U.S. patent application number 12/365122 was filed with the patent office on 2009-10-01 for sequence analysis of the tetravalent rotavirus vaccine.
This patent application is currently assigned to THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by the Department of Health and Human. Invention is credited to Deborah A. Buonagurio, Alice F. Georgiu, Robert A. Lerch, Bruce B. Mason, Shridhara C. Murthy, Ruth S. Rappaport, Mohinder S. Sidhu, Stephen A. Udem, Timothy J. Zamb.
Application Number | 20090246829 12/365122 |
Document ID | / |
Family ID | 27766169 |
Filed Date | 2009-10-01 |
United States Patent
Application |
20090246829 |
Kind Code |
A1 |
Buonagurio; Deborah A. ; et
al. |
October 1, 2009 |
SEQUENCE ANALYSIS OF THE TETRAVALENT ROTAVIRUS VACCINE
Abstract
Isolated nucleic acid molecules comprising a gene segment from a
rhesus rotavirus (RRV) or from one of three rhesus:human
reassortant viruses are disclosed, including isolated nucleic acid
molecules having a sequence selected from the group consisting of:
SEQ ID NO: 1-14, inclusive, and isolated nucleic acid molecules
encoding a protein having a sequence selected from the group
consisting of SEQ ID NO: 15-28, inclusive, as well as variants of
the isolated nucleic acid molecules.
Inventors: |
Buonagurio; Deborah A.;
(Rye, NY) ; Georgiu; Alice F.; (Montgomery,
NY) ; Lerch; Robert A.; (New Hempstead, NY) ;
Mason; Bruce B.; (Downington, PA) ; Murthy; Shridhara
C.; (Ann Arbor, MI) ; Rappaport; Ruth S.;
(Strafford, PA) ; Sidhu; Mohinder S.; (New City,
NY) ; Udem; Stephen A.; (New York, NY) ; Zamb;
Timothy J.; (Nyack, NJ) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, 8TH FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
THE GOVERNMENT OF THE UNITED STATES
OF AMERICA, as represented by the Department of Health and
Human
Rockville
MD
Services
|
Family ID: |
27766169 |
Appl. No.: |
12/365122 |
Filed: |
February 3, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10505818 |
Jan 18, 2005 |
7485415 |
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PCT/US03/05172 |
Feb 19, 2003 |
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12365122 |
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60359960 |
Feb 27, 2002 |
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Current U.S.
Class: |
435/69.1 ;
435/252.33; 435/254.2; 435/320.1; 435/348; 435/358; 435/365;
536/23.72 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2720/12322 20130101; C12N 7/00 20130101; A61K 2039/5254
20130101; C12Q 1/701 20130101; C12N 2720/12362 20130101; A61K 39/00
20130101; A61K 2039/525 20130101 |
Class at
Publication: |
435/69.1 ;
536/23.72; 435/320.1; 435/252.33; 435/348; 435/254.2; 435/358;
435/365 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C12N 15/11 20060101 C12N015/11; C12N 15/00 20060101
C12N015/00; C12N 1/21 20060101 C12N001/21; C12N 5/00 20060101
C12N005/00; C12N 1/19 20060101 C12N001/19; C12N 5/06 20060101
C12N005/06 |
Claims
1. An isolated nucleic acid molecule having a sequence selected
from the group consisting of: SEQ ID NO:1-14, inclusive.
2. An isolated nucleic acid molecule encoding a polypeptide having
a sequence selected from the group consisting of: SEQ ID NO: 15-28,
inclusive.
3. An isolated nucleic acid molecule having the sequence of SEQ ID
NO: 1 and having a nucleotide change to a G at nucleotide 2120
4. An isolated nucleic acid molecule having the sequence of SEQ ID
NO: 2 and having one or more of the following nucleotide changes: a
C at nucleotide 493, and a T at nucleotide 947.
5. An isolated nucleic acid molecule having the sequence of SEQ ID
NO:3 and having one or more of the following nucleotide changes: an
A at nucleotide 169, a C at nucleotide 283, a C at nucleotide 448,
a C at nucleotide 874, a C at nucleotide 1306, and a C at
nucleotide 2388.
6. An isolated nucleic acid molecule having the sequence of SEQ ID
NO:4 and having one or more of the following nucleotide changes: a
C at nucleotide 119, a G at nucleotide 417, a G at nucleotide 809,
a C at nucleotide 977, an A at nucleotide 1463, a C at nucleotide
1481, a C at nucleotide 1608, a C at nucleotide 1755, and an A at
nucleotide 1953.
7. An isolated nucleic acid molecule having the sequence of SEQ ID
NO:5 and having one or more of the following nucleotide changes: an
A at nucleotide 75, a T at nucleotide 84, a C at nucleotide 347, a
T at nucleotide 667, a C at nucleotide 1186, a G at nucleotide
1219, and an A at nucleotide 1204.
8. An isolated nucleic acid molecule having the sequence of SEQ ID
NO: 6 and having one or more of the following nucleotide changes: a
C at nucleotide 376, an A at nucleotide 756, an A at nucleotide
1008, and a G at nucleotide 1041.
9. An isolated nucleic acid molecule having the sequence of SEQ ID
NO: 7 and having a nucleotide change to a G at nucleotide 387.
10. An isolated nucleic acid molecule having the sequence of SEQ ID
NO:10 and having one or more of the following nucleotide changes:
an A at nucleotide 92, an A at nucleotide 174, and a G at
nucleotide 218.
11. An isolated nucleic acid molecule having the sequence of SEQ ID
NO: 11 and having a nucleotide change to A at nucleotide 180.
12. An isolated nucleic acid molecule having the sequence of SEQ ID
NO: 12 and having a nucleotide change to an A at nucleotide
556.
13. An isolated nucleic acid molecule having the sequence of SEQ ID
NO: 14 and having a nucleotide change to a G at nucleotide 263.
14. A vector comprising an isolated nucleic acid molecule of claim
1.
15. A recombinant host cell comprising a vector of claim 14.
16. A method for producing a polypeptide encoded by an isolated
nucleic acid molecule, comprising culturing the recombinant host
cell of claim 15 under conditions suitable for expression of said
nucleic acid molecule.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Application No.
60/359,960, filed Feb. 27, 2002. The entire teachings of the above
application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Rotavirus is a segmented, double-stranded RNA virus that is
the major cause of severe gastroenteritis in infants and young
children. Profound fluid and electrolyte loss often leads to
hospitalization for life-saving rehydration therapy. Where access
to medical care is limited or unavailable, volume depletion, shock
and death can occur. Annually, nearly one million childhood deaths
in developing countries have been ascribed to inadequately treated
rotavirus gastroenteritis. In the industrialized world, more than
30% of the children admitted to hospitals with acute
gastroenteritis have rotavirus infections.
SUMMARY OF THE INVENTION
[0003] The invention is drawn to isolated nucleic acid molecules
comprising a gene segment from a rhesus rotavirus (RRV) or from one
of three rhesus:human reassortant viruses. In one embodiment, the
isolated nucleic acid molecule has a sequence selected from the
group consisting of: SEQ ID NO: 1-14, inclusive. In another
embodiment, the isolated nucleic acid molecule encodes a protein
having a sequence selected from the group consisting of: SEQ ID NO:
15-28, inclusive. In yet another embodiment, the isolated nucleic
acid molecule is a variant of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 10,
11, 12, or 14, such as one of the following: a nucleic acid
molecule having the sequence of SEQ ID NO: 1 (gene 1) and having a
nucleotide change to a G at nucleotide 2120; a nucleic acid
molecule having the sequence of SEQ ID NO: 2 (gene 2) and having
one or more of the following nucleotide changes: a C at nucleotide
493, and a T at nucleotide 947; a nucleic acid molecule having the
sequence of SEQ ID NO:3 (gene 3) and having one or more of the
following nucleotide changes: an A at nucleotide 169, a C at
nucleotide 283, a C at nucleotide 448, a C at nucleotide 874, a C
at nucleotide 1306, and a C at nucleotide 2388; a nucleic acid
molecule having the sequence of SEQ ID NO:4 (gene 4) and having one
or more of the following nucleotide changes: a C at nucleotide 119,
a G at nucleotide 417, a G at nucleotide 809, a C at nucleotide
977, an A at nucleotide 1463, a C at nucleotide 1481, a C at
nucleotide 1608, a C at nucleotide 1755, and an A at nucleotide
1953; a nucleic acid molecule having the sequence of SEQ ID NO:5
(gene 5) and having one or more of the following nucleotide
changes: an A at nucleotide 75, a T at nucleotide 84, a C at
nucleotide 347, a T at nucleotide 667, a C at nucleotide 1186, a G
at nucleotide 1219, and an A at nucleotide 1204; a nucleic acid
molecule having the sequence of SEQ ID NO: 6 (gene 6) and having
one or more of the following nucleotide changes: a C at nucleotide
376, an A at nucleotide 756, an A at nucleotide 1008, and a G at
nucleotide 1041; a nucleic acid molecule having the sequence of SEQ
ID NO: 7 (gene 7) and having a nucleotide change to a G at
nucleotide 387; a nucleic acid molecule having the sequence of SEQ
ID NO:10 (gene 10) and having one or more of the following
nucleotide changes: an A at nucleotide 92, an A at nucleotide 174,
and a G at nucleotide 218; a nucleic acid molecule having the
sequence of SEQ ID NO: 11 (gene 11) and having a nucleotide change
to A at nucleotide 180; a nucleic acid molecule having the sequence
of SEQ ID NO: 12 (DxRRV (serotype 1)) and having a nucleotide
change to an A at nucleotide 556; and a nucleic acid molecule
having the sequence of SEQ ID NO: 14 (ST3xRRV (serotype 4)) and
having a nucleotide change to a G at nucleotide 263. Each gene
variant can have one, more than one, or all of the nucleotide
changes enumerated for that particular genie. Other variants of any
one of nucleic acid molecules having SEQ ID NO:1-14, or encoding a
polypeptide of SEQ ID NO: 15-28, are also included.
DETAILED DESCRIPTION OF THE INVENTION
[0004] Human rotavirus serotypes G1-G4 are the major causes of
diarrheal gastroenteritis in humans (Gentsch, et al., 1995). The
serotypes are determined by epitopic differences in the outer
capsid of the virus particle encoded by the VP7 gene. The
ROTAMUNE.TM. (ROTASHIELD.TM.) vaccine is a live virus vaccine
comprised of four different rotaviruses, each containing the outer
capsid protein, VP7, of one of the major serotypes G1-G4 known to
cause disease in humans. The foundation of this vaccine is a virus
isolated from a rhesus macaque, rhesus rotavirus (RRV). The virus
is sufficiently similar to human strains to permit limited
replication in human intestinal tracts and thereby elicit
protective immune responses to human rotaviruses. The ROTAMUNE.TM.
vaccine includes RRV (serotype G3, for which VP7 is 96% homologous
to VP7 from human serotype 3 viruses); and three rhesus:human
reassortant viruses (serotypes G1, G2 and G4). The reassortants are
comprised of the rhesus virus genetic background (10 gene
segments), but replace the gene segment encoding VP7 with the
corresponding gene segments from the human serotype 1 (D strain), 2
(DS 1 strain) or 4 (ST3 strain) viruses. Applicants have, for the
first time, identified the nucleic acid sequence of all 11 gene
segments of each of the four virus strains, including the 10 common
gene segments and the four independent gene segments (VP7 gene),
for a total of 14 gene segments.
Nucleic Acids of the Invention
[0005] Accordingly, the invention pertains to an isolated nucleic
acid molecule comprising a gene segment from the rhesus rotavirus
(RRV) or from one of the three rhesus:human reassortant viruses.
The term, "gene segment," as used herein, refers to a nucleotide
sequence, preferably which encodes a polypeptide or protein, and
preferably which contains regulatory, non-coding nucleotide
sequence(s) present at the 3' and/or 5' end of each gene segment.
In a preferred embodiment, the gene segment is selected from the
group consisting of the nucleotide sequences shown in SEQ ID
NO:1-14, inclusive, as described in Table 1, below.
TABLE-US-00001 TABLE 1 SEQ ID NO: 1-14, Nucleic Acids of the
Invention SEQ ID NO: Description 1 Gene 1 (RRV) - VP1 2 Gene 2
(RRV) - VP2 3 Gene 3 (RRV) - VP3 4 Gene 4 (RRV) - VP4 5 Gene 5
(RRV) - NSP1 6 Gene 6 (RRV) - VP6 7 Gene 7 (RRV) - NSP3 8 Gene 8
(RRV) - NSP2 9 Gene 9 (RRV) (Serotype 3) - VP7 10 Gene 10 (RRV) -
NSP4 11 Gene 11 (RRV) - NSP5 12 Gene 9 (DxRRV) (Serotype 1) - VP7
13 Gene 9 (DS1xRRV) (Serotype 2) - VP7 14 Gene 9 (ST3xRRV)
(Serotype 4) - VP7
[0006] Due to differences in electrophoretic mobility, numerical
gene assignments differ among RRV and the reassortant viruses.
These differences involve only genes 7, 8 and 9. For the purpose of
comparison and discussion, segment 7 is designated as the segment
coding for NSP3, segment 8 as the segment coding for NSP2 and
segment 9 as the segment coding for outer capsid viral protein 7
(VP7).
[0007] The isolated nucleic acid molecules of the present invention
can be RNA, for example, mRNA, or DNA, such as cDNA. The RNA or DNA
molecules can be double-stranded or single-stranded; single
stranded RNA or DNA can be either the coding, or sense, strand or
the non-coding, or antisense, strand. The nucleic acid molecule can
include all or a portion of the coding sequence of the gene segment
and can further comprise additional non-coding sequences such as
non-coding. 3' and 5' sequences (including regulatory sequences,
for example). Additionally, the nucleic acid molecule can be fused
to a marker sequence, for example, a sequence that encodes a
polypeptide to assist in isolation or purification of the protein.
Such sequences include, but are not limited to, those which encode
a glutathione-S-transferase (GST) fusion protein and those which
encode a hemagglutinin A (HA) polypeptide marker from
influenza.
[0008] An "isolated" nucleic acid molecule, as used herein, is one
that is separated from nucleic acids which normally flank the gene
or nucleotide sequence and/or has been completely or partially
purified from other transcribed sequences (e.g., as in an RNA
library). For example, an isolated nucleic acid of the invention
may be substantially isolated with respect to the complex cellular
milieu (e.g., the virus) in which it naturally occurs, or culture
medium when produced by recombinant techniques, or chemical
precursors or other chemicals when chemically synthesized. In some
instances, the isolated material will form part of a composition
(for example, a crude extract containing other substances), buffer
system or reagent mix. In other circumstances, the material may be
purified to essential homogeneity, for example as determined by
PAGE or column chromatography such as HPLC.
[0009] Preferably, an isolated nucleic acid molecule comprises at
least about 50, 80 or 90% (on a molar basis) of all macromolecular
species present.
[0010] The nucleic acid molecule can be fused to other coding or
regulatory sequences and still be considered isolated. Thus,
recombinant nucleic acid contained in a vector is included in the
definition of "isolated" as used herein. Also, isolated nucleic
acid molecules include recombinant nucleic acid molecules in
heterologous host cells, as well as partially or substantially
purified nucleic acid molecules in solution. "Isolated" nucleic
acid molecules also encompass in vivo and in vitro RNA transcripts
(e.g., of cDNA) of the present invention. An isolated nucleic acid
molecule or nucleotide sequence can include a nucleic acid molecule
or nucleotide sequence which is synthesized chemically or by
recombinant means. Therefore, recombinant nucleic acids contained
in a vector are included in the definition of "isolated" as used
herein. Also, isolated nucleotide sequences include recombinant
nucleic acid molecules in heterologous organisms, as well as
partially or substantially purified nucleic acid molecules in
solution. In vivo and in vitro RNA transcripts of the DNA molecules
(e.g., cDNA) of the present invention are also encompassed by
"isolated" nucleotide sequences. Such isolated nucleotide sequences
are useful in the manufacture of the encoded protein, to raise
anti-protein antibodies using DNA immunization techniques, and as
an antigen to raise anti-DNA antibodies or elicit immune responses,
or for detecting expression of the gene in tissue (e.g., human
tissue), such as by Northern blot analysis, indicating the presence
of infection.
[0011] The present invention also pertains to nucleic acid
molecules which are not necessarily found in nature but which
encode a protein described herein. Thus, for example, nucleic acid
molecules which comprise a sequence that is different from the
naturally-occurring nucleotide sequence but which, due to the
degeneracy of the genetic code, encode a protein that is the same
as a protein encoded by a gene segment of the present invention,
are also the subject of this invention (e.g., a nucleic acid
molecule that encodes a protein having as a sequence any one of SEQ
ID NO:15-28, as described in Table 2, below).
TABLE-US-00002 TABLE 2 SEQ ID NO: 15-28, Proteins Encoded by
Nucleic Acids of the Invention SEQ ID Illustrative Nucleic Acid NO:
Description Encoding the Protein 15 Protein 1 (RRV) - VP1 1 16
Protein 2 (RRV) - VP2 2 17 Protein 3 (RRV) - VP3 3 18 Protein 4
(RRV) - VP4 4 19 Protein 5 (RRV) - NSP1 5 20 Protein 6 (RRV) - VP6
6 21 Protein 7 (RRV) - NSP3 7 22 Protein 8 (RRV) - NSP2 8 23
Protein 9 (RRV) (Serotype 3) - 9 VP7 24 Protein 10 (RRV) - NSP4 10
25 Protein 11 (RRV) - NSP5 11 26 Protein 9 (DxRRV) (Serotype 1) -
12 VP7 27 Protein 9 (DS1xRRV) (Serotype 13 2) - VP7 28 Protein 9
(ST3xRRV) (Serotype 14 4) - VP7
[0012] The invention also encompasses variants of certain
nucleotide sequences of the invention. For example, in one
embodiment, the variant nucleotide sequences of the invention
comprise the nucleotide differences set forth in Table 4 or Table
8, below.
[0013] That is, representative valiant embodiments include: a
nucleic acid molecule having the sequence of SEQ ID NO: 1 (gene 1)
and having a nucleotide change to a G at nucleotide 2120; a nucleic
acid molecule having the sequence of SEQ ID NO: 2 (gene 2) and
having one or more of the following nucleotide changes: a C at
nucleotide 493, and a T at nucleotide 947; a nucleic acid molecule
having the sequence of SEQ ID NO:3 (gene 3) and having one or more
of the following nucleotide changes: an A at nucleotide 169, a C at
nucleotide 283, a C at nucleotide 448, a C at nucleotide 874, a C
at nucleotide 1306, and a C at nucleotide 2388; a nucleic acid
molecule having the sequence of SEQ ID NO:4 (gene 4) and having one
or more of the following nucleotide changes: a C at nucleotide 119,
a G at nucleotide 417, a G at nucleotide 809, a C at nucleotide
977, an A at nucleotide 1463, a C at nucleotide 1481, a C at
nucleotide 1608, a C at nucleotide 1755, and an A at nucleotide
1953; a nucleic acid molecule having the sequence of SEQ ID NO:5
(gene 5) and having one or more of the following nucleotide
changes: an A at nucleotide 75, a T at nucleotide 84, a C at
nucleotide 347, a T at nucleotide 667, a C at nucleotide 1186, a G
at nucleotide 1219, and an A at nucleotide 1204; a nucleic acid
molecule having the sequence of SEQ ID NO: 6 (gene 6) and having
one or more of the following nucleotide changes: a C at nucleotide
376, an A at nucleotide 756, an A at nucleotide 1008, and a G at
nucleotide 1041; a nucleic acid molecule having the sequence of SEQ
ID NO: 7 (gene 7) and having a nucleotide change to a G at
nucleotide 387; a nucleic acid molecule having the sequence of SEQ
ID NO:10 (gene 10) and having one or more of the following
nucleotide changes: an A at nucleotide 92, an A at nucleotide 174,
and a G at nucleotide 218; a nucleic acid molecule having the
sequence of SEQ ID NO: 11 (gene 11) and having a nucleotide change
to A at nucleotide 180; a nucleic acid molecule having the sequence
of SEQ ID NO: 12 (DxRRV (serotype 1)) and having a nucleotide
change to an A at nucleotide 556; and a nucleic acid molecule
having the sequence of SEQ ID NO: 14 (ST3xRRV (serotype 4)) and
having a nucleotide change to a G at nucleotide 263. Each gene
variant can have one of the nucleotide changes, or can have more
than one, or all, of the nucleotide changes enumerated for that
particular gene.
[0014] Such variants can be naturally-occurring, such as in the
case of allelic variation or base substitution in a clinical
isolate, or non-naturally-occurring, such as those induced by
various mutagens and mutagenic processes. Other intended variations
can also be included in any one of the isolated nucleic acids of
the invention (e.g., in any one of SEQ ID NO: 1-14 or in a nucleic
acid molecule encoding a polypeptide of any one of SEQ ID
NO:15-28); such intended variations include, but are not limited
to, addition, deletion and substitution of one or more nucleotides
which can result in conservative or non-conservative amino acid
changes, including additions and deletions. Preferably the
nucleotide (and/or resultant amino acid) changes are silent or
conserved; that is, they do not alter the characteristics or
activity of protein encoded by the gene segment of the
invention.
[0015] Other alterations of the nucleic acid molecules of the
invention can include, for example, labeling, methylation,
internucleotide modifications such as uncharged linkages (e.g.,
methyl phosphonates, phosphotriesters, phosphoamidates,
carbamates), charged linkages (e.g., phosphorothioates,
phosphorodithioates), pendent moieties (e.g., polypeptides),
intercalators (e.g., acridine, psoralen), chelators, alkylators,
and modified linkages (e.g., alpha anomeric nucleic acids). Also
included are synthetic molecules that mimic nucleic acid molecules
in the ability to bind to designated sequences via hydrogen bonding
and other chemical interactions. Such molecules include, for
example, those in which peptide linkages substitute for phosphate
linkages in the backbone of the molecule.
[0016] In a related aspect, the nucleic acid molecules of the
invention are used as probes or primers in assays. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid molecules. Such probes include
polypeptide nucleic acids, as described in Nielsen et al., Science,
254, 1497-1500 (1991). Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75, consecutive nucleotides of a nucleic
acid molecule comprising a nucleotide sequence selected from SEQ ID
NO: 1-14, and the complement of SEQ ID NO: 1-14. More typically,
the probe further comprises a label, e.g., radioisotope,
fluorescent compound, enzyme, or enzyme co-factor.
[0017] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides.
[0018] The nucleic acid molecules of the invention such as those
described above can be identified and isolated using standard
molecular biology techniques and the sequence information provided
in SEQ ID NO: 1-14. For example, nucleic acid molecules can be
amplified and isolated by the polymerase chain reaction using
synthetic oligonucleotide primers designed based on one or more of
the sequences provided in SEQ ID NO: 1-14 and/or the complement of
SEQ ID NO: 1-14. See generally PCR Technology: Principles and
Applications for DNA Amplification (ed. H. A. Erlich, Freeman
Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and
Applications (Eds. Innis, et al., Academic Press, San Diego,
Calif., 1990); Mattila et al., Nucleic Acids Res., 19:4967 (1991);
Eckert et al., PCR Methods and Applications, 1:17 (1991); PCR (eds.
McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202.
The nucleic acid molecules can be amplified using cDNA, RNA, or
mRNA as a template, cloned into an appropriate vector and
characterized by DNA sequence analysis.
[0019] Other suitable amplification methods include the ligase
chain reaction (LCR) (see Wu and Wallace, Genomics, 4:560 (1989),
Landegren et al., Science, 241:1077 (1988), transcription
amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173
(1989)), and self-sustained sequence replication (Guatelli et al.,
Proc. Nat. Acad. Sci. USA, 87:1874 (1990)) and nucleic acid based
sequence amplification (NASBA). The latter two amplification
methods involve isothermal reactions based on isothermal
transcription, which produce both single stranded RNA (ssRNA) and
double stranded DNA (dsDNA) as the amplification products in a
ratio of about 30 or 100 to 1, respectively.
[0020] Antisense nucleic acid molecules of the invention can be
designed using the nucleotide sequences of SEQ ID NO: 1-14 and/or
the complement of SEQ ID NO: 1-14, or a portion of the nucleotide
sequence of SEQ ID NO: 1-14 and/or the complements thereof, and
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid molecule (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides call be used. Alternatively, the
antisense nucleic acid molecule can be produced biologically using
an expression vector into which a nucleic acid molecule has been
subcloned in an antisense orientation (i.e., RNA transcribed from
the inserted nucleic acid molecule will be of an antisense
orientation to a target nucleic acid of interest).
[0021] Another aspect of the invention pertains to nucleic acid
constructs containing a nucleic acid molecule selected from the
group consisting of SEQ ID NO: 1-14 and the complement of SEQ ID
NO: 1-14 (or a portion thereof). The constructs comprise a vector
(e.g., an expression vector) into which a sequence of the invention
has been inserted hi a sense or antisense orientation. As used
herein, the term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. However, the
invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses, negative
strand RNA virus vectors, VEE vectors) that serve equivalent
functions.
[0022] Preferred recombinant expression vectors of the invention
comprise a nucleic acid molecule of the invention in a form
suitable for expression of the nucleic acid molecule in a host
cell. This means that the recombinant expression vectors include
one or more regulatory sequences, selected on the basis of the host
cells to be used for expression, which is operably linked to the
nucleic acid sequence to be expressed. Within a recombinant
expression vector, "operably linked" is intended to mean that the
nucleotide sequence of interest is linked to the regulatory
sequence(s) in a manner which allows for expression of the
nucleotide sequence (e.g., in an in vitro transcription/translation
system or in a host cell when the vector is introduced into the
host cell). The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those which direct constitutive
expression of a nucleotide sequence in many types of host cell and
those which direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed and the level of expression of
protein desired. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins, including
fusion proteins, encoded by nucleic acid molecules as described
herein.
[0023] The recombinant expression vectors of the invention can be
designed for expression of a protein described herein, in
prokaryotic or eukaryotic cells, e.g., bacterial cells such as E.
coli, insect cells (using baculovirus expression vectors), yeast
cells or mammalian cells. Suitable host cells are discussed further
in Goeddel, supra. Alternatively, the recombinant expression vector
can be transcribed and translated in Vitro, for example using T7
promoter regulatory sequences and T7 polymerase.
[0024] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0025] A host cell can be any prokaryotic or eukaryotic cell. For
example, a nucleic acid molecule of the invention can be expressed
in bacterial cells (e.g., E. coli), insect cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS
cells). Other suitable host cells are known to those skilled in the
art.
[0026] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing a foreign nucleic acid molecule (e.g., DNA) into a host
cell, including calcium phosphate or calcium chloride
co-precipitation, DEAE-dextran-mediated transfection, lipofection,
or electroporation. Suitable methods for transforming or
transfecting host cells can be found in Sambrook, et al. (supra),
and other laboratory manuals.
[0027] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those that confer resistance to drugs, such as
G418, hygromycin and methotrexate. Nucleic acid molecules encoding
a selectable marker can be introduced into a host cell on the same
vector as the nucleic acid molecule of the invention or can be
introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid molecule can be identified by drug
selection (e.g., cells that have incorporated the selectable marker
gene will survive, while the other cells die).
[0028] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) a protein described herein. Accordingly, the invention
further provides methods for producing a protein using the host
cells of the invention. In one embodiment, the method comprises
culturing the host cell of the invention (into which a recombinant
expression vector encoding a protein described herein has been
introduced) in a suitable medium such that the protein is produced.
In another embodiment, the method further comprises isolating the
protein from the medium or the host cell.
[0029] The invention will be further described by the following
non-limiting examples. The teachings of all publications cited
herein are incorporated herein by reference in their entirety.
EXAMPLES
Example 1
Consensus Sequencing Materials and Methods
[0030] Viral Stocks
[0031] A rotavirus seed bank system was developed for the
ROTAMUNE.TM. vaccine consisting of a Master Virus Seed (MVS) Bank,
a Primary Virus Seed (PVS) Bank and a Manufacturer's Working Virus
Seed (MWVS) Bank. The nucleotide sequence of all 11 gene segments
of each of the four strains comprising the commercial virus seed
(MWVS-5, 6, 7 and 8) were identified and the sequence identity
(genetic equivalence) between the commercial virus seeds and the
clinical virus seeds (MWVS-1, 2, 3, and 4) were demonstrated.
[0032] A subset of Manufacturer's Working Virus Seed (WVS),
representing clinical lots (MWVS 1-4) and commercial lots (MWVS
5-8), was used. The strains used in this study are:
TABLE-US-00003 1) D .times. RRV Lot MWVS-5 Type-1 2) DS1 .times.
RRV Lot MWVS-6 Type-2 3) RRV Lot MWVS-7 Type-3 4) ST3 .times. RRV
Lot MWVS-8 Type-4 5) RRV Lot MWVS-1 6) DS1 .times. RRV Lot MWVS-2
7) D .times. RRV Lot MWVS-3 8) ST3 .times. RRV Lot MWVS-4
[0033] RNA Isolation
[0034] Genomic RNAs from the aliquots of each MWVS were extracted
using Trizol-LS.TM. reagent (Life Technologies, Grand Island,
N.Y.). RNA was resuspended in RNase-free water and used for all
genomic amplifications.
[0035] Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Amplification RT-PCR amplifications spanning individual full length
RNA gene segments of the rotavirus genome were performed by using
the GeneAmp XL RNA PCR Kit (Perkin-Elmer) and primer pairs specific
to each individual rotavirus gene segment. Common RRV primers were
used for all segments of the three reassortants, except for gene 9
where primers specific to gene 9 of the rhesus or of the different
human rotavirus sequences (RRV, HRV-D, HRV-DS1 and GRV-ST3) were
used. Primers are shown in Table 3A.
TABLE-US-00004 TABLE 3A Primers Used For Amplification of Genomic
Fragments SEQ Gene Primer Name Nucleotide Sequence 5' .fwdarw. 3'
ID 1 RRV-101 1-27 GGCTATTAAAGCTGTACAATGGGGAAG 29 RRV-102 3265-3294
CTAAGCGTTCTAATCTTGAAAGAAGTTTGC 30 2 RRV-201 1-27
GGCTATTAAAGGCTCAATGGCGTACAG 31 RRV-202 2683-2708
GGTCATATCTCCACAATGGGGTTGGC 32 3 *RRV-301A 1-25
GGCTATTAAAGCAGTACGAGTAGTG 33 *RRV-302A 2571-2591
GGTCACATCATGACTAGTGTG 34 4 RRV-401 1-21 GGCTATAAAATGGCTTCGCTC 35
RRV-402 2341-2362 GGTCACATCCTCTGGAAATTGC 36 5 *RRV-501A 1-29
GGCTTTTTTTTGAAATGTCTTGTGTTAGC 37 *RRV-502A 1576-1599
GGTCACAGTTTTTGCTGGCTAGGC 38 6 RRV-601 1-26
GGCTTTTAAACGAAGTCTTCAACATG 39 RRV-602 1332-1356
GGTCACATCCTCTCACTATACCATC 40 7 RRV-701 1-25
GGCATTTAATGCTTTTCAGTGGTTG 41 RRV-702 1056-1077
GGTCACATAACGCCCCTATAGC 42 8 RRV-801 1-29
GGCTTTTAAAGCGTCTCAGTCGCCGTTCG 43 RRV-802 1037-1059
GGTCACATAAGCGCTTTCTATTC 44 9 **RRV-901 1-24
GGCTTTAAAAGCGAGAATTTCCGT 45 RRV-902 1041-1062
GGTCACATCATACATTTCTAAC 46 D-RRV-902 1036-1062
GGTCACATCGAACAATTCTAATCTAAG 47 DS1-RRV-902 1035-1062
GGTCACATCGAACAATTCTGACCAAATC 48 ST3-RRV-902 1036-1062
GGTCACATCATACATTTCTATTTTAGG 49 10 RRV-1001 1-27
GGCTTTTTAAAAGTTCTGTTCCGAGAG 50 RRV-1002 728-750
GGTCACATTAAGACCGTTCCTTC 51 11 RRV-1101 1-24
GGCTTTTAAAGCGCTACAGTGATG 52 RRV-1102 644-667
GGTGACATAACTGGAGTGGGGAGC 53 Primers named . . . 01 are (+) sense
and primers named . . . 02 are (-) sense. *Primers 301A, 302A,
501A, and 502A reflect the actual termini sequence as determined.
**Primer 901 was used to amp1ify all four serotypes, while primer
902 was designed specifically for each.
[0036] RT-PCR amplification steps were as follows:
[0037] RT Reaction
TABLE-US-00005 RNA (RNA + water) 10 .mu.l RNase-free water -- 5X XL
RT buffer 4 .mu.l dNTP mix (2.5 mM each dNTP) 1.6 .mu.l rTth
polymerase 2.5 .mu.l 25 mM Mn(OAc2) 0.88 .mu.l RT (downstream)
primer (20 pmoles/.mu.l) 1 .mu.l TOTAL: 20 .mu.l
[0038] RNA (100-500 ng per reaction) was mixed with water and
5.times.SL RT buffer in a Gene-amp tube. The mixture was denatured
at 96.degree. C. for 5-6 minutes in a pre-heated thermal cycler,
and then placed on ice immediately for 3 minutes to quick cool.
Samples were pulse-spun in microfuge to remove any condensation on
caps, and the remaining ingredients were added. A drop of mineral
oil was added to each tube, and samples were placed in pre-heated
thermal cycler at 40.degree. C. The RT thermal cycle was 40.degree.
C. for 60 minutes, followed by 45.degree. C. for 60 minutes and
then 4.degree. C., soak. If several different fragments from the
same RNA template were amplified, the entire reaction was scaled up
in one tube, eliminating only the primer from the mix. One .mu.l of
the RT (+sense) primer was added to each reaction tube, and then 19
.mu.l of the scaled-up RT mix were added.
[0039] PCR Reaction
TABLE-US-00006 RNase-free water 58.8 .mu.l 5X XL Chelating Buffer
16 .mu.l 25 mM Mg(OAc2) 3.2 .mu.l PCR (upstream) primer (20
pmoles/.mu.l) 1.5 .mu.l RT (downstream) primer (20 pmoles/.mu.l)
0.5 .mu.l TOTAL 80 .mu.l.
[0040] The reagents were mixed for each sample; 80 .mu.l PCR mix
were added to the 20 .mu.l RT reaction under the oil, and mixed by
pipetting up and down several times. The samples were placed in
pre-heated thermal cycler (hot-start) at 94.degree. C. and cycled
as follows: 94.degree. C. denaturation for 3 minutes, followed by
40 cycles of denaturation at 94.degree. C. for 1 minute, primer
annealing at 40.degree. C. for 30 seconds, and extension at
70.degree. C. for five minutes. A final extension step was run at
70.degree. C. for 10 minutes, followed by a soak cycle at 4.degree.
C.
[0041] Ten .mu.l of reaction were run on agarose gel. Products were
purified using Promega's Wizard PCR preps DNA purification system
(Madison Wis.) either directly or using gel purification from
low-melting agarose.
[0042] RNA Ligation RT-PCR to Determine Consensus Nucleotide
sequence of the Gene Termini
[0043] The nucleotide sequence of the absolute 3' and 5' termini of
the eleven RNA gene segments of the rotavirus vaccine strains was
determined using an RNA ligation reverse transcription-PCR protocol
modified from Sidhu et al. (Virology 193:66-72, 1993) for use on
double-stranded RNA. Genomic RNA was extracted using Trizol-LS.TM.
reagent (Life Technologies) and 2-3 .mu.g was treated with Tobacco
Acid Pyrophosphatase (TAP) at 37.degree. C. for 1 hour in a 40
.mu.l volume according to the manufacturer's directions (Epicentre
Technologies). This step was used to remove the CAP structure on
the 5' end of the plus strand of the genomic RNA segments, and
leave behind a 5' monophosphate that was used for RNA ligation. The
TAP-treated RNA was extracted with phenol/chloroform/isoamyl
alcohol (25:24:1) and ethanol-precipitated. The RNA pellet was
air-dried and resuspended in 13.7 .mu.L of RNase-free water. The
double-stranded RNA was then denatured at 96.degree. C. for 4
minutes and quick-chilled on ice for 2 minutes. The reagents
required for RNA ligation in at 20 .mu.l volume were then added.
The final ligation reaction conditions were 1.times.RNA ligase
buffer (New England Biolabs), 10% DMSO (Sigma D-2650), 20 U Promega
RNasin, and 36 U NEB T4 RNA ligase. Ligation was performed at
16.degree. C. overnight (approximately 16 hours). The ligated RNA
was phenol extracted and ethanol precipitated as described above,
and the RNA pellet was resuspended in 15 .mu.l of water. One .mu.l
of ligated RNA was seeded into each of 11 RT-PCR reactions
containing rotavirus gene-specific primers (shown in Table 3B)
designed to amplify across the ligated RNA junction.
TABLE-US-00007 TABLE 3B Rotavirus Primers Used to Amplify across
Ligated RNA Junctions (Gene Termini) Gene Primer Nucleotide
Sequence 5' .fwdarw. 3' SEQ ID 1 RRV-101T 206-229
CCCAAGCTTGTGTGGCATTCTCTATAACATCGC 54 RRV-102T 3163-3186
CCCGGATCCGGCTCATGGATAAGCTTATTCTGC 55 RRV-103T 301-324
CCCAAGCTTCAAATCTGCTTCTAGCGGCTTACC 56 RRV-104T 3084-3107
CCCGGATCCTAAAGGAAAGATACCAGCTGTCAC 57 2 RRV-201T 147-170
CCCAAGCTTCCTTTTGAGATAGCACTTTCTCTG 58 RRV-202T 2524-2547
CCCGGATCCACCACAACAATTTGATTTTAGAGC 59 RRV-203T 270-293
CTTCTTTTTGATGTTCTTCCTTAG 60 RRV-204T 2470-2493
TGTAGCGAATTATGACTGGGTTCG 61 3 RRV-301T 128-151
CCCAAGCTTAAGAAATGCATTCTCGTAACTGTC 62 RRV-302T 2415-2438
CCCGGATCCGTCAATCTAGAATGTTTATTCCAC 63 RRV-303T 227-250
TTGAATCTCAACAGCTGCAATTCC 64 RRV-304T 2344-2367
AAACGTTAGTGGAGTTCTAGCGAC 65 4 RRV-401T 137-160
CCCAAGCTTCCCAGTTAACTGGAGCATAACCTG 66 RRV-402T 2145-2168
CCCGGATCCAACTGACTCTCCGGTCATCTCAGC 67 RRV-403T 208-231
GAACGTTGTTGGTTGATAAGGACC 68 RRV-404T 2049-2072
AGTCTTTGAAGCGGGAACAGATGG 69 5 RRV-501T 155-178
CCCAAGCTTATTGACAACACTCAATGCACCACC 70 RRV-502T 1396-1419
CCCGGATCCGAATTAGATCACTTGCCGTTATGC 71 RRV-503T 214-237
GATGCACCACTGACAAACATGAGC 72 RRV-504T 1302-1325
GATACTGGAAACCGAGGCTCTTCC 73 6 RRV-601T 166-189
CCCAAGCTTTCGGCAGATTACCAATTCCTCCAG 74 RRV-602T 1170-1193
CCCGGATCCCAGCGTGTATTTACAGTGGCTTCC 75 RRV-603T 246-269
ACGGGCCGTTTCGACATAGTTAGC 76 RRV-604T 1076-1099
AGAATACGCGATACCAGTTGGACC 77 7 RRV-705T 94-117
CCCAAGCTTTCAAGAGTAGAAGTTGCAGCAACC 78 RRV-706T 923-946
CCCGGATCCAAAGGATTATTGCAGCAATGCAAC 79 RRV-703T 146-169
ACTCTTTACTCTAGTATATACCTC 80 RRV-704T 800-823
AAATCAGACATTGAACAACAGCTG 81 RRV-707T 285-311
TAGCTACAGTTCGAGAGTCAGTCATCC 82 RRV-708T 756-782
CAATAGAATGGTATCTAAGATCGATGG 83 8 RRV-801T 194-217
CCCAAGCTTGAATTGTGGCGGTGGTGCGATACC 84 RRV-802T 920-943
CCCGGATCCAAAATGAAGCGGGAAAGTAATCCG 85 RRV-803T 284-307
CGCTTCACAAATTAACACCGCCAC 86 RRV-804T 847-870
GAACTGGTATGCGTTTACATCCTC 87 9 (RRV) RRV-901T 227-250
CCCAAGCTTCGTATGCAGTGTCCATTGAACCAG 88 RRV-902T 905-928
CCCGGATCCGCATTAATTGGAAGAAATGGTGGC 89 RRV-903T 304-327
TATTTCTGTTGCAGCTTCAGTTGG 90 RRV-904T 835-858
GTTGGAGGTTCTGATGTTCTCGAC 91 9 (DSI) DS1-901T 134-157
CCCAAGCTTACCTGAAAATTATGTAGTCCATCG 92 DS1-902T 882-905
CCCGGATCCCCCACAAGTTCAAAGAATCATGCG 93 DS1-903T 220-243
AGCGTCTAGTGACCCCGTTATTGG 94 DS1-904T 828-851
AATTCAAGTTGGTGGACCGAACGC 95 9 (D) D-901T 122-145
CCCAAGCTTTGTAGTCCATTATTCGAGTCACTG 96 D-902T 886-909
CCCGGATCCCAAACTGAGAGAATGATGAGAGTG 97 D-903T 220-243
AGCGTCCATTGATCCTGTTATTGG 98 D-904T 819-842 TGTAGCTGTAATACAAGTTGGTGG
99 9 (ST3) ST3-901T 219-242 CCCAAGCTTGTATCCATAGATCCAGTAATTGGC 100
ST3-902T 920-943 CCCGGATCCAATGGTGGCAAGTATTCTACACTG 101 ST3-903T
304-327 AATTTGAGTTGGAGCTTCTGATGG 102 ST3-904T 861-884
AACAGCTGATCCCACAACTTCTCC 103 10 RRV-1005T 141-168
GTACAGTTAGGACAGAAGCAATGTATGG 104 RRV-1006T 628-655
ATCGGACCTGATGACTGGTTGAGAAGCC 105 RRV-1007T 359-386
CTTATCAATCATTTCCAGCTGACGTCTC 184 RRV-1008T 533-559
TCCATATGAACCAAAAGAGGTGACTGC 185 11 RRV-1101T 124-147
CCCAAGCTTTGAAACGTACTGTTCACTCCTACC 106 RRV-1102T 470-493
CCCGGATCCTTGAAGCAGATTCCGATTCAGACG 107 RRV-1103T 205-228
CCCAAGCTTGTCGTTTGAAGCAGAATCAGATGG 108 RRV-1104T 403-426
CCCGGATCCGTATCAACAGTTTCCAAGAAGGAG 109 Odd numbered primers are
negative sense; Even numbered primers are positive sense. Primers
ending with 03, 04 or 07, 08 (genes 7 and 10) are used for reverse
transcription and first round PCR. Primers ending with 01, 02 or
05, 06 (genes 7 and 10) are used for second round (nested) PCR and
sequencing. Primers ending with 01 and 103T, 1103T, 705T contain 9
nucleotides at the 5' end that include a HindIII site. Primers
ending with 02 and 104T, 1104T, 706T contain 9 nucleotides at the
5' end that include a BamHI site.
The RT step and the first round of PCR was done using the
Perkin-Elmer GeneAmp Thermostable rTth Reverse Transcriptase RNA
PCR Kit (catalog #N808-0069) as per the manufacturer's
specifications with modification.
[0044] RT mix for one reaction (multiply .mu.l volumes by number of
reactions needed)
TABLE-US-00008 10X RT buffer 2 2.5 mM dNTP mix* 1.6 10 mM MnCl2 2
rTth DNA polymerase (2.5 U/.mu.l) 2 RNase-free water 7.4 TOTAL: 15
.mu.l *prepared by mixing equal volumes of each 10 mM dNTP stock
(dATP, dGTP, dCTP, dTTP).
Fifteen .mu.l of RT mix were combined with 2 .mu.l of upstream and
2 .mu.l of downstream first round PCR primers (each are 20
pmoles/.mu.l) and 1 .mu.l of ligated RNA in a 0.5 ml thin-walled
GeneAmp tube and overlaid with 2 drops of Sigma mineral oil. Since
both plus and minus RNA strands of the genome have been ligated,
each of the first round PCR primers could be used for cDNA
synthesis during reverse transcription. For each different primer
pair used, a negative control was set up that contained 1 .mu.l of
water in place of the ligated RNA. The 0.5 ml reaction tubes were
loaded in the PE thermal cycler 480 at 4.degree. C. and the cycler
was quickly ramped to 80.degree. C. and then ramped back to
45.degree. C. RT was performed at 45.degree. C. for 30 minutes,
followed by 50.degree. C. for 30 minutes. Following RT, 80 .mu.l of
first-round PCR mix was added to each RT reaction over the oil and
the tubes were pulse spun in a microcentrifuge.
[0045] First Round PCR Mix for one reaction (multiply III volumes
by the number of reactions needed):
TABLE-US-00009 2.5 mM dNTP mix 2.4 25 mM MgCl2 9 10x chelating
buffer 8 RNase-free water 60.0 TOTAL 80 .mu.l
Thermal cycling profile was as follows: 94.degree. C. for 2
minutes; 40 cycles of 94.degree. C. for 1 minute, 45.degree. C. for
1 minute, 72.degree. C. for 1 minute; 72.degree. C., for 10
minutes; and 4.degree. C., soak. Following each first round PCR, a
second round (nested) PCR amplification was performed using
Perkin-Elmer reagents and AmpliTaq Gold.TM. DNA polymerase (catalog
#N808-0241).
[0046] Second Round (nested) PCR mix for one reaction (multiply
.mu.l volumes by the number of reactions needed)
TABLE-US-00010 10x PCR buffer II (catalog #N808-0010) 10 25 mM
MgCl2 8 2.5 mM dNTP mix 8 AmpliTaq Gold DNA polymerase (5 U/.mu.l)
0.5 RNase-free water 65.5 TOTAL: 92 .mu.l
The second round PCR mix (92 .mu.l) was combined with 2 .mu.l of
upstream and 2 .mu.l of downstream second round PCR primers (each
are 20 pmoles/.mu.l) and 4 .mu.l of the first round PCR reaction
(including negative controls). The reactions were overlaid with 2
drops of Sigma mineral oil and pulse spun. The thermal cycling
profile was the same as for the first round PCR except the initial
step at 94.degree. C. for 2 minutes was extended to 12 minutes to
activate the AmpliTaq Gold.TM. DNAP.
[0047] Second round PCR products (10 .mu.l) were analyzed by
agarose gel electrophoresis with ethidium bromide. The ligation PCR
products were gel-purified using the Promega Wizard.TM. PCR preps
DNA purification system. A consensus sequence for the PCR amplified
products was determined. If necessary to resolve nucleotide
sequence ambiguities, the PCR products were cloned using pGEM-T
Easy Vector System I Promega, Madison, Wis.) and multiple clones
were sequenced.
[0048] DNA Sequencing
[0049] A consensus sequence for the PCR amplified products was
generated by using the Applied Biosystems-PRISM fluorescent dye
terminator cycle sequencing kit with AmpliTaq DNA Polymerase-FS,
and the Applied Biosystems 377 DNA sequencer (ABI-Perkin-Elmer).
Over 100 primers spaced approximately 200 nucleotides apart on each
strand were used for sequencing both strands of the PCR products.
When needed, gel purified PCR products were cloned by using pGEM-T
Easy Vector System I (Promega, Madison, Wis.). Positive clones were
selected by T7/SP6 primer-specific PCR screening and the amplified
PCR products of the positive clones were directly sequenced as
described above. Sequences were analyzed by using MacVector gene
analysis program (Oxford Molecular, Oxford, UK).
[0050] Results
[0051] Eleven full length gene segments were amplified for each
virus strain using high fidelity RNA-PCR amplification reactions as
described above. Both strands of the amplified products were
sequenced directly by using RRV-specific primers, except for gene
segment 9, for which strain specific primers were used. The
sequences for each of the eleven RRV (MWVS-7) genes are SEQ ID NO:
1-11, respectively as shown in Table 1 above. Gene segment 9
sequences for the three reassortants are SEQ ID NO:12 (DxRRV
(MWVS-5)), SEQ ID NO:13 (DS1xRRV (MWVS-6)), and SEQ ID NO: 14
(ST3xRRV (MWVS-8)). The putative protein sequences for each of
these are SEQ ID NO: 15-28, respectively, as shown in Table 2
above.
[0052] Table 4 lists nucleotide differences identified between the
parent RRV (MWVS-7) strain and the three reassortant viruses
D.times.RRV (MWVS-5), DS1.times.RRV (MWVS-6), and ST3.times.RRV
(MWVS-8). These nucleotide differences were common to both clinical
(MWVS 1-4) and commercial (MWVS 5-8) seeds.
TABLE-US-00011 TABLE 4 List of nucleotide changes identified in the
gene segments of the three reassortants as compared to the
progenitor RRV strain MWVS-5 MWVS-6 & MWVS-8 & MWVS- MWVS-2
& MWVS- Nucleotide 3 (D .times. (DS1 .times. 4 (ST3 .times.
Amino Gene Position RRV) RRV) RRV) Acid 1 2120 A.fwdarw.G -- --
Lys.fwdarw.Arg 2 493* -- -- A.fwdarw.C silent 947 C.fwdarw.T -- --
Leu.fwdarw.Phe 4 119 T.fwdarw.C T.fwdarw.C -- Leu.fwdarw.Pro 809
A.fwdarw.G A.fwdarw.G -- Tyr.fwdarw.Cys 977 T.fwdarw.C -- --
Met.fwdarw.Thr 1463 -- -- C.fwdarw.A Thr.fwdarw.Asn 1755 T.fwdarw.C
-- -- silent 5 84 C.fwdarw.T -- -- silent 347 -- T.fwdarw.C --
Leu.fwdarw.Ser 6 376 -- -- A.fwdarw.C Lys.fwdarw.Thr 756 G.fwdarw.A
G.fwdarw.A G.fwdarw.A Ala.fwdarw.Thr 1008 G.fwdarw.A -- --
Ala.fwdarw.Thr 1041 A.fwdarw.G -- -- Lys.fwdarw.Glu 7 387 --
A.fwdarw.G A.fwdarw.G Asn.fwdarw.Ser 10 174 G/A mix.dagger. -- G/A
mix.dagger. Ala/Thr mix 218 A.fwdarw.G -- -- silent 11 180
G.fwdarw.A -- -- silent -- Identical to RRV. .dagger.Position 174
is an `A` in RRV (MWVS-1 and MWVS-7).
Example 2
Clonal Analysis
[0053] Materials and Methods
[0054] Viral Stocks
[0055] Viral stocks were from the Wyeth Laboratories, Inc., in
Marietta, Pa., USA, rotavirus seed bank system. The strains used in
this study are:
Working virus seeds for commercial vaccine production:
TABLE-US-00012 1) D .times. RRV Lot MWVS-5 Type-1, Serotype G1 2)
DS1 .times. RRV Lot MWVS-6 Type-2, Serotype G1 3) RRV Lot MWVS-7
Type-3, Serotype G3 4) ST3 .times. RRV Lot MWVS-8 Type-4, Serotype
G4
Working virus seeds for clinical vaccine production:
TABLE-US-00013 5) RRV Lot MWVS-1 (ST3), Serotype G3 6) DS1 .times.
RRV Lot MWVS-2 (TS2), Serotype G2 7) D .times. RRV Lot MWVS-3
(ST1), Serotype G1 8) ST3 .times. RRV Lot MWVS-4 (ST4), Serotype
G4
Commercial vaccine lots used in clonal analysis were as
follows:
Serotype G1: I973020, I973017, I983003, I983026
Serotype G2: I973030, I973029, I983008, I983037
Serotype G3: I973026, I983021, I983032
Serotype G4: I97003, I973040, I983030, I973034.
[0056] All clinical isolates were plaque purified three times,
except for isolates 37 and 38 (purified twice).
[0057] Serotype G1 clinical isolates: clones 1, 16, 22 and 29
[0058] Serotype G2 clinical isolates: clones 3, 4, 8, 9 and 19
[0059] Serotype G3 clinical isolates: clones 5, 6, 10, 11, 12, 20,
21 and 25
[0060] Serotype G4 clinical isolates: clones 37 and 38.
[0061] RNA Isolation
[0062] Total RNA was extracted from clinical samples, aliquots of
virus seed or aliquots of vaccine virus using Trizol-LS.TM. reagent
(Life Technologies, Grand Island, N.Y.). RNA was resuspended in
nuclease-free water and used for all RT/PCR amplifications.
[0063] Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Amplification
[0064] Clonal analysis was carried out to determine the following:
1) the micro-heterogeneity in MWVS-1, 2, 3 and 4 at the 7
nucleotide positions (position 2388 in gene 3, positions 1674 and
1953 in gene 4, positions 75, 667 and 1204 in gene 5, and position
556 in gene 9) which were found to differ from consensus sequence
in more than one clinical isolate; 2) the micro-heterogeneity in
the four seed strains (MWVS-1, 2, 3 and 4) at position 174 in gene
10, found to contain a base mixture in serotypes G1 and G4 by
consensus sequencing; and 3) the micro-heterogeneity in genes 3 and
10 at nucleotide positions 2388 and 174, respectively, in the
commercial vaccine lots and their seeds (MWVS-5, 6, 7 and 8).
[0065] A portion of each gene spanning the nucleotide position(s)
of interest was amplified by RT-PCR using the GeneAmp XL RNA PCR
Kit (Perkin-Elmer) and primer pairs listed in Table 5.
TABLE-US-00014 TABLE 5 Primer Pairs Used in RT/PCR Amplification of
Clinical and Commercial Seeds for Clonal Analysis Nucleotide Primer
Primer Sequence SEQ Gene Of Interest Name (5' -> 3') ID 3 2388
RRV-307 GCTGATATAGAAGGTGGAAAG 110 RRV-302A GGTCACATCATGACTAGTGTG
111 4 1674 RRV-406 GAGCACCATAGATGCAGCT 112 RRV-409
CTGACAGATGAAGAAACATC 113 1953 RRV-406 GAGCACCATAGATGCAGCT 114
RRV-408 GAGTCAGTTACTAGATCTGC 115 5 75, 667, RRV-501A
GGCTTTTTTTTGAAATGTCT 116 1204 TGTGTTAGC RRV-507
GTGCATAACGGCAAGTGATC 117 9 (G1) 556 RRV-901 GGCTTTAAAAGCGAGAATTT
118 CCGT DxRRV- GGTCACATCGAACAATTCTA 119 902 ATCTAAG 10 174
RRV-1001 GGCTTTTAAAAGTTCTGTTC 120 CGAGAG RRV-1002
GGTCACATTAAGACCGTTCC 121 TTC
Primers common to all four serotypes were used for RT-PCR
amplification of fragments from all genes except gene 9 where
primers specific to gene 9 of human rotavirus serotype G1 (HRV-D)
were used. RT-PCR amplification was carried out as follows:
[0066] RT Reaction
TABLE-US-00015 RNA (RNA + water) 10 .mu.l RNase-free water -- 5X XL
RT buffer 4 .mu.l dNTP mix (2.5 mM each dNTP) 1.6 .mu.l rTth
polymerase 2.5 .mu.l 25 mM Mn (OAc2) 0.88 .mu.l RT (downstream)
primer (20 pmoles/.mu.l) 1 .mu.l TOTAL: 20 .mu.l
RNA (100-500 ng per reaction) was mixed with water and 5.times. SL
RT buffer in a Gene-amp tube. The mixture was denatured at
96.degree. C. for 4 minutes in a preheated thermal cycler, and then
placed on ice immediately for 3 minutes to quick cool. Samples were
pulse-spun in microfuge to remove any condensation on caps, and the
remaining ingredients were added. A drop of mineral oil was added
to each tube, and samples were placed in pre-heated thermal cycler
at 40.degree. C. The RT thermal cycle was 40.degree. C. for 60
minutes, followed by 45.degree. C. for 60 minutes and then
4.degree. C., soak. If several different fragments from the same
RNA template were amplified, the entire reaction was scaled up in
one tube, eliminating only the primer from the mix. One .mu.l of
the RT (+sense) primer was added to each reaction tube, and then 19
.mu.l of the scaled-up RT mix were added.
[0067] PCR Reaction
TABLE-US-00016 RNase-free water 58.8 .mu.l 5X XL Chelating Buffer
16 .mu.l 25 mM Mg (OAc2) 3.2 .mu.l PCR (upstream) primer (20
pmoles/.mu.l) 1.5 .mu.l RT (downstream) primer (20 pmoles/.mu.l)
0.5 .mu.l TOTAL 80 .mu.l.
The reagents were mixed for each sample; 80 .mu.l PCR mix were
added to the 20 .mu.l RT reaction under the oil, and mixed by
pipetting up and down several times. The samples were placed in
pre-heated thermal cycler (hot-start) at 94.degree. C. and cycled
as follows: 94.degree. C. denaturation for 3 minutes, followed by
40 cycles of denaturation at 94.degree. C. for 1 minutes primer
annealing at 40.degree. C. for 30 seconds, and extension at
70.degree. C. for two minutes. A final extension step was run at
70.degree. C. for 10 minutes, followed by a soak cycle at 4.degree.
C.
[0068] Ten .mu.l of reaction were run on 1% agarose gel. Products
were purified using Promega's Wizard PCR preps DNA purification
system (Madison Wis.) either directly or using gel purification
from low-melting agarose.
[0069] Cloning of RT-PCR Products
[0070] The RT-PCR products from genes 3, 4, 5, 9 and 10 of MWVS-1,
2, 3 and 4 were cloned into the appropriate restriction
endonuclease sites of either pGEM-3Zf(+).TM., pGEM-5Zf(+).TM., or
pGEM T-Easy.TM. (T/A cloning) plasmid vectors using standard
cloning methods.
[0071] Screening for Positive Clones
[0072] Approximately 40-100 colonies from each plasmid construct
were screened for the presence of cloned RRV sequences by PCR using
primers specific to the SP6 and T7 promoter sequences
(SP6:5'TATTTAGGTGACACTATAG3') (SEQ ID NO.: 122)
(T7:5'TAATACGACTCACTATAGGG3') (SEQ ID NO.: 123) flanking the
cloning sites of each vector as follows: a single colony was
transferred to 10 .mu.l of water in a 0.5 ml Gene-Amp.RTM. tube
using a sterile inoculating needle, and overlaid with a drop of
mineral oil. The tubes were placed in a pre-heated thermal cycler
at 96.degree. C. for 10 minutes to lyse bacterial cells, and cooled
to 4.degree. C. for 3-4 minutes. Then, 40 .mu.l PCR mix was added
to each tube underneath the oil and pipetted several times to mix
the sample.
[0073] PCR MIX (50 reactions):
TABLE-US-00017 10X PCR buffer with MgCl.sub.2 (Perkin-Elmer) 200
.mu.l dNTP mix (2.5 mM each dNTP) (Perkin-Elmer) 250 .mu.l SP6
promoter primer (20 pmole/.mu.l) 50 .mu.l T7 promoter primer (20
pmole/.mu.l) 50 .mu.l Nuclease-free water 1438 .mu.l Taq DNA
polymerase (Perkin-Elmer) 5 U/.mu.l 12 .mu.l TOTAL 2000 .mu.l
The mixture was placed in a pre-heated thermal cycler (hot-start)
at 94.degree. C. and cycled as follows: initial 94.degree. C.
denaturation for 3 minutes, followed by 40 cycles of denaturation
at 94.degree. C. for 1 minute, primer annealing at 42.degree. C.
for 1 minute, and extension at 72.degree. C. for 2 minutes,
terminating with a soak file at 4.degree. C. Each PCR product (10
.mu.l) was run on a 1% agarose gel to confirm the presence of an
insert of the appropriate size.
[0074] Sequence Determination of Positive Clones
[0075] PCR products from 25-30 positive clones were directly
purified from the PCR reaction using the PCR Preps Kit (Promega)
and eluted in 25 .mu.l nuclease-free water. Three .mu.l of the
purified product were then sequenced using Taq Cycle Sequencing
terminator mix, FS (ABI) and primers specific for the SP6 and T7
promoters which flank the MCS of each plasmid vector.
[0076] Sequence Configuration of Clinical Virus Isolates
[0077] RNA was extracted from 400 .mu.l of each clinical virus
isolate as previously described. For each virus, a fragment
containing the nucleotide position(s) of interest was amplified
from 200-600 ng of RNA using the GeneAmp XL RNA PCR Kit
(Perkin-Elmer) and primer pairs shown in Table 6. Consensus
sequence of the resulting RT/PCR product, including the nucleotide
of interest, was determined using Taq cycle sequencing and the ABI
377 DNA sequencer with primers denoted in Table 6.
TABLE-US-00018 TABLE 6 Primers Used for RT/PCR Amplification and
Sequence Confirmation of Clinical Virus Isolates Gene Clone # #
(Serotype) NT # Primer Pairs For RT/PCR and Sequencing 3 3 (G2) 283
301A .fwdarw. GGCTATTAAAGCAGTACGAGTAGTGTG SID 313 .fwdarw.
CGTGCTATCGGTAAAGAAGTAGT SID 4 (G2) 2288 307 .fwdarw.
GCTGATATAGAAGGTGGAAAG 124 302A .fwdarw. GGTCACATCATGACTAGTGTG 125 5
(G3) 1306 305 .fwdarw. GACTGCTATGGATTTAGAGC 126 311 .fwdarw.
GATAATGCGTATAATGCCAC 127 6 (G3) 169 301A .fwdarw.
GGCTATTAAAGCAGTACGAGTAGTGTG 128 314 .fwdarw. CTCAACAGCTGCAATTCCTG
129 9 (G2) 2388 307 .fwdarw. GCTGATATAGAAGGTGGAAAG 130 302A
.fwdarw. GGTCACATCATGACTAGTGTG 131 21 (G3) 2388 307 .fwdarw.
GCTGATATAGAAGGTGGAAAG 132 302A .fwdarw. GGTCACATCATGACTAGTGTG 133
25 (G3) 308 301A .fwdarw. GGCTATTAAAGCAGTACGAGTAGTGTG 134 313
.fwdarw. CGTGCTATCGGTAAAGAAGTAGT 135 29 (G1) 874 304 .fwdarw.
GTCTCAGTTGGACATTGGAC 136 312 .fwdarw. GAATATGGAGTGTCAAGTGGGTC 137
38 (G4) 448 301A .fwdarw. GGCTATTAAAGCAGTACGAGTAGTGTG 138 313
.fwdarw. CGTGCTATCGGTAAAGAAGTAGT 139 4 1 (G1) 1953 406 .fwdarw.
GAGCACCATAGATGCAGCT 140 408 .fwdarw. GAGTCAGTTACTAGATCTGC 141 6
(G3) 1608 405 .fwdarw. CAGTAATGACTGGCGGAGCAGT 142 409 .fwdarw.
CTGACAGATGAAGAAACATC 143 11 (G3) 417 401 .fwdarw.
GGCTATAAAATGGCTTCGCTC 144 412 .fwdarw. GTCACAAAATGCTGTCATG 145 12
(G3) 1481 405 .fwdarw. CAGTAATGACTGGCGGAGCAGT 146 409 .fwdarw.
CTGACAGATGAAGAAACATC 147 16 (G1) 1953 406 .fwdarw.
GAGCACCATAGATGCAGCT 148 408 .fwdarw. GAGTCAGTTACTAGATCTGC 149 19
(G2) 1674 405 .fwdarw. CAGTAATGACTGGCGGAGCAGT 150 409 .fwdarw.
CTGACAGATGAAGAAACATC 151 20 (G3) 1674 405 .fwdarw.
CAGTAATGACTGGCGGAGCAGT 152 402 .fwdarw. GGTCACATCCTCTGGAAATTGG 153
5 6 (G3) 75 501A .fwdarw. GGCTTTTTTTTGAAATGTCTTGTGTTAGC 154 509
.fwdarw. GTCTATATGGCAAATCTATGC 155 20 (G3) 75 501A .fwdarw.
GGCTTTTTTTTGAAATGTCTTGTGTTAGC 156 509 .fwdarw.
GTCTATATGGCAAATCTATGC 157 3 (G2) 1204 504 .fwdarw.
CTCAAACTGATTTACATCATG 158 507 .fwdarw. GTGCATAACGGCAAGTGATC 159 4
(G2) 1204 504 .fwdarw. CTCAAACTGATTTACATCATG 160 507 .fwdarw.
GTGCATAACGGCAAGTGATC 161 5 (G3) 394 501A .fwdarw.
GGCTTTTTTTTGAAATGTCTTGTGTTAGC 162 509 .fwdarw.
GTCTATATGGCAAATCTATGC 163 8 (G2) 667 503 .fwdarw.
CAGAGGAAATGTAGAAATGAG 164 508 .fwdarw. CAATCCATGTCTCTGAATGC 165 9
(G2) 667 503 .fwdarw. CAGAGGAAATGTAGAAATGAG 166 508 .fwdarw.
CAATCCATGTCTCTGAATGC 167 10 (G3) 1186 504 .fwdarw.
CTCAAACTGATTTACATCATG 168 507 .fwdarw. GTGCATAACGGCAAGTGATC 169 29
(G1) 1219 504 .fwdarw. CTCAAACTGATTTACATCATG 170 507 .fwdarw.
GTGCATAACGGCAAGTGATC 171 9 22 (G1) 556 901 .fwdarw.
GGCTTTAAAAGCGAGAATTTCCGT 172 D-902 .fwdarw.
GGTCACATCGAACAATTCTAATCTAAG 173 29 (G1) 556 901 .fwdarw.
GGCTTTAAAAGCGAGAATTTCCGT 174 D-902 .fwdarw.
GGTCACATCGAACAATTCTAATCTAAG 175 37 (G4) 263 901 .fwdarw.
GGGTTTAAAAGCGAGAATTTCCGT 176 ST3-905 .fwdarw. GTACATGATGATCCCATTGA
177 10 22 (G1) 92 1001 .fwdarw. GGCTTTTTAAAAGTTCTGTTCCGAGAG 178
1002 .fwdarw. GGTCACATTAAGACCGTTCCTTC 179 22 (G1) 174 1001 .fwdarw.
GGCTTTTTAAAAGTTCTGTTCCGAGAG 180 1002 .fwdarw.
GGTCACATTAAGACCGTTCCTTC 181 22 (G1) 218 1001 .fwdarw.
GGCTTTTTAAAAGTTCTGTTCCGAGAG 182 1002 .fwdarw.
GGTCACATTAAGACCGTTCCTTC 183
Results
[0078] Analysis of Micro-Heterogeneity in Clinical Virus Seeds
(MWVS-1, 2, 3 and 4)
[0079] Sequence analysis of genes 3, 4, 5, 9 and 10 of the 19
clinical viral isolates obtained from the stools of ROTAMUNE.TM.
recipients had heterogeneity at 24 nucleotide positions when
compared to consensus sequence of the clinical seeds (MWVS-1, 2, 3
and 4). One of these sites, nucleotide position 174 of gene 10,
contains a base mixture in the serotype G1 and G4 viruses. In
addition, 7 of the 24 nucleotide base substitutions identified in
the clinical virus isolates (i.e., position 2388 in gene 3;
positions 1674 and 1953 in gene 4; positions 75, 667 and 1204 in
gene 5; and position 556 in gene 9) were observed in more than one
virus sample. To establish the precise level of microheterogeneity
present at nucleotide position 174 of gene 10, as well as to
determine whether the seven nucleotide base substitutions found in
more than one clinical virus isolate were the result of sequence
micro-heterogeneity in the clinical virus seeds, clonal analysis
was carried out on each of the four seeds at these eight nucleotide
positions.
[0080] The clonal analysis revealed micro-heterogeneity at position
2388 in gene 3 of the G2 clinical seed (MWVS-2), and at position
174 in gene 10 of the G1, G21 and G4 virus seeds (MWVS-3, 2 and 4,
respectively). Twenty-six percent of the viral genomes in the G2
clinical seed were found to contain minor species C at position
2388 of gene 3, while the remaining 74% of the genomes contained T
at this position. As predicted by consensus sequence, heterogeneity
(i.e., G>A) was observed for position 174 of gene 10 in the G1
(MWVS-3) and G4 (MWVS-4) clinical seeds, where the minor species A
was found in 12% and 7% of the genomes, respectively. In contrast,
the genomes of the RRV parental strain contained solely A at this
position. The analysis also revealed a minor population of 5% G at
the same position in the G2 virus seed (MWVS-2) which was not
detected by consensus sequencing.
[0081] The remainder of the nucleotide substitutions which occur in
more than one of the clinical isolates (i.e., positions 1674 and
1953 of gene 4; positions 75, 667 and 1204 of gene 5; and position
556 of gene 9) apparently do not result from measurable sequence
micro-heterogeneity within the virus seeds, since mixtures of bases
were not found at these positions by clonal analysis. Table 7
summarizes the micro-heterogeneity found in the clinical virus
seeds (MWVS-1, 2, 3 and 4) at the variable nucleotide positions.
The micro-heterogeneity at these positions may exist at levels
below the detection limits, or the substitutions observed in the
clinical isolates may represent adaption within the human
gastrointestinal tract.
[0082] Sequence Confirmation of Clinical Virus Isolates
[0083] The sequence of the clinical virus isolates at each of the
24 nucleotide positions where the sequence had been found to
diverge from consensus was re-examined. Table 8 lists the 24
nucleotide positions of heterogeneity.
[0084] Analysis of the Genetic Stability of RRV at Positions 2388
and 174 in Genes 3 and 10, Respectively
[0085] Consensus sequencing of the commercial seeds (MWVS-5, 6, 7
and 8) revealed heterogeneity at positions 2388 and 174 in genes 3
and 10, respectively. To compare the precise levels of
micro-heterogeneity in the clinical and commercial virus seeds at
these positions, as well as to analyze the genetic stability of
these nucleotide positions during vaccine manufacture, clonal
analysis of these positions was carried out in the commercial virus
seeds and several vaccine lots generated from them.
[0086] The data showed that a similar level of micro-heterogeneity
existed in the commercial (MWVS-5, 6, 7 and 8) and clinical (WVS-1,
2, 3 and 4) vaccine seeds at positions 2388 and 174 in genes 3 and
10, respectively. By clonal analysis, 15% of the minor species (C)
was observed at nucleotide position 2388 in the G2 commercial virus
seed (MWVS-2), compared to 26% C in the G2 clinical virus seed
(MWVS-2), as
Serotype 1-MWVS-3=DxRRV; Serotype 2=MWVS-2=DS1xRRV; Serotype
3=MWVS-1=RRV; Serotype 4=MWVS-4=ST3xRRV
TABLE-US-00019 TABLE 7 Micro-Heterogeneity within clinical virus
seeds (MWVS 1, 2, 3 and 4) Nucleotide Nucleotide Representing
Representing Genomic Consensus Minor Number of Serotype Nucleotide
(% Variant (% Clones Gene (Virus Seed) Position population)
population) Sequenced 3 1 (MWVS-3) 2388 T (100%) C (0%) 28 2
(MWVS-2) T (74%) C (26%) 27 3 (MWVS-1) T (100%) C (0%) 60 4
(MWVS-4) T (100%) C (0%) 26 4 1 (MWVS-3) 1674 A (100%) G (0%) 26 2
(MWVS-2) A (100%) G (0%) 24 3 (MWVS-1) A (100%) G (0%) 33 4
(MWVS-4) A (100%) G (0%) 31 1 (MWVS-3) 1953 C (100%) A (0%) 28 5 1
(MWVS-3) 75 C (100%) A (0%) 25 2 (MWVS-2) C (100%) A (0%) 20 3
(MWVS-1) C (100%) A (0%) 20 4 (MWVS-4) C (100%) A (0%) 20 1
(MWVS-3) 667 C (100%) T (0%) 26 2 (MWVS-2) C (100%) T (0%) 19 3
(MWVS-1) C (100%) T (0%) 20 4 (MWVS-4) C (100%) T (0%) 20 1
(MWVS-3) 1204 G (100%) A (0%) 26 2 (MWVS-2) G (100%) A (0%) 20 3
(MWVS-1) G (100%) A (0%) 20 4 (MWVS-4) G (100%) A (0%) 21 9 1
(MWVS-3) 556 G (100%) A (0%) 41 10 1 (MWVS-3) 174 *G (88%) *A (12%)
26 2 (MWVS-2) A (95%) G (5%) 22 3 (MWVS-1) A (100%) G (0%) 21 4
(MWVS-4) *G (93%) *A (7%) 28 *Mixed base depicted in MWVS-3 and
MWVS-4 by consensus sequencing (see Table 4).
TABLE-US-00020 TABLE 8 Nucleotide Sequence Differences Between
Clinical Virus Isolates and Consensus Sequence of MWVS. Clone #
Gene # (Serotype) NT # NT Change AA # AA Change Gene 3 3 (G2) 283
TGC (TGT).sup.1 4 (G2) 2288 No change.sup.3 747 5 (G3) 1306 AGC
(AGT) 6 (G3) 169 ACA (ACG) 9 (G2) 2388 TCA (TTA) 780 Phe
(Leu).sup.2 21 (G3) 2388 No change 780 25 (G3) 308 No change 87 29
(G1) 874 TTC (TTA) 38 (G4) 448 GAC (GAT) Gene 4 1 (G1) 1953 TCA
(TCC) 6 (G3) 1608 GGC (GGT) 11 (G3) 417 ACG (ACA) 12 (G3) 1481 ACA
(AGA) 501 Thr (Arg) 16 (G1) 1953 TCA (TCC) 19 (G2) 1674 No change
20 (G3) 1674 No change Gene 5 6 (G3) 75 AAA (ACA) 22 Lys (Thr) 20
(G3) 75 AAA (ACA) 22 ys (Thr) 3 (G2) 1204 GAA (GAG) 4 (G2) 1204 GAA
(GAG) 5 (G3) 394 No change 8 (G2) 667 TTT (TTC) 9 (G2) 667 TTT
(TTC) 10 (G3) 1186 TAC (TAT) 29 (G1) 1219 ACG (ACA) Gene 9 22 (G1)
556 ATA (GTA) 170 Ile (Val) 29 (G1) 556 ATA (GTA) 170 Ile (Val) 37
(G4) 263 CGA (CAA) 72 Arg (Gln) Gene 10 22 (G1) 92 ATA (ATG) 17 Ile
(Met) 22 (G1) 174 ACA (GCA) 45 Thr (Ala) 22 (G1) 218 AAG (AAA)
.sup.1Underlined nucleotide is different in the clinical isolate
when compared to the Manufacture's Working Virus Seed (MWVS).
Triplet in parentheses is the consensus sequence of MWVS.
.sup.2Amino acid (aa) in parentheses is present in the relevant
MWVS. .sup.3Initial clonal sequence analysis had indicated
heterogeneity at the positions listed as "No change". Conseusus
sequence analysis of these isolates revealed sequence identity with
the corresponding MWVS. Boldface type indicates that the nucleotide
change was identified in more than one clinical isolate.
shown in Table 9. Analysis of the micro-heterogeneity present in
the commercial seeds at nucleotide position 174 in gene 10 revealed
25% and 23% of the minor species (A) in the G1 and G4 viruses,
respectively, as shown in Table 10; these were similar to the
levels observed in the clinical virus seed bank (12% and 7%
respectively). As observed in the clinical seeds, the RRV G3
commercial seed strain (MWVS-7) contained solely A at nucleotide
position 174. The G2 strain of the commercial seed bank (MWVS-6),
however, did not retain the same minor population observed in the
G2 clinical strain (5% G), but instead, resembled the G3 RRV strain
at this position, harboring 100% A at this site by clonal
analysis.
[0087] Determination of the heterogeneity at nucleotides 2388 and
174 of genes 3 and 10, respectively, in several vaccine lots
produced from the commercial manufacturer's working virus seed
allowed the monitoring of the genetic stability of these positions
after passage in vitro. Four vaccine lots of each serotype,
generated from the commercial virus seeds (MWVS-5, 6, 7 and 8) were
analyzed by clonal analysis. Four G2 vaccine lots (I973029,
I983008, I983037 and I973030) were analyzed for heterogeneity at
nucleotide position 2388 in gene 3, and in each case, the level of
the minor variant (4%, 8%, 8% and 16% C) was found to be similar to
the 15% observed in the G2 commercial seed (MWVS-6) (Table 9). For
nucleotide 174 in gene 10, each of the four G1 and G4 vaccine lots
contained the minor species (A) at a level similar to that seen in
its corresponding commercial seed. The four G1 vaccine lots
(I973020, I973017, I983026 and I983003) contained 22%, 6%, 33% and
4% A, respectively, at nucleotide position 174 compared to the 25%
A observed in the G1 commercial (MWVS-5) seed. Likewise, the G4
vaccine lots (I973034, I973004, I973030 and I983030) retained 9%,
14%, 19% and 23% A, respectively, at this position, similar to the
23% A found in the G4 (MWVS-8) seed (Table 10). These data indicate
that conditions used in the vaccine manufacturing process preserve
the identity of the four RRV vaccine strains as reflected in their
genomic nucleotide sequence.
TABLE-US-00021 TABLE 9 Base Composition at Nucleotide 2388 in Gene
3 for Serotype 2 Manufacturer's Working Virus Seed and Vaccine
Concentrates Nucleotide representing consensus/variant (%
population) Manufacturer's Working Virus Seed - Commercial RRV
Commercial Vaccine Concentrates and Harvests Serotype (MWVS-6) Lot
#I973030 Lot #I973029 Lot #I983008 Lot #I983037 2 T/C (85/15) T/C
(84/16) T/C (96/4) T/C (92/8) T/C (92/8)
TABLE-US-00022 TABLE 10 Base Composition at Nucleotide 174 in Gene
10 for Manufacturer's Working Virus Seeds and Vaccine Concentrates
Nucleotide Representing Consensus/Variant (% Population)
Manufacturer's Working Virus Serotype Seeds RRV Commercial Vaccine
Concentrates and Harvests* 1 G/A (75/25).sup.24 G/A (78/22).sup.23
G/A (94/6).sup.32 G/A (67/33).sup.24 G/A (96/4).sup.24 2 A/G
(100/0).sup.29 A/G (100/0).sup.26 A/G (100/0).sup.21 A/G
(100/0).sup.21 A/G (100/0).sup.24 3 A/G (100/0).sup.30 A/G
(100/0).sup.43 A/G (100/0).sup.23 A/G (100/0).sup.37 Not Determined
4 G/A (77/23).sup.30 G/A (91/9).sup.23 G/A (86/14).sup.28 G/A
(81/19).sup.21 G/A (77/23).sup.71 Manufacturer's Working Virus
Seeds = MWVS-5, 6, 7 and 8 for Serotypes 1, 2, 3 and 4,
respectively. *Vaccine Concentrates and Harvests: Serotype 1 - Lot
# I973020, I973017, I983026 and I983003, respectively. Serotype 2 -
Lot # I973030, I973029, I983008 and I983037, respectively. Serotype
3 - Lot # I973026, I983021 and I983032, respectively. Serotype 4 -
Lot # I973034, I973004, I973040 and I983030, respectively.
Superscripts denote the number of clones sequenced for each virus
lot
Example 3
Mutant Analysis by PCR and Restriction Enzyme Cleavage (MAPREC)
[0088] A sensitive and direct method to monitor the levels of
micro-heterogeneity at nucleotide 2388 of gene 3, Mutant Analysis
by PCR and Restriction Enzyme Cleavage (MAPREC), was developed.
[0089] Materials
[0090] The following materials were used: Life Technologies
(Gibco-BRL) SuperScript.TM. Preamplification for First Strand cDNA
Synthesis kit; RNase-free water; RRV Serotype 2 total RNA; Gene 3
nucleotide 2388 100% T DNA control template (1/30 dilution of
stock); Gene 3 nucleotide 2388 100% C DNA control template (1/30
dilution of stock); Perkin Elmer Thermal Cycler PE 480; primer
RRV-G3-EcoRI (20 pmole/.mu.l); primer RRV-302A-flourescein (20
pmole/.mu.l); Perkin-Elmer Taq DNA polymerase; mineral oil (Sigma),
molecular biology grade 5; Perkin-Elmer 0.5 ml Gene-Amp reaction
tubes; Pharmacia G-40 AutoSeq spin columns; restriction
endonuclease Eco RI (10 U/.mu.l) (Roche Molecular Biochemicals);
glycerol; bromophenol blue; xylene cyanol; Bio-Rad 40%
acrylamide:bis-acrylamide (38:2) liquid; distilled water;
10.times.TBE (Gibco-BRL); TEMED; ammonium persulfate; two
20.times.20 cm vertical polyacrylamide gel electrophoresis
apparatus with 0.75 mm, 20-well combs and 0.75 mm spacers; flat
head gel loading pipet tips; Molecular Dynamics Flourimager
595.
[0091] Reverse Transcription
[0092] For each sample, 500-1000 ng RNA and water were added to a
0.5 .mu.l microfuge tube for a final volume of 11 .mu.l. It was
preferable to use 1000 ng RNA per reaction if the RNA is
concentrated enough; however, 500 ng per reaction was usually
sufficient to generate product. The RNA and water mixture was
heated in a preheated thermal cycler at 96.degree. C. for 4
minutes, and then immediately placed on ice for 2-3 minutes. During
that time, the RT mix was made as follows:
TABLE-US-00023 10X PCR Buffer (Superscript II kit) 2 .mu.l 25 mM
Magnesium chloride (Superscript II kit) 2 .mu.l 10 mM dNTP mix
(Superscript II kit) 1 .mu.l 0.1 M DTT (Superscript II kit) 2 .mu.l
RV-302A-Flouroscein primer (20 pmole/.mu.l) 1 .mu.l TOTAL: 8
.mu.l.
[0093] After cooling, the chilled RNA and water mixture was briefly
centrifuged to spin down any condensation on the tube cap. Then 8
.mu.l RT mix (above) was added to each tube of RNA and water
mixture, and mixed by pipeting up and down several times. A drop of
mineral oil was overlaid in the reaction tube, and the tube was
then incubated in a pre-heated thermal cycler at 50.degree. C. for
5 minutes. Subsequently, 1 .mu.l Superscript II Reverse
Transcriptase 200 U/.mu.l (kit) was added to each reaction tube
underneath the oil layer. The reverse transcription process was
allowed to proceed at 50.degree. C., for 60 minutes, followed by
70.degree. C. for 15 minutes to inactivate the RT; the mixture was
then soaked at 4.degree. C. One .mu.l RNase H (Superscript II kit)
was added to each reaction underneath the oil, and the reaction was
incubated in a thermal cycler at 37.degree. C. for 20 minutes. The
first-strand cDNA resulting from this procedure could either be
transferred to 4.degree. C. and used directly in the PCR reaction,
or stored at -20.degree. C.
[0094] PCR Step
[0095] It should be noted that in addition to the vaccine samples,
each assay must be accompanied by two DNA control reactions
containing either 100% T or 100% C DNA templates, to validate each
assay.
[0096] PCR mix was prepared as follows:
TABLE-US-00024 10X PCR Buffer (Superscript II kit) 5 .mu.l 25 mM
magnesium chloride (Superscript II kit) 3 .mu.l 10 mM dNTP mix
(Superscript II kit) 1 .mu.l RRV-302A-Flouroscein primer (20
pmole/.mu.l) 1.5 .mu.l RRV-G3-EcoRI primer (20 pmole/.mu.l) 1.5
.mu.l Taq DNA polymerase (Perkin-Elmer) 0.5 .mu.l Nuclease-free
water 35 .mu.l TOTAL: 47.5 .mu.l.
[0097] The PCR mix was aliquoted to a fresh 0.5 ml tube for each
sample. Three .mu.l of the first strand cDNA (from the RT
reaction), 3 .mu.l of 1/30 dilution of 100% T DNA, or 3 .mu.l of
1/40 dilution of 100% C DNA were added to each PCR reaction as a
template, and the reaction was then overlaid with a drop of mineral
oil. The reaction was placed in a pre-heated thermal cycler, and
cycled for 94.degree. C. for 1 minutes (1 cycle), followed by
94.degree. C. for 30 seconds and 60.degree. C. for 3 minutes (40
cycles), and then a 4.degree. C. soak.
[0098] Since the PCR products being generated are fluorescent,
their exposure to light should be minimized by placing a piece of
aluminum foil over the thermal cycler cover during cycling. Storage
of the PCR products should always be in light tight containers.
[0099] Purification and Digestion of PCR Products
[0100] A Pharmacia G-40 spin column was prepared for use in the
purification of each PCR product. Each column was vortexed for 2-3
seconds to thoroughly resuspend the Sephadex beads; the screw cap
was loosened one-half turn; the bottom of the column was snapped
off and discarded, and then the screw cap was removed and discarded
as well. Each column was placed in an empty 1.5 ml Eppendorf tube
and spun in an Eppendorf microfuge at 3200 rpm (approximately
200.times.g) for 1 minute. Columns were then used immediately to
avoid drying of the resin. The PCR reaction described above was
removed from each tube, being careful to transfer as little oil as
possible. The G-50 spin column was removed from its tube, and the
entire 50 .mu.l PCR reaction was slowly loaded onto the center of
the angled resin in the column. The loaded column was then placed
into a fresh, labeled 1.5 ml Eppendorf microfuge tube. The tubes
were spun at 3200 rpm for 1 minute to collect the effluent
containing the purified PCR product, and column was discarded. This
purification step allows subsequent digestion of the PCR product
with EcoRI to take place in a proper restriction enzyme buffer, as
digestion with EcoRI in other buffers results in non-specific
digestion of PCR products. Eight .mu.l of each purified PCR
reaction was removed to a new 0.5 ml Gene-amp tube, and 1 .mu.l of
Restriction Buffer H (Roche Mol. Biochemicals) provided with the
EcoRI enzyme was added. One .mu.l of EcoRI restriction enzyme (10
U/.mu.l) was added to each sample and pipetted up and down several
times to thoroughly mix reaction contents. The reaction tubes were
then placed in a thermal cycler at 37.degree. C. for 3-4 hours. The
reactions were spun down by pulsing in microfuge to spin down any
condensation on the tube cap, and 2 .mu.l of 6.times. loading dye
(40% glycerol, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol) were
added to each digested sample, and then the samples were stored at
4.degree. C. in light tight containers until loaded onto the
gel.
[0101] Polyacrylamide Gel Electrophoresis of Digested PCR
Products
[0102] During the final hour of the digestion step (above),
polyacrylamide gels were prepared for analyses of the digested PCR
products. Gel plates were washed with Alconox detergent, followed
by a final rinse with ethanol, and allowed to air dry. The plates
were assembled, and a 6% non-denaturing polyacrylamide gel mixture
was prepared as follows:
TABLE-US-00025 Distilled water 45 ml 10X TBE 6 ml 40%
acrylamide:bis (38:2) 9 ml TOTAL: 60 ml
(This gel recipe was sufficient to pour two 20.times.20 cm gels;
each gel accommodated nine samples.)
[0103] For polymerization, 50 .mu.l TEMED and 500 .mu.l freshly
prepared 10% ammonium persulfate were added to the gel mixture,
swirling gently to mix reagents. The gel was immediately poured,
the comb inserted, and clamps placed on the wells. Polymerization
was allowed to occur for approximately one hour, after which the
comb was removed and the wells rinsed with 1.times.TBE. The bottom
buffer chamber was filled with 1.times.TBE. An entire 12 .mu.l of
each sample was loaded onto the gel, and the gel was run at 200-220
volts until the xylene cyanol was approximately 2 cm from the
bottom of the gel (approximately 3 hours).
[0104] Quantitation of Undigested and Digested Band Density by
Flourimaging
[0105] The gels were transferred to an overhead transparency, and
then to a glass sample plate on the Flourimager. The gel was
scanned on the Flourimager with the following settings: voltage
(PMI)=600; filter 1=530 dF30 agarose; wavelength=488 in. Once the
gel was scanned, the image could be modified and quantitated using
ImageQuant2 software. The percent C at nucleotide 2388 was then
calculated: [0106] a) Corrected volume of undigested product
(CU)=undigested band volume-background volume. [0107] b) Corrected
volume of digested product (CD)=digested band volume-background
volume. [0108] c) Percent 2388 "C"=[CD/(CD+CU)].times.100. To
ensure validity of each individual determination, all of the
following conditions were met: 1) the value of % 2388 C in the 100%
C DNA control sample must have been .gtoreq.905; 2) the value of %
2388 C in the 100% T DNA control sample must have been less than
1.5%; and 3) the total bands of fluorescence (indicated by the
volume) present in the undigested and digested bands must have been
.gtoreq.500,000.
Results
[0109] RNA was extracted as described above from each of 15
serotype G2 commercial vaccine lots, and for each vaccine lot, 5
individual determinations of the level of variant "C" at nucleotide
position 2388 were carried out. The RNA from each virus sample was
used to synthesize first strand cDNA (reverse transcription) in two
independent experiments carried out on separate days. Three
independent determinations of % C at nucleotide position 2388 were
made using cDNA derived from the first reverse transcription
reaction as PCR template, while the remaining two independent
determinations were carried out using cDNA derived from the second
reverse transcription reaction. For each sample, the Mean and
Standard Deviation of the five determinations was calculated.
[0110] Following the protocols described above, a short region of
gene 3 of RRV serotype G2 virus, encompassing nucleotide position
2388, was amplified by RT/PCR using the Superscript
Pre-amplification System for First Strand cDNA synthesis (Life
Technologies, Rockville, Md.). One of the primers used for
amplification was homologous to the sequence immediately upstream
of nucleotide 2388 and was designed to create an Eco RI restriction
enzyme site if a cytosine residue was present at position 2388
(RRV-G3-EcoRI primer, GTTAGTGGAGTTCTAGCGACATATTTTAAAATGTAGAAT (SEQ
ID NO: 186), corresponding to plus sense nucleotides 2347-2386).
The second primer (RRV-302A-5'Flourescein, GGTCACATCATGACTAGTGTG
(SEQ ID NO: 187), corresponding to negative sense nucleotides
2571-2591) was labeled with Flourescein (at the 5' end), enabling
the resulting PCR product to be visualized and quantitated using
polyacrylamide gel electrophoresis and flourimaging techniques.
[0111] Following amplification, the PCR product was purified and
digested. PCR products derived from genomes containing cytidine (C)
at nucleotide position 2388 were digested, resulting in 34 and 207
bp digested fragments; of these two fragments, only the 207 bp
fragment retains the flourescein tag and was detected by PAGE).
Those genomes containing thymidine (T) at nucleotide position 2388
did not generate an EcoRI site, and thus yielded a 244 bp uncleaved
product. The products were separated on a 6% polyacrylamide gel,
and the relative densities of bands representing the undigested and
digested products were quantitated using the Flourimager 595
(Molecular Dynamics), and a measurement of background fluorescence
was taken. The percentage of viral genomes in the sample containing
"C" at nucleotide 2388 was then calculated.
[0112] Base mixtures at nucleotide position 2388 in gene 3 and
nucleotide position 174 in gene 10 were consistently detected in
vaccine virus at the master working virus seed (MWVS) and vaccine
monopool bulk concentrate stages. Thus, measurement of the inherent
heterogeneity at these positions in the vaccine virus lots can be
used as the foundation for a product consistency assay. The MAPREC
analysis was used to determine the base composition at nucleotide
2388. The low level of variation observed in the fifteen G2
commercial vaccine lots at this nucleotide position demonstrates,
not only the stability of the subpopulation containing C at
nucleotide, but also the consistency of the vaccine manufacturing
process. The average percentage of C at nucleotide position 2388
ranged from 2/75% to 6.68%, resulting in a variation of no more
than 3.92% when any of the fifteen vaccine lots were compared
(Table 11). The first three MAPREC determinations, revealed
slightly higher levels of the minor species (C) compared to the
remaining twelve G2 vaccine lots. However, three additional
determinations carried out from a new RT reaction yielded levels of
C consistent with those observed in the other twelve lots.
Reanalyzing the data to exclude the first three MAPREC
determinations, the range of variation observed for the fifteen
vaccine lots was significantly reduced (i.e., a low of 2.76% and a
high of 4.90%, equivalent to a variation of just 2.14%). In each
case, excluding the three aberrant determinations, the standard
deviation observed across five MAPREC determinations of the
percentage C at nucleotide position 2388 of gene 3 in the
commercial vaccine lots fell well below 1%, with over 50% of the
samples resulting in standard deviations less than 0.5%. Taking
into account that the variation observed was nearly 4 times greater
by clonal analyses
TABLE-US-00026 TABLE 11 MAPREC Analysis of % C at Nucleotide
Position 2388 in RRV Gene 3 Sample Standard Name Trial#1 Trial#2
Trial#3 Trial#4 {circumflex over ( )}Trial#5 *Trial#5B {circumflex
over ( )}Trial#6 Average Duration 100% T- 1.13 1.04 0.85 0.47 0.86
0.87 0.25 DNA Control 100% C- 92.24 93.38 93.85 93.94 93.33 93.35
0.68 DNA Control Serotype 3.60 4.16 3.56 4.88 4.20 3.12 3.11 3.80
0.64 2-I97328 Serotype 3.38 3.08 4.16 5.20 4.97 3.39 3.17 3.91 0.88
2-I973029 Serotype 4.06 3.11 4.72 3.38 4.62 4.04 3.38 3.90 0.63
2-I973030 Serotype 4.16 4.60 3.82 3.32 3.23 2.58 3.76 3.64 0.66
2-I973032 Serotype 7.69 5.38 12.31 4.97 5.41 4.34 6.68 2.98
2-I983005 Serotype 6.67 4.33 9.00 4.12 5.25 5.11 5.75 1.83
2-I983006 Serotype 6.68 5.20 6.11 4.14 4.04 4.31 5.08 1.11
2-I983007 Serotype 2.97 3.97 3.46 2.79 2.85 3.65 3.28 0.48
2-I983008 Serotype 3.76 3.72 3.63 3.06 3.46 3.60 3.54 0.26
2-I983009 Serotype 3.65 4.36 3.83 4.01 4.13 3.81 3.97 0.26
2-I983011 Serotype 3.14 3.52 3.30 2.82 2.95 2.76 3.08 0.29
2-I983012 Serotype 3.32 3.99 3.55 3.48 2.55 3.72 3.44 0.49
2-I983013 Serotype 3.36 3.35 3.82 3.83 3.97 3.67 0.29 2-I983037
Serotype 4.16 4.67 4.76 4.05 4.56 4.44 0.32 2-I983038 Serotype 4.88
3.75 3.84 3.38 4.49 4.07 0.61 2-I983039 *A second digestion sample
taken from the PCR rxn generated in trial 5. (A digestion
duplicate) {circumflex over ( )}Trials 5 and 6 were PCR rxns.
seeded from the same RT rxn. and carried out on the same day.
than was found using MAPREC (8% vs 2.14%, respectively), the levels
of the minor species (C) in the G2 vaccine pools as measured by
either method are comparable (average of 4% for MAPREC vs 8% for
clonal analysis). These data indicate that MAPREC analyses of base
composition at nucleotide 2388 in gene 3 of RRV were both accurate
and reproducible.
[0113] While this invention has been particularly shown and
described with reference to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
Sequence CWU 1
1
18713302DNARotavirus 1ggctattaaa gctatacaat ggggaagtat aatctaatct
tgtcagaata tttatcattc 60atatataact cacaatccgc agttcaaatt ccaatttact
attcttccaa tagtgaatta 120gagaatagat gtattgaatt tcattctaaa
tgcttagaaa actcaaagaa tggactatca 180ttgaaaaagc tctttgttga
atatagcgat gttatagaga atgccacact gttgtcaata 240ttatcgtact
cttatgataa atataacgct gttgaaagga aattagtaaa atatgcaaaa
300ggtaagccgc tagaagcaga tttgacagtg aatgagttgg attatgaaaa
taacaagata 360acatctgaac tattcccaac ggcagaggaa tatactgatt
tattgatgga tccagcaatt 420ttaacttcat tatcatcgaa tttaaatgca
gttatgttct ggttggaaaa acatgaaaat 480gacgttgctg aaaaactcaa
aatttacaaa aggagattag acttatttac tatagtagct 540tcaacagtaa
ataaatatgg tgtaccaagg cacaatgcga aatatagata tgaatatgaa
600gtaatgaaag ataagccgta ctacttggtg acatgggcaa attcttcaat
tgaaatgctg 660atgtcagttt tttctcatga agattattta attgcgagag
aactgatagt actgtcatat 720tctaatagat cgactctggc aaaactggtg
tcatcaccaa tgtcaattct ggtagcttta 780gtggatataa acggaacgtt
cattacgaat gaagaactag agctagagtt ttcaaacaaa 840tatgtacgag
caatagttcc tgaccaaaca tttgatgaat taaaacaaat gcttgacaat
900atgagaaaag ctgggttaac tgacatacct aagatgatac aggactggtt
ggtcgattgc 960tctattgaaa aatttccatt gatggctaaa atatattcgt
ggtcatttca tgtcggattc 1020aggaaacaga aaatgttgga cgccgcacta
gatcaattga aaactgagta tacagaagat 1080gtagatgacg aaatgtatcg
agaatacaca atgctaataa gagatgaagt tgtgaaaatg 1140cttgaggaac
cagtaaagca tgatgaccat ttgttacagg attctgaatt agctggttta
1200ctatcaatgt catcagcgtc gaatggtgaa tcaagacaac taaaatttgg
tagaaagaca 1260attttttcga ctaaaaagaa tatgcatgta atggatgaca
tggctaatgg aagatacacg 1320ccaggcataa taccaccagt gaatgtcgat
aaaccgatac cattaggaag gagagatgta 1380ccaggaagac ggactagaat
aatatttatt ttaccatatg aatatttcat agcacaacat 1440gctgtagttg
aaaaaatgct aatttatgcg aaacatacta gagaatatgc tgaattctac
1500tcacagtcaa atcagttatt gtcttatggt gatgttacac gctttttatc
taataactct 1560atggtactat atacagacgt gtcccagtgg gactcatctc
aacacaatac gcagccattt 1620aggaaaggga taattatggg attggacatg
ctagccaata tgactaatga tgctagagtt 1680atacagacgc taaacttgta
taaacagacg caaattaatc taatggattc atacgttcaa 1740ataccagatg
gtaatgttat taagaagata caatatgggg ctgtagcgtc aggagagaaa
1800cagacgaaag cagcgaattc aatagcaaat ttagcactga ttaaaacggt
tttatcacgc 1860atttctaaca aatattcatt cgcgacgaag ataataagag
ttgacggaga tgacaattac 1920gcagtattgc aattcaatac agaagtaact
aaacaaatgg ttcaagatgt gtcaaacgac 1980gtgagagaaa catatgcgcg
aatgaatact aaagttaaag ccttagtatc tacagtggga 2040atagaaatag
ctaaaaggta tattgcaggt gggaaaatat tctttagggc tggaataaat
2100ttactgaata atgaaaaaaa aggacaaagc acacagtggg accaagcagc
tgtcctatat 2160tcgaactata ttgtgaatag acttcgagga tttgaaactg
acagagagtt cattttaact 2220aaaataatgc aaatgacgtc agttgctatt
accggatcgc taagactctt tccttctgaa 2280cgcgtgttaa ccacgaactc
tacatttaaa gtatttgact cggaggactt tattatagag 2340tatgggacaa
ctgacgatga agtatacata caaagagcgt tcatgtcttt atctagtcag
2400aagtcaggaa tagctgatga gatagctgca tcatcgacgt ttaagaatta
tgtgtctaga 2460ttatctgagc agctgttgtt ttcaaagaat aatatagtgt
ctagaggaat agcattgact 2520gaaaaggcaa agttgaactc atacgcacca
atatcacttg agaaaagacg tgcgcaaata 2580tcagctttgc tgactatgct
acaaaaaccg gttactttta aatcaagtaa aataacaata 2640aatgatatac
ttagagatat aaagccattt ttcactgtaa acgaagcaca tttgccgata
2700caatatcaaa aatttatgcc aactttacca gacaatgtgc agtatataat
tcagtgtata 2760ggatccagaa cctaccaaat tgaagacgac ggttcaaagt
cagcaatatc tcgactaata 2820tcaaagtatt cagtttacaa accgtcaatc
gaagagttat acaaagtaat ttcactacac 2880gagaatgaaa tacaactata
tttgatctca ctaggcatac cgaaaataga cgctgatacg 2940tacgtcggat
cgaaaattta ttctcaagat aaatacagga tattagagtc gtatgtatat
3000aacttattat ctattaatta tggatgttat caactattcg actttaattc
accagatcta 3060gaaaagttga tcagaatacc gtttaaagga aagataccag
ctgtcacttt tatattgcat 3120ttatacgcta agctagaagt tataaatcat
gccatcaaaa atggctcatg gataagctta 3180ttctgcaact acccaaaatc
agaaatgata aaattatgga agaaaatgtg gaacattaca 3240tcactacgtt
caccgtatac caatgcaaac ttctttcaag attagagcgc ttagatgtga 3300cc
330222708DNARotavirus 2ggctattaaa ggctcaatgg cgtacagaaa gcgtggagcg
cgtcgtgaga cgaatttaaa 60acaagatgat cgaatgcaag aaaaagaaga aaataaaaac
gtaaatacta atagtgaaaa 120taaaaatgct acaaaacctc aattatcaga
gaaagtgcta tctcaaaagg aagaggtgat 180tacagataat caagaagaaa
ttaaaatagc tgatgaagtt aaaaaatcta ataaagagga 240atcaaaacaa
ctattagagg ttctaaaaac taaggaagaa catcaaaaag aagttcaata
300cgaaatacta cagaaaacaa tacctacttt tgaaccaaaa gaatctatac
taaagaaatt 360agaagatatt aaaccagagc aagttaaaaa gcaaaccaaa
ttattcagaa tatttgaacc 420taggcaacta ccagtataca gagcaaacgg
agagaaagaa ctacgcaaca gatggtattg 480gaagttaaaa cgagacacct
tgcccgatgg agattatgat gtgagagaat attttttaaa 540tttgtatgat
caagtactaa ctgaaatgcc agattattta ctacttaaag acatggctgt
600tgaaaacaag aattctagag atgcaggcaa agttgttgac tctgaaactg
cagccatttg 660cgatgcgatt ttccaagatg aagaaaccga aggtgtagta
agaagattca tagcagaaat 720gaggcaaaga gtacaagctg atcgaaatgt
agttaattac ccatcaatat tgcatccaat 780tgatcatgcc tttaatgagt
attttttaca gcatcagtta gttgaaccat tgaataatga 840tataatattc
aattacatac ctgaaagaat acggaatgac gtaaactata tactgaacat
900ggatcgaaat ttaccttcaa cagctagata cattagacca aatttacttc
aagatagact 960caatttgcat gataacttcg aatccttatg ggatactata
actacatcaa actacatctt 1020agctagatca gttgtgccag accttaagga
actagtgtcc accgaggctc aaatacaaaa 1080aatgtcacag gatttgcaat
tagaggcgct gacaattcaa tcagaaacac aatttttaac 1140aggtatcaat
tcacaggcag ctaatgactg ctttaagacg ttaattgccg ctatgcttag
1200tcaacgtact atgtctttag atttcgtcac tacaaactac atgtcattaa
tttcagggat 1260gtggttatta acagttgtac ctaatgatat gttcatacgt
gaatccctag tagcatgtca 1320attggcaata attaatacca tcatatatcc
agcctttgga atgcagagaa tgcattacag 1380aaatggtgac ccccaaactc
cttttcagat cgctgaacaa caaattcaga actttcaggt 1440ggccaattgg
ctacattttg ttaacaataa tcaatttaga caagtagtaa ttgatggagt
1500attaaaccaa gttctgaatg ataatataag aaatggacat gtagttaatc
aattgatgga 1560agctttaatg caattgtcac gacaacaatt cccaaccatg
ccagtagatt ataaaagatc 1620aatacagaga ggaatattac ttctatcgaa
tagattagga caattggttg acctaactag 1680gctattggca tataattatg
aaactctgat ggcgtgcatt accatgaaca tgcaacatgt 1740acaaactcta
actactgaaa agttgcaatt aacatcagtt acttccttat gtatgttaat
1800aggaaatgct acagttatac caagtccaca aacattattt cattattata
acgtcaacgt 1860caattttcat tcaaattaca atgaaagaat aaatgacgca
gtagcaatca taaccgccgc 1920aaatagattg aatttgtatc agaaaaagat
gaagtcgata gttgaagatt tcttaaagag 1980actacaaata ttcgacattt
ctagagttcc agatgatcaa atgtacagac tcagggatag 2040attgagatta
ctcccagttg aaattagaag attagatata tttaatttaa tattgatgaa
2100tatggagcag attgaacgcg catcggataa aattgcccag ggagtgatta
tagcttatag 2160agacatgcag ttagagagag atgaaatgta tggctacgtt
aacatagctc gtaatttaga 2220cggttttcag cagataaatt tagaggagtt
gatgagaacg ggagattatg cacaaattac 2280taatatgcta ctaaataatc
agccagtggc attagtagga gcactaccat ttataacaga 2340ctcatcagtt
atctcattgg tagctaaatt agacgctact gtctttgcac aaattgttaa
2400gctcaggaag gttgatactt taaagccaat cctgtataaa ataaattctg
attcaaatga 2460tttttatctt gtagcgaatt atgactgggt tccaacgtct
acaacaaaag tttataaaca 2520aataccacaa caatttgatt ttagagcatc
tatgcatatg ttaacgtcta atttgacttt 2580cactgtatat tccgaccttc
ttgcattcgt ttcagcagac actgttgaac caattaatgc 2640tgttgcattt
gacaatatgc gcatcatgaa cgaactgtaa acgccaaccc cactgtggag 2700atatgacc
270832591DNARotavirus 3ggctattaaa gcagtacgag tagtgtgttt tacctctaat
ggtgtaaaca tgaaagtact 60agctttaaga cacggtgtgg ctcaggtgta tgcagacacc
caaatctata ctcatgatga 120tactaaagac agttacgaga atgcatttct
tatatctaat cttacaacgc ataacatctt 180atatttaaat tacagtatca
aaacgcttga aatattgaat aaatcaggaa ttgcagctgt 240tgagattcaa
tctcttgaag aattattcac tttaattaga tgtaatttta cttatgatta
300tgaaaacaat ataatttact tgcatgacta ctcatactac actaataatg
aaataagaac 360tgatcaacat tgggttacaa agactgatat tgaggaatac
ttgttaccag gatggaaatt 420aacatacgta ggatataatg ggagtgatac
tagaggacat tataacttct cattcacatg 480ccagaatgct gcaaccgatg
atgacttaat aatagaatac atttattccg aagcattgga 540ctttcagaat
ttcatgttaa agaaaattaa agaaagaatg actacttctt taccgatagc
600acgtttatca aatagagttt ttagagataa attatttcca ttactaagtg
aaaaacatca 660gcgtatagtg aacattggac cgagaaatga atcaatgttt
accttcttaa attttccatc 720aattaagcaa ttttcaaatg gaccatattt
agttaaagat actattaaat tgaagcaaga 780aagatggttg gggaaaagag
tgtctcagtt cgacattgga caatacaaga acatgatgaa 840cgtcataaca
actgtatatt attattataa cttatatcag aaaaaaccta ttatatatat
900ggttggttca gctccttcat actggattta tgatgtcaaa caatattctg
attttatgtt 960tgagacttgg gacccacttg acactccata ttcatcagtg
catcataaag aattattttt 1020tgagaaggac ataactagat taaaggatga
ttcaatattg tatattgata tcaggactga 1080tcgtggaaac acggattgga
aagaatggag gaaaatagtt gaggcgcaaa ctattagtaa 1140ccttaaactt
gcataccgat acttatctgg tggtaagtcg aaggtatgtt gtgttaaaat
1200gactgctatg gatttagagc ttcccatatc tgcaaagtta ttgcatcatc
caactactga 1260aatccgatca gagttttatc ttcttctgga catctgggac
attagtaatg tcaaaagatt 1320tattccaaag ggagtattat attcattcat
aaataacgtt actactgaaa atgtattcat 1380acaaccgccg ttcaaaatca
aaccgtttaa gaatgattat attgtggcat tatacgcatt 1440atcaaatgat
tttaatgata gaacggatgt aattaactta attaacaatc agaaacaatc
1500gctcattact gtaagaatta ataacacatt taaagatgaa ccaaaggtag
ggtttaagaa 1560tatatatgat tggacctttc taccaacaga ttttactaca
actgatgcca taataacctc 1620atacgatgga tgtttaggta tatttggatt
atcaatatcc ttagcttcaa agcctacggg 1680aaataatcac ttgtttatct
taaatggaac cgataagtat tataaattgg atcaattcgc 1740aaaccatact
ggcatttcca gaagatcaca ccaaattaga ttttcagaat ccgcaacatc
1800gtattcagga tacatattca gagatttatc taacaacaat tttaacttga
ttgggacaaa 1860tgtagaaaat tcagtttcag gacatgtata taatgcgtta
atttattata gatataacta 1920ctcttttgac ttaaaaagat ggatatactt
acactcgata gaaaaagctg atatagaagg 1980tggaaagtat tatgaacatg
ctccgataga attgatttat gcctgtagat cagcaaaaga 2040attcgcttta
ttacaagatg atcttactgt attacgttat gctaatgaaa tcgagagcta
2100tataaataaa gtatatagta taacatatgc agatgatcca aattacttta
taggtattaa 2160attcagacac attccctatg aatatgatgt taaaattcca
catttgacat ttggagtatt 2220atttatttca gataatatga ttccagatgt
agtggagatc atgaaaatta tgaaaaagga 2280attatttgaa atggatataa
ccactagtta cacatatatg ttatctgatg gaatatatgt 2340agcaaacgtt
agtggagttc tagcgacata ttttaaaatg tataatttat tttataagag
2400tcagattaca ttcggtcaat ctagaatgtt tattccacat ataacactaa
gttttagtaa 2460taataaaaca gtaagaatag aaagtactag gttaaagatt
agctcaatat atttaagaaa 2520gattaaagga gatacggtgt ttgatatgtc
tgagtgagct agaaacttaa cacactagtc 2580atgatgtgac c
259142362DNARotavirus 4ggctataaaa tggcttcgct catttataga caattgctta
caaattcata taccgttgac 60ttatctgatg aaatacaaga aattggatct acaaaaacgc
aaaatgtcac tattaatcta 120ggtccttttg ctcaaacagg ttatgctcca
gttaactggg gtcctggtga aactaatgat 180tctactactg tagaaccggt
acttgatggt ccttatcaac caacaacgtt caatccacca 240gtagattatt
ggatgctatt agcacctaca gcagctggag tagtagtaga aggtactaat
300aatacagacc gatggctagc tacaatttta gttgagccaa acgtaacatc
agaaaccaga 360agttatacgc tatttggaac gcaagagcaa attacaatag
ctaatgcttc ccaaacacaa 420tggaaattta ttgatgtcgt taaaactaca
caaaatggaa gctattcaca atacggacca 480ttacaatcta ctccaaaact
ctatgccgtg atgaaacata atggtaaaat ttatacatat 540aatggagaaa
ctccgaatgt gaccactaag tactactcaa ctacaaatta tgattcagta
600aacatgacag cattttgtga cttttatatt atacctagag aagaagaatc
aacatgtacc 660gagtacatta ataacgggtt acctccgatt cagaatacac
gaaacattgt tccattggcg 720ctttcagcta gaaatataat atcacataga
gctcaagcga atgaagatat cgttgtgtca 780aagacatcac tttggaaaga
gatgcaatac aatagagaca tcacaattcg atttaaattc 840gcaagttcaa
ttgttaaatc cggtgggcta ggttataaat ggtcagagat ttcatttaaa
900ccagcaaact atcaatatac gtatacacga gatggagagg aggtgacagc
tcacacgacg 960tgctccgtaa acggaatgaa cgattttaat ttcaatgggg
gatcgttacc aacggatttt 1020gtaatatcaa gatatgaagt aattaaagag
aattcttatg tttatgttga ttactgggat 1080gattcacaag ccttcaggaa
catggtttat gtaaggtcat tagctgctaa tttaaactct 1140gttatatgta
ctgggggtga ttatagcttt gcattaccgg ttggtcaatg gccagtaatg
1200actggcggag cagtgtcatt gcattcagct ggtgttacgc tatccacaca
gttcacagat 1260tttgtatcat taaattcttt aaggttcagg tttagactaa
ctgttgaaga gccatcattc 1320tcgatcacca gaactagagt tagtagattg
tatgggttac ctgcagctaa cccaaataat 1380ggaaaagaat attatgaagt
ggctggcaga ttctcactaa tatcattggt accatctaat 1440gacgattacc
agacaccaat aactaattca gttacagtca gacaagattt agaacgacag
1500ttgggtgaac ttagagaaga attcaacgct ctctcacaag agatagccat
gtcgcagcta 1560attgatttgg cattacttcc attggatatg ttttcgatgt
tttccggtat taagagcacc 1620atagatgcag ctaaatcaat ggctactagt
gtaatgaaga aatttaagaa atcaggttta 1680gctaactctg tatctacatt
aacagactca ctgtccgacg cagcttcttc aatttcaaga 1740ggagcatcta
ttcgttcagt tggatcatca gcatcagcat ggacggatgt ctcaacacaa
1800atcactgatg tttcttcatc tgtcagttcg atctcgacac agacttcaac
tattagtaga 1860cggctacgac taaaagaaat ggctacgcaa acagaaggga
tgaatttcga tgatatatct 1920gctgcagtat tgaagactaa aattgatcga
tccactcaaa tatctccaaa cacattacca 1980gatatagtca ctgaagcttc
agagaagttc attcctaata gagcgtacag agtaataaat 2040aatgatgaag
tctttgaagc gggaacagat ggaagatttt ttgcgtatcg tgttgaaacg
2100ttcgatgaaa taccttttga tgtgcaaaag tttgcagatc tagtaactga
ctctccggtc 2160atctcagcca taatagactt taagacacta aagaatctaa
acgacaatta tggtattagt 2220aggcaacaag catttaatct gctaagatcc
gatccaagag tattacgtga atttatcaat 2280caagacaatc caataattcg
taacagaatt gaacagttaa taatgcagtg tagactgtaa 2340gcaatttcta
gaggatgtga cc 236251599DNARotavirus 5ggcttttttt tgaaaagtct
tgtgttagcc atggcaacct ttaaggatgc ttgctttcat 60tatagaaggg ttacaaaact
aaacagagaa ttgctgagaa ttggagcaaa ttcagtatgg 120actccagtct
cttcgaataa aattaaaatt aaagggtggt gcattgagtg ttgtcaatta
180actggattga ctttttgtca cggatgttcg ctagctcatg tttgtcagtg
gtgcatccaa 240aacaaacgtt gcttcttgga caatgaacca catcttttaa
aattaagaac ttttgaatct 300ccaataacga aggaaaaatt acaatgcatt
attaatttat atgaattact atttccaatt 360aatcatgggg ttatcaataa
atttaaaaaa acaataaaac agaggaaatg tagaaatgag 420tttgacaaat
catggtataa tcagctactg cttccaatta ctttaaatgc tgcagttttc
480aagtttcact caagggatgt ttatgttttt ggattttatg aaggatcatc
accatgcata 540gatttgccat atagacttgt aaattgcatt gatttatatg
ataaactatt gttagatcaa 600gtaaactttg aaaggatgag ttctcttcca
gataatttac aatccatcta tgcaaacaaa 660tacttcaaat taagtagact
tccttcaatg aagctaaaac gaatctatta ctcagatttc 720tccaaacaga
atttgattaa taagtacaag actaaaagtc gcatagttct taggaatctt
780actgaattca cctgggattc tcaaactgat ttacatcatg atctgattaa
tgataaagat 840aaaatacttg ccgcattatc aacatcatca ttaaaacaat
ttgaaacaca tgatttaaat 900ttggggagaa taaaagctga catttttgaa
cttggacatc actgcaaacc aaattacatc 960tcatcaaatc attggcaacc
agcatcaaaa atttctaaat gtaaatggtg taatgtaaaa 1020tatgcattca
gagacatgga ttggaagatg gaatcaatgt acaatgaact tttaagcttt
1080atccaatctt gctataaaag taatgttaat gtaggacatt gtagttcaat
tgaaaaagct 1140tatccattag ttaaagatat actttggcat tcaattactg
aatatattga tcaaactgtt 1200gagaaattgt ttaatacaat gaatccagtg
caagtaaatg aacagcaggt aataaagttc 1260tgttggcaaa tagatatcgc
attatatatg cacattaaaa tgatactgga aaccgaggct 1320cttccattta
ctttcacatt gaatcagttc aattctataa ttaaagggat tgtgaaccaa
1380tggtgtgatg ttgctgaatt agatcacttg ccgttatgca ctgaacagac
tgatgcattg 1440gttaaattgg aagaagaagg aaaactatct gaagaatatg
agcttctgat ctcggactct 1500gaagatgacg actaatgatt gaattaacta
tcaccacagt ttttgccatc acaagacctt 1560ctggactaga gtagcgccta
gccagcaaaa actgtgacc 159961356DNARotavirus 6ggcttttaaa cgaagtcttc
aacatggatg tcctgtactc cttgtcaaaa actcttaaag 60atgctagaga caaaattgtc
gaaggcacat tatactccaa tgtaagtgat ctaattcaac 120aatttaatca
aatgataatt actatgaatg gaaatgaatt tcaaactgga ggaattggta
180atctgccgat tagaaattgg aattttgatt ttggattact tggaacaact
ctactaaatt 240tagatgctaa ctatgtcgaa acggcccgta atacaattga
ttattttgta gattttgtag 300ataatgtatg catggacgaa atggttagag
aatcacaaag aaatggaatt gcaccacaat 360cagactcact tagaaagttg
tcaggcatta aatttaaaag aataaatttt gacaattcat 420cagaatacat
tgagaactgg aatttgcaaa acagaagaca aagaacgggt tttacatttc
480ataaaccaaa cattttccct tattcagctt cattcacact gaacagatca
caaccggctc 540atgataactt gatgggtacg atgtggctca atgcgggatc
agaaattcag gtcgctggat 600tcgactattc atgtgcaata aatgcgccag
ctaatataca acaatttgag catattgtac 660agcttcgaag ggtgttgact
acagctacaa taactcttct accagatgca gaaagattta 720gttttccaag
agtgattaat tcagctgacg gagcggctac atggtacttt aatccagtga
780ttcttagacc aaataacgtt gaagtagaat ttctactaaa cgggcagata
ataaatactt 840accaagcaag atttggaacg ataatagcca gaaattttga
tacaattaga ttgtcatttc 900agttaatgag accaccaaat atgacaccag
cggtagcggc gttatttcca aatgcgcaac 960catttgaaca tcatgcaaca
gtaggactca cgcttagaat cgaatctgca gtttgtgaat 1020cagtacttgc
cgacgcaagc aaaacaatgc tagcgaacgt gacatctgtt agacaagaat
1080acgcgatacc agttggacca gtttttccac caggtatgaa ttggactgat
ttgatcacta 1140actattcacc atctagagag gataacttgc agcgtgtatt
tacagtggct tccattagaa 1200gcatgcttgt caaatgagga ccaagctaac
cacttggtat ccgactttga tgagtatgta 1260gcttcgtcaa gctgtttgaa
ctctgtaagt aaggatgcgt ccacgtattc gctacacaga 1320gtaatcactc
agatgacgta gtgagaggat gtgacc 135671078DNARotavirus 7ggcatttaat
gcttttcagt ggttgatgct caagatggag tctactcagc agatggcttc 60ttctattatt
aattcttcat ttgaagctgc agtggttgct gcaacttcta ctcttgaatt
120gatgggtatt caatatgact acaatgaggt atatactaga gtaaagagta
aatttgattt 180agttatggat gattctggtg taaaaaataa cttaataggt
aaagcaatta ctattgatca 240agctttgaat ggaaaattta gttcagcgat
taggaataga aattggatga ctgactctcg 300aactgtagct aaattagatg
aggatgtaaa taaactaaga attatgctat catcaaaagg 360aatcgatcag
aaaatgagag tgcttaatgc ttgttttagt gtcaagagaa tacctgggaa
420atcatcatct atagttaaat gtactagact gatgaaagac aaattagaac
gtggtgaagt 480tgaagttgat gattcctttg ttgaagagaa aatggaagta
gatacaattg attggaaatc 540aagatatgaa cagttagaaa agagatttga
gtcactgaaa catcgggtta atgagaagta 600taatcattgg gttcttaaag
ctagaaaggt aaatgaaaat atgaattctc ttcaaaatgt 660gatttctcaa
caacaagcac acattaatga actacaaatg tataataata aattagaacg
720tgatttgcaa tccaaaattg gatctgttgt gtcatcaata
gaatggtatc taagatcgat 780ggaattatct gatgatgtaa aatcagacat
tgaacaacag ctgaattcaa tagatcaatt 840aaatccagtt aatgcaatag
atgattttga atcaatactt cgcaatttaa tttctgatta 900tgataggcta
tttataatgt ttaaaggatt attgcagcaa tgcaactaca cttatactta
960tgagtaattg aatgaacaat tcaatactat taccatctac acgtaaccct
ctatgagcac 1020aatagttaaa agctaacact gtcaaaaacc taaatggcta
taggggcgtt atgtgacc 107881059DNARotavirus 8ggcttttaaa gcgtctcagt
cgccgtttga gccttgcggt gtagccatgg ctgagctagc 60ttgcttttgt tatccccatt
tggagaacga tagctataga ttcattccat tcaatagttt 120ggctataaaa
tgtatgttga cagcaaaagt agataaaaaa gatcaggata aattttacaa
180ttcaataatt tatggtatcg caccaccgcc acaattcaaa aaacgttata
acacaaatga 240caattcaaga ggaatgaatt atgaaactcc aatgtttaat
aaagtggcgg tgttaatttg 300tgaagcgttg aattcaatta aagttactca
atctgatgtt gcgaatgtac tttcaaaagt 360agtttctgta agacatctag
agaatttggt actgagaaga gagaatcatc aggacgtgct 420ttttcattca
aaagagttgt tgctgaaatc agtactaata gctattggtc actcaaaaga
480aattgaaaca actgccaccg ctgaaggagg ggaaatagtt tttcaaaatg
cagcttttac 540aatgtggaaa ttgacatacc tggaacatag actaatgcca
attttggatc aaaattttat 600tgaatataaa ataacagtga atgaagataa
accgatttca gaatcacatg ttaaagaact 660cattgctgag ttgcggtggc
aatacaacaa atttgcagta attacacatg gtaaaggtca 720ctacagagtt
gtaaaatatt catcagttgc gaatcatgca gatagagttt acgctacttt
780caagagcaat aataagaatg gtaatgtgct agagtttaat ctacttgatc
aaagagtaat 840atggcagaac tggtatgcgt ttacatcctc aatgaaacaa
ggtaacactc ttgaaatatg 900caagaaacta ctgttccaaa aaatgaagcg
ggaaagtaat ccgtttaagg gactgtcaac 960tgatagaaag atggatgagg
tttctcaaat aggaatttaa ttcgttatca atttgagagt 1020gggtatgaca
aagtaagaat agaaagcgct tatgtgacc 105991062DNARotavirus 9ggctttaaaa
gcgagaattt ccgtttggct agcggttagc tccttttaat gtatggtatt 60gaatatacca
cagttctaac ctttctgata tcgctcattt tattgaatta tattttaaaa
120tctttgacta gaatgatgga ctttattatt tacagatttc tttttattgt
agttattttg 180tcaccattac taaaagccca aaattatgga attaatctac
caattactgg ttcaatggac 240actgcatacg ctaactctac acaggaagag
acttttctca catctacttt gtgtctatat 300tatccaactg aagctgcaac
agaaataaat gataattcgt ggaaggatac actctcacaa 360ttattcttga
ctaaaggatg gccaactgga tcagtttatt ttaaagaata cacggatatt
420gcttcctttt cagttgatcc acaactatat tgtgattata acgtggtact
aatgaaatat 480gatgcgactt tgcagctgga catgtctgaa ctagctgatt
taatactgaa tgaatggctg 540tgcaatccaa tggatattac tctatattat
tatcaacaaa cagacgaagc taacaaatgg 600atttctatgg gatcttcctg
tacaataaaa gtatgtccac ttaatacaca gactcttgga 660attgggtgtt
tgactactga tacggcaaca tttgaagaag tcgctacagc tgaaaaactg
720gtgattactg acgttgtcga tggtgtgaat cataaacttg atgttacaac
tgctacttgc 780actatcagaa actgcaaaaa attaggacca agggaaaatg
tagcagtaat tcaagttgga 840ggttctgatg ttctcgacat aacggctgat
ccaaccacag caccacaaac tgaacgaatg 900atgcgcatta attggaagaa
atggtggcaa gttttttata ccgtagtcga ctatgtgaat 960caaataattc
aagcaatgtc caaaagatca cgatcactta actctgctgc attctattat
1020agaatatagg tatagctttg gttagaattg tatgatgtga cc
106210750DNARotavirus 10ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa
gatggaaaag cttaccgacc 60tcaactacac attgagtgta gtcactctca tgaatgatac
tttacatacc ataatggagg 120atcctggaat ggcgtatttt ccatacattg
cttctgtcct aactgtacta tttacattac 180ataaggcctc ggttccaacc
atgaagattg ctcttaaaac gtcaaagtgt tcatataaag 240taatcaaata
ctgcattgtg tcaattttta acactctatt gaaactggct ggatataaag
300aacaaattac tactaaagat gaaattgaaa ggcaaatgga cagagttgta
aaagaaatga 360gacgtcagct ggaaatgatt gataagctaa ccactagaga
gattgagcaa gtcgaactac 420ttaaacgaat tcatgatatg ttgataatta
aaccagttga caaaattgat atgtcacaag 480aatttaatca gaaatatttc
aaaacgctaa atgattgggc tgaaggtgaa aatccatatg 540aaccaaaaga
ggtgactgca tcattgtgag aggttgagct gccgtcgtct gtctgcggaa
600gcggcggagt tcttaacagt aagccccatc ggacctgatg actggttgag
aagccacaac 660cagtcatatc gcgtgtgact cagtcttaat cccgtttaac
caatccagcc agcgctggac 720gttaatggaa ggaacggtct taatgtgacc
75011667DNARotavirus 11ggcttttaaa gcgctacagt gatgtctctc agtattgacg
tgacaagtct tccatctatt 60tcctccagca tttataaaca tgaatcatct tcaacgacgt
caactctttc tggaaaatct 120attggtagga gtgaacagta cgtttcacca
gatgcagaag cattcaataa gtacatgttg 180tcgaagtctc cagaggatat
tggaccatct gattctgctt caaacgaccc actcaccagt 240ttttcgatta
gatcgaatgc agttaagaca aatgcagacg ctggcgtgtc tatggattca
300tcgacacaat cacgaccttc aagtaacgtt ggatgcgatc aagtggattt
ctccttaagt 360aaaggcatta aagtaaacgc taatttagat tcatctattt
cagtatcaac agtttccaag 420aaggagaaat ccaaatcaga tcataaaaat
aggaaacact acccgagaat tgaagcagat 480tccgattcag acgaatatgt
acttgatgat tcagatagtg atgatggtaa gtgtaaaaac 540tgtaaatata
agaaaaagta tttcgcactt agaatgagaa tgaagcaagt cgcaatgcag
600ttgattgaag atttgtaagt ctaacctgag gactcactag gaagctcccc
acttccgttt 660tgtgacc 667121062DNARotavirus 12ggctttaaaa gagagaattt
ccgtctggct aacggttagc tccttttaat gtatggtatt 60gaatatacca caattctaat
ctttttgata tcaatcattc tactcaacta tatattaaaa 120tcagtgactc
gaataatgga ctacattata tatagatttt tgttgattac tgtagcatta
180tttgctttga caagagctca gaattatgga cttaacttac caataacagg
atcaatggac 240gctgtatata ctaactctac tcaagaagaa gtgtttctaa
cttctacgtt atgtctgtat 300tatccaactg aagcaagtac tcaaatcaat
gatggtgact ggaaagactc attgtcgcaa 360atgtttctta caaagggttg
gccaacagga tctgtttact ttaaagagta ctcaagtatt 420gttgattttt
ctgttgaccc acagctgtat tgtgactata atttagtact tatgaaatat
480gaccaaagtc ttgaattaga tatgtcggag ttagctgatt taatattgaa
tgaatggtta 540tgtaacccaa tggatgtaac attatactat tatcaacaat
cgggagaatc aaataagtgg 600atatcgatgg gatcatcatg taccgtgaaa
gtgtgtccgc taaatacaca aacgttaggg 660ataggttgtc aaacaacaaa
cgtagactca tttgaaatga ttgctgagaa tgagaaatta 720gctatagtgg
atgtcgttga tgggataaat cataaaataa atttaacaac tacgacatgt
780actattcgaa attgtaagaa attaggtcca agagaaaatg tagctgtaat
acaagttggt 840ggttctaatg tattagacat aacagcagat ccaacaacta
atccacaaac tgagagaatg 900atgagagtga attggaaaaa gtggtggcaa
gtattttata ctatagtaga ttatattaat 960caaattgtac aggtaatgtc
caagagatca agatcattaa attctgcagc tttttattat 1020agagtataga
tatatcttag attagaattg ttcgatgtga cc 1062131062DNARotavirus
13ggctttaaaa acgagaattt ccgtctggct agcggttagc tctttttaat gtatggtatt
60gaatatacca caattctgac cattttaata tctatcatat tattgaatta tatattaaaa
120actataacta atacgatgga ctacataatt ttcaggtttt tactactcat
tgctttaata 180tcaccatttg taaggacaca aaattatggt atgtatttac
caataacggg gtcactagac 240gctgtatata cgaattcgac tagtggagag
ccatttttaa cttcgacgct atgtttatac 300tatccagcag aagctaaaaa
tgagatttca gatgatgaat gggaaaatac tctatcacaa 360ttatttttaa
ctaaaggatg gccaattgga tcagtttatt ttaaagacta caatgatatt
420aacacatttt ctgtgaatcc acaactgtat tgtgattata atgtagtatt
gatgagatat 480gacaatacat ctgaattaga tgcatcagag ttagcagatc
ttatattgaa tgaatggctg 540tgcaatccta tggacatatc actttactat
tatcaacaaa gtagcgaatc aaataaatgg 600atatcgatgg gaacagactg
cacggtaaaa gtttgtccac tcaatacaca aaccttaggg 660attggatgca
aaactacgga cgtaaacaca tttgagattg ttgcgtcgtc tgaaaaatta
720gtaattactg acgttgtaaa tggtgttaat cataagataa atatttcaat
aaatacgtgc 780actatacgta actgtaataa attaggacca cgagaaaatg
ttgctataat tcaagttggt 840ggaccgaacg cattagatat cactgctgat
ccaacaacag tcccacaagt tcaaagaatc 900atgcgaataa attggaaaaa
atggtggcaa gtattttata cagtagttga ctatattaac 960caagttatac
aagtcatgtc caaacgatca agatcattag acgcagctgc tttttattat
1020agaatttaga tatagatttg gtcagatttg tatgatgtga cc
1062141062DNARotavirus 14ggctttaaaa gagagaattt ccgtttggct
agcggatagc tccttttaat gtatggtatt 60gaatatacca cagttctatt ttatttgata
tcgttcgttc ttgtgagtta tattctgaaa 120accataataa agataatgga
ctatattatt tatagaataa catttgtaat tgtagtatta 180tcagtattat
cgaatgcaca aaattatgga ataaatttgc caattactgg atctatggat
240acagcatatg ctaactcaac acaagacaat aattttttat tttcaacttt
atgtctatat 300tatccatcag aagctccaac tcaaattagt gacactgaat
ggaaagatac actatctcag 360ctgtttttaa ccaaaggatg gccgacaggt
tcagtttatt ttaatgaata ttcaaacgtt 420ttagaatttt ccatcgaccc
aaagctatac tgtgattata atgttgtgct aattagattc 480gtttctggtg
aggagttgga catatctgaa ttagctgatc taatactgaa tgagtggtta
540tgtaatccaa tggatataac attatattat taccaacaaa ctggagaggc
aaacaaatgg 600atatcaatgg gatcatcatg taccgttaaa gtgtgtccat
taaatactca gacattagga 660attggatgtc aaacgacaaa tacagctact
tttgaaacag ttgctgatag cgaaaaattg 720gcaataattg atgttgtcga
cagcgtaaat cataaattaa atatcacatc tactacatgt 780acaatacgga
attgtaataa actaggaccg agagaaaatg tggctataat acaggttggc
840ggttctaata tattagatat aacagctgat cccacaactt ctccacaaac
agaacgaatg 900atgcgcgtaa actggaaaaa atggtggcaa gtattctaca
ctgtagttga ttacattgat 960cagatagtac aagtaatgtc caaaagatca
agatcgttag atttgtcatc tttctattat 1020agagtgtaga tatatcctaa
aatagaactg tttgatgtga cc 1062151078PRTRotavirus 15Met Gly Lys Tyr
Asn Leu Ile Leu Ser Glu Tyr Leu Ser Phe Ile Tyr 1 5 10 15Asn Ser
Gln Ser Ala Val Gln Ile Pro Ile Tyr Tyr Ser Ser Asn Ser 20 25 30Glu
Leu Glu Asn Arg Cys Ile Glu Phe His Ser Lys Cys Leu Glu Asn 35 40
45Ser Lys Asn Gly Leu Ser Leu Lys Lys Leu Phe Val Glu Tyr Ser Asp
50 55 60Val Ile Glu Asn Ala Thr Leu Leu Ser Ile Leu Ser Tyr Ser Tyr
Asp65 70 75 80Lys Tyr Asn Ala Val Glu Arg Lys Leu Val Lys Tyr Ala
Lys Gly Lys 85 90 95Pro Leu Glu Ala Asp Leu Thr Val Asn Glu Leu Asp
Tyr Glu Asn Asn 100 105 110Lys Ile Thr Ser Glu Leu Phe Pro Thr Ala
Glu Glu Tyr Thr Asp Leu 115 120 125Leu Met Asp Pro Ala Ile Leu Thr
Ser Leu Ser Ser Asn Leu Asn Ala 130 135 140Val Met Phe Trp Leu Glu
Lys His Glu Asn Asp Val Ala Glu Lys Leu145 150 155 160Lys Ile Tyr
Lys Arg Arg Leu Asp Leu Phe Thr Ile Val Ala Ser Thr 165 170 175Val
Asn Lys Tyr Gly Val Pro Arg His Asn Ala Lys Tyr Arg Tyr Glu 180 185
190Tyr Glu Val Met Lys Asp Lys Pro Tyr Tyr Leu Val Thr Trp Ala Asn
195 200 205Ser Ser Ile Glu Met Leu Met Ser Val Phe Ser His Glu Asp
Tyr Leu 210 215 220Ile Ala Arg Glu Leu Ile Val Leu Ser Tyr Ser Asn
Arg Ser Thr Leu225 230 235 240Ala Lys Leu Val Ser Ser Pro Met Ser
Ile Leu Val Val Asp Ile Asn 245 250 255Gly Thr Phe Ile Thr Asn Glu
Glu Leu Glu Leu Glu Phe Ser Asn Lys 260 265 270Tyr Val Arg Ala Ile
Val Pro Asp Gln Thr Phe Asp Glu Leu Lys Gln 275 280 285Met Leu Asp
Asn Met Arg Lys Ala Gly Leu Thr Asp Ile Pro Lys Met 290 295 300Ile
Gln Asp Trp Leu Val Asp Cys Ser Ile Glu Lys Phe Pro Leu Met305 310
315 320Ala Lys Ile Tyr Ser Trp Ser Phe His Val Gly Phe Arg Lys Gln
Lys 325 330 335Met Leu Asp Ala Ala Leu Asp Gln Leu Lys Thr Glu Tyr
Thr Glu Asp 340 345 350Val Asp Asp Glu Met Tyr Arg Glu Tyr Thr Met
Leu Ile Arg Asp Glu 355 360 365Val Val Lys Met Leu Glu Glu Pro Val
Lys His Asp Asp His Leu Leu 370 375 380Gln Asp Ser Glu Leu Ala Gly
Leu Leu Ser Met Ser Ser Asn Gly Glu385 390 395 400Ser Arg Gln Leu
Lys Phe Gly Arg Lys Thr Ile Phe Ser Thr Lys Lys 405 410 415Asn Met
His Val Met Asp Asp Met Ala Asn Gly Arg Tyr Thr Pro Gly 420 425
430Ile Ile Pro Pro Val Asn Val Asp Lys Pro Ile Pro Leu Gly Arg Arg
435 440 445Asp Val Pro Gly Arg Arg Thr Arg Ile Ile Phe Ile Leu Pro
Tyr Glu 450 455 460Tyr Phe Ile Ala Gln His Ala Val Val Glu Lys Met
Leu Ile Tyr Ala465 470 475 480Lys His Thr Arg Glu Tyr Ala Glu Phe
Tyr Ser Gln Ser Asn Gln Leu 485 490 495Leu Ser Tyr Gly Asp Val Thr
Arg Phe Leu Ser Asn Asn Ser Met Val 500 505 510Leu Tyr Thr Asp Val
Ser Gln Trp Asp Ser Ser Gln His Asn Thr Gln 515 520 525Pro Phe Arg
Lys Gly Ile Ile Met Gly Leu Asp Met Leu Ala Asn Met 530 535 540Thr
Asn Asp Ala Arg Val Ile Gln Thr Leu Asn Leu Tyr Lys Gln Thr545 550
555 560Gln Ile Asn Leu Met Asp Ser Tyr Val Gln Ile Pro Asp Gly Asn
Val 565 570 575Ile Lys Lys Ile Gln Tyr Gly Ala Val Ala Ser Gly Glu
Lys Gln Thr 580 585 590Lys Ala Ala Asn Ser Ile Ala Asn Leu Ala Leu
Ile Lys Thr Val Leu 595 600 605Ser Arg Ile Ser Asn Lys Tyr Ser Phe
Ala Thr Lys Ile Ile Arg Val 610 615 620Asp Gly Asp Asp Asn Tyr Ala
Val Leu Gln Phe Asn Thr Glu Val Thr625 630 635 640Lys Gln Met Val
Gln Asp Val Ser Asn Asp Val Arg Glu Thr Tyr Ala 645 650 655Arg Met
Asn Thr Lys Val Lys Ala Leu Val Ser Thr Val Gly Ile Glu 660 665
670Ile Ala Lys Arg Tyr Ile Ala Gly Gly Lys Ile Phe Phe Arg Ala Gly
675 680 685Ile Asn Leu Leu Asn Asn Glu Lys Lys Gly Gln Ser Thr Gln
Trp Asp 690 695 700Gln Ala Ala Val Lys Asn Tyr Ile Val Asn Arg Leu
Arg Gly Phe Glu705 710 715 720Thr Asp Arg Glu Phe Ile Leu Thr Lys
Ile Met Gln Met Thr Ser Val 725 730 735Ala Ile Thr Gly Ser Leu Arg
Leu Phe Pro Ser Val Leu Thr Thr Asn 740 745 750Ser Thr Phe Lys Val
Phe Asp Ser Glu Asp Phe Ile Ile Glu Tyr Gly 755 760 765Thr Thr Asp
Asp Glu Val Tyr Ile Gln Arg Ala Phe Met Ser Leu Ser 770 775 780Ser
Gln Lys Ser Gly Ile Ala Asp Glu Ile Ala Ala Ser Ser Thr Phe785 790
795 800Lys Asn Tyr Val Ser Arg Leu Ser Glu Gln Leu Leu Phe Ser Lys
Asn 805 810 815Asn Ile Val Ser Arg Gly Ile Ala Leu Thr Glu Lys Ala
Lys Leu Asn 820 825 830Ser Tyr Ala Pro Ile Ser Leu Glu Lys Arg Arg
Ala Gln Ile Ser Ala 835 840 845Leu Leu Thr Met Leu Gln Lys Pro Val
Thr Phe Lys Ser Ser Lys Ile 850 855 860Thr Ile Asn Asp Ile Leu Arg
Asp Ile Lys Pro Phe Phe Thr Val Asn865 870 875 880Glu Ala His Leu
Pro Ile Gln Tyr Gln Lys Phe Met Pro Thr Leu Pro 885 890 895Asp Asn
Val Gln Tyr Ile Ile Gln Cys Ile Gly Ser Arg Thr Tyr Gln 900 905
910Ile Glu Asp Asp Gly Ser Lys Ser Ala Ile Ser Arg Leu Ile Ser Lys
915 920 925Tyr Ser Val Tyr Lys Pro Ser Ile Glu Glu Leu Tyr Lys Val
Ile Ser 930 935 940Leu His Glu Asn Glu Ile Gln Leu Tyr Leu Ile Ser
Leu Gly Ile Pro945 950 955 960Lys Ile Asp Ala Asp Thr Tyr Val Gly
Ser Lys Ile Tyr Ser Gln Asp 965 970 975Lys Tyr Arg Ile Ser Tyr Val
Tyr Asn Leu Leu Ser Ile Asn Tyr Gly 980 985 990Cys Tyr Gln Leu Phe
Asp Phe Asn Ser Pro Asp Leu Glu Lys Leu Ile 995 1000 1005Arg Ile
Pro Phe Lys Gly Lys Ile Pro Ala Val Thr Phe Ile Leu His 1010 1015
1020Leu Tyr Ala Lys Leu Glu Val Ile Asn His Ala Ile Lys Asn Gly
Ser1025 1030 1035 1040Trp Ile Ser Leu Phe Cys Asn Tyr Pro Lys Ser
Glu Met Ile Lys Leu 1045 1050 1055Trp Lys Lys Met Trp Asn Ile Thr
Ser Leu Arg Ser Pro Tyr Thr Asn 1060 1065 1070Ala Asn Phe Phe Gln
Asp 107516885PRTRotavirus 16Met Ala Tyr Arg Lys Arg Gly Ala Arg Arg
Glu Thr Asn Leu Lys Gln 1 5 10 15Asp Asp Arg Met Gln Glu Lys Glu
Glu Asn Lys Asn Val Asn Thr Asn 20 25 30Ser Glu Asn Lys Asn Ala Thr
Lys Pro Gln Leu Ser Glu Lys Val Leu 35 40 45Ser Gln Lys Glu Glu Val
Ile Thr Asp Asn Gln Glu Glu Ile Lys Ile 50 55 60Ala Asp Glu Val Lys
Lys Ser Asn Lys Glu Glu Ser Lys Gln Leu Leu65 70 75 80Glu Val Leu
Lys Thr Lys Glu Glu His Gln Lys Glu Val Gln Tyr Glu 85 90 95Ile Leu
Gln Lys Thr Ile Pro Thr Phe Glu Pro Lys Glu Ser Ile Leu 100 105
110Lys Lys Leu Glu Asp Ile Lys Pro Glu Gln Val Lys Lys Gln Thr Lys
115 120 125Leu Phe Arg Ile Phe Glu Pro Arg Gln Leu Pro Val Tyr Arg
Ala Asn 130 135 140Gly Glu Lys Glu Leu Arg Asn Arg Trp Tyr Trp Lys
Leu Lys Arg Asp145 150 155 160Thr Leu Pro Asp Gly Asp Tyr Asp Val
Arg Glu Tyr Phe Leu Asn Leu 165 170 175Tyr Asp Gln Val Leu Thr Glu
Met Pro Asp
Tyr Leu Leu Leu Lys Asp 180 185 190Met Ala Val Glu Asn Lys Asn Ser
Arg Asp Ala Gly Lys Val Val Asp 195 200 205Ser Glu Thr Ala Ala Ile
Cys Asp Ala Ile Phe Gln Asp Glu Glu Thr 210 215 220Glu Gly Val Val
Arg Arg Phe Ile Ala Glu Met Arg Gln Arg Val Gln225 230 235 240Ala
Asp Arg Asn Val Val Asn Tyr Pro Ser Ile Leu His Pro Ile Asp 245 250
255His Ala Phe Asn Glu Tyr Phe Leu Gln His Gln Leu Val Glu Pro Leu
260 265 270Asn Asn Asp Ile Ile Phe Asn Tyr Ile Pro Glu Arg Ile Arg
Asn Asp 275 280 285Val Asn Tyr Ile Leu Asn Met Asp Arg Asn Leu Pro
Ser Thr Ala Arg 290 295 300Tyr Ile Arg Pro Asn Leu Leu Gln Asp Arg
Leu Asn Leu His Asp Asn305 310 315 320Phe Glu Ser Leu Trp Asp Thr
Ile Thr Thr Ser Asn Tyr Ile Leu Ala 325 330 335Arg Ser Val Val Pro
Asp Leu Lys Glu Leu Val Ser Thr Glu Ala Gln 340 345 350Ile Gln Lys
Met Ser Gln Asp Leu Gln Leu Glu Ala Leu Thr Ile Gln 355 360 365Ser
Glu Thr Gln Phe Leu Thr Gly Ile Asn Ser Gln Ala Ala Asn Asp 370 375
380Cys Phe Lys Thr Leu Ile Ala Ala Met Leu Ser Gln Arg Thr Met
Ser385 390 395 400Leu Asp Phe Val Thr Thr Asn Tyr Met Ser Leu Ile
Ser Gly Met Trp 405 410 415Leu Leu Thr Val Val Pro Asn Asp Met Phe
Ile Arg Glu Ser Leu Val 420 425 430Ala Cys Gln Leu Ala Ile Ile Asn
Thr Ile Ile Tyr Pro Ala Phe Gly 435 440 445Met Gln Arg Met His Tyr
Arg Asn Gly Asp Pro Gln Thr Pro Phe Gln 450 455 460Ile Ala Glu Gln
Gln Ile Gln Asn Phe Gln Val Ala Asn Trp Leu His465 470 475 480Phe
Val Asn Asn Asn Gln Phe Arg Gln Val Val Ile Asp Gly Val Leu 485 490
495Asn Gln Val Leu Asn Asp Asn Ile Arg Asn Gly His Val Val Asn Gln
500 505 510Leu Met Glu Ala Leu Met Gln Leu Ser Arg Gln Gln Phe Pro
Thr Met 515 520 525Pro Val Asp Tyr Lys Arg Ser Ile Gln Arg Gly Ile
Leu Leu Leu Ser 530 535 540Asn Arg Leu Gly Gln Leu Val Asp Leu Thr
Arg Leu Leu Ala Tyr Asn545 550 555 560Tyr Glu Thr Leu Met Ala Cys
Ile Thr Met Asn Met Gln His Val Gln 565 570 575Thr Leu Thr Thr Glu
Lys Leu Gln Leu Thr Ser Val Thr Ser Leu Cys 580 585 590Met Leu Ile
Gly Asn Ala Thr Val Ile Pro Ser Pro Gln Thr Leu Phe 595 600 605His
Tyr Tyr Asn Val Asn Val Asn Phe His Ser Asn Tyr Asn Glu Arg 610 615
620Ile Asn Asp Ala Val Ala Ile Ile Thr Ala Ala Asn Arg Leu Asn
Leu625 630 635 640Tyr Gln Lys Lys Met Lys Ser Ile Val Glu Asp Phe
Leu Lys Arg Leu 645 650 655Gln Ile Phe Asp Ile Ser Arg Val Pro Asp
Asp Gln Met Tyr Arg Leu 660 665 670Arg Asp Arg Leu Arg Leu Leu Pro
Val Glu Ile Arg Arg Leu Asp Ile 675 680 685Phe Asn Leu Ile Leu Met
Asn Met Glu Gln Ile Glu Arg Ala Ser Asp 690 695 700Lys Ile Ala Gln
Gly Val Ile Ile Ala Tyr Arg Asp Met Gln Leu Glu705 710 715 720Arg
Asp Glu Met Tyr Gly Tyr Val Asn Ile Ala Arg Asn Leu Asp Gly 725 730
735Phe Gln Gln Ile Asn Leu Glu Glu Leu Met Arg Thr Gly Asp Tyr Ala
740 745 750Gln Ile Thr Asn Met Leu Leu Asn Asn Gln Pro Val Val Gly
Ala Leu 755 760 765Pro Phe Ile Thr Asp Ser Ser Val Ile Ser Leu Val
Ala Lys Leu Asp 770 775 780Ala Thr Val Phe Ala Gln Ile Val Lys Leu
Arg Lys Val Asp Thr Leu785 790 795 800Lys Pro Ile Leu Tyr Lys Ile
Asn Ser Asp Ser Asn Asp Phe Tyr Leu 805 810 815Val Ala Asn Tyr Asp
Trp Val Pro Thr Ser Thr Thr Lys Val Tyr Lys 820 825 830Gln Ile Pro
Gln Gln Phe Asp Phe Arg Ala Ser Met His Met Leu Thr 835 840 845Ser
Asn Leu Thr Phe Thr Val Tyr Ser Asp Leu Leu Ala Phe Val Ser 850 855
860Ala Asp Thr Val Glu Pro Ile Asn Ala Val Ala Phe Asp Asn Met
Arg865 870 875 880Ile Met Asn Glu Leu 88517831PRTRotavirus 17Met
Lys Val Leu Ala Leu Arg His Gly Val Ala Gln Val Tyr Ala Asp 1 5 10
15Thr Gln Ile Tyr Thr His Asp Asp Thr Lys Asp Ser Tyr Glu Asn Ala
20 25 30Phe Leu Ile Ser Asn Leu Thr Thr His Asn Ile Leu Tyr Leu Asn
Tyr 35 40 45Ser Ile Lys Thr Leu Glu Ile Leu Asn Lys Ser Gly Ile Ala
Ala Val 50 55 60Glu Ile Gln Ser Leu Glu Glu Leu Phe Thr Leu Ile Arg
Cys Asn Phe65 70 75 80Thr Tyr Asp Tyr Glu Asn Asn Ile Ile Tyr Leu
His Asp Tyr Ser Tyr 85 90 95Tyr Thr Asn Asn Glu Ile Arg Thr Asp Gln
His Trp Val Thr Lys Thr 100 105 110Asp Ile Glu Glu Tyr Leu Leu Pro
Gly Trp Lys Leu Thr Tyr Val Gly 115 120 125Tyr Asn Gly Ser Asp Thr
Arg Gly His Tyr Asn Phe Ser Phe Thr Cys 130 135 140Gln Asn Ala Ala
Thr Asp Asp Asp Leu Ile Ile Glu Tyr Ile Tyr Ser145 150 155 160Glu
Ala Leu Asp Phe Gln Asn Phe Met Leu Lys Lys Ile Lys Glu Arg 165 170
175Met Thr Thr Ser Leu Pro Ile Ala Arg Leu Ser Asn Arg Val Phe Arg
180 185 190Asp Lys Leu Phe Pro Leu Leu Ser Glu Lys His Gln Arg Ile
Val Asn 195 200 205Ile Gly Pro Arg Asn Glu Ser Met Phe Thr Phe Leu
Asn Phe Pro Ser 210 215 220Ile Lys Gln Phe Ser Asn Gly Pro Tyr Leu
Val Lys Asp Thr Ile Lys225 230 235 240Leu Lys Gln Glu Arg Trp Leu
Gly Lys Arg Val Ser Gln Phe Asp Ile 245 250 255Gly Gln Tyr Lys Asn
Met Met Asn Val Ile Thr Thr Val Tyr Tyr Tyr 260 265 270Tyr Asn Leu
Tyr Gln Lys Lys Pro Ile Ile Tyr Met Val Gly Ser Ala 275 280 285Pro
Ser Tyr Trp Ile Tyr Asp Val Lys Gln Tyr Ser Asp Phe Met Phe 290 295
300Glu Thr Trp Asp Pro Leu Asp Thr Pro Tyr Ser Ser Val His His
Lys305 310 315 320Glu Leu Phe Phe Glu Lys Asp Ile Thr Arg Leu Lys
Asp Asp Ser Ile 325 330 335Leu Tyr Ile Asp Ile Arg Thr Asp Arg Gly
Asn Thr Asp Trp Lys Glu 340 345 350Trp Arg Lys Ile Val Glu Ala Gln
Thr Ile Ser Asn Leu Lys Leu Ala 355 360 365Tyr Arg Tyr Leu Ser Gly
Gly Lys Ser Lys Val Cys Cys Val Lys Met 370 375 380Thr Ala Met Asp
Leu Glu Leu Pro Ile Ser Ala Lys Leu Leu His His385 390 395 400Pro
Thr Thr Glu Ile Arg Ser Glu Phe Tyr Leu Leu Leu Asp Ile Trp 405 410
415Asp Ile Ser Asn Val Lys Arg Phe Ile Pro Lys Gly Val Lys Phe Ile
420 425 430Asn Asn Val Thr Thr Glu Asn Val Phe Ile Gln Pro Pro Phe
Lys Ile 435 440 445Lys Pro Phe Lys Asn Asp Tyr Ile Val Tyr Ala Leu
Ser Asn Asp Phe 450 455 460Asn Asp Arg Thr Asp Val Ile Asn Leu Ile
Asn Asn Gln Lys Gln Ser465 470 475 480Leu Ile Thr Val Arg Ile Asn
Asn Thr Phe Lys Asp Glu Pro Lys Val 485 490 495Gly Phe Lys Asn Ile
Tyr Asp Trp Thr Phe Leu Pro Thr Asp Phe Thr 500 505 510Thr Thr Asp
Ala Ile Ile Thr Ser Tyr Asp Gly Cys Leu Gly Ile Phe 515 520 525Gly
Leu Ser Ile Ser Leu Ala Ser Lys Pro Thr Gly Asn Asn His Leu 530 535
540Phe Ile Leu Asn Gly Thr Asp Lys Tyr Tyr Lys Leu Asp Gln Phe
Ala545 550 555 560Asn His Thr Gly Ile Ser Arg Arg Ser His Gln Ile
Arg Phe Ser Glu 565 570 575Ser Ala Thr Ser Tyr Ser Gly Tyr Ile Phe
Arg Asp Leu Ser Asn Asn 580 585 590Asn Phe Asn Leu Ile Gly Thr Asn
Val Glu Asn Ser Val Ser Gly His 595 600 605Val Tyr Asn Ala Leu Ile
Tyr Tyr Arg Tyr Asn Tyr Ser Phe Asp Leu 610 615 620Lys Arg Trp Ile
Tyr Leu His Ser Ile Glu Lys Ala Asp Ile Glu Gly625 630 635 640Gly
Lys Tyr Tyr Glu His Ala Pro Ile Glu Leu Ile Tyr Ala Cys Arg 645 650
655Ser Ala Lys Glu Phe Ala Leu Leu Gln Asp Asp Leu Thr Val Leu Arg
660 665 670Tyr Ala Asn Glu Ile Glu Ser Tyr Ile Asn Lys Val Tyr Ser
Ile Thr 675 680 685Tyr Ala Asp Asp Pro Asn Tyr Phe Ile Gly Ile Lys
Phe Arg His Ile 690 695 700Pro Tyr Glu Tyr Asp Val Lys Ile Pro His
Leu Thr Phe Gly Val Leu705 710 715 720Phe Ile Ser Asp Asn Met Ile
Pro Asp Val Val Glu Ile Met Lys Ile 725 730 735Met Lys Lys Glu Leu
Phe Glu Met Asp Ile Thr Thr Ser Tyr Thr Tyr 740 745 750Met Leu Ser
Asp Gly Ile Tyr Val Ala Asn Val Ser Gly Val Leu Ala 755 760 765Thr
Tyr Phe Lys Met Tyr Asn Leu Phe Tyr Lys Ser Gln Ile Thr Phe 770 775
780Gly Gln Ser Arg Met Phe Ile Pro His Ile Thr Leu Ser Phe Ser
Asn785 790 795 800Asn Lys Thr Val Arg Ile Glu Ser Thr Arg Leu Lys
Ile Ser Ser Ile 805 810 815Tyr Leu Arg Lys Ile Lys Gly Asp Thr Val
Phe Asp Met Ser Glu 820 825 83018776PRTRotavirus 18Met Ala Ser Leu
Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Thr Val 1 5 10 15Asp Leu
Ser Asp Glu Ile Gln Glu Ile Gly Ser Thr Lys Thr Gln Asn 20 25 30Val
Thr Ile Asn Leu Gly Pro Phe Ala Gln Thr Gly Tyr Ala Pro Val 35 40
45Asn Trp Gly Pro Gly Glu Thr Asn Asp Ser Thr Thr Val Glu Pro Val
50 55 60Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Asn Pro Pro Val Asp
Tyr65 70 75 80Trp Met Leu Leu Ala Pro Thr Ala Ala Gly Val Val Val
Glu Gly Thr 85 90 95Asn Asn Thr Asp Arg Trp Leu Ala Thr Ile Leu Val
Glu Pro Asn Val 100 105 110Thr Ser Glu Thr Arg Ser Tyr Thr Leu Phe
Gly Thr Gln Glu Gln Ile 115 120 125Thr Ile Ala Asn Ala Ser Gln Thr
Gln Trp Lys Phe Ile Asp Val Val 130 135 140Lys Thr Thr Gln Asn Gly
Ser Tyr Ser Gln Tyr Gly Pro Leu Gln Ser145 150 155 160Thr Pro Lys
Leu Tyr Ala Val Met Lys His Asn Gly Lys Ile Tyr Thr 165 170 175Tyr
Asn Gly Glu Thr Pro Asn Val Thr Thr Lys Tyr Tyr Ser Thr Thr 180 185
190Asn Tyr Asp Ser Val Asn Met Thr Ala Phe Cys Asp Phe Tyr Ile Ile
195 200 205Pro Arg Glu Glu Glu Ser Thr Cys Thr Glu Tyr Ile Asn Asn
Gly Leu 210 215 220Pro Pro Ile Gln Asn Thr Arg Asn Ile Val Pro Leu
Ala Leu Ser Ala225 230 235 240Arg Asn Ile Ile Ser His Arg Ala Gln
Ala Asn Glu Asp Ile Val Val 245 250 255Ser Lys Thr Ser Leu Trp Lys
Glu Met Gln Tyr Asn Arg Asp Ile Thr 260 265 270Ile Arg Phe Lys Phe
Ala Ser Ser Ile Val Lys Ser Gly Gly Leu Gly 275 280 285Tyr Lys Trp
Ser Glu Ile Ser Phe Lys Pro Ala Asn Tyr Gln Tyr Thr 290 295 300Tyr
Thr Arg Asp Gly Glu Glu Val Thr Ala His Thr Thr Cys Ser Val305 310
315 320Asn Gly Met Asn Asp Phe Asn Phe Asn Gly Gly Ser Leu Pro Thr
Asp 325 330 335Phe Val Ile Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser
Tyr Val Tyr 340 345 350Val Asp Tyr Trp Asp Asp Ser Gln Ala Phe Arg
Asn Met Val Tyr Val 355 360 365Arg Ser Leu Ala Ala Asn Leu Asn Ser
Val Ile Cys Thr Gly Gly Asp 370 375 380Tyr Ser Phe Ala Leu Pro Val
Gly Gln Trp Pro Val Met Thr Gly Gly385 390 395 400Ala Val Ser Leu
His Ser Ala Gly Val Thr Leu Ser Thr Gln Phe Thr 405 410 415Asp Phe
Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Arg Leu Thr Val 420 425
430Glu Glu Pro Ser Phe Ser Ile Thr Arg Thr Arg Val Ser Arg Leu Tyr
435 440 445Gly Leu Pro Ala Ala Asn Pro Asn Asn Gly Lys Glu Tyr Tyr
Glu Val 450 455 460Ala Gly Arg Phe Ser Leu Ile Ser Leu Val Pro Ser
Asn Asp Asp Tyr465 470 475 480Gln Thr Pro Ile Thr Asn Ser Val Thr
Val Arg Gln Asp Leu Glu Arg 485 490 495Gln Leu Gly Glu Leu Arg Glu
Glu Phe Asn Ala Leu Ser Gln Glu Ile 500 505 510Ala Met Ser Gln Leu
Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe 515 520 525Ser Met Phe
Ser Gly Ile Lys Ser Thr Ile Asp Ala Ala Lys Ser Met 530 535 540Ala
Thr Ser Val Met Lys Lys Phe Lys Lys Ser Gly Leu Ala Asn Ser545 550
555 560Val Ser Thr Leu Thr Asp Ser Leu Ser Asp Ala Ala Ser Ser Ile
Ser 565 570 575Arg Gly Ala Ser Ile Arg Ser Val Gly Ser Ser Ala Ser
Ala Trp Thr 580 585 590Asp Val Ser Thr Gln Ile Thr Asp Val Ser Ser
Ser Val Ser Ser Ile 595 600 605Ser Thr Gln Thr Ser Thr Ile Ser Arg
Arg Leu Arg Leu Lys Glu Met 610 615 620Ala Thr Gln Thr Glu Gly Met
Asn Phe Asp Asp Ile Ser Ala Ala Val625 630 635 640Leu Lys Thr Lys
Ile Asp Arg Ser Thr Gln Ile Ser Pro Asn Thr Leu 645 650 655Pro Asp
Ile Val Thr Glu Ala Ser Glu Lys Phe Ile Pro Asn Arg Ala 660 665
670Tyr Arg Val Ile Asn Asn Asp Glu Val Phe Glu Ala Gly Thr Asp Gly
675 680 685Arg Phe Phe Ala Tyr Arg Val Glu Thr Phe Asp Glu Ile Pro
Phe Asp 690 695 700Val Gln Lys Phe Ala Asp Leu Val Thr Asp Ser Pro
Val Ile Ser Ala705 710 715 720Ile Ile Asp Phe Lys Thr Leu Lys Asn
Leu Asn Asp Asn Tyr Gly Ile 725 730 735Ser Arg Gln Gln Ala Phe Asn
Leu Leu Arg Ser Asp Pro Arg Val Leu 740 745 750Arg Glu Phe Ile Asn
Gln Asp Asn Pro Ile Ile Arg Asn Arg Ile Glu 755 760 765Gln Leu Ile
Met Gln Cys Arg Leu 770 77519492PRTRotavirus 19Met Ala Thr Phe Lys
Asp Ala Cys Phe His Tyr Arg Arg Val Thr Lys 1 5 10 15Leu Asn Arg
Glu Leu Leu Arg Ile Gly Ala Asn Ser Val Trp Thr Pro 20 25 30Val Ser
Ser Asn Lys Ile Lys Ile Lys Gly Trp Cys Ile Glu Cys Cys 35 40 45Gln
Leu Thr Gly Leu Thr Phe Cys His Gly Cys Ser Leu Ala His Val 50 55
60Cys Gln Trp Cys Ile Gln Asn Lys Arg Cys Phe Leu Asp Asn Glu Pro65
70 75 80His Leu Leu Lys Leu Arg Thr Phe Glu Ser Pro Ile Thr Lys Glu
Lys 85 90 95Leu Gln Cys Ile Ile Asn Leu Tyr Glu Leu Leu Phe Pro Ile
Asn His 100 105 110Gly Val Ile Asn Lys Phe Lys Lys Thr Ile Lys Gln
Arg Lys Cys Arg 115 120 125Asn Glu Phe Asp Lys Ser Trp Tyr Asn Gln
Leu Leu Leu Pro Ile Thr 130 135 140Leu Asn Ala Ala Val Phe Lys Phe
His Ser Arg Asp Val Tyr Val Phe145 150
155 160Gly Phe Tyr Glu Gly Ser Ser Pro Cys Ile Asp Leu Pro Tyr Arg
Leu 165 170 175Val Asn Cys Ile Asp Leu Tyr Asp Lys Leu Leu Leu Asp
Gln Val Asn 180 185 190Phe Glu Arg Met Ser Ser Leu Pro Asp Asn Leu
Gln Ser Ile Tyr Ala 195 200 205Asn Lys Tyr Phe Lys Leu Ser Arg Leu
Pro Ser Met Lys Leu Lys Arg 210 215 220Ile Tyr Tyr Ser Asp Phe Ser
Lys Gln Asn Leu Ile Asn Lys Tyr Lys225 230 235 240Thr Lys Ser Arg
Ile Val Leu Arg Asn Leu Thr Glu Phe Thr Trp Asp 245 250 255Ser Gln
Thr Asp Leu His His Asp Leu Ile Asn Asp Lys Asp Lys Ile 260 265
270Leu Ala Ala Leu Ser Thr Ser Ser Leu Lys Gln Phe Glu Thr His Asp
275 280 285Leu Asn Leu Gly Arg Ile Lys Ala Asp Ile Phe Glu Leu Gly
His His 290 295 300Cys Lys Pro Asn Tyr Ile Ser Ser Asn His Trp Gln
Pro Ala Ser Lys305 310 315 320Ile Ser Lys Cys Lys Trp Cys Asn Val
Lys Tyr Ala Phe Arg Asp Met 325 330 335Asp Trp Lys Met Glu Ser Met
Tyr Asn Glu Leu Leu Ser Phe Ile Gln 340 345 350Ser Cys Tyr Lys Ser
Asn Val Asn Val Gly His Cys Ser Ser Ile Glu 355 360 365Lys Ala Tyr
Pro Leu Val Lys Asp Ile Leu Trp His Ser Ile Thr Glu 370 375 380Tyr
Ile Asp Gln Thr Val Glu Lys Leu Phe Asn Thr Met Asn Pro Val385 390
395 400Gln Val Asn Glu Gln Gln Val Ile Lys Phe Cys Trp Gln Ile Asp
Ile 405 410 415Ala Leu Tyr Met His Ile Lys Met Ile Thr Glu Ala Leu
Pro Phe Thr 420 425 430Phe Thr Leu Asn Gln Phe Asn Ser Ile Ile Lys
Gly Ile Val Asn Gln 435 440 445Trp Cys Asp Val Ala Glu Leu Asp His
Leu Pro Leu Cys Thr Glu Gln 450 455 460Thr Asp Ala Leu Val Lys Leu
Glu Glu Glu Gly Lys Leu Ser Glu Glu465 470 475 480Tyr Glu Leu Leu
Ile Ser Asp Ser Glu Asp Asp Asp 485 49020393PRTRotavirus 20Met Asp
Val Lys Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys Ile 1 5 10
15Val Glu Gly Thr Lys Asn Val Ser Asp Leu Ile Gln Gln Phe Asn Gln
20 25 30Met Ile Ile Thr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly Ile
Gly 35 40 45Asn Leu Pro Ile Arg Asn Trp Asn Phe Asp Phe Gly Leu Leu
Gly Thr 50 55 60Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr Ala
Arg Asn Thr65 70 75 80Ile Asp Tyr Phe Val Asp Phe Val Asp Asn Val
Cys Met Asp Glu Met 85 90 95Val Arg Glu Ser Gln Arg Asn Gly Ile Ala
Pro Gln Ser Asp Ser Leu 100 105 110Arg Lys Leu Ser Gly Ile Lys Phe
Lys Arg Ile Asn Phe Asp Asn Ser 115 120 125Ser Glu Tyr Ile Glu Asn
Trp Asn Leu Gln Asn Arg Arg Gln Arg Thr 130 135 140Gly Phe Thr Phe
His Lys Pro Asn Ile Phe Pro Tyr Ser Ala Ser Phe145 150 155 160Thr
Leu Asn Arg Ser Gln Pro Ala His Asp Asn Leu Met Gly Thr Met 165 170
175Trp Leu Asn Ala Gly Ser Glu Ile Gln Val Ala Gly Phe Asp Tyr Ser
180 185 190Cys Ala Ile Asn Ala Pro Ala Asn Ile Gln Gln Phe Glu His
Ile Val 195 200 205Gln Leu Arg Arg Val Leu Thr Thr Ala Thr Ile Thr
Leu Leu Pro Asp 210 215 220Ala Glu Arg Phe Ser Phe Pro Arg Val Ile
Asn Ser Ala Asp Gly Ala225 230 235 240Ala Thr Trp Tyr Phe Asn Pro
Val Ile Leu Arg Pro Asn Asn Val Glu 245 250 255Val Glu Phe Leu Leu
Asn Gly Gln Ile Ile Asn Thr Tyr Gln Ala Arg 260 265 270Phe Gly Thr
Ile Ile Ala Arg Asn Phe Asp Thr Ile Arg Leu Ser Phe 275 280 285Gln
Leu Met Arg Pro Pro Asn Met Thr Pro Ala Val Ala Ala Leu Phe 290 295
300Pro Asn Ala Gln Pro Phe Glu His His Ala Thr Val Gly Leu Thr
Leu305 310 315 320Arg Ile Glu Ser Ala Val Cys Glu Ser Val Leu Ala
Asp Ala Ser Lys 325 330 335Thr Met Leu Ala Asn Val Thr Ser Val Arg
Gln Glu Tyr Ala Ile Pro 340 345 350Val Gly Pro Val Phe Pro Pro Gly
Met Asn Trp Thr Asp Leu Ile Thr 355 360 365Asn Tyr Ser Pro Ser Arg
Glu Asp Asn Leu Gln Arg Val Phe Thr Val 370 375 380Ala Ser Ile Arg
Ser Met Leu Val Lys385 39021313PRTRotavirus 21Met Leu Lys Met Glu
Ser Thr Gln Gln Met Ala Ser Ser Ile Ile Asn 1 5 10 15Ser Ser Phe
Glu Ala Ala Val Val Ala Ala Thr Ser Thr Leu Glu Leu 20 25 30Met Gly
Ile Gln Tyr Asp Tyr Asn Glu Val Tyr Thr Arg Val Lys Ser 35 40 45Lys
Phe Asp Leu Val Met Asp Asp Ser Gly Val Lys Asn Asn Leu Ile 50 55
60Gly Lys Ala Ile Thr Ile Asp Gln Ala Leu Asn Gly Lys Phe Ser Ser65
70 75 80Ala Ile Arg Asn Arg Asn Trp Met Thr Asp Ser Arg Thr Val Ala
Lys 85 90 95Leu Asp Glu Asp Val Asn Lys Leu Arg Ile Met Leu Ser Ser
Lys Gly 100 105 110Ile Asp Gln Lys Met Arg Val Leu Asn Ala Cys Phe
Ser Val Lys Arg 115 120 125Ile Pro Gly Lys Ser Ser Ser Ile Val Lys
Cys Thr Arg Leu Met Lys 130 135 140Asp Lys Leu Glu Arg Gly Glu Val
Glu Val Asp Asp Ser Phe Val Glu145 150 155 160Glu Lys Met Glu Val
Asp Thr Ile Asp Trp Lys Ser Arg Tyr Glu Gln 165 170 175Leu Glu Lys
Arg Phe Glu Ser Leu Lys His Arg Val Asn Glu Lys Tyr 180 185 190Asn
His Trp Val Leu Lys Ala Arg Lys Val Asn Glu Asn Met Asn Ser 195 200
205Leu Gln Asn Val Ile Ser Gln Gln Gln Ala His Ile Asn Glu Leu Gln
210 215 220Met Tyr Asn Asn Lys Leu Glu Arg Asp Leu Gln Ser Lys Ile
Gly Ser225 230 235 240Val Val Ser Ser Ile Glu Trp Tyr Leu Arg Ser
Met Glu Leu Ser Asp 245 250 255Asp Val Lys Ser Asp Ile Glu Gln Gln
Leu Asn Ser Ile Asp Gln Leu 260 265 270Asn Pro Val Asn Ala Ile Asp
Asp Phe Glu Ser Ile Leu Arg Asn Leu 275 280 285Ile Ser Asp Tyr Asp
Arg Leu Phe Ile Met Phe Lys Gly Leu Leu Gln 290 295 300Gln Cys Asn
Tyr Thr Tyr Thr Tyr Glu305 31022317PRTRotavirus 22Met Ala Glu Leu
Ala Cys Phe Cys Tyr Pro His Leu Glu Asn Asp Ser 1 5 10 15Tyr Arg
Phe Ile Pro Phe Asn Ser Leu Ala Ile Lys Cys Met Leu Thr 20 25 30Ala
Lys Val Asp Lys Lys Asp Gln Asp Lys Phe Tyr Asn Ser Ile Ile 35 40
45Tyr Gly Ile Ala Pro Pro Pro Gln Phe Lys Lys Arg Tyr Asn Thr Asn
50 55 60Asp Asn Ser Arg Gly Met Asn Tyr Glu Thr Pro Met Phe Asn Lys
Val65 70 75 80Ala Val Leu Ile Cys Glu Ala Leu Asn Ser Ile Lys Val
Thr Gln Ser 85 90 95Asp Val Ala Asn Val Leu Ser Lys Val Val Ser Val
Arg His Leu Glu 100 105 110Asn Leu Val Leu Arg Arg Glu Asn His Gln
Asp Val Leu Phe His Ser 115 120 125Lys Glu Leu Leu Leu Lys Ser Val
Leu Ile Ala Ile Gly His Ser Lys 130 135 140Glu Ile Glu Thr Thr Ala
Thr Ala Glu Gly Gly Glu Ile Val Phe Gln145 150 155 160Asn Ala Ala
Phe Thr Met Trp Lys Leu Thr Tyr Leu Glu His Arg Leu 165 170 175Met
Pro Ile Leu Asp Gln Asn Phe Ile Glu Tyr Lys Ile Thr Val Asn 180 185
190Glu Asp Lys Pro Ile Ser Glu Ser His Val Lys Glu Leu Ile Ala Glu
195 200 205Leu Arg Trp Gln Tyr Asn Lys Phe Ala Val Ile Thr His Gly
Lys Gly 210 215 220His Tyr Arg Val Val Lys Tyr Ser Ser Val Ala Asn
His Ala Asp Arg225 230 235 240Val Tyr Ala Thr Phe Lys Ser Asn Asn
Lys Asn Gly Asn Val Leu Glu 245 250 255Phe Asn Leu Leu Asp Gln Arg
Val Ile Trp Gln Asn Trp Tyr Ala Phe 260 265 270Thr Ser Ser Met Lys
Gln Gly Asn Thr Leu Glu Ile Cys Lys Lys Leu 275 280 285Leu Phe Gln
Lys Met Lys Arg Glu Ser Asn Pro Phe Lys Gly Leu Ser 290 295 300Thr
Asp Arg Lys Met Asp Glu Val Ser Gln Ile Gly Ile305 310
31523326PRTRotavirus 23Met Tyr Gly Ile Glu Tyr Thr Thr Val Leu Thr
Phe Leu Ile Ser Leu 1 5 10 15Ile Leu Leu Asn Tyr Ile Leu Lys Ser
Leu Thr Arg Met Met Asp Phe 20 25 30Ile Ile Tyr Arg Phe Leu Phe Ile
Val Val Ile Leu Ser Pro Leu Leu 35 40 45Lys Ala Gln Asn Tyr Gly Ile
Asn Leu Pro Ile Thr Gly Ser Met Asp 50 55 60Thr Ala Tyr Ala Asn Ser
Thr Gln Glu Glu Thr Phe Leu Thr Ser Thr65 70 75 80Leu Cys Leu Tyr
Tyr Pro Thr Glu Ala Ala Thr Glu Ile Asn Asp Asn 85 90 95Ser Trp Lys
Asp Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro 100 105 110Thr
Gly Ser Val Tyr Phe Lys Glu Tyr Thr Asp Ile Ala Ser Phe Ser 115 120
125Val Asp Pro Gln Leu Tyr Cys Asp Tyr Asn Val Val Leu Met Lys Tyr
130 135 140Asp Ala Thr Leu Gln Leu Asp Met Ser Glu Leu Ala Asp Leu
Ile Leu145 150 155 160Asn Glu Trp Leu Cys Asn Pro Met Asp Ile Thr
Leu Tyr Tyr Tyr Gln 165 170 175Gln Thr Asp Glu Ala Asn Lys Trp Ile
Ser Met Gly Ser Ser Cys Thr 180 185 190Ile Lys Val Cys Pro Leu Asn
Thr Gln Thr Leu Gly Ile Gly Cys Leu 195 200 205Thr Thr Asp Thr Ala
Thr Phe Glu Glu Val Ala Thr Ala Glu Lys Leu 210 215 220Val Ile Thr
Asp Val Val Asp Gly Val Asn His Lys Leu Asp Val Thr225 230 235
240Thr Ala Thr Cys Thr Ile Arg Asn Cys Lys Lys Leu Gly Pro Arg Glu
245 250 255Asn Val Ala Val Ile Gln Val Gly Gly Ser Asp Val Leu Asp
Ile Thr 260 265 270Ala Asp Pro Thr Thr Ala Pro Gln Thr Glu Arg Met
Met Arg Ile Asn 275 280 285Trp Lys Lys Trp Trp Gln Val Phe Tyr Thr
Val Val Asp Tyr Val Asn 290 295 300Gln Ile Ile Gln Ala Met Ser Lys
Arg Ser Arg Ser Leu Asn Ser Ala305 310 315 320Ala Phe Tyr Tyr Arg
Ile 32524175PRTRotavirus 24Met Glu Lys Leu Thr Asp Leu Asn Tyr Thr
Leu Ser Val Val Thr Leu 1 5 10 15Met Asn Asp Thr Leu His Thr Ile
Met Glu Asp Pro Gly Met Ala Tyr 20 25 30Phe Pro Tyr Ile Ala Ser Val
Leu Thr Val Leu Phe Thr Leu His Lys 35 40 45Ala Ser Val Pro Thr Met
Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser 50 55 60Tyr Lys Val Ile Lys
Tyr Cys Ile Val Ser Ile Phe Asn Thr Leu Leu65 70 75 80Lys Leu Ala
Gly Tyr Lys Glu Gln Ile Thr Thr Lys Asp Glu Ile Glu 85 90 95Arg Gln
Met Asp Arg Val Val Lys Glu Met Arg Arg Gln Leu Glu Met 100 105
110Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys
115 120 125Arg Ile His Asp Met Leu Ile Ile Lys Pro Val Asp Lys Ile
Asp Met 130 135 140Ser Gln Glu Phe Asn Gln Lys Tyr Phe Lys Thr Leu
Asn Asp Trp Ala145 150 155 160Glu Gly Glu Asn Pro Tyr Glu Pro Lys
Glu Val Thr Ala Ser Leu 165 170 17525196PRTRotavirus 25Met Ser Leu
Ser Ile Asp Val Thr Ser Leu Pro Ser Ile Ser Ser Ser 1 5 10 15Ile
Tyr Lys His Glu Ser Ser Ser Thr Thr Ser Thr Leu Ser Gly Lys 20 25
30Ser Ile Gly Arg Ser Glu Gln Tyr Val Ser Pro Asp Ala Glu Ala Phe
35 40 45Asn Lys Tyr Met Leu Ser Lys Ser Pro Glu Asp Ile Gly Pro Ser
Asp 50 55 60Ser Asn Asp Pro Leu Thr Ser Phe Ser Ile Arg Ser Asn Ala
Val Lys65 70 75 80Thr Asn Ala Asp Ala Gly Val Ser Met Asp Ser Ser
Thr Gln Ser Arg 85 90 95Pro Ser Ser Asn Val Gly Cys Asp Gln Val Asp
Phe Ser Leu Ser Lys 100 105 110Gly Ile Lys Val Asn Ala Asn Leu Asp
Ser Ser Ile Ser Val Ser Thr 115 120 125Val Ser Lys Lys Glu Lys Ser
Lys Ser Asp His Lys Asn Arg Lys His 130 135 140Tyr Pro Arg Ile Glu
Ala Asp Ser Asp Ser Asp Glu Tyr Val Leu Asp145 150 155 160Asp Ser
Asp Ser Asp Asp Gly Lys Cys Lys Asn Cys Lys Tyr Lys Lys 165 170
175Lys Tyr Phe Ala Leu Arg Met Arg Met Lys Gln Val Ala Met Gln Leu
180 185 190Ile Glu Asp Leu 19526322PRTRotavirus 26Met Tyr Gly Ile
Glu Tyr Thr Thr Ile Leu Ile Phe Leu Ile Ser Ile 1 5 10 15Ile Leu
Leu Asn Tyr Ile Leu Lys Ser Val Thr Arg Ile Met Asp Tyr 20 25 30Ile
Ile Tyr Arg Phe Leu Leu Ile Thr Val Phe Ala Leu Thr Arg Ala 35 40
45Gln Asn Tyr Gln Leu Pro Ile Thr Gly Ser Met Asp Ala Val Tyr Thr
50 55 60Asn Ser Thr Gln Glu Glu Val Phe Leu Thr Ser Thr Leu Cys Leu
Tyr65 70 75 80Tyr Pro Thr Glu Ala Ser Thr Gln Ile Asn Asp Gly Asp
Trp Lys Asp 85 90 95Ser Leu Ser Gln Met Phe Leu Thr Lys Gly Trp Pro
Thr Gly Ser Val 100 105 110Tyr Phe Lys Glu Tyr Ser Ser Ile Val Asp
Phe Ser Val Asp Pro Gln 115 120 125Leu Tyr Cys Asp Tyr Asn Leu Val
Leu Met Lys Tyr Asp Gln Ser Leu 130 135 140Glu Leu Asp Met Ser Glu
Leu Ala Asp Leu Ile Leu Asn Glu Trp Leu145 150 155 160Cys Asn Pro
Met Asp Val Thr Leu Tyr Tyr Tyr Gln Gln Ser Gly Glu 165 170 175Ser
Asn Lys Trp Ile Ser Met Gly Ser Ser Cys Thr Val Lys Val Cys 180 185
190Pro Leu Asn Thr Gln Thr Leu Gly Ile Gly Cys Gln Thr Thr Asn Val
195 200 205Asp Ser Phe Glu Met Ile Ala Glu Asn Glu Lys Leu Ala Ile
Val Asp 210 215 220Val Val Asp Gly Ile Asn His Lys Ile Asn Leu Thr
Thr Thr Thr Cys225 230 235 240Thr Ile Arg Asn Cys Lys Lys Leu Gly
Pro Arg Glu Asn Val Ala Val 245 250 255Ile Gln Val Gly Gly Ser Asn
Val Leu Asp Ile Thr Ala Asp Pro Thr 260 265 270Thr Asn Pro Gln Thr
Glu Arg Met Met Arg Val Asn Trp Lys Lys Trp 275 280 285Trp Gln Val
Phe Tyr Thr Ile Val Asp Tyr Ile Asn Gln Ile Val Gln 290 295 300Val
Met Ser Lys Arg Ser Arg Ser Leu Asn Ser Ala Ala Phe Tyr Tyr305 310
315 320Arg Val27326PRTRotavirus 27Met Tyr Gly Ile Glu Tyr Thr Thr
Ile Leu Thr Ile Leu Ile Ser Ile 1 5 10 15Ile Leu Leu Asn Tyr Ile
Leu Lys Thr Ile Thr Asn Thr Met Asp Tyr 20 25 30Ile Ile Phe Arg Phe
Leu Leu Leu Ile Ala Leu Ile Ser Pro Phe Val 35 40 45Arg Thr Gln Asn
Tyr Gly Met Tyr Leu Pro Ile Thr Gly Ser Leu Asp 50 55 60Ala Val Tyr
Thr Asn Ser Thr Ser Gly Glu Pro Phe Leu Thr Ser Thr65 70 75 80Leu
Cys Leu Tyr Tyr
Pro Ala Glu Ala Lys Asn Glu Ile Ser Asp Asp 85 90 95Glu Trp Glu Asn
Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro 100 105 110Ile Gly
Ser Val Tyr Phe Lys Asp Tyr Asn Asp Ile Asn Thr Phe Ser 115 120
125Val Asn Pro Gln Leu Tyr Cys Asp Tyr Asn Val Val Leu Met Arg Tyr
130 135 140Asp Asn Thr Ser Glu Leu Asp Ala Ser Glu Leu Ala Asp Leu
Ile Leu145 150 155 160Asn Glu Trp Leu Cys Asn Pro Met Asp Ile Ser
Leu Tyr Tyr Tyr Gln 165 170 175Gln Ser Ser Glu Ser Asn Lys Trp Ile
Ser Met Gly Thr Asp Cys Thr 180 185 190Val Lys Val Cys Pro Leu Asn
Thr Gln Thr Leu Gly Ile Gly Cys Lys 195 200 205Thr Thr Asp Val Asn
Thr Phe Glu Ile Val Ala Ser Ser Glu Lys Leu 210 215 220Val Ile Thr
Asp Val Val Asn Gly Val Asn His Lys Ile Asn Ile Ser225 230 235
240Ile Asn Thr Cys Thr Ile Arg Asn Cys Asn Lys Leu Gly Pro Arg Glu
245 250 255Asn Val Ala Ile Ile Gln Val Gly Gly Pro Asn Ala Leu Asp
Ile Thr 260 265 270Ala Asp Pro Thr Thr Val Pro Gln Val Gln Arg Ile
Met Arg Ile Asn 275 280 285Trp Lys Lys Trp Trp Gln Val Phe Tyr Thr
Val Val Asp Tyr Ile Asn 290 295 300Gln Val Ile Gln Val Met Ser Lys
Arg Ser Arg Ser Leu Asp Ala Ala305 310 315 320Ala Phe Tyr Tyr Arg
Ile 32528326PRTRotavirus 28Met Tyr Gly Ile Glu Tyr Thr Thr Val Leu
Phe Tyr Leu Ile Ser Phe 1 5 10 15Val Leu Val Ser Tyr Ile Leu Lys
Thr Ile Ile Lys Ile Met Asp Tyr 20 25 30Ile Ile Tyr Arg Ile Thr Phe
Val Ile Val Val Leu Ser Val Leu Ser 35 40 45Asn Ala Gln Asn Tyr Gly
Ile Asn Leu Pro Ile Thr Gly Ser Met Asp 50 55 60Thr Ala Tyr Ala Asn
Ser Thr Gln Asp Asn Asn Phe Leu Phe Ser Thr65 70 75 80Leu Cys Leu
Tyr Tyr Pro Ser Glu Ala Pro Thr Gln Ile Ser Asp Thr 85 90 95Glu Trp
Lys Asp Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro 100 105
110Thr Gly Ser Val Tyr Phe Asn Glu Tyr Ser Asn Val Leu Glu Phe Ser
115 120 125Ile Asp Pro Lys Leu Tyr Cys Asp Tyr Asn Val Val Leu Ile
Arg Phe 130 135 140Val Ser Gly Glu Glu Leu Asp Ile Ser Glu Leu Ala
Asp Leu Ile Leu145 150 155 160Asn Glu Trp Leu Cys Asn Pro Met Asp
Ile Thr Leu Tyr Tyr Tyr Gln 165 170 175Gln Thr Gly Glu Ala Asn Lys
Trp Ile Ser Met Gly Ser Ser Cys Thr 180 185 190Val Lys Val Cys Pro
Leu Asn Thr Gln Thr Leu Gly Ile Gly Cys Gln 195 200 205Thr Thr Asn
Thr Ala Thr Phe Glu Thr Val Ala Asp Ser Glu Lys Leu 210 215 220Ala
Ile Ile Asp Val Val Asp Ser Val Asn His Lys Leu Asn Ile Thr225 230
235 240Ser Thr Thr Cys Thr Ile Arg Asn Cys Asn Lys Leu Gly Pro Arg
Glu 245 250 255Asn Val Ala Ile Ile Gln Val Gly Gly Ser Asn Ile Leu
Asp Ile Thr 260 265 270Ala Asp Pro Thr Thr Ser Pro Gln Thr Glu Arg
Met Met Arg Val Asn 275 280 285Trp Lys Lys Trp Trp Gln Val Phe Tyr
Thr Val Val Asp Tyr Ile Asp 290 295 300Gln Ile Val Gln Val Met Ser
Lys Arg Ser Arg Ser Leu Asp Leu Ser305 310 315 320Ser Phe Tyr Tyr
Arg Val 3252927DNAArtificial SequencePrimer 29ggctattaaa gctgtacaat
ggggaag 273030DNAArtificial SequencePrimer 30ctaagcgttc taatcttgaa
agaagtttgc 303127DNAArtificial SequencePrimer 31ggctattaaa
ggctcaatgg cgtacag 273226DNAArtificial SequencePrimer 32ggtcatatct
ccacaatggg gttggc 263325DNAArtificial SequencePrimer 33ggctattaaa
gcagtacgag tagtg 253421DNAArtificial SequencePrimer 34ggtcacatca
tgactagtgt g 213521DNAArtificial SequencePrimer 35ggctataaaa
tggcttcgct c 213622DNAArtificial SequencePrimer 36ggtcacatcc
tctggaaatt gc 223729DNAArtificial SequencePrimer 37ggcttttttt
tgaaatgtct tgtgttagc 293824DNAArtificial SequencePrimer
38ggtcacagtt tttgctggct aggc 243926DNAArtificial SequencePrimer
39ggcttttaaa cgaagtcttc aacatg 264025DNAArtificial SequencePrimer
40ggtcacatcc tctcactata ccatc 254125DNAArtificial SequencePrimer
41ggcatttaat gcttttcagt ggttg 254222DNAArtificial SequencePrimer
42ggtcacataa cgcccctata gc 224329DNAArtificial SequencePrimer
43ggcttttaaa gcgtctcagt cgccgttcg 294423DNAArtificial
SequencePrimer 44ggtcacataa gcgctttcta ttc 234524DNAArtificial
SequencePrimer 45ggctttaaaa gcgagaattt ccgt 244622DNAArtificial
SequencePrimer 46ggtcacatca tacatttcta ac 224727DNAArtificial
SequencePrimer 47ggtcacatcg aacaattcta atctaag 274828DNAArtificial
SequencePrimer 48ggtcacatcg aacaattctg accaaatc 284927DNAArtificial
SequencePrimer 49ggtcacatca tacatttcta ttttagg 275027DNAArtificial
SequencePrimer 50ggctttttaa aagttctgtt ccgagag 275123DNAArtificial
SequencePrimer 51ggtcacatta agaccgttcc ttc 235224DNAArtificial
SequencePrimer 52ggcttttaaa gcgctacagt gatg 245324DNAArtificial
SequencePrimer 53ggtcacataa ctggagtggg gagc 245433DNAArtificial
SequencePrimer 54cccaagcttg tgtggcattc tctataacat cgc
335533DNAArtificial SequencePrimer 55cccggatccg gctcatggat
aagcttattc tgc 335633DNAArtificial SequencePrimer 56cccaagcttc
aaatctgctt ctagcggctt acc 335733DNAArtificial SequencePrimer
57cccggatcct aaaggaaaga taccagctgt cac 335833DNAArtificial
SequencePrimer 58cccaagcttc cttttgagat agcactttct ctg
335933DNAArtificial SequencePrimer 59cccggatcca ccacaacaat
ttgattttag agc 336024DNAArtificial SequencePrimer 60cttctttttg
atgttcttcc ttag 246124DNAArtificial SequencePrimer 61tgtagcgaat
tatgactggg ttcc 246233DNAArtificial SequencePrimer 62cccaagctta
agaaatgcat tctcgtaact gtc 336333DNAArtificial SequencePrimer
63cccggatccg tcaatctaga atgtttattc cac 336424DNAArtificial
SequencePrimer 64ttgaatctca acagctgcaa ttcc 246524DNAArtificial
SequencePrimer 65aaacgttagt ggagttctag cgac 246633DNAArtificial
SequencePrimer 66cccaagcttc ccagttaact ggagcataac ctg
336733DNAArtificial SequencePrimer 67cccggatcca actgactctc
cggtcatctc agc 336824DNAArtificial SequencePrimer 68gaacgttgtt
ggttgataag gacc 246924DNAArtificial SequencePrimer 69agtctttgaa
gcgggaacag atgg 247033DNAArtificial SequencePrimer 70cccaagctta
ttgacaacac tcaatgcacc acc 337133DNAArtificial SequencePrimer
71cccggatccg aattagatca cttgccgtta tgc 337224DNAArtificial
SequencePrimer 72gatgcaccac tgacaaacat gagc 247324DNAArtificial
SequencePrimer 73gatactggaa accgaggctc ttcc 247433DNAArtificial
SequencePrimer 74cccaagcttt cggcagatta ccaattcctc cag
337533DNAArtificial SequencePrimer 75cccggatccc agcgtgtatt
tacagtggct tcc 337624DNAArtificial SequencePrimer 76acgggccgtt
tcgacatagt tagc 247724DNAArtificial SequencePrimer 77agaatacgcg
ataccagttg gacc 247833DNAArtificial SequencePrimer 78cccaagcttt
caagagtaga agttgcagca acc 337933DNAArtificial SequencePrimer
79cccggatcca aaggattatt gcagcaatgc aac 338024DNAArtificial
SequencePrimer 80actctttact ctagtatata cctc 248124DNAArtificial
SequencePrimer 81aaatcagaca ttgaacaaca gctg 248227DNAArtificial
SequencePrimer 82tagctacagt tcgagagtca gtcatcc 278327DNAArtificial
SequencePrimer 83caatagaatg gtatctaaga tcgatgg 278433DNAArtificial
SequencePrimer 84cccaagcttg aattgtggcg gtggtgcgat acc
338533DNAArtificial SequencePrimer 85cccggatcca aaatgaagcg
ggaaagtaat ccg 338624DNAArtificial SequencePrimer 86cgcttcacaa
attaacaccg ccac 248724DNAArtificial SequencePrimer 87gaactggtat
gcgtttacat cctc 248833DNAArtificial SequencePrimer 88cccaagcttc
gtatgcagtg tccattgaac cag 338933DNAArtificial SequencePrimer
89cccggatccg cattaattgg aagaaatggt ggc 339024DNAArtificial
SequencePrimer 90tatttctgtt gcagcttcag ttgg 249124DNAArtificial
SequencePrimer 91gttggaggtt ctgatgttct cgac 249233DNAArtificial
SequencePrimer 92cccaagctta cctgaaaatt atgtagtcca tcg
339333DNAArtificial SequencePrimer 93cccggatccc ccacaagttc
aaagaatcat gcg 339424DNAArtificial SequencePrimer 94agcgtctagt
gaccccgtta ttgg 249524DNAArtificial SequencePrimer 95aattcaagtt
ggtggaccga acgc 249633DNAArtificial SequencePrimer 96cccaagcttt
gtagtccatt attcgagtca ctg 339733DNAArtificial SequencePrimer
97cccggatccc aaactgagag aatgatgaga gtg 339824DNAArtificial
SequencePrimer 98agcgtccatt gatcctgtta ttgg 249924DNAArtificial
SequencePrimer 99tgtagctgta atacaagttg gtgg 2410033DNAArtificial
SequencePrimer 100cccaagcttg tatccataga tccagtaatt ggc
3310133DNAArtificial SequencePrimer 101cccggatcca atggtggcaa
gtattctaca ctg 3310224DNAArtificial SequencePrimer 102aatttgagtt
ggagcttctg atgg 2410324DNAArtificial SequencePrimer 103aacagctgat
cccacaactt ctcc 2410428DNAArtificial SequencePrimer 104gtacagttag
gacagaagca atgtatgg 2810528DNAArtificial SequencePrimer
105atcggacctg atgactggtt gagaagcc 2810633DNAArtificial
SequencePrimer 106cccaagcttt gaaacgtact gttcactcct acc
3310733DNAArtificial SequencePrimer 107cccggatcct tgaagcagat
tccgattcag acg 3310833DNAArtificial SequencePrimer 108cccaagcttg
tcgtttgaag cagaatcaga tgg 3310933DNAArtificial SequencePrimer
109cccggatccg tatcaacagt ttccaagaag gag 3311021DNAArtificial
SequencePrimer 110gctgatatag aaggtggaaa g 2111121DNAArtificial
SequencePrimer 111ggtcacatca tgactagtgt g 2111219DNAArtificial
SequencePrimer 112gagcaccata gatgcagct 1911320DNAArtificial
SequencePrimer 113ctgacagatg aagaaacatc 2011419DNAArtificial
SequencePrimer 114gagcaccata gatgcagct 1911520DNAArtificial
SequencePrimer 115gagtcagtta ctagatctgc 2011629DNAArtificial
SequencePrimer 116ggcttttttt tgaaatgtct tgtgttagc
2911720DNAArtificial SequencePrimer 117gtgcataacg gcaagtgatc
2011824DNAArtificial SequencePrimer 118ggctttaaaa gcgagaattt ccgt
2411927DNAArtificial SequencePrimer 119ggtcacatcg aacaattcta
atctaag 2712026DNAArtificial SequencePrimer 120ggcttttaaa
agttctgttc cgagag 2612123DNAArtificial SequencePrimer 121ggtcacatta
agaccgttcc ttc 2312219DNAArtificial SequencePrimer 122tatttaggtg
acactatag 1912320DNAArtificial SequencePrimer 123taatacgact
cactataggg 2012421DNAArtificial SequencePrimer 124gctgatatag
aaggtggaaa g 2112521DNAArtificial SequencePrimer 125ggtcacatca
tgactagtgt g 2112620DNAArtificial SequencePrimer 126gactgctatg
gatttagagc 2012720DNAArtificial SequencePrimer 127gataatgcgt
ataatgccac 2012827DNAArtificial SequencePrimer 128ggctattaaa
gcagtacgag tagtgtg 2712920DNAArtificial SequencePrimer
129ctcaacagct gcaattcctg 2013021DNAArtificial SequencePrimer
130gctgatatag aaggtggaaa g 2113121DNAArtificial SequencePrimer
131ggtcacatca tgactagtgt g 2113221DNAArtificial SequencePrimer
132gctgatatag aaggtggaaa g 2113321DNAArtificial SequencePrimer
133ggtcacatca tgactagtgt g 2113427DNAArtificial SequencePrimer
134ggctattaaa gcagtacgag tagtgtg 2713523DNAArtificial
SequencePrimer 135cgtgctatcg gtaaagaagt agt 2313620DNAArtificial
SequencePrimer 136gtctcagttc gacattggac 2013723DNAArtificial
SequencePrimer 137gaatatggag tgtcaagtgg gtc 2313827DNAArtificial
SequencePrimer 138ggctattaaa gcagtacgag tagtgtg
2713923DNAArtificial SequencePrimer 139cgtgctatcg gtaaagaagt agt
2314019DNAArtificial SequencePrimer 140gagcaccata gatgcagct
1914120DNAArtificial SequencePrimer 141gagtcagtta ctagatctgc
2014222DNAArtificial SequencePrimer 142cagtaatgac tggcggagca gt
2214320DNAArtificial SequencePrimer 143ctgacagatg aagaaacatc
2014421DNAArtificial SequencePrimer 144ggctataaaa tggcttcgct c
2114519DNAArtificial SequencePrimer 145gtcacaaaat gctgtcatg
1914622DNAArtificial SequencePrimer 146cagtaatgac tggcggagca gt
2214720DNAArtificial SequencePrimer 147ctgacagatg aagaaacatc
2014819DNAArtificial SequencePrimer 148gagcaccata gatgcagct
1914920DNAArtificial SequencePrimer 149gagtcagtta ctagatctgc
2015022DNAArtificial SequencePrimer 150cagtaatgac tggcggagca gt
2215120DNAArtificial SequencePrimer 151ctgacagatg aagaaacatc
2015222DNAArtificial SequencePrimer 152cagtaatgac tggcggagca gt
2215322DNAArtificial SequencePrimer 153ggtcacatcc tctggaaatt gc
2215429DNAArtificial SequencePrimer 154ggcttttttt tgaaatgtct
tgtgttagc 2915521DNAArtificial SequencePrimer 155gtctatatgg
caaatctatg c 2115629DNAArtificial SequencePrimer 156ggcttttttt
tgaaatgtct tgtgttagc 2915721DNAArtificial SequencePrimer
157gtctatatgg caaatctatg c
2115821DNAArtificial SequencePrimer 158ctcaaactga tttacatcat g
2115920DNAArtificial SequencePrimer 159gtgcataacg gcaagtgatc
2016021DNAArtificial SequencePrimer 160ctcaaactga tttacatcat g
2116120DNAArtificial SequencePrimer 161gtgcataacg gcaagtgatc
2016229DNAArtificial SequencePrimer 162ggcttttttt tgaaatgtct
tgtgttagc 2916321DNAArtificial SequencePrimer 163gtctatatgg
caaatctatg c 2116421DNAArtificial SequencePrimer 164cagaggaaat
gtagaaatga g 2116520DNAArtificial SequencePrimer 165caatccatgt
ctctgaatgc 2016621DNAArtificial SequencePrimer 166cagaggaaat
gtagaaatga g 2116720DNAArtificial SequencePrimer 167caatccatgt
ctctgaatgc 2016821DNAArtificial SequencePrimer 168ctcaaactga
tttacatcat g 2116920DNAArtificial SequencePrimer 169gtgcataacg
gcaagtgatc 2017021DNAArtificial SequencePrimer 170ctcaaactga
tttacatcat g 2117120DNAArtificial SequencePrimer 171gtgcataacg
gcaagtgatc 2017224DNAArtificial SequencePrimer 172ggctttaaaa
gcgagaattt ccgt 2417327DNAArtificial SequencePrimer 173ggtcacatcg
aacaattcta atctaag 2717424DNAArtificial SequencePrimer
174ggctttaaaa gcgagaattt ccgt 2417527DNAArtificial SequencePrimer
175ggtcacatcg aacaattcta atctaag 2717624DNAArtificial
SequencePrimer 176ggctttaaaa gcgagaattt ccgt 2417720DNAArtificial
SequencePrimer 177gtacatgatg atcccattga 2017827DNAArtificial
SequencePrimer 178ggctttttaa aagttctgtt ccgagag
2717923DNAArtificial SequencePrimer 179ggtcacatta agaccgttcc ttc
2318027DNAArtificial SequencePrimer 180ggctttttaa aagttctgtt
ccgagag 2718123DNAArtificial SequencePrimer 181ggtcacatta
agaccgttcc ttc 2318227DNAArtificial SequencePrimer 182ggctttttaa
aagttctgtt ccgagag 2718323DNAArtificial SequencePrimer
183ggtcacatta agaccgttcc ttc 2318428DNAArtificial SequencePrimer
184cttatcaatc atttccagct gacgtctc 2818527DNAArtificial
SequencePrimer 185tccatatgaa ccaaaagagg tgactgc
2718639DNAArtificial SequencePrimer 186gttagtggag ttctagcgac
atattttaaa atgtagaat 3918721DNAArtificial SequencePrimer
187ggtcacatca tgactagtgt g 21
* * * * *