
containing one or more genes or genome segments. Genome segments
can correspond to structural and/or functional domains, for
example, cytoplasmic, transmembrane or ectodomains of proteins,
active sites such as sites that mediate binding or other
biochemical interactions with different proteins, epitopic sites,
for example, sites that stimulate antibody binding and/or humoral
or cell mediated immune responses, etc. Useful genome segments in
this regard range from about 15-35 nucleotides in the case of
genome segments encoding small functional domains of proteins, for
example, epitopic sites, to about 50, about 75, about 100, about
200-500, and about 500-1,500 or more nucleotides.
[0192] The ability to introduce defined mutations into infectious
recombinant HMPV has many applications, including the manipulation
of HMPV pathogenic and immunogenic mechanisms. For example, the
functions of HMPV proteins, including the N, P, M, F, M2, SH, G
and/or L proteins can be manipulated by introducing mutations which
ablate or reduce the level of protein expression, or which yield
mutant protein. Various genome RNA structural features, such as
promoters, intergenic regions, and transcription signals, can also
be routinely manipulated within the methods and compositions of the
current disclosure. The effects of trans-acting proteins and
cis-acting RNA sequences can be readily determined, for example,
using a complete antigenome cDNA in parallel assays employing HMPV
minigenomes (Dimock et al., J. Virol. 67:2772-8, 1993 in its
entirety), whose rescue-dependent status is useful in
characterizing those mutants that may be too inhibitory to be
recovered in replication-independent infectious virus.
[0193] Certain substitutions, insertions, deletions or
rearrangements of genes or genome segments within recombinant HMPV
(for example, substitutions of a genome segment encoding a selected
protein or protein region, for instance a cytoplasmic tail,
transmembrane domain or ectodomain, an epitopic site or region, a
binding site or region, an active site or region containing an
active site, etc.) are made in structural or functional relation to
an existing, "counterpart" gene or genome segment from the same or
different HMPV or other source. Such modifications yield novel
recombinants having desired phenotypic changes compared to wild
type or parental HMPV or other viral strains. For example,
recombinants of this type may express a chimeric protein having a
cytoplasmic tail and/or transmembrane domain of one HMPV, or of a
non-HMPV virus such as PIV or RSV, fused to an ectodomain of a
rHMPV of the current disclosure. Other exemplary recombinants of
this type express duplicate protein regions, such as duplicate
immunogenic regions.
[0194] As used herein, "counterpart" genes, genome segments,
proteins or protein regions, are typically from heterologous
sources (for example, from different HMPV genes, or representing
the same (that is, homologous or allelic) gene or genome segment in
different HMPV types or strains). Typical counterparts selected in
this context share gross structural features, for example, each
counterpart can encode a comparable protein or protein structural
domain, such as a cytoplasmic domain, transmembrane domain,
ectodomain, binding site or region, epitopic site or region, etc.
Counterpart domains and their encoding genome segments embrace an
assemblage of species having a range of size and sequence
variations defined by a common biological activity among the domain
or genome segment variants.
[0195] Counterpart genes and genome segments, as well as other
polynucleotides disclosed herein for producing recombinant HMPV,
often share substantial sequence identity with a selected
polynucleotide "reference sequence," for example, with another
selected counterpart sequence. As used herein, a "reference
sequence" is a defined sequence used as a basis for sequence
comparison, for example, a segment of a full-length cDNA or gene,
or a complete cDNA or gene sequence. Generally, a reference
sequence is at least 20 nucleotides in length, frequently at least
25 nucleotides in length, and often at least 50 nucleotides in
length. Since two polynucleotides may each (1) include a sequence
(that is, a portion of the complete polynucleotide sequence) that
is similar between the two polynucleotides, and (2) can further
include a sequence that is divergent between the two
polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of
the two polynucleotides over a "comparison window" to identify and
compare local regions of sequence similarity. A "comparison window"
as used herein, refers to a conceptual segment of at least 20
contiguous nucleotide positions wherein a polynucleotide sequence
may be compared to a reference sequence of at least 20 contiguous
nucleotides and wherein the portion of the polynucleotide sequence
in the comparison window may comprise additions or deletions (that
is, gaps) of 20 percent or less as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. Optimal alignment of
sequences for aligning a comparison window may be conducted by the
local homology algorithm of Smith & Waterman, (Adv. Appl. Math.
2:4821-1981), by the homology alignment algorithm of Needleman
& Wunsch, (J. Mol. Biol. 48:443, 1970), by the search for
similarity method of Pearson & Lipman, (Proc. Natl. Acad. Sci.
U.S.A. 85:2444, 1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection, and the best
alignment (that is, resulting in the highest percentage of sequence
similarity over the comparison window) generated by the various
methods is selected. The term "sequence identity" means that two
polynucleotide sequences are identical (that is, on a
nucleotide-by-nucleotide basis) over the window of comparison. The
term "percentage of sequence identity" is calculated by comparing
two optimally aligned sequences over the window of comparison,
determining the number of positions at which the identical nucleic
acid base (for example, A, T, C, G, U, or I) occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison (that is, the window size), and multiplying
the result by 100 to yield the percentage of sequence identity. The
terms "substantial identity" as used herein denotes a
characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence that has at least 85 percent
sequence identity, preferably at least 90 to 95 percent sequence
identity, more usually at least 99 percent sequence identity as
compared to a reference sequence over a comparison window of at
least 20 nucleotide positions, frequently over a window of at least
25-50 nucleotides, wherein the percentage of sequence identity is
calculated by comparing the reference sequence to the
polynucleotide sequence which may include deletions or additions
which total 20 percent or less of the reference sequence over the
window of comparison. The reference sequence may be a subset of a
larger sequence.
[0196] In addition to these polynucleotide sequence relationships,
proteins and protein regions encoded by recombinant HMPV are also
typically selected to have conservative relationships, that is, to
have substantial sequence identity or sequence similarity, with
selected reference polypeptides. As applied to polypeptides, the
term "sequence identity" means peptides share identical amino acids
at corresponding positions. The term "sequence similarity" means
peptides have identical or similar amino acids (that is,
conservative substitutions) at corresponding positions. The term
"substantial sequence identity" means that two peptide sequences,
when optimally aligned, such as by the programs GAP or BESTFIT
using default gap weights, share at least 80 percent sequence
identity, such as at least 90 percent sequence identity, at least
95 percent sequence identity or more (for example, 97, 98, or 99
percent sequence identity). The term "substantial similarity" means
that two peptide sequences share corresponding percentages of
sequence similarity. Preferably, residue positions that are not
identical differ by conservative amino acid substitutions.
Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Exemplary conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. Abbreviations for the twenty naturally
occurring amino acids used herein follow conventional usage
(Immunology--A Synthesis, 2nd ed., E. S. Golub & D. R. Gren,
eds., Sinauer Associates, Sunderland, Mass., 1991). Stereoisomers
(for example, D-amino acids) of the twenty conventional amino
acids, unnatural amino acids such as .alpha.,.alpha.-disubstituted
amino acids, N-alkyl amino acids, lactic acid, and other
unconventional amino acids may also be suitable components for
polypeptides of the current disclosure. Examples of unconventional
amino acids include: 4-hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .omega.-N-methylarginine, and
other similar amino acids and iminio acids (for example,
4-hydroxyproline). Moreover, amino acids may be modified by
glycosylation, phosphorylation and the like.
[0197] To select candidate viruses, the criteria of viability,
attenuation and immunogenicity are determined according to
well-known methods. Viruses that will be most desired in
immunogenic compositions must maintain viability, have a stable
attenuation phenotype, exhibit replication in an immunized host
(albeit at lower levels), and effectively elicit production of an
immune response in a recipient sufficient to elicit a desired
immune response. The recombinant HMPVs are not only viable and
appropriately attenuated, they are more stable genetically in vivo
retaining the ability to stimulate an immune response and in some
instances to expand the immune response elicited by multiple
modifications, for example, induce an immune response against
different viral strains or subgroups, or to stimulate a response
mediated by a different immunologic basis, for example, secretory
versus serum immunoglobulins, cellular immunity, and the like.
[0198] Recombinant HMPVs can be tested in various well-known and
generally accepted in vitro and in vivo models to confirm adequate
attenuation, resistance to phenotypic reversion, and
immunogenicity. In in vitro assays, the modified virus (for
example, a multiply attenuated, biologically derived or recombinant
HMPV) is tested, for example, for temperature sensitivity of virus
replication, that is, ts phenotype, and for the small plaque or
other desired phenotype. Modified viruses are further tested in
animal models of HMPV infection. A variety of animal models have
been described and are summarized in various references
incorporated herein. HMPV model systems, including rodents and
non-human primates, for evaluating attenuation and immunogenic
activity of HMPV candidates are widely accepted in the art, and the
data obtained there from correlate well with HMPV infection,
attenuation and immunogenicity in humans.
[0199] In accordance with the foregoing description, the present
disclosure also provides isolated, infectious recombinant HMPV for
use in immunogenic compositions. The attenuated virus which is a
component of an immunogenic composition is in an isolated and
typically purified form. By "isolated" is meant to refer to HMPV
which is in other than a native environment of a wild type virus,
such as the nasopharynx of an infected individual. More generally,
isolated is meant to include the attenuated virus as a component of
a cell culture or other artificial medium where it can be
propagated and characterized in a controlled setting. For example,
attenuated HMPV can be produced by an infected cell culture,
separated from the cell culture and added to a stabilizer.
[0200] For use in immunogenic compositions, recombinant HMPV
produced according to the current disclosure can be used directly
in formulations, or lyophilized, as desired, using lyophilization
protocols well known to the artisan. Lyophilized virus will
typically be maintained at about 4.degree. C. When ready for use
the lyophilized virus is reconstituted in a stabilizing solution,
for example, saline or comprising SPG, Mg.sup.++ and HEPES, with or
without adjuvant, as further described herein.
[0201] HMPV-based immunogenic compositions contain as an active
ingredient an immunogenically effective amount of a recombinant
HMPV produced as described herein. The modified virus can be
introduced into a host with a physiologically acceptable carrier
and/or adjuvant. Useful carriers are well known in the art, and
include, for example, water, buffered water, 0.4%, saline, 0.3%,
glycine, hyaluronic acid and the like. The resulting aqueous
solutions may be packaged for use as is or lyophilized, the
lyophilized preparation being combined with a sterile solution
prior to administration, as mentioned above. The compositions may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, sorbitan
monoolaurate, triethanolamine oleate, and the like. Acceptable
adjuvants include incomplete Freund's adjuvant, MPL.TM.
(3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton IN) and
Il-12 ((Genetics Institute, Cambridge Mass.), among many other
suitable adjuvants well known in the art.
[0202] Upon immunization with a recombinant HMPV composition, via
aerosol, droplet, oral, topical or other route, the immune system
of the host responds to the immunogenic composition by producing
antibodies specific for HMPV proteins, for example, F and G
glycoproteins. As a result of the immunization with an
immunogenically effective amount of a recombinant HMPV produced as
described herein, the host becomes at least partially or completely
immune to infection by the targeted HMPV or non-HMPV pathogen, or
resistant to developing moderate or severe infection there from,
particularly of the lower respiratory tract.
[0203] The host to which the immunogenic compositions are
administered can be any mammal which is susceptible to infection by
HMPV or a selected non-HMPV pathogen and which host is capable of
generating an immune response to the antigens of the immunizing
strain. Accordingly, methods are provided for creating immunogenic
compositions for a variety of human and veterinary uses.
[0204] The compositions containing the recombinant HMPV are
administered to a host susceptible to or otherwise at risk for HMPV
infection to enhance the host's own immune response capabilities.
Such an amount is defined to be a "immunogenically effective dose."
In this use, the precise amount of recombinant HMPV to be
administered within an effective dose will depend on the host's
state of health and weight, the mode of administration, the nature
of the formulation, etc., but will generally range from about
10.sup.3 to about 10.sup.7 plaque forming units (PFU) or more of
virus per host, more commonly from about 10.sup.4 to 10.sup.6 PFU
virus per host. In any event, the formulations should provide a
quantity of modified HMPV of the current disclosure sufficient to
elicit a detectable immune response in the host patient against the
subject pathogens.
[0205] The recombinant HMPV can be combined with viruses of other
HMPV serotypes or strains to elicit a desired immune response
against multiple HMPV serotypes or strains. Alternatively, an
immune response against multiple HMPV serotypes or strains can be
achieved by combining protective epitopes of multiple serotypes or
strains engineered into one virus, as described herein. Typically
when different viruses are administered they will be in admixture
and administered simultaneously, but they call also be administered
separately. Immunization with one strain may immunize against
different strains of the same or different serotype.
[0206] In some instances it may be desirable to combine the
recombinant HMPV immunogenic compositions with immunogenic
compositions that induce immune responses to other agents,
particularly other childhood viruses. In another aspect of the
current disclosure the recombinant HMPV can be employed as a vector
for protective antigens of other pathogens, such as RSV, by
incorporating the sequences encoding those protective antigens into
the recombinant HMPV genome or antigenome that is used to produce
infectious virus, as described herein.
[0207] In all subjects, the precise amount of recombinant HMPV
administered, and the timing and repetition of administration, will
be determined using conventional methods based on the patient's
state of health and weight, the mode of administration, the nature
of the formulation, etc. Dosages will generally range from about
10.sup.3 to about 10.sup.7 plaque forming units (PFU) or more of
virus per patient, more commonly from about 10.sup.4 to 10.sup.6
PFU virus per patient. In any event, the formulations should
provide a quantity of attenuated recombinant HMPV sufficient to
effectively stimulate or induce an anti-HMPV or other
anti-pathogenic immune response, for example, as can be determined
by hemagglutination inhibition, complement fixation, plaque
neutralization, and/or enzyme-linked immunosorbent assay, among
other methods. In this regard, individuals are also monitored for
signs and symptoms of upper respiratory illness. As with
administration to chimpanzees, the attenuated virus of grows in the
nasopharynx of recipients at levels approximately 10-fold or more
lower than wild type virus, or approximately 10-fold or more lower
when compared to levels of incompletely attenuated virus.
[0208] In neonates and infants, multiple administrations can be
required to elicit sufficient levels of immunity. Administration
should begin within the first month of life, and at intervals
throughout childhood, such as at two months, six months, one year
and two years, as necessary to maintain an immune response against
native (wild type) HMPV infection. Similarly, adults who are
particularly susceptible to repeated or serious HMPV infection,
such as, for example, health care workers, day care workers, family
members of young children, the elderly, individuals with
compromised cardiopulmonary function, can require multiple
immunizations to establish and/or maintain immune responses. Levels
of induced immunity can be monitored by measuring amounts of
neutralizing secretory and serum antibodies, and dosages adjusted
or vaccinations repeated as necessary to maintain desired levels of
immune response. Further, different candidate viruses may be
indicated for administration to different recipient groups. For
example, an engineered HMPV expressing a cytokine or an additional
protein rich in T cell epitopes may be particularly advantageous
for adults rather than for infants.
[0209] HMPV-based immunogenic compositions can be combined with
viruses expressing antigens of another subgroup or strain of HMPV
to elicit an immune response against multiple HMPV subgroups or
strains. Alternatively, the candidate virus can incorporate
protective epitopes of multiple HMPV strains or subgroups
engineered into one HMPV clone as described herein.
[0210] The recombinant HMPV immunogenic compositions elicit
production of an immune response that reduces or alleviates serious
lower respiratory tract disease, such as pneumonia and
bronchiolitis when the individual is subsequently infected with
wild type HMPV. While the naturally circulating virus is still
capable of causing infection, particularly in the upper respiratory
tract, there is a very greatly reduced possibility of rhinitis as a
result of the vaccination. Boosting of resistance by subsequent
infection by wild type virus can occur. Following immunization,
there are delectable levels of host engendered serum and secretory
antibodies which are capable of neutralizing homologous (of the
same subgroup) wild type virus in vitro and in vivo.
[0211] In one embodiment, recombinant HMPV candidates exhibit a
very substantial diminution of virulence when compared to wild type
virus that naturally infects humans. The virus is sufficiently
attenuated so that symptoms of infection will not occur in most
immunized individuals. In some instances the attenuated virus can
still be capable of dissemination to nonimmunized individuals.
However, its virulence is sufficiently abrogated such that severe
lower respiratory tract infections in the immunized host do not
occur.
[0212] The level of attenuation of recombinant HMPV candidates can
be determined by, for example, quantifying the amount of virus
present in the respiratory tract of an immunized host and comparing
the amount to that produced by wild type HMPV or other attenuated
HMPV which have been evaluated as candidate strains. For example,
the attenuated virus will have a greater degree of restriction of
replication in the upper respiratory tract of a highly susceptible
host, such as a chimpanzee, compared to the levels of replication
of wild type virus, for example, 10- to 1000-fold less. In order to
further reduce the development of rhinorrhea, which is associated
with the replication of virus in the upper respiratory tract, a
useful candidate virus should exhibit a restricted level of
replication in both the upper and lower respiratory tract. However,
the attenuated viruses must be sufficiently infectious and
immunogenic in humans to elicit a desired immune response in
immunized individuals. Methods for determining levels of HMPV in
the nasopharynx of an infected host are well known in the
literature and facilitated by the methods and compositions
disclosed herein.
[0213] Levels of induced immunity provided by the immunogenic
compositions of the current disclosure can also be monitored by
measuring amounts of neutralizing secretory and serum antibodies.
Based on these measurements, dosages can be adjusted or
immunizations repeated as necessary to maintain desired levels of
immune response. Further, different candidate viruses can be
advantageous for different recipient groups. For example, an
engineered recombinant HMPV strain expressing an additional protein
rich in T cell epitopes may be particularly advantageous for adults
rather than for infants.
[0214] In another embodiment, the recombinant HMPV is employed as a
vector for transient gene therapy of the respiratory tract.
According to this embodiment the recombinant HMPV genome or
antigenome incorporates a sequence that is capable of encoding a
gene product of interest. The gene product of interest is under
control of the same or a different promoter from that which
controls HMPV expression. The infections recombinant HMPV produced
by coexpressing the recombinant HMPV genome or antigenome with the
N, P, L and other desired HMPV proteins, and containing a sequence
encoding the gene product of interest, is administered to a
patient. Administration is typically by aerosol, nebulize, or other
topical application to the respiratory tract of the patient being
treated. Recombinant HMPV is administered in an amount sufficient
to result in the expression of therapeutic or prophylactic levels
of the desired gene product. Representative gene products that may
be administered within this method are preferably suitable for
transient expression, including, for example, interleukin-2,
interleukin-4, gamma-interferon, GM-CSF, G-CSF, erythropoietin, and
other cytokines, glucocerebrosidase, phenylalanine hydroxylase,
cystic fibrosis transmembrane conductance regulator (CFTR),
hypoxanthine-guanine phosphoribosyl-transferase, cytotoxins, tumor
suppressor genes, antisense RNAs, and other candidate antigens.
[0215] The following examples are provided by way of illustration,
not limitation. These examples describe the development of a novel
reverse genetics system for the recovery of HMPV from cDNA, and the
use of this system for construction of novel recombinant HMPV
candidates. Thus, the subject matter of the current disclosure is
illustrated by the following non-limiting Examples.
EXAMPLES
[0216] The mononegaviruses generally have a genome that comprises a
single negative-sense strand of RNA tightly encapsidated in a
nucleocapsid. There is a virally encoded polymerase that associates
with the nucleocapsid and directs transcription of the genome by a
process that initiates at the 3' end and copies the linear array of
genes by a sequential stop-start mechanism that yields subgenomic
mRNAs. Replication involves the synthesis of a complete positive
sense copy called the antigenome, which in turn serves as the
template for producing progeny genomes. Newly synthesized viral
glycoproteins are transported to and accumulate at the plasma
membrane. Progeny virus is formed when the nucleocapsid associates
with these areas of modified plasma membrane and acquires a lipid
envelope by budding.
[0217] RSV is the leading cause of hospitalization for viral lower
respiratory tract disease in infants and young children, followed
by HPIV3 (Collins et al., 4th ed. In "Fields Virology," D. M.
Knipe, P. M. Howley, Eds., Vol. 1, pp. 1443-1485,
Lippincott-Williams and Wilkins Publishers, Philadelphia, 2001;
Crowe et al., Vaccine 13:415421, 1995; Marx et al., J. Infect. Dis.
176:1423-1427, 1997). HPIV1 and HPIV2 are the principal etiologic
agents of laryngotracheobronchitis (croup), and can also cause
pneumonia and bronchiolitis (Chanock et al., 4th ed. In "Fields
Virology," D. M. Knipe, P. M. Howley. Eds., Vol. 1 pp. 1341-1:379,
Lippincott-Williams and Wilkins Publishers, Philadelphia, 2001).
RSV and the PIVs also are important causes of respiratory tract
disease in adults. The available evidence indicates that HMPV is
also a significant agent of human respiratory tract disease,
particularly in young infants and children but occurring in all age
groups (Boivin et al. J. Infect. Dis. 186:1330-6, 2002).
[0218] HMPV replicates slowly and inefficiently in cell culture,
rendering it relatively challenging to maintain and study in
laboratory. In addition, trypsin reportedly must be present in the
medium in order to activate infectivity of the virus, which
complicates the lengthy incubations that are necessary to
successfully propagate the virus. Indeed, the difficulty in
propagating HMPV provides an explanation for why it previously
escaped detection as an important respiratory pathogen despite
several decades of widespread cultivation of respiratory tract
specimens in cell culture in many hospitals and research facilities
across the world.
[0219] Methods for the production of RSV and PIV are known in the
art, and compositions to induce an immune response against these
viruses have been described (see Durbin et al., Virology
235:323-332, 1997; Skiadopoulos et al., J. Virol. 72:1762-1768,
1998; Skiadopoulos et al., J. Virol. 73:1374-1381, 1999; Tao et a.,
Vaccine 19:3620-3631, 2001; Durbin et al., J. Virol. 74:6821-6831,
2000; U.S. patent application Ser. No. 09/083,793, filed May 22,
1998; U.S. patent application Ser. No. 09/458,813, filed Dec. 10,
1999; U.S. patent application Ser. No. 09/459,062, filed Dec. 10,
1999; U.S. Provisional Application No. 60/047,575, filed May 23,
1997 (corresponding to International Publication No. WO 98/53078),
U.S. Provisional Application No. 60/059,385, filed Sep. 19, 1997;
U.S. Provisional Application No. 60/170,195 filed Dec. 10, 1999;
and U.S. patent application Ser. No. 09/733,692, filed Dec. 8, 2000
(corresponding to International Publication No. WO 01/42445A2),
each incorporated herein by reference). However, HMPV provides a
contrasting situation in which there are no available reference
reagents such as monoclonal antibodies or available reference viral
strains, and no established methods for the detection and analysis
of HMPV. Knowledge concerning the molecular biology of HMPV has
remained rudimentary and was previously based largely on
extrapolation from other viruses. Because HMPV has only recently
been identified and its characterization had been very incomplete,
there were many challenges to generating suitably attenuated,
immunogenic and genetically stable for use in immunogenic and
diagnostic compositions. To facilitate these goals, it is necessary
to produce recombinant infectious HMPV from cDNA to serve as a
starting point for manipulations that include the development and
staged introduction of predetermined attenuating mutations; the
deletion, modification or rearrangement of existing genes, the
introduction of foreign genes, the swapping of protective antigens
between strains, and the swapping of attenuating or other desired
mutations between HMPV strains and between HMPV and other related
viruses, and to provide additional tools to generate immunogenic
compositions, vectors and immunization methods. The present
disclosure describes the production of recombinant infectious HMPV
from cDNA, and attenuating mutations with this cDNA, as well as the
specific deletions and substitutions within existing genes, the
introduction heterologous nucleic acid sequences, and additional
methods and compositions that are of use in methods designed to
generate an immune response against HMPV.
Example 1
Determination of a Complete Consensus Sequence for the Genome of
HMPV
[0220] The present Example provides the complete genomic sequence
of a HMPV. The subject viral strain for this analysis is strain
CAN97-83 (Peret et al., J. Infect. Dis. 185:1660-3, 2002),
hereafter referred to as strain 83 or CAN83. The sequence was
determined by direct analysis of uncloned RT-PCR products of viral
RNA and thus represents a consensus or majority sequence of a
viable virus. The virulence, and hence wild type status, of this
virus was confirmed by the ability to induce respiratory tract
disease signs in chimpanzees following intranasal inoculation, as
described in a following Example. The determination of a complete
authentic wild type HMPV sequence is a necessary step in developing
a system to produce HMPV of defined sequence and characteristics
for use in immunogenic compositions and methods.
[0221] Cell Lines and Viruses
[0222] HEp-2 (ATCC CCL 23), Vero (ATCC CCL-81) and LLC-MK2 (ATCC
CCL 7.1) cells were maintained in OptiMEM I (Invitrogen GIBCO)
supplemented with 5% fetal bovine serum and, in some instances,
gentamicin sulfate (50 .mu.g/mL). BSR T7/5 cells are baby hamster
kidney 21 (BHK-21) cells that have been transformed and
constitutively express T7 RNA polymerase (Buchholz et al., J.
Virol. 73:251-9). They were maintained in Glasgow MEM supplemented
with glutamine and amino acids (Invitrogen) and 5% fetal bovine
serum. The BSR T7/5 cells were subjected to geneticine (1 mg/ml)
selection every second passage. Biological and recombinant HMPV
were propagated in Vero, LLC-MK2, or BSR T7/5 cells in the absence
of serum and the presence of 5 .mu.g/ml of trypsin.
[0223] Virion RNA Isolation
[0224] Confluent monolayers of LLC-MK2 cells were infected with
HMPV and incubated at 32.degree. C. in the presence of 5 .mu.g/ml
trypsin. 10-14 days post-infection, clarified supernatants were
harvested. Virion RNA (vRNA) was isolated directly from the
clarified supernatants using the QIAamp viral RNA purification kit
(Qiagen) according to the manufacturer's instructions.
Alternatively, virus was purified by sucrose gradient
centrifugation. Supernatant and infected cells were harvested by
scraping with a rubber policeman, mixed with HEPES buffer and
MgSO.sub.4 at a final concentration of 50 mM and 0.1 M,
respectively, and then submitted to three cycles of freezing and
thawing. The mixture was clarified by low-speed centrifugation and
the virus was pelleted by centrifugation at 7000 rpm overnight at
4.degree. C. The virus pellet was resuspended in OptiMEM
(Invitrogen) containing 50 mM of HEPES buffer and 0.1 M of
MgSO.sub.4. The viral suspension was layered on a 30 to 60% sucrose
gradient and was centrifuged for 90 min at 26,000 rpm in an SW28
rotor. The upper virus-containing fraction was collected, diluted
in TEN (10 mM Tris-HCl [pH 7.4], 0.1 M NaCl, 1 mM EDTA) and virus
particles were pelleted by centrifugation at 25,000 rpm in an SW28
rotor for 90 min. The virus pellet was resuspended in TEN and vRNA
was isolated directly from an aliquot using the RNeasy kit (Qiagen)
according to the manufacturer's instructions.
[0225] Reverse Transcription, Polymerase Chain Reaction and
Nucleotide Sequencing
[0226] vRNA was reverse transcribed using specific primers
generated from the published partial sequence of HMPV strain 00-1.
In most cases these failed to yield RT-PCR products, presumably
reflecting nucleotide sequence differences between these two
strains. Therefore additional primers were designed based on the
regions between ORFs in the 00-1 sequence that, by analogy to other
mononegaviruses, would likely contain cis-acting signals that would
be more highly conserved between HMPV strains. This strategy
provided RT-PCR products for strain 83 that, upon sequence
analysis, provided information for designing strain 83-specific
primers. Purified RNA was mixed with 50 pmol of different primers
in a final volume of 14 .mu.l in water and incubated for 5 min at
70.degree. C. and 25 min at 60.degree. C. for denaturing and
annealing steps. Then, 5 .mu.l of 5.times. first-strand buffer 11,
1 .mu.l of 10 mM deoxynucleoside triphosphates (dNTPs), 2 .mu.l of
0.1 M dithiothreitol, 1 .parallel.l of RNaseOUT (40 U/.mu.l;
Invitrogen) and 2 .mu.l of Superscript II reverse transcriptase
(Invitrogen) were added to the reaction. Synthesis of cDNA by RT
was conducted at 44.degree. C. for 1 h and 30 min at 51.degree. C.
to minimize the formation of RNA secondary structures. PCR was
carried out on the reverse transcribed cDNA product using 50 pmol
each of specific forward and reverse primers, 5 .mu.l of 10.times.
Pfx amplification buffer, 1 .mu.l of dNTP mixture, 3 .mu.l of
MgSO.sub.4 and 1 .mu.l of Platinum Pfx DNA polymerase in a total
volume of 50 .mu.l. The PCRs were conducted on a Peltier Thermal
Cycler (PTC-200; MJ Research) as follows: 3 min at 94.degree. C.
and then 40 s at 94.degree. C., 40 s at 55.degree. C. and 1 min/Kb
at 68.degree. C. for 30 cycles and a final extension reaction for 7
min at 68.degree. C. RT and PCR primers designed from the 00-1
strain sequence to prime within ORFs usually were not successful
generating RT-PCR products from the 83 strain, presumably
reflecting sequence differences relative to the 00-1 strain.
Therefore, primers were designed from the semi-conserved sequence
motifs flanking the viral ORFs (see FIG. 8), which resulted in the
successful generation of cDNA. The nucleotide sequences of cDNA
products were determined by direct sequence analysis of the RT-PCR
products using a ABI 3100 sequencer with the Big-Dye termintator
ready reaction kit v1.1 (Applied Biosystems). Finally, the sequence
was assembled from overlapping RT-PCR products.
[0227] To map and sequence the 3' end of the HMPV genome, vRNA was
converted into cDNA and amplified using the 3' RACE System for
Rapid Amplification of cDNA Ends (Invitrogen, Inc.) as specified by
the manufacturer. Briefly, vRNA was polyadenylated at its 3'-end
using poly A polymerase (Invitrogein. Inc.) followed by
first-strand cDNA synthesis primed with oligo (dT) and PCR using an
HMPV specific reverse primer and a forward UAP primer supplied with
the kit. Then, RACE products were used as template for a nested-PCR
using an interial HMPV specific reverse primer and the AUAP primer
supplied with the kit to improve the specificity of the reaction.
Finally, the amplified cDNA nested-PCR products were sequenced
directly.
[0228] To map and sequence the 5' end of tie HMPV genome, vRNA was
processed by first-strand cDNA synthesis, terminal transferase
tailing, and PCR amplification as specified by the 5' RACE System
for Rapid Amplificatiom of cDNA 5' end Version 2.0 (Invitrogen,
Inc.) using the AAP primer supplied with the kit. Then, the
amplified cDNA RACE products were used as template for a nested-PCR
using an internal HMPV specific primer and the AUAP primer supplied
with the kit to improve the specificity of tie reaction. Finally,
the amplified cDNA nested-PCR products were sequenced directly.
[0229] The 5' and 3' ends of the HMPV genome also were confirmed by
genomic RNA ligation and RT-PCR followed by sequencing. Briefly,
vRNA, isolated from sucrose gradient purified virus using the
RNeasy kit (Qiagen), was ligated in presence of 10 U of T4 RNA
ligase (Epicentre) for 3 h at 25.degree. C. and 3 h at 37.degree.
C. A cDNA corresponding to the junction of the ligated ends was
amplified by RT-PCR using HMPV specific primers. The RT-PCR
products were used as template for a nested-PCR using HMPV internal
specific primers to improve the specificity of the reaction.
Finally, the nested-PCR products were cloned into a blunt vector
(pSTBlue-1; Novagen) and 20 clones were sequenced, thereby
confirming the length, content and sequence of the two
previously-uncharacterized ends of the HMPV genome.
[0230] The complete sequence of HMPV strain 83 was determined to be
13335 nucleotides in length, and is set forth in FIGS. 37A-37D.
This sequence was determined directly from uncloned RT-PCR products
and thus represents the authentic majority, consensus sequence of a
virulent, wild type virus. This discovery provides a necessary step
in the development of a reverse genetics system for recovering
complete infectious virus and recombinantly engineered derivatives
from cloned cDNAs. A partial sequence had been published
representing a portion of the genome of HMPV strain 00-1 (van den
Hoogen et al., Virology 295:119-132, 2002).
[0231] A genomic map for HMPV 83 is shown in FIG. 1. Compared to
RSV, the prototype pneumovirus, HMPV lacks the NS1 and NS2 genes
and contains a significant difference in gene order. Specifically,
the SH-G gene pair precedes the F-M2 gene pair in RSV, whereas SH-G
follows F-M2 in HMPV. Identification of HMPV genes was made based
on the presence of ORFs whose predicted protein products exhibit
significant amino acid sequence relatedness with counterpart ORFs
in RSV (N, P, M, F, M2-1 and L) or, in cases where sequence
relatedness was unclear, gene identification were made by genome
position and by the characteristics of the predicted protein (M2-2,
G, , SH). In these analyses, RSV was chosen for comparison because
all of its proteins have been directly identified and in most cases
functions have been assigned. In a number of cases, the size of the
predicted HMPV protein was substantially different from that of its
RSV counterpart. For example, the putative HMPV SH protein was 180%
larger than that of RSV ( 179 amino acids compared to 64) and the
HMPV G protein was 27% shorter (219 amino acids compared to 298)
and lacked certain characteristic structural features such as the
conserved cysteine noose (Johnson et al., Proc. Natl Acad.Sci. USA
84:5625-9, 1987) and CX3C chemokine domain (Tripp et al., Nat. Med.
2:732-8, 2001). Even the putative HMPV P and L proteins exhibited
significant differences in length, being 22% larger and 7% shorter,
respectively, than the proposed RSV counterparts.
[0232] The ends of the HMPV genome were mapped and sequenced. The
deduced sequences of the 3' leader and 5' trailer regions are shown
in FIG. 2. This analysis ruled out the possibility that one or more
additional genes might follow the L gene or precede the N gene,
comparable to the NS1 and NS genes of RSV. Comparison of the leader
and trailer regions of HMPV 83 with that of APV (Randhawa et al.,
J. Virol. 71:9849-9854, 1997) and RSV (Mink et al., Virology
185:615-624, 1991) showed a high degree of sequence identity
between the first 22-23 nucleotides at each end, consistent with
the idea that these conserved sequences represent important
functional domains. In the case of RSV, it was directly
demonstrated that the first 11 nucleotides of the RSV genome
represent the core promoter essential for both transcription and
RNA replication (Fearns et al., J. Virol. 76:1663-1672, 2002).
[0233] Thus, the published partial sequence for strain 00-1 appears
to lack the core element of both the genomic and antigenomic
promoters, and probably lacks any HMPV promoter whatsoever. It is
noteworthy that, within the first 12 nucleotides at either end of
the HMPV genome, there were at least one (3' leader) or two (5'
trailer) sequence differences compared to the other pneumoviruses.
Thus, the expedient of using the termini of APV or RSV to
supplement the incomplete HMPV 00-1 sequence by constructing a
chimeric molecule would not have yielded an authentic HMPV sequence
at either end. While the functional significance of these
particular differences have yet to be fully elucidated, studies
with RSV showed that any point mutations within the genomic
promoter affected transcription or RNA replication or both, and in
most cases these effects were severe (Fearns et al., J. Virol.
76:1663-1672, 2002). In any event, for the development of a genetic
system to engineer recombinant HMPV for use in immunogenic
compositions, it is critical to begin with a verified authentic
wild type sequence, as provided herein. This sequence was used as a
starting point; confirmation that the sequence indeed is authentic
and encodes a functional virus is provided by the recovery of
infectious, wild type-like recombinant virus. This demonstration is
provided in examples below.
[0234] Another important feature of mononegavirus genomic RNA is
that the 3' and 5' ends typically exhibit terminal complementarity,
meaning that the two ends are complementary when aligned in an
antiparallel fashion. This reflects the presence of a conserved
sequence at each end that probably reflects a conserved promoter
core. This terminal complementarity is shown for HMPV 83 and APIV
in FIG. 3. For typical mononegaviruses, the two ends exhibit a high
degree of complementarity for at least the first 12 nucleotides and
often more, and typically within this region the complementarity is
exact or has at most one mismatch. As shown in FIG. 3, the APIV
genome indeed has an exact match for the first 13 nucleotides
whereas, surprisingly, the HMPV 83 genome has two adjacent
mismatches. This further illustrates that extrapolation from other
pneumoviruses to develop a HMPV genetic system based on an
incomplete sequence could not have yielded an authentic HMPV
sequence.
[0235] The sequence of HMPV 83 contained a number of differences
compared to that published for strain 00-1. As examples, FIG. 4
shows that the SH ORF terminated four codons earlier in the 83
sequence than in that for strain 00-1, yielding a shorter SH
protein. Also, the G ORF in the HMPV 83 sequence contained a
deletion that shortened the G protein by 17 amino acids, and the
intergenic region between the F and M2 genes was 13 nucleotides for
83 instead of41 nucleotides. The significance of differences such
as shown in FIG. 4 can be directly evaluated with the reverse
genetics system of this current disclosure.
[0236] The percent amino acid sequence identity between the
predicted proteins of HMPV strain 83 and those of other
pneumoviruses is shown in FIG. 5. It should be noted that a
subsequent Example provides a more extensive comparison between
strain 83 and another HMPV strain, 75, that forms part of the
current disclosure, demonstrating the extent of sequence diversity
between and within the proposed HMPV subgroups. It is noteworthy
that, among all of the pneumoviruses shown in FIG. 5, reverse
genetics systems for the recovery of complete infectious virus have
been reported only in the case of bovine RSV (BRSV) and human RSV
subgroup A. This is despite that fact that PVM has been known for
almost 65 years and has been the subject of extensive molecular
analysis beginning in the late 1980's, and despite the fact that
APV has been known for almost 25 years and also is the subject of
molecular analysis. The fact that reverse genetics systems are not
yet available despite considerable interest in their development
illustrates the difficulty of producing such systems. As has
already been noted, HRSV and BRSV differ from HMPV with regard to
the lack of two genes (NS1 and NS2) and the rearrangement of other
genes (SH and G). HRSV and BRSV also differ extensively from HMPV
with regard to amino acid sequence identity of the predicted HMPV
proteins. Three of the potential HMPV proteins (SH, G, and M2-2),
share only 6-15% sequence identity with their putative HRSV
counterparts, whereas low sequence identity values (for example,
less than 25% identity) are generally considered at best to
correspond to remote homologs or, alternatively, to proteins that
are unrelated. Conversely, the highest value for percent identity
is only 46%, in the case of the L protein. Thus, HMPV is quite
distinct from any virus for which a reverse genetics system for the
recovery of virus has been developed. It should be further noted in
this context that the function of a protein in HMPV cannot be
reliably predicted by extrapolation from results obtained for a
related protein of a related virus, not even for a closely related
virus. Briefly, there is initial uncertainty in deducing proteins
from nucleotide sequence. For example, despite the reported
existence of an M2-2 ORF in APV, the available evidence suggests
that this ORF is not expressed (Ahmadian et al., J. Gen. Virol.
80:2011-2016, 1999), whereas it clearly is expressed in RSV and has
been shown to have a role in regulating RNA synthesis (Bermingham
and Collins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999; Jin
et al., J. Virol. 74:74-82, 2000). Thus, the presence of an ORF is
not proof alone of significance. As another example, in the
Filovirus family, both Ebola and Marburg viruses express a protein
called VP30 that is highly related between the two viruses.
However, these two proteins have different functions (Weik et al.
J. Virol. 76:8532-9, 2002; Muhlberger et al., J. Virol. 73:2333-42,
1999). These are but two examples of the uncertainty in predicting
the existence and function of a protein based on nucleotide
sequence alone. This highlights the importance of direct, relevant
assays as provided hereon for the demonstration of protein
function. These are presented in the current disclosure in two
forms: (i) the development of a mini-replicon that is capable of
transcription and replication when supplied with appropriate viral
proteins, and importantly (ii) the development of a system for the
recovery of complete infectious virus completely from cDNA, which
provides the basis for analysis of the function of all elements of
the genome and its encoded RNA and protein products and infectious
virus.
[0237] The amino acid sequence of the predicted M protein of HMPV
83 was used to estimate the phylogenetic relationships between HMPV
and other members of the Paramyxovirus family, as illustrated in
FIG. 6. As shown in this figure, the Paramyxovirus family consists
of two subfamilies Pneumovirinae and Paramyxovirinae, herein
referred to as pneumoviruses and paramyxoviruses, respectively.
These subfamilies are further divided into genera, the genus of
HMPV being the Metapneumoviruses. A system for producing infectious
recombinant virus did not exist previously for any member of this
genus. This analysis provided further evidence of evolutionary
distance and distinction from pneumoviruses such as RSV and, in
particular, from paramyxovirus such as Sendai, measles, mumps,
human parainfluenza, and Newcastle disease viruses that constitute
the more well-known and more extensively-characterized members of
the Paramyxovirus family.
[0238] Example 2
Assembly of a Full-Length HMPV Antigenomic cDNA Clone Containing a
GFP Transcription Cassette and Recovery of Infectious Recombinant
HMPV
[0239] The complete, authentic, consensus sequence (FIGS. 37A-37D)
determined for HMPV strain 83 provided the basis for designing a
system for producing infectious recombinant virus. FIG. 7
illustrates the construction of a cDNA clone encoding the complete
antigenome of HMPV strain 83 designed from the complete consensus
sequence. Three separate cloned subgenomic fragments were created:
fragment 1 contains the putative N, P and M genes and is bordered
on the upstream (left) side by an added promoter for bacteriophage
T7 RNA polymerase (T7pr) and on the right hand side by an NheI site
that was added to the putative N-F intergenic region as a marker.
The T7 promoter was designed to add three nonviral G residues to
the 5' end of the antigenome, a configuration chosen to improve the
efficiency of the T7 promoter. Fragment 2 included the putative 1,
M2, SH and G genes and is bordered on the upstream side by the
added NheI site and on the downstream side by a naturally-occurring
Acc65I site. The NheI site serves as a marker to distinguish
between cDNA-derived and biologically-derived HMPV. Fragment 3
consists of the putative L gene followed by half of the hepatitis
delta virus ribozyme (HDVribo) bordered by an RsrII site that
occurs naturally within the ribozyme. The sequence of each cloned
cDNA fragment was confirmed. The vector for cloning and expressing
the HMPV 83 antigenome cDNA is pBSKSII, which contains the
hepatitis delta virus ribozyme followed by a terminator for T7 RNA
polymerase (17t) and was derived from vector p3/7 that was used
previously for the recovery of recombinant HPIV1, HPIV2 and HPIV3
(Durbin et al., Virology 235:323-332, 1997; Schmidt et al., J.
Virol. 74:8922-9, 2000; Skiadopoulos et al., J. Virol. 77:270-9,
2003; Newman et al., Virus Genes 24:77-92, 2002). This vector was
modified by the insertion of a polylinker containing AvaI, AaII,
NheI, and Acc65I sites, which in turn served to accept the fragment
1, 2 and 3 restriction sites. Specifically, the polylinker consists
of the following sequence,
CCCGGGGACGTCCTAGCTAGCTAGGGTACCCCGCTCGAGCGGTCCG (SEQ ID NO: 33; SmaI
and RsrII sites italicized), and was inserted between the SmaI and
RsrII sites of the vector p3/7 of Durbin et al. (Durbin et al.,
Virology 235:323-332, 1997). The final cDNA encodes the complete
13335-nucleotide HMPV 83 antigenome containing three added nonviral
G residues at the 5' end. The complete pHMPV-83 recombinant plasmid
("antigenome plasmid") contains 16333 bp.
[0240] It was anticipated that the poor growth and lack of
established reference reagents and assays for HMPV could complicate
the recovery and identification of recombinant HMPV recovered from
cDNA. Therefore, the strategy was adopted to modify the antigenomic
cDNA to insert a foreign marker gene encoding a detectable label,
whereby expression of the marker gene could be readily monitored.
The gene that was chosen encodes the well-known marker or label
GFP, a cnidarian protein whose presence can be visualized by green
fluorescent emission when observed under a fluorescent microscope.
The GFP cDNA was the Enhanced GFP cDNA of Clonetech, Inc.
Expression of a detectable marker in this manner, directly linked
with HMPV production, makes it possible to monitor expression of
GFP by recombinant HMPV in living cells without loss of viability
or sterility, and to thereby detect and quantify the recovery of
rHMPV at all stages of transfection and passage.
[0241] Among the obstacles to this strategy were the lack of direct
information on HMPV transcription and the lack of direct mapping of
gene boundaries. It therefore was necessary to identify sequence
that, when attached to the foreign ORF, would render it capable of
being expressed during the HMPV transcriptional program. It also
was necessary to identify a site within the HMPV genome that could
accommodate the insertion of a foreign gene without disrupting HMPV
growth. The transcription of a typical monomegavirus genome
initiates at or near the 3' end and proceeds by a sequential
start-stop mechanism by which the individual genes are transcribed
into individual, separate mRNAs. Typically, mononegavirus
transcription is guided by short sequence motifs that flank the
viral genes. For example, the genes of RSV, which is the most
thoroughly characterized pneumovirus, contain a highly-conserved
10-nucleotide GS signal on the upstream end of each gene and a
semi-conserved 12- to 13-nucleotide GE signal at the downstream end
of each gene. These motifs were first noticed when RSV mRNAs were
isolated from infected cells and their termini were directly mapped
and sequenced. It is thought that the GS signal directs initiation
of transcription of the individual gene, and the GE signal directs
polyadenylation and termination. The polymerase remains bound to
the template and crosses the intergenic region to resume
transcription at the next gene. In the case of RSV, the analysis of
the mRNA ends showed that the arrangement of genes was unexpectedly
complex. Specifically, the GS signal of the L gene was found to
differ from that of the others, and furthermore was found to be
located within its upstream neighbor rather than downstream of it.
As another example, the M2-1 and M2-2 ORFs were found to be
contained in a single mRNA rather than two. Also, there was no
consistency of spacing of the RSV GS and GE signals with respect to
the ORFs, such that there were instances where the GS signals
overlapped the ORFs or, alternatively, were separated from the ORFs
by as many as 85 nucleotides. Similarly, certain RSV GE signals
overlapped the respective ORF or were separated by as many as 173
nucleotides. Furthermore, the spacing between the RSV genes was
irregular, ranging from the overlapped genes mentioned above to
ones separated by as many as 56 nucleotides. This indicates the
uncertainty of predicting gene boundaries based on the locations of
putative ORFs and potential sequence motifs. However, the methods
of this current disclosure provide for the direct identification of
appropriate transcription signals and insertion sites.
[0242] The complete sequence of HMPV strain 83 was examined for
potential GS and GE motifs, as shown in FIG. 8. These motifs were
identified on the basis of being located between ORFs and by
exhibiting partial sequence conservation among themselves. For
example, the putative HMPV GS motif has the sequence
GgGAcAAgTgaaaATG, where the nucleotide assigmnents in upper case
are conserved in each of the putative genes. In sharp contrast to
RSV, the putative GS signals were found in close proximity to the
start of their respective putative ORFs, with the putative
initiation ATG codon located at positions 14-16 relative to each
putative GS signal. It should be noted that the L ORF was preceded
by two potential GS sequences, either of which was a reasonable fit
for the proposed consensus sequence. One of these is shown in FIG.
8, while the second is located 18 nt upstream
(7102-GGGCAAAACAGCATCC). Typically mononegavirus genes each have a
single GS site, and it is not known which one of these is
functional for the HMPV L gene. However, this can now be readily
determined by the methods of this current disclosure, by mapping
the 5' end of the putative L mRNA by RT-PCR, or by testing each
potential GS signal for functionality in a mini-replicon system, or
by destroying each potential signal in the complete antigenome and
evaluating each mutant for virus recovery and efficiency of
growths.
[0243] The putative GE motif has the sequence AGTtaattaAAAA, where
the upper case assignments are conserved in each of the putative
genes. The limited number of highly-conserved nucleotide
assignments in these motifs made their identification speculative,
particularly since not a single HMPV signal had been directly
identified to use as a benchmark. While these conserved motifs
appear to contain the core of the GS and GE signals, it would not
be surprising to find that either sequence as described here might
be shortened at one or both ends by one or several nucleotides
without complete loss of activity, or that one or more additional
nticleotides added to either side might affect the activity of
either signal. As an indication of the uncertainty of deducing
transcription signals from sequence data alone, van den Hoogen et
al. noted two different types of consensus sequences between genes,
one of which corresponds to that noted above (van den Hoogen et
al., Virology 295:119-132,2002). In this regard, these authors
concluded that they could clearly identify this particular
consensus sequence for two genes, F and L (although, as noted
above, there would appear to be two candidates to be the GS signal
for the L gene), found "variants" in several other genes, failed to
detect a counterpart for the G gene, and noted that "another
[different] repeated sequence . . . was found downstream of each of
the hMPV ORFs except G", raising further uncertainty as to which,
if any, of these sequence motifs might function as a transcription
signal. Thus, it was important to directly identify transcription
signals that could render a foreign sequence competent for
expression by HMPV.
[0244] In order to directly identify sequence signals that can
confer gene expression, the 16-nucleotide putative GS sequence and
13-nucleotide putative GE sequence of the N gene were attached to a
cDNA containing the GFP ORF. In this configuration, the ATG present
in the GS signal replaced that of the GFP ORF. As shown in FIG. 9,
this transcription-cassette was inserted into the HMPV antigenomic
cDNA following nucleotide 41, resulting in an antigenome in which
the GFP gene was placed first in the 5' to 3' order. In the
corresponding HMPV-GFP genome, the GFP gene would be the most
promoter-proximal of the genes. The complete sequence of the
antigenomic rHMPV-GFP cDNA (FIGS. 38A-38D) and flanking plasmid
regions was confirmed by sequence analysis.
[0245] Initially four of the ORFs of HMPV, namely the putative N,
P, L and M2-1 ORFs, were selected to be expressed in separate
plasmids to complement recovery. This initial recovery protocol
focused on proteins predicted to be "internal" proteins (as opposed
to components of the envelope). Examination of the HMPV genome
identified ORFs whose potential products share 41%, 31%, 36%, and
46% identity with the RSV N, P, M2-1 and L proteins, respectively
(FIG. 5), and these ORFs were selected as candidates for inclusion.
The putative M2-1 HMPV ORF also was included in the recovery
because it is a possible internal protein, and studies with RSV,
Ebola virus and Marburg virus indicate that possible M2-1
counterparts can be involved in RNA synthesis. In this regard it is
noted that the presumed M2-1 counterparts appear to play different
roles in Ebola and Marburg viruses despite their high degree of
relatedness (Weik et al. J. Virol. 76:8532-9, 2002: Muhlberger et
al., J. Virol. 73:2333-42, 1999). Also, the putative HMPV M2-1
protein contains a Cys-7/Cys-15/Cys-21/His-25 motif (numbered
according to the complete deduced HMPV M2-1 amino acid sequence,
designated herein the Cys3-1-His1 motif) that is a potential zinc
binding domain that might indicate a role in interacting with
nucleotides or nucleic acid. On the other hand, the putative M2-2
ORF was not included in the initial recovery system, and there is
evidence from APIV that a seemingly-corresponding M2-2 ORF is not
expressed (Ahmadian et al., J. Gen. Virol. 80:2011-2016, 1999).
[0246] The ORF s for the G, F and SH proteins of HMPV were not
included in the initial recovery system, because these ORFs were
predicted to encode transmembrane surface proteins--based on the
presence of putative hydrophobic signals and anchors in the deduced
amino acid sequences. Notably, the predicted HMPV F protein shares
36% amino acid sequence relatedness to that of RSV. The percent
amino acid identity for the putative G and SH proteins between HMPV
and RSV are 15% and 6%, respectively. Despite this substantial
departure in primary structure, other features of the amino acid
sequences, such as the presence of potential glycosylation sites
support the identification of these proteins herein. It is
generally acknowledged in the field that the envelope proteins do
not have a known role in genome transcription and RNA replication.
This does not preclude the possibility that the expression of these
additional ORFs might facilitate recovery, perhaps by increasing
the efficiency of virion assembly. However, an important aspect of
the current disclosure involves identification of a minimal
complement of support plasmids necessary for HMPV recovery. Once
minimal recovery elements were shown to be successful, additional
embodiments can now be constructed and evaluated to optimize the
efficiency of recovery. For example, although the expression of the
M2-1 ORF from a separate plasmid is shown herein to be
non-essential for recovery, it may nonetheless be a desired
complement that could function to influence transcription or RNA
replication or some other aspect of HMPV replication. Accordingly,
expression plasmids were constructed in which the N, P, L and M2-1
ORFs were placed under the control of a T7 transcription promoter
and terminator.
[0247] Support plasmids encoding putative HMPV nucleocapsid and
polymerase proteins were prepared as follows (FIG. 10): to prepare
a support plasmid expressing the putative N protein, vRNA was
subjected to RT-PCR using a positive sense primer designed to
hybridize at beginning of the putative N ORF (nucleotides 55 to 76
in the HMPV genome) and a reverse primer designed to hybridize at
the end of the putative N ORF (nucleotides 1239 to 1220 in the HMPV
genome). The PCR product was digested with XhoI and subjected to a
partial digestion with AflIII, which was done because the putative
N ORF contains a naturally-occurring AflIII and a partial digest
would yield some product that had been cleaved at the terminal site
and not at the internal site. The products were into pTMI (Durbin
et al., Virology 235:323-332, 1997; Durbin et al, Virology
234:74-83, 1997; Elroy-Stein et al., Proc. Natl. Acad. Sci. USA.
86:6126-30, 1998) that was digested with NcoI (which leaves
overhangs compatible with AflIII) and XhoI, and clones containing
the complete putative N ORF were selected and the sequence
confirmed.
[0248] To construct a support plasmid containing the putative P
ORF: of HMPV (pT17-P) (FIG. 10), vRNA was subjected to RT-PCR using
a positive sense primer designed to hybridize at beginning of the
putative P ORF (nucleotides 1263 to 1279 in the HMPV genome) and a
reverse primer designed to hybridize at the end of the putative P
ORF (nucleotides 2147 to 2132 in the HMPV genome). The PCR product
was digested with AflIII and XhoI and cloned into pTM1 that was
digested with NcoI and XhoI. In the final cloned recombinant
plasmid, the sequence of the HMPV insert and flanking plasmid
regions was confirmed by sequence analysis.
[0249] A comparable putative M2-1 support plasmid for HMPV
(pT17-M2-1) was generated (FIG. 10). vRNA was subjected to RT-PCR
using a positive sense primer designed to hybridize at beginning of
the putative M2-1 ORF (nucleotides 4724 to 4740 in the HMPV genome)
and a reverse primer designed to hybridize at the end of the
putative M2-1 ORF (nucleotides 5287 to 5272 in the HMPV genome).
The PCR product was digested with AflIII and BamHI and cloned into
pTM1 that was digested with NcoI and BamHI. In the final cloned
recombinant plasmid, the sequence of the HMPV insert and flanking
plasmid regions was confirmed by sequence analysis.
[0250] A support plasmid containing the putative L ORF of HMPV
(pT7-L; FIG. 10) was made by RT-PCR amplification of two
overlapping fragments and cloned separately into pTM1 vector. The
first fragment was amplified by RT-PCR using a positive sense
primer designed to hybridize at the beginning of the putative L ORF
(nucleotides 7133 to 7157 in the HMPV genome) and a reverse primer
designed to hybridize downstream to a unique AvrII (nucleotides
10892 to 10897 in the HMPV genome) restriction site (nucleotides
11037 to 11018 in the HMPV genome), and the second using a positive
sense primer designed to hybridize upstream to the unique AvrII
site (nucleotides 10845 to 10866 in the HMPV genome) and a reverse
primer designed to hybridize at the end of the putative L ORF
(nucleotides 13150 to 13124 in the HMPV genome). Then, the first
fragment was treated by BamHI and the Klenow fragment of the E.
coli polymerase I to generate a blunt end, digested by AvrII and
cloned into pTMI that was treated by NcoI and Klenow enzyme and
digested by AvrII. Finally, the second fragment of the putative L
ORF was digested by AvrII and XhoI and cloned in the intermediate
construct digested by the same enzymes, leading to the final
construct pT7-L. In the final cloned recombinant plasmid, the
sequence of the HMPV insert and flanking plasmid regions was
confirmed by sequence analysis.
[0251] To provide an initial functional evaluation of these support
plasmids, a mini-replicon system was developed. A cloned cDNA was
constructed so that its expression from a T7 promoter yielded a
negative-sense mini-replicon RNA, or minigenome, that contained, in
3'-to 5' order: the leader end of the HMPV genome and adjoining
putative N GS signal followed by a negative sense copy of the ORF
encoding bacterial chloramphenicol acetyl transferase (CAT),
followed by the putative L GE signal and the trailer end of the
HMPV genome. The correct 3' end was generated by a HDV ribozyme as
described above. In this minigenome, the CAT gene would be under
the control of putative HMPV GS and GE signals, and would be
expressed as a subgenomic mRNA. The minigenome plasmid( was
transfected into HEp-2 cells together with the N, P, M2-1 and L
support plasmids with simultaneous coinfection by vTF-7, a vaccinia
virus recombinant that expresses T7 RNA polymerase. 48 h post
transfection, intracellular RNA was isolated and analyzed by
Northern blot hybridization using a double stranded probe specific
to the CAT cDNA. This analysis demonstrated that coexpression of
the N, P, and L genes with the minigenome plasmid yielded RNA
species of the appropriate size to represent progeny minigenomes,
antigenomes, and subgenomic CAT mRNA. In this assay, the further
addition of the M2-1 gene did not alter the profile of RNA
products. This indicated that in this assay N, P and L coexpression
was sufficient to reconstitute RNA replication and transcription.
These findings are in sharp contrast to RSV, where the expression
of M2-1 is necessary to achieve efficient synthesis of mRNA
(Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85, 1996; Fearns
and Collins, J. Virol. 73:5852-64, 1999). These preliminary results
do not rule out any role for M2-1 in HMPV RNA synthesis, since it
is possible that M2-1 might have one or more functions that are not
required for minigenome replication and transcription but might be
essential to launch infection by complete recombinant virus. The
current disclosure provides the methods to expeditiously evaluate
the role of M2-1 (and M2-2) in viral recovery, gene expression, and
replication.
[0252] To test the ability to generate infectious recombinant HMPV
from cDNA, the full-length HMPV-GFP antigenome plasmid was
cotransfected with the N, P, M2-1 and L plasmids into BSR T7/5
cells, which are baby hamster kidney-21 (BHK-21) cells that
constitutively express T7 RNA polymerase (Buchholz et al. J. Virol.
73:251-59, 1999). Transfections were done in 6-well dishes with 5
.mu.g of antigenome plasmid, 2 .mu.g each of N and P support
plasmid, and 1 .mu.g each of M2-1 and L support plasmid per well.
Transfections were done with SuperFect (Qiagen) or Lipofectamine
2000 (Invitrogen) in medium without trypsin or serum. One day post
transfection trypsin was added to 5 .mu.g/ml. Also, to maintain the
cells in an active state, they typically were split and reseeded at
a 1:3 ratio one or two days post transfection. In another,
alternative permutation, fresh trypsin was added one day post
transfection, the cells were incubated for one or more hours, the
cells were scraped into the medium, and the total suspension was
passaged to fresh LLC-MK2 or Vero cells. Approximately 12-24 h
later the medium was replaced with fresh medium containing 5
.mu.g/ml trypsin (unless otherwise noted, 5 .mu.g/ml trypsin was
the concentration that was routinely used).
[0253] When examined by fluorescent microscopy on successive days
post-transfection, green cells were visualized that initially
consisted of scattered isolated cells and subsequently formed small
foci that consisted of two or more cells and exhibited cytopathic
effect consistent with HMPV. When the transfection monolayer was
split into new cultures, single green cells were visualized within
one day and developed over successive days into multicellular
syncytia. The expression of GFP was monitored by fluorescent
microscopy and photographed 8 days post-transfection. The nature of
the cytopathology was indistinguishable from that produced by
biologically-derived HMPV. The recovered virus replicated in
LLC-MK2 cells with kinetics and cytopathogenicity consistent with
HMPV.
[0254] The ability to monitor the infection in live cells without
compromising sterility can be used to expeditiously examine
conditions to improve growth. For example, it was found that the
replenishment of trypsin by the addition of further amounts during
incubation resulted in a greater number of infected cells. In a
preliminary experiment, the addition of fresh trypsin to a
concentration of 2.5 or 5 .mu.g/ml at two-day intervals resulted in
the highest level of GFP expression and virus spread. However, the
higher level also was associated with increased syncytium
formation, which has the potential to cause more rapid cell death
and decreased yield. Further experimentation, combined with assay
of released infectious virus (an assay that also is facilitated by
the expression of the GFP marker), will serve to identify the
condition of incubation and level of trypsin that is optimal for
the production of infectious virus. The most optimal schedule found
to date has been to add 5 .mu.g/ml fresh trypsin at intervals of 2
or 3 days.
[0255] The expression of GFP by cells infected with the recovered
virus provided evidence that the virus was cDNA-derived and was not
contaminating biologically-derived HMPV. Further identification of
the virus as recombinant HMPV was made in two ways, by
immunofluorescence and by detection of the NheI restriction site
marker. In the immunofluorescence assay, antibodies from hamsters
that had been infected with biologically-derived HMPV reacted
specifically with cells infected with the putative recovered
rHMPV-GFP. For this assay, BSR T7/5 cells were transfected for the
recovery of recombinant HMPV-GFP as described above. Four days
later the cells were split again at a ratio of 1:3 and incubated on
a coverslip for nine more days. Incubations were in the presence of
5 .mu.g/ml trypsin. The cells were fixed with 80% acetone for 15
min at 4.degree. C., followed by incubation with serum from
hamsters that had been infected with HMPV, followed by incubation
with goat antibodies that were specific to hamster IgG and had been
labeled with the fluorescent tag Alexa-488. The same field of cells
was visualized by confocal microscopy with visible light and under
conditions for fluorescence. This demonstrated strong fluorescence
that was specific to foci in HMPV-infected cultures.
[0256] In a related assay series, RT-PCR was performed to amplify
nucleotides 2719 to 3894 (numbered according to their position in
the wild type HMPV sequence exclusive of GFP) in the rHMPV-GFP
genomic RNA (FIG. 12A). This RT-PCR procedure yielded a product of
the expected size when the template was RNA from cells infected
with biologically-derived HMPV or recombinant HMPV-GFP, but only in
the latter case was the cDNA cleaved by NheI, confirming the
presence of the added NheI site specific to the recombinant
antigenomic cDNA from which the recombinant virus was derived.
Amplification of RT-PCR products was dependent on the addition of
RT, indicating that the template was indeed viral RNA and not
contaminating DNA. Infectious recombinant HMPV-GFP also was
recovered when the M2-1 support plasmid was omitted from the panel
of support plasmids. Although these results suggest that M2-1 is
not required for recovery, it is possible that M2-1 is expressed
from the antigenome plasmid, as was found in the case of RSV
(Collins et al. Virology 295: 251-255, 1999). Also, it may be that
co-expression of M2-1 will have some qualitative or quantitative
effect on recovery. This is suggested by the observation that, in a
typical experiment, three times more infectious virus was recovered
in the presence of M2-1. It is believed that the minimal complement
of proteins required for recovery of HMPV is N, P and L.
[0257] The expression of the inserted GFP coding sequence also was
characterized by Northern blot hybridization. Cells were infected
with biologically-derived HMPV83, rHMPV, rHMPV-GFP, or were mock
infected, and total intracellular RNA was isolated three days later
and analyzed by Northern blot hybridization with double-stranded
DNA probes specific to the GFP or M genes. As shown in FlG. 12B,
the GFP-specific probe hybridized only with RNA from
rHMPV-GFP-infected cells (lane 4), and detected an abundant RNA
band of the appropriate size to be the predicted 746-nt (exclusive
of polyA) GFP mRNA transcribed from the inserted transcription
cassette. This showed that the putative HMPV GS and GE
transcription signals indeed functioned in the context of the
foreign ORF to direct the efficient synthesis of a monocistronic
GFP RNA. The GFP probe also hybridized to several larger RNAs that
were of low abundance and appeared to represent GFP-N, GFP-N-P and
GFP-N-P-M read-through mRNAs, as well as to a large, faint band
that was of the appropriate size to contain rHMPV-GFP genome and
antigenome RNA. The very low levels of read-though mRNAs indicated
that termination at the end of the GFP transcription cassette was
very efficient, and hence an HMPV GE signal indeed had been
correctly identified. Analysis with the M-specific probe identified
in each of the viruses bands of the appropriate sizes to be the
predicted 853-nt monocistronic M mRNA as well as bands representing
the P-M and N-P-M read-through mRNAs. The M probe also detected the
genome and antigenome RNA band for each virus, which in the case of
rHMPV-GFP would be 748 nt larger than HMPV83 or rHMPV due to the
presence of the GFP transcription cassette (FIG. 12B, lanes 6, 7
and 8).
[0258] To test the ability to recover recombinant HMPV that lacked
the GFP gene, plasmid expressing the HMPV antigenome depicted in
FIG. 11 but lacking the GFP insert was transfected into BRS T7/5
cells together with N, P, M2-1 and L support plasmids. The recovery
of recombinant HMPV was confirmed by the detection of viral antigen
in single cells and in foci by indirect immunofluorescence assay as
described above.
[0259] The recovered recombinant HMPV strain 83 was compared to the
biologically-derived strain 83 isolate with regard to the
efficiency of multi-cycle replication in vitro. LLC-MK2 cells were
inoculated with 0.01 plaque forming units (PFU) per cell and
incubated at 32.degree. C. in the presence of 5 .mu.g/ml trypsin.
Aliquots of the medium were taken at 24 h intervals and flash
frozen. The aliquots were subsequently diluted in medium containing
fresh trypsin and analyzed in parallel by plaque assay to determine
the infectivity titer. As shown in FIG. 13, the recombinant HMPV
replicated efficiently over the 10 day period tested. The
efficiency of replication of rHMPV was essentially
indistinguishable from that of its biologically-derived
counterpart. The ability to recover a wild type-like virus entirely
from cloned cDNA showed that the sequences used to construct these
cDNAs are fully functional, and thus define an authentic,
functional, prototype wild type HMPV and its encoded proteins. This
is noteworthy, since these viruses exhibit a high frequency of
mutation (and thus a sequence cannot be assumed to be functional).
The rHMPV antigenomic cDNA had been sequenced in its entirety
following construction, as noted above, with the result that its
sequence was confirmed to be exactly as designed. Thus, this
experiment established (i) the exact genomic sequence of a
prototypic HMPV, and (ii) conditions for recovering cloned cDNAs
representing this sequence. This verified prototypic system
provides the basis for the expedited recovery of other strains of
HMPV, as well as for the systematic modification of HMPV via
changes introduced into the cloned cDNA intermediate. Finally, the
ability to precisely reconstruct a wild type recombinant HMPV
indicates the robustness and precision of the methods disclosed
herein. This is further demonstrated by the ability to recovery
numerous engineered derivatives described in subsequent
examples.
[0260] Next, recombinant HMPV-GFP was compared to rHMPV with regard
to the efficiency of multi-cycle growth in vitro to evaluate the
effect of the addition of a transcription cassette. As shown in
FIG. 14, rHMPV-GFP replicated over the tested 10-day period with an
efficiency that, at times, was reduced more than 10-fold compared
to rHMPV. However, at the end of the 10 day period the final titers
were very similar. Thus, a foreign insert can be accommodated by
HMPV without a drastic effect on in vitro replication, such that a
recombinant virus containing such an insert can be feasibly
manufactured. It also is possible that such an insert will prove to
be attenuated in vivo, as can now be expeditiously determined in
experiments employing experimental animals that were identified as
supporting HMPV replication in experiments described in a
subsequent Example.
Example 3
The Development of Recombinant HMPV Derivatives Containing
Pre-Determined Mutations Specifying Desired Phenotypic Changes
[0261] The ability to produce infectious rHMPV from cDNA provides
the basis for the planned introduction of mutations into infectious
HMPV to develop recombinant viral candidates possessing desirable
characteristics for use in immunogenic compositions, such as
temperature-sensitivity, cold adaptation, host range restriction,
improved replication in vitro, reduced reactogenicity, increased
safety, increased antigen expression, increased genetic or
phenotypic stability, broader immunogenic coverage, increased
immunogenicity, and attenuation. Mutations might be devised that
have advantages in other applications as well, such as to yield
increased antigen expression and/or to facilitate preparation of
purified viral protein. Each antigenome can be assayed for the
ability to direct the recovery of infectious virus, and recovered
mutant viruses can be evaluated for in vitro affects on growth and
cytopathogenicity, temperature sensitivity, plaque morphology, and
other characteristics. Appropriate mutant viruses can then be
evaluated for growth efficiency, antigen expression, temperature
sensitivity, plaque morphology, cytopathogenesis, attenuation,
pathogenicity, tropism, genetic and phenotypic stability,
immunogenicity, safety, and protective efficacy in cell culture and
predictive animal models for HMPV activity in human subjects,
including mice, hamsters, cotton rats, and non-human primates, as
well as in clinical studies.
[0262] One type of desirable mutation involves the deletion of
sequence from the HMPV genome. For example, each of the HMPV genes
or ORfs or parts thereof can be systematically deleted either in
its entirety or in part, either alone or in combination with other
genes or ORFs, to obtain virus with improved properties,
particularly for use within immunogenic compositions. Exemplary
genes or ORFs include the SH, G, M2-2 and M2-1 ORFs, but can
involve any gene or ORF or genome region or part thereof, including
non-ORF sequences such as noncoding genes or extragenic regions or
cis-acting signals in whole or in part . As an example of the
strategy of deleting entire genes, antigenomic cDNAs were designed
in which the putative SH and G ORFs, each with their
surrounding-set of putative GS and GE signals, were deleted singly
or together (FIG. 15). To make these constructs, the wild type GFP
was modified to insert BsiWI and BsrGI sites at positions 5459 and
6099 (numbered relative to the complete antigenome exclusive of
GFP, a convention that will be followed throughout this document).
These positions are within the M2-SH and SH-G intergenic regions,
respectively. Cleavage at either of these two sites creates the
overhang GTAC, which also is the case for the naturally-occurring
Acc65I site located at position 6959, within the G-L intergenic
region (FIG. 15). Thus, the presence of these three compatible
restriction sites flanking the SH and G genes facilitate deletion
of either or both genes by cleavage with the appropriate pairs of
enzymes followed by religation. This resulted in cDNAs encoding
rHMPV-GFP antigenomes that lacked the SH (.DELTA.SH) or G
(.DELTA.G) genes or both (.DELTA.SH/G). Each antigenomic cDNA was
evaluated for the ability to direct the recovery of infectious
virus in BSR-T7 cells in the presence of the N, P, L and M2-1
support plasmids. In each case, infectious virus was readily and
successfully recovered. These could then be evaluated with regard
to kinetics and efficiency of growth in vitro and in experimental
animals to define advantageous properties including attenuation.
The presence of the expected deletion in each virus was confirmed
by RT-PCR analysis of the SH-G region of each recovered genome, as
shown in FIG. 16. Also, limited nucleotide sequencing of the RT-PCR
products confirmed the sequences at the junctions of the
deletions.
[0263] The rHMPV-GFP.DELTA.SH, .DELTA.G and .DELTA.SH/G mutants
were compared with rHMPV-GFP and rHMPV with regard to the
efficiency of multi-cycle replication LLC-MK2 cells following
infection with an input of 0.01 PFU per cell (FIG. 17A). In this
experiment, the gene-deletion mutants contained the GFP marker gene
and thus should be compared to rHMPV-GFP. Remarkably, rHMPV-GFP
containing the deletion of a single gene, either SH or G,
replicated 3- to 10-fold more efficiently than rHMPV-GFP alone.
Thus, these genes are not essential for replication in vitro and,
indeed, their loss improved replicative fitness in vitro. This may
reflect a growth advantage due to the shorter length of the genome
and loss of a transcriptional unit, although it also is possible
that the absence of the SH or G protein might somehow improve
growth. This can readily be investigated by the methods disclosed
herein by ablating translation of SH and/or G by making nucleotide
substitutions that remove translational start codons and introduce
termination codons, thereby ablating expression of the protein
while maintaining the gene number, the genome length, and the
number of transcribed mRNAs. rHMPV-GFP lacking both SH and G
replicated with an efficiency similar to that of complete
rHMPV-GFP, suggesting as one interpretation that whatever gain
might be made from deleting genes and shortening the genome was
counteracted by a slight loss of replicative fitness due to the
absence of both SH and G. Thus, production of an HMPV immunogenic
composition might be improved by deletion of SH or G, but deletion
of both might not be optimal. Also, deletion of either gene might
increase the ability of rHMPV to accommodate one or more added
foreign genes. Given the efficient replication of these viruses in
vitro, it will now be possible to evaluate the effects of these
gene deletions on HMPV replication, immunogenicity, and
pathogenesis in experimental animals and human volunteers.
[0264] A second, parallel set of .DELTA.SH, .DELTA.G, and
.DELTA.SH/G mutants was made in an rHMPV strain 83 backbone lacking
the GFP marker gene. Each of the viruses from this second set of
mutants was readily recovered and propagated in vitro. These could
be evaluated with regard to kinetics and efficiency of growth in
vitro and in experimental animals to define advantageous properties
including immunogenicity and attenuation. While the presence of the
GFP gene can facilitate characterization of mutant viruses in vitro
and in vivo, the GFP, gene preferably would not be included in
candidates for developing immunogenic compositions. Hence, it is
advantageous to be able to expeditiously produce mutants with or
without GFP, or both, as appropriate.
[0265] The set of rHMPV.DELTA.SH and .DELTA.G viruses lacking the
GFP marker gene were evaluated for replication in vivo (FIG. 17B).
Golden Syrian hamsters were infected intranasally with
biologically-derived HMPV83, rHMPV, rHMPV.DELTA.SH, rHMPV.DELTA.G,
or rHMPV.DELTA.SH/G. Six animals from each group were sacrificed
three or five days later and the nasal turbinates and lungs were
harvested, homogenized, and analyzed by limiting dilution to
determine the viral titers(FIG. 17B). There were several instances
where differences in titer were observed between days 3 and 5, the
most notable being the higher titers of HMPV 83, rHMPV and
rHMPV.DELTA.SH in the lungs on day 3 versus day 5. However, in
general the titers for the two days followed a consistent pattern.
Recombinantly-derived HMPV replicated in vivo with an efficiency
similar to that of its biologically-derived parent HMPV83. Thus,
rHMPV appeared to have wild type-like growth properties in vivo as
well as in vitro and represents a suitable starting point for
developing attenuated derivatives as candidate vaccines.
[0266] Interestingly, the replication of HMPV was not significantly
reduced by deletion of the SH gene (FIG. 17B). Indeed, the
.DELTA.SH virus replicated marginally better than its wild type
counterpart, perhaps due to the shorter genome length and reduced
gene number. In contrast, deletion of the G gene resulted in a 2.9
(day 5) to 3.2 (day 3) log.sub.10 decrease in replication in the
nasal turbinates, and a 0.3 (day 5) to 2.3 (day 3) log.sub.10
decrease in the lungs. The rHMPV.DELTA.SH/G double-deletion virus
exhibited a similar marked reduction in virus titer. Thus, these
mutant viruses are highly attenuated in vivo.
[0267] In order to evaluate possible immunogenicity and protective
efficacy, the rHMPV, rHMPV.DELTA.SH, rHMPV.DELTA.G and
rHMPV.DELTA.SH/G viruses were administered intranasally to
additional hamsters (FIG. 17C). Serum samples were taken 27 days
later and analyzed for the ability to neutralize HMPV infectivity
in vitro (FIG. 17C). This showed that the highly attenuated
.DELTA.G and .DELTA.SH/G viruses induced high titers of
HMPV-neutralizing serum antibodies even though they were highly
attenuated. On day 28 post immunization, the animals were
challenged intranasally with wild type HMPV, and the animals were
sacrificed three days later and nasal turbinates and lungs were
harvested and virus titers were determined by limiting dilution
(FIG. 17C). This showed that no challenge virus replication could
be detected in animals that had been infected with rHMPV or
rHMPV.DELTA.SH. Interestingly, animals that had been infected with
rHMPV.DELTA.G or rHMPV.DELTA.SH/G also did not have detectable
challenge virus replication in the lungs, and only a low level of
challenge virus replication was detected in the nasal turbinates.
Thus, despite their strongly attenuated nature, the rHMPV.DELTA.G
and rHMPV.DELTA.SH/G viruses were immunogenic and highly protective
against HMPV challenge. These viruses represent promising vaccine
candidates. In particular, it was remarkable that deletion of the G
gene would yield a promising vaccine candidate, since the G protein
is presumed to be an attachment protein important in initiating
infection, and the corresponding deletion in HRSV yielded a virus
that did not replicate in mice (Teng et al, Virology 289,
283-296,2001) and was over attenuated in humans (Karron et al.,
Proc. Natl. Acad. Sci. USA 94: 13961-13966).
[0268] The deletion or otherwise modification of intergenic regions
of HMPV offers an important strategy for achieving improved
phenotypic properties. For example, the intergenic regions of HMPV
strain 83 range in length from 2 nt (N/P) to 190 nt (G/L). Previous
work with RSV indicated that plaque size was decreased if a single
intergenic region was longer than 100 nt in length (Bukreyev et
al., J. Virol. 74:11017-26, 2000). Two of the naturally-occurring
intergenic regions of HMPV strain 83 are greater than 100 nt in
length, namely SH/G (124 nt) and G/L (190 nt). Thus, reducing the
length of these or other intergenic regions offers a strategy to
improve replication in vitro, while increasing intergenic length
offers a strategy for attenuating the virus, as desired.
[0269] As another example, recombinant HMPV was recovered in which
the M2-2 ORF was silenced (FIG. 18A). The proposed M2-1 ORF
initiates at nucleotide position 4724 and terminates at position
5287. The proposed M2-2 ORF potentially initiates at positions 5235
or 5247 and terminates at 5450, and thus overlaps the M2-1 ORF. To
create a mutant HMPV in which the M2-2 ORF is silenced, single
nucleotide substitutions were introduced at positions 5236 and
5248, which removed the two potential translational start codons
without changing the amino acid coding assignment in the
overlapping M2-1 ORF (FIG. 18A). A third nucleotide substitution
was made at position 5272, which introduced a translational stop
codon into the M2-2 ORF. In addition, nucleotides 5289-5440 were
deleted, which deleted most of he M2-2 ORF. These changes were
introduced into the wild type rHMPV antigenomic cDNA as well as
into the rHMPV-GFP antigenomic cDNA. In each case, the .DELTA.M2-2
virus was recovered successfully from cDNA. The rHMPV.DELTA.M2-2
and rHMPV-GFP.DELTA.M2-2 viruses were compared to their respective
parents, rHMPV and rHMPV-GFP, with regard to multi-step growth
kinetics in vitro (FIG. 18B). This showed that each .DELTA.M2-2
virus replicated somewhat less efficiently than its rHMPV-GFP
parent at many of the time points, although by the end of the
experiment at 13 hours, the titers were very similar. These
mutations are of interest for vaccine design, since they do not
greatly affect growth in vitro (which is necessary for vaccine
manufacture) and are likely attenuating in vivo. They are evaluated
in the hamster model noted in FIGS. 17B and C, and described more
completely in a subsequent Example below. Northern blot
hybridization was then used to monitor the synthesis of
intracellular RNA by the .DELTA.M2-2 virus. As shown in FIG. 18C,
the synthesis of genome and antigenome by the .DELTA.M2-2 virus was
not greatly different than that of wild type HMPV. However, the
.DELTA.M2-2 virus directed a level of mRNA synthesis that, when
normalized to the amount of genome from replicate gel lanes, was up
to 8.7-fold that of wild type HMPV. The up-regulation of protein
synthesis without a concomitant increase in virus replication would
be highly desirable for a vaccine virus. This phenotype resembles,
but is not identical to, that of the .DELTA.M2-2 mutant of human
RSV. For RSV, the .DELTA.M2-2 mutant exhibited a delay in the
accumulation of mRNA that was not observed here, and exhibited a
delay and reduction in the accumulation of genome and antigenome
that also was not observed for HMPV. Further unanticipated
differences between the HMPV and RSV are described herein, namely
the ability to disrupt the HMPV M2-1 ORF, or to delete M2-1 and
M2-2 altogether, without loss of viral viability for HMPV whereas
either mutation was lethal for RSV.
[0270] In another application, illustrated in FIG. 19, a series of
single amino acid substitutions was introduced into the
cysteine-histidine motif present in the predicted M2-1 protein of
rHMPV-GFP. This "Cys3-His1" motif consists of three cysteine
residues at M2-1 amino acid positions 7, 15, and 21, and a
histidine residue at position 25 (FIG. 19). This motif is exactly
conserved in RSV and other pneumoviruses. In RSV, the integrity of
this motif is essential for the transcriptional processivity and
anti-termination function of M2-1, and is essential for the
recovery of infectious recombinant virus (Hardy and Wertz J. Virol.
74:5880-5885, 2000; Tang et al., J. Virol. 75:11328-11335, 2001).
The amino acid substitutions introduced into the M2- 1 protein of
rHMPV-GFP were as follows: Cysteine-7-Serine (C7S),
Cysteine-15-Serine (C15S), and Histidine-25-Serine (H25S). As
controls, two additional mutants were made involving amino acids
that are found in the vicinity of the motif but are not considered
to be part of the motif: Tyrosine-9-Serine (Y6S) and
Asparagine-25-Serine (N25S). Each of these mutants was readily
recovered as infectious virus. The C7S, Y19S, C15S, N16S, H25S and
.DELTA.M2-1 mutants of rHMPV-GFP were evaluated for multi-cycle
growth in LLC-MK2 cells in parallel with rHMPV and rHMPV-GFP, as
shown in FIG. 20. The mutants exhibited a range of growth
efficiency relative to the rHMPV-GFP parent, with the Y9S mutant
replicating more efficiently, the C7S mutant replicating
comparably, and the others replicating somewhat less efficiently.
Nonetheless, with the exception of the H25S mutant, the final
titers at 13 days were mostly comparable to that of the rHMPV-GFP
parent.
[0271] In another mutation involving the M2-1 ORF of rHMPV-GFP, the
ATG translational start site was changed to a TAG termination
codon, as shown in FIG. 21, resulting in the mutant designated
rHMPV-GFP.DELTA.M2-1. The next ATG codon is located far down the
ORF, at codon 134 out of 187 codons, and hence this mutation should
ablate synthesis of M2-1. In this same mutant, additional
termination codons were introduced closely downstream in each of
the three reading frames to further preclude possible ribosomal
entry and passage down the ORF. The HMPV-GFP-.DELTA.M2-1 mutant was
readily recovered as infectious virus.
[0272] The ability to rapidly and effectively recover this panel of
mutants in the M2-1 ORF was a further demonstration that the
cDNA-based recovery system is a robust method for expeditiously
developing rHMPV variants bearing predetermined changes. The
finding that a number of these mutants replicated more efficiently
that their direct parent rHMPV-GFP suggests that such mutants would
be valuable for use in immunogenic compositions, since improved
replication in vitro would greatly facilitate the production of
rHMPV and also might be an improved source of HMPV antigen for a
protein-based immunogenic composition. In these embodiments,
rHMPV-GFP was used as a parent because the expression of the GFP
marker facilitates in vitro characterization. These forms also can
be directly evaluated in experimental animals, and the presence of
the marker would facilitate analysis of possible effects on virus
localization in vivo. In addition, non-GFP versions can readily be
generated, as was done above for the .DELTA.SH, .DELTA.G and
.DELTA.SH/G viruses, which would be appropriate for clinical
evaluation. The finding that the Cys3His1 motif, and the M2-1 ORF
altogether, are not required for efficient growth in vitro was
completely unanticipated, particularly since with the Cys3-His1
motif and M2-1 protein were previously shown to be essential for
the recovery of infectious RSV (Tang et al., J. Virol.
75:11328-11335, 2001). In addition, it was independently confirmed
that mutations in the Cys3-His1 motif of RSV, or silencing the M2-1
ORF of RSV, prevented the recovery of recombinant RSV. Although the
Cys3-His1 motif is not required for efficient HMPV replication in
vitro, it is reasonable to anticipate that this motif and this
protein have been conserved in HMPV for functional reasons and do
contribute to some aspect of HMPV replication or interaction with
its host. Thus, it is reasonable to anticipate that some of these
mutations will be found to be attenuating in vivo and useful for
inclusion in a live attenuated HMPV immunogenic composition.
[0273] In another exemplary embodiment the HMPV M2 gene was deleted
altogether from rHMPV-GFP, including its GS and GE signals and both
the M2-1 and M2-2 ORFs (FIG. 22A). The resulting
rHMPV-GFP-.DELTA.M2(1+2) virus was readily recovered and propagated
in vitro. The .DELTA.M2-2, .DELTA.M2-1, and .DELTA.M2(1+2) viruses
were compared with wild type HMPV with regard to the efficiency of
multi-cycle replication in LLC-MK2 and Vero cells (FIG. 22B, upper
and lower panels, respectively). This experiment was performed with
versions of the viruses that express GFP in order to facilitate
visualization of growth but the expression of GFP was not otherwise
relevant to the experiment. In LLC-MK2 cells, each of the M2 mutant
viruses replicated more than 10-fold less efficiently than wild
type. In contrast, in Vero cells each of the M2 deletion viruses
grew to a final titer that equaled or exceeded that of wild type
HMPV. One of the important differences between LLC-MK2 and Vero
cells is that the latter lack the structural genes for type I
interferon. Thus, this result suggested that that the M2 deletion
viruses were more sensitive to interferon than wild type HMPV. In
addition, the observation that the M2 deletion viruses replicated
efficiently in Vero cells is important, since this is a cell
substrate that is acceptable for preparing vaccines for humans.
[0274] To directly evaluate the interferon-sensitivity of the M2
deletion viruses, Vero cells were pre-treated overnight with a
range of concentrations of type I interferon in order to induce an
antiviral state. The ability of the .DELTA.M2(1+2) and .DELTA.M2-2
viruses to replicate was evaluated in the interferon-treated cells
compared to wild type HMPV and wild type HRSV. Each of the viruses
used in this experiment expressed GFP for the purpose of monitoring
the infections, but the presence of GFP was not otherwise relevant
to the results. Cell monolayers were infected at an moi of 1 PFU
per cell (or a multiplicity of infection (moi) of 0.01 in the case
of RSV, reflecting its more efficient growth) and incubated for 4
days. Control monolayers were mock-interferon-treated, infected and
processed in parallel. The media supernatants were harvested and
analyzed by plaque assay to determine virus titers. The fold
reduction between each interferon-treated culture and its
corresponding mock-interferon-treated control was calculated and is
shown in FIG. 22C. Thus, the replication of wild type rHMPV-GFP
were reduced from 5-fold to 1680-fold by increasing amounts of
interferon, compared to mock-interferon-treated controls. In
comparison, the .DELTA.M2(1+2) and .DELTA.M2-2 viruses were more
sensitive and were reduced 19-fold and 13-fold, respectively at the
lowest concentration and were completely inhibited at-the highest
concentration. Viruses that exhibit increased sensitivity to
interferon typically are attenuated in vivo. Thus, these results
suggest that these viruses are candidates to be attenuated
derivatives.
[0275] Another exemplary embodiment of the current disclosure
involves changing gene order to achieve attenuation, increase
antigen expression, or achieve some other desirable phenotype. In a
subsequent Example, the F protein of HMPV is identified as a major
protective antigen, and the G and SH are also identified as virion
surface proteins, which makes them likely protective antigens. In
the wild type gene order, SH, G and F are located at gene positions
6, 7 and 4, respectively. It is anticipated that HMPV transcription
will exhibit a polar gradient, in which promoter-proximal genes are
expressed more efficiently than downstream ones. Thus, placement of
putative protective antigen genes such as SH, G, and F proximal to
the promoter, either singly, as a pair, or as a triplet, will
result in more efficient expression. In this application, the genes
can be moved to this position from their wild type location, such
that the virus retains a single copy of each, or the genes can be
inserted as a second copy, such that the virus now expresses two
copies of one or more of the HMPV protective antigens. The change
in gene expression due to the relocation of genes or to the
addition of a second copy can have other desirable effects, such as
increased or decreased growth in vitro or in vivo.
[0276] Exemplifying this strategy of "shifting" gene position for
developing HMPV derivatives with improved properties, the position
of the SH-G gene pair was altered in rHMPV-GFP (FIG. 23A). In one
construct, designated "Order #1", the SH-G gene pair was moved from
its position following the M2 gene to a position preceding the F
gene. This results in a gene order in that region of the genome
that mimics that of RSV, namely: M-SH-G-F -M2-L. Hence, the
positions of four genes were altered in "Order 1", namely SH and G
(each moved upstream by two positions) and F and M2 (each moved
downstream by two positions). Accordingly, any gene or combination
of genes can be moved within the genome, obtaining changes in gene
expression that can provide desirable phenotypes including
increased antigen expression, improved growth in vitro, and
attenuation. As shown in FIG. 23B, the kinetics and magnitude of
replication of the mutant called RSV Order #1 was indistinguishable
from that of its direct parent rHMPV-GFP. This showed that changes
in gene order can readily be achieved, resulting in infectious
virus that replicates efficiently in vitro and can be evaluated for
desirable properties such as attenuation or improved antigen
expression in vivo.
[0277] As another permutation shown in FIG. 23A, the SH-G pair was
moved to be upstream of the F gene in rHMPV-GFP, and a second copy
of the pair was inserted, resulting in the construct "Order #2".
Thus, this construct contains two copies of the SH and G genes,
which are potential protective antigens. In this case, the F-M2
gene pair has been moved a total of four positions downstream
relative to the viral promoter, and the L gene is now two positions
further downstream compared to the parental wild type HMPV. Yet
another strategy of "gene shifting" involves moving the presumed
protective antigen G and F genes from their normal positions as the
7.sup.th and 4.sup.th genes, respectively, in the HMPV gene map to
promoter-proximal positions. Each gene can be moved individually or
as a pair, as shown in FIG. 24A. In the case of the constructs
shown in FIG. 24A, the promoter-proximal insertion site will be the
same as was used for insertion of the GFP transcription cassette to
make the rHMPV-GFP construct (FIG. 9), from which efficient
expression has been demonstrated. While these represent preferred
signals and insertion sites, other signals and insertion sites can
readily be evaluated and used by the methods of this current
disclosure. Preferred insertion sites would be ones in proposed
intergenic regions or regions of genes that are outside of the ORFs
and putative transcription signals and thus more likely to
accommodate engineering without affecting HMPV replication. A
subsequent Example provides identification of genome regions that
are poorly conserved between divergent strains of HMPV, suggesting
that they are less likely to be essential, which provides further
guidance to identify preferred insertion sites. The transcription
cassette should preferably be designed so that, following its
insertion into the antigenomic cDNA clone, the inserted ORF is
flanked by a set of GS and GE signals and each gene is the backbone
similarly is flanked by a set of transcription signals. In the
gene-shift constructs shown in FIG. 24A, insertion of the G and/or
F gene into a promoter proximal position was accompanied by
deletion of the corresponding copy from the normal antigenome
position: in instances where G was shifted to the first or second
position, nucleotides 6101-6960 were deleted to delete G from its
natural location, and in instances where F was shifted, nucleotides
3053-4710 were deleted to delete the naturally-occurring copy of F.
Analysis of the multi-step growth kinetics of these viruses in
vitro (FIG. 24B) showed that each replicated somewhat less
efficiently than the wild type parent, although the Final titers of
the single-gene shift mutants (rHMPV-G1 and F1) were identical to
that of the rHMPV parent, while the double-gene shift mutants were
only slightly lower.
[0278] A further set of exemplary viruses were constructed in which
the G and F genes were inserted into the promoter proximal
positions, but the copies of the genes in their natural positions
were left undisturbed, as shown in FIG. 25A. Thus, rHMPV+G1 and +F1
contains two copies of the indicated gene (note that the symbol "+"
in the designation indicates that the genes were added rather than
"shifted"). Similarly, rHMPV+G1F2 and +F1G2 contained an additional
copy each of G and F in the indicated position, and rHMPV+G1F2F3
contained an additional copy of G and two additional copies of F in
the indicated positions. Interestingly, the multi-cycle growth
kinetics of these viruses were somewhat reduced compared to their
rHMPV parent (FIG. 25B), but among themselves the various addition
mutations grew with similar kinetics. Thus, while the addition of
one gene was somewhat attenuating, as had also been observed with
GFP, the further addition of one or two more genes had little
effect, even in the case of the relatively large F gene. The level
of replication in this particular experiment had not plateaued, and
the final virus yields were not determined. In these particular
constructs, the added genes were of the same virus strain. However,
it also should be feasible to incorporate one or more genes from a
different strain, such as one possessing antigenic differences.
Thus, for example, a rHMPV+G1F2 virus could be made that expresses
F and G pairs representing two different strains, resulting in a
single bivalent recombinant virus that would elicit a broader
immune response.
[0279] The presence and intactness of the additional genes in the
genome of recovered rHMPV+G1F23 were investigated by RT-PCR
performed on total RNA from recovered virus following three
passages in Vero cells. RT-PCR was performed with a forward primer
hybridizing to the HMPV leader region and a reverse primer
hybridizing to N sequence, which yielded a single, major band of
approximately 4.1 kb, the appropriate size to contain the tandem
G1F2F3 supernumerary genes (not shown). The sequence of the
supernumerary G1F2F3 genes could not be determined directly by
RT-PCR consensus sequencing of viral RNA because primers specific
to G or F would prime on each of the two copies of G and three
copies of F, precluding analysis of each gene individually.
Therefore, the supernumerary genes were first amplified by RT-PCR
in three separate, overlapping segments. The first RT-PCR segment
used a forward (positive-sense) primer (which also served as the RT
primer) from the leader region and a reverse primer from the F gene
(840 nt downstream of the F GS signal): although this latter primer
would prime on each copy of F, priming from the promoter-proximal
copy (F2, numbered as in rHMPV+G1F23) would be the most efficient
in combination with this forward primer, and yielded a .about.1.6
kb fragment that contained the leader, the G1 gene, and about 840
bp of the F2 gene, and was purified by gel electrophoresis for
sequence analysis. The second segment was amplified by RT-PCR with
an RT/forward primer located about 670 nt downstream of the F GS,
and a reverse primer located 554 nt downstream of the F GS:
although the forward primer would prime in all three copies of F
and the reverse primer in all three copies of F, priming in F2 and
F3 would be the only combination to result in successful
amplification of a PCR fragment (.about.1.5 kb) that contained the
downstream 978 bp part of the F2 gene, and a 554 bp upstream part
of F3, and was purified by gel electrophoresis for sequence
analysis. The third fragment was generated with an RT/forward
primer from the upstream end of F ( 14 nt downstream the F GS
signal) and a reverse primer from the upstream end of N: the
forward primer would prime in all three copies of F, but priming in
F3 would be the most efficient and would yield a .about.1.75 kb
product that contained most of the F3 gene, and 120 bp of the N
gene, and was purified by gel electrophoresis for sequence
analysis. This provided an RT-PCR consensus sequence of the G1F-2F3
supernumerary genes that was free of mutations, and all GS/GE
signals and intergenic sequences were correct, indicating that
these added genes were stably recovered in recombinant virus.
[0280] To determine the effect of the gene additions on viral gene
expression, cells were mock-infected or infected with
biologically-derived HMPV83, rHMPV, or rHMPV+G1F23, and total
intracellular RNA was isolated three days later and subjected to
Northern blot analysis with double stranded probes to the F, G or M
genes (FIG. 25C). The F probe detected a major band of the
appropriate size to be the 1644-nt F mRNA, as well as a fainter
band of genome and antigenome, which in the case of rHMPV+G1F23
would be 4008 nt larger compared to HMPV83 or rHMPV due to the
presence of the three added gene copies (FIG. 25C, lane 4). There
were only trace amounts of readthrough mRNAs detected with the F
probe. Phosphorimager analysis indicated that the amount of genome
plus antigenome for rHPMV+G1F23 compared to HMPV83 and rHMPV was
0.9 and 0.5, respectively (corrected for the two extra copies of
the F gene present in rHMPV+G1F23). In comparison, the relative
amount of F mRNA for rHMPV+G1F23 compared to HMPV83 and rHMPV was 6
in each case. Normalized to the respective value of genome plus
antigenome, this corresponded to a 6.6- to 12-fold higher level of
F mRNA for rHMPV+G1F23 compared to HMPV83 and rHMPV.
[0281] Northern blot analysis with the G probe detected, for each
of the three viruses, a band of the appropriate size to be the
711-nt G mRNA as well as bands corresponding to SH-G and M-SH-G
readthrough mRNAs and a band corresponding to antigenome and genome
RNAs (FIG. 25C, lanes 6, 7 and 8). Phosphorimager analysis
indicated that the amount of genome plus antigenome for rHMPV+G1F23
compared to HMPV83 and rHMPV was 1.0 and 0.5, respectively
(corrected for the extra copy of the G gene present in
rHMPV+G1F23), values very similar to that observed with the F probe
noted above. In comparison the relative amount of G mRNA for
rHMPV-G1F23 compared to HMPV83 and rHMPV was 14-fold and 15-fold,
respectively. This would correspond to a 14- to 30-fold higher
level of G mRNA for rHMPV+G1F23 compared to HMPV83 and rHMPV. Thus,
the expression of the G and F genes by the rHMPV+G1F23 virus indeed
was greatly increased.
[0282] Yet another strategy for designing improved immunogenic
compositions against HMPV is to modify the ORF s encoding
protective antigens so that the codon usage is consistent with
efficient translation. Specifically, for many of the codons in a
given ORF, the degeneracy of the genetic code allows for more that
one choice of codon without changing the amino acid coding
assignment. In a number of cases, specific codon choices have been
shown to be associated with efficient translation while,
conversely, other codon choices are associated with decreased
efficiency of translation. Thus, the ORF s encoding HMPV antigens
can be re-engineered to contain codon choices consistent with
efficient translation. This can be done for ORFs in their natural
genome positions as well as for ORFs that have been shifted or for
heterologous ORFs that have been inserted.
[0283] Another exemplary embodiment of the current disclosure
involves using a single attenuated HMPV backbone to make
immunogenic compositions against more than one HMPV strain,
subgroup or serotype. In this application, the protective antigen
genes of an attenuated derivative of HMPV, such as strain 83, are
replaced singly or in toto by the corresponding genes of a
heterologous HMPV. Provided that most of the attenuating mutations
of the attenuated parent HMPV lie in genes other than the
protective antigen genes, this results in a chimeric virus in which
the attenuated backbone of the parent bears the protective antigens
of the heterologous strain. This chimeric virus can then be
administered on its own, or can be combined with the original
parent to make a two-virus bivalent immunogenic composition against
the two viruses. This process can be repeated for additional
heterologous strains as necessary.
[0284] Yet another exemplary embodiment of the current disclosure
involves the addition of one or more transcription cassettes
encoding one or more foreign ORFs, as exemplified in Example II by
GFP. In this application, the foreign gene is placed under the
control of HMPV transcription signals and is inserted into the
genome in a location that does not interfere with virus viability,
such as an intergenic region or a gene noncoding region. In this
way, a variety of foreign proteins can be expressed, such as ones
encoding protective antigens of heterologous pathogens, or
immunomodulatory molecules.
[0285] In yet another exemplary embodiment, rHMPV-GFP was
recombinantly modified to incorporate a Phenylalanine to Leucine
mutation at amino acid position 456 in the L protein, a mutation
designated F456L. This particular mutation was suggested by amino
acid sequence alignment as corresponding to a position that encodes
attenuating mutations in other, heterologous mutant nonsegmented
negative stranded RNA viruses. The sequence alignment in FIG. 26A
shows a phenylalanine-521 to Leucine (F521L) substitution in the L
protein specified by a mutation in the L gene of an RSV
cold-passage temperature-sensitive (cpts) mutant cpts530 (Juhasz et
al, J. Virol. 71:5814-9, 1999). The wild type F521 residue in the
RSV sequence is exactly conserved among other mononegaviruses
examined and, in particular, is present in HMPV. It should be noted
that the amino acid position of the corresponding residue is not
the same in each L protein, reflecting differences in length as
well as small deletions and insertions elsewhere in the various
individual molecules. Thus, the corresponding residue is F456 in
HMPV. From this comparative mapping analysis, the F456 residue in
HMPV is identified as a target for either an identical,
conservative, or even non-conservative amino acid substitution (for
example, substitution of the F456 residue by a leucine, or by a
conservative or non-conservative amino acid as compared to
leucine). In this particular Example, the F456L mutation was
introduced into rHMPV-GFP and was readily recovered in a
recombinant virus, designated rHMPV-GFP-F456L. As shown in FIG. 27,
this mutant replicated in vitro with an efficiency that was
indistinguishable from that of its direct parent, rHMPV-GFP, a
characteristic that is important for efficient viral recovery,
evaluation and manufacture. Thus, this mutant is an excellent
candidate for further evaluation to determine its phenotypic
characteristics in vitro and in vivo. Further description of this
general strategy, including discussion of FIGS. 26B-26F and 28, is
provided above, along with other strategies for the expeditious
design of attenuated derivatives based on the methods of this
current disclosure.
[0286] Yet another strategy for making live attenuated vaccine
candidates is the "Jennerian" approach, which involves the use of a
mammalian or avian virus to immunize against an antigenically
related human virus. This approach is named after Edward Jenner's
successful use of cowpox as the initial vaccine against smallpox in
humans. This approach is based on the idea that a virus that has
evolved to replicate efficiently in its natural animal host often
will replicate inefficiently and thus be attenuated in the
non-natural human host, reflecting a host range restriction. Such a
host range restriction typically is a very stable phenotype, being
the aggregate effect of the many nucleotide and amino acid sequence
differences between the animal and human viral counterpart. A
phenotype that is based on many loci would be refractory to
drift.
[0287] Other examples of "Jennerian" vaccines include the use of
bovine rotavirus or bovine PIV as candidate vaccines against the
respective human viruses. This can involve using the animal virus
as it is provided by nature, as illustrated by the use of bovine
PIV directly as a vaccine (Karron et al., Pediatr Infect Dis J
15:650-654, 1996). Alternatively, the virus can be modified so that
some of the genes are derived from the animal virus and some from
the human virus. In such a case, it is preferable to have the major
protective antigens be derived from the human virus, such that the
immunity that is induced will be maximally effective against the
human virus, and one or more other genes be derived from the animal
virus such that they confer host range restriction. One such
situation involves chimeras between human and bovine parainfluenza
type3 viruses: in one case, the attenuated bovine backbone was
modified so that the protective HN and F antigen genes were
replaced by those of the human virus, thus combining the attenuated
bovine viral backbone with the antigenic determinants of the human
virus (Schmidt et al., J. Virol. 74: 8922-8929, 2000), and in a
second case the human virus was attenuated by replacing a single
gene, that of the nucleocapsid N protein, with that of its bovine
viral counterpart (Bailly et al., J. Virol. 74:3188-3195, 2000).
Surprisingly, each of these bovine/human chimeric viruses was
satisfactorily attenuated and immunogenic in rodent and nonhuman
primates, and both are being prepared for clinical trials.
[0288] FIG. 30A illustrates chimeric HMPV vaccine candidates that
were constricted by individually replacing the N, ) or M ORFs of
HMPV with the corresponding ORFs from APIV subtype C. At the amino
acid level, the N, P and M genes of HMPV and APV-C are 90, 69, and
89% identical, respectively. For comparison, the N protein of
bovine parainfluenza type 3 virus, whose substitution into the
human PIV backbone provided a promising vaccine candidate, also was
within this range, being 85% identical with its human viral
counterpart. Furthermore, there are A, B and D subtypes of APV that
are less closely related to HMPV, providing a source of additional
replacements ORFs whose greater degree of divergence might provide
a higher degree of attenuation.
[0289] FIG. 30 Panels B, C and D illustrate the construction of
chimeric N (Part B), P (Part C), and M (Part D) genes. In each
panel, the upper sequence is that of a cDNA containing the native
N, P or M HMPV gene, and the bottom sequence of each panel is a
cDNA in which the HMPV ORF has been replaced by its APV
counterpart. Both the native HMPV and chimeric HMPV/APV cDNAs have
been constructed to be flanked by restriction recognition sites for
enzymes (BbsI, BsmBI, BfuAI, as indicated) that cleave on the inner
side of each recognition sequence to yield 4-nt overhangs. Since
the actual cut site of these particular enzymes are not sequence
specific, the cut sites can be designed to have the native HMPV
sequence. This makes it possible to ligate the N, P and M genes
together in a single step, as in FIG. 30E. Any combination of HMPV
and HMPV/APV genes can be ligated. For example, ligation of
HMPV/APV N with HMPV P and HMPV M would yield a virus in which the
N ORF alone was derived from APV. Single, double, or triple ORF
replacements can readily be made. This represents one strategy for
introducing attenuating host range mutations, namely a strategy in
which the backbone is derived from HMPV and one or more individual
ORFs from APV are introduced. An alternative strategy that also can
be achieved by the methods of this current disclosure involves
starting with a full length APV genome and replacing the putative
protective antigen genes, namely G, F and SH, by their HMPV
counterparts in whatever combination is desired. This would combine
the antigenic determinants of HMPV with the attenuated backbone of
APV.
Example 4
Analysis of Genetic Diversity Between Distinct Antigenic Subgroups
of HMPV for Development of Improved Immunogenic Compositions
[0290] When van den Hoogen et al. first described the isolation of
HMPV, they proposed that there was significant diversity among the
various isolates (also called strains) and noted that "it is
tempting to speculate that these subgroups of hMPV isolates
represent different serotypes of hMPV" (Nat. Med. 7:719-24, 2001).
"Serotype" means that there would be insignificant antigenic
cross-reactivity between HMPV subgroups following a primary
immunization, and would necessitate the development of a separate
vaccine for each. Even if the HMPV subgroups do exhibit antigenic
cross reactivity, it could be that sufficient antigenic diversity
exists such that neutralization and protection across subgroups is
significantly reduced compared to within a subgroup, and it might
be necessary to have both such subgroups included in an effective
immunogenic composition against HMPV. Sequence diversity also was
reported by Peret et al., albeit following analysis of a small
segment of the genome who proposed that there may be two "major
groups or lineages" of HMPV (J. Infect. Dis. 185:1660-3, 2002).
This also is supported by recent sequence analysis of the N, P, M
and F genes of a number of HMPV isolates (Bastien et al., Virus
Res. 93:51-62, 2003). The extent of sequence and antigenic
diversity among circulating HMPV strains is of considerable
importance to epidemiology, viral detection, and development of
immunogenic compositions. Strains 83 and 00-1 as described herein
are members of one of the proposed subgroups, and therefore a third
strain was analyzed (75) that represents the second subgroup.
Comparison of the three strains provided a measure of the degree of
genetic diversity between subgroups (strain 83 or 00-1 versus 75)
or within a subgroup (strain 83 versus 75).
[0291] A complete consensus sequence (FIGS. 39A-39D) was determined
for the genome of strain 75 using the general procedures described
in Example I above for strain 83. In this analysis, the sequence of
the genomic termni of strain 75 were determined by 3' and 5' RACE
as described in Example, I. Whereas strain 83 had yielded an
unambiguous sequence for its entirety, strain 75 yielded a sequence
in which a number of positions in the SH and G genes contained a
mixture of two assignments. This suggested that the isolate was a
mixture of two or more viruses that likely reflected quasispecies
variants, either present in the original clinical sample or arising
during in vitro passage. Analysis of cloned RT-PCR products
spanning the SH and G genes produced two closely related sets of
sequence that likely represented two different viruses. To obtain a
sequence that could be unambiguously attributed to a single
replication-competent HMPV 75 virus, the isolate was subjected to
biological cloning by plaque isolation. Eight independent
plaque-purified preparations yielded identical consensus sequences
in the region of SH and G that was consistent with that of one of
the two groups of cloned cDNA sequences. This sequence appears to
represent the major constituent and was taken to be HMPV 75.
[0292] The genome of strain 75 is 13,280 nucleotides in length,
compared to 13,335 for strain 83 and an estimated length of 13,378
for strain 00-1. The gene maps of strains 83 and 75, showing the
cross-subgroup comparison, are shown in FIG. 29. Overall, the two
genomes shared 80% nucleotide identity, compared to 81% between the
A2 and B1 strains of RSV representing the two RSV antigenic
subgroups A and B, respectively (Genbank accession numbers M74568
and AF013254, respectively). Similarly, the nucleotide sequence of
isolate 75 (FIGS. 39A-39D) was 80% identical to that of isolate
00-1 (excluding the regions for which sequence was unavailable for
00-1), representing a second cross-subgroup comparison. The
sequences of isolate 83 (FIGS. 37A-37D) and 00-1, representing an
intra-subgroup comparison, were 92% identical. As will be detailed
further herein, the level of divergence between the two HMPV
subgroups bears considerable similarity to that between the two
antigenic subgroups of RSV. This finding was unpredicted and is of
conisiderable value because there is extensive information on the
significance of RSV diversity for epidemiology and development of
inmunogenic compositions, and this provides a very useful context
for consideration of the divergence between HMPV subgroups,
particularly since the overall magnitude is similar. As will be
noted herein, there were some differences between the diversity of
HMPV versus RSV, but even here RSV provides a useful benchmark for
evaluating these differences.
[0293] For most genera of the Paramyxovirus family, there is a
"rule of six" whereby the nucleotide length of each genome is an
even multiple of six (Kolakofsky et al., J. Virol. 72:891-899,
1998). In many cases, the placement of cis-acting signals relative
to hexamer spacing also follows a conserved pattern. However, HMPV
replication and gene expression do not appear to be ruled by a
phasing requirement based on the following observations: (i) the
sequences for the three available isolates differ in length, as
mentioned above; (ii) no integer from 2 to 9 is evenly divisible
into each of the three genomes, and (iii) there was no apparent
pattern of phasing for the gene boundaries within or between
isolates. The absence of a phasing requirement also is suggested by
the ability to recover a number of recombinant viral mutants
possessing a wide variety of lengths, as shown in previous
Examples. The absence of such a requirement was unpredicted, and
will greatly facilitate the construction of recombinant HMPVs.
[0294] The availability of complete genome sequences representing
the two HMPV genetic subgroups provides an opportunity to examine
the relatedness of the proposed HMPV transcription signals between
subgroups. This is illustrated in FIG. 31, which compares the
putative GS, GE and intergenic sequences between the two subgroups.
The GS and GE signals exhibited extensive sequence identity among
the difference genes and between the two subgroups, and a number of
residues were exactly conserved in all sequences. In contrast, the
intergenic regions exhibited only 48% identity between subgroups,
and non-coding gene regions that were not part of the putative GS
and GE signal exhibited only 54% identity. This sharp distinction
in the pattern of sequence identity, combined with the functional
analysis for transcription activity illustrated by the the GFP
expression cassette in Example II above, provides guidance and an
experimental method for a clear delineation of these important
cis-acting sequences. Conversely, the identification of regions
such as the intergenic regions and the non-translated, non-signal
gene regions points out areas where the likehood is increased that
modifications involved in gene insertions, gene rearrangements and
other genetic engineering would be tolerated, providing guidance
that will facilitate production of recombinant virues for use in
immunogenic compositions.
[0295] The conserved sequence at the up-stream end of each gene,
which would include the GS signal, consists of 16 nucleotides,
consensus: GGGACAAnTnnnAATG (FIG. 31). One unusual feature of the
GS signal of all HMPV isolates sequenced to date is the presence of
ATG at positions 14-16 (FIGS. 8 and 31), which initiates the major
ORF. In the SH gene of strains 75 and 00-1, there is an additional
upstream ATG at GS positions 8-10: however, this start site is
unlikely to be used because of its unfavorable sequence context.
The methods of the current disclosure allow for a determination to
be made whether the ATG at positions 14-16 is a functional element
of the HMPV GS signal or whether the conserved spacing of this
translational start site plays some other role in gene expression.
This can readily be accomplished using either complete recombinant
virus or the minigenome system. The downstream end of each HMPV
gene is delineated by the putative 12- to 15-nucleotide GE motif
(consensus: AGTTAtnnnAAAAA), consisting of (in positive sense) a
highly conserved AGTTA pentamer, followed by a poorly conserved
A/T-rich trinucleotide followed by a tract of4 to 7 A residues. A
sequence that resembles the GE motif also is present near the
downstream end of the HMPV leader region (FIG. 31), and the methods
of the current disclosure will clarify whether this functions like
a GE signal.
[0296] FIG. 32 shows the percent nucleotide identity and amino acid
sequence identity between the two HMPV subgroups (strain 83 versus
75) and within a subgroup (strain 83 versus 00-1) for the various
individual ORF s and their encoded proteins. With regard to
nucleotide sequence identity between the two subgroups, the HMPV
ORFS shared 81-87% nucleotide identity except for SH and G, which
were substantially more divergent (69% and 59% identity,
respectively). This pattern resembled that of the two subgroups of
HRSV, for which the percent nucleotide identity for the various
ORFs was 81-85% identical except for the more divergent M2-2, SH
and G ORFs (69%, 77%, and 67% identity, respectively; FIG. 32). As
noted above, the intergenic regions and the noncoding gene regions
(exclusive of GS and GE signals) were 48% and 54% identical,
respectively, between subgroups, compared to values of 42% and
approximately 50%, respectively, for RSV (Johnson et al., J. Gen.
Virol. 69:2901-2906, 1988). Thus, the coding sequence for HMPV G
was only marginally more conserved than HMPV noncoding gene
sequence in general (59% versus 54%). Extensive nucleotide sequence
divergence for the ORFs encoding the SH protein and, in particular,
the G protein also was observed between two strains of the same
subgroup (strain 83 versus 00-1, FIG. 32).
[0297] FIG. 32 also shows the amino acid sequence relatedness of
the various HMPV proteins between the two subgroups (strain 83
versus strain 75), and within a subgroup (strain 83 versus 00-1).
With regard to the total proteome, the HMPV subgroups shared 90%
amino acid sequence identity, compared to 88% identity between the
RSV subgroups. With regard to individual proteins, the most
conserved HMPV proteins were N, M, F, M2-1, and L (.gtoreq.94%
identity between subgroups), followed by P and M2-2 (85%-89%),
followed by the divergent SH (59%) and G (37%) proteins. SH and G
are the only ones for which the percent relatedness at the amino
acid level was less than at the nucleotide level, suggesting that
amino acid substitutions were preferentially retained.
[0298] The pattern of HMPV sequence diversity between subgroups has
general similarities to that of RSV (FIG. 32). For RSV, the
"internal" virion proteins and the F protein constituted a group of
highly conserved species, while the M2-2, SH and G proteins were
more divergent. Three differences between HMPV and RSV were
particularly noteworthy: (i) the HMPV M2-2 protein was markedly
more conserved (89% amino acid identity between subgroups) than its
RSV counterpart (61%); (ii) the HMPV F protein also was more
conserved than was the case for RSV (95% identity between subgroups
versus 89%); and (iii) the HMPV SH and G proteins were markedly
more divergent than their RSV counterparts (59% and 37%, identity
between subgroups, respectively, compared to 72% and 55%,
respectively). FIG. 32 also compares the HMPV proteins within a
subgroup (strain 83 versus 00-1). For most of the proteins, the
percent amino acid identity was very high (.ltoreq.95%), with the
SH and G proteins being more divergent (85% and 70% identity,
respectively). The value of 70% sequence identity for G within an
HMPV subgroup is much lower that the value of 91% reported for RSV
G from the analysis of seven subgroup B strains (Sullender et al;,
J. Virol. 65:5425-5434, 1991). Thus, the HMPV SH protein and, in
particular, the G protein were markedly more divergent within and
between subgroups than was the case for their HRSV counterparts.
This finding was unpredicted and is important because it indicates
these antigens would be ones for which it will be important to have
both subgroups represented in an immunogenic composition once they
are identified as significant protective antigens. A test for
evaluating whether or not an HMPV protein is a protective antigen
is presented below.
[0299] It was also surprising to find that the HMPV M2-2 ORF and
protein exhibited a high level of identity between the two putative
subgroups, since M2-2 of RSV is poorly conserved (FIG. 32). In the
case of RSV, the M2-2 protein was identified as a functional gene
product by the observation that disruption of its ORF in infectious
recombinant virus resulted in altered virus growth and RNA
synthesis (Bermingham and Collins, Proc. Natl. Acad. Sci. USA
96:11259-11264, 1999; Jin et al., J. Virol. 74:74-78, 2000). As
noted above, rHMPV in which the M2-2 ORF has rendered nonfunctional
remains viable in vitro, although its phenotype remains to be fully
defined. The high level of sequence conservation for the M2-2 ORF
between HMPV subgroups suggests that it encodes a significant
product, the absence of which might prove to be attenuating and
useful in immunogenic compositions. The RSV M2-2 ORF is expressed
by a ribosomal stop-restart mechanism, whereby ribosomes that exit
the M2-1 ORF apparently double back and initiate translation at the
overlapping M2-2 ORF (Ahmadian et al., EMBO J. 19:2681-2689). For
the HMPV M2-2 ORF, there are two possible methionyl translational
start sites located 38 and 50 nucleotides upstream of the
translational termination codon of the M2- 1 ORF. These two sites
are exactly conserved among all three strains compared here, and
are embedded in highly conserved sequence. This is another example
where the availability of the cross-subgroup comparison is useful
for identifying structural features that are of potential
significance, and whose possible functional roles can be tested
using complete recombinant virus or minigenomes by the methods of
this current disclosure.
[0300] The HMPV F, SH and G proteins appear to correspond to the
three RSV surface glycoproteins F, SH and G, although there was
clear sequence relatedness between HMPV and HRSV counterparts only
for F (33% amino acid identity). For RSV, G and F are the only
significant neutralization antigens and are the major protective
antigens. The HMPV F protein is identified as a neutralization and
protective antigen of HMPV in an Example below. The status of the
remaining HMPV proteins as potential neutralization and protective
antigens can be determined by the same method. The RSV F protein
has a high degree of antigenic relatedness between subgroups
(Johnson et al., J. Virol. 61:3163-3166), consistent with its
sequence relatedness (FIG. 32), and is the major contributor to
HRSV cross-subgroup neutralization and protection. For HMPV F,
amino acid sequence identity between subgroups was even higher than
for RSV, and it is reasonable to anticipate that this similarly
will make a substantial contribution to cross-neutralization and
cross-protection between the HMPV subgroups. Conversely, the high
degree of divergence of the G protein between and within HMPV
subgroups likely will compromise its contribution to
cross-neutralization and cross-protection to an even greater degree
than is the case for RSV.
[0301] FIG. 33 shows amino acid sequence alignments between and
within a subgroup for the SH (FIG. 33A) and G (FIG. 33B) proteins.
The HMPV SH protein, like its RSV counterpart, is predicted to be a
type II glycoprotein that is inserted in the plasma membrane by a
hydrophobic signal/anchor sequence located near its amino terminus
(FIG. 33A, boxed sequence), with a cytoplasmic amino terminus and
an extracellular carboxy terminus. The SH proteins of strains 75,
83, and 00-1 varied in length (177, 179, and 183 amino acids,
respectively; FIG. 33A) and were considerably longer than their 64-
or 65-amino acid counterparts in RSV. The predicted extracellular
domain has 2-4 motifs for N-linked glycosylation, one of which is
conserved in all three isolates, as well as 3 or 4 potential sites
for O-glycosylation (Hansen et al., Glycoconj. J. 15:115-130, 1998)
that are clustered within residues 75-81 in all three isolates
(FIG. 33A). In addition, the SH proteins of the three isolates
contain 9 or 10 cysteine residues, which are mostly in the
extracellular domain, 9 of which are conserved among all three
strains. The differences in length and amino acid sequence between
the SH proteins of the different isolates were concentrated in the
extracellular domain.
[0302] The HMPV G protein (FIG. 33B) also is a type II surface
protein, bearing a general resemblance (but no significant sequence
relatedness) to the G protein of RSV. The amino acid lengths of the
G proteins of strains 75, 83, and 00-1 were 236, 219, and 236,
considerably shorter than their 289- to 299-amino acid counterparts
in RSV. The HMPV G protein, like its RSV counterpart, contains a
high percentage of serine plus threonine residues (32-35% for the
three isolates, compared to a data base average of 13%) and a
somewhat elevated level of proline residues (7-8.5%, compared to an
average of 5%). Strains 83, -75 and 00-1 had 1, 4 and 4,
respectively, potential acceptor sites for N-linked glycosylation
in the ectodomain. Each HMPV G protein contained more than 40
predicted acceptor sites for O-linked carbohydrate (not shown): by
analogy to RSV it is likely that not all of these are used, and
usage might be heterogeneous. The serine, threonine, proline
residues and the predicted sites for O-linked sugars were
concentrated in the predicted extracellular domain, suggesting that
this region has a long, extended, heavily glycosylated "mucin-like"
structure, as is thought to be the case for RSV.
[0303] The HMPV G protein also resembles that of RSV in having most
of the amino acid divergence localized to the extracellular domain
(FIG. 33B). Specifically, the cytoplasmic and transmembrane domains
combined were 64% and 96% identical between and within subgroups,
respectively, compared to the remarkably low values for the
extracellular domain of 25% and 61% identity between and within
subgroups. For comparison, the value of 25% amino acid sequence
identity is comparable to that observed for the attachment
hemagglutinin-neuraminidase protein of PIVs from different genera,
such as Genus Paramyxovirus versus Genus Rubulavirus or Avulavirus,
and is substantially lower than the value of 42-66% identity
observed for the influenza A hemagglutinin glycoprotein between
subtypes H1, H2 and H3.
[0304] The HMPV G protein lacks several of the prominent features
of its RSV counterpart. HMPV G lacks the conserved 13-amino acid
domain in the RSV G extracellular domain that is exactly conserved
among all RSV strains and is partially conserved in bovine RSV
(Johnson et al., Proc. Natl. Acad. Sci. USA 84:5625-5629, 1987;
Teng and Collins, J. Virol. 76:6164-6171, 2002). Indeed, the HMPV G
ectodomain did not contain more than 3 adjacent amino acids
conserved among all three isolates. HMPV G also lacks the four
conserved, closely-spaced cysteine residues in HRSV G that
partially overlap the 13-amino acid conserved domain, form a
cystine noose, and include a CX3C chemokine motif (Tripp et al.,
Nat. Immunol. 2:732-738 2001). The three HMPV G proteins in FIG.
33B each contained only 1 or 2 cysteine residues, with the only
conserved one being at the inner face of the cytoplasmic domain,
representing a potential acceptor site for fatty acid acylation.
HMPV G also lacks a counterpart to the second methionyl
translational start codon in the HRSV G ORF, which gives rise to a
secreted form and is conserved between HRSV subgroups (Roberts et
al. J. Virol. 68:45384546, 1994). Only isolate CAN75 has an
appropriately located potential counterpart (methionine-43), and it
is the fourth rather than the second methionyl residue. Thus,
comparison of sequence features whose presence or absence is
consistent across subgroups helps guide the identification of
structural features as possible targets for mutational analysis and
construction of chimeric recombinant viruses for use in immunogenic
compositions according to the methods of this current
disclosure.
[0305] The high level of divergence in HMPV SH and G proteins is
consistent with the idea that there is a preference for retaining
amino acid changes in these proteins. It is particularly noteworthy
that the divergence was concentrated in the extracellular domain of
each protein (FIG. 33A, B). A codon-by-codon examination of the
nucleotide coding sequences for the extracellular domains of F, G
and SH ORFs indicated that 75% of the codons containing a single
nucleotide change were associated with amino acid substitutions,
compared to 50% for SH and 7% for F. Based on the findings herein,
it is suggested that the extraordinary frequency of amino acid
substitution per nucleotide substitution in HMPV G (and to a lesser
extent SH) reflects two factors: (i) selective pressure for amino
acid change, which might come from host immunity, and (ii) the
ability of the protein to tolerate substitution, which might be due
to its proposed extended, unfolded nature. In contrast. HMPV F
appears to resemble HRSV F, which is one of the more highly
conserved proteins despite its status as a major HRSV
neutralization and protective antigen. This likely reflects
functional and structural constraints on amino acid substitution in
this folded, globular glycoprotein. The time scale of nucleotide
and amino acid substitution for HMPV is not known, but it was noted
that, for HRSV, it is difficult to detect antigenic drift and thus
the differences probably reflect a slow accumulation over years.
These findings indicate that diversity in SH and G is an important
consideration for HMPV epidemiology, virus detection, and
development of effective immunogenic compositions.
[0306] In conclusion, the present Example defines the amount
genetic diversity that exists between the two proposed HMPV genetic
subgroups an(d shows that, overall, the diversity between subgroups
is similar in magnitude to that observed between the
well-characterized RSV antigenic subgroups, although certain
specific differences were observed. By comparing the two divergent
HMPV subgroups, the identification of transcription signal motifs
was further substantiated. Conversely, the identification of
poorly-conserved regions such as the non-signal, non-protein-coding
gene regions and the intergenic regions provides guidance in
selecting these sites as being likely to be amenable to genetic
manipulation. Importantly, the F glycoprotein was shown to be
highly conserved between the two subgroups, which highlights the
usefulness of this surface protein for incorporation in recombinant
HMPV and chimeric recombinant HMPV of the current disclosure for
production of broadly cross-reactive immunogenic compositions.
Conversely, the other two viral surface proteins, SH and G, were
demonstrated to be highly divergent between the subgroups, and will
therefore be employed within recombinant HMPV and chimeric
recombinant HMPV to contribute to subgroup-specific, rather than
cross-subgroup, immunity (although they will often be used in
multivalent immunogenic compositions as described herein above).
The diversity of the two genetic subgroups at the antigenic level
is-characterized herein, and the importance of the F protein as a
major neutralization and protective antigen is demonstrated.
Example 5
The Use of Experimental Animals to Evaluate HMPV Infectivity and
Immunogenicity
[0307] Generation of Hamster Polyclonal Anti-HMPV Antibodies to the
75 and 83 Strains
[0308] To demonstrate that HMPV is infectious in a rodent animal
model and can elicit an immune response characterized by production
of HMPV-specific antibodies in the model host, three groups of 8-
to 10-week-old golden Syrian hamsters (Mesocricetus auratus) were
infected intranasally (IN) with approximately 10.sup.3-10.sup.4
TCID.sub.50 of: a mixture of HMPV strains 75 and 83, HMPV 75 alone,
or HMPV 83 alone. As described in the previous Example, strains 75
and 83 (also referred to as CAN75 and CAN83, respectively)
represent the two genetic subgroups of HMPV and thus provide an
evaluation of the biological characteristics of viruses from each
subgroup and, in particular, give a measurement of the antigenic
diversity between subgroups. Serum samples were collected on day 28
or 44 post-immunization and examined for their ability to detect
HMPV infected monkey kidney cells (LLC-MK2) by indirect
immunofluorescence. LLC-MK2 cells grown in OptiMEM (Invitrogen
GIBCO) on glass slides were infected with a mixture of HMPV strains
75 and 83, strain 75 alone, or strain 83 alone, and were incubated
at 32.degree. C. in OptiMEM medium with 5 .mu.g/ml trypsin. At
approximately 70 hours post-infection the cells were fixed and
permeabilized with 3% formaldehyde and 0.1% Triton-X100 in PBS, as
described previously (Skiadopoulos and McBride, J. Virol.
70:1117-24, 1996). Indirect immunofluorescence was performed with
pooled hamster sera from animals that had been inoculated with HMPV
75+83, and fluorescein (FITC)-conjugated goat anti-Syrian Hamster
IgG antibodies (Jackson ImmunoResearch Laboratories, Inc, West
Grove, Pa.) were used for indirect immunofluorescence. As a
control, serum from an adult human seropositive for HMPV was also
used to detect HMPV infected cells in a comparable indirect
immunofluorescence assay. Immunofluorescence was detected and
images were collected on a Leica TCS-SP2 (Leica Microsystems,
Mannheim, Germany). Images were processed using the Leica TCS-NT/SP
software (version 1.6.587), Imaris 3.1.1 (Bitplane AG, Zurich
Switzerland), and Adobe Photoshop 5.5 (Adobe systems). Both the
human serum and the serum from hamsters infected with HMPV strains
75 and 83 were able to detect LLC-MK2 cells infected with either
HMPV strain 75 or 83, demonstrating that IN-administered HMPV is
infectious and immunogenic in hamsters. Sera from animals that
received strain 75 alone or strain 83 alone were further analyzed
as described herein.
[0309] Generation of Rabbit Polyclonal Antibodies to HMPV Strains
75 and 83
[0310] Specific pathogen free rabbits (Harlan Sprague Dawley) were
surgically implanted with sterilized plastic wiffle balls and were
immunized by injection of approximately 10.sup.5 TCID.sub.50 of
HMPV strain 75 or 83 into the wiffle ball, as described previously
(Clemons et al., Lab. Anim. Sci. 42:307-11, 1992). Fluid present in
the chambers was collected on day 29, and the animals received two
boosts by injection of HMPV preparations that had been purified and
concentrated by centrifugation in sucrose gradient following
conventional procedures: the first boost was on day 29 and the
second was on day 57. Fluid present in the chambers was collected
again on day 71. These sera were analyzed as described herein.
[0311] Virus Titration and Serum Antibody Infectivity
Neutralization Assays
[0312] Viral titer was quantified by serial 10-fold dilutions of
virus applied to LLC-MK2 monolayer cultures on 96-well plates that
were then incubated for 7 days at 37.degree. C. in OptiMEM medium
with 5 82 g/ml added trypsin, as described previously (Newman et
al., Virus Genes 24:77-92, 2002). Virus infected monolayers were
detected by incubation with hamster polyclonal antiserum, which had
been raised by TN infection with a mixture of strains 75 and 83 as
described above, followed by a second antibody consisting of
peroxidase-conjugated rabbit anti-Syrian hamster IgG. Bound
antibody-antigen complexes were detected by immunoperoxidase
staining achieved with ECL chromogenic substrate.
[0313] The susceptibility of HMPV to neutralization by
HMPV-specific antiserum was determined using an endpoint dilution
neutralization assay performed with heat inactivated hamster,
rabbit or chimpanzee sera. The hamster and rabbit sera were
prepared as described above. The chimpanzee sera were from 31 caged
animals, as described further herein. Briefly, replicate aliquots
each containing approximately 100-200 TCID.sub.50 of HMPV 75 or 83
were mixed with aliquots from a dilution series prepared from a
heat-inactivated serum sample and incubated at 37.degree. C. for 1
hr. The virus-antiserum mixtures were then transferred to LLC-MK2
monolayers in 96-well plates and the cells were incubated for 1
hour at 32.degree. C. The monolayers were washed two times with
OptiMEM to remove serum and were overlaid with OptiMEM supplemented
with 5 .mu.g/ml added trypsin. The cells were then incubated for 7
days at 32.degree. C. and infected monolayers were detected by
immunoperoxidase staining with the polyclonal hamster anti-HMPV
75+83 antiserum and peroxidase-conjugated rabbit anti-Syrian
hamster IgG and ECL chromogenic substrate, as described above. The
neutralization titer is read as the highest dilution of antibody at
which the cultures were 50% reduced for infection. This assay was
used to determine the HMPV neutralization titer of serum from
infected animals, and also was used to screen animals to determine
whether they had previously been exposed to HMPV.
[0314] With the availability of the HMPV-GFP virus, the
neutralization assay can now be simplified. In this case, replicate
aliquots of HMPV-GFP virus (approximately 100-200 TCID.sub.50)
would be mixed with aliquots of a dilution series of serum as
above, incubated for 1 hour at 32.degree. C., and transferred to
LLC-MK2 cells. The cells would be washed to remove serum, overlaid
with OptiMEM supplemented with 5 .mu.g/ml added trypsin, and
incubated for 7 days at 32.degree. C. Infected cells could be
visualized directly by fluorescent microscopy. The neutralization
endpoint would be indicated by the appearance of green
fluorescence, indicative of infection. Many of the details of this
assay can be varied as necessary, such as the period of incubation
of HMPV-GFP and antibody, the choice of cell substrate, and the
period of incubation of the cells. In particular, the use of
HMP-GFP means that the cells can be monitored by fluorescent
microscopy to select an optimal time at which to read the assay.
The efficiency of virus titration was compared by visualization of
GFP verus immunostaining and found to be identical. However,
detection by GFP was easier because the progression of the virus
infection can be monitored and the plates read at an optimal time.
In contrast, immunostaining can only be done once, and the timing
is determined by estimating cytopathology by eye, which is more
qualitative and not as precise. A further advantage is that GFP can
be read directly, whereas immunostaining is a multi-step,
time-consuming process requiring antibodies and development
reagents. Furthermore, the washing steps need in immunostaining
frequently result in damage to or removal of the cell monolayer,
which can complicate or ruin the assay. Thus, using rHMPV-GFP as an
indicator virus for antibody titration (or evaluation of antiviral
agents) provides a better method for titration.
[0315] Evaluation of the Antigenic Relatedness of Strains 83 and
75
[0316] As described in the previous Example, it has been suggested
that HMPV isolates can be segregated into two genetic subgroups
(van den Hoogen et al., Nat. Med. 7:719-24; Peret et al., J.
Infect. Dis. 185:1660-3, 2002). The previous Example described in
detail the level of genetic diversity between the two proposed
subgroups. The present Example (and also Example VI) describes the
amount of antigenic difference between the two subgroups. The
extent of antigenic diversity among circulating HMPV strains is of
considerable importance to epidemiology and for development of
effective immunogenic compositions.
[0317] The antigenic relatedness between strains 75 and 83 was
investigated by a cross neutralization study. As described above,
antiserum was raised in rabbits by parenteral immunization of
gradient-purified HMPV 75 or 83 followed by a boost of the same
material, with serum obtained following each step. In addition, as
described above, hamsters were infected IN with strain 75 or 83 and
sera were obtained. These individual sera were assayed for the
ability to neutralize both strain 75 and 83. Thus, this would
compare the ability of antibodies from an animal exposed to either
strain to neutralize the infectivity of the homologous strain
versus its ability to neutralize the heterologous strain. If
extensive antigenic differences exit, each serum should be much
less effective in neutralizing the heterologous strain compared to
the homologous strain.
[0318] As shown in Table 1, hamsters were immunized by intranasal
infection with strain 83 or 75 and the sera were collected on day
44 and evaluated for the ability to neutralize each strain in
vitro. Of the eleven animals that were infected with strain 75,
there was no consistent pattern in which the homologous 75 strain
was neutralized more efficiently than the heterologous 83 strain:
four animals neutralized the homologous strain more efficiently (up
to 3.0 log.sub.2 difference, with animal 2164 exhibiting the
greatest difference), but one (animal 2168) showed no difference
and the remainder neutralized the homologous strain less
efficiently (up to 3.0 log.sub.2 difference, animal 2174).
Conversely, all ten animals infected with strain 83 yielded sera
that neutralized the homologous 83 strain more efficiently than the
heterologous 75 strain, with a difference ranging from 0.4
log.sub.2 (animal 2203) to 3.4 log.sub.2 (animal 2201). Thus, each
of the HMPV strains representing the two heterologous subgroups
induced antibodies that efficiently neutralized the homologous and
heterologous viruses, although antibodies from animals infected
with strain 83 appeared to be somewhat less effective in
neutralizing the heterologous versus the homologous virus.
[0319] Also as shown in Table 1, rabbits were immunized with either
strain of HMPV and then boosted with the same strain, and
post-primary immunization and post-boost sera were collected and
tested for the ability to neutralize the homologous and
heterologous subgroup virus. The initial immunization of rabbits
resulted in sera that had relatively low neutralizing titers
against either strain of HMPV, with titers in the range of
.ltoreq.4.3 log.sub.2 to 8.4 log.sub.2. With these primary sera,
there was no consistent difference between the efficiency of
neutralization of the homologous versus the heterologous strain.
Following the boost, the titers were substantially higher, in the
range of 6.9 log.sub.2 to 11.4 log.sub.2, and in most cases the
neutralization liter against the homologous strain was greater than
against the heterologous strain. However, the difference was
relatively modest, ranging from either no difference to a
(difference of 2.6 log.sub.2. For example, animal R030, which had
been immunized and boosted with strain 75, had a titer of 11.4
log.sub.2 against the homologous strain 75 and a titer of 9.9
log.sub.2 against the heterologous strain 83, corresponding to a
1.5 log.sub.2 difference. The other rabbit (R031) that was
immunized and boosted with strain 75 yielded serum that neutralized
the homologous strain 75 four times (2.0 log.sub.2) more
efficiently than the heterologous strain 83. In the converse
situation, with animals (R032 and R033) that had been immunized and
boosted with strain 83, the titers following the boost either
showed no difference for the heterologous versus homologous strain
(animal R03), or showed 0.5 log.sub.2 higher homologous versus
heterologous titer (animal R033). Thus, out of the eight
post-immunization and post-boost rabbit sera, one had a lower
homologous versus heterologous titer, three had no difference, and
four had a greater homologous versus heterologous titer, with the
greatest difference being-2.6 log.sub.2. This indicates a trend
towards greater homologous versus heterologous reactivity, but the
inconsistent nature of the pattern and the low magnitude is
consistent with the interpretation that the two strain exhibit a
very high degree of cross-neutralization in vitro. Thus, the high
degree of antigenic relatedness observed with post-infection
hamster sera was confirmed with rabbit sera both upon initial
immunization and following a boost.
[0320] The high degree of antigenic cross-reactivity observed in
vitro indicates that at least one major HMPV neutralization antigen
or major epitope is conserved between the HMPV subgroups. The
genetic analysis presented in the previous Example showed that, of
the three HMPV surface proteins F, SH and G, the F glycoprotein was
highly conserved between subgroups. In contrast, the SH and G
glycoproteins were highly variable and neither protein had no more
than three contiguous conserved amino acid in the extracellular
domain, suggesting that conserved antigenic sites would be rare or
nonexistent. The HMPV neutralization and protective antigens had
yet been identified previously. In the case of RSV, the F and G
surface proteins are the only significant neutralization antigens
and the major protective antigens (Connors et al., J. Virol.
65:1634-1637 1991), and in the case of the human PIVs the two
surface proteins F and HN also are the neutralization and major
protective antigens. A subsequent Example (Example VI) will
illustrate identification of a major HMPV neutralization and
protective antigen effective in conferring protection against
infection with either genetic subgroup.
1TABLE 1 Homologous and heterologous HMPV antibody responses in
rodents following immunization with HMPV strain CAN75 or CAN83
Serum-neutralizing antibody titer.sup.c Rodent species Immunizing
(recip. log.sub.2) against: and number virus.sup.a,b CAN75 CAN83
Hamsters 2163 CAN75 8.9 6.9 2164 CAN75 8.3 5.3 2165 CAN75 7.9 5.9
2169 CAN75 9.9 10.4 2170 CAN75 8.4 7.3 2166 CAN75 8.4 8.9 2167
CAN75 8.9 9.9 2168 CAN75 8.4 8.4 2173 CAN75 9.9 12.7 2174 CAN75 9.4
12.4 2175 CAN75 9.9 11.9 2201 CAN83 7.3 10.7 2202 CAN83 8.4 11.3
2203 CAN83 10.9 11.3 2204 CAN83 7.9 10.3 2205 CAN83 7.9 10.3 2206
CAN83 8.9 11.7 2207 CAN83 7.9 10.3 2208 CAN83 8.9 11.7 2209 CAN83
9.9 12.3 2210 CAN83 8.9 10.3 Rabbits R030 CAN75 .ltoreq.4.3.sup.d
5.9.sup.d R030 (p.b.) CAN75 11.4.sup.e 9.9.sup.e R031 CAN75
.ltoreq.4.3.sup.d .ltoreq.4.3.sup.d R031 (p.b.) CAN75 11.4.sup.e
9.4.sup.e R032 CAN83 .ltoreq.4.3.sup.d 6.9.sup.d R032 (p.b.) CAN83
6.9.sup.e 6.9.sup.e R033 CAN83 8.4.sup.d 8.4.sup.d R033 (p.b.)
CAN83 8.4.sup.e 8.9.sup.e .sup.aHamsters were inoculated
intranasally with 10.sup.4 TCID.sub.50 of the indicated strain of
HMPV. .sup.bRabbits were immunized parenterally with 10.sup.5
TCID.sub.50 on day 0 with the indicated strain of HMPV, and were
boosted on day 28 with 10.sup.7 TCID.sub.50 of gradient-purified
virus of the same strain of HMPV. .sup.cHamster neutralizing
antibody titer, day 44 post-immunization serum. All animals had
pre-immunization neutralizing antibody titers of .ltoreq.1.0.
.sup.dRabbit neutralizing antibody titer, day 28 post-immunization.
All animals had pre-immunization neutralizing antibody titers of
.ltoreq.1.0. .sup.eRabbit neutralizing antibody titer post-boost,
day 70 following the initial immunization. All animals had
pre-immunization neutralizing antibody titers of .ltoreq.1.0. p.b.:
post-boost
[0321] Prevalence of HMPV Serum Antibodies in Chimpanzees
[0322] Thirty-one captive chimpanzees (pan troglodytes) were
screened for the presence of serum neutralizing antibodies to HMPV
strain 75 or 83, using an in vitro infectivity neutralization assay
with HMPV strain 75 or strain 83 as the antigen, (Table 2).
Nineteen animals (61%) were found to be seropositive for either one
or both HMPV strains. Serum neutralizing antibody titers to HMPV
strain 75 ranged from undetectable (.ltoreq.1.0 log.sub.2) to
.gtoreq.9.6 log.sub.2. Serum neutralizing antibody titers to HMPV
strain 83 ranged from .ltoreq.1.0 log.sub.2 to 8.6 log.sub.2.
Twelve animals (39%) did not have detectable HMPV-75 neutralizing
liters: these 12 animals also were seronegative to strain 83. Four
additional animals were seropositive to strain 75 but seronegative
to strain 83. These four animals had very low neutralizing antibody
titers (2.6-4.0 log.sub.2), and their specificity is provisional.
The prevalence of HMPV antibodies in chimpanzees indicates that
these animals can become readily infected with HMPV in captivity,
most likely from their human handlers. It also is possible, and
indeed likely, that there is animal-to-animal transmission. The
ability of these animals to be infected by contact from humans or
other animals is a strong indication of permissiveness to HMPV. The
finding that very few animals were seropositive for only one of the
two strains suggests that these strains are highly related
antigenically in primates and likely represent only a single HMPV
serotype.
2TABLE 2 Prevalence of HMPV-neutralizing antibodies in chimpanzee
sera Serum neutralization titer (recip. log.sub.2) to Sample
Chimpanzee indicated HMPV strain.sup.1 number ID No. 75 83 1 99A005
6.6 4.6 2 A0A001 7.0 5.6 3 A0A002 5.6 4.0 4 A1A005 1.0 .ltoreq.1.0
5 A1A007 .ltoreq.1.0 .ltoreq.1.0 6 A1A001 2.6 3.6 7 A1A002 5.6 3.6
8 A1A003 3.6 .ltoreq.1.0 9 A1A006 .ltoreq.1.0 .ltoreq.1.0 10 A0A006
4.0 .ltoreq.1.0 11 A0A007 4.6 3.6 12 A0A005 2.6 .ltoreq.1.0 13
A0A009 3.6 .ltoreq.1.0 14 AOA003 7.6 4.6 15 99A004 7.6 6.6 16
A2A001 .ltoreq.1.0 .ltoreq.1.0 17 A2A004 .ltoreq.1.0 .ltoreq.1.0 18
A2A006 .ltoreq.1.0 .ltoreq.1.0 19 A2A009 .ltoreq.1.0 .ltoreq.1.0 20
A2A011 6.0 4.6 21 A2A013 6.6 4.0 22 A2A014 6.6 5.6 23 98A008 8.6
6.0 24 99A001 8.0 5.0 25 99A002 8.0 6.6 26 99A003 9.6 8.6 27 A1A010
.ltoreq.1.0 .ltoreq.1.0 28 A1A012 .ltoreq.1.0 .ltoreq.1.0 29 A1A013
.ltoreq.1.0 .ltoreq.1.0 30 A2A007 .ltoreq.1.0 .ltoreq.1.0 31 A2A008
.ltoreq.1.0 .ltoreq.1.0 Control serum .ltoreq.1.0 .ltoreq.1.0
(pre)2 Control serum 8.3 8.3 (post)3 .sup.1Numbers in parentheses
are the titers expressed as the reciprocal dilution log.sub.2. 2.
pre-immune serum from hamsters before inoculation with both HMPV 75
and 83. 3. Post-immunization serum obtained from hamsters following
inoculation with both HMPV 75 and 83.
[0323] HMPV Infection of Chimpanzees and Monkeys: Virus
Replication, Immunogenicity and Effect of Previous Exposure to
HMPV
[0324] The ability of HMPV strains 75 and 83 to infect chimpanzees
identified as seropositive or seronegative by the HMPV
neutralization assay described above was evaluated. As shown in
Table 3, eight animals were organized into four groups (two animals
per group): group 1 was seronegative for strain 75 and 83. Group 2
and group 4 were seropositive for both strains. Group 3 was
seronegative for 83 and had low or no detectable neutralization
titers to strain 75. Groups 1 and 2 were administered strain 75,
and groups 3 and 4 were administered strain 83. The animals were
inoculated intranasally (IN) and intratracheally (IT) with 1 ml per
site of 1.times.10.sup.5.2 TCID.sub.50/ml of the indicated virus.
Administration was by both the intranasal (IN) and intratracheal
(IT) routes to provide the opportunity for the virus to replicate
at each site (Hall et al., Virus Res. 22:173-84, 1992; Crow et al.,
Vaccine 12:783-790, 1994). Animals were housed and specimens
collected as previously described (Durbin et al., J. Infect. Dis.
179:1345-51, 1999; Skiadopoulos et al., Virology 260:125-35, 1999).
The animals were observed clinically for 16 days post-inoculation.
Nasopharyngeal swabs were collected on days 1 to 10 and 12
post-infection and tracheal lavages were collected on days 2, 4, 6,
8, 10, and 12 post-infection. Each of the four seronegative animals
(groups 1 and 3) had nasal discharge on days 7, 8, 9 and 10
post-infection. As a precautionary measure, groups 1 and 3 were
started on penicillin on day 8, because of evidence of congestion
and noise in the lungs. One of the animals in group 1 had thick
mucus in the day 8 post-infection TL sample and had decreased
appetite. The seropositive animals (groups 2 and 4) showed no signs
of illness throughout the course of the study. Thus, HMPV
seronegative chimpanzees can develop illness following infection
with the 75 and 83 HMPV strains demonstrating that these viruses
are infectious and virulent in chimpanzees. Since the recombinant
HMPV strain 83 virus differs from its biologically-derived parent
by only four nucleotide differences involved in creating the NheI
marker in the M/F intergenic region (FIG. 7), it is also considered
a "wild type" virus in terms of genomic and phenotypic
characteristics. Importantly, animals that were seropositive to
both strains were protected from disease following challenge with
either strain. This appears to be the first observation
demonstrating the feasibility of immunizing against HMPV: since
natural exposure protects against disease, it is reasonable to
expect immunization with an appropriate immunogenic composition
comprising a recombinant HMPV of the current disclosure will elicit
a desired immune response as well. These results indicate that
serum neutralizing antibody titers of 4.6 (animal AOA003) to 7.0
(animal AOA001) log.sub.10 are sufficient to confer protection from
HMPV disease. Results obtained with chimpanzees are particularly
relevant given the close evolutionary relationship between
chimpanzees and humans.
3TABLE 3 Infection of chimpanzees with HMPV Serum neutralization
titer to clinical signs Chimpanzee HMPV strain: HMPV (day
post-infection) Group ID No. 75 83 administered.sup.1 rhinorrhea
cough 1 A1A005 .ltoreq.1.0 .ltoreq.1.0 75 6, 7, 8, 9, 10, 12 A1A007
.ltoreq.1.0 .ltoreq.1.0 7, 8, 9, 10 2 99A005 6.6 4.6 75 A0A001 7.0
5.6 3 A1A006 .ltoreq.1.0 .ltoreq.1.0 83 7, 8, 9, 10 A0A005 2.6
.ltoreq.1.0 7, 8, 9, 10 5 4 AOA003 7.6 4.6 83 99A004 7.6 6.6
.sup.1The animals were inoculated intranasally (IN) and
intratracheally (IT) with 1 ml per site of 10.sup.5.2
TCID.sub.50/ml of HMPV strain 75 or strain 83.
[0325] The virus titers in the nasopharyngeal swabs and tracheal
lavages taken from the HMPV-infected chimpanzees described in Table
3 were determined by an end point dilution assay with viral
infectivity detected by immunostaining. As shown in Table 4, the
chimpanzees shed relatively low levels of virus. This was
particularly noteworthy for the animals in group 1, which were
seronegative for both strains, and group 3, which was seronegative
for strain 83: animals even though all of the animals in these
groups exhibited clinical disease signs in response to infection,
the mean peak titers of virus recovered from the nasopharyngeal
swabs ranged from 1.9 to 3.2 log.sub.10, and the titers from the
tracheal lavage were 1.8 to 2.0 log.sub.10. The swabs and lavages
from animals in groups 2 and 4, which were seropositive to HMPV,
either lacked detectable HMPV or had a very low titer (1.5
log.sub.10). Thus, the lack of disease signs correlated with a
reduced or undetectable level of virus shedding. The apparent
incongruity between the low level of HMPV shedding in the presence
of disease symptoms might reflect difficulty in retrieving
infectious virus from secretions, possibly due to inactivation by
host immune factors or a high level of cell-association of HMPV in
the epithelial cells lining the respiratory tract of the
chimpanzees. Since the chimpanzee is so closely related to humans,
this raises the possibility that detection of HMPV in human
secretions might also be inefficient. The inability to detect
substantial HMPV shedding was unexpected, since RSV is known to be
labile and cell-associated in vitro, but nonetheless is shed from
RSV infected chimpanzees presenting with RSV-associated illness at
a level that is 100 to 1000-fold greater than was the case for
HMPV-infected chimpanzees (Belshe et al., J. Med. Virol. 1:157-162,
1977). Alternatively, the level of HMPV replication in chimpanzees
may indeed be low, but infection may trigger immune- or
chemokine-mediated pathology resulting in development of disease
symptoms (Glass et al., Curr. Opin. Allergy Clin. Immunol.
3:467473).
[0326] Serum samples taken four to six weeks post infection
contained high levels of serum neutralizing antibodies for every
experimental group, with no clear difference between animals that
had been seronegative or seropositive prior to the experimental
infection. The post-immunization titers ranged from 7.4 to 10.2
log.sub.10, considerably higher than the range of 4.6 to 7.0
log.sub.10 that was associated with protection from clinical
disease. Interesting, in this small sample there was no evidence of
antigenic differences between strains 75 and 83. Specifically,
seronegative animals that were immunized with either strain (groups
1 and 3) developed antibodies with titers that were somewhat higher
against strain 83 than against strain 83, irrespective of the
immunizing virus.
[0327] The animals that were seronegative for the strain used in
the primary infection were next challenged with the heterologous
HMPV strain. As shown in Table 4, both groups of animals were
completely protected from cross-challenge, indicating that previous
infection by one HMPV lineage protects chimpanzees from subsequent
infection by the heterologous lineage. Thus, in the experimental
animal that most closely resembles the human, immunity induced by
an HMPV strain representing either genetic subgroup protected
against infection with an HMPV strain from the heterologous
subgroup.
[0328] It also was of interest to investigate the replication and
immunogenicity of HMPV 75 and 83 in monkeys, which are more
plentiful, less expensive and easier to house than chimpanzees.
Twenty-two rhesus macaques and twenty African green monkeys were
screened for serum neutralizing antibodies to HMPV, and each animal
was seronegative to HMPV (.ltoreq.1.0 log.sub.2). This limited
sampling suggested that these two species of monkeys are not
readily infected with HMPV by exposure in captivity. Four animals
of each species were infected and intratracheally with 10.sup.5.2
TCID.sub.50 of either HMPV strain CAN75 or CAN83. Four animals of
each species were infected and intratracheally with 10.sup.5.2
TCID.sub.50 of either HMPV strain CAN75 or CAN83 at each site, and
nasopharyngeal swab Samples were taken at days 1 to 10 and 12
post-infection, and tracheal lavage samples were taken at days 2,
4, 6, 8, 10 and 12 post-infection (Table 5). The samples were
assayed for virus titer by end point dilution. Illness did not
develop in any of the monkeys. The infected rhesus monkeys had very
low titers of virus, ranging from 0.9 to 2.6 log.sub.10 TCID.sub.50
(Table 5). Furthermore, the post-immunization titers of serum
neutralizing antibodies were also relatively low, ranging from 6.3
to 8.4 10g.sub.2. Thus, rhesus monkeys support the replication of
HMPV, but the level of replication is low.
[0329] Infected African green monkeys (Table 5) shed titers of HMPV
ranging from 2.2 to 4.9 log.sub.10 TCID.sub.50, and developed serum
neutralizing antibody titers that ranged from 9.4 to 10.9
log.sub.2. The time course of HMPV replication in samples collected
from the nasopharynx and the trachea of the African green monkeys
is shown in FIG. 35, and indicates that virus shedding occurred
over a broad window of more than six days. These data showed that
African green monkeys are permissive for HMPV replication and, in
particular, represent a suitable primate host in which to evaluate
the replication and immunogenicity of rHMPV derivatives for use in
immunogenic compositions. In particular, the highest titers of
virus and antibody were obtained with strain 83, which is the one
represented by recombinant HMPV of this current disclosure. In this
regard, the ability to monitor HMPV replication tip to almost 5
log.sub.10 with concomitant immunogenicity provides a wide window
for assessing effects on replication and immunogenicity due to the
incorporation of attenuating mutations.
[0330] Thirty days following the first infection, the African green
monkeys were challenged with the heterologous HMPV strain (Table
5). Both groups of animals were completely protected from
cross-challenge, indicating that previous infection by one HMPV
lineage confers resistance to subsequent infection by the
heterologous strain. These data indicate that African green monkeys
represent a suitable primate host in which to evaluate the
protective efficacy of HMPV immunogenic compositions.
[0331] The studies described above provide data on the primary
antibody response to strain 75 or 83 in seronegative animals, and
describe the ability of the resulting antibodies to neutralize the
homologous versus heterologous subgroup virus. These data were
analyzed statistically by the Archetti-Horsfall formula (Archetti
and Horsfall, J. Exp. Med. 92:441-462, 1950) to determine the
degree of antigenic relatedness based on in vitro neutralization.
Each species was calculated separately, showing that the percent
relatedness of the two HMPV subgroups was 48%, 64% and 99%,
respectively, based on the data from hamsters, Rhesus monkeys and
African green monkeys, respectively. This compares with a value of
25% and 31% for the two subgroups of RSV, based on analysis of
animal and human sera, respectively (Johnson et al. J. Virol.
61:3163-3166, 1987; Hendry et al., J. Infect. Dis. 157:640-647,
1988). These data indicate that the two HMPV genetic subgroups are
highly related antigenically and do not represent distinct
serotypes or, likely, not even significant antigenic subgroups.
This high degree of relatedness for the two HMPV subgroups is based
on in vitro neutralization, and thus needs to be taken with some
caution, since cross-neutralization in vitro might be mediated by a
single highly conserved antigen, whereas cross-protection in vivo
might require the contribution of more than a single antigen, or
might be augmented by the inclusion of additional antigens.
Nonetheless, the present findings provide the first indication that
the genetic diversity of HMPV can readily be accommodated by an
HMPV vaccine, and that such a vaccine might need to represent only
a single HMPV strain.
4TABLE 4 Level Of Replication, Immunogenicity And Cross-Protection
Of HMPV Strains CAN75 And CAN83 In Chimpanzees Post-immunization
Serostatus at Pre-immunization serum Level of virus (day 28) serum
Level of challenge Group Virus time neutralization titer.sup.d to:
replication.sup.e in: neutralization titer.sup.d to: virus
replication.sup.f in: no..sup.a administered of inoculation CAN75
CAN83 NP swab TL CAN75 CAN83 NP swab TL 1 CAN75 negative.sup.b
.ltoreq.1.0 .+-. 0.0 .ltoreq.1.0 .+-. 0.0 1.9 .+-. 0.4 2.0 .+-. 0.8
8.7 .+-. 0.3 10.2 .+-. 0.3 .ltoreq.0.5 .+-. 0.0 .ltoreq.0.5 .+-.
0.0 2 CAN83 negative.sup.b 3.8 .+-. 1.5 .ltoreq.1.0 .+-. 0.0 3.2
.+-. 0.5 1.8 .+-. 0.3 8.9 .+-. 1.0 10.2 .+-. 0.3 .ltoreq.0.5 .+-.
0.0 .ltoreq.0.5 .+-. 0.0 3 CAN75 positive.sup.c 6.8 .+-. 0.2 5.1
.+-. 0.5 1.5 .+-. 0.0 .ltoreq.0.5 .+-. 0.0 7.4 .+-. 0.5 9.2 .+-.
0.2 nd nd 4 CAN83 positive.sup.c 7.6 .+-. 0.0 5.6 .+-. 1.0
.ltoreq.0.5 .+-. 0.0 1.5 .+-. 0.3 8.4 .+-. 1.5 10.2 .+-. 0.2 nd nd
.sup.aAnimals in groups of two were inoculated IN and
intratracheally (IT) with 10.sup.5.2 TCID.sub.50 of the indicated
virus in a 1 ml inoculum at each site. .sup.bChimpanzees in this
group were seronegative (mean antibody titer of <1:4) for the
homologous infecting HMPV strain at the start of the study.
.sup.cChimpanzees in this group were seropositive (mean antibody
titer of >1:20) for the homologous infecting HMPV strain at the
start of the study. .sup.dSerum neutralization antibody titer to
the indicated virus is expressed as the mean reciprocal log.sub.2
.+-. standard error. .sup.eLevel of virus replication is expressed
as the mean of the peak virus titers (log.sub.10 TCID.sub.50/ml
.+-. standard error) for the animals in each group irrespective of
sampling day. The NP titers on day 0 were .ltoreq.0.5 log.sub.10
TCID.sub.50/ml. The lower limit of detection is 1.0 log.sub.10
TCID.sub.50/ml. A value of .ltoreq.0.5 log.sub.10 TCID.sub.50/ml is
assigned to samples with no detectable virus. .sup.fOn day 35,
animals were challenged IN and IT with 10.sup.5.2 TCID.sub.50 of
the heterologous strain of HMPV in a 1 ml inoculum at each site.
Level of virus replication is expressed as the mean of the peak
virus titers (log.sub.10 TCID.sub.50/ml .+-. standard error) for
the animals in each group irrespective of sampling day. The NP
titers on day 0 were .ltoreq.0.5 log.sub.10 TCID.sub.50/ml. The
lower limit of detection is 1.0 log.sub.10 TCID.sub.50/ml. nd: not
done
[0332]
5TABLE 5 Level Of Replication, Immunogenicity And Cross-Protection
Of HMPV Strains CAN75 And CAN83 In Two Species Of Monkeys
Post-immunization Level of virus (day 28) serum Percent Level of
challenge Primate No. of Virus replication.sup.b in: neutralization
titer.sup.c to: antigenic virus replication.sup.e in: species
animals inoculum.sup.a: NP swab TL CAN75 CAN83 relatedness.sup.d NP
swab TL Rhesus 4 CAN75 1.3 .+-. 0.1 0.9 .+-. 0.2 6.4 .+-. 0.6 7.2
.+-. 0.3 64 nd nd Rhesus 4 CAN83 1.6 .+-. 0.1 2.6 .+-. 0.3 6.3 .+-.
0.1 8.4 .+-. 0.3 nd nd African Green 4 CAN75 2.6 .+-. 0.5 3.2 .+-.
0.4 9.4 .+-. 0.3 10.8 .+-. 0.4 99 1.4 .+-. 0.1 0.8 .+-. 0.1 African
Green 4 CAN83 2.2 .+-. 0.2 4.9 .+-. 0.1 9.5 .+-. 0.5 10.9 .+-. 0.4
0.6 .+-. 0.1 0.9 .+-. 0.2 .sup.aAnimals were inoculated IN and
intratracheally (IT) with 10.sup.5.2 TCID.sub.50 of the indicated
virus in a 1 ml inoculum at each site. .sup.bThe level of virus
replication is expressed as the mean of the peak virus titers
(log.sub.10 TCID.sub.50/ml .+-. standard error) for the animals in
each group irrespective of sampling day. Virus titrations were
performed on LLC-MK2 cells. The lower limit of detection is 1.0
log.sub.10 TCID.sub.50/ml. A value of .ltoreq.0.5 log.sub.10
TCID.sub.50/ml is assigned to samples with no detectable virus.
.sup.cMean serum neutralization antibody titer to the indicated
HMPV strain is expressed as the mean reciprocal log.sub.2 .+-.
standard error. All animals were seronegative for both strains of
HMPV at the start of the study (that is, day 0 reciprocal
neutralization titer of .ltoreq.1.0 log.sub.2). .sup.dPercent
antigenic relatedness was calculated using the formula of Archetti
and Horsfall. .sup.eOn day 30, animals were challenged IN and IT
with 10.sup.5.2 TCID.sub.50 of the heterologous virus in a 1 ml
inoculum at each site. Level of virus replication is expressed as
the mean of the peak virus titers (log.sub.10 TCID.sub.50/ml .+-.
standard error) for the animals in each group irrespective of
sampling day. The lower limit of detection is 1.0 log.sub.10
TCID.sub.50/ml. nd: not done
[0333] Development of a Small Animal Model of HMPV Replication
[0334] The evaluation of HMPV and rHMPV derivatives is further
facilitated by the availability of a small animal model for
evaluating replication and immunogenicity in vivo. Hamsters were
infected intranasally with 10.sup.6 TCID.sub.50 of either strain 83
or 75, and animals were sacrificed on days 3, 4, 5, 6 or 7 and the
nasal turbinates and lungs were harvested and assayed for virus
titer. As shown in Table 6, each virus replicated to a peak titer
greater than 6 log.sub.10 in the nasal turbinates and 3.6 to 4.4
log.sub.10 in the lower respiratory tract. Thus, hamsters were
permissive for both subgroup strains. As shown in Table 1 (and in
Table 7), infection with strain 83 induced serum neutralizing
antibody titers of 10.3 to 12.3 log.sub.10 against the homologous
strain, and infection with strain 75 induced titers of 7.9 to 9.9
log.sub.10 against the homologous strain. The basis for the
apparent lower immunogenicity of strain 75 is not known, although
this finding will need to be confirmed in a further study in which
virus replication and immunogenicity are measured for each strain
in the same study. The present data show that hamsters are
relatively permissive for HMPV replication and provide a convenient
small animal model for evaluating HMPV replication and
immunogenicity.
[0335] The hamster model was used to evaluate cross-protection
between the two HMPV genetic subgroups, as had also been
investigated in chimpanzees as shown in Table 4. Groups of hamsters
were infected with either strain of HMPV or with L15 diluent as a
negative control. After 6 weeks the hamsters were challenged with
either strain of HMPV and the level of pulmonary challenge virus
replication in the respiratory tract on day 4 was determined, as
described above. As shown in Table 7, previous infection by either
HMPV strain induced a high level of protection against the
homologous and heterologous HMPV strain in the upper respiratory
tract. The lower respiratory tract was completely protected from
challenge with either strain. In addition, sera were collected 31
days following the first infection and assayed to determine the
neutralizing titer against each strain (Table 7). The results were
very similar to those shown previously in Table 1: infection with
CAN83 induced antibodies that were modestly more effective in
neutralizing the homologous versus heterologous strain, but
antibodies induced by infection with CAN75 neutralized the
homologous and heterologous strains with similar efficiencies.
These cross-protection data and reciprocal-neutralization assays
indicated that CAN83 and CAN75 are antigenically highly
related.
6TABLE 6 DAILY LEVEL OF REPLICATION OF HMPV STRAINS CAN75 AND CAN83
IN THE UPPER AND LOWER RESPIRATORY TRACT OF INFECTED HAMSTERS Mean
virus titer (log.sub.10 TCID.sub.50/g .+-. S.E.) on day.sup.b:
Virus inoculum.sup.a Tissue 3 4 5 6 7 HMPV-CAN83 Nasal turbinates
6.0 .+-. 0.5 5.7 .+-. 0.3 5.1 .+-. 0.3 3.7 .+-. 1.2 1.7 .+-. 0.5
Lungs 3.6 .+-. 1.0 3.1 .+-. 0.3 3.0 .+-. 0.3 .ltoreq.1.5 .+-. 0.0
.ltoreq.1.5 .+-. 0.0 HMPV-CAN75 Nasal turbinates 6.6 .+-. 0.4 6.4
.+-. 0.4 6.4 .+-. 0.6 5.2 .+-. 0.5 4.2 .+-. 0.7 Lungs 4.4 .+-. 0.6
4.3 .+-. 0.7 3.3 .+-. 0.2 .ltoreq.1.5 .+-. 0.0 .ltoreq.1.5 .+-. 0.0
.sup.aHamsters in groups of 30 animals were inoculated intranasally
with 10.sup.6 TCID.sub.50 of the indicated virus. .sup.bNasal
turbinates and lung tissues from six animals from each group where
harvested on days 3 through 7 post-infection. Virus present in
tissue homogenates was quantified by serial dilution on LLC-MK2
monolayers at 32.degree. C. Infected cultures were detected with
hamster anti-HMPV antibodies and immunoperoxidase staining. Mean
virus titer for each group of hamsters is expressed as log.sub.10
TCID.sub.50/gram .+-. standard error, S.E.
[0336]
7TABLE 7 Hamsters Infected With HMPV Strain CAN83 Or CAN75 Are
Protected Against Challenge With The Homologous And Heterologous
HMPV Strains Mean virus titer (log.sub.10 Mean serum-
TCID.sub.50/g) in neutralizing antibody nasal turbinates of Animal
titer.sup.b (reciprocal hamsters administered the immunized
log.sub.2) against: indicated challenge virus.sup.c: with.sup.a
CAN75 CAN83 CAN75 CAN83 CAN83 9.2 .+-. 0.2 10.4 .+-. 0.3 2.0 .+-.
0.2 1.7 .+-. 0.2 CAN75 10.9 .+-. 0.2 7.7 .+-. 0.2 .ltoreq.1.5 .+-.
0.0 .sup. 2.4 .+-. 0.5 L15 medium .ltoreq.1.0 .+-. 0.0.sup.
.ltoreq.1.0 .+-. 0.0.sup. 5.3 .+-. 0.1 5.6 .+-. 0.2 .sup.aHamsters
in groups of 12 were immunized by IN infection with 10.sup.5.5
TCID.sub.50 of the indicated virus or mock-infected with L15
medium. .sup.bSera were collected 2-3 days before and 31 days
following the first infection, and the neutralizing titer against
each of the two strains was determined. The pre-infection anti-HMPV
serum titers were .ltoreq.1.0 (reciprocal log.sub.2) for all
animals in the study. .sup.cOn day 42, six hamsters from each group
were challenged IN with 10.sup.5.5 TCID.sub.50 of CAN83 and the
remaining six were challenged with CAN75. Nasal turbinates and
lungs were harvested four days later, and the virus titer was
determined on LLC-MK2 cells. The mean virus titer for each group of
hamsters is expressed as log.sub.10 TCID.sub.50/gram of nasal
turbinate .+-. standard error.
Example 6
Construction of a Human Parainfluenza Type I Virus (HPIV1) that
Induces Neutralizing Antibodies and Protective Efficacy Against
Both HMPV Genetic Subgroups
[0337] To examine the ability of the HMPV F protein alone to induce
serum neutralizing antibodies and to confer protection against HMPV
challenge, a recombinant HPIV1 expressing the CAN83 F protein
(rHPIV1-F.sub.83) was recovered in vitro from an HPIV1 antigenomic
cDNA containing the CAN83 F protein ORF inserted upstream of the
HPIV1 N gene and flanked by HPIV1 GS and GE transcription signals
(FIG. 36A). Expression of the F protein was confirmed by indirect
immunofluorescence of infected cells (FIG. 36 B). Thus,
rHPIV1-F.sub.83 would express all of the proteins of HPIV1 as well
as the F protein of HMPV.
[0338] Groups of hamsters were immunized with CAN83, wild type
rHPIV1, rHPIV1-F.sub.83 or with L15 medium, and 33 days later sera
were collected and tested for the ability to neutralize the CAN75
and CAN83 strains. As shown in Table 8, sera from animals immunized
with CAN83 or rHPIV1-F.sub.83 efficiently neutralized both strains
of virus. In each case, neutralization of the homologous CAN83
strain was somewhat more efficient than for the heterologous CAN75
strain. The mean neutralization titer of sera from
rHPIV1-F.sub.83-infected animals was 1.2 log.sub.2 lower than that
observed from CAN83-infected animals, which corresponds well with
the lower titer of rHPIV1 replication in hamsters in this
experiment compared to CAN83. Thus, when adjusted for the level of
virus replication, the rHPIV1-F.sub.83 vector, expressing only a
single CAN83 antigen, was as efficient in inducing
CAN83-neutralizing antibodies as CAN83 itself.
[0339] Fifty days following immunization, the animals were
challenged by intranasal infection with rHPIV1, CAN75 or CAN83. As
shown in Table 8, animals previously infected with rHPIV1-F.sub.83
had a 125-fold and 158-fold reduction in the upper respiratory
tract in the level of replication of the CAN75 and CAN83 challenge
virus, respectively. Animals were completely protected in the lower
respiratory tract. The rHPIV1-F.sub.83 virus also protected against
HPIV1 challenge, indicating that this virus was able to protect
against the three respiratory tract pathogens, namely CAN75, CAN83
and HPIV1. This method also can now be used to determine the
relative contribution of each of the other HMPV proteins to the
induction of neutralizing antibodies and resistance to challenge
virus replication. Importantly, these findings identify the F
protein of HMPV as a major neutralization and protective antigen.
Furthermore, the F protein of the CAN83 strain induced neutralizing
antibodies and protection that were effective against both CAN83
and CAN75. This highlights the importance of this specific antigen
in developing vaccines effective against all strains of HMPV.
8TABLE 8 A Recombinant HPIV1 Expressing The CAN83 F Protein
(RHPIV1-F.sub.83) Equally Protects Hamsters Against Challenge With
HMPV Subgroup CAN75 And CAN83 As Well As Against HPIV1 Mean level
of replication of Mean serum neutralization titer (log.sub.2) to
the indicated challenge virus Virus indicated virus.sup.b: in the
nasal turbinates.sup.c inoculum.sup.a rHPIV1 CAN75 CAN83 rHPIV1
CAN75 CAN83 L15 medium .ltoreq.1.0 .+-. 0.0 .ltoreq.1.0 .+-. 0.0
.ltoreq.1.0 .+-. 0.0 4.9 .+-. 0.3 5.0 .+-. 0.2.sup. 6.1 .+-.
0.3.sup. CAN83 .ltoreq.1.0 .+-. 0.0 8.3 .+-. 0.2 9.8 .+-. 0.2 4.6
.+-. 0.1 1.5 .+-. 0.0.sup. 1.5 .+-. 0.0.sup. rHPIV1 wt 6.1 .+-. 0.3
.ltoreq.1.0 .+-. 0.0 .ltoreq.1.0 .+-. 0.0 2.0 .+-. 0.2 4.4 .+-.
0.2.sup.d 5.8 .+-. 0.2.sup.e rHPIV1-F.sub.83 4.7 .+-. 0.2 6.0 .+-.
0.3 8.6 .+-. 0.2 1.9 .+-. 0.2 2.9 .+-. 0.3.sup.d 3.9 .+-. 0.1.sup.e
.sup.aGroups of 18 hamsters were infected IN with 10.sup.5
TCID.sub.50 of the indicated virus or with L15 medium as a negative
control. .sup.bThirty-three days following the first infection,
sera were collected and assayed to determine the neutralization
titers against HPIV1, CAN75 and CAN83. 50 days following the first
infection, six hamsters from each group were challenged IN with
10.sup.5 TCID.sub.50 of the indicated virus and the nasal
turbinates were harvested after four days. .sup.cVirus present in
the tissue homogenates was quantified and is expressed as the mean
log.sub.10 TCID.sub.50/g for each group .+-. standard error.
.sup.dStatistically significant difference between indicated
values; p < 0.01 (unpaired t-test). .sup.eStatistically
significant difference between indicated values; p < 0.0001
(unpaired t-test)
Example 7
Construction of HMPV Recombinant Virus Expressing Protective
Antigens for Both Putative Antigenic Subgroups of HMPV
[0340] As described above, different strains of HMPV can exhibit
genetic diversity, especially in the SH and G proteins, and there
appear to be two major genetic subgroups. The preceding Example
identified the HMPV F protein as a major neutralization and
protective antigen effective against both subgroups. This, it is a
preferred antigen for including in an HMPV vaccine. The preceding
Examples showed that it is possible to achieve a high degree of
protective immunity against both genetic subgroups by infection
with a single HMPV from either subgroup. Furthermore, the F protein
was identified as a major neutralization and protective antigen
effective against both subgroups. However, it may be that optimal
immunogenicity and protective efficacy will be achieved by
including the SH or G protein from either or both subgroups, or by
including F protein from both subgroups. In addition, the available
information indicates that HMPV is a significant cause of serious
respiratory tract disease early in life, approximately coincident
with RSV and the PIVs. Thus, an immunogenic composition against
HMPV likely would be administered at approximately the same time in
life (optionally in a combinatorial formulation or coordinate
immunization protocol for infants, and certain adult subjects) as
immunogenic compositions directed against RSV and the PIVs.
Accordingly, certain aspects of the current disclosure provide for
broad coverage of an HMPV immunogenic composition, yielding
increased effectiveness against multiple, antigenically-distinct
strains. It is also advantageous in certain embodiments to decrease
the number of separate viruses that must be administered: an
immunogenic composition comprised of multiple separate viruses is
more difficult to develop and formulate since each virus must be
verified to be safe and immunogenic separately and in combination,
and issues such as viral interference can complicate formulation.
The methods of the current disclosure offer a flexibility and
versatility in design of immunogenic compositions that addressees
these and related problems.
[0341] In one exemplary strategy, one or more ORFs each encoding a
heterologous, immunogenic protein or antigenic determinant (for
example, fragment or epitope) thereof can be engineered into
transcription cassettes and inserted into the genome of HMPV. For
example, as illustrated in FIG. 34, an ORF encoding the G, F or SH
surface protein of HMPV strain 75 is engineered to be flanked by GS
and GE signals from strain 83, and is inserted into the HMPV strain
83 backbone. This results in a chimeric virus that contains an
additional gene and expresses all of the genes of strain 83 as well
as a surface protein antigen of strain 75. This chimeric virus
therefore expresses antigens representing both HMPV antigenic
subgroups. This addresses the need to broaden coverage of
immunogenic compositions, as well as meeting the need to minimize
the number of separate viruses that must be included in such
compositions. A preferred heterologous strain HMPV gene for
insertion into an HMPV backbone would be the F gene, since F has
been directly identified as a major HMPV neutralization and
protective antigen. A second preferred gene is the G gene, since
its encoded protein appears to he highly divergent between HMPV
strains, exhibiting only 70% amino acid identity between strain
00-1 and 83 compared lo 98% identify for the F protein. Similarly,
the SH protein is a third preferred gene for insertion into
chimeric virus, since it also is relatively divergent and exhibits
85% sequence identity between strains 00-1 and 83. The example
illustrated in FIG. 34 employed the same transcription signals and
genome insertion site that was used for expression of GFP. However,
the methods of the current disclosure allow other signals or
insertion sites to be tested. Also, the Example of FIG. 34 involved
an ORF from a heterologous strain of HMPV, but the same strategy
can be applied to other heterologous ORFs, such as ones from RSV,
HPIV1, 2 or 3, other heterologous viruses or pathogens, and other
molecules such as immunomodulatory proteins such as
granulocyte-macrophage colony stimulating factor. In this way, an
HMPV backbone can be used to create additional viruses bearing the
surface proteins of heterologous, non-HMPVs. Chimeric viruses of
the current disclosure can then be administered on their own, or
can be combined with the original parent or other rHMPV to make a
multi-virus or multivalent immunogenic composition.
Example 8
Methods of Screening for Compounds that Inhibit HMPV
[0342] The current disclosure demonstrates that expression of the
N, P, L and M2-1 proteins alone are sufficient to direct HMPV
transcription and RNA replication. This was documented in two ways.
First, expression of these proteins was sufficient to direct
transcription and RNA replication of a cDNA-encoded mini-replicon
derived from the HMPV genome and carrying a reporter gene. Second,
expression of these proteins was sufficient to launch the rescue of
complete infectious HMPV from a cDNA-encoded antigenome. The
antigenome is by definition the complement of the genome and is not
able to serve as the direct template for transcription. Hence, both
RNA replication and transcription must have occurred under the
direction of the supplied proteins in order for a productive
infection to be launched, indicating that N, P, L, and M2-1 are
sufficient to direct all phases of HMPV RNA synthesis. Therefore,
the current disclosure provides novel and readily practiced methods
for screening, identifying, designing, and characterizing compounds
that inhibit one or more activities of HMPV (for example,
transcription, translation, growth, infectivity, virulence,
immunogenicity, etc.)
[0343] In addition, the current disclosure provides new,
facilitated methods of monitoring infection by complete HMPV which
provide for the screening, identification and design of antiviral
compounds that act at any stage in the HMPV replicative cycle. The
current disclosure also provides methods for identifying the viral
targets of antiviral compounds, thereby providing for improved
design of compounds effective in inhibiting HMPV.
[0344] Potential antiviral compounds effective against HMPV include
monoclonal or polyclonal antibodies obtained by biological or
recombinant methods, peptides or protein fragments (Lambert et al.,
Proc. Natl. Acad. Sci. USA 93:2186-2191, 1996), sugar derivatives,
polyionic polymers, anti-sense RNA, double stranded RNAs capable of
meditating RNA interference RNA (RNAi) (Bitko and Barik, BMC Mirco.
1:34-44 2001), and small molecule compounds (De Clercq, Antimicrob.
Agents 7:193-202, 1996; Prince, Ex. Opin. Invest. Drugs 10:
197-308, 2002). In general, the methods will apply to any antiviral
compounds capable of inhibiting HMPV in solution, in cell extracts,
in cell culture, and in vivo. Antiviral compounds call act, for
example, by inhibiting attachment, penetration (fusion), RNA
synthesis, and virion assembly, and in other cases their specific
target of action may not be fully elucidated (De Clercq,
Antimicrob. Agents 7:193-202, 1996; Prince, Ex. Opin. Invest. Drugs
10:197-308,2002). Antiviral agents also can act by enhancing the
host immune response, such as by stimulating the
interferon-mediated antiviral state (Player et al., Proc. Natl.
Acad. Sci. USA 95:8874-9, 1998). In addition, an antiviral agent
can be combined with an anti-inflammatory agent to effect a further
reduction in disease (Prince et al., J. Infect. Dis.
182:1326-1330,2000).
[0345] In one embodiment of the current disclosure, rHMPV-GFP is
used to screen antiviral compounds. In one exemplary strategy,
rHMPV-GFP) is treated with a test compound or library of compounds
(for example, a compound or library of compounds prospectively
including one or more antiviral agents) and compared with
mock-treated control rHMPV-GFP for the ability to infect cells.
Following a period of absorption, the inoculum containing the
potential antiviral compound can be removed by washing if desired.
Infectivity is typically assayed by observation or measurement of a
detectable label or signal whose presence or level of expression or
detection is correlated with one or more activities of HMPV, for
example as indicated by the development of green fluorescence due
to expression of the GFP marker gene by recombinant HMPV-GFP, a
method that can be monitored in living cells without compromising
sterility over the course of the infection. Alternatively, the
antiviral agent can be included in the medium overlay for the
duration of the infection. Alternatively, infection can be
initiated in the absence of the compound, which can be subsequently
added. In addition to identifying compounds capable of inhibiting
one or more activities of HMPV, these assays can give an indication
of the mode of action of a compound, such as whether it acts by
directly neutralizing virus, or inhibiting subsequent gene
expression, or inhibiting viral spread. While GFP is provided as an
exemplary label or marker, a wide range of useful "reporter
sequences" (for example, reporter genes) and other sequences that
direct expression of detectable labels and markers are known in the
art and can be readily employed as detectable "signal" agents whose
presence, level of activity or expression correlate with one or
more selected activities of HMPV. Exemplary alternative markers
and/or labels in this context include, inter alia, chloramphenicol
acetyl transferase, luciferase, secreted alkaline phosphatase, and
a large number of other known reporter sequences routinely used in
the art. This provides the ability to design a reporter virus or
subviral construct that will be useful in a variety of high
throughput screening protocols.
[0346] Within related aspects of the current disclosure, the
availability of rHMPV variants that each lack one or more of the
viral ORFs, as described above, provides particularly useful tools
and methods to identify possible viral targets of antiviral
compounds. For example, if a compound inhibits the infectivity of
complete HMPV, it will be tested (for example, for its efficacy
and/or target gene or protein specificity) against a panel of rHMPV
lacking various genes, such as the .DELTA.SH, .DELTA.G,
.DELTA.SH/G, and .DELTA.M2-2 variants mentioned previously. An
alternative and complementary method of identifying targets for an
antiviral compound involves forcing the virus to grow in the
presence of the compound (present at minimal or suboptimal
inhibitory concentrations if necessary). Under these conditions, it
is expected that a large panel of useful variants will emerge that
have developed increased resistance to the subject inhibitory
compound. Sequence analysis of such variants can identify mutations
that are potentially responsible for the increased resistance. By
the methods of this current disclosure, mutations suspected of
conferring increased resistance can be introduced individually or
in combinations into rHMPV, and the resistant rHMPV expressing a
convenient reporter gene such as GFP will provide yet additional
tools and methods to facilitate other phases of HMPV control and
antiviral development. For example, the introduction of mutations
identified in resistant viruses provides a method of directly
confirming that they confer resistance and thus distinguishes them
from adventitious mutation that occur during passage. This provides
direct identification of viral genes involved in drug resistance,
and is an important tool for further drug development. As another
example, as noted above, HMPV-GFP can be used to assay quickly and
accurately to detect antibodies to HMPV. This can be advantageous
in studies to develop monoclonal or polyclonal antibodies capable
of neutralizing HMPV as immunoprophylaxic or therapeutic agents.
This also has applications in epidemiologic studies for example to
determine the serostatus of individuals in a population.
Furthermore, it provides a facilitated method to monitor changes in
the level of HMPV antibodies in experimental animals or clinical
subjects, such as in response to experimental immunogenic
compositions, or in situations were the efficacy of an antiviral
compound is being evaluated. Also, the expression of a reporter
gene such as GFP provides a useful marker to detect and quantify
HMPV infection and immunity in individuals, including experimental
animals, thereby providing an assay for viral disease detection,
tropism and/or to identify and evaluate additional antiviral
agents.
[0347] In other embodiments of the current disclosure, rHMPV
expressing a convenient reporter gene can be readily employed
within screening compositions and methods based on cell-free in
vitro assays. For example, a nucleocapsid preparation can be made
from cells infected with rHMPV expressing a reporter gene, such as
alkaline phosphatase or some other sensitively-detected enzyme or
marker. Replicate in vitro assays can be constructed and used to
screen compounds for antiviral activity in a high throughput format
designed to detect the expressed reporter gene. This can be based
on the expression of a convenient reporter gene or, alternatively,
can be based on the detection of RNA representing the marker gene
or one or more HMPV genes. For example, mRNA produced in such an in
vitro reaction mixture can be captured in situ by matrix-bound
oligodT and, following washing to remove the reaction mixture, the
resulting hybrids can be detected by secondary hybridization with
probes tagged with reporter enzymes or ligands. Such assays can be
further modified to specifically target, for example, products of
RNA replication or read-through of gene junctions as a measure of
altered transcription.
[0348] The identification of the N, P, L, and optionally, M2-1 as
proteins useful and/or sufficient to direct HMPV transcription and
RNA replication provides additional new methods for screening
compounds for anti-viral activity. In one embodiment, these four
proteins can be expressed intracellularly together with a
mini-replicon that encodes a reporter gene, resulting in
reconstituted HMPV transcription and RNA. This can be used to
screen for compounds, particularly those that are effective against
HMPV transcription, RNA replication, and nucleotide assembly and
function. Alternatively, the N, P, L, and M2-1 proteins can be
expressed at high levels, using recombinant baculovirus, vaccinia
virus, bacteria or any other suitable expression system, and
purified to use in reconstituted assays such as in vitro RNA
synthesis, protein-protein interactions, or protein-RNA
interactions. Alternatively, the N, P. L and M2-1 proteins can be
tagged recombinantly by the incorporation of, for example, a
hexa-Histindine tag or an epitope recognized by a monoclonal
antibody, such as the well known FLAG tag (Nilsson et al., Prot.
Exp. Pur. 11: 1-16, 1997). These provide the basis for purification
of these components by affinity chromatography, facilitating the
development of in vitro assays. In addition, when such tagged genes
are expressed from the context of infection by recombinant HMPV, it
provides a method of isolating and identifying possible cellular
and viral binding partners. Such binding partners can be identified
by a variety of methods. For example, in some cases, binding
partners will be present in sufficient quantity that they can be
visualized by gel electrophoresis and identified by conventional
methods of protein sequencing. Low abundance binding partners can
be identified from complex mixtures by mass spectrometric methods
coupled with trypsin cleavage and protein sequencing. The
identification of viral and cellular binding partners provides
additional information to guide the development of compounds that
inhibit HMPV.
[0349] While this disclosure has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments may
be used and it is intended that the disclosure may be practiced
otherwise than as specifically described herein. Accordingly, this
disclosure includes all modifications encompassed within the spirit
and scope of the disclosure as defined by the claims below.
Sequence CWU 1
1
36 1 13335 DNA human metapneumovirus 1 acgcgaaaaa aacgcgtata
aattaagtta caaaaaaaca tgggacaagt gaaaatgtct 60 cttcaaggga
ttcacctgag tgatctatca tacaagcatg ctatattaaa agagtctcag 120
tatacaataa agagagatgt aggcacaaca acagcagtga caccctcatc attgcaacaa
180 gaaataacac tattgtgtgg agaaattcta tatgctaagc atgctgatta
caaatatgct 240 gcagaaatag gaatacaata tattagcaca gctctaggat
cagagagagt acagcagatt 300 ctaagaaact caggcagtga agtccaagtg
gttttaacca gaacgtactc cttggggaaa 360 gttaaaaaca acaaaggaga
agatttacag atgttagaca tacacggagt agagaaaagc 420 tgggtggaag
agatagacaa agaagcaaga aaaacaatgg caactttgct taaagaatca 480
tcaggcaata ttccacaaaa tcagaggcct tcagcaccag acacacctat aatcttatta
540 tgtgtaggtg ccttaatatt taccaaacta gcatcaacta tagaagtggg
attagagacc 600 acagtcagaa gagctaaccg tgtactaagt gatgcactca
aaagataccc taggatggac 660 ataccaaaaa tcgctagatc tttctatgat
ttatttgaac aaaaagtgta ttacagaagt 720 ttgttcattg agtatggcaa
agcattaggc tcatcctcta caggcagcaa agcagaaagt 780 ttattcgtta
atatattcat gcaagcttac ggtgctggtc aaacaatgct gaggtgggga 840
gtcattgcca ggtcatctaa caatataatg ttaggacatg tatctgtcca agctgagtta
900 aaacaagtca cagaagtcta tgacctggtg cgagaaatgg gccctgaatc
tgggctccta 960 catttaaggc aaagcccaaa agctggactg ttatcactag
ccaattgtcc caactttgca 1020 agtgttgttc tcggcaatgc ctcaggctta
ggcataatag gtatgtatcg cgggagagtg 1080 ccaaacacag aactattttc
agcagcagaa agctatgcca agagtttgaa agaaagcaat 1140 aaaattaact
tttcttcatt aggactcaca gatgaagaaa aagaggctgc agaacacttt 1200
ctaaatgtga gtgacgacag tcaaaatgat tatgagtaat taaaaaagtg ggacaagtca
1260 aaatgtcatt ccctgaagga aaagatattc ttttcatggg taatgaagcg
gcaaaattgg 1320 cagaagcttt ccaaaaatca ttaagaaaac ctagtcataa
aagatctcaa tctattatag 1380 gagaaaaagt gaacactgta tctgaaacat
tggaattacc tactatcagt agacctacca 1440 aaccgaccat attgtcagag
ccgaagttag catggacaga caaaggtggg gcaatcaaaa 1500 ctgaagcaaa
gcaaacaatc aaagttatgg atcctattga agaagaagag tttactgaga 1560
aaagggtgct gccctccagt gatgggaaaa ctcctgcaga aaagaagttg aaaccatcaa
1620 ccaatactaa aaagaaggtc tcatttacac caaatgaacc aggaaaatac
acaaagttgg 1680 agaaagatgc tctagacttg ctttcagaca atgaagaaga
agatgcagaa tcctcaatct 1740 taaccttcga agaaagagat acttcatcat
taagcattga agccagacta gaatcgattg 1800 aggagaaatt aagcatgata
ttagggctat taagaacact caacattgct acagcaggac 1860 ccacagcagc
aagagatggg atcagagatg caatgattgg cataagggag gaactaatag 1920
cagacataat aaaagaagcc aagggaaaag cagcagaaat gatggaagaa gaaatgaacc
1980 agcggacaaa aataggaaac ggtagtgtaa aattaactga aaaggcaaag
gagctcaaca 2040 aaattgttga agacgagagc acaagtggtg aatccgaaga
agaagaagaa ctaaaagaca 2100 cacaggaaaa taatcaagaa gatgacattt
accagttaat tatgtagttt aataaaaata 2160 aaaaatggga caagtgaaaa
tggagtccta tctggtagac acctatcaag gcatccctta 2220 cacagcagct
gttcaagttg atctagtaga aaaggacctg ttacctgcaa gcctaacaat 2280
atggttcccc ctgtttcagg ccaatacacc accagcagtt ctgcttgatc agctaaagac
2340 tctgactata actactctgt atgctgcatc acaaagtggt ccaatactaa
aagtgaatgc 2400 atcggcccag ggtgcagcaa tgtctgtact tcccaaaaag
tttgaagtca atgcgactgt 2460 agcacttgac gaatatagca aattagaatt
tgacaaactt acagtctgtg aagtaaaaac 2520 agtttactta acaaccatga
aaccatatgg gatggtatca aagtttgtga gctcggccaa 2580 accagttggc
aaaaaaacac atgatctaat cgcattatgc gattttatgg atctagaaaa 2640
gaacacacca gttacaatac cagcatttat caaatcagtt tctatcaagg agagtgaatc
2700 agccactgtt gaagctgcaa taagcagtga agcagaccaa gctctaacac
aagccaaaat 2760 tgcaccttat gcgggactga tcatgattat gaccatgaac
aatcccaaag gcatattcaa 2820 gaagcttgga gctgggaccc aagttatagt
agaactagga gcatatgtcc aggctgaaag 2880 cataagtaaa atatgcaaga
cttggagcca tcaaggaaca agatatgtgc tgaagtccag 2940 ataacagcca
agcaacctga ccaagaacta ccaactctat tctatagact aaaaagtcgc 3000
cattttagtt atataaaaat caagttagaa taagaattaa atcaatcaag aacgggacaa
3060 ataaaaatgt cttggaaagt ggtgatcatt ttttcattgc taataacacc
tcaacacggt 3120 cttaaagaga gctacctaga agaatcatgt agcactataa
ctgagggata tcttagtgtt 3180 ctgaggacag gttggtatac caacgttttt
acattagagg tgggtgatgt agaaaacctt 3240 acatgttctg atggacctag
cctaataaaa acagaattag atctgaccaa aagtgcacta 3300 agagagctca
aaacagtctc tgctgaccaa ttggcaagag aggaacaaat tgagaatccc 3360
agacaatcta ggtttgttct aggagcaata gcactcggtg ttgcaacagc agctgcagtc
3420 acagcaggtg ttgcaattgc caaaaccatc cggcttgaga gtgaagtcac
agcaattaag 3480 aatgccctca aaacgaccaa tgaagcagta tctacattgg
ggaatggagt tcgagtgttg 3540 gcaactgcag tgagagagct gaaagacttt
gtgagcaaga atttaactcg tgcaatcaac 3600 aaaaacaagt gcgacattga
tgacctaaaa atggccgtta gcttcagtca attcaacaga 3660 aggtttctaa
atgttgtgcg gcaattttca gacaatgctg gaataacacc agcaatatct 3720
ttggacttaa tgacagatgc tgaactagcc agggccgttt ctaacatgcc gacatctgca
3780 ggacaaataa aattgatgtt ggagaaccgt gcgatggtgc gaagaaaggg
gttcggaatc 3840 ctgatagggg tctacgggag ctccgtaatt tacatggtgc
agctgccaat ctttggcgtt 3900 atagacacgc cttgctggat agtaaaagca
gccccttctt gttccggaaa aaagggaaac 3960 tatgcttgcc tcttaagaga
agaccaaggg tggtattgtc agaatgcagg gtcaactgtt 4020 tactacccaa
atgagaaaga ctgtgaaaca agaggagacc atgtcttttg cgacacagca 4080
gcgggaatta atgttgctga gcaatcaaag gagtgcaaca tcaacatatc cactacaaat
4140 tacccatgca aagtcagcac aggaagacat cctatcagta tggttgcact
gtctcctctt 4200 ggggctctgg ttgcttgcta caaaggagta agctgttcca
ttggcagcaa cagagtaggg 4260 atcatcaagc agctgaacaa gggttgctcc
tatataacca accaagatgc agacacagtg 4320 acaatagaca acactgtata
tcagctaagc aaagttgagg gtgaacagca tgttataaaa 4380 ggcagaccag
tgtcaagcag ctttgatcca atcaagtttc ctgaagatca attcaatgtt 4440
gcacttgacc aagtttttga gaacattgaa aacagccagg ccttggtaga tcaatcaaac
4500 agaatcctaa gcagtgcaga gaaagggaat actggcttca tcattgtaat
aattctaatt 4560 gctgtccttg gctctagcat gatcctagtg agcatcttca
ttataatcaa gaaaacaaag 4620 aaaccaacgg gagcacctcc agagctgagt
ggtgtcacaa acaatggctt cataccacac 4680 agttagttaa ttaaaaataa
aataaaattt gggacaaatc ataatgtctc gcaaggctcc 4740 atgcaaatat
gaagtgcggg gcaaatgcaa cagaggaagt gagtgtaagt ttaaccacaa 4800
ttactggagt tggccagata gatacttatt aataagatca aactatctat taaatcagct
4860 tttaaggaac actgatagag ctgatggcct atcaataata tcaggcgcag
gcagagaaga 4920 cagaacgcaa gattttgttc taggttccac caatgtggtt
caaggttata ttgatgataa 4980 ccaaagcata acaaaagctg cagcctgcta
cagtctacac aacataatca agcaactaca 5040 agaagttgaa gttaggcagg
ctagagatag caaactatct gacagcaagc atgtggcact 5100 ccataactta
atcttatctt acatggagat gagcaaaact cccgcatctt taatcaacaa 5160
tctcaaaaga ctgccgagag aaaaactgaa aaaattagca aagctgataa ttgacttatc
5220 agcaggcgct gacaatgact cttcatatgc cctgcaagac agtgaaagca
ttaatcaagt 5280 gcagtgagca tggtcctgtt ttcattacta tagaggttga
tgaaatgata tggactcaaa 5340 aagaattaaa agaagctttg tccgatggga
tagtgaagtc tcacaccaac atttacaatt 5400 gttatttaga aaacatagaa
attatatatg tcaaggctta cttaagttag taaaaacaca 5460 tcagagtggg
ataagtgaca atgataacat tagatgtcat taaaagtgat gggtcttcaa 5520
aaacatgtac tcacctcaaa aaaataatca aagaccattc tggtaaagtg cttattgcac
5580 ttaagttaat attagcttta ctaacatttt tcacaataac aatcactata
aattacataa 5640 aagtagaaaa caatctacaa atatgccagt caaaaactga
atcagacaaa gaagactcac 5700 catcaaatac cacatccgtc acaaccaaga
ccactctaga ccatgatata acacagtatt 5760 ttaaaagatt aattcaaagg
tatacagatt ctgtaataaa caaggacaca tgctggaaaa 5820 taagcagaaa
tcaatgcaca aatataacaa catataaatt tttatgcttt aaacctgagg 5880
actcaaaaat caacagttgt gatagactga cagatctatg cagaaacaaa tcaaaatcag
5940 cagctgaagc atatcataca gtagaatgcc attgcatata cacaattgag
tggaagtgct 6000 atcaccactc aatagattaa acccaatctt gaatgttaaa
actagactag gatccgtcta 6060 agactatcag ttcaatagtt tagttatttt
aaaatatttg agaataggta agtttctatg 6120 gcacttcata gcaataggta
ataattaaca gcttaattat aattaaaaca ttatttaaaa 6180 tcgtaactat
ttaatttaca aagtaaaaac aaaaatatgg gacaagtagt tatggaggtg 6240
aaagtagaga acattcgagc aatagacatg ctcaaagcaa gagtgaaaaa tcgtgtggca
6300 cgtagcaaat gctttaaaaa tgcttcttta atcctcatag gaataactac
actgagtata 6360 gctctcaata tctatctgat cataaactac acaatacaaa
aaacctcatc tgaatcagaa 6420 caccacacca gctcaccacc cacagaatcc
aacaaggaag cttcaacaat ctccacagac 6480 aacccagaca tcaatccaaa
ctcacagcat ccaactcaac agtccacaga aaaccccaca 6540 ctcaaccccg
cagcatcagt gagcccatca gaaacagaac cagcatcaac accagacaca 6600
acaaaccgcc tgtcctccgt agacaggtcc acagcacaac caagtgaaag cagaacaaag
6660 acaaaaccga cagtccacac aagaaacaac ccaagcacag cttccagtac
acaatcccca 6720 ccacgggcaa caacgaaggc aatccgcaga gccaccactt
tccgcatgag cagcacagga 6780 aaaagaccaa ccacaacatc agtccagtcc
gacagcagca ccacaaccca aaatcatgaa 6840 gaaacaggtt cagcgaaccc
acaggcatct gtaagcacaa tgcaaaacta gcacaccaac 6900 aatataaaac
caaattagtt aacaaaaaat acgagatagc tctaaagtaa aacatgtagg 6960
taccaacaat caagaaacca aaagacaact cacaatctcc ctaaaacagc aacgacacca
7020 tgtcagcttt gctcaaatct ctctgggaga aacttttgcc cacatactaa
caacatcaca 7080 accatctcaa gaaaagaaac tgggcaaaac agcatccaag
agacaaatag caatggatcc 7140 tcttaatgaa tccactgtta atgtctatct
ccctgattcg taccttaaag gagtaatttc 7200 ttttagtgaa actaatgcaa
ttggttcatg tctcttaaaa agaccctact taaaaaatga 7260 caacactgca
aaagttgcca tagagaatcc tgttattgag catgtgagac tcaaaaatgc 7320
agtcaattct aaaatgaaaa tatcagatta caaggtagta gagccagtaa acatgcaaca
7380 tgaaataatg aagaatgtac acagttgtga gctcacacta ttgaaacagt
ttttaacaag 7440 gagtaaaaac attagcactc tcaaattgaa tatgatatgt
gattggctgc aattaaagtc 7500 tacatcagat gatacctcaa tcctaagttt
catagatgta gaatttatac ctagttgggt 7560 aagcaactgg tttagtaatt
ggtacaatct caataagtta attttggaat tcagaagaga 7620 ggaagtaata
agaaccggtt caatcttatg caggtcattg ggtaaattag tttttattgt 7680
atcatcatat ggatgtatcg tcaagagcaa caaaagcaaa agagtgagct tcttcacata
7740 caatcaactg ttaacatgga aagatgtgat gttaagtaga tttaatgcga
atttttgtat 7800 atgggtaagc aatagtctga atgaaaatca ggaagggcta
gggttgagaa gtaatctgca 7860 aggtatgtta actaataaac tatatgaaac
tgtagattat atgctaagct tatgttgcaa 7920 tgaaggtttc tcacttgtga
aagagttcga aggttttatt atgagtgaga tccttaggat 7980 tactgaacat
gctcaattca gtactagatt tagaaatact ttattgaatg gattaacaga 8040
tcaattaaca aaattaaaaa ataaaaacag actcagagtt catagtaccg tattagaaaa
8100 taatgattat ccaatgtatg aagttgtact taaattatta ggagatactt
tgagatgtat 8160 caaattatta atcaataaaa acttagagaa tgctgcagaa
ttatactata tattcagaat 8220 ttttggtcat ccaatggtag atgaaagaga
tgcaatggat gctgtcaaat taaacaatga 8280 aatcacaaaa atcctaaggt
tggagagctt gacagaacta agaggggcat tcatattaag 8340 gattatcaaa
ggatttgtgg acaacaacaa aaggtggccc aaaattaaaa acttaaaagt 8400
gcttagcaaa agatggacta tgtacttcaa agctaaaaat taccccagtc aactcgaatt
8460 aagtgaacaa gactttctag agcttgctgc aatacaattt gaacaagagt
tttctgttcc 8520 tgaaaaaacc aatcttgaga tggtattaaa tgacaaagcc
atatcacctc ctaaaagatt 8580 aatatggtct gtgtatccaa agaattactt
acctgagacg ataaaaaatc gatatttaga 8640 agaaactttc aatgcgagtg
atagtctcaa aacaagaaga gtactagagt actatttaaa 8700 agataataaa
tttgatcaaa aggaacttaa aagttatgta gttagacaag aatatttaaa 8760
cgataaggag cacattgtct cattaactgg aaaagaaaga gaattaagtg taggtagaat
8820 gtttgctatg caaccaggaa aacagcgaca aatacaaata ttggcagaaa
aattgttagc 8880 tgataacatt gtacctttct tcccggaaac cttaacaaag
tatggtgatc tagatcttca 8940 gagaataatg gaaatcaaat cagaactttc
ttctatcaaa accagaagaa atgatagtta 9000 taataattac attgcaagag
catccatagt aacagatttg agcaagttca accaagcctt 9060 tagatatgaa
actacagcga tctgtgcgga tgtagcagac gaattacatg gaacacaaag 9120
cttattctgt tggttacatc ttatcgttcc tatgactaca atgatatgtg cctatagaca
9180 tgcaccacca gaaacaaaag gtgaatatga tatagataag atagaagagc
aaagtggtct 9240 atatagatat cacatgggcg gtattgaagg atggtgtcaa
aaactctgga caatggaagc 9300 tatatcttta ttggatgttg tatctgtaaa
gacacggtgt caaatgacat ctttattaaa 9360 cggtgacaac caatcaatag
atgtaagtaa accagtcaag ttatctgaag gtttagatga 9420 agtgaaggca
gattatcgct tagcagtaaa aatgctaaaa gaaataagag atgcatacag 9480
aaatataggc cataaactta aagaagggga aacatatata tcaagggatc ttcagtttat
9540 aagcaaggtg attcaatctg aaggagtgat gcatcctacc cctataaaaa
aggtcttgag 9600 agtaggacca tggataaaca caatattaga tgacattaaa
actagtgctg agtcaatagg 9660 gagtctatgt caagaattag aatttagggg
agaaagcata atagttagtc tgatattaag 9720 aaacttctgg ctgtataact
tatacatgca tgaatcaaag caacatcctt tggcagggaa 9780 acagttattc
aaacaactaa ataaaacatt aacatcagtg cagagatttt ttgaaattaa 9840
aagggaaaat gaggtagtag atctatggat gaacatacca atgcaatttg gaggaggaga
9900 tccagtagtc ttctatagat ctttctatag aaggacccct gattttttaa
ctgaggcaat 9960 cagccatgta gatattctgt taaaaatatc agctaacata
aaaaatgaaa cgaaagtaag 10020 tttcttcaaa gccttactat caatagaaaa
aaatgaacgt gctacactga caacactaat 10080 gagagatcct caagctgttg
gatcagaacg acaagcaaaa gtaacaagtg acatcaatag 10140 aacagcagtt
accagtatct taagtctttc cccaaatcaa cttttcagtg atagtgctat 10200
acactacagc agaaatgaag aagaagtggg aatcattgca gaaaacataa cacctgttta
10260 tcctcatggg ctgagagtat tatatgaatc attgcccttt cacaaagctg
aaaaagttgt 10320 aaacatgata tcagggacaa aatctataac caacttatta
cagagaacat ccgctattaa 10380 tggtgaagat attgacaggg ctgtatctat
gatgttggag aatctaggat tattatctag 10440 aatattgtca gtagttgttg
atagtataga aattccaatc aaatctaatg gtaggctgat 10500 atgttgtcaa
atctctagga ctttaagaga gacatcatgg aataatatgg aaatagttgg 10560
agtaacatct cctagcatca ctacatgtat ggatgtcata tatgcaacta gttctcattt
10620 gaaagggata attatagaaa agttcagcac tgacagaact acaaggggtc
aaagaggtcc 10680 aaaaagccct tgggtagggt cgagtactca agagaaaaaa
ttagtacctg tttataacag 10740 acaaattctc tcaaaacaac aaagagaaca
gctagaagca attggaaaaa tgagatgggt 10800 gtataaaggg acaccaggct
tgcgacgatt actcaacaag atctgtcttg ggagtttagg 10860 cattagctac
aaatgtgtaa aacctttatt acctaggttt atgagtgtaa atttcttaca 10920
tagattatct gtcagtagta gacctatgga attcccagca tcagttccag cttatagaac
10980 aacaaattac catttcgaca ctagtcctat taatcaagca ctaagtgaga
gatttgggaa 11040 tgaagatatt aacttggtct tccaaaatgc aatcagctgt
ggaattagca taatgagtgt 11100 agtagaacaa ttaacaggta gaagcccaaa
acagttagtt ttaatacccc aattagaaga 11160 aatagacatt atgccaccac
cagtgtttca agggaaattc aattataaat tagtagataa 11220 gataacttct
gatcaacata tctttagtcc ggacaaaata gatatgttaa cactagggaa 11280
aatgctcatg cccactataa aaggtcagaa aacagatcag ttcttaaata agagagaaaa
11340 ttatttccat ggaaacaatc ttattgagtc tttatcagca gcattagcat
gtcattggtg 11400 tgggatatta acagaacaat gcatagaaaa taatattttc
aagaaagact ggggtgacgg 11460 gtttatatca gatcatgctt ttatggactt
caaaatattc ctatgtgtct ttaaaactaa 11520 acttttatgt agttggggat
ctcaagggaa aaacattaaa gatgaagata tagtagatga 11580 atcaatagat
aaattgttaa ggattgacaa tactttttgg agaatgttca gcaaagttat 11640
gtttgaacca aaggttaaga aaaggataat gttatatgat gtaaaattcc tatcactagt
11700 aggctacata gggtttaaga actggtttat agagcagttg agatcagctg
aattgcatga 11760 aataccttgg attgtcaatg ccgaaggtga tttggttgag
atcaagtcaa ttaaaatcta 11820 tttgcaactg atagaacaaa gcttattttt
aagaataact gttttgaact atacagatat 11880 ggcacatgct ctcacacgat
taatcagaaa gaagttaatg tgtgataatg cactgttaac 11940 cccaatttca
tccccaatgg ttaacttaac tcaagttatt gatcccacaa cacaattaga 12000
ttacttcccc aagataacat tcgaaaggct aaaaaattat gacacaagtt caaattatgc
12060 taaagggaag ctaacaagaa attacatgat actattgcca tggcagcatg
ttaatagata 12120 taactttgtc tttagttcta ctggatgtaa agttagtctg
aaaacatgta ttggaaaact 12180 tatgaaagac ctaaatccta aagttttgta
ctttattgga gaaggagcag gaaattggat 12240 ggccagaaca gcatgtgaat
atcctgatat taaatttgta tatagaagtc tgaaagatga 12300 ccttgatcat
cattatcctc tggaatacca gagagtgata ggtgaattaa gcagaatcat 12360
agatagtggt gaaggacttt caatggaaac aacagacgca actcaaaaaa ctcattggga
12420 tttgatacac agggtaagca aagatgcttt attaataact ttatgtgatg
cagaatttaa 12480 ggacagagat gattttttta agatggtaat tctatggaga
aaacatgtat tatcatgcag 12540 aatttgcact acttatggga cggacctcta
tttattcgca aagtatcatg ctaaagactg 12600 caatgtaaaa ttaccttttt
ttgtgagatc agttgctact ttcattatgc agggtagtaa 12660 gctgtcaggt
tcagaatgct acatactctt aacactaggc caccacaaca gtttaccttg 12720
ccatggagaa atacaaaatt ctaagatgaa aatagcagtg tgtaatgatt tttatgctgc
12780 aaaaaaactc gacaataaat caattgaagc taattgtaaa tcacttttgt
cagggctaag 12840 aatacctata aataagaagg aactagatag acagagaaga
ttattaacac tacaaagcaa 12900 tcattcttct gtagcaacag ttggcggtag
caagatcata gagtctaagt ggttaacaaa 12960 caaagcaagt acaataattg
attggttaga acatatttta aattctccaa agggcgaatt 13020 aaattatgat
ttttttgaag cattggagaa cacttaccct aatatgatta aactaataga 13080
taacttaggg aatgcagaga ttaaaaaact gatcaaagta acaggataca tgcttgtaag
13140 taaaaaatga aaaatgatga agatgacaaa atagatgaca acttcatact
attctaaatt 13200 aattatttga ttatgcaatt atatgatagt taattaaaat
taaaaattaa aaatcaaaag 13260 ttaaaattta aaacctatca ttaagtttat
taaaaataag aaattataat tgaatgtata 13320 cggttttttt gccgt 13335 2
13280 DNA human metapneumovirus 2 acgcgaaaaa aacgcgtata aattaaattc
caaacaaaac gggacaaata aaaatgtctc 60 ttcaagggat tcacctaagt
gatctgtcat ataaacatgc tatattaaaa gagtctcaat 120 acacaataaa
aagagatgta ggcaccacaa ctgcagtgac accttcatca ttgcagcaag 180
agataacact tttgtgtgga gagattcttt acactaaaca tactgattac aaatatgctg
240 cagagatagg gatacaatat atttgcacgg ctctaggatc agaaagagta
caacagattt 300 taagaaattc aggcagtgaa gttcaggtgg ttctaaccaa
gacatactct ttagggaaag 360 gtaaaaatag taaaggggaa gagttgcaaa
tgttagatat acatggagtg gaaaagagtt 420 gggtagaaga aatagacaaa
gaggcaagaa aaacaatggt gactttgcta aaggaatcat 480 caggtaacat
cccacaaaac cagaggcctt cagcaccaga cacaccaata attttattat 540
gtgtaggtgc tttaatattc actaaactag catcaacaat agaagttgga ctagagacta
600 cagttagaag agctaacaga gtgctaagtg atgcgctcaa aagataccct
agggtagata 660 taccgaagat tgctagatct ttctatgaac tatttgagca
gaaagtgtat tacaggagtc 720 tattcattga gtatgggaaa gctttaggct
catcttcaac aggaagcaaa gcagaaagtt 780 tgtttgtaaa tatatttatg
caagcttatg gagccggtca aacaatgcta aggtggggtg 840 tcattgccag
atcatctaac aacataatgc taggacatgt gtctgtgcaa gctgaattga 900
agcaagttac agaggtttat gatttggtga gagaaatggg tcctgaatct gggcttttac
960 atctaagaca aagtccaaag gcaggactgt tatcgttggc caattgcccc
gattttgcta 1020 gtgttgttct tggtaatgct tcaggtctag gtataatcgg
aatgtacaga ggaagagtgc 1080 caaacacaga gctattttct gcagcagaaa
gttatgccag aagcttaaaa gaaagcaaca 1140 aaatcaactt ctcctcatta
gggctcacag acgaagaaaa agaagctgca gaacacttct 1200 taaacatgag
tgatgacaat caagatgatt atgagtaatt aaaaaactgg gacaagtcaa 1260
aatgtcattc cctgaaggaa aagatatcct gttcatgggt aatgaagcag caaaaatagc
1320 agaagctttc cagaaatcac taaaaagatc aggtcacaaa agaacccagt
ctattgtagg 1380 ggaaaaagta aacactatat cagaaactct agagctacct
accatcagca aacctgcacg 1440 atcatctaca ctgctagagc caaaattggc
atgggcagac agcagcagag ccaccaaaac 1500 cacagaaaaa caaacaacca
aaacaacaga tcctgttgaa gaagaggaac tcaatgaaaa 1560 gaagatatca
ccttccagtg atgggaagac tcccgcagag aaaaaatcaa aatctccaac
1620 caatgtaaaa aagaaagttt ccttcacatc aaatgaacca gggaaatata
ccaaactaga 1680 aaaagatgcc ctagatttgc tctcagacaa tgaggaagaa
gacgcagagt cctcaatctt 1740 aacctttgaa gagagagaca catcatcact
aagcattgag gctagactag aatcaataga 1800 agagaagcta agcatgatat
taggactgct tcgtacactt aacattgcaa cagcaggacc 1860 aacggctgca
agagatggaa tcagagatgc aatgattggt ataagagaag aactaatagc 1920
agaaataata aaagaagcaa agggaaaagc agctgaaatg atggaagagg aaatgaatca
1980 aaggtcaaaa ataggtaatg gcagtgtaaa actaaccgag aaggcaaaag
aacttaataa 2040 aattgttgaa gacgagagca caagcggtga atcagaagaa
gaagaagaac caaaagaaac 2100 tcaggataac aatcaaggag aagatattta
ccagttaatc atgtagttta ataaaaataa 2160 acaatgggac aagtcaagat
ggagtcctat ctagtggaca cttatcaagg cattccctac 2220 acagctgctg
ttcaagttga tctggtagaa aaagacttac taccagcaag tttgacaata 2280
tggtttcctc tattccaagc caacacacca ccagcggttt tgctcgatca gctaaagacc
2340 ttgacaataa caactctgta tgctgcatca cagaatggtc caatactcaa
ggtaaatgca 2400 tcagctcagg gtgctgctat gtctgtactt cccaaaaaat
tcgaagtaaa tgcaactgtg 2460 gcacttgatg aatacagcaa acttgacttt
gacaagttaa cggtttgcga tgttaaaaca 2520 gtttatttga caaccatgaa
accatatggg atggtgtcaa aatttgtgag ttcagccaaa 2580 tcagttggca
acaagacaca tgatctaatt gcactgtgtg acttcatgga cctagagaaa 2640
aatatacctg tgacaatacc agcattcata aagtcagttt caatcaaaga gagtgagtca
2700 gccactgttg aagctgcaat aagcagtgag gccgaccaag cattaacaca
agccaaaatt 2760 gcaccctatg caggactaat catgatcatg accatgaaca
atccaaaagg tatattcaag 2820 aaactaggag ctggaacaca agtgatagta
gagctagggg catatgttca agccgagagc 2880 atcagcagga tctgcaagag
ctggagtcac caaggaacaa gatatgtact aaaatccaga 2940 taaaaataac
tgtcctaatc aataattgct tatataatct taaagatcaa tgagcttatt 3000
attatagtta tataaaaaaa tttagaacta ggaaggtatt aatagaaagc gggacaagta
3060 aaaatgtctt ggaaagtgat gattatcatt tcgttactca taacacctca
gcacggacta 3120 aaggaaagtt atttagaaga atcatgtagt actataactg
aaggatatct cagtgtttta 3180 agaacaggtt ggtacaccaa tgtctttaca
ttagaagttg gtgatgttga aaatcttaca 3240 tgtactgatg gacctagctt
aatcaaaaca gaacttgacc taaccaaaag tgctctaaga 3300 gaactcaaaa
cagtttctgc tgatcagtta gcgagagaag aacaaattga aaatcccaga 3360
caatcaaggt ttgtcctagg tgcaatagct cttggtgttg ccacagcagc agcagtcaca
3420 gcaggcattg cgatagccaa aaccataagg cttgagagtg aagtgaatgc
aatcaaaggt 3480 gctctcaaaa caaccaatga ggcagtatcc acactaggaa
atggagtgcg agtcctagcc 3540 accgcagtaa gagagctgaa agaatttgtg
agcaaaaacc tgactagtgc aattaacaag 3600 aacaaatgtg acattgctga
tctgaagatg gctgtcagct tcagtcaatt caacagaaga 3660 ttcctaaatg
ttgtgcggca gttttcagac aatgcaggga taacaccagc aatatcattg 3720
gacctaatga ctgatgctga gctggccaga gctgtatcat acatgccaac atctgcagga
3780 cagataaaac taatgttaga gaaccgtgca atggtgagga gaaaaggatt
tggaatcttg 3840 ataggggtct acggaagctc cgtgatttac atggtccagc
tgccgatctt tggtgtcata 3900 gatacacctt gttggataat caaggcagct
ccctcttgtt cagaaaaaga tggaaactat 3960 gcttgcctcc taagagagga
tcaagggtgg tattgtaaaa atgcaggatc cactgtttac 4020 tacccaaata
aaaaagactg cgaaacaaga ggtgatcatg ttttttgtga cacagctgca 4080
gggatcaatg ttgctgagca atcaagagaa tgcaacatca acatatctac aaccaactac
4140 ccatgcaaag tcagcacagg aagacaccct atcagcatgg ttgcactatc
acctctcggt 4200 gctttggtgg cttgctacaa aggggttagc tgttcaattg
gcagtaatcg ggttggaata 4260 atcaaacaac tacctaaagg ctgctcatac
ataactaacc aggacgcaga cactgtaaca 4320 attgacaaca ctgtgtatca
actaagcaaa gttgagggtg aacagcatgt aataaaaggg 4380 agaccagttt
caagcagttt cgatccaatc aagtttcctg aggatcagtt caatgttgcg 4440
cttgatcaag tctttgaaag cattgaaaac agtcaagcac tagtggacca gtcaaacaaa
4500 attctgaaca gtgcagaaaa aggaaacact ggcttcatta ttgtaataat
tttgattgct 4560 gttcttgggt taaccatgat ttcagtgagc atcatcatca
taatcaaaaa aacaaggaaa 4620 cccacagggg cacctccaga gctgaatggt
gttaccaacg gcggttttat accgcatagt 4680 tagttaatta aaaaatggga
caaatcatca tgtctcgtaa agctccatgc aaatatgaag 4740 tacggggcaa
gtgcaacagg ggaagtgagt gcaaattcaa ccacaattac tggagttggc 4800
ctgataggta tttattgtta agatcaaatt atctcttgaa tcagctttta agaaacactg
4860 ataaggctga tggtttgtca ataatatcag gagcaggtag agaagacagg
actcaagact 4920 ttgttcttgg ttctactaat gtggttcaag ggtacattga
tgacaatcaa ggaataacaa 4980 aggctgcagc ttgctatagt ctacataaca
taataaaaca gctacaagaa atagaagtaa 5040 gacaggccag agataataag
ctttctgaca gcaaacatgt ggcacttcac aacttgatat 5100 tatcctatat
ggagatgagc aaaactcctg catccctgat taataaccta aagaaactac 5160
caagagaaaa actgaaaaaa ttagcgaaat taataattga tttatcagca ggaactgata
5220 atgactcttc atatgccttg caagacagtg aaagcactaa tcaagtgcag
taagcatggt 5280 cccaaattca tcaccataga ggcagatgat atgatatgga
cacacaaaga attaaaagag 5340 acactgtctg atgggatagc aaaatcacac
accaatattt acagttgtta tttagaaaat 5400 atagaaataa tatatgttaa
agcttactta agttagtaaa aaataaatag aatgggataa 5460 atgacaatga
aaacattaga tgtcataaaa agtgacggat cctcagaaac atgtaatcaa 5520
ctcaaaaaaa taataaaaaa acactcaggt aaattgctta ttgcatcaaa accgacattg
5580 gccttattga cgtccttcac agtaacaatt actgtcaact atacaaaagt
agaaaataat 5640 ttgcaggcat gtcaattaaa aaatgaatca gacaaaaagg
acacaaagct aaataccaca 5700 tcaacaacaa tcagacccat tcctgatcta
aatgcagtac agtacctgaa aaggctgatt 5760 cagaaacaca ccaactctgt
cacaaaagac agagatacct gttggagaat acacacgaat 5820 caatgcacaa
atataaaaat atataagttc ttatgttttg ggtctatgaa ttcaacaaat 5880
acagactgtg aagaaccaac agttctatgc gacaaaaagt caaaaaccat gacagaaaaa
5940 cataggaaag cagagtgtca ccgtccacat acaaccgagt ggtggtgcca
ttatctttaa 6000 gagaaaactc agttttcaac attaaaatca gaacaaatca
tatctagatc tattaatata 6060 atagtctagt tatttaaaaa ctctaaatat
tgtctagact tcacaacacc ctgcggtcat 6120 atgcaataat caatggtcaa
accactgttg caaacccacc tataatacaa tcactgagta 6180 atacaaaaca
agaaaatggg acaagtggcc atggaagcaa gagtggagaa cattcgggca 6240
atagacatgt tcaaagcaaa gatgaaaaac cgtataagaa gtagcaagtg ccatagaaat
6300 gctacactga tccttattgg atcaacagca ccaagtatgg cactcaacac
ccttttaatc 6360 attgatcatg caacatcaaa aaacatgacc aaagtggaac
actgtgtcaa catgccgccg 6420 gtagaaccaa gcaagaagac cccaatgacc
tctgcagcag acccaaacac caaacccaat 6480 ccacagcagg caacacagct
gaccacagag gattcaacat ctctagcagc aaccctagag 6540 gaccatctac
acacagggac aactccaaca ccagatgcaa cagtctccca gcaaaccaca 6600
gacgagcaca caacactgct gagatcaacc aacagacaga ccacccaaac aaccgcagag
6660 aaaaagccaa ccagagcaac aaccaaaaaa gaaaccacaa ctcgaaccac
aagcacagct 6720 gcaacccaaa cactcaacac caccaaccaa actagcaatg
gaagagaggc aaccacaaca 6780 tctgccagat ccagaaacaa tgccacaact
caaagcagcg atcaaacaac ccaggcagca 6840 gacccaagct cccaatcaca
acatacacag aaaagcacaa caacaacaca caacacagac 6900 acatcttctc
caagtagtta acaaaaaaac tataaaataa ccatgaaaac caaaaaacta 6960
gaaaagttaa tttgaactca gaaaagaaca caaacactat atgaattgtt tgagcgtata
7020 tactaatgaa atagcatctg tttgtgcatc aataatacca tcattattta
agaaataaga 7080 agaagctaaa attcaaggga caaataacaa tggatccgtt
ttgtgaatcc actgtcaatg 7140 tctatcttcc tgattcatat ctcaaaggag
taatatcttt cagtgaaacc aatgcaattg 7200 gctcatgcct tttgaaaaga
ccctatctta aaaaagataa cactgctaaa gttgctgtag 7260 aaaaccctgt
tgttgaacat gtcagactta gaaatgcagt catgaccaaa atgaagatat 7320
cagattataa agtggttgaa ccaattaata tgcagcatga aataatgaaa aatatacaca
7380 gttgtgagct cacattatta aaacaattct taacaagaag taaaaacatt
agctccctaa 7440 aattaagtat gatatgtgat tggttacagt taaaatccac
ctcagataac acatcaattc 7500 ttaattttat agatgtggag tttatacccg
tttgggtgag caattggttt agtaactggt 7560 ataatctcaa taaattaatc
ttagagttta gaagagagga agtaataaga actggttcaa 7620 ttttatgcag
atcactaggc aagttagttt tcattgtatc atcttatggg tgtgtagtaa 7680
aaagcaacaa aagtaaaaga gtaagttttt tcacatataa ccaactgtta acatggaaag
7740 atgtgatgtt aagtaggttc aatgcaaact tttgtatatg ggtaagtaac
aacctgaaca 7800 aaaatcaaga aggactagga tttagaagta atctacaagg
tatgttaact aataaattat 7860 atgaaactgt tgattatatg ttaagtctat
gtagcaatga agggttctca ctagtgaaag 7920 agttcgaagg ctttattatg
agtgaaattc ttaaaattac tgagcatgct caattcagta 7980 ctaggtttag
gaatacttta ttaaatgggt tgactgaaca attatcaatg ttgaaagcta 8040
aaaacagatc tagagttctt ggcactatat tagaaaacaa tgattacccc atgtatgaag
8100 tagtacttaa attattaggg gacactttga aaagtataaa attattaatt
aacaagaatt 8160 tagaaaatgc tgcagaatta tattatatat tcagaatttt
tggacaccct atggtagatg 8220 agagggaagc aatggatgct gttaaattaa
ataatgagat tacaaaaatt cttaaactgg 8280 agagcttaac agaactaaga
ggagcattta tactaagaat tataaaaggg tttgtagata 8340 ataataaaag
atggcctaaa attaagaatt taaaagtgct cagtaaaaga tgggttatgt 8400
atttcaaagc taaaagttac cctagccaac ttgagctaag tgtacaagat tttttagaac
8460 ttgctgcagt acaattcgaa caggaatttt ctgtccctga aaaaaccaat
cttgagatgg 8520 tattaaatga taaagcaata tctccaccaa aaaagttaat
atggtcggta tatccaaaaa 8580 attatctacc tgaaattata aaaaatcaat
atttagaaga ggtcttcaat gcaagtgaca 8640 gtcaaagaac gaggagagtc
ttagaatttt acttaaaaga ttgcaaattt gatcaaaaag 8700 acctcaaacg
ttatgtaact aaacaagagt atctaaatga caaagaccac attgtctcat 8760
taactgggaa agaaagagaa ttaagtgtag gcaggatgtt tgcaatgcaa cctggcaaac
8820 aaagacaaat acagatacta gccgagaaac ttttagctga taatattgta
ccctttttcc 8880 cagaaacttt aacaaagtat ggtgacttgg atctccaaag
aattatggaa atgaaatcag 8940 aactttcttc cattaaaact aggaagaatg
atagttacaa caattatatt gcaagagcct 9000 ccatagtaac agacctaagt
aaattcaatc aagcctttag atatgaaacc acagctatct 9060 gcgcagacgt
agcagatgag ttacatggca cgcaaagctt attttgttgg ttacatctta 9120
ttgttcccat gaccacaatg atatgtgcat acagacatgc accaccagaa acaaaggggg
9180 agtatgatat agacaaaata gaagagcaaa gtgggctata cagataccat
atgggaggga 9240 ttgaagggtg gtgtcagaag ttatggacaa tggaggcgat
atccttgtta gatgtagtat 9300 ctgttaagac tcgttgtcag atgacctctc
tattaaacgg agacaatcaa tcaatagatg 9360 tcagtaaacc agtaaaattg
tctgaaggta tagatgaagt aaaagcagat tatagcttag 9420 caattaaaat
gcttaaagag ataagagatg cctataaaaa cattggccat aaactcaaag 9480
aaggtgaaac atatatatca agagatcttc aatttataag taaggtgatt caatctgagg
9540 gggtcatgca tcctaccccc ataaaaaaga tattaagggt aggtccctgg
ataaatacaa 9600 tactagatga cattaaaact agtgcagaat caatagggag
tctgtgtcaa gaactagagt 9660 tcagaggaga aagtatacta gttagcttga
tattaaggaa tttctggctg tataacttat 9720 acatgcatga gtcaaaacag
catccgttag ctggaaaaca actgtttaaa caattgaaca 9780 aaacactaac
atctgtgcaa agattttttg agctgaagaa agaaaatgat gtggttgacc 9840
tatggatgaa tataccaatg cagtttggag ggggagaccc agtagttttt tacagatctt
9900 tttacagaag gactcctgat ttcttgactg aagcaatcag ccatgtggat
ttactgttaa 9960 aagtttcaaa caatattaaa aatgagacta agatacgatt
ctttaaagcc ttattatcta 10020 tagaaaagaa tgaacgtgct acattaacaa
cactaatgag agacccccag gcggtaggat 10080 cggaaagaca agctaaggta
acaagtgata taaatagaac agcagttact agcatactga 10140 gtctatctcc
gaatcagcta ttttgtgata gtgctataca ctatagcaga aatgaagaag 10200
aagtagggat cattgcagac aacataacac ctgtttatcc tcacggattg agagtgctct
10260 atgaatcact accttttcat aaggctgaaa aggttgtcaa tatgatatca
ggtacaaagt 10320 ctataactaa cctattgcag agaacatctg ctatcaatgg
tgaagatatt gatagagcag 10380 tgtctatgat gttagagaac ttagggttgt
tatctaggat attgtcagta ataattaata 10440 gtatagaaat accaattaag
tccaatggca gattgatatg ctgtcaaatt tctaagactt 10500 tgagagaaaa
atcatggaac aatatggaaa tagtaggagt gacatctcca agtattgtaa 10560
catgtatgga tgttgtgtat gcgactagtt ctcatttaaa aggaataatt attgaaaaat
10620 tcagtactga caagaccaca agaggtcaga ggggaccaaa aagcccttgg
gtaggatcaa 10680 gcactcaaga gaaaaaatta gttcctgttt ataacagaca
aattctttca aaacaacaaa 10740 aagagcaact ggaagcaata ggaaaaatga
ggtgggtgta taaaggaact ccagggctaa 10800 gaagattgct caataagatt
tgcataggaa gtttaggtat tagctataaa tgtgtaaaac 10860 ctctattacc
aagatttatg agtgtaaact tcttacatag gttatctgtt agtagcagac 10920
ccatggaatt cccagcttct gttccagctt ataggacaac aaattaccac tttgacacta
10980 gtccaatcaa ccaagcatta agtgagaggt tcgggaacga agacattaat
ctagtgttcc 11040 aaaatgcaat cagctgcgga attagtataa tgagtgttgt
agaacagtta actggtagaa 11100 gcccaaaaca attagtctta atcccccaat
tagaagagat agatattatg ccccctcctg 11160 tatttcaagg aaaattcaat
tataaactag ttgataaaat aacctccgat caacacatct 11220 tcagtcctga
caaaatagac atattaacac tagggaagat gcttatgcct actataaaag 11280
gtcaaaaaac tgatcagttc ttaaataaga gagaaaacta tttccatgga aataatttaa
11340 ttgaatcttt atctgcagca cttgcatgcc attggtgtgg aatattaaca
gaacagtgtg 11400 tagaaaacaa tatctttagg aaagactggg gtgatgggtt
catatcagat catgccttca 11460 tggatttcaa gatatttcta tgtgtattta
aaaccaaact tttatgtagt tggggatccc 11520 aagggaaaaa tgtaaaagat
gaagatataa tagatgaatc cattgacaaa ttattaagaa 11580 ttgacaacac
tttttggaga atgttcagca aagtcatgtt tgaatcaaag gtcaaaaaaa 11640
gaataatgtt atatgatgta aaattcctat cattagtagg ttatatagga tttaaaaact
11700 ggtttataga gcagttaaga gtagtagaat tgcatgaagt accctggatt
gtcaatgctg 11760 aaggggagct agttgaaatt aaaccaatca aaatttattt
gcagttaata gaacaaagtc 11820 tatctttaag aataactgtt ttgaattata
cagacatggc acatgctctt acacgattaa 11880 ttaggaagaa attgatgtgt
gataatgcac tctttaatcc aagttcatca ccaatgttta 11940 gtctaactca
agttattgat cctacaacac agctagacta ttttcctaag gtaatatttg 12000
aaaggttaaa aagttatgac accagttcag actacaacaa agggaagtta acaagaaatt
12060 acatgacatt attaccatgg cagcacgtaa acaggtataa ttttgtcttt
agttcaacag 12120 gatgtaaaat cagcttgaag acatgcatcg ggaaattgat
aaaggactta aaccctaagg 12180 ttctttactt tattggagaa ggagcaggta
actggatggc aagaacagca tgtgagtatc 12240 ctgacataaa atttgtatat
aggagtttaa aggatgatct tgatcaccat tacccattag 12300 aatatcaaag
ggtaataggt gatttaaata gagtaataga tggtggtgaa ggattatcaa 12360
tggagaccac agatgcaact caaaagactc attgggactt gatacacaga ataagtaaag
12420 atgctttatt gataacattg tgtgatgcag aattcaaaaa cagagatgat
ttctttaaaa 12480 tggtaattct ttggagaaaa catgtattat catgtagaat
ctgtacagct tatggaacag 12540 atctttactt atttgcaaag tatcatgcga
cggactgcaa tataaagtta ccattttttg 12600 taaggtctgt agctactttt
attatgcaag gaagcaaatt gtcaggatca gaatgttaca 12660 tacttttaac
attaggtcat cacaataatc tgccatgtca cggagaaata caaaattcca 12720
aaatgagaat agcagtgtgt aatgatttcc atgcctcaaa aaaactagac aacaaatcaa
12780 ttgaagcaaa ctgcaaatct cttctatcag gattaagaat accaataaac
aaaaaagagt 12840 taaatagaca aaagaaactg ttaacactac aaagcaatca
ttcttccata gcaacagttg 12900 gcggaagtaa gattatagaa tccaaatggt
taaagaataa agcaagtaca ataattgatt 12960 ggttagagca tatcttgaat
tctccaagag gtgaattaaa ctatgatttc tttgaagcat 13020 tagagaacac
atatcccaat atgatcaagc ttatagataa cctgggaaat gcagagataa 13080
aaaaactaat caaagttacc gggtatatgc ttgtgagtga gaagtaataa taataataat
13140 aatcaaccat aatctcacac aactgagaaa atgatcatct aacagtttaa
ttgaccatta 13200 gttaattaaa aattataaat tagtaactaa ttgataaaaa
ataagaaatt gaaattgaat 13260 gtatacggtt tttttgccgt 13280 3 14083 DNA
Artificial Sequence HMPV strain 83 with GFP inserted prior to N
gene. 3 acgcgaaaaa aacgcgtata aattaagtta caaaaaaaca tgggacaagt
gaaaatggtg 60 agcaagggcg aggagctgtt caccggggtg gtgcccatcc
tggtcgagct ggacggcgac 120 gtaaacggcc acaagttcag cgtgtccggc
gagggcgagg gcgatgccac ctacggcaag 180 ctgaccctga agttcatctg
caccaccggc aagctgcccg tgccctggcc caccctcgtg 240 accaccctga
cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 300
gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
360 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
cctggtgaac 420 cgcatcgagc tgaagggcat cgacttcaag gaggacggca
acatcctggg gcacaagctg 480 gagtacaact acaacagcca caacgtctat
atcatggccg acaagcagaa gaacggcatc 540 aaggtgaact tcaagatccg
ccacaacatc gaggacggca gcgtgcagct cgccgaccac 600 taccagcaga
acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 660
agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
720 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
gtaagttaat 780 taaaaaagtg ggacaagtga aaatgtctct tcaagggatt
cacctgagtg atctatcata 840 caagcatgct atattaaaag agtctcagta
tacaataaag agagatgtag gcacaacaac 900 agcagtgaca ccctcatcat
tgcaacaaga aataacacta ttgtgtggag aaattctata 960 tgctaagcat
gctgattaca aatatgctgc agaaatagga atacaatata ttagcacagc 1020
tctaggatca gagagagtac agcagattct aagaaactca ggcagtgaag tccaagtggt
1080 tttaaccaga acgtactcct tggggaaagt taaaaacaac aaaggagaag
atttacagat 1140 gttagacata cacggagtag agaaaagctg ggtggaagag
atagacaaag aagcaagaaa 1200 aacaatggca actttgctta aagaatcatc
aggcaatatt ccacaaaatc agaggccttc 1260 agcaccagac acacctataa
tcttattatg tgtaggtgcc ttaatattta ccaaactagc 1320 atcaactata
gaagtgggat tagagaccac agtcagaaga gctaaccgtg tactaagtga 1380
tgcactcaaa agatacccta ggatggacat accaaaaatc gctagatctt tctatgattt
1440 atttgaacaa aaagtgtatt acagaagttt gttcattgag tatggcaaag
cattaggctc 1500 atcctctaca ggcagcaaag cagaaagttt attcgttaat
atattcatgc aagcttacgg 1560 tgctggtcaa acaatgctga ggtggggagt
cattgccagg tcatctaaca atataatgtt 1620 aggacatgta tctgtccaag
ctgagttaaa acaagtcaca gaagtctatg acctggtgcg 1680 agaaatgggc
cctgaatctg ggctcctaca tttaaggcaa agcccaaaag ctggactgtt 1740
atcactagcc aattgtccca actttgcaag tgttgttctc ggcaatgcct caggcttagg
1800 cataataggt atgtatcgcg ggagagtgcc aaacacagaa ctattttcag
cagcagaaag 1860 ctatgccaag agtttgaaag aaagcaataa aattaacttt
tcttcattag gactcacaga 1920 tgaagaaaaa gaggctgcag aacactttct
aaatgtgagt gacgacagtc aaaatgatta 1980 tgagtaatta aaaaagtggg
acaagtcaaa atgtcattcc ctgaaggaaa agatattctt 2040 ttcatgggta
atgaagcggc aaaattggca gaagctttcc aaaaatcatt aagaaaacct 2100
agtcataaaa gatctcaatc tattatagga gaaaaagtga acactgtatc tgaaacattg
2160 gaattaccta ctatcagtag acctaccaaa ccgaccatat tgtcagagcc
gaagttagca 2220 tggacagaca aaggtggggc aatcaaaact gaagcaaagc
aaacaatcaa agttatggat 2280 cctattgaag aagaagagtt tactgagaaa
agggtgctgc cctccagtga tgggaaaact 2340 cctgcagaaa agaagttgaa
accatcaacc aatactaaaa agaaggtctc atttacacca 2400 aatgaaccag
gaaaatacac aaagttggag aaagatgctc tagacttgct ttcagacaat 2460
gaagaagaag atgcagaatc ctcaatctta accttcgaag aaagagatac ttcatcatta
2520 agcattgaag ccagactaga atcgattgag gagaaattaa gcatgatatt
agggctatta 2580 agaacactca acattgctac agcaggaccc acagcagcaa
gagatgggat cagagatgca 2640 atgattggca taagggagga actaatagca
gacataataa aagaagccaa gggaaaagca 2700 gcagaaatga tggaagaaga
aatgaaccag cggacaaaaa taggaaacgg tagtgtaaaa 2760 ttaactgaaa
aggcaaagga gctcaacaaa attgttgaag acgagagcac aagtggtgaa 2820
tccgaagaag aagaagaact aaaagacaca caggaaaata atcaagaaga tgacatttac
2880 cagttaatta tgtagtttaa taaaaataaa aaatgggaca agtgaaaatg
gagtcctatc 2940 tggtagacac ctatcaaggc atcccttaca cagcagctgt
tcaagttgat ctagtagaaa 3000 aggacctgtt acctgcaagc ctaacaatat
ggttccccct gtttcaggcc aatacaccac 3060 cagcagttct gcttgatcag
ctaaagactc tgactataac tactctgtat gctgcatcac 3120 aaagtggtcc
aatactaaaa gtgaatgcat cggcccaggg tgcagcaatg tctgtacttc 3180
ccaaaaagtt tgaagtcaat gcgactgtag cacttgacga atatagcaaa ttagaatttg
3240 acaaacttac agtctgtgaa gtaaaaacag tttacttaac
aaccatgaaa ccatatggga 3300 tggtatcaaa gtttgtgagc tcggccaaac
cagttggcaa aaaaacacat gatctaatcg 3360 cattatgcga ttttatggat
ctagaaaaga acacaccagt tacaatacca gcatttatca 3420 aatcagtttc
tatcaaggag agtgaatcag ccactgttga agctgcaata agcagtgaag 3480
cagaccaagc tctaacacaa gccaaaattg caccttatgc gggactgatc atgattatga
3540 ccatgaacaa tcccaaaggc atattcaaga agcttggagc tgggacccaa
gttatagtag 3600 aactaggagc atatgtccag gctgaaagca taagtaaaat
atgcaagact tggagccatc 3660 aaggaacaag atatgtgctg aagtccagat
aacagccaag caacctgacc aagaactacc 3720 aactctattc tatagactaa
aaagtcgcca ttttagttat ataaaaatca agttagaata 3780 agaatgctag
caatcaagaa cgggacaaat aaaaatgtct tggaaagtgg tgatcatttt 3840
ttcattgcta ataacacctc aacacggtct taaagagagc tacctagaag aatcatgtag
3900 cactataact gagggatatc ttagtgttct gaggacaggt tggtatacca
acgtttttac 3960 attagaggtg ggtgatgtag aaaaccttac atgttctgat
ggacctagcc taataaaaac 4020 agaattagat ctgaccaaaa gtgcactaag
agagctcaaa acagtctctg ctgaccaatt 4080 ggcaagagag gaacaaattg
agaatcccag acaatctagg tttgttctag gagcaatagc 4140 actcggtgtt
gcaacagcag ctgcagtcac agcaggtgtt gcaattgcca aaaccatccg 4200
gcttgagagt gaagtcacag caattaagaa tgccctcaaa acgaccaatg aagcagtatc
4260 tacattgggg aatggagttc gagtgttggc aactgcagtg agagagctga
aagactttgt 4320 gagcaagaat ttaactcgtg caatcaacaa aaacaagtgc
gacattgatg acctaaaaat 4380 ggccgttagc ttcagtcaat tcaacagaag
gtttctaaat gttgtgcggc aattttcaga 4440 caatgctgga ataacaccag
caatatcttt ggacttaatg acagatgctg aactagccag 4500 ggccgtttct
aacatgccga catctgcagg acaaataaaa ttgatgttgg agaaccgtgc 4560
gatggtgcga agaaaggggt tcggaatcct gataggggtc tacgggagct ccgtaattta
4620 catggtgcag ctgccaatct ttggcgttat agacacgcct tgctggatag
taaaagcagc 4680 cccttcttgt tccggaaaaa agggaaacta tgcttgcctc
ttaagagaag accaagggtg 4740 gtattgtcag aatgcagggt caactgttta
ctacccaaat gagaaagact gtgaaacaag 4800 aggagaccat gtcttttgcg
acacagcagc gggaattaat gttgctgagc aatcaaagga 4860 gtgcaacatc
aacatatcca ctacaaatta cccatgcaaa gtcagcacag gaagacatcc 4920
tatcagtatg gttgcactgt ctcctcttgg ggctctggtt gcttgctaca aaggagtaag
4980 ctgttccatt ggcagcaaca gagtagggat catcaagcag ctgaacaagg
gttgctccta 5040 tataaccaac caagatgcag acacagtgac aatagacaac
actgtatatc agctaagcaa 5100 agttgagggt gaacagcatg ttataaaagg
cagaccagtg tcaagcagct ttgatccaat 5160 caagtttcct gaagatcaat
tcaatgttgc acttgaccaa gtttttgaga acattgaaaa 5220 cagccaggcc
ttggtagatc aatcaaacag aatcctaagc agtgcagaga aagggaatac 5280
tggcttcatc attgtaataa ttctaattgc tgtccttggc tctagcatga tcctagtgag
5340 catcttcatt ataatcaaga aaacaaagaa accaacggga gcacctccag
agctgagtgg 5400 tgtcacaaac aatggcttca taccacacag ttagttaatt
aaaaataaaa taaaatttgg 5460 gacaaatcat aatgtctcgc aaggctccat
gcaaatatga agtgcggggc aaatgcaaca 5520 gaggaagtga gtgtaagttt
aaccacaatt actggagttg gccagataga tacttattaa 5580 taagatcaaa
ctatctatta aatcagcttt taaggaacac tgatagagct gatggcctat 5640
caataatatc aggcgcaggc agagaagaca gaacgcaaga ttttgttcta ggttccacca
5700 atgtggttca aggttatatt gatgataacc aaagcataac aaaagctgca
gcctgctaca 5760 gtctacacaa cataatcaag caactacaag aagttgaagt
taggcaggct agagatagca 5820 aactatctga cagcaagcat gtggcactcc
ataacttaat cttatcttac atggagatga 5880 gcaaaactcc cgcatcttta
atcaacaatc tcaaaagact gccgagagaa aaactgaaaa 5940 aattagcaaa
gctgataatt gacttatcag caggcgctga caatgactct tcatatgccc 6000
tgcaagacag tgaaagcatt aatcaagtgc agtgagcatg gtcctgtttt cattactata
6060 gaggttgatg aaatgatatg gactcaaaaa gaattaaaag aagctttgtc
cgatgggata 6120 gtgaagtctc acaccaacat ttacaattgt tatttagaaa
acatagaaat tatatatgtc 6180 aaggcttact taagttagta aaaacacatc
agagtgggat aagtgacaat gataacatta 6240 gatgtcatta aaagtgatgg
gtcttcaaaa acatgtactc acctcaaaaa aataatcaaa 6300 gaccattctg
gtaaagtgct tattgcactt aagttaatat tagctttact aacatttttc 6360
acaataacaa tcactataaa ttacataaaa gtagaaaaca atctacaaat atgccagtca
6420 aaaactgaat cagacaaaga agactcacca tcaaatacca catccgtcac
aaccaagacc 6480 actctagacc atgatataac acagtatttt aaaagattaa
ttcaaaggta tacagattct 6540 gtaataaaca aggacacatg ctggaaaata
agcagaaatc aatgcacaaa tataacaaca 6600 tataaatttt tatgctttaa
acctgaggac tcaaaaatca acagttgtga tagactgaca 6660 gatctatgca
gaaacaaatc aaaatcagca gctgaagcat atcatacagt agaatgccat 6720
tgcatataca caattgagtg gaagtgctat caccactcaa tagattaaac ccaatcttga
6780 atgttaaaac tagactagga tccgtctaag actatcagtt caatagttta
gttattttaa 6840 aatatttgag aataggtaag tttctatggc acttcatagc
aataggtaat aattaacagc 6900 ttaattataa ttaaaacatt atttaaaatc
gtaactattt aatttacaaa gtaaaaacaa 6960 aaatatggga caagtagtta
tggaggtgaa agtagagaac attcgagcaa tagacatgct 7020 caaagcaaga
gtgaaaaatc gtgtggcacg tagcaaatgc tttaaaaatg cttctttaat 7080
cctcatagga ataactacac tgagtatagc tctcaatatc tatctgatca taaactacac
7140 aatacaaaaa acctcatctg aatcagaaca ccacaccagc tcaccaccca
cagaatccaa 7200 caaggaagct tcaacaatct ccacagacaa cccagacatc
aatccaaact cacagcatcc 7260 aactcaacag tccacagaaa accccacact
caaccccgca gcatcagtga gcccatcaga 7320 aacagaacca gcatcaacac
cagacacaac aaaccgcctg tcctccgtag acaggtccac 7380 agcacaacca
agtgaaagca gaacaaagac aaaaccgaca gtccacacaa gaaacaaccc 7440
aagcacagct tccagtacac aatccccacc acgggcaaca acgaaggcaa tccgcagagc
7500 caccactttc cgcatgagca gcacaggaaa aagaccaacc acaacatcag
tccagtccga 7560 cagcagcacc acaacccaaa atcatgaaga aacaggttca
gcgaacccac aggcatctgt 7620 aagcacaatg caaaactagc acaccaacaa
tataaaacca aattagttaa caaaaaatac 7680 gagatagctc taaagtaaaa
catgtaggta ccaacaatca agaaaccaaa agacaactca 7740 caatctccct
aaaacagcaa cgacaccatg tcagctttgc tcaaatctct ctgggagaaa 7800
cttttgccca catactaaca acatcacaac catctcaaga aaagaaactg ggcaaaacag
7860 catccaagag acaaatagca atggatcctc ttaatgaatc cactgttaat
gtctatctcc 7920 ctgattcgta ccttaaagga gtaatttctt ttagtgaaac
taatgcaatt ggttcatgtc 7980 tcttaaaaag accctactta aaaaatgaca
acactgcaaa agttgccata gagaatcctg 8040 ttattgagca tgtgagactc
aaaaatgcag tcaattctaa aatgaaaata tcagattaca 8100 aggtagtaga
gccagtaaac atgcaacatg aaataatgaa gaatgtacac agttgtgagc 8160
tcacactatt gaaacagttt ttaacaagga gtaaaaacat tagcactctc aaattgaata
8220 tgatatgtga ttggctgcaa ttaaagtcta catcagatga tacctcaatc
ctaagtttca 8280 tagatgtaga atttatacct agttgggtaa gcaactggtt
tagtaattgg tacaatctca 8340 ataagttaat tttggaattc agaagagagg
aagtaataag aaccggttca atcttatgca 8400 ggtcattggg taaattagtt
tttattgtat catcatatgg atgtatcgtc aagagcaaca 8460 aaagcaaaag
agtgagcttc ttcacataca atcaactgtt aacatggaaa gatgtgatgt 8520
taagtagatt taatgcgaat ttttgtatat gggtaagcaa tagtctgaat gaaaatcagg
8580 aagggctagg gttgagaagt aatctgcaag gtatgttaac taataaacta
tatgaaactg 8640 tagattatat gctaagctta tgttgcaatg aaggtttctc
acttgtgaaa gagttcgaag 8700 gttttattat gagtgagatc cttaggatta
ctgaacatgc tcaattcagt actagattta 8760 gaaatacttt attgaatgga
ttaacagatc aattaacaaa attaaaaaat aaaaacagac 8820 tcagagttca
tagtaccgta ttagaaaata atgattatcc aatgtatgaa gttgtactta 8880
aattattagg agatactttg agatgtatca aattattaat caataaaaac ttagagaatg
8940 ctgcagaatt atactatata ttcagaattt ttggtcatcc aatggtagat
gaaagagatg 9000 caatggatgc tgtcaaatta aacaatgaaa tcacaaaaat
cctaaggttg gagagcttga 9060 cagaactaag aggggcattc atattaagga
ttatcaaagg atttgtggac aacaacaaaa 9120 ggtggcccaa aattaaaaac
ttaaaagtgc ttagcaaaag atggactatg tacttcaaag 9180 ctaaaaatta
ccccagtcaa ctcgaattaa gtgaacaaga ctttctagag cttgctgcaa 9240
tacaatttga acaagagttt tctgttcctg aaaaaaccaa tcttgagatg gtattaaatg
9300 acaaagccat atcacctcct aaaagattaa tatggtctgt gtatccaaag
aattacttac 9360 ctgagacgat aaaaaatcga tatttagaag aaactttcaa
tgcgagtgat agtctcaaaa 9420 caagaagagt actagagtac tatttaaaag
ataataaatt tgatcaaaag gaacttaaaa 9480 gttatgtagt tagacaagaa
tatttaaacg ataaggagca cattgtctca ttaactggaa 9540 aagaaagaga
attaagtgta ggtagaatgt ttgctatgca accaggaaaa cagcgacaaa 9600
tacaaatatt ggcagaaaaa ttgttagctg ataacattgt acctttcttc ccggaaacct
9660 taacaaagta tggtgatcta gatcttcaga gaataatgga aatcaaatca
gaactttctt 9720 ctatcaaaac cagaagaaat gatagttata ataattacat
tgcaagagca tccatagtaa 9780 cagatttgag caagttcaac caagccttta
gatatgaaac tacagcgatc tgtgcggatg 9840 tagcagacga attacatgga
acacaaagct tattctgttg gttacatctt atcgttccta 9900 tgactacaat
gatatgtgcc tatagacatg caccaccaga aacaaaaggt gaatatgata 9960
tagataagat agaagagcaa agtggtctat atagatatca catgggcggt attgaaggat
10020 ggtgtcaaaa actctggaca atggaagcta tatctttatt ggatgttgta
tctgtaaaga 10080 cacggtgtca aatgacatct ttattaaacg gtgacaacca
atcaatagat gtaagtaaac 10140 cagtcaagtt atctgaaggt ttagatgaag
tgaaggcaga ttatcgctta gcagtaaaaa 10200 tgctaaaaga aataagagat
gcatacagaa atataggcca taaacttaaa gaaggggaaa 10260 catatatatc
aagggatctt cagtttataa gcaaggtgat tcaatctgaa ggagtgatgc 10320
atcctacccc tataaaaaag gtcttgagag taggaccatg gataaacaca atattagatg
10380 acattaaaac tagtgctgag tcaataggga gtctatgtca agaattagaa
tttaggggag 10440 aaagcataat agttagtctg atattaagaa acttctggct
gtataactta tacatgcatg 10500 aatcaaagca acatcctttg gcagggaaac
agttattcaa acaactaaat aaaacattaa 10560 catcagtgca gagatttttt
gaaattaaaa gggaaaatga ggtagtagat ctatggatga 10620 acataccaat
gcaatttgga ggaggagatc cagtagtctt ctatagatct ttctatagaa 10680
ggacccctga ttttttaact gaggcaatca gccatgtaga tattctgtta aaaatatcag
10740 ctaacataaa aaatgaaacg aaagtaagtt tcttcaaagc cttactatca
atagaaaaaa 10800 atgaacgtgc tacactgaca acactaatga gagatcctca
agctgttgga tcagaacgac 10860 aagcaaaagt aacaagtgac atcaatagaa
cagcagttac cagtatctta agtctttccc 10920 caaatcaact tttcagtgat
agtgctatac actacagcag aaatgaagaa gaagtgggaa 10980 tcattgcaga
aaacataaca cctgtttatc ctcatgggct gagagtatta tatgaatcat 11040
tgccctttca caaagctgaa aaagttgtaa acatgatatc agggacaaaa tctataacca
11100 acttattaca gagaacatcc gctattaatg gtgaagatat tgacagggct
gtatctatga 11160 tgttggagaa tctaggatta ttatctagaa tattgtcagt
agttgttgat agtatagaaa 11220 ttccaatcaa atctaatggt aggctgatat
gttgtcaaat ctctaggact ttaagagaga 11280 catcatggaa taatatggaa
atagttggag taacatctcc tagcatcact acatgtatgg 11340 atgtcatata
tgcaactagt tctcatttga aagggataat tatagaaaag ttcagcactg 11400
acagaactac aaggggtcaa agaggtccaa aaagcccttg ggtagggtcg agtactcaag
11460 agaaaaaatt agtacctgtt tataacagac aaattctctc aaaacaacaa
agagaacagc 11520 tagaagcaat tggaaaaatg agatgggtgt ataaagggac
accaggcttg cgacgattac 11580 tcaacaagat ctgtcttggg agtttaggca
ttagctacaa atgtgtaaaa cctttattac 11640 ctaggtttat gagtgtaaat
ttcttacata gattatctgt cagtagtaga cctatggaat 11700 tcccagcatc
agttccagct tatagaacaa caaattacca tttcgacact agtcctatta 11760
atcaagcact aagtgagaga tttgggaatg aagatattaa cttggtcttc caaaatgcaa
11820 tcagctgtgg aattagcata atgagtgtag tagaacaatt aacaggtaga
agcccaaaac 11880 agttagtttt aataccccaa ttagaagaaa tagacattat
gccaccacca gtgtttcaag 11940 ggaaattcaa ttataaatta gtagataaga
taacttctga tcaacatatc tttagtccgg 12000 acaaaataga tatgttaaca
ctagggaaaa tgctcatgcc cactataaaa ggtcagaaaa 12060 cagatcagtt
cttaaataag agagaaaatt atttccatgg aaacaatctt attgagtctt 12120
tatcagcagc attagcatgt cattggtgtg ggatattaac agaacaatgc atagaaaata
12180 atattttcaa gaaagactgg ggtgacgggt ttatatcaga tcatgctttt
atggacttca 12240 aaatattcct atgtgtcttt aaaactaaac ttttatgtag
ttggggatct caagggaaaa 12300 acattaaaga tgaagatata gtagatgaat
caatagataa attgttaagg attgacaata 12360 ctttttggag aatgttcagc
aaagttatgt ttgaaccaaa ggttaagaaa aggataatgt 12420 tatatgatgt
aaaattccta tcactagtag gctacatagg gtttaagaac tggtttatag 12480
agcagttgag atcagctgaa ttgcatgaaa taccttggat tgtcaatgcc gaaggtgatt
12540 tggttgagat caagtcaatt aaaatctatt tgcaactgat agaacaaagc
ttatttttaa 12600 gaataactgt tttgaactat acagatatgg cacatgctct
cacacgatta atcagaaaga 12660 agttaatgtg tgataatgca ctgttaaccc
caatttcatc cccaatggtt aacttaactc 12720 aagttattga tcccacaaca
caattagatt acttccccaa gataacattc gaaaggctaa 12780 aaaattatga
cacaagttca aattatgcta aagggaagct aacaagaaat tacatgatac 12840
tattgccatg gcagcatgtt aatagatata actttgtctt tagttctact ggatgtaaag
12900 ttagtctgaa aacatgtatt ggaaaactta tgaaagacct aaatcctaaa
gttttgtact 12960 ttattggaga aggagcagga aattggatgg ccagaacagc
atgtgaatat cctgatatta 13020 aatttgtata tagaagtctg aaagatgacc
ttgatcatca ttatcctctg gaataccaga 13080 gagtgatagg tgaattaagc
agaatcatag atagtggtga aggactttca atggaaacaa 13140 cagacgcaac
tcaaaaaact cattgggatt tgatacacag ggtaagcaaa gatgctttat 13200
taataacttt atgtgatgca gaatttaagg acagagatga tttttttaag atggtaattc
13260 tatggagaaa acatgtatta tcatgcagaa tttgcactac ttatgggacg
gacctctatt 13320 tattcgcaaa gtatcatgct aaagactgca atgtaaaatt
accttttttt gtgagatcag 13380 ttgctacttt cattatgcag ggtagtaagc
tgtcaggttc agaatgctac atactcttaa 13440 cactaggcca ccacaacagt
ttaccttgcc atggagaaat acaaaattct aagatgaaaa 13500 tagcagtgtg
taatgatttt tatgctgcaa aaaaactcga caataaatca attgaagcta 13560
attgtaaatc acttttgtca gggctaagaa tacctataaa taagaaggaa ctagatagac
13620 agagaagatt attaacacta caaagcaatc attcttctgt agcaacagtt
ggcggtagca 13680 agatcataga gtctaagtgg ttaacaaaca aagcaagtac
aataattgat tggttagaac 13740 atattttaaa ttctccaaag ggcgaattaa
attatgattt ttttgaagca ttggagaaca 13800 cttaccctaa tatgattaaa
ctaatagata acttagggaa tgcagagatt aaaaaactga 13860 tcaaagtaac
aggatacatg cttgtaagta aaaaatgaaa aatgatgaag atgacaaaat 13920
agatgacaac ttcatactat tctaaattaa ttatttgatt atgcaattat atgatagtta
13980 attaaaatta aaaattaaaa atcaaaagtt aaaatttaaa acctatcatt
aagtttatta 14040 aaaataagaa attataattg aatgtatacg gtttttttgc cgt
14083 4 187 PRT human metapneumovirus 4 Met Ser Arg Lys Ala Pro Cys
Lys Tyr Glu Val Arg Gly Lys Cys Asn 1 5 10 15 Arg Gly Ser Glu Cys
Lys Phe Asn His Asn Tyr Trp Ser Trp Pro Asp 20 25 30 Arg Tyr Leu
Leu Ile Arg Ser Asn Tyr Leu Leu Asn Gln Leu Leu Arg 35 40 45 Asn
Thr Asp Arg Ala Asp Gly Leu Ser Ile Ile Ser Gly Ala Gly Arg 50 55
60 Glu Asp Arg Thr Gln Asp Phe Val Leu Gly Ser Thr Asn Val Val Gln
65 70 75 80 Gly Tyr Ile Asp Asp Asn Gln Ser Ile Thr Lys Ala Ala Ala
Cys Tyr 85 90 95 Ser Leu His Asn Ile Ile Lys Gln Leu Gln Glu Val
Glu Val Arg Gln 100 105 110 Ala Arg Asp Ser Lys Leu Ser Asp Ser Lys
His Val Ala Leu His Asn 115 120 125 Leu Ile Leu Ser Tyr Met Glu Met
Ser Lys Thr Pro Ala Ser Leu Ile 130 135 140 Asn Asn Leu Lys Arg Leu
Pro Arg Glu Lys Leu Lys Lys Leu Ala Lys 145 150 155 160 Leu Ile Ile
Asp Leu Ser Ala Gly Ala Asp Asn Asp Ser Ser Tyr Ala 165 170 175 Leu
Gln Asp Ser Glu Ser Ile Asn Gln Val Gln 180 185 5 179 PRT human
metapneumovirus 5 Met Ile Thr Leu Asp Val Ile Lys Ser Asp Gly Ser
Ser Lys Thr Cys 1 5 10 15 Thr His Leu Lys Lys Ile Ile Lys Asp His
Ser Gly Lys Val Leu Ile 20 25 30 Ala Leu Lys Leu Ile Leu Ala Leu
Leu Thr Phe Phe Thr Ile Thr Ile 35 40 45 Thr Ile Asn Tyr Ile Lys
Val Glu Asn Asn Leu Gln Ile Cys Gln Ser 50 55 60 Lys Thr Glu Ser
Asp Lys Glu Asp Ser Pro Ser Asn Thr Thr Ser Val 65 70 75 80 Thr Thr
Lys Thr Thr Leu Asp His Asp Ile Thr Gln Tyr Phe Lys Arg 85 90 95
Leu Ile Gln Arg Tyr Thr Asp Ser Val Ile Asn Lys Asp Thr Cys Trp 100
105 110 Lys Ile Ser Arg Asn Gln Cys Thr Asn Ile Thr Thr Tyr Lys Phe
Leu 115 120 125 Cys Phe Lys Pro Glu Asp Ser Lys Ile Asn Ser Cys Asp
Arg Leu Thr 130 135 140 Asp Leu Cys Arg Asn Lys Ser Lys Ser Ala Ala
Glu Ala Tyr His Thr 145 150 155 160 Val Glu Cys His Cys Ile Tyr Thr
Ile Glu Trp Lys Cys Tyr His His 165 170 175 Ser Ile Asp 6 219 PRT
human metapneumovirus 6 Met Glu Val Lys Val Glu Asn Ile Arg Ala Ile
Asp Met Leu Lys Ala 1 5 10 15 Arg Val Lys Asn Arg Val Ala Arg Ser
Lys Cys Phe Lys Asn Ala Ser 20 25 30 Leu Ile Leu Ile Gly Ile Thr
Thr Leu Ser Ile Ala Leu Asn Ile Tyr 35 40 45 Leu Ile Ile Asn Tyr
Thr Ile Gln Lys Thr Ser Ser Glu Ser Glu His 50 55 60 His Thr Ser
Ser Pro Pro Thr Glu Ser Asn Lys Glu Ala Ser Thr Ile 65 70 75 80 Ser
Thr Asp Asn Pro Asp Ile Asn Pro Asn Ser Gln His Pro Thr Gln 85 90
95 Gln Ser Thr Glu Asn Pro Thr Leu Asn Pro Ala Ala Ser Val Ser Pro
100 105 110 Ser Glu Thr Glu Pro Ala Ser Thr Pro Asp Thr Thr Asn Arg
Leu Ser 115 120 125 Ser Val Asp Arg Ser Thr Ala Gln Pro Ser Glu Ser
Arg Thr Lys Thr 130 135 140 Lys Pro Thr Val His Thr Arg Asn Asn Pro
Ser Thr Ala Ser Ser Thr 145 150 155 160 Gln Ser Pro Pro Arg Ala Thr
Thr Lys Ala Ile Arg Arg Ala Thr Thr 165 170 175 Phe Arg Met Ser Ser
Thr Gly Lys Arg Pro Thr Thr Thr Ser Val Gln 180 185 190 Ser Asp Ser
Ser Thr Thr Thr Gln Asn His Glu Glu Thr Gly Ser Ala 195 200 205 Asn
Pro Gln Ala Ser Val Ser Thr Met Gln Asn 210 215 7 2005 PRT human
metapneumovirus 7 Met Asp Pro Leu Asn Glu Ser Thr Val Asn Val Tyr
Leu Pro Asp Ser 1 5 10 15 Tyr Leu Lys Gly Val Ile Ser Phe Ser Glu
Thr Asn Ala Ile Gly Ser 20 25 30 Cys Leu Leu Lys Arg Pro Tyr Leu
Lys Asn Asp Asn Thr Ala Lys Val 35 40 45 Ala Ile Glu Asn Pro Val
Ile Glu His Val Arg Leu Lys Asn Ala Val 50 55 60 Asn Ser Lys Met
Lys Ile Ser Asp Tyr Lys Val Val Glu Pro Val Asn 65 70 75 80 Met Gln
His Glu Ile Met Lys Asn Val His
Ser Cys Glu Leu Thr Leu 85 90 95 Leu Lys Gln Phe Leu Thr Arg Ser
Lys Asn Ile Ser Thr Leu Lys Leu 100 105 110 Asn Met Ile Cys Asp Trp
Leu Gln Leu Lys Ser Thr Ser Asp Asp Thr 115 120 125 Ser Ile Leu Ser
Phe Ile Asp Val Glu Phe Ile Pro Ser Trp Val Ser 130 135 140 Asn Trp
Phe Ser Asn Trp Tyr Asn Leu Asn Lys Leu Ile Leu Glu Phe 145 150 155
160 Arg Arg Glu Glu Val Ile Arg Thr Gly Ser Ile Leu Cys Arg Ser Leu
165 170 175 Gly Lys Leu Val Phe Ile Val Ser Ser Tyr Gly Cys Ile Val
Lys Ser 180 185 190 Asn Lys Ser Lys Arg Val Ser Phe Phe Thr Tyr Asn
Gln Leu Leu Thr 195 200 205 Trp Lys Asp Val Met Leu Ser Arg Phe Asn
Ala Asn Phe Cys Ile Trp 210 215 220 Val Ser Asn Ser Leu Asn Glu Asn
Gln Glu Gly Leu Gly Leu Arg Ser 225 230 235 240 Asn Leu Gln Gly Met
Leu Thr Asn Lys Leu Tyr Glu Thr Val Asp Tyr 245 250 255 Met Leu Ser
Leu Cys Cys Asn Glu Gly Phe Ser Leu Val Lys Glu Phe 260 265 270 Glu
Gly Phe Ile Met Ser Glu Ile Leu Arg Ile Thr Glu His Ala Gln 275 280
285 Phe Ser Thr Arg Phe Arg Asn Thr Leu Leu Asn Gly Leu Thr Asp Gln
290 295 300 Leu Thr Lys Leu Lys Asn Lys Asn Arg Leu Arg Val His Ser
Thr Val 305 310 315 320 Leu Glu Asn Asn Asp Tyr Pro Met Tyr Glu Val
Val Leu Lys Leu Leu 325 330 335 Gly Asp Thr Leu Arg Cys Ile Lys Leu
Leu Ile Asn Lys Asn Leu Glu 340 345 350 Asn Ala Ala Glu Leu Tyr Tyr
Ile Phe Arg Ile Phe Gly His Pro Met 355 360 365 Val Asp Glu Arg Asp
Ala Met Asp Ala Val Lys Leu Asn Asn Glu Ile 370 375 380 Thr Lys Ile
Leu Arg Leu Glu Ser Leu Thr Glu Leu Arg Gly Ala Phe 385 390 395 400
Ile Leu Arg Ile Ile Lys Gly Phe Val Asp Asn Asn Lys Arg Trp Pro 405
410 415 Lys Ile Lys Asn Leu Lys Val Leu Ser Lys Arg Trp Thr Met Tyr
Phe 420 425 430 Lys Ala Lys Asn Tyr Pro Ser Gln Leu Glu Leu Ser Glu
Gln Asp Phe 435 440 445 Leu Glu Leu Ala Ala Ile Gln Phe Glu Gln Glu
Phe Ser Val Pro Glu 450 455 460 Lys Thr Asn Leu Glu Met Val Leu Asn
Asp Lys Ala Ile Ser Pro Pro 465 470 475 480 Lys Arg Leu Ile Trp Ser
Val Tyr Pro Lys Asn Tyr Leu Pro Glu Thr 485 490 495 Ile Lys Asn Arg
Tyr Leu Glu Glu Thr Phe Asn Ala Ser Asp Ser Leu 500 505 510 Lys Thr
Arg Arg Val Leu Glu Tyr Tyr Leu Lys Asp Asn Lys Phe Asp 515 520 525
Gln Lys Glu Leu Lys Ser Tyr Val Val Arg Gln Glu Tyr Leu Asn Asp 530
535 540 Lys Glu His Ile Val Ser Leu Thr Gly Lys Glu Arg Glu Leu Ser
Val 545 550 555 560 Gly Arg Met Phe Ala Met Gln Pro Gly Lys Gln Arg
Gln Ile Gln Ile 565 570 575 Leu Ala Glu Lys Leu Leu Ala Asp Asn Ile
Val Pro Phe Phe Pro Glu 580 585 590 Thr Leu Thr Lys Tyr Gly Asp Leu
Asp Leu Gln Arg Ile Met Glu Ile 595 600 605 Lys Ser Glu Leu Ser Ser
Ile Lys Thr Arg Arg Asn Asp Ser Tyr Asn 610 615 620 Asn Tyr Ile Ala
Arg Ala Ser Ile Val Thr Asp Leu Ser Lys Phe Asn 625 630 635 640 Gln
Ala Phe Arg Tyr Glu Thr Thr Ala Ile Cys Ala Asp Val Ala Asp 645 650
655 Glu Leu His Gly Thr Gln Ser Leu Phe Cys Trp Leu His Leu Ile Val
660 665 670 Pro Met Thr Thr Met Ile Cys Ala Tyr Arg His Ala Pro Pro
Glu Thr 675 680 685 Lys Gly Glu Tyr Asp Ile Asp Lys Ile Glu Glu Gln
Ser Gly Leu Tyr 690 695 700 Arg Tyr His Met Gly Gly Ile Glu Gly Trp
Cys Gln Lys Leu Trp Thr 705 710 715 720 Met Glu Ala Ile Ser Leu Leu
Asp Val Val Ser Val Lys Thr Arg Cys 725 730 735 Gln Met Thr Ser Leu
Leu Asn Gly Asp Asn Gln Ser Ile Asp Val Ser 740 745 750 Lys Pro Val
Lys Leu Ser Glu Gly Leu Asp Glu Val Lys Ala Asp Tyr 755 760 765 Arg
Leu Ala Val Lys Met Leu Lys Glu Ile Arg Asp Ala Tyr Arg Asn 770 775
780 Ile Gly His Lys Leu Lys Glu Gly Glu Thr Tyr Ile Ser Arg Asp Leu
785 790 795 800 Gln Phe Ile Ser Lys Val Ile Gln Ser Glu Gly Val Met
His Pro Thr 805 810 815 Pro Ile Lys Lys Val Leu Arg Val Gly Pro Trp
Ile Asn Thr Ile Leu 820 825 830 Asp Asp Ile Lys Thr Ser Ala Glu Ser
Ile Gly Ser Leu Cys Gln Glu 835 840 845 Leu Glu Phe Arg Gly Glu Ser
Ile Ile Val Ser Leu Ile Leu Arg Asn 850 855 860 Phe Trp Leu Tyr Asn
Leu Tyr Met His Glu Ser Lys Gln His Pro Leu 865 870 875 880 Ala Gly
Lys Gln Leu Phe Lys Gln Leu Asn Lys Thr Leu Thr Ser Val 885 890 895
Gln Arg Phe Phe Glu Ile Lys Arg Glu Asn Glu Val Val Asp Leu Trp 900
905 910 Met Asn Ile Pro Met Gln Phe Gly Gly Gly Asp Pro Val Val Phe
Tyr 915 920 925 Arg Ser Phe Tyr Arg Arg Thr Pro Asp Phe Leu Thr Glu
Ala Ile Ser 930 935 940 His Val Asp Ile Leu Leu Lys Ile Ser Ala Asn
Ile Lys Asn Glu Thr 945 950 955 960 Lys Val Ser Phe Phe Lys Ala Leu
Leu Ser Ile Glu Lys Asn Glu Arg 965 970 975 Ala Thr Leu Thr Thr Leu
Met Arg Asp Pro Gln Ala Val Gly Ser Glu 980 985 990 Arg Gln Ala Lys
Val Thr Ser Asp Ile Asn Arg Thr Ala Val Thr Ser 995 1000 1005 Ile
Leu Ser Leu Ser Pro Asn Gln Leu Phe Ser Asp Ser Ala Ile 1010 1015
1020 His Tyr Ser Arg Asn Glu Glu Glu Val Gly Ile Ile Ala Glu Asn
1025 1030 1035 Ile Thr Pro Val Tyr Pro His Gly Leu Arg Val Leu Tyr
Glu Ser 1040 1045 1050 Leu Pro Phe His Lys Ala Glu Lys Val Val Asn
Met Ile Ser Gly 1055 1060 1065 Thr Lys Ser Ile Thr Asn Leu Leu Gln
Arg Thr Ser Ala Ile Asn 1070 1075 1080 Gly Glu Asp Ile Asp Arg Ala
Val Ser Met Met Leu Glu Asn Leu 1085 1090 1095 Gly Leu Leu Ser Arg
Ile Leu Ser Val Val Val Asp Ser Ile Glu 1100 1105 1110 Ile Pro Ile
Lys Ser Asn Gly Arg Leu Ile Cys Cys Gln Ile Ser 1115 1120 1125 Arg
Thr Leu Arg Glu Thr Ser Trp Asn Asn Met Glu Ile Val Gly 1130 1135
1140 Val Thr Ser Pro Ser Ile Thr Thr Cys Met Asp Val Ile Tyr Ala
1145 1150 1155 Thr Ser Ser His Leu Lys Gly Ile Ile Ile Glu Lys Phe
Ser Thr 1160 1165 1170 Asp Arg Thr Thr Arg Gly Gln Arg Gly Pro Lys
Ser Pro Trp Val 1175 1180 1185 Gly Ser Ser Thr Gln Glu Lys Lys Leu
Val Pro Val Tyr Asn Arg 1190 1195 1200 Gln Ile Leu Ser Lys Gln Gln
Arg Glu Gln Leu Glu Ala Ile Gly 1205 1210 1215 Lys Met Arg Trp Val
Tyr Lys Gly Thr Pro Gly Leu Arg Arg Leu 1220 1225 1230 Leu Asn Lys
Ile Cys Leu Gly Ser Leu Gly Ile Ser Tyr Lys Cys 1235 1240 1245 Val
Lys Pro Leu Leu Pro Arg Phe Met Ser Val Asn Phe Leu His 1250 1255
1260 Arg Leu Ser Val Ser Ser Arg Pro Met Glu Phe Pro Ala Ser Val
1265 1270 1275 Pro Ala Tyr Arg Thr Thr Asn Tyr His Phe Asp Thr Ser
Pro Ile 1280 1285 1290 Asn Gln Ala Leu Ser Glu Arg Phe Gly Asn Glu
Asp Ile Asn Leu 1295 1300 1305 Val Phe Gln Asn Ala Ile Ser Cys Gly
Ile Ser Ile Met Ser Val 1310 1315 1320 Val Glu Gln Leu Thr Gly Arg
Ser Pro Lys Gln Leu Val Leu Ile 1325 1330 1335 Pro Gln Leu Glu Glu
Ile Asp Ile Met Pro Pro Pro Val Phe Gln 1340 1345 1350 Gly Lys Phe
Asn Tyr Lys Leu Val Asp Lys Ile Thr Ser Asp Gln 1355 1360 1365 His
Ile Phe Ser Pro Asp Lys Ile Asp Met Leu Thr Leu Gly Lys 1370 1375
1380 Met Leu Met Pro Thr Ile Lys Gly Gln Lys Thr Asp Gln Phe Leu
1385 1390 1395 Asn Lys Arg Glu Asn Tyr Phe His Gly Asn Asn Leu Ile
Glu Ser 1400 1405 1410 Leu Ser Ala Ala Leu Ala Cys His Trp Cys Gly
Ile Leu Thr Glu 1415 1420 1425 Gln Cys Ile Glu Asn Asn Ile Phe Lys
Lys Asp Trp Gly Asp Gly 1430 1435 1440 Phe Ile Ser Asp His Ala Phe
Met Asp Phe Lys Ile Phe Leu Cys 1445 1450 1455 Val Phe Lys Thr Lys
Leu Leu Cys Ser Trp Gly Ser Gln Gly Lys 1460 1465 1470 Asn Ile Lys
Asp Glu Asp Ile Val Asp Glu Ser Ile Asp Lys Leu 1475 1480 1485 Leu
Arg Ile Asp Asn Thr Phe Trp Arg Met Phe Ser Lys Val Met 1490 1495
1500 Phe Glu Pro Lys Val Lys Lys Arg Ile Met Leu Tyr Asp Val Lys
1505 1510 1515 Phe Leu Ser Leu Val Gly Tyr Ile Gly Phe Lys Asn Trp
Phe Ile 1520 1525 1530 Glu Gln Leu Arg Ser Ala Glu Leu His Glu Ile
Pro Trp Ile Val 1535 1540 1545 Asn Ala Glu Gly Asp Leu Val Glu Ile
Lys Ser Ile Lys Ile Tyr 1550 1555 1560 Leu Gln Leu Ile Glu Gln Ser
Leu Phe Leu Arg Ile Thr Val Leu 1565 1570 1575 Asn Tyr Thr Asp Met
Ala His Ala Leu Thr Arg Leu Ile Arg Lys 1580 1585 1590 Lys Leu Met
Cys Asp Asn Ala Leu Leu Thr Pro Ile Ser Ser Pro 1595 1600 1605 Met
Val Asn Leu Thr Gln Val Ile Asp Pro Thr Thr Gln Leu Asp 1610 1615
1620 Tyr Phe Pro Lys Ile Thr Phe Glu Arg Leu Lys Asn Tyr Asp Thr
1625 1630 1635 Ser Ser Asn Tyr Ala Lys Gly Lys Leu Thr Arg Asn Tyr
Met Ile 1640 1645 1650 Leu Leu Pro Trp Gln His Val Asn Arg Tyr Asn
Phe Val Phe Ser 1655 1660 1665 Ser Thr Gly Cys Lys Val Ser Leu Lys
Thr Cys Ile Gly Lys Leu 1670 1675 1680 Met Lys Asp Leu Asn Pro Lys
Val Leu Tyr Phe Ile Gly Glu Gly 1685 1690 1695 Ala Gly Asn Trp Met
Ala Arg Thr Ala Cys Glu Tyr Pro Asp Ile 1700 1705 1710 Lys Phe Val
Tyr Arg Ser Leu Lys Asp Asp Leu Asp His His Tyr 1715 1720 1725 Pro
Leu Glu Tyr Gln Arg Val Ile Gly Glu Leu Ser Arg Ile Ile 1730 1735
1740 Asp Ser Gly Glu Gly Leu Ser Met Glu Thr Thr Asp Ala Thr Gln
1745 1750 1755 Lys Thr His Trp Asp Leu Ile His Arg Val Ser Lys Asp
Ala Leu 1760 1765 1770 Leu Ile Thr Leu Cys Asp Ala Glu Phe Lys Asp
Arg Asp Asp Phe 1775 1780 1785 Phe Lys Met Val Ile Leu Trp Arg Lys
His Val Leu Ser Cys Arg 1790 1795 1800 Ile Cys Thr Thr Tyr Gly Thr
Asp Leu Tyr Leu Phe Ala Lys Tyr 1805 1810 1815 His Ala Lys Asp Cys
Asn Val Lys Leu Pro Phe Phe Val Arg Ser 1820 1825 1830 Val Ala Thr
Phe Ile Met Gln Gly Ser Lys Leu Ser Gly Ser Glu 1835 1840 1845 Cys
Tyr Ile Leu Leu Thr Leu Gly His His Asn Ser Leu Pro Cys 1850 1855
1860 His Gly Glu Ile Gln Asn Ser Lys Met Lys Ile Ala Val Cys Asn
1865 1870 1875 Asp Phe Tyr Ala Ala Lys Lys Leu Asp Asn Lys Ser Ile
Glu Ala 1880 1885 1890 Asn Cys Lys Ser Leu Leu Ser Gly Leu Arg Ile
Pro Ile Asn Lys 1895 1900 1905 Lys Glu Leu Asp Arg Gln Arg Arg Leu
Leu Thr Leu Gln Ser Asn 1910 1915 1920 His Ser Ser Val Ala Thr Val
Gly Gly Ser Lys Ile Ile Glu Ser 1925 1930 1935 Lys Trp Leu Thr Asn
Lys Ala Ser Thr Ile Ile Asp Trp Leu Glu 1940 1945 1950 His Ile Leu
Asn Ser Pro Lys Gly Glu Leu Asn Tyr Asp Phe Phe 1955 1960 1965 Glu
Ala Leu Glu Asn Thr Tyr Pro Asn Met Ile Lys Leu Ile Asp 1970 1975
1980 Asn Leu Gly Asn Ala Glu Ile Lys Lys Leu Ile Lys Val Thr Gly
1985 1990 1995 Tyr Met Leu Val Ser Lys Lys 2000 2005 8 177 PRT
human metapneumovirus 8 Met Lys Thr Leu Asp Val Ile Lys Ser Asp Gly
Ser Ser Glu Thr Cys 1 5 10 15 Asn Gln Leu Lys Lys Ile Ile Lys Lys
His Ser Gly Lys Leu Leu Ile 20 25 30 Ala Ser Lys Pro Thr Leu Ala
Leu Leu Thr Ser Phe Thr Val Thr Ile 35 40 45 Thr Val Asn Tyr Thr
Lys Val Glu Asn Asn Leu Gln Ala Cys Gln Leu 50 55 60 Lys Asn Glu
Ser Asp Lys Lys Asp Thr Lys Leu Asn Thr Thr Ser Thr 65 70 75 80 Thr
Ile Arg Pro Ile Pro Asp Leu Asn Ala Val Gln Tyr Leu Lys Arg 85 90
95 Leu Ile Gln Lys His Thr Asn Ser Val Thr Lys Asp Arg Asp Thr Cys
100 105 110 Trp Arg Ile His Thr Asn Gln Cys Thr Asn Ile Lys Ile Tyr
Lys Phe 115 120 125 Leu Cys Phe Gly Ser Met Asn Ser Thr Asn Thr Asp
Cys Glu Glu Pro 130 135 140 Thr Val Leu Cys Asp Lys Lys Ser Lys Thr
Met Thr Glu Lys His Arg 145 150 155 160 Lys Ala Glu Cys His Arg Pro
His Thr Thr Glu Trp Trp Cys His Tyr 165 170 175 Leu 9 236 PRT human
metapneumovirus 9 Met Glu Ala Arg Val Glu Asn Ile Arg Ala Ile Asp
Met Phe Lys Ala 1 5 10 15 Lys Met Lys Asn Arg Ile Arg Ser Ser Lys
Cys His Arg Asn Ala Thr 20 25 30 Leu Ile Leu Ile Gly Ser Thr Ala
Pro Ser Met Ala Leu Asn Thr Leu 35 40 45 Leu Ile Ile Asp His Ala
Thr Ser Lys Asn Met Thr Lys Val Glu His 50 55 60 Cys Val Asn Met
Pro Pro Val Glu Pro Ser Lys Lys Thr Pro Met Thr 65 70 75 80 Ser Ala
Ala Asp Pro Asn Thr Lys Pro Asn Pro Gln Gln Ala Thr Gln 85 90 95
Leu Thr Thr Glu Asp Ser Thr Ser Leu Ala Ala Thr Leu Glu Asp His 100
105 110 Leu His Thr Gly Thr Thr Pro Thr Pro Asp Ala Thr Val Ser Gln
Gln 115 120 125 Thr Thr Asp Glu His Thr Thr Leu Leu Arg Ser Thr Asn
Arg Gln Thr 130 135 140 Thr Gln Thr Thr Ala Glu Lys Lys Pro Thr Arg
Ala Thr Thr Lys Lys 145 150 155 160 Glu Thr Thr Thr Arg Thr Thr Ser
Thr Ala Ala Thr Gln Thr Leu Asn 165 170 175 Thr Thr Asn Gln Thr Ser
Asn Gly Arg Glu Ala Thr Thr Thr Ser Ala 180 185 190 Arg Ser Arg Asn
Asn Ala Thr Thr Gln Ser Ser Asp Gln Thr Thr Gln 195 200 205 Ala Ala
Asp Pro Ser Ser Gln Ser Gln His Thr Gln Lys Ser Thr Thr 210 215 220
Thr Thr His Asn Thr Asp Thr Ser Ser Pro Ser Ser 225 230 235 10 183
PRT human metapneumovirus 10 Met Ile Thr Leu Asp Val Ile Lys Ser
Asp Gly Ser Ser Lys Thr Cys 1 5 10 15 Thr His Leu Lys Lys Ile Ile
Lys Asp His Ser Gly Lys Val Leu Ile 20 25 30 Val Leu Lys Leu Ile
Leu Ala Leu Leu Thr Phe Leu Thr Val Thr Ile 35 40 45 Thr Ile Asn
Tyr Ile Lys Val Glu Asn Asn Leu Gln Ile Cys Gln Ser 50 55 60 Lys
Thr Glu Ser Asp Lys Lys Asp Ser Ser Ser Asn Thr Thr Ser Val 65 70
75
80 Thr Thr Lys Thr Thr Leu Asn His Asp Ile Thr Gln Tyr Phe Lys Ser
85 90 95 Leu Ile Gln Arg Tyr Thr Asn Ser Ala Ile Asn Ser Asp Thr
Cys Trp 100 105 110 Lys Ile Asn Arg Asn Gln Cys Thr Asn Ile Thr Thr
Tyr Lys Phe Leu 115 120 125 Cys Phe Lys Ser Glu Asp Thr Lys Thr Asn
Asn Cys Asp Lys Leu Thr 130 135 140 Asp Leu Cys Arg Asn Lys Pro Lys
Pro Ala Val Gly Val Tyr His Ile 145 150 155 160 Val Glu Cys His Cys
Ile Tyr Thr Val Lys Trp Lys Cys Tyr His Tyr 165 170 175 Pro Thr Asp
Glu Thr Gln Ser 180 11 236 PRT human metapneumovirus 11 Met Glu Val
Lys Val Glu Asn Ile Arg Thr Ile Asp Met Leu Lys Ala 1 5 10 15 Arg
Val Lys Asn Arg Val Ala Arg Ser Lys Cys Phe Lys Asn Ala Ser 20 25
30 Leu Val Leu Ile Gly Ile Thr Thr Leu Ser Ile Ala Leu Asn Ile Tyr
35 40 45 Leu Ile Ile Asn Tyr Lys Met Gln Lys Asn Thr Ser Glu Ser
Glu His 50 55 60 His Thr Ser Ser Ser Pro Met Glu Ser Ser Arg Glu
Thr Pro Thr Val 65 70 75 80 Pro Thr Asp Asn Ser Asp Thr Asn Ser Ser
Pro Gln His Pro Thr Gln 85 90 95 Gln Ser Thr Glu Gly Ser Thr Leu
Tyr Phe Ala Ala Ser Ala Ser Ser 100 105 110 Pro Glu Thr Glu Pro Thr
Ser Thr Pro Asp Thr Thr Asn Arg Pro Pro 115 120 125 Phe Val Asp Thr
His Thr Thr Pro Pro Ser Ala Ser Arg Thr Lys Thr 130 135 140 Ser Pro
Ala Val His Thr Lys Asn Asn Pro Arg Thr Ser Ser Arg Thr 145 150 155
160 His Ser Pro Pro Arg Ala Thr Thr Arg Thr Ala Arg Arg Thr Thr Thr
165 170 175 Leu Arg Thr Ser Ser Thr Arg Lys Arg Pro Ser Thr Ala Ser
Val Gln 180 185 190 Pro Asp Ile Ser Ala Thr Thr His Lys Asn Glu Glu
Ala Ser Pro Ala 195 200 205 Ser Pro Gln Thr Ser Ala Ser Thr Thr Arg
Ile Gln Arg Lys Ser Val 210 215 220 Glu Ala Asn Thr Ser Thr Thr Tyr
Asn Gln Thr Ser 225 230 235 12 2005 PRT human metapneumovirus 12
Met Asp Pro Leu Asn Glu Ser Thr Val Asn Val Tyr Leu Pro Asp Ser 1 5
10 15 Tyr Leu Lys Gly Val Ile Ser Phe Ser Glu Thr Asn Ala Ile Gly
Ser 20 25 30 Cys Leu Leu Lys Arg Pro Tyr Leu Lys Asn Asp Asn Thr
Ala Lys Val 35 40 45 Ala Ile Glu Asn Pro Val Ile Glu His Val Arg
Leu Lys Asn Ala Val 50 55 60 Asn Ser Lys Met Lys Ile Ser Asp Tyr
Lys Ile Val Glu Pro Val Asn 65 70 75 80 Met Gln His Glu Ile Met Lys
Asn Val His Ser Cys Glu Leu Thr Leu 85 90 95 Leu Lys Gln Phe Leu
Thr Arg Ser Lys Asn Ile Ser Thr Leu Lys Leu 100 105 110 Asn Met Ile
Cys Asp Trp Leu Gln Leu Lys Ser Thr Ser Asp Asp Thr 115 120 125 Ser
Ile Leu Ser Phe Ile Asp Val Glu Phe Ile Pro Ser Trp Val Ser 130 135
140 Asn Trp Phe Ser Asn Trp Tyr Asn Leu Asn Lys Leu Ile Leu Glu Phe
145 150 155 160 Arg Lys Glu Glu Val Ile Arg Thr Gly Ser Ile Leu Cys
Arg Ser Leu 165 170 175 Gly Lys Leu Val Phe Val Val Ser Ser Tyr Gly
Cys Ile Val Lys Ser 180 185 190 Asn Lys Ser Lys Arg Val Ser Phe Phe
Thr Tyr Asn Gln Leu Leu Thr 195 200 205 Trp Lys Asp Val Met Leu Ser
Arg Phe Asn Ala Asn Phe Cys Ile Trp 210 215 220 Val Ser Asn Ser Leu
Asn Glu Asn Gln Glu Gly Leu Gly Leu Arg Ser 225 230 235 240 Asn Leu
Gln Gly Ile Leu Thr Asn Lys Leu Tyr Glu Thr Val Asp Tyr 245 250 255
Met Leu Ser Leu Cys Cys Asn Glu Gly Phe Ser Leu Val Lys Glu Phe 260
265 270 Glu Gly Phe Ile Met Ser Glu Ile Leu Arg Ile Thr Glu His Ala
Gln 275 280 285 Phe Ser Thr Arg Phe Arg Asn Thr Leu Leu Asn Gly Leu
Thr Asp Gln 290 295 300 Leu Thr Lys Leu Lys Asn Lys Asn Arg Leu Arg
Val His Gly Thr Val 305 310 315 320 Leu Glu Asn Asn Asp Tyr Pro Met
Tyr Glu Val Val Leu Lys Leu Leu 325 330 335 Gly Asp Thr Leu Arg Cys
Ile Lys Leu Leu Ile Asn Lys Asn Leu Glu 340 345 350 Asn Ala Ala Glu
Leu Tyr Tyr Ile Phe Arg Ile Phe Gly His Pro Met 355 360 365 Val Asp
Glu Arg Asp Ala Met Asp Ala Val Lys Leu Asn Asn Glu Ile 370 375 380
Thr Lys Ile Leu Arg Trp Glu Ser Leu Thr Glu Leu Arg Gly Ala Phe 385
390 395 400 Ile Leu Arg Ile Ile Lys Gly Phe Val Asp Asn Asn Lys Arg
Trp Pro 405 410 415 Lys Ile Lys Asn Leu Lys Val Leu Ser Lys Arg Trp
Thr Met Tyr Phe 420 425 430 Lys Ala Lys Ser Tyr Pro Ser Gln Leu Glu
Leu Ser Glu Gln Asp Phe 435 440 445 Leu Glu Leu Ala Ala Ile Gln Phe
Glu Gln Glu Phe Ser Val Pro Glu 450 455 460 Lys Thr Asn Leu Glu Met
Val Leu Asn Asp Lys Ala Ile Ser Pro Pro 465 470 475 480 Lys Arg Leu
Ile Trp Ser Val Tyr Pro Lys Asn Tyr Leu Pro Glu Lys 485 490 495 Ile
Lys Asn Arg Tyr Leu Glu Glu Thr Phe Asn Ala Ser Asp Ser Leu 500 505
510 Lys Thr Arg Arg Val Leu Glu Tyr Tyr Leu Lys Asp Asn Lys Phe Asp
515 520 525 Gln Lys Glu Leu Lys Ser Tyr Val Val Lys Gln Glu Tyr Leu
Asn Asp 530 535 540 Lys Asp His Ile Val Ser Leu Thr Gly Lys Glu Arg
Glu Leu Ser Val 545 550 555 560 Gly Arg Met Phe Ala Met Gln Pro Gly
Lys Gln Arg Gln Ile Gln Ile 565 570 575 Leu Ala Glu Lys Leu Leu Ala
Asp Asn Ile Val Pro Phe Phe Pro Glu 580 585 590 Thr Leu Thr Lys Tyr
Gly Asp Leu Asp Leu Gln Arg Ile Met Glu Ile 595 600 605 Lys Ser Glu
Leu Ser Ser Ile Lys Thr Arg Arg Asn Asp Ser Tyr Asn 610 615 620 Asn
Tyr Ile Ala Arg Ala Ser Ile Val Thr Asp Leu Ser Lys Phe Asn 625 630
635 640 Gln Ala Phe Arg Tyr Glu Thr Thr Ala Ile Cys Ala Asp Val Ala
Asp 645 650 655 Glu Leu His Gly Thr Gln Ser Leu Phe Cys Trp Leu His
Leu Ile Val 660 665 670 Pro Met Thr Thr Met Ile Cys Ala Tyr Arg His
Ala Pro Pro Glu Thr 675 680 685 Lys Gly Glu Tyr Asp Ile Asp Lys Ile
Glu Glu Gln Ser Gly Leu Tyr 690 695 700 Arg Tyr His Met Gly Gly Ile
Glu Gly Trp Cys Gln Lys Leu Trp Thr 705 710 715 720 Met Glu Ala Ile
Ser Leu Leu Asp Val Val Ser Val Lys Thr Arg Cys 725 730 735 Gln Met
Thr Ser Leu Leu Asn Gly Asp Asn Gln Ser Ile Asp Val Ser 740 745 750
Lys Pro Val Lys Leu Ser Glu Gly Leu Asp Glu Val Lys Ala Asp Tyr 755
760 765 Ser Leu Ala Val Lys Met Leu Lys Glu Ile Arg Asp Ala Tyr Arg
Asn 770 775 780 Ile Gly His Lys Leu Lys Glu Gly Glu Thr Tyr Ile Ser
Arg Asp Leu 785 790 795 800 Gln Phe Ile Ser Lys Val Ile Gln Ser Glu
Gly Val Met His Pro Thr 805 810 815 Pro Ile Lys Lys Ile Leu Arg Val
Gly Pro Trp Ile Asn Thr Ile Leu 820 825 830 Asp Asp Ile Lys Thr Ser
Ala Glu Ser Ile Gly Ser Leu Cys Gln Glu 835 840 845 Leu Glu Phe Arg
Gly Glu Ser Ile Ile Val Ser Leu Ile Leu Arg Asn 850 855 860 Phe Trp
Leu Tyr Asn Leu Tyr Met His Glu Ser Lys Gln His Pro Leu 865 870 875
880 Ala Gly Lys Gln Leu Phe Lys Gln Leu Asn Lys Thr Leu Thr Ser Val
885 890 895 Gln Arg Phe Phe Glu Ile Lys Lys Glu Asn Glu Val Val Asp
Leu Trp 900 905 910 Met Asn Ile Pro Met Gln Phe Gly Gly Gly Asp Pro
Val Val Phe Tyr 915 920 925 Arg Ser Phe Tyr Arg Arg Thr Pro Asp Phe
Leu Thr Glu Ala Ile Ser 930 935 940 His Val Asp Ile Leu Leu Arg Ile
Ser Ala Asn Ile Arg Asn Glu Ala 945 950 955 960 Lys Ile Ser Phe Phe
Lys Ala Leu Leu Ser Ile Glu Lys Asn Glu Arg 965 970 975 Ala Thr Leu
Thr Thr Leu Met Arg Asp Pro Gln Ala Val Gly Ser Glu 980 985 990 Arg
Gln Ala Lys Val Thr Ser Asp Ile Asn Arg Thr Ala Val Thr Ser 995
1000 1005 Ile Leu Ser Leu Ser Pro Asn Gln Leu Phe Ser Asp Ser Ala
Ile 1010 1015 1020 His Tyr Ser Arg Asn Glu Glu Glu Val Gly Ile Ile
Ala Asp Asn 1025 1030 1035 Ile Thr Pro Val Tyr Pro His Gly Leu Arg
Val Leu Tyr Glu Ser 1040 1045 1050 Leu Pro Phe His Lys Ala Glu Lys
Val Val Asn Met Ile Ser Gly 1055 1060 1065 Thr Lys Ser Ile Thr Asn
Leu Leu Gln Arg Thr Ser Ala Ile Asn 1070 1075 1080 Gly Glu Asp Ile
Asp Arg Ala Val Ser Met Met Leu Glu Asn Leu 1085 1090 1095 Gly Leu
Leu Ser Arg Ile Leu Ser Val Val Val Asp Ser Ile Glu 1100 1105 1110
Ile Pro Thr Lys Ser Asn Gly Arg Leu Ile Cys Cys Gln Ile Ser 1115
1120 1125 Arg Thr Leu Arg Glu Thr Ser Trp Asn Asn Met Glu Ile Val
Gly 1130 1135 1140 Val Thr Ser Pro Ser Ile Thr Thr Cys Met Asp Val
Ile Tyr Ala 1145 1150 1155 Thr Ser Ser His Leu Lys Gly Ile Ile Ile
Glu Lys Phe Ser Thr 1160 1165 1170 Asp Arg Thr Thr Arg Gly Gln Arg
Gly Pro Lys Ser Pro Trp Val 1175 1180 1185 Gly Ser Ser Thr Gln Glu
Lys Lys Leu Val Pro Val Tyr Asn Arg 1190 1195 1200 Gln Ile Leu Ser
Lys Gln Gln Arg Glu Gln Leu Glu Ala Ile Gly 1205 1210 1215 Lys Met
Arg Trp Val Tyr Lys Gly Thr Pro Gly Leu Arg Arg Leu 1220 1225 1230
Leu Asn Lys Ile Cys Leu Gly Ser Leu Gly Ile Ser Tyr Lys Cys 1235
1240 1245 Val Lys Pro Leu Leu Pro Arg Phe Met Ser Val Asn Phe Leu
His 1250 1255 1260 Arg Leu Ser Val Ser Ser Arg Pro Met Glu Phe Pro
Ala Ser Val 1265 1270 1275 Pro Ala Tyr Arg Thr Thr Asn Tyr His Phe
Asp Thr Ser Pro Ile 1280 1285 1290 Asn Gln Ala Leu Ser Glu Arg Phe
Gly Asn Glu Asp Ile Asn Leu 1295 1300 1305 Val Phe Gln Asn Ala Ile
Ser Cys Gly Ile Ser Ile Met Ser Val 1310 1315 1320 Val Glu Gln Leu
Thr Gly Arg Ser Pro Lys Gln Leu Val Leu Ile 1325 1330 1335 Pro Gln
Leu Glu Glu Ile Asp Ile Met Pro Pro Pro Val Phe Gln 1340 1345 1350
Gly Lys Phe Asn Tyr Lys Leu Val Asp Lys Ile Thr Ser Asp Gln 1355
1360 1365 His Ile Phe Ser Pro Asp Lys Ile Asp Met Leu Thr Leu Gly
Lys 1370 1375 1380 Met Leu Met Pro Thr Ile Lys Gly Gln Lys Thr Asp
Gln Phe Leu 1385 1390 1395 Asn Lys Arg Glu Asn Tyr Phe His Gly Asn
Asn Leu Ile Glu Ser 1400 1405 1410 Leu Ser Ala Ala Leu Ala Cys His
Trp Cys Gly Ile Leu Thr Glu 1415 1420 1425 Gln Cys Ile Glu Asn Asn
Ile Phe Lys Lys Asp Trp Gly Asp Gly 1430 1435 1440 Phe Ile Ser Asp
His Ala Phe Met Asp Phe Lys Ile Phe Leu Cys 1445 1450 1455 Val Phe
Lys Thr Lys Leu Leu Cys Ser Trp Gly Ser Gln Gly Lys 1460 1465 1470
Asn Ile Lys Asp Glu Asp Ile Val Asp Glu Ser Ile Asp Lys Leu 1475
1480 1485 Leu Arg Ile Asp Asn Thr Phe Trp Arg Met Phe Ser Lys Val
Met 1490 1495 1500 Phe Glu Ser Lys Val Lys Lys Arg Ile Met Leu Tyr
Asp Val Lys 1505 1510 1515 Phe Leu Ser Leu Val Gly Tyr Ile Gly Phe
Lys Asn Trp Phe Ile 1520 1525 1530 Glu Gln Leu Arg Ser Ala Glu Leu
His Glu Val Pro Trp Ile Val 1535 1540 1545 Asn Ala Glu Gly Asp Leu
Val Glu Ile Lys Ser Ile Lys Ile Tyr 1550 1555 1560 Leu Gln Leu Ile
Glu Gln Ser Leu Phe Leu Arg Ile Thr Val Leu 1565 1570 1575 Asn Tyr
Thr Asp Met Ala His Ala Leu Thr Arg Leu Ile Arg Lys 1580 1585 1590
Lys Leu Met Cys Asp Asn Ala Leu Leu Thr Pro Ile Pro Ser Pro 1595
1600 1605 Met Val Asn Leu Thr Gln Val Ile Asp Pro Thr Glu Gln Leu
Ala 1610 1615 1620 Tyr Phe Pro Lys Ile Thr Phe Glu Arg Leu Lys Asn
Tyr Asp Thr 1625 1630 1635 Ser Ser Asn Tyr Ala Lys Gly Lys Leu Thr
Arg Asn Tyr Met Ile 1640 1645 1650 Leu Leu Pro Trp Gln His Val Asn
Arg Tyr Asn Phe Val Phe Ser 1655 1660 1665 Ser Thr Gly Cys Lys Val
Ser Leu Lys Thr Cys Ile Gly Lys Leu 1670 1675 1680 Met Lys Asp Leu
Asn Pro Lys Val Leu Tyr Phe Ile Gly Glu Gly 1685 1690 1695 Ala Gly
Asn Trp Met Ala Arg Thr Ala Cys Glu Tyr Pro Asp Ile 1700 1705 1710
Lys Phe Val Tyr Arg Ser Leu Lys Asp Asp Leu Asp His His Tyr 1715
1720 1725 Pro Leu Glu Tyr Gln Arg Val Ile Gly Glu Leu Ser Arg Ile
Ile 1730 1735 1740 Asp Ser Gly Glu Gly Leu Ser Met Glu Thr Thr Asp
Ala Thr Gln 1745 1750 1755 Lys Thr His Trp Asp Leu Ile His Arg Val
Ser Lys Asp Ala Leu 1760 1765 1770 Leu Ile Thr Leu Cys Asp Ala Glu
Phe Lys Asp Arg Asp Asp Phe 1775 1780 1785 Phe Lys Met Val Ile Leu
Trp Arg Lys His Val Leu Ser Cys Arg 1790 1795 1800 Ile Cys Thr Thr
Tyr Gly Thr Asp Leu Tyr Leu Phe Ala Lys Tyr 1805 1810 1815 His Ala
Lys Asp Cys Asn Val Lys Leu Pro Phe Phe Val Arg Ser 1820 1825 1830
Val Ala Thr Phe Ile Met Gln Gly Ser Lys Leu Ser Gly Ser Glu 1835
1840 1845 Cys Tyr Ile Leu Leu Thr Leu Gly His His Asn Asn Leu Pro
Cys 1850 1855 1860 His Gly Glu Ile Gln Asn Ser Lys Met Lys Ile Ala
Val Cys Asn 1865 1870 1875 Asp Phe Tyr Ala Ala Lys Lys Leu Asp Asn
Lys Ser Ile Glu Ala 1880 1885 1890 Asn Cys Lys Ser Leu Leu Ser Gly
Leu Arg Ile Pro Ile Asn Lys 1895 1900 1905 Lys Glu Leu Asn Arg Gln
Arg Arg Leu Leu Thr Leu Gln Ser Asn 1910 1915 1920 His Ser Ser Val
Ala Thr Val Gly Gly Ser Lys Val Ile Glu Ser 1925 1930 1935 Lys Trp
Leu Thr Asn Lys Ala Asn Thr Ile Ile Asp Trp Leu Glu 1940 1945 1950
His Ile Leu Asn Ser Pro Lys Gly Glu Leu Asn Tyr Asp Phe Phe 1955
1960 1965 Glu Ala Leu Glu Asn Thr Tyr Pro Asn Met Ile Lys Leu Ile
Asp 1970 1975 1980 Asn Leu Gly Asn Ala Glu Ile Lys Lys Leu Ile Lys
Val Thr Gly 1985 1990 1995 Tyr Met Leu Val Ser Lys Lys 2000 2005 13
2165 PRT human respiratory syncytial virus strain A2 13 Met Asp Pro
Ile Ile Asn Gly Asn Ser Ala Asn Val Tyr Leu Thr Asp 1 5 10 15 Ser
Tyr Leu Lys Gly Val Ile Ser Phe Ser Glu Cys Asn Ala Leu Gly 20 25
30 Ser Tyr Ile Phe Asn Gly Pro Tyr Leu Lys Asn Asp Tyr Thr Asn Leu
35 40 45 Ile Ser Arg Gln Asn Pro Leu Ile Glu His Met Asn Leu Lys
Lys Leu 50 55 60 Asn Ile Thr Gln Ser Leu Ile Ser Lys
Tyr His Lys Gly Glu Ile Lys 65 70 75 80 Leu Glu Glu Pro Thr Tyr Phe
Gln Ser Leu Leu Met Thr Tyr Lys Ser 85 90 95 Met Thr Ser Ser Glu
Gln Ile Ala Thr Thr Asn Leu Leu Lys Lys Ile 100 105 110 Ile Arg Arg
Ala Ile Glu Ile Ser Asp Val Lys Val Tyr Ala Ile Leu 115 120 125 Asn
Lys Leu Gly Leu Lys Glu Lys Asp Lys Ile Lys Ser Asn Asn Gly 130 135
140 Gln Asp Glu Asp Asn Ser Val Ile Thr Thr Ile Ile Lys Asp Asp Ile
145 150 155 160 Leu Ser Ala Val Lys Asp Asn Gln Ser His Leu Lys Ala
Asp Lys Asn 165 170 175 His Ser Thr Lys Gln Lys Asp Thr Ile Lys Thr
Thr Leu Leu Lys Lys 180 185 190 Leu Met Cys Ser Met Gln His Pro Pro
Ser Trp Leu Ile His Trp Phe 195 200 205 Asn Leu Tyr Thr Lys Leu Asn
Asn Ile Leu Thr Gln Tyr Arg Ser Asn 210 215 220 Glu Val Lys Asn His
Gly Phe Thr Leu Ile Asp Asn Gln Thr Leu Ser 225 230 235 240 Gly Phe
Gln Phe Ile Leu Asn Gln Tyr Gly Cys Ile Val Tyr His Lys 245 250 255
Glu Leu Lys Arg Ile Thr Val Thr Thr Tyr Asn Gln Phe Leu Thr Trp 260
265 270 Lys Asp Ile Ser Leu Ser Arg Leu Asn Val Cys Leu Ile Thr Trp
Ile 275 280 285 Ser Asn Cys Leu Asn Thr Leu Asn Lys Ser Leu Gly Leu
Arg Cys Gly 290 295 300 Phe Asn Asn Val Ile Leu Thr Gln Leu Phe Leu
Tyr Gly Asp Cys Ile 305 310 315 320 Leu Lys Leu Phe His Asn Glu Gly
Phe Tyr Ile Ile Lys Glu Val Glu 325 330 335 Gly Phe Ile Met Ser Leu
Ile Leu Asn Ile Thr Glu Glu Asp Gln Phe 340 345 350 Arg Lys Arg Phe
Tyr Asn Ser Met Leu Asn Asn Ile Thr Asp Ala Ala 355 360 365 Asn Lys
Ala Gln Lys Asn Leu Leu Ser Arg Val Cys His Thr Leu Leu 370 375 380
Asp Lys Thr Val Ser Asp Asn Ile Ile Asn Gly Arg Trp Ile Ile Leu 385
390 395 400 Leu Ser Lys Phe Leu Lys Leu Ile Lys Leu Ala Gly Asp Asn
Asn Leu 405 410 415 Asn Asn Leu Ser Glu Leu Tyr Phe Leu Phe Arg Ile
Phe Gly His Pro 420 425 430 Met Val Asp Glu Arg Gln Ala Met Asp Ala
Val Lys Ile Asn Cys Asn 435 440 445 Glu Thr Lys Phe Tyr Leu Leu Ser
Ser Leu Ser Met Leu Arg Gly Ala 450 455 460 Phe Ile Tyr Arg Ile Ile
Lys Gly Phe Val Asn Asn Tyr Asn Arg Trp 465 470 475 480 Pro Thr Leu
Arg Asn Ala Ile Val Leu Pro Leu Arg Trp Leu Thr Tyr 485 490 495 Tyr
Lys Leu Asn Thr Tyr Pro Ser Leu Leu Glu Leu Thr Glu Arg Asp 500 505
510 Leu Ile Val Leu Ser Gly Leu Arg Phe Tyr Arg Glu Phe Arg Leu Pro
515 520 525 Lys Lys Val Asp Leu Glu Met Ile Ile Asn Asp Lys Ala Ile
Ser Pro 530 535 540 Pro Lys Asn Leu Ile Trp Thr Ser Phe Pro Arg Asn
Tyr Met Pro Ser 545 550 555 560 His Ile Gln Asn Tyr Ile Glu His Glu
Lys Leu Lys Phe Ser Glu Ser 565 570 575 Asp Lys Ser Arg Arg Val Leu
Glu Tyr Tyr Leu Arg Asp Asn Lys Phe 580 585 590 Asn Glu Cys Asp Leu
Tyr Asn Cys Val Val Asn Gln Ser Tyr Leu Asn 595 600 605 Asn Pro Asn
His Val Val Ser Leu Thr Gly Lys Glu Arg Glu Leu Ser 610 615 620 Val
Gly Arg Met Phe Ala Met Gln Pro Gly Met Phe Arg Gln Val Gln 625 630
635 640 Ile Leu Ala Glu Lys Met Ile Ala Glu Asn Ile Leu Gln Phe Phe
Pro 645 650 655 Glu Ser Leu Thr Arg Tyr Gly Asp Leu Glu Leu Gln Lys
Ile Leu Glu 660 665 670 Leu Lys Ala Gly Ile Ser Asn Lys Ser Asn Arg
Tyr Asn Asp Asn Tyr 675 680 685 Asn Asn Tyr Ile Ser Lys Cys Ser Ile
Ile Thr Asp Leu Ser Lys Phe 690 695 700 Asn Gln Ala Phe Arg Tyr Glu
Thr Ser Cys Ile Cys Ser Asp Val Leu 705 710 715 720 Asp Glu Leu His
Gly Val Gln Ser Leu Phe Ser Trp Leu His Leu Thr 725 730 735 Ile Pro
His Val Thr Ile Ile Cys Thr Tyr Arg His Ala Pro Pro Tyr 740 745 750
Ile Gly Asp His Ile Val Asp Leu Asn Asn Val Asp Glu Gln Ser Gly 755
760 765 Leu Tyr Arg Tyr His Met Gly Gly Ile Glu Gly Trp Cys Gln Lys
Leu 770 775 780 Trp Thr Ile Glu Ala Ile Ser Leu Leu Asp Leu Ile Ser
Leu Lys Gly 785 790 795 800 Lys Phe Ser Ile Thr Ala Leu Ile Asn Gly
Asp Asn Gln Ser Ile Asp 805 810 815 Ile Ser Lys Pro Ile Arg Leu Met
Glu Gly Gln Thr His Ala Gln Ala 820 825 830 Asp Tyr Leu Leu Ala Leu
Asn Ser Leu Lys Leu Leu Tyr Lys Glu Tyr 835 840 845 Ala Gly Ile Gly
His Lys Leu Lys Gly Thr Glu Thr Tyr Ile Ser Arg 850 855 860 Asp Met
Gln Phe Met Ser Lys Thr Ile Gln His Asn Gly Val Tyr Tyr 865 870 875
880 Pro Ala Ser Ile Lys Lys Val Leu Arg Val Gly Pro Trp Ile Asn Thr
885 890 895 Ile Leu Asp Asp Phe Lys Val Ser Leu Glu Ser Ile Gly Ser
Leu Thr 900 905 910 Gln Glu Leu Glu Tyr Arg Gly Glu Ser Leu Leu Cys
Ser Leu Ile Phe 915 920 925 Arg Asn Val Trp Leu Tyr Asn Gln Ile Ala
Leu Gln Leu Lys Asn His 930 935 940 Ala Leu Cys Asn Asn Lys Leu Tyr
Leu Asp Ile Leu Lys Val Leu Lys 945 950 955 960 His Leu Lys Thr Phe
Phe Asn Leu Asp Asn Ile Asp Thr Ala Leu Thr 965 970 975 Leu Tyr Met
Asn Leu Pro Met Leu Phe Gly Gly Gly Asp Pro Asn Leu 980 985 990 Leu
Tyr Arg Ser Phe Tyr Arg Arg Thr Pro Asp Phe Leu Thr Glu Ala 995
1000 1005 Ile Val His Ser Val Phe Ile Leu Ser Tyr Tyr Thr Asn His
Asp 1010 1015 1020 Leu Lys Asp Lys Leu Gln Asp Leu Ser Asp Asp Arg
Leu Asn Lys 1025 1030 1035 Phe Leu Thr Cys Ile Ile Thr Phe Asp Lys
Asn Pro Asn Ala Glu 1040 1045 1050 Phe Val Thr Leu Met Arg Asp Pro
Gln Ala Leu Gly Ser Glu Arg 1055 1060 1065 Gln Ala Lys Ile Thr Ser
Glu Ile Asn Arg Leu Ala Val Thr Glu 1070 1075 1080 Val Leu Ser Thr
Ala Pro Asn Lys Ile Phe Ser Lys Ser Ala Gln 1085 1090 1095 His Tyr
Thr Thr Thr Glu Ile Asp Leu Asn Asp Ile Met Gln Asn 1100 1105 1110
Ile Glu Pro Thr Tyr Pro His Gly Leu Arg Val Val Tyr Glu Ser 1115
1120 1125 Leu Pro Phe Tyr Lys Ala Glu Lys Ile Val Asn Leu Ile Ser
Gly 1130 1135 1140 Thr Lys Ser Ile Thr Asn Ile Leu Glu Lys Thr Ser
Ala Ile Asp 1145 1150 1155 Leu Thr Asp Ile Asp Arg Ala Thr Glu Met
Met Arg Lys Asn Ile 1160 1165 1170 Thr Leu Leu Ile Arg Ile Leu Pro
Leu Asp Cys Asn Arg Asp Lys 1175 1180 1185 Arg Glu Ile Leu Ser Met
Glu Asn Leu Ser Ile Thr Glu Leu Ser 1190 1195 1200 Lys Tyr Val Arg
Glu Arg Ser Trp Ser Leu Ser Asn Ile Val Gly 1205 1210 1215 Val Thr
Ser Pro Ser Ile Met Tyr Thr Met Asp Ile Lys Tyr Thr 1220 1225 1230
Thr Ser Thr Ile Ser Ser Gly Ile Ile Ile Glu Lys Tyr Asn Val 1235
1240 1245 Asn Ser Leu Thr Arg Gly Glu Arg Gly Pro Thr Lys Pro Trp
Val 1250 1255 1260 Gly Ser Ser Thr Gln Glu Lys Lys Thr Met Pro Val
Tyr Asn Arg 1265 1270 1275 Gln Val Leu Thr Lys Lys Gln Arg Asp Gln
Ile Asp Leu Leu Ala 1280 1285 1290 Lys Leu Asp Trp Val Tyr Ala Ser
Ile Asp Asn Lys Asp Glu Phe 1295 1300 1305 Met Glu Glu Leu Ser Ile
Gly Thr Leu Gly Leu Thr Tyr Glu Lys 1310 1315 1320 Ala Lys Lys Leu
Phe Pro Gln Tyr Leu Ser Val Asn Tyr Leu His 1325 1330 1335 Arg Leu
Thr Val Ser Ser Arg Pro Cys Glu Phe Pro Ala Ser Ile 1340 1345 1350
Pro Ala Tyr Arg Thr Thr Asn Tyr His Phe Asp Thr Ser Pro Ile 1355
1360 1365 Asn Arg Ile Leu Thr Glu Lys Tyr Gly Asp Glu Asp Ile Asp
Ile 1370 1375 1380 Val Phe Gln Asn Cys Ile Ser Phe Gly Leu Ser Leu
Met Ser Val 1385 1390 1395 Val Glu Gln Phe Thr Asn Val Cys Pro Asn
Arg Ile Ile Leu Ile 1400 1405 1410 Pro Lys Leu Asn Glu Ile His Leu
Met Lys Pro Pro Ile Phe Thr 1415 1420 1425 Gly Asp Val Asp Ile His
Lys Leu Lys Gln Val Ile Gln Lys Gln 1430 1435 1440 His Met Phe Leu
Pro Asp Lys Ile Ser Leu Thr Gln Tyr Val Glu 1445 1450 1455 Leu Phe
Leu Ser Asn Lys Thr Leu Lys Ser Gly Ser His Val Asn 1460 1465 1470
Ser Asn Leu Ile Leu Ala His Lys Ile Ser Asp Tyr Phe His Asn 1475
1480 1485 Thr Tyr Ile Leu Ser Thr Asn Leu Ala Gly His Trp Ile Leu
Ile 1490 1495 1500 Ile Gln Leu Met Lys Asp Ser Lys Gly Ile Phe Glu
Lys Asp Trp 1505 1510 1515 Gly Glu Gly Tyr Ile Thr Asp His Met Phe
Ile Asn Leu Lys Val 1520 1525 1530 Phe Phe Asn Ala Tyr Lys Thr Tyr
Leu Leu Cys Phe His Lys Gly 1535 1540 1545 Tyr Gly Lys Ala Lys Leu
Glu Cys Asp Met Asn Thr Ser Asp Leu 1550 1555 1560 Leu Cys Val Leu
Glu Leu Ile Asp Ser Ser Tyr Trp Lys Ser Met 1565 1570 1575 Ser Lys
Val Phe Leu Glu Gln Lys Val Ile Lys Tyr Ile Leu Ser 1580 1585 1590
Gln Asp Ala Ser Leu His Arg Val Lys Gly Cys His Ser Phe Lys 1595
1600 1605 Leu Trp Phe Leu Lys Arg Leu Asn Val Ala Glu Phe Thr Val
Cys 1610 1615 1620 Pro Trp Val Val Asn Ile Asp Tyr His Pro Thr His
Met Lys Ala 1625 1630 1635 Ile Leu Thr Tyr Ile Asp Leu Val Arg Met
Gly Leu Ile Asn Ile 1640 1645 1650 Asp Arg Ile His Ile Lys Asn Lys
His Lys Phe Asn Asp Glu Phe 1655 1660 1665 Tyr Thr Ser Asn Leu Phe
Tyr Ile Asn Tyr Asn Phe Ser Asp Asn 1670 1675 1680 Thr His Leu Leu
Thr Lys His Ile Arg Ile Ala Asn Ser Glu Leu 1685 1690 1695 Glu Asn
Asn Tyr Asn Lys Leu Tyr His Pro Thr Pro Glu Thr Leu 1700 1705 1710
Glu Asn Ile Leu Ala Asn Pro Ile Lys Ser Asn Asp Lys Lys Thr 1715
1720 1725 Leu Asn Asp Tyr Cys Ile Gly Lys Asn Val Asp Ser Ile Met
Leu 1730 1735 1740 Pro Leu Leu Ser Asn Lys Lys Leu Ile Lys Ser Ser
Ala Met Ile 1745 1750 1755 Arg Thr Asn Tyr Ser Lys Gln Asp Leu Tyr
Asn Leu Phe Pro Met 1760 1765 1770 Val Val Ile Asp Arg Ile Ile Asp
His Ser Gly Asn Thr Ala Lys 1775 1780 1785 Ser Asn Gln Leu Tyr Thr
Thr Thr Ser His Gln Ile Ser Leu Val 1790 1795 1800 His Asn Ser Thr
Ser Leu Tyr Cys Met Leu Pro Trp His His Ile 1805 1810 1815 Asn Arg
Phe Asn Phe Val Phe Ser Ser Thr Gly Cys Lys Ile Ser 1820 1825 1830
Ile Glu Tyr Ile Leu Lys Asp Leu Lys Ile Lys Asp Pro Asn Cys 1835
1840 1845 Ile Ala Phe Ile Gly Glu Gly Ala Gly Asn Leu Leu Leu Arg
Thr 1850 1855 1860 Val Val Glu Leu His Pro Asp Ile Arg Tyr Ile Tyr
Arg Ser Leu 1865 1870 1875 Lys Asp Cys Asn Asp His Ser Leu Pro Ile
Glu Phe Leu Arg Leu 1880 1885 1890 Tyr Asn Gly His Ile Asn Ile Asp
Tyr Gly Glu Asn Leu Thr Ile 1895 1900 1905 Pro Ala Thr Asp Ala Thr
Asn Asn Ile His Trp Ser Tyr Leu His 1910 1915 1920 Ile Lys Phe Ala
Glu Pro Ile Ser Leu Phe Val Cys Asp Ala Glu 1925 1930 1935 Leu Ser
Val Thr Val Asn Trp Ser Lys Ile Ile Ile Glu Trp Ser 1940 1945 1950
Lys His Val Arg Lys Cys Lys Tyr Cys Ser Ser Val Asn Lys Cys 1955
1960 1965 Met Leu Ile Val Lys Tyr His Ala Gln Asp Asp Ile Asp Phe
Lys 1970 1975 1980 Leu Asp Asn Ile Thr Ile Leu Lys Thr Tyr Val Cys
Leu Gly Ser 1985 1990 1995 Lys Leu Lys Gly Ser Glu Val Tyr Leu Val
Leu Thr Ile Gly Pro 2000 2005 2010 Ala Asn Ile Phe Pro Val Phe Asn
Val Val Gln Asn Ala Lys Leu 2015 2020 2025 Ile Leu Ser Arg Thr Lys
Asn Phe Ile Met Pro Lys Lys Ala Asp 2030 2035 2040 Lys Glu Ser Ile
Asp Ala Asn Ile Lys Ser Leu Ile Pro Phe Leu 2045 2050 2055 Cys Tyr
Pro Ile Thr Lys Lys Gly Ile Asn Thr Ala Leu Ser Lys 2060 2065 2070
Leu Lys Ser Val Val Ser Gly Asp Ile Leu Ser Tyr Ser Ile Ala 2075
2080 2085 Gly Arg Asn Glu Val Phe Ser Asn Lys Leu Ile Asn His Lys
His 2090 2095 2100 Met Asn Ile Leu Lys Trp Phe Asn His Val Leu Asn
Phe Arg Ser 2105 2110 2115 Thr Glu Leu Asn Tyr Asn His Leu Tyr Met
Val Glu Ser Thr Tyr 2120 2125 2130 Pro Tyr Leu Ser Glu Leu Leu Asn
Ser Leu Thr Thr Asn Glu Leu 2135 2140 2145 Lys Lys Leu Ile Lys Ile
Thr Gly Ser Leu Leu Tyr Asn Phe His 2150 2155 2160 Asn Glu 2165 14
2233 PRT human parainfluenza virus 3 14 Met Asp Thr Glu Ser Asn Asn
Gly Thr Val Ser Asp Ile Leu Tyr Pro 1 5 10 15 Glu Cys His Leu Asn
Ser Pro Ile Val Lys Gly Lys Ile Ala Gln Leu 20 25 30 His Thr Ile
Met Ser Leu Pro Gln Pro Tyr Asp Met Asp Asp Asp Ser 35 40 45 Ile
Leu Val Ile Thr Arg Gln Lys Ile Lys Leu Asn Lys Leu Asp Lys 50 55
60 Arg Gln Arg Ser Ile Arg Arg Leu Lys Leu Ile Leu Thr Glu Lys Val
65 70 75 80 Asn Asp Leu Gly Lys Tyr Thr Phe Ile Arg Tyr Pro Glu Met
Ser Lys 85 90 95 Glu Met Phe Lys Leu His Ile Pro Gly Ile Asn Ser
Lys Val Thr Glu 100 105 110 Leu Leu Leu Lys Ala Asp Arg Thr Tyr Ser
Gln Met Thr Asp Gly Leu 115 120 125 Arg Asp Leu Trp Ile Asn Val Leu
Ser Lys Leu Ala Ser Lys Asn Asp 130 135 140 Gly Ser Asn Tyr Asp Leu
Asn Glu Glu Ile Asn Asn Ile Ser Lys Val 145 150 155 160 His Thr Thr
Tyr Lys Ser Asp Lys Trp Tyr Asn Pro Phe Lys Thr Trp 165 170 175 Phe
Thr Ile Lys Tyr Asp Met Arg Arg Leu Gln Lys Ala Arg Asn Glu 180 185
190 Val Thr Phe Asn Met Gly Lys Asp Tyr Asn Leu Leu Glu Asp Gln Lys
195 200 205 Asn Phe Leu Leu Ile His Pro Glu Leu Val Leu Ile Leu Asp
Lys Gln 210 215 220 Asn Tyr Asn Gly Tyr Leu Ile Thr Pro Glu Leu Val
Leu Pro Tyr Cys 225 230 235 240 Asp Val Val Glu Gly Arg Trp Asn Ile
Ser Ala Cys Ala Lys Leu Asp 245 250 255 Pro Lys Leu Gln Ser Met Tyr
Gln Lys Gly Asn Asn Leu Trp Glu Val 260 265 270 Ile Asp Lys Leu Phe
Pro Ile Met Gly Glu Lys Thr Phe Asp Val Ile 275 280 285 Ser Leu Leu
Glu Pro Leu Ala Leu Ser Leu Ile Gln Thr His Asp Pro 290 295 300 Val
Lys Gln Leu Arg Gly Ala Phe Leu Asn His Val Leu Ser Glu Met 305 310
315
320 Glu Leu Ile Phe Glu Ser Arg Glu Ser Ile Lys Glu Phe Leu Ser Val
325 330 335 Asp Tyr Ile Asp Lys Ile Leu Asp Ile Phe Asn Lys Ser Thr
Ile Asp 340 345 350 Glu Ile Ala Glu Ile Phe Ser Phe Phe Arg Thr Phe
Gly His Pro Pro 355 360 365 Leu Glu Ala Ser Ile Ala Ala Glu Lys Val
Arg Lys Tyr Met Tyr Ile 370 375 380 Gly Lys Gln Leu Lys Phe Asp Thr
Ile Asn Lys Cys His Ala Ile Phe 385 390 395 400 Cys Thr Ile Ile Ile
Asn Gly Tyr Arg Glu Arg His Gly Gly Gln Trp 405 410 415 Pro Pro Val
Thr Leu Pro Asp His Ala His Glu Phe Ile Ile Asn Ala 420 425 430 Tyr
Gly Ser Asn Ser Ala Ile Ser Tyr Glu Asn Ala Val Asp Tyr Tyr 435 440
445 Gln Ser Phe Ile Gly Ile Lys Phe Asn Lys Phe Ile Glu Pro Gln Leu
450 455 460 Asp Glu Asp Leu Thr Ile Tyr Met Lys Asp Lys Ala Leu Ser
Pro Lys 465 470 475 480 Lys Ser Asn Trp Asp Thr Val Ser Pro Ala Ser
Asn Leu Leu Tyr Arg 485 490 495 Thr Asn Ala Ser Asn Glu Ser Arg Arg
Leu Val Glu Lys Phe Ile Ala 500 505 510 Asp Ser Lys Phe Asp Pro Asn
Gln Ile Leu Asp Tyr Val Glu Ser Gly 515 520 525 Asp Trp Leu Asp Asp
Pro Glu Phe Asn Ile Ser Tyr Ser Leu Lys Glu 530 535 540 Lys Glu Ile
Lys Gln Glu Gly Arg Leu Phe Ala Lys Met Thr Tyr Lys 545 550 555 560
Met Arg Ala Thr Gln Val Leu Ser Glu Thr Leu Leu Ala Asn Asn Ile 565
570 575 Gly Lys Phe Phe Gln Glu Asn Gly Met Val Lys Gly Glu Ile Glu
Leu 580 585 590 Leu Lys Arg Leu Thr Thr Ile Ser Ile Ser Gly Val Pro
Arg Tyr Asn 595 600 605 Glu Val Tyr Asn Asn Ser Lys Ser His Thr Asp
Asp Leu Lys Thr Tyr 610 615 620 Asn Lys Ile Ser Asn Leu Asn Leu Ser
Ser Asn Gln Lys Ser Lys Lys 625 630 635 640 Phe Glu Phe Lys Ser Thr
Asp Ile Tyr Asn Asp Gly Tyr Glu Thr Val 645 650 655 Ser Cys Phe Leu
Thr Thr Asp Leu Lys Lys Tyr Cys Leu Asn Trp Arg 660 665 670 Tyr Glu
Ser Thr Ala Leu Phe Gly Glu Thr Cys Asn Gln Ile Phe Gly 675 680 685
Leu Asn Lys Leu Phe Asn Trp Leu His Pro Arg Leu Glu Gly Ser Thr 690
695 700 Ile Tyr Val Gly Asp Pro Tyr Cys Pro Pro Ser Asp Lys Glu His
Ile 705 710 715 720 Ser Leu Glu Asp His Pro Asp Ser Gly Phe Tyr Val
His Asn Pro Arg 725 730 735 Gly Gly Ile Glu Gly Phe Cys Gln Lys Leu
Trp Thr Leu Ile Ser Ile 740 745 750 Ser Ala Ile His Leu Ala Ala Val
Arg Ile Gly Val Arg Val Thr Ala 755 760 765 Met Val Gln Gly Asp Asn
Gln Ala Ile Ala Val Thr Thr Arg Val Pro 770 775 780 Asn Asn Tyr Asp
Tyr Arg Val Lys Lys Glu Ile Val Tyr Lys Asp Val 785 790 795 800 Val
Arg Phe Phe Asp Ser Leu Arg Glu Val Met Asp Asp Leu Gly His 805 810
815 Glu Leu Lys Leu Asn Glu Thr Ile Ile Ser Ser Lys Met Phe Ile Tyr
820 825 830 Ser Lys Arg Ile Tyr Tyr Asp Gly Arg Ile Leu Pro Gln Ala
Leu Lys 835 840 845 Ala Leu Ser Arg Cys Val Phe Trp Ser Glu Thr Val
Ile Asp Glu Thr 850 855 860 Arg Ser Ala Ser Ser Asn Leu Ala Thr Ser
Phe Ala Lys Ala Ile Glu 865 870 875 880 Asn Gly Tyr Ser Pro Val Leu
Gly Tyr Ala Cys Ser Ile Phe Lys Asn 885 890 895 Ile Gln Gln Leu Tyr
Ile Ala Leu Gly Met Asn Ile Asn Pro Thr Ile 900 905 910 Thr Gln Asn
Ile Lys Asp Leu Tyr Phe Arg Asn Pro Asn Trp Met Gln 915 920 925 Tyr
Ala Ser Leu Ile Pro Ala Ser Val Gly Gly Phe Asn Tyr Met Ala 930 935
940 Met Ser Arg Cys Phe Val Arg Asn Ile Gly Asp Pro Ser Val Ala Ala
945 950 955 960 Leu Ala Asp Ile Lys Arg Phe Ile Lys Ala Asn Leu Leu
Asp Arg Ser 965 970 975 Val Leu Tyr Arg Ile Met Asn Gln Glu Pro Gly
Glu Ser Ser Phe Leu 980 985 990 Asp Trp Ala Ser Asp Pro Tyr Ser Cys
Asn Leu Pro Gln Ser Gln Asn 995 1000 1005 Ile Thr Thr Met Ile Lys
Asn Ile Thr Ala Arg Asn Val Leu Gln 1010 1015 1020 Asp Ser Pro Asn
Pro Leu Leu Ser Gly Leu Phe Thr Asn Thr Met 1025 1030 1035 Ile Glu
Glu Asp Glu Glu Leu Ala Glu Phe Leu Met Asp Arg Lys 1040 1045 1050
Val Ile Leu Pro Arg Val Ala His Asp Ile Leu Asp Asn Ser Leu 1055
1060 1065 Thr Gly Ile Arg Asn Ala Ile Ala Gly Met Leu Asp Thr Thr
Lys 1070 1075 1080 Ser Leu Ile Arg Val Gly Ile Asn Arg Gly Gly Leu
Thr Tyr Ser 1085 1090 1095 Leu Leu Arg Lys Ile Ser Asn Tyr Asp Leu
Val Gln Tyr Glu Thr 1100 1105 1110 Leu Ser Arg Thr Leu Arg Leu Ile
Val Ser Asp Lys Ile Arg Tyr 1115 1120 1125 Glu Asp Met Cys Ser Val
Asp Leu Ala Ile Ala Leu Arg Gln Lys 1130 1135 1140 Met Trp Ile His
Leu Ser Gly Gly Arg Met Ile Ser Gly Leu Glu 1145 1150 1155 Thr Pro
Asp Pro Leu Glu Leu Leu Ser Gly Val Ile Ile Thr Gly 1160 1165 1170
Ser Glu His Cys Lys Ile Cys Tyr Ser Ser Asp Gly Thr Asn Pro 1175
1180 1185 Tyr Thr Trp Met Tyr Leu Pro Gly Asn Ile Lys Ile Gly Ser
Ala 1190 1195 1200 Glu Thr Gly Ile Ser Ser Leu Arg Val Pro Tyr Phe
Gly Ser Val 1205 1210 1215 Thr Asp Glu Arg Ser Glu Ala Gln Leu Gly
Tyr Ile Lys Asn Leu 1220 1225 1230 Ser Lys Pro Ala Lys Ala Ala Ile
Arg Ile Ala Met Ile Tyr Thr 1235 1240 1245 Trp Ala Phe Gly Asn Asp
Glu Ile Ser Trp Met Glu Ala Ser Gln 1250 1255 1260 Ile Ala Gln Thr
Arg Ala Asn Phe Thr Leu Asp Ser Leu Lys Ile 1265 1270 1275 Leu Thr
Pro Val Ala Thr Ser Thr Asn Leu Ser His Arg Leu Lys 1280 1285 1290
Asp Thr Ala Thr Gln Met Lys Phe Ser Ser Thr Ser Leu Ile Arg 1295
1300 1305 Val Ser Arg Phe Ile Thr Met Ser Asn Asp Asn Met Ser Ile
Lys 1310 1315 1320 Glu Ala Asn Glu Thr Lys Asp Thr Asn Leu Ile Tyr
Gln Gln Ile 1325 1330 1335 Met Leu Thr Gly Leu Ser Val Phe Glu Tyr
Leu Phe Arg Leu Glu 1340 1345 1350 Glu Thr Thr Gly His Asn Pro Ile
Val Met His Leu His Ile Glu 1355 1360 1365 Asp Glu Cys Cys Ile Lys
Glu Ser Phe Asn Asp Glu His Ile Asn 1370 1375 1380 Pro Glu Ser Thr
Leu Glu Leu Ile Arg Tyr Pro Glu Ser Asn Glu 1385 1390 1395 Phe Ile
Tyr Asp Lys Asp Pro Leu Lys Asp Val Asp Leu Ser Lys 1400 1405 1410
Leu Met Val Ile Lys Asp His Ser Tyr Thr Ile Asp Met Asn Tyr 1415
1420 1425 Trp Asp Asp Thr Asp Ile Ile His Ala Ile Ser Ile Cys Thr
Ala 1430 1435 1440 Ile Thr Ile Ala Asp Thr Met Ser Gln Leu Asp Arg
Asp Asn Leu 1445 1450 1455 Lys Glu Ile Ile Val Ile Ala Asn Asp Asp
Asp Ile Asn Ser Leu 1460 1465 1470 Ile Thr Glu Phe Leu Thr Leu Asp
Ile Leu Val Phe Leu Lys Thr 1475 1480 1485 Phe Gly Gly Leu Leu Val
Asn Gln Phe Ala Tyr Thr Leu Tyr Ser 1490 1495 1500 Leu Lys Thr Glu
Gly Arg Asp Leu Ile Trp Asp Tyr Ile Met Arg 1505 1510 1515 Thr Leu
Arg Asp Thr Ser His Ser Ile Leu Lys Val Leu Ser Asn 1520 1525 1530
Ala Leu Ser His Pro Lys Val Phe Lys Arg Phe Trp Asp Cys Gly 1535
1540 1545 Val Leu Asn Pro Ile Tyr Gly Pro Asn Thr Ala Ser Gln Asp
Gln 1550 1555 1560 Ile Lys Leu Ala Leu Ser Ile Cys Glu Tyr Ser Leu
Asp Leu Phe 1565 1570 1575 Met Arg Glu Trp Leu Asn Gly Val Ser Leu
Glu Ile Tyr Ile Cys 1580 1585 1590 Asp Ser Asp Met Glu Val Ala Asn
Asp Arg Lys Gln Ala Phe Ile 1595 1600 1605 Ser Arg His Leu Ser Phe
Val Cys Cys Leu Ala Glu Ile Ala Ser 1610 1615 1620 Phe Gly Pro Asn
Leu Leu Asn Leu Thr Tyr Leu Glu Arg Leu Asp 1625 1630 1635 Leu Leu
Lys Gln Tyr Leu Glu Leu Asn Ile Lys Asp Asp Pro Thr 1640 1645 1650
Leu Lys Tyr Val Gln Ile Ser Gly Leu Leu Ile Lys Ser Phe Pro 1655
1660 1665 Ser Thr Val Thr Tyr Val Arg Lys Thr Ala Ile Lys Tyr Leu
Arg 1670 1675 1680 Ile Arg Gly Ile Ser Pro Pro Glu Val Ile Asp Asp
Trp Asp Pro 1685 1690 1695 Ile Glu Asp Glu Asn Met Leu Asp Asn Ile
Val Lys Thr Ile Asn 1700 1705 1710 Asp Asn Cys Asn Lys Asp Asn Lys
Gly Asn Lys Ile Asn Asn Phe 1715 1720 1725 Trp Gly Leu Ala Leu Lys
Asn Tyr Gln Val Leu Lys Ile Arg Ser 1730 1735 1740 Ile Thr Ser Asp
Ser Asp Asn Asn Asp Arg Ser Asp Ala Ser Thr 1745 1750 1755 Gly Gly
Leu Thr Leu Pro Gln Gly Gly Asn Tyr Leu Ser His Gln 1760 1765 1770
Leu Arg Leu Phe Gly Ile Asn Ser Thr Ser Cys Leu Lys Ala Leu 1775
1780 1785 Glu Leu Ser Gln Ile Leu Met Lys Glu Val Asn Lys Asp Gln
Asp 1790 1795 1800 Arg Leu Phe Leu Gly Glu Gly Ala Gly Ala Met Leu
Ala Cys Tyr 1805 1810 1815 Asp Ala Thr Leu Gly Pro Ala Val Asn Tyr
Tyr Asn Ser Gly Leu 1820 1825 1830 Asn Ile Thr Asp Val Ile Gly Gln
Arg Glu Leu Lys Ile Phe Pro 1835 1840 1845 Ser Glu Val Ser Leu Val
Gly Lys Lys Leu Gly Asn Val Thr Gln 1850 1855 1860 Ile Leu Asn Arg
Val Lys Val Leu Phe Asn Gly Asn Pro Asn Ser 1865 1870 1875 Thr Trp
Ile Gly Asn Met Glu Cys Glu Thr Leu Ile Trp Ser Glu 1880 1885 1890
Leu Asn Asp Lys Ser Ile Gly Leu Val His Cys Asp Met Glu Gly 1895
1900 1905 Ala Ile Gly Lys Ser Glu Glu Thr Val Leu His Glu His Tyr
Ser 1910 1915 1920 Val Ile Arg Ile Thr Tyr Leu Ile Gly Asp Asp Asp
Val Val Leu 1925 1930 1935 Ile Ser Lys Ile Ile Pro Thr Ile Thr Pro
Asn Trp Ser Arg Ile 1940 1945 1950 Leu Tyr Leu Tyr Lys Leu Tyr Trp
Lys Asp Val Ser Ile Ile Ser 1955 1960 1965 Leu Lys Thr Ser Asn Pro
Ala Ser Thr Glu Leu Tyr Leu Ile Ser 1970 1975 1980 Lys Asp Ala Tyr
Cys Thr Ile Met Glu Pro Ser Glu Val Val Leu 1985 1990 1995 Ser Lys
Leu Lys Arg Leu Ser Leu Leu Glu Glu Asn Asn Leu Leu 2000 2005 2010
Lys Trp Ile Ile Leu Ser Lys Lys Lys Asn Asn Glu Trp Leu His 2015
2020 2025 His Glu Ile Lys Glu Gly Glu Arg Asp Tyr Gly Val Met Arg
Pro 2030 2035 2040 Tyr His Met Ala Leu Gln Ile Phe Gly Phe Gln Ile
Asn Leu Asn 2045 2050 2055 His Leu Ala Lys Glu Phe Leu Ser Thr Pro
Asp Leu Thr Asn Ile 2060 2065 2070 Asn Asn Ile Ile Gln Ser Phe Gln
Arg Thr Ile Lys Asp Val Leu 2075 2080 2085 Phe Glu Trp Ile Asn Ile
Thr His Asp Gly Lys Arg His Lys Leu 2090 2095 2100 Gly Gly Arg Tyr
Asn Ile Phe Pro Leu Lys Asn Lys Gly Lys Leu 2105 2110 2115 Arg Leu
Leu Ser Arg Arg Leu Val Leu Ser Trp Ile Ser Leu Ser 2120 2125 2130
Leu Ser Thr Arg Leu Leu Thr Gly Arg Phe Pro Asp Glu Lys Phe 2135
2140 2145 Glu His Arg Ala Gln Thr Gly Tyr Val Ser Leu Pro Asp Thr
Asp 2150 2155 2160 Leu Glu Ser Leu Lys Leu Leu Ser Lys Asn Thr Ile
Lys Asn Tyr 2165 2170 2175 Arg Glu Cys Ile Gly Ser Ile Ser Tyr Trp
Phe Leu Thr Lys Glu 2180 2185 2190 Val Lys Ile Leu Met Lys Leu Ile
Gly Gly Ala Lys Leu Leu Gly 2195 2200 2205 Ile Pro Arg Gln Tyr Lys
Glu Pro Glu Glu Gln Leu Leu Glu Asp 2210 2215 2220 Tyr Asn Gln His
Asp Glu Phe Asp Ile Asp 2225 2230 15 2223 PRT human parainfluenza
virus 1 15 Met Asp Lys Gln Glu Ser Thr Gln Asn Ser Ser Asp Ile Leu
Tyr Pro 1 5 10 15 Glu Cys His Leu Asn Ser Pro Ile Val Lys Ser Lys
Ile Ala Gln Leu 20 25 30 His Val Leu Leu Asp Ile Asn Gln Pro Tyr
Asp Leu Lys Asp Asn Ser 35 40 45 Ile Ile Asn Ile Thr Lys Tyr Lys
Ile Arg Asn Gly Gly Leu Ser Pro 50 55 60 Arg Gln Ile Lys Ile Arg
Ser Leu Gly Lys Ile Leu Lys Gln Glu Ile 65 70 75 80 Lys Asp Ile Asp
Arg Tyr Thr Phe Glu Pro Tyr Pro Ile Phe Ser Leu 85 90 95 Glu Leu
Leu Arg Leu Asp Ile Pro Glu Ile Cys Asp Lys Ile Arg Ser 100 105 110
Ile Phe Ser Val Ser Asp Arg Leu Ile Arg Glu Leu Ser Ser Gly Phe 115
120 125 Gln Glu Leu Trp Leu Asn Ile Leu Arg Gln Leu Gly Cys Val Glu
Gly 130 135 140 Lys Glu Gly Phe Asp Ser Leu Lys Asp Val Asp Ile Ile
Pro Asp Ile 145 150 155 160 Thr Asp Lys Tyr Asn Lys Asn Thr Trp Tyr
Arg Pro Phe Leu Thr Trp 165 170 175 Phe Ser Ile Lys Tyr Asp Met Arg
Trp Met Gln Lys Asn Lys Ser Gly 180 185 190 Asn His Leu Asp Val Ser
Asn Ser His Asn Phe Leu Asp Cys Lys Ser 195 200 205 Tyr Ile Leu Ile
Ile Tyr Arg Asp Leu Val Ile Ile Ile Asn Lys Leu 210 215 220 Lys Leu
Thr Gly Tyr Val Leu Thr Pro Glu Leu Val Leu Met Tyr Cys 225 230 235
240 Asp Val Val Glu Gly Arg Trp Asn Met Ser Ser Ala Gly Arg Leu Asp
245 250 255 Lys Arg Ser Ser Lys Ile Thr Cys Lys Gly Glu Glu Leu Trp
Glu Leu 260 265 270 Ile Asp Ser Leu Phe Pro Asn Leu Gly Glu Asp Val
Tyr Asn Ile Ile 275 280 285 Ser Leu Leu Glu Pro Leu Ser Leu Ala Leu
Ile Gln Leu Asp Asp Pro 290 295 300 Val Thr Asn Leu Lys Gly Ala Phe
Met Arg His Val Leu Thr Glu Leu 305 310 315 320 His Thr Ile Leu Ile
Lys Asp Asn Ile Tyr Thr Asp Ser Glu Ala Asp 325 330 335 Ser Ile Met
Glu Ser Leu Ile Lys Ile Phe Arg Glu Thr Ser Ile Asp 340 345 350 Glu
Lys Ala Glu Ile Phe Ser Phe Phe Arg Thr Phe Gly His Pro Ser 355 360
365 Leu Glu Ala Ile Thr Ala Ala Asp Lys Val Arg Thr His Met Tyr Ser
370 375 380 Ser Lys Lys Ile Ile Leu Lys Thr Leu Tyr Glu Cys His Ala
Ile Phe 385 390 395 400 Cys Ala Ile Ile Ile Asn Gly Tyr Arg Glu Arg
His Gly Gly Gln Trp 405 410 415 Pro Pro Cys Glu Phe Pro Asn His Val
Cys Leu Glu Leu Lys Asn Ala 420 425 430 Gln Gly Ser Asn Ser Ala Ile
Ser Tyr Glu Cys Ala Val Asp Asn Tyr 435 440 445 Ser Ser Phe Ile Gly
Phe Lys Phe Leu Lys Phe Ile Glu Pro Gln Leu 450 455 460 Asp Glu Asp
Leu Thr Ile Tyr Met Lys Asp Lys Ala Leu Ser Pro Arg 465 470 475 480
Lys Ala Ala Trp Asp Ser Val Tyr Pro Asp Ser Asn Leu Tyr Tyr Lys 485
490 495 Val Pro Glu Ser Glu Glu Thr Arg Arg Leu Ile Glu
Val Phe Ile Asn 500 505 510 Asp Asn Asn Phe Asn Pro Ala Asp Ile Ile
Asn Tyr Val Glu Ser Gly 515 520 525 Glu Trp Leu Asn Asp Asp Ser Phe
Asn Ile Ser Tyr Ser Leu Lys Glu 530 535 540 Lys Glu Ile Lys Gln Glu
Gly Arg Leu Phe Ala Lys Met Thr Tyr Lys 545 550 555 560 Met Arg Ala
Val Gln Val Leu Ala Glu Thr Leu Leu Ala Lys Gly Val 565 570 575 Gly
Glu Leu Phe Ser Glu Asn Gly Met Val Lys Gly Glu Ile Asp Leu 580 585
590 Leu Lys Arg Leu Thr Thr Leu Ser Val Ser Gly Val Pro Arg Ser Asn
595 600 605 Ser Val Tyr Asn Asn Pro Ile Leu His Glu Lys Leu Ile Lys
Asn Met 610 615 620 Asn Lys Cys Asn Ser Asn Gly Tyr Trp Asp Glu Arg
Lys Lys Ser Lys 625 630 635 640 Asn Glu Phe Lys Ala Ala Asp Ser Ser
Thr Glu Gly Tyr Glu Thr Leu 645 650 655 Ser Cys Phe Leu Thr Thr Asp
Leu Lys Lys Tyr Cys Leu Asn Trp Arg 660 665 670 Phe Glu Ser Thr Ala
Leu Phe Gly Gln Arg Cys Asn Glu Ile Phe Gly 675 680 685 Phe Lys Thr
Phe Phe Asn Trp Met His Pro Ile Leu Glu Lys Ser Thr 690 695 700 Ile
Tyr Val Gly Asp Pro Tyr Cys Pro Val Pro Asp Arg Met His Lys 705 710
715 720 Glu Leu Gln Asp His Asp Asp Thr Gly Ile Phe Ile His Asn Pro
Arg 725 730 735 Gly Gly Ile Glu Gly Tyr Cys Gln Lys Leu Trp Thr Leu
Ile Ser Ile 740 745 750 Ser Ala Ile His Leu Ala Ala Val Lys Val Gly
Val Arg Val Ser Ala 755 760 765 Met Val Gln Gly Asp Asn Gln Ala Ile
Ala Val Thr Ser Arg Val Pro 770 775 780 Val Thr Gln Thr Tyr Lys Gln
Lys Lys Thr His Val Tyr Glu Glu Ile 785 790 795 800 Thr Arg Tyr Phe
Gly Ala Leu Arg Glu Val Met Phe Asp Ile Gly His 805 810 815 Glu Leu
Lys Leu Asn Glu Thr Ile Ile Ser Ser Lys Met Phe Val Tyr 820 825 830
Ser Lys Arg Ile Tyr Tyr Asp Gly Lys Ile Leu Pro Gln Cys Leu Lys 835
840 845 Ala Leu Thr Arg Cys Val Phe Trp Ser Glu Thr Leu Val Asp Glu
Asn 850 855 860 Arg Ser Ala Cys Ser Asn Ile Ala Thr Ser Ile Ala Lys
Ala Ile Glu 865 870 875 880 Asn Gly Tyr Ser Pro Ile Leu Gly Tyr Cys
Ile Ala Leu Phe Lys Thr 885 890 895 Cys Gln Gln Val Cys Ile Ser Leu
Gly Met Thr Ile Asn Pro Thr Ile 900 905 910 Thr Ser Thr Ile Lys Asp
Gln Tyr Phe Lys Gly Lys Asn Trp Leu Arg 915 920 925 Cys Ala Ile Leu
Ile Pro Ala Asn Ile Gly Gly Phe Asn Tyr Met Ser 930 935 940 Thr Ala
Arg Cys Phe Val Arg Asn Ile Gly Asp Pro Ala Val Ala Ala 945 950 955
960 Leu Ala Asp Leu Lys Arg Phe Ile Lys Ala Gly Leu Leu Asp Lys Gln
965 970 975 Val Leu Tyr Arg Val Met Asn Gln Glu Pro Gly Asp Ser Ser
Phe Leu 980 985 990 Asp Trp Ala Ser Asp Pro Tyr Ser Cys Asn Leu Pro
His Ser Gln Ser 995 1000 1005 Ile Thr Thr Ile Ile Lys Asn Val Thr
Ala Arg Ser Val Leu Gln 1010 1015 1020 Glu Ser Pro Asn Pro Leu Leu
Ser Gly Leu Phe Ser Glu Ser Ser 1025 1030 1035 Ser Glu Glu Asp Leu
Asn Leu Ala Ser Phe Leu Met Asp Arg Lys 1040 1045 1050 Ala Ile Leu
Pro Arg Val Ala His Glu Ile Leu Asp Asn Ser Leu 1055 1060 1065 Thr
Gly Val Arg Glu Ala Ile Ala Gly Met Leu Asp Thr Thr Lys 1070 1075
1080 Ser Leu Val Arg Ala Ser Val Arg Arg Gly Gly Leu Ser Tyr Ser
1085 1090 1095 Ile Leu Arg Arg Leu Ile Asn Tyr Asp Leu Leu Gln Tyr
Glu Thr 1100 1105 1110 Leu Thr Arg Thr Leu Arg Lys Pro Val Lys Asp
Asn Ile Glu Tyr 1115 1120 1125 Glu Tyr Met Cys Ser Val Glu Leu Ala
Ile Gly Leu Arg Gln Lys 1130 1135 1140 Met Trp Phe His Leu Thr Tyr
Gly Arg Pro Ile His Gly Leu Glu 1145 1150 1155 Thr Pro Asp Pro Leu
Glu Leu Leu Arg Gly Ser Phe Ile Glu Gly 1160 1165 1170 Ser Glu Ile
Cys Lys Phe Cys Arg Ser Glu Gly Asn Asn Pro Met 1175 1180 1185 Tyr
Thr Trp Phe Tyr Leu Pro Asp Asn Ile Asp Leu Asp Thr Leu 1190 1195
1200 Ser Asn Gly Ser Pro Ala Ile Arg Ile Pro Tyr Phe Gly Ser Ala
1205 1210 1215 Thr Asp Glu Arg Ser Glu Ala Gln Leu Gly Tyr Val Lys
Asn Leu 1220 1225 1230 Ser Lys Pro Ala Lys Ala Ala Ile Arg Ile Ala
Met Val Tyr Thr 1235 1240 1245 Trp Ala Tyr Gly Thr Asp Glu Ile Ser
Trp Met Glu Ala Ala Leu 1250 1255 1260 Ile Ala Gln Thr Arg Ala Asn
Leu Ser Leu Glu Asn Leu Lys Leu 1265 1270 1275 Leu Thr Pro Val Ser
Thr Ser Thr Asn Leu Ser His Arg Leu Arg 1280 1285 1290 Asp Thr Ala
Thr Gln Met Lys Phe Ser Ser Ala Thr Leu Val Arg 1295 1300 1305 Ala
Ser Arg Phe Ile Thr Ile Ser Asn Asp Asn Met Ala Leu Lys 1310 1315
1320 Glu Ala Gly Glu Ser Lys Asp Thr Asn Leu Val Tyr Gln Gln Ile
1325 1330 1335 Met Leu Thr Gly Leu Ser Leu Phe Glu Phe Asn Met Arg
Tyr Lys 1340 1345 1350 Gln Gly Ser Leu Ser Lys Pro Met Ile Leu His
Leu His Leu Asn 1355 1360 1365 Asn Lys Cys Cys Ile Ile Glu Ser Pro
Gln Glu Leu Asn Ile Pro 1370 1375 1380 Pro Arg Ser Thr Leu Asp Leu
Glu Ile Thr Gln Glu Asn Asn Lys 1385 1390 1395 Leu Ile Tyr Asp Pro
Asp Pro Leu Lys Asp Ile Asp Leu Glu Leu 1400 1405 1410 Phe Ser Lys
Val Arg Asp Val Val His Thr Ile Asp Met Asn Tyr 1415 1420 1425 Trp
Ser Asp Asp Glu Ile Ile Arg Ala Thr Ser Ile Cys Thr Ala 1430 1435
1440 Met Thr Ile Ala Asp Thr Met Ser Gln Leu Asp Arg Asp Asn Leu
1445 1450 1455 Lys Glu Met Ile Ala Leu Ile Asn Asp Asp Asp Ile Asn
Ser Leu 1460 1465 1470 Ile Thr Glu Phe Met Val Ile Asp Ile Pro Leu
Phe Cys Ser Thr 1475 1480 1485 Phe Gly Gly Ile Leu Ile Asn Gln Phe
Ala Tyr Ser Leu Tyr Gly 1490 1495 1500 Leu Asn Val Arg Gly Arg Asp
Glu Ile Trp Gly Tyr Val Ile Arg 1505 1510 1515 Ile Ile Lys Asp Thr
Ser His Ala Val Leu Lys Val Leu Ser Asn 1520 1525 1530 Ala Leu Ser
His Pro Lys Ile Phe Lys Arg Phe Trp Asp Ala Gly 1535 1540 1545 Val
Val Glu Pro Val Tyr Gly Pro Asn Leu Ser Asn Gln Asp Lys 1550 1555
1560 Ile Leu Leu Ala Ile Ser Val Cys Glu Tyr Ser Val Asp Leu Phe
1565 1570 1575 Met Arg Asp Trp Gln Glu Gly Ile Pro Leu Glu Ile Phe
Ile Cys 1580 1585 1590 Asp Asn Asp Pro Asn Ile Ala Glu Met Arg Lys
Leu Ser Phe Leu 1595 1600 1605 Ala Arg His Leu Ala Tyr Leu Cys Ser
Leu Ala Glu Ile Ala Lys 1610 1615 1620 Glu Gly Pro Lys Leu Glu Ser
Met Thr Ser Leu Glu Arg Leu Glu 1625 1630 1635 Ser Leu Lys Glu Tyr
Leu Glu Leu Thr Phe Leu Asp Asp Pro Ile 1640 1645 1650 Leu Arg Tyr
Ser Gln Leu Thr Gly Leu Val Ile Lys Ile Phe Pro 1655 1660 1665 Ser
Thr Leu Thr Tyr Ile Arg Lys Ser Ser Ile Lys Val Leu Arg 1670 1675
1680 Val Arg Gly Ile Gly Ile Pro Glu Val Leu Glu Asp Trp Asp Pro
1685 1690 1695 Asp Ala Asp Ser Met Leu Leu Asp Asn Ile Thr Ala Glu
Val Gln 1700 1705 1710 His Asn Ile Pro Leu Lys Lys Asn Glu Arg Thr
Pro Phe Trp Gly 1715 1720 1725 Leu Arg Val Ser Lys Ser Gln Val Leu
Arg Leu Arg Gly Tyr Glu 1730 1735 1740 Glu Ile Lys Arg Glu Glu Arg
Gly Arg Ser Gly Val Gly Leu Thr 1745 1750 1755 Leu Pro Phe Asp Gly
Arg Tyr Leu Ser His Gln Leu Arg Leu Phe 1760 1765 1770 Gly Ile Asn
Ser Thr Ser Cys Leu Lys Ala Leu Glu Leu Thr Tyr 1775 1780 1785 Leu
Leu Asn Pro Leu Val Asn Lys Asp Lys Asp Arg Leu Tyr Leu 1790 1795
1800 Gly Glu Gly Ala Gly Ala Met Leu Ser Cys Tyr Asp Ala Thr Leu
1805 1810 1815 Gly Pro Cys Met Asn Tyr Tyr Asn Ser Gly Val Asn Ser
Cys Asp 1820 1825 1830 Leu Asn Gly Gln Arg Glu Leu Asn Ile Tyr Pro
Ser Glu Val Ala 1835 1840 1845 Leu Val Gly Lys Lys Leu Asn Asn Val
Thr Ser Leu Cys Gln Arg 1850 1855 1860 Val Lys Val Leu Phe Asn Gly
Asn Pro Gly Ser Thr Trp Ile Gly 1865 1870 1875 Asn Asp Glu Cys Glu
Thr Leu Ile Trp Asn Glu Leu Gln Asn Asn 1880 1885 1890 Ser Ile Gly
Phe Ile His Cys Asp Met Glu Gly Gly Glu His Lys 1895 1900 1905 Cys
Asp Gln Val Val Leu His Glu His Tyr Ser Val Ile Arg Ile 1910 1915
1920 Ala Tyr Leu Val Gly Asp Lys Asp Val Ile Leu Val Ser Lys Ile
1925 1930 1935 Ala Pro Arg Leu Gly Thr Asp Trp Thr Lys Gln Leu Ser
Leu Tyr 1940 1945 1950 Leu Arg Tyr Trp Arg Asp Val Ser Leu Ile Val
Leu Lys Thr Ser 1955 1960 1965 Asn Pro Ala Ser Thr Glu Met Tyr Leu
Ile Ser Lys Asp Pro Lys 1970 1975 1980 Ser Asp Ile Ile Glu Asp Ser
Asn Thr Val Leu Ala Asn Leu Leu 1985 1990 1995 Pro Leu Ser Lys Glu
Asp Ser Ile Lys Ile Glu Lys Trp Ile Leu 2000 2005 2010 Val Glu Lys
Ala Lys Val His Asp Trp Ile Val Arg Glu Leu Lys 2015 2020 2025 Glu
Gly Ser Ala Ser Ser Gly Met Leu Arg Pro Tyr His Gln Ala 2030 2035
2040 Leu Gln Ile Phe Gly Phe Glu Pro Asn Leu Asn Lys Leu Cys Arg
2045 2050 2055 Asp Phe Leu Ser Thr Leu Asn Ile Val Asp Thr Lys Asn
Cys Ile 2060 2065 2070 Ile Thr Phe Asp Arg Val Leu Arg Asp Thr Ile
Phe Glu Trp Thr 2075 2080 2085 Arg Ile Lys Asp Ala Asp Lys Lys Leu
Arg Leu Thr Gly Lys Tyr 2090 2095 2100 Asp Leu Tyr Pro Leu Arg Asp
Ser Gly Lys Leu Lys Val Ile Ser 2105 2110 2115 Arg Arg Leu Val Ile
Ser Trp Ile Ala Leu Ser Met Ser Thr Arg 2120 2125 2130 Leu Val Thr
Gly Ser Phe Pro Asp Ile Lys Phe Glu Ser Arg Leu 2135 2140 2145 Gln
Leu Gly Ile Val Ser Ile Ser Ser Arg Glu Ile Lys Asn Leu 2150 2155
2160 Arg Val Ile Ser Lys Ile Val Ile Asp Lys Phe Glu Asp Ile Ile
2165 2170 2175 His Ser Val Thr Tyr Arg Phe Leu Thr Lys Glu Ile Lys
Ile Leu 2180 2185 2190 Met Lys Ile Leu Gly Ala Val Lys Leu Phe Gly
Ala Arg Gln Ser 2195 2200 2205 Thr Ser Ala Asp Ile Thr Asn Ile Asp
Thr Ser Asp Ser Ile Gln 2210 2215 2220 16 2233 PRT bovine
parainfluenza virus 3 16 Met Asp Thr Glu Ser His Ser Gly Thr Thr
Ser Asp Ile Leu Tyr Pro 1 5 10 15 Glu Cys His Leu Asn Ser Pro Ile
Val Lys Gly Lys Ile Ala Gln Leu 20 25 30 His Thr Ile Met Ser Leu
Pro Gln Pro Tyr Asp Met Asp Asp Asp Ser 35 40 45 Ile Leu Ile Ile
Thr Arg Gln Lys Ile Lys Leu Asn Lys Leu Asp Lys 50 55 60 Arg Gln
Arg Ser Ile Arg Lys Leu Arg Ser Val Leu Met Glu Arg Val 65 70 75 80
Ser Asp Leu Gly Lys Tyr Thr Phe Ile Arg Tyr Pro Glu Met Ser Ser 85
90 95 Glu Met Phe Gln Leu Cys Ile Pro Gly Ile Asn Asn Lys Ile Asn
Glu 100 105 110 Leu Leu Ser Lys Ala Ser Lys Thr Tyr Asn Gln Met Thr
Asp Gly Leu 115 120 125 Arg Asp Leu Trp Val Thr Ile Leu Ser Lys Leu
Ala Ser Lys Asn Asp 130 135 140 Gly Ser Asn Tyr Asp Ile Asn Glu Asp
Ile Ser Asn Ile Ser Asn Val 145 150 155 160 His Met Thr Tyr Gln Ser
Asp Lys Trp Tyr Asn Pro Phe Lys Thr Trp 165 170 175 Phe Thr Ile Lys
Tyr Asp Met Arg Arg Leu Gln Lys Ala Lys Asn Glu 180 185 190 Ile Thr
Phe Asn Arg His Lys Asp Tyr Asn Leu Leu Glu Asp Gln Lys 195 200 205
Asn Ile Leu Leu Ile His Pro Glu Leu Val Leu Ile Leu Asp Lys Gln 210
215 220 Asn Tyr Asn Gly Tyr Ile Met Thr Pro Glu Leu Val Leu Met Tyr
Cys 225 230 235 240 Asp Val Val Glu Gly Arg Trp Asn Ile Ser Ser Cys
Ala Lys Leu Asp 245 250 255 Pro Lys Leu Gln Ser Met Tyr Tyr Lys Gly
Asn Asn Leu Trp Glu Ile 260 265 270 Ile Asp Gly Leu Phe Ser Thr Leu
Gly Glu Arg Thr Phe Asp Ile Ile 275 280 285 Ser Leu Leu Glu Pro Leu
Ala Leu Ser Leu Ile Gln Thr Tyr Asp Pro 290 295 300 Val Lys Gln Leu
Arg Gly Ala Phe Leu Asn His Val Leu Ser Glu Met 305 310 315 320 Glu
Leu Ile Phe Ala Ala Glu Cys Thr Thr Glu Glu Ile Pro Asn Val 325 330
335 Asp Tyr Ile Asp Lys Ile Leu Asp Val Phe Lys Glu Ser Thr Ile Asp
340 345 350 Glu Ile Ala Glu Ile Phe Ser Phe Phe Arg Thr Phe Gly His
Pro Pro 355 360 365 Leu Glu Ala Ser Ile Ala Ala Glu Lys Val Arg Lys
Tyr Met Tyr Thr 370 375 380 Glu Lys Cys Leu Lys Phe Asp Thr Ile Asn
Lys Cys His Ala Ile Phe 385 390 395 400 Cys Thr Ile Ile Ile Asn Gly
Tyr Arg Glu Arg His Gly Gly Gln Trp 405 410 415 Pro Pro Val Thr Leu
Pro Val His Ala His Glu Phe Ile Ile Asn Ala 420 425 430 Tyr Gly Ser
Asn Ser Ala Ile Ser Tyr Glu Asn Ala Val Asp Tyr Tyr 435 440 445 Lys
Ser Phe Ile Gly Ile Lys Phe Asp Lys Phe Ile Glu Pro Gln Leu 450 455
460 Asp Glu Asp Leu Thr Ile Tyr Met Lys Asp Lys Ala Leu Ser Pro Lys
465 470 475 480 Lys Ser Asn Trp Asp Thr Val Tyr Pro Ala Ser Asn Leu
Leu Tyr Arg 485 490 495 Thr Asn Val Ser His Asp Ser Arg Arg Leu Val
Glu Val Phe Ile Ala 500 505 510 Asp Ser Lys Phe Asp Pro His Gln Val
Leu Asp Tyr Val Glu Ser Gly 515 520 525 Tyr Trp Leu Asp Asp Pro Glu
Phe Asn Ile Ser Tyr Ser Leu Lys Glu 530 535 540 Lys Glu Ile Lys Gln
Glu Gly Arg Leu Phe Ala Lys Met Thr Tyr Lys 545 550 555 560 Met Arg
Ala Thr Gln Val Leu Ser Glu Thr Leu Leu Ala Asn Asn Ile 565 570 575
Gly Lys Phe Phe Gln Glu Asn Gly Met Val Lys Gly Glu Ile Glu Leu 580
585 590 Leu Lys Arg Leu Thr Thr Ile Ser Met Ser Gly Val Pro Arg Tyr
Asn 595 600 605 Glu Val Tyr Asn Asn Ser Lys Ser His Thr Glu Glu Leu
Gln Ala Tyr 610 615 620 Asn Ala Ile Ser Ser Ser Asn Leu Ser Ser Asn
Gln Lys Ser Lys Lys 625 630 635 640 Phe Glu Phe Lys Ser Thr Asp Ile
Tyr Asn Asp Gly Tyr Glu Thr Val 645 650 655 Ser Cys Phe Leu Thr Thr
Asp Leu Lys Lys Tyr Cys Leu Asn Trp Arg 660 665 670 Tyr Glu Ser Thr
Ala Leu Phe Gly Asp Thr Cys Asn Gln Ile Phe Gly 675 680 685 Leu Lys
Glu Leu Phe Asn Trp Leu His Pro Arg Leu Glu Lys Ser
Thr 690 695 700 Ile Tyr Val Gly Asp Pro Tyr Cys Pro Pro Ser Asp Ile
Glu His Leu 705 710 715 720 Pro Leu Asp Asp His Pro Asp Ser Gly Phe
Tyr Val His Asn Pro Lys 725 730 735 Gly Gly Ile Glu Gly Phe Cys Gln
Lys Leu Trp Thr Leu Ile Ser Ile 740 745 750 Ser Ala Ile His Leu Ala
Ala Val Lys Ile Gly Val Arg Val Thr Ala 755 760 765 Met Val Gln Gly
Asp Asn Gln Ala Ile Ala Val Thr Thr Arg Val Pro 770 775 780 Asn Asn
Tyr Asp Tyr Lys Val Lys Lys Glu Ile Val Tyr Lys Asp Val 785 790 795
800 Val Arg Phe Phe Asp Ser Leu Arg Glu Val Met Asp Asp Leu Gly His
805 810 815 Glu Leu Lys Leu Asn Glu Thr Ile Ile Ser Ser Lys Met Phe
Ile Tyr 820 825 830 Ser Lys Arg Ile Tyr Tyr Asp Gly Arg Ile Leu Pro
Gln Ala Leu Lys 835 840 845 Ala Leu Ser Arg Cys Val Phe Trp Ser Glu
Thr Ile Ile Asp Glu Thr 850 855 860 Arg Ser Ala Ser Ser Asn Leu Ala
Thr Ser Phe Ala Lys Ala Ile Glu 865 870 875 880 Asn Gly Tyr Ser Pro
Val Leu Gly Tyr Val Cys Ser Ile Phe Lys Asn 885 890 895 Ile Gln Gln
Leu Tyr Ile Ala Leu Gly Met Asn Ile Asn Pro Thr Ile 900 905 910 Thr
Gln Asn Ile Lys Asp Gln Tyr Phe Arg Asn Ile His Trp Met Gln 915 920
925 Tyr Ala Ser Leu Ile Pro Ala Ser Val Gly Gly Phe Asn Tyr Met Ala
930 935 940 Met Ser Arg Cys Phe Val Arg Asn Ile Gly Asp Pro Thr Val
Ala Ala 945 950 955 960 Leu Ala Asp Ile Lys Arg Phe Ile Lys Ala Asn
Leu Leu Asp Arg Gly 965 970 975 Val Leu Tyr Arg Ile Met Asn Gln Glu
Pro Gly Glu Ser Ser Phe Leu 980 985 990 Asp Trp Ala Ser Asp Pro Tyr
Ser Cys Asn Leu Pro Gln Ser Gln Asn 995 1000 1005 Ile Thr Thr Met
Ile Lys Asn Ile Thr Ala Arg Asn Val Leu Gln 1010 1015 1020 Asp Ser
Pro Asn Pro Leu Leu Ser Gly Leu Phe Thr Ser Thr Met 1025 1030 1035
Ile Glu Glu Asp Glu Glu Leu Ala Glu Phe Leu Met Asp Arg Arg 1040
1045 1050 Ile Ile Leu Pro Arg Val Ala His Asp Ile Leu Asp Asn Ser
Leu 1055 1060 1065 Thr Gly Ile Arg Asn Ala Ile Ala Gly Met Leu Asp
Thr Thr Lys 1070 1075 1080 Ser Leu Ile Arg Val Gly Ile Ser Arg Gly
Gly Leu Thr Tyr Asn 1085 1090 1095 Leu Leu Arg Lys Ile Ser Asn Tyr
Asp Leu Val Gln Tyr Glu Thr 1100 1105 1110 Leu Ser Lys Thr Leu Arg
Leu Ile Val Ser Asp Lys Ile Lys Tyr 1115 1120 1125 Glu Asp Met Cys
Ser Val Asp Leu Ala Ile Ser Leu Arg Gln Lys 1130 1135 1140 Met Trp
Met His Leu Ser Gly Gly Arg Met Ile Asn Gly Leu Glu 1145 1150 1155
Thr Pro Asp Pro Leu Glu Leu Leu Ser Gly Val Ile Ile Thr Gly 1160
1165 1170 Ser Glu His Cys Arg Ile Cys Tyr Ser Thr Glu Gly Glu Ser
Pro 1175 1180 1185 Tyr Thr Trp Met Tyr Leu Pro Gly Asn Leu Asn Ile
Gly Ser Ala 1190 1195 1200 Glu Thr Gly Ile Ala Ser Leu Arg Val Pro
Tyr Phe Gly Ser Val 1205 1210 1215 Thr Asp Glu Arg Ser Glu Ala Gln
Leu Gly Tyr Ile Lys Asn Leu 1220 1225 1230 Ser Lys Pro Ala Lys Ala
Ala Ile Arg Ile Ala Met Ile Tyr Thr 1235 1240 1245 Trp Ala Phe Gly
Asn Asp Glu Ile Ser Trp Met Glu Ala Ser Gln 1250 1255 1260 Ile Ala
Gln Thr Arg Ala Asn Phe Thr Leu Asp Ser Leu Lys Ile 1265 1270 1275
Leu Thr Pro Val Thr Thr Ser Thr Asn Leu Ser His Arg Leu Lys 1280
1285 1290 Asp Thr Ala Thr Gln Met Lys Phe Ser Ser Thr Ser Leu Ile
Arg 1295 1300 1305 Val Ser Arg Phe Ile Thr Ile Ser Asn Asp Asn Met
Ser Ile Lys 1310 1315 1320 Glu Ala Asn Glu Thr Lys Asp Thr Asn Leu
Ile Tyr Gln Gln Val 1325 1330 1335 Met Leu Thr Gly Leu Ser Val Phe
Glu Tyr Leu Phe Arg Leu Glu 1340 1345 1350 Glu Ser Thr Gly His Asn
Pro Met Val Met His Leu His Ile Glu 1355 1360 1365 Asp Gly Cys Cys
Ile Lys Glu Ser Tyr Asn Asp Glu His Ile Asn 1370 1375 1380 Pro Glu
Ser Thr Leu Glu Leu Ile Lys Tyr Pro Glu Ser Asn Glu 1385 1390 1395
Phe Ile Tyr Asp Lys Asp Pro Leu Lys Asp Ile Asp Leu Ser Lys 1400
1405 1410 Leu Met Val Ile Arg Asp His Ser Tyr Thr Ile Asp Met Asn
Tyr 1415 1420 1425 Trp Asp Asp Thr Asp Ile Val His Ala Ile Ser Ile
Cys Thr Ala 1430 1435 1440 Val Thr Ile Ala Asp Thr Met Ser Gln Leu
Asp Arg Asp Asn Leu 1445 1450 1455 Lys Glu Leu Val Val Ile Ala Asn
Asp Asp Asp Ile Asn Ser Leu 1460 1465 1470 Ile Thr Glu Phe Leu Thr
Leu Asp Ile Leu Val Phe Leu Lys Thr 1475 1480 1485 Phe Gly Gly Leu
Leu Val Asn Gln Phe Ala Tyr Thr Leu Tyr Gly 1490 1495 1500 Leu Lys
Ile Glu Gly Arg Asp Pro Ile Trp Asp Tyr Ile Met Arg 1505 1510 1515
Thr Leu Lys Asp Thr Ser His Ser Val Leu Lys Val Leu Ser Asn 1520
1525 1530 Ala Leu Ser His Pro Lys Val Phe Lys Arg Phe Trp Asp Cys
Gly 1535 1540 1545 Val Leu Asn Pro Ile Tyr Gly Pro Asn Thr Ala Ser
Gln Asp Gln 1550 1555 1560 Val Lys Leu Ala Leu Ser Ile Cys Glu Tyr
Ser Leu Asp Leu Phe 1565 1570 1575 Met Arg Glu Trp Leu Asn Gly Ala
Ser Leu Glu Ile Tyr Ile Cys 1580 1585 1590 Asp Ser Asp Met Glu Ile
Ala Asn Asp Arg Arg Gln Ala Phe Leu 1595 1600 1605 Ser Arg His Leu
Ala Phe Val Cys Cys Leu Ala Glu Ile Ala Ser 1610 1615 1620 Phe Gly
Pro Asn Leu Leu Asn Leu Thr Tyr Leu Glu Arg Leu Asp 1625 1630 1635
Glu Leu Lys Gln Tyr Leu Asp Leu Asn Ile Lys Glu Asp Pro Thr 1640
1645 1650 Leu Lys Tyr Val Gln Val Ser Gly Leu Leu Ile Lys Ser Phe
Pro 1655 1660 1665 Ser Thr Val Thr Tyr Val Arg Lys Thr Ala Ile Lys
Tyr Leu Arg 1670 1675 1680 Ile Arg Gly Ile Asn Pro Pro Glu Thr Ile
Glu Asp Trp Asp Pro 1685 1690 1695 Ile Glu Asp Glu Asn Ile Leu Asp
Asn Ile Val Lys Thr Val Asn 1700 1705 1710 Asp Asn Cys Ser Asp Asn
Gln Lys Arg Asn Lys Ser Ser Tyr Phe 1715 1720 1725 Trp Gly Leu Ala
Leu Lys Asn Tyr Gln Val Val Lys Ile Arg Ser 1730 1735 1740 Ile Thr
Ser Asp Ser Glu Val Asn Glu Ala Ser Asn Val Thr Thr 1745 1750 1755
His Gly Met Thr Leu Pro Gln Gly Gly Ser Tyr Leu Ser His Gln 1760
1765 1770 Leu Arg Leu Phe Gly Val Asn Ser Thr Ser Cys Leu Lys Ala
Leu 1775 1780 1785 Glu Leu Ser Gln Ile Leu Met Arg Glu Val Lys Lys
Asp Lys Asp 1790 1795 1800 Arg Leu Phe Leu Gly Glu Gly Ala Gly Ala
Met Leu Ala Cys Tyr 1805 1810 1815 Asp Ala Thr Leu Gly Pro Ala Ile
Asn Tyr Tyr Asn Ser Gly Leu 1820 1825 1830 Asn Ile Thr Asp Val Ile
Gly Gln Arg Glu Leu Lys Ile Phe Pro 1835 1840 1845 Ser Glu Val Ser
Leu Val Gly Lys Lys Leu Gly Asn Val Thr Gln 1850 1855 1860 Ile Leu
Asn Arg Val Arg Val Leu Phe Asn Gly Asn Pro Asn Ser 1865 1870 1875
Thr Trp Ile Gly Asn Met Glu Cys Glu Ser Leu Ile Trp Ser Glu 1880
1885 1890 Leu Asn Asp Lys Ser Ile Gly Leu Val His Cys Asp Met Glu
Gly 1895 1900 1905 Ala Ile Gly Lys Ser Glu Glu Thr Val Leu His Glu
His Tyr Ser 1910 1915 1920 Ile Ile Arg Ile Thr Tyr Leu Ile Gly Asp
Asp Asp Val Val Leu 1925 1930 1935 Val Ser Lys Ile Ile Pro Thr Ile
Thr Pro Asn Trp Ser Lys Ile 1940 1945 1950 Leu Tyr Leu Tyr Lys Leu
Tyr Trp Lys Asp Val Ser Val Val Ser 1955 1960 1965 Leu Lys Thr Ser
Asn Pro Ala Ser Thr Glu Leu Tyr Leu Ile Ser 1970 1975 1980 Lys Asp
Ala Tyr Cys Thr Val Met Glu Pro Ser Asn Leu Val Leu 1985 1990 1995
Ser Lys Leu Lys Arg Ile Ser Ser Ile Glu Glu Asn Asn Leu Leu 2000
2005 2010 Lys Trp Ile Ile Leu Ser Lys Arg Lys Asn Asn Glu Trp Leu
Gln 2015 2020 2025 His Glu Ile Lys Glu Gly Glu Arg Asp Tyr Gly Ile
Met Arg Pro 2030 2035 2040 Tyr His Thr Ala Leu Gln Ile Phe Gly Phe
Gln Ile Asn Leu Asn 2045 2050 2055 His Leu Ala Arg Glu Phe Leu Ser
Thr Pro Asp Leu Thr Asn Ile 2060 2065 2070 Asn Asn Ile Ile Gln Ser
Phe Thr Arg Thr Ile Lys Asp Val Met 2075 2080 2085 Phe Glu Trp Val
Asn Ile Thr His Asp Asn Lys Arg His Lys Leu 2090 2095 2100 Gly Gly
Arg Tyr Asn Leu Phe Pro Leu Lys Asn Lys Gly Lys Leu 2105 2110 2115
Arg Leu Leu Ser Arg Arg Leu Val Leu Ser Trp Ile Ser Leu Ser 2120
2125 2130 Leu Ser Thr Arg Leu Leu Thr Gly Arg Phe Pro Asp Glu Lys
Phe 2135 2140 2145 Glu Asn Arg Ala Gln Thr Gly Tyr Val Ser Leu Ala
Asp Ile Asp 2150 2155 2160 Leu Glu Ser Leu Lys Leu Leu Ser Arg Asn
Ile Val Lys Asn Tyr 2165 2170 2175 Lys Glu His Ile Gly Leu Ile Ser
Tyr Trp Phe Leu Thr Lys Glu 2180 2185 2190 Val Lys Ile Leu Met Lys
Leu Ile Gly Gly Val Lys Leu Leu Gly 2195 2200 2205 Ile Pro Lys Gln
Tyr Lys Glu Leu Glu Asp Arg Ser Ser Gln Gly 2210 2215 2220 Tyr Glu
Tyr Asp Asn Glu Phe Asp Ile Asp 2225 2230 17 29 RNA human
metapneumovirus 17 cauauuuaau cuaagguuuu uuuauaccc 29 18 44 RNA
human metapneumovirus 18 ugcgcuuuuu uugcgcauau uuaauucaau
guuuuuuugu accc 44 19 44 RNA avian metapneumovirus 19 ugcucuuuuu
uugcguaagu ucguccaaga ucuuuuuauu accc 44 20 47 RNA human
respiratory syncytial virus strain A2 20 ugcgcuuuuu uacgcauguu
guuugaacgu auuugguuuu uuuaccc 47 21 33 RNA human metapneumovirus 21
ccguauacau ucaauuauaa uuucuuauuu uua 33 22 45 RNA human
metapneumovirus 22 acggcaaaaa aaccguauac auucaauuau aauuucuuau
uuuua 45 23 45 RNA avian metapneumovirus 23 acgagaaaaa aaccguauuc
aucaaauuuu uagcuuuuag uuuuu 45 24 46 RNA human respiratory
syncytial virus strain A2 24 acgagaaaaa aagugucaaa aacuaauauc
ucguaauuua guuaau 46 25 20 DNA Artificial Sequence HMPV strain 83
gene start consensus sequence. 25 atgggacaag tgaaaatgtc 20 26 20
DNA Artificial Sequence HMPV strain 83 gene start consensus
sequence with alternative assignments. 26 gcgagataaa tagttatgga 20
27 20 DNA Artificial Sequence HMPV strain 83 gene start consensus
sequence with alternative assignments. 27 tagggacaag tcacaatgat 20
28 17 DNA Artificial Sequence HMPV strain 83 gene end consensus
sequence. 28 ttagttaatt aaaaata 17 29 17 DNA Artificial Sequence
HMPV strain 83 gene end consensus sequence with alternative
assignments. 29 agagtattaa taaaacc 17 30 17 DNA Artificial Sequence
HMPV strain 83 gene end consensus sequence with alternative
assignments. 30 gaagttagct aaaaagt 17 31 10 DNA human respiratory
syncytial virus 31 ggggcaaata 10 32 17 DNA Artificial Sequence HMPV
strain 83 gene start consensus sequence. 32 tgagacaagt gaaaatg 17
33 46 DNA Artificial Sequence Synthetic oligonucleotide. 33
cccggggacg tcctagctag ctagggtacc ccgctcgagc ggtccg 46 34 13 DNA
Artificial Sequence HMPV gene end consensus sequence. 34 agttannnaa
aaa 13 35 16 DNA Artificial Sequence HMPV gene start consensus
sequence. 35 gggacaantn nnaatg 16 36 13350 DNA human
metapneumovirus 36 gtataaatta gattccaaaa aaatatggga caagtgaaaa
tgtctcttca agggattcac 60 ctgagtgatt tatcatacaa gcatgctata
ttaaaagagt ctcagtacac aataaaaaga 120 gatgtgggta caacaactgc
agtgacaccc tcatcattgc aacaagaaat aacactgttg 180 tgtggagaaa
ttctgtatgc taaacatgct gactacaaat atgctgcaga aataggaata 240
caatatatta gcacagcttt aggatcagag agagtgcagc agattctgag gaactcaggc
300 agtgaagtcc aagtggtctt aaccagaacg tactctctgg ggaaaattaa
aaacaataaa 360 ggagaagatt tacagatgtt agacatacac ggggtagaga
agagctgggt agaagagata 420 gacaaagaag caaggaaaac aatggcaacc
ttgcttaagg aatcatcagg taatatccca 480 caaaatcaga ggccctcagc
accagacaca cccataatct tattatgtgt aggtgcctta 540 atattcacta
aactagcatc aaccatagaa gtgggactag agaccacagt cagaagggct 600
aaccgtgtac taagtgatgc actcaagaga taccctagaa tggacatacc aaagattgcc
660 agatccttct atgacttatt tgaacaaaaa gtgtatcaca gaagtttgtt
cattgagtat 720 ggcaaagcat taggctcatc atctacaggc agcaaagcag
aaagtctatt tgttaatata 780 ttcatgcaag cttatggggc cggtcaaaca
atgctaaggt ggggggtcat tgccaggtca 840 tccaacaata taatgttagg
acatgtatcc gtccaagctg agttaaaaca ggtcacagaa 900 gtctatgact
tggtgcgaga aatgggccct gaatctggac ttctacattt aaggcaaagc 960
ccaaaagctg gactgttatc actagccaac tgtcccaact ttgcaagtgt tgttctcgga
1020 aatgcctcag gcttaggcat aatcggtatg tatcgaggga gagtaccaaa
cacagaatta 1080 ttttcagcag ctgaaagtta tgccaaaagt ttgaaagaaa
gcaataaaat aaatttctct 1140 tcattaggac ttacagatga agagaaagag
gctgcagaac atttcttaaa tgtgagtgac 1200 gacagtcaaa atgattatga
gtaattaaaa aagtgggaca agtcaaaatg tcattccctg 1260 aaggaaaaga
tattcttttc atgggtaatg aagcagcaaa attagcagaa gctttccaga 1320
aatcattaag aaaaccaggt cataaaagat ctcaatctat tataggagaa aaagtgaata
1380 ctgtatcaga aacattggaa ttacctacta tcagtagacc tgcaaaacca
accataccgt 1440 cagaaccaaa gttagcatgg acagataaag gtggggcaac
caaaactgaa ataaagcaag 1500 caatcaaagt catggatccc attgaagaag
aagagtctac cgagaagaag gtgctaccct 1560 ccagtgatgg gaaaacccct
gcagaaaaga aactgaaacc atcaactaac accaaaaaga 1620 aggtttcatt
tacaccaaat gaaccaggga aatatacaaa gttggaaaaa gatgctctag 1680
atttgctctc agataatgaa gaagaagatg cagaatcttc aatcttaacc tttgaagaaa
1740 gagatacttc atcattaagc attgaggcca gattggaatc aatagaggag
aaattaagca 1800 tgatattagg gctattaaga acactcaaca ttgctacagc
aggacccaca gcagcaagag 1860 atgggatcag agatgcaatg attggcgtaa
gagaggaatt aatagcagac ataataaagg 1920 aagctaaagg gaaagcagca
gaaatgatgg aagaggaaat gagtcaacga tcaaaaatag 1980 gaaatggtag
tgtaaaatta acagaaaaag caaaagagct caacaaaatt gttgaagatg 2040
aaagcacaag tggagaatcc gaagaagaag aagaaccaaa agacacacaa gacaatagtc
2100 aagaagatga catttaccag ttaattatgt agtttaataa aaataaacaa
tgggacaagt 2160 aaaaatggag tcctacctag tagacaccta tcaaggcatt
ccttacacag cagctgttca 2220 agttgatcta atagaaaagg acctgttacc
tgcaagccta acaatatggt tccctttgtt 2280 tcaggccaac acaccaccag
cagtgctgct cgatcagcta aaaaccctga caataaccac 2340 tctgtatgct
gcatcacaaa atggtccaat actcaaagtg aatgcatcag cccaaggtgc 2400
agcaatgtct gtacttccca aaaaatttga agtcaatgcg actgtagcac tcgatgaata
2460 tagcaaactg gaatttgaca aactcacagt ctgtgaagta aaaacagttt
acttaacaac 2520 catgaaacca tacgggatgg tatcaaaatt tgtgagctca
gccaaatcag ttggcaaaaa 2580 aacacatgat ctaatcgcac tatgtgattt
tatggatcta gaaaagaaca cacctgttac 2640 aataccagca ttcatcaaat
cagtttcaat caaagagagt gagtcagcta ctgttgaagc 2700 tgctataagc
agtgaagcag accaagctct aacacaggcc aaaattgcac cttatgcggg 2760
attaattatg atcatgacta tgaacaatcc caaaggcata ttcaaaaagc ttggagctgg
2820 gactcaagtc atagtagaac taggagcata tgtccaggct gaaagcataa
gcaaaatatg 2880 caagacttgg agccatcaag ggacaagata tgtcttgaag
tccagataac aaccaagcac 2940 cttggccaag agctactaac cctatctcat
agatcataaa gtcaccattc tagttatata 3000 aaaatcaagt tagaacaaga
attaaatcaa tcaagaacgg gacaaataaa aatgtcttgg 3060 aaagtggtga
tcattttttc attgttaata acacctcaac acggtcttaa agagagctac 3120
ttagaagagt catgtagcac tataactgaa ggatatctca
gtgttctgag gacaggttgg 3180 tacaccaatg tttttacact ggaggtaggc
gatgtagaga accttacatg tgccgatgga 3240 cccagcttaa taaaaacaga
attagacctg accaaaagtg cactaagaga gctcagaaca 3300 gtttctgctg
atcaactggc aagagaggag caaattgaaa atcccagaca atctagattc 3360
gttctaggag caatagcact cggtgttgca actgcagctg cagttacagc aggtgttgca
3420 attgccaaaa ccatccggct tgaaagtgaa gtaacagcaa ttaagaatgc
cctcaaaaag 3480 accaatgaag cagtatctac attggggaat ggagttcgtg
tgttggcaac tgcagtgaga 3540 gagctgaaag attttgtgag caagaatcta
acacgtgcaa tcaacaaaaa caagtgcgac 3600 attgctgacc tgaaaatggc
cgttagcttc agtcaattca acagaaggtt cctaaatgtt 3660 gtgcggcaat
tttcagacaa cgctggaata acaccagcaa tatctttgga cttaatgaca 3720
gatgctgaac tagccagagc tgtttccaac atgccaacat ctgcaggaca aataaaactg
3780 atgttggaga accgtgcaat ggtaagaaga aaagggttcg gattcctgat
aggagtttac 3840 ggaagctccg taatttacat ggtgcaactg ccaatctttg
gggttataga cacgccttgc 3900 tggatagtaa aagcagcccc ttcttgttca
ggaaaaaagg gaaactatgc ttgcctctta 3960 agagaagacc aaggatggta
ttgtcaaaat gcagggtcaa ctgtttacta cccaaatgaa 4020 aaagactgtg
aaacaagagg agaccatgtc ttttgcgaca cagcagcagg aatcaatgtt 4080
gctgagcagt caaaggagtg caacataaac atatctacta ctaattaccc atgcaaagtt
4140 agcacaggaa gacatcctat cagtatggtt gcactatctc ctcttggggc
tttggttgct 4200 tgctacaagg gagtgagctg ttccattggc agcaacagag
tagggatcat caagcaactg 4260 aacaaaggct gctcttatat aaccaaccaa
gacgcagaca cagtgacaat agacaacact 4320 gtataccagc taagcaaagt
tgaaggcgaa cagcatgtta taaaaggaag gccagtgtca 4380 agcagctttg
acccagtcaa gtttcctgaa gatcaattca atgttgcact tgaccaagtt 4440
ttcgagagca ttgagaacag tcaggccttg gtggatcaat caaacagaat cctaagcagt
4500 gcagagaaag gaaacactgg cttcatcatt gtaataattc taattgctgt
ccttggctct 4560 accatgatcc tagtgagtgt ttttatcata ataaagaaaa
caaagaaacc cacaggagca 4620 cctccagagc tgagtggtgt cacaaacaat
ggcttcatac cacataatta gttaattaaa 4680 aataaagtaa attaaaataa
attaaaatta aaaataaaaa tttgggacaa atcataatgt 4740 ctcgcaaggc
tccgtgcaaa tatgaagtgc ggggcaaatg caatagagga agtgagtgca 4800
agtttaacca caattactgg agttggccag atagatactt attaataaga tcaaattatt
4860 tattaaatca acttttaagg aacactgata gagctgatgg cttatcaata
atatcaggag 4920 caggcagaga agataggaca caagattttg tcctaggttc
caccaatgtg gttcaaggtt 4980 atattgatga taaccaaagc ataacaaaag
ctgcagcctg ttacagtcta cataatataa 5040 tcaaacaact acaagaagtt
gaagttaggc aggctagaga taacaaacta tctgacagca 5100 aacatgtagc
acttcacaac ttagtcctat cttatatgga gatgagcaaa actcctgcat 5160
ctttaatcaa caatctcaag agactgccga gagagaaact gaaaaaatta gcaaagctca
5220 taattgactt atcagcaggt gctgaaaatg actcttcata tgccttgcaa
gacagtgaaa 5280 gcactaatca agtgcagtga gcatggtcca gttttcatta
ctatagaggt tgatgacatg 5340 atatggactc acaaggactt aaaagaagct
ttatctgatg ggatagtgaa gtctcatact 5400 aacatttaca attgttattt
agaaaacata gaaattatat atgtcaaggc ttacttaagt 5460 tagtaaaaac
acatcagagt gggataaatg acaatgataa cattagatgt cattaaaagt 5520
gatgggtctt caaaaacatg tactcacctc aaaaaaataa ttaaagacca ctctggtaaa
5580 gtgcttattg tacttaagtt aatattagct ttactaacat ttctcacagt
aacaatcacc 5640 atcaattata taaaagtgga aaacaatctg caaatatgcc
agtcaaaaac tgaatcagac 5700 aaaaaggact catcatcaaa taccacatca
gtcacaacca agactactct aaatcatgat 5760 atcacacagt attttaaaag
tttgattcaa aggtatacaa actctgcaat aaacagtgac 5820 acatgctgga
aaataaacag aaatcaatgc acaaatataa caacatacaa atttttatgt 5880
tttaaatctg aagacacaaa aaccaacaat tgtgataaac tgacagattt atgcagaaac
5940 aaaccaaaac cagcagttgg agtgtatcac atagtagaat gccattgtat
atacacagtt 6000 aaatggaagt gctatcatta cccaaccgat gaaacccaat
cctaaatgtt aacaccagat 6060 taggatccat ccaagtctgt tagttcaaca
atttagttat ttaaaaatat tttgaaaaca 6120 agtaagtttc tatgatactt
cataataata agtaataatt aattgcttaa tcatcatcac 6180 aacattattc
gaaaccataa ctattcaatt taaaaagtaa aaaacaataa catgggacaa 6240
gtagttatgg aggtgaaagt ggagaacatt cgaacaatag atatgctcaa agcaagagta
6300 aaaaatcgtg tggcacgcag caaatgcttt aaaaatgcct ctttggtcct
cataggaata 6360 actacattga gtattgccct caatatctat ctgatcataa
actataaaat gcaaaaaaac 6420 acatctgaat cagaacatca caccagctca
tcacccatgg aatccagcag agaaactcca 6480 acggtcccca cagacaactc
agacaccaac tcaagcccac agcatccaac tcaacagtcc 6540 acagaaggct
ccacactcta ctttgcagcc tcagcaagct caccagagac agaaccaaca 6600
tcaacaccag atacaacaaa ccgcccgccc ttcgtcgaca cacacacaac accaccaagc
6660 gcaagcagaa caaagacaag tccggcagtc cacacaaaaa acaacccaag
gacaagctct 6720 agaacacatt ctccaccacg ggcaacgaca aggacggcac
gcagaaccac cactctccgc 6780 acaagcagca caagaaagag accgtccaca
gcatcagtcc aacctgacat cagcgcaaca 6840 acccacaaaa acgaagaagc
aagtccagcg agcccacaaa catctgcaag cacaacaaga 6900 atacaaagga
aaagcgtgga ggccaacaca tcaacaacat acaaccaaac tagttaacaa 6960
aaaatacaaa ataactctaa gataaaccat gcagacacca acaatggaga agccaaaaga
7020 caattcacaa tctccccaaa aaggcaacaa caccatatta gctctgccca
aatctccctg 7080 gaaaaaacac tcgcccatat accaaaaata ccacaaccac
cccaagaaaa aaactgggca 7140 aaacaacacc caagagacaa ataacaatgg
atcctctcaa tgaatccact gttaatgtct 7200 atcttcctga ctcatatctt
aaaggagtga tttcctttag tgagactaat gcaattggtt 7260 catgtctctt
aaaaagacct tacctaaaaa atgacaacac tgcaaaagtt gccatagaga 7320
atcctgttat cgagcatgtt agactcaaaa atgcagtcaa ttctaagatg aaaatatcag
7380 attacaagat agtagagcca gtaaacatgc aacatgaaat tatgaagaat
gtacacagtt 7440 gtgagctcac attattaaaa cagtttttaa caaggagtaa
aaatattagc actctcaaat 7500 taaatatgat atgtgattgg ctgcagttaa
agtctacatc agatgatacc tcaatcctaa 7560 gttttataga tgtagaattt
atacctagct gggtaagcaa ttggtttagt aattggtaca 7620 atctcaacaa
gttgattctg gaattcagga aagaagaagt aataagaact ggttcaatct 7680
tgtgtaggtc attgggtaaa ttagtttttg ttgtatcatc atatggatgt atagtcaaga
7740 gcaacaaaag caaaagagtg agcttcttca catacaatca actgttaaca
tggaaagatg 7800 tgatgttaag tagattcaat gcaaattttt gtatatgggt
aagcaacagt ctgaatgaaa 7860 atcaagaagg gctagggttg agaagtaatc
tgcaaggcat attaactaat aagctatatg 7920 aaactgtaga ttatatgctt
agtttatgtt gcaatgaagg tttctcactt gtgaaagagt 7980 tcgaaggctt
tattatgagt gaaattctta ggattactga acatgctcaa ttcagtacta 8040
gatttagaaa tactttatta aatggattaa ctgatcaatt aacaaaatta aaaaataaaa
8100 acagactcag agttcatggt accgtgttag aaaataatga ttatccaatg
tacgaagttg 8160 tacttaagtt attaggagat actttgagat gtattaaatt
attaatcaat aaaaacttag 8220 agaatgctgc tgaattatac tatatattta
gaatattcgg tcacccaatg gtagatgaaa 8280 gagatgcaat ggatgctgtc
aaattaaaca atgaaatcac aaaaatcctt aggtgggaga 8340 gcttgacaga
actaagaggg gcattcatat taaggattat caaaggattt gtagacaaca 8400
acaaaagatg gcccaaaatt aaaaacttaa aagtgcttag taagagatgg actatgtact
8460 tcaaagcaaa aagttacccc agtcaacttg aattaagcga acaagatttt
ttagagcttg 8520 ctgcaataca gtttgaacaa gagttttctg tccctgaaaa
aaccaacctt gagatggtat 8580 taaatgataa agctatatca cctcctaaaa
gattaatatg gtctgtgtat ccaaaaaatt 8640 acttacctga gaaaataaaa
aatcgatatc tagaagagac tttcaatgca agtgatagtc 8700 tcaaaacaag
aagagtacta gagtactatt tgaaagataa taaattcgac caaaaagaac 8760
ttaaaagtta tgttgttaaa caagaatatt taaatgataa ggatcatatt gtctcgctaa
8820 ctggaaaaga aagagaatta agtgtaggta gaatgtttgc tatgcaacca
ggaaaacagc 8880 gacaaataca aatattggct gaaaaattgt tagctgataa
tattgtacct tttttcccag 8940 aaaccttaac aaagtatggt gatctagatc
ttcagagaat aatggaaatc aaatcggaac 9000 tttcttctat taaaactaga
agaaatgata gttataataa ttacattgca agagcatcca 9060 tagtaacaga
tttaagtaag ttcaaccaag cctttaggta tgaaactaca gcgatctgtg 9120
cggatgtagc agatgaacta catggaacac aaagcctatt ctgttggtta catcttatcg
9180 tccctatgac aacaatgata tgtgcctata gacatgcacc accagaaaca
aaaggtgaat 9240 atgatataga taagatagaa gagcaaagtg gtttatatag
atatcatatg ggtggtattg 9300 aaggatggtg tcaaaaactc tggacaatgg
aagctatatc tctattagat gttgtatctg 9360 taaaaacacg atgtcaaatg
acatctttat taaacggtga caaccaatca atagatgtaa 9420 gtaaaccagt
taagttatct gagggtttag atgaagtgaa agcagattat agcttggctg 9480
taaaaatgtt aaaagaaata agagatgcat acagaaatat aggccataaa cttaaagaag
9540 gggaaacata tatatcaaga gatcttcagt ttataagtaa ggtgattcaa
tctgaaggag 9600 taatgcatcc tacccctata aaaaagatct taagagtggg
accatggata aacacaatat 9660 tagatgacat taaaaccagt gcagagtcaa
tagggagtct atgtcaggaa ttagaattta 9720 ggggggaaag cataatagtt
agtctgatat taaggaattt ttggctgtat aatttataca 9780 tgcatgaatc
aaagcaacac cccctagcag ggaagcagtt attcaaacaa ctaaataaaa 9840
cattaacatc agtgcagaga ttttttgaaa taaaaaagga aaatgaagta gtagatctat
9900 ggatgaacat accaatgcag tttggaggag gagatccagt agtcttctat
agatctttct 9960 atagaaggac ccctgatttt ttaactgaag caatcagtca
tgtggatatt ctgttaagaa 10020 tatcagccaa cataagaaat gaagcgaaaa
taagtttctt caaagcctta ctgtcaatag 10080 aaaaaaatga acgtgctaca
ctgacaacac taatgagaga tcctcaagct gttggctcag 10140 agcgacaagc
aaaagtaaca agtgatatca atagaacagc agttaccagc atcttaagtc 10200
tttctccaaa tcaacttttc agcgatagtg ctatacacta cagtagaaat gaagaagagg
10260 tcggaatcat tgctgacaac ataacacctg tttatcctca tggactgaga
gttttgtatg 10320 aatcattacc ttttcataaa gctgaaaaag ttgtgaatat
gatatcagga acgaaatcca 10380 taaccaactt attacagaga acatctgcta
ttaatggtga agatattgac agagctgtat 10440 ccatgatgct ggagaaccta
ggattattat ctagaatatt gtcagtagtt gttgatagta 10500 tagaaattcc
aaccaaatct aatggtaggc tgatatgttg tcagatatct agaaccctaa 10560
gggagacatc atggaataat atggaaatag ttggagtaac atcccctagc atcactacat
10620 gcatggatgt catatatgca actagctctc atttgaaagg gataatcatt
gaaaagttca 10680 gcactgacag aactacaaga ggtcaaagag gtccaaagag
cccttgggta gggtcgagca 10740 ctcaagagaa aaaattagtt cctgtttata
acagacaaat tctttcaaaa caacaaagag 10800 aacagctaga agcaattgga
aaaatgagat gggtatataa agggacacca ggtttaagac 10860 gattactcaa
taagatttgt cttggaagtt taggcattag ttacaaatgt gtaaaacctt 10920
tattacctag gtttatgagt gtaaatttcc tacacaggtt atctgtcagt agtagaccta
10980 tggaattccc agcatcagtt ccagcttata gaacaacaaa ttaccatttt
gacactagtc 11040 ctattaatca agcactaagt gagagatttg ggaatgaaga
tattaatttg gtcttccaaa 11100 atgcaatcag ctgtggaatt agcataatga
gtgtagtaga acaattaact ggtaggagtc 11160 caaaacagtt agttttaata
cctcaattag aagaaataga cattatgcca ccaccagtgt 11220 ttcaagggaa
attcaattat aagctagtag ataagataac ttctgatcaa catatcttca 11280
gtccagacaa aatagatatg ttaacactgg ggaaaatgct catgcccact ataaaaggtc
11340 agaaaacaga tcagttcctg aacaagagag agaattattt ccatgggaat
aatcttattg 11400 agtctttgtc agcagcgtta gcatgtcatt ggtgtgggat
attaacagag caatgtatag 11460 aaaataatat tttcaagaaa gactggggtg
acgggttcat atcggatcat gcttttatgg 11520 acttcaaaat attcctatgt
gtctttaaaa ctaaactttt atgtagttgg gggtcccaag 11580 ggaaaaacat
taaagatgaa gatatagtag atgaatcaat agataaactg ttaaggattg 11640
ataatacttt ttggagaatg ttcagcaagg ttatgtttga atcaaaggtt aagaaaagga
11700 taatgttata tgatgtaaaa tttctatcat tagtaggtta tatagggttt
aagaattggt 11760 ttatagaaca gttgagatca gctgagttgc atgaggtacc
ttggattgtc aatgccgaag 11820 gtgatctggt tgagatcaag tcaattaaaa
tctatttgca actgatagag caaagtttat 11880 ttttaagaat aactgttttg
aactatacag atatggcaca tgctctcaca agattaatca 11940 gaaagaagtt
gatgtgtgat aatgcactat taactccgat tccatcccca atggttaatt 12000
taactcaagt tattgatcct acagaacaat tagcttattt ccctaagata acatttgaaa
12060 ggctaaaaaa ttatgacact agttcaaatt atgctaaagg aaagctaaca
aggaattaca 12120 tgatactgtt gccatggcaa catgttaata gatataactt
tgtctttagt tctactggat 12180 gtaaagttag tctaaaaaca tgcattggaa
aacttatgaa agatctaaac cctaaagttc 12240 tgtactttat tggagaaggg
gcaggaaatt ggatggccag aacagcatgt gaatatcctg 12300 acatcaaatt
tgtatacaga agtttaaaag atgaccttga tcatcattat cctttggaat 12360
accagagagt tataggagaa ttaagcagga taatagatag cggtgaaggg ctttcaatgg
12420 aaacaacaga tgcaactcaa aaaactcatt gggatttgat acacagagta
agcaaagatg 12480 ctttattaat aactttatgt gatgcagaat ttaaggacag
agatgatttt tttaagatgg 12540 taattctatg gaggaaacat gtattatcat
gcagaatttg cactacttat gggacagacc 12600 tctatttatt cgcaaagtat
catgctaaag actgcaatgt aaaattacct ttttttgtga 12660 gatcagtagc
cacctttatt atgcaaggta gtaaactgtc aggctcagaa tgctacatac 12720
tcttaacact aggccaccac aacaatttac cctgccatgg agaaatacaa aattctaaga
12780 tgaaaatagc agtgtgtaat gatttttatg ctgcaaaaaa acttgacaat
aaatctattg 12840 aagccaactg taaatcactt ttatcagggc taagaatacc
gataaataag aaagaattaa 12900 atagacagag aaggttatta acactacaaa
gcaaccattc ttctgtagca acagttggag 12960 gtagcaaggt catagagtct
aaatggttaa caaacaaggc aaacacaata attgattggt 13020 tagaacatat
tttaaattct ccaaaaggtg aattaaatta tgattttttt gaagcattag 13080
aaaatactta ccctaatatg attaaactaa tagataatct agggaatgca gagataaaaa
13140 aactgatcaa agtaactgga tatatgcttg taagtaaaaa atgaaaaatg
ataaaaatga 13200 taaaataggt gacaacttca tactattcca aagtaatcat
ttgattatgc aattatgtaa 13260 tagttaatta aaaactaaaa atcaaaagtt
agaaactaac aactgtcatt aagtttatta 13320 aaaataagaa attataattg
gatgtatacg 13350
* * * * *