U.S. patent application number 12/995024 was filed with the patent office on 2011-07-14 for expression and assembly of human group c rotavirus-like particles and uses thereof.
This patent application is currently assigned to The Government of the US, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Baoming Jiang.
Application Number | 20110171316 12/995024 |
Document ID | / |
Family ID | 41398797 |
Filed Date | 2011-07-14 |
United States Patent
Application |
20110171316 |
Kind Code |
A1 |
Jiang; Baoming |
July 14, 2011 |
EXPRESSION AND ASSEMBLY OF HUMAN GROUP C ROTAVIRUS-LIKE PARTICLES
AND USES THEREOF
Abstract
Group C rotaviruses are a cause of acute gastroenteritis in
children and adults that is distinct from group A RV. However,
human group C rotaviruses cannot be grown in culture, resulting in
a lack of tools for detection and treatment of GrpC RV disease.
Consequently, the burden of GpC RV disease has not been clearly
established. Isolated recombinant human rotavirus group C
virus-like particles are provided according to embodiments of the
present invention along with methods of their production and use
in, inter alia, detection of Grp C RV infection, diagnostic assays
and immunogenic compositions.
Inventors: |
Jiang; Baoming; (Duluth,
GA) |
Assignee: |
The Government of the US, as
represented by the Secretary, Department of Health and Human
Services
Atlanta
GA
|
Family ID: |
41398797 |
Appl. No.: |
12/995024 |
Filed: |
May 29, 2009 |
PCT Filed: |
May 29, 2009 |
PCT NO: |
PCT/US09/45688 |
371 Date: |
January 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61111425 |
Nov 5, 2008 |
|
|
|
61130615 |
May 29, 2008 |
|
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Current U.S.
Class: |
424/499 ;
424/215.1; 435/29; 435/69.3; 530/350 |
Current CPC
Class: |
A61P 37/04 20180101;
C07K 14/005 20130101; A61P 31/14 20180101; C12N 2720/12323
20130101; A61K 39/12 20130101; C12N 2720/12334 20130101; A61K
2039/5258 20130101; C12N 2710/14143 20130101; C12N 2720/12322
20130101 |
Class at
Publication: |
424/499 ;
530/350; 424/215.1; 435/69.3; 435/29 |
International
Class: |
A61K 39/15 20060101
A61K039/15; C07K 14/14 20060101 C07K014/14; A61K 9/14 20060101
A61K009/14; C12P 21/00 20060101 C12P021/00; A61P 37/04 20060101
A61P037/04; A61P 31/14 20060101 A61P031/14; C12Q 1/02 20060101
C12Q001/02 |
Goverment Interests
GOVERNMENT SPONSORSHIP
[0002] This invention was made by the Centers for Disease Control
and Prevention, an agency of the United States Government.
Claims
1. An isolated recombinant human rotavirus group C virus-like
particle comprising human rotavirus group C VP6 protein and a human
rotavirus group C VP7 protein.
2. The isolated recombinant human rotavirus group C virus-like
particle of claim 1 further comprising human rotavirus group C VP2
protein.
3. The isolated recombinant human rotavirus group C virus-like
particle of claim 1 wherein the virus-like particle is free of
human rotavirus group C VP1, VP3, VP4 and human rotavirus group C
non-structural proteins NSP1, NSP2, NPS3, NSP4, NSP5, NSP6 and
NSP7.
4. The isolated recombinant human rotavirus group C virus-like
particle of claim 1 wherein the human rotavirus group C VP6 protein
comprises the amino acid sequence of SEQ ID No. 32.
5. The isolated recombinant human rotavirus group C virus-like
particle of claim 1 wherein the human rotavirus group C VP7 protein
comprises the amino acid sequence of SEQ ID No. 34.
6. The isolated recombinant human rotavirus group C virus-like
particle of claim 2 wherein the human rotavirus group C VP2 protein
comprises the amino acid sequence of SEQ ID No. 1.
7. (canceled)
8. The isolated recombinant human rotavirus group C virus-like
particle according to claim 1 admixed with a pharmaceutically
acceptable carrier.
9. The isolated recombinant human rotavirus group C virus-like
particle of claim 1, further comprising a cargo moiety in an
internal space defined by the isolated recombinant human rotavirus
group C virus-like particle.
10. The isolated recombinant human rotavirus group C virus-like
particle according to claim 9, wherein the cargo moiety is selected
from the group consisting of: a label, an antigen, a nucleic acid
sequence encoding a protein or peptide, and a therapeutic
agent.
11. (canceled)
12. The isolated recombinant human rotavirus group C virus-like
particle of claim 1 attached to a solid substrate.
13. (canceled)
14. The isolated recombinant human rotavirus group C virus-like
particle of claim 1, further comprising an immunological
adjuvant.
15. A process of generating an immunological response in a human
comprising administering an isolated recombinant human rotavirus
group C virus-like particle comprising human rotavirus group C VP6
protein and a human rotavirus group C VP7 protein. to a human.
16. The process of claim 15, wherein the step of administering the
immunological composition is administered to a mucosal surface.
17-25. (canceled)
26. A process of forming a human group C rotavirus-like particle
comprising: constructing a first vector comprising a nucleic acid
molecule comprising a sequence encoding a human group C rotavirus
VP6 capsid protein operably linked to a promoter that drives
expression of said protein in an insect cell; constructing a second
vector comprising a nucleic acid molecule comprising a sequence
encoding a human group C rotavirus VP7 capsid protein operably
linked to a promoter that drives expression of said protein in an
insect cell; infecting an insect cell culture with said first and
second baculovirus vector under conditions that promote expression
of the VP6 capsid protein and VP7 capsid protein and association to
form the human group C rotavirus-like particle.
27. The process of claim 26 further comprising: constructing a
third vector comprising a nucleic acid molecule comprising a
sequence encoding a human group C rotavirus VP2 core protein
operably linked to a promoter that drives expression of said
protein in an insect cell; and infecting an insect cell culture
with said first baculovirus vector, second baculovirus vector, and
third baculovirus vector under conditions that promote expression
of the VP6 capsid protein, and VP7 capsid protein and said VP2 core
protein.
28. The process of claim 26 further comprising determining that
human group C rotavirus-like particle is present in said
culture.
29. The process of claim 26 further comprising isolating the human
group C rotavirus-like particle from said culture.
30. The process of claim 26, wherein said first vector and said
second vector both have baculovirus promoters.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application Ser. No. 61/130,615 filed May 29, 2008 and U.S.
Provisional Patent Application Ser. No. 61/111,425 filed Nov. 5,
2008, the entire content of both are incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The present invention relates to group C rotaviruses (GpC
RV) and rotavirus-like particles, methods of producing immunogenic
rotavirus-like particles, immunogenic compositions inclusive of
rotavirus-like particles and methods for eliciting an immune
response using compositions inclusive of rotavirus-like particles,
as well as producing a diagnostic for rotavirus infection.
BACKGROUND OF THE INVENTION
[0004] Rotaviruses are a diverse set of pathogens classified into
groups A through G based on the distinct characteristics of the
inner capsid protein, VP6. Among these, GpC RV has been identified
as a pathogen in humans and attributed to outbreaks and sporadic
cases of gastroenteritis worldwide in young children <3 years of
age (8, 13, 15, 25, 26, 29, 30) and in older children and adults
(2, 15, 20, 21, 25, 26, 30, 32). While some studies have reported
low detection rates in children with diarrhea (1, 2, 4, 26, 29),
seroprevalence studies have demonstrated that GpC RV is a
commonplace pathogen with a much higher occurrence in adults (4, 6,
10, 20, 27, 31).
[0005] One possible cause of low GpC RV detection is the
unavailability of adequate diagnostic tools. While PCR is a
frequently employed technique, it is often insensitive for
diagnosis of GpC RV due to the instability of its capsid proteins
and the degradation of its RNA genome. It is also not an accessible
technique to many clinical laboratories that are involved in
diagnostics of samples from patients with gastroenteritis. If a
more practical and economical tool, like a GpC RV-specific enzyme
immunoassay (EIA), was available, testing of large numbers of
samples could be performed to better estimate GpC RV disease
burden. Propagation of the Cowden strain, a prototype porcine GpC
RV, has been successful and antibodies to this virus have been
employed for GpC RV diagnostics (13, 28, 30, 33, 34). However their
specificity and sensitivity to human GpC RV is questionable.
Progress in the development of a sensitive and specific EIA for
human GpC RV has been stunted by its fastidious growth in cell
culture. To circumvent the prior art problems of GpC RV fastidious
growth problem, VP6 from a human GpC RV was expressed in insect
cells using the Baculovirus System and antibody to this recombinant
protein was employed in seroprevalence studies (6, 11, 31). To the
best of our knowledge, these reagents have not been utilized for
viral detection in human specimens and their specificity to GpC RV
remains questionable.
[0006] GpC RV are a cause of acute gastroenteritis in children and
adults that is distinct from group A RV. Human group A RV detection
methods are well established and widely available while group C RV
diagnostics are only available in a few reference laboratories.
Since native human group C RV are unstable and cannot be grown in
cell culture, reagents from animal group C RV have been used for
diagnostics. However these diagnostic tools may not be sensitive or
specific enough for human strains. Thus, sensitive and specific
detection methods and reagents for human group C RV are not readily
available. Consequently, the burden of GpC RV disease has not been
clearly established.
[0007] Thus, there exists a need for a human specific group C
rotavirus diagnostic. There also exists a need for a human group C
RV-like particle for use in such a diagnostic and for eliciting an
immune response as a vaccine.
SUMMARY OF THE INVENTION
[0008] An isolated recombinant human rotavirus group C virus-like
particle including human rotavirus group C VP6 protein and a human
rotavirus group C VP7 protein is provided according to embodiments
of the present invention. In further embodiments, an isolated
recombinant human rotavirus group C virus-like particle including
human rotavirus group C VP6 protein, a human rotavirus group C VP7
protein and a human rotavirus group C VP2 protein is provided.
According to certain embodiments, the isolated recombinant human
rotavirus group C virus-like particles of the present invention are
free of other human rotavirus group C proteins such as VP1, VP3,
VP4, NSP1, NSP2, NPS3, NSP4, NSP5, NSP6 and NSP7.
[0009] Isolated recombinant human rotavirus group C virus-like
particles are provided according to embodiments of the present
invention which include human rotavirus group C VP6 protein
including the amino acid sequence of SEQ ID No. 32. Isolated
recombinant human rotavirus group C virus-like particles are
provided according to embodiments of the present invention which
include human rotavirus group C VP7 protein including the amino
acid sequence of SEQ ID No. 34. In particular embodiments, isolated
recombinant human rotavirus group C virus-like particles are
provided according to embodiments of the present invention which
include human rotavirus group C VP6 protein including the amino
acid sequence of SEQ ID No. 32 and human rotavirus group C VP7
protein including the amino acid sequence of SEQ ID No. 34.
[0010] Isolated recombinant human rotavirus group C virus-like
particles are provided according to embodiments of the present
invention which include the human rotavirus group C VP2 protein
including the amino acid sequence of SEQ ID No. 1. In further
embodiments, isolated recombinant human rotavirus group C
virus-like particles are provided according to embodiments of the
present invention which include human rotavirus group C VP6 protein
including the amino acid sequence of SEQ ID No. 32, human rotavirus
group C VP7 protein including the amino acid sequence of SEQ ID No.
34 and human rotavirus group C VP2 protein including the amino acid
sequence of SEQ ID No. 1.
[0011] Processes for detection of a human rotavirus group C
antibody in a biological sample are provided according to
embodiments of the present invention which include contacting a
first biological sample with a plurality of isolated recombinant
human rotavirus group C virus-like particles and detecting the
formation of a complex between an anti-human rotavirus group C
antibody present in the first biological sample and the plurality
of isolated recombinant human rotavirus group C virus-like
particles, to obtain a first signal indicative of the presence of
an anti-human rotavirus group C antibody.
[0012] Anti-human rotavirus group C vaccines are provided according
to embodiments of the present invention which includes isolated
recombinant human rotavirus group C virus-like particles admixed
with a pharmaceutically acceptable carrier.
[0013] Processes of delivering a cargo moiety to a cell are
provided according to embodiments of the present invention which
include introducing a cargo moiety into an internal space defined
by an isolated recombinant human rotavirus group C virus-like
particle and contacting the isolated recombinant human rotavirus
group C virus-like particle and a cell.
[0014] Exemplary cargo moieties are a label, an antigen, a nucleic
acid sequence encoding a protein or peptide, and a therapeutic
agent.
[0015] Anti-human rotavirus group C antibody assay kits are
provided according to embodiments of the present invention which
include isolated recombinant human rotavirus group C virus-like
particles and at least one ancillary reagent. Optionally, the
virus-like particles are attached to a solid substrate.
[0016] Immunogenic compositions are provided according to
embodiments of the present invention which include an isolated
recombinant human rotavirus group C virus-like particle described
herein and a pharmaceutically acceptable carrier. Optionally, an
inventive immunogenic composition includes an immunological
adjuvant.
[0017] Processes of generating an immunological response in a human
including administering an immunogenic composition including an
inventive human rotavirus group C virus-like particle to a human
are provided according to embodiments of the present invention.
Optionally, an inventive process includes administering the
immunological composition to a mucosal surface.
[0018] An isolated polypeptide including an amino acid sequence of:
a) an amino acid sequence having at least 98% to the amino acid
sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence);
b) an amino acid sequence having at least 99% to the amino acid
sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence);
c) an amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2
amino acid sequence); or d) an amino acid sequence set forth in SEQ
ID NO: 32 (S 1 VP6 amino acid sequence) is provided according to
embodiments of the invention. An isolated nucleic acid molecule
including a nucleotide sequence encoding the isolated polypeptide
including an amino acid sequence of: a) an amino acid sequence
having at least 98% to the amino acid sequence set forth in SEQ ID
NO: 1 (ASP88 VP2 amino acid sequence); b) an amino acid sequence
having at least 99% to the amino acid sequence set forth in SEQ ID
NO: 1 (ASP88 VP2 amino acid sequence); c) an amino acid sequence
set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); or d) an
amino acid sequence set forth in SEQ ID NO: 32 (S 1 VP6 amino acid
sequence) is provided according to embodiments of the
invention.
[0019] Immunogenic compositions are provided according to
embodiments of the present invention including a polypeptide
including at least one amino acid sequence of any of SEQ ID NOS:
1-13 wherein said amino sequence is an antigenic epitope recognized
by an antibody. Optionally, such an immunogenic composition further
includes a rotavirus-like particle described herein.
[0020] An antibody is provided according to embodiments of the
present invention that is specific for a group C rotavirus and
which does not recognize a group A rotavirus. In particular
embodiments, an antibody according to embodiments of the present
invention is specific for an amino acid sequence of any of SEQ ID
NOS: 3-13 and does not recognize a group A rotavirus.
[0021] An isolated polypeptide is provided according to embodiments
of the present invention that includes at least one amino acid
sequence of SEQ ID NO: 3 or 8. An isolated nucleic acid molecule
including a nucleotide sequence encoding the isolated polypeptide
that includes at least one amino acid sequence of SEQ ID NO: 3 or 8
is provided according to embodiments of the present invention.
[0022] Vectors including an isolated nucleic acid molecule
including a nucleotide sequence encoding the isolated polypeptide
that includes at least one amino acid sequence of SEQ ID NO: 3 or 8
are provided according to embodiments of the present invention.
Isolated host cells including a vector of the present invention are
provided according to particular embodiments.
[0023] Processes of forming a human group C rotavirus-like particle
are provided according to embodiments of the present invention
which include constructing a first vector comprising a nucleic acid
molecule comprising a sequence encoding a human group C rotavirus
VP6 capsid protein operably linked to a promoter that drives
expression of said protein in an insect cell; constructing a second
vector comprising a nucleic acid molecule comprising a sequence
encoding a human group C rotavirus VP7 capsid protein operably
linked to a promoter that drives expression of said protein in an
insect cell; and infecting an insect cell culture with said first
and second baculovirus vector under conditions that promote
expression of the VP6 capsid protein and VP7 capsid protein and
association to form the human group C rotavirus-like particle. In
further embodiments, processes of forming a human group C
rotavirus-like particle include constructing a third vector
comprising a nucleic acid molecule comprising a sequence encoding a
human group C rotavirus VP2 core protein operably linked to a
promoter that drives expression of said protein in an insect cell;
and infecting an insect cell culture with said first baculovirus
vector, second baculovirus vector, and third baculovirus vector
under conditions that promote expression of the VP6 capsid protein,
and VP7 capsid protein and said VP2 core protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A is an image of an electrophoretic gel showing the
kinetics of GpC RV VP6 and VP7 expression in Sf9 cells in different
media;
[0025] FIG. 1B is an image of an electrophoretic gel showing the
kinetics of GpC RV VP6 and VP7 expression in Hi5 (B) cells in
different media;
[0026] FIG. 2A is an image of an electron micrograph of human GpC
RV VLPs formed by self-assembly of recombinant human rotavirus C
VP2, VP6, and VP7 proteins expressed in Sf9 cells infected with
recombinant baculoviruses at an MOI of 1 each;
[0027] FIG. 2B is an image of an electron micrograph of human GpC
RV VLPs formed by self-assembly of recombinant human rotavirus C
VP6 and VP7 proteins expressed in Sf9 cells infected with
recombinant baculoviruses at an MOI of 1.4 each;
[0028] FIG. 3A is an image showing Coomassie blue staining of major
structural viral proteins from GpA RV YK-1 and human rotavirus GpC
VLPs;
[0029] FIG. 3B is an image of an immunoblot showing the comparison
of major structural viral proteins from GpA RV YK-1 and human
rotavirus GpC VLPs;
[0030] FIG. 4A is an image of an immunoelectron micrograph showing
human GpC RV VLPs immunostained with GpC-specific rabbit
hyperimmune serum;
[0031] FIG. 4B is an image of an immunoelectron micrograph showing
human GpC RV VLPs immunostained with GpA-specific rabbit
hyperimmune serum;
[0032] FIG. 4C is an image of an immunoelectron micrograph showing
GpA RV immunostained with GpC-specific rabbit hyperimmune
serum;
[0033] FIG. 4D is an image of an immunoelectron micrograph showing
GpA RV immunostained with GpA-specific rabbit hyperimmune
serum;
[0034] FIG. 5 is an amino acid sequence alignment for Group C
rotavirus VP2 proteins from human strain ASP88 (SEQ ID NO: 1);
"Bristol" human strain (SEQ ID NO: 16, Accession CAC 44890) and
"Cowden" porcine strain (SEQ ID NO: 17, Accession M74217);
[0035] FIG. 6 is a nucleotide sequence alignment of sequences
encoding human Group C VP-2 for inventive strain ASP88 (SEQ ID NO:
18), "Cowden" porcine strain (SEQ ID No. 44) and Bristol (SEQ ID
NO: 19, Accession AJ303139);
[0036] FIG. 7 is a nucleotide sequence alignment of sequences
encoding human Group C VP-6 for inventive strain S-1 (SEQ ID NO:
31) relative to conventional strains Bristol (SEQ ID NO: 25,
Accession CAA42504); Jajeri (SEQ ID NO: 26, Accession AAK26534);
CMH004 (SEQ ID NO: 27, Accession ABR31794); V508 (SEQ ID NO: 28,
Accession AAX13496); China (SEQ ID NO: 29, Accession BAB83829); and
BCN6 (SEQ ID NO: 30, Accession CAJ41549);
[0037] FIG. 8 is an amino acid sequence alignment of sequences
encoding human Group C VP-6 for inventive strain S-1 (SEQ ID NO:
32) relative to conventional strains Bristol (SEQ ID NO: 35,
Accession CAA42504); Jajeri (SEQ ID NO: 36, Accession AAK26534);
CMH004 (SEQ ID NO: 37, Accession ABR31794); V508 (SEQ ID NO: 38,
Accession AAX13496); China (SEQ ID NO: 39, Accession BAB83829); and
BCN6 (SEQ ID NO: 40, Accession CAJ41549);
[0038] FIG. 9 is a nucleotide sequence of human rotavirus VP6
protein from S-1 strain (SEQ ID No. 46) including an open reading
frame, 5' and 3' non-coding sequences; and
[0039] FIG. 10 is a nucleotide sequence alignment of sequences
encoding human Group C VP-6 and including 5' and 3' non-coding
sequences for inventive strain S-1 (SEQ ID NO: 46) and Bristol VP6
(SEQ ID No. 47).
DETAILED DESCRIPTION OF THE INVENTION
[0040] Group C rotavirus (GpC RV) is a causative agent of acute
gastroenteritis in children and adults. Characterization of GpC RV
has only been accomplished to date with porcine and bovine strains
that can be grown in cell culture. Because human GpC RVs are
unstable and cannot be cultivated in cell culture, reagents and
sensitive and specific detection methods are not available.
Consequently, the impact of GpC RV on diarrheal disease has not
been clearly established.
[0041] Demonstrated herein is the expression of the major inner and
outer capsid human GpC proteins VP6 and VP7 and the human GpC core
protein VP2 and the self-assembly of human GpC VP6/7 virus-like
particles (VLPs) or human GpC VP2/6/7 VLPs. Antibodies to these
human GpC RV VLPs show highly specific reactivities with the
corresponding GpC but not GpA RV.
[0042] The ability to produce large amounts of human GpC RV
antigenic materials, such as human GpC RV proteins and VLPs, and
the availability of high quality antibody reagents provide
sensitive and specific diagnostic assays and provide tools for
investigation of the epidemiology and disease burden of GpC RV in
humans.
[0043] The instant invention has numerous uses including, but not
limited to, detection of human rotavirus C antibodies in biological
samples, diagnosis of human rotavirus C infection, identification
of individuals previously or currently infected with human
rotavirus C, as an antigen for generation of antibodies and for the
development of therapeutics for prophylaxis or treatment of disease
associated with human rotavirus C infection.
[0044] In accordance with the present invention, various techniques
and terms including, but not limited to, conventional molecular
biology, microbiology, immunology and recombinant DNA techniques
and terms, may be used which are known by those of skill in the
art. Such techniques and terms are described and/or defined in
detail in standard references such as J. Sambrook and D. W.
Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 3rd Ed., 2001; F. M. Ausubel et al., Eds., Short
Protocols in Molecular Biology, Current Protocols, Wiley, 2002;
Wild, D., The Immunoassay Handbook, 3rd Ed., Elsevier Science,
2005; Gosling, J. P., Immunoassays: A Practical Approach, Practical
Approach Series, Oxford University Press, 2005; and Harlow, E. and
Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 1988; F. Breitling and S. Diibel, Recombinant
Antibodies, John Wiley & Sons, New York, 1999; H. Zola,
Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies
and Engineered Antibody Derivatives, Basics: From Background to
Bench, BIOS Scientific Publishers, 2000; B.K.C. Lo, Antibody
Engineering: Methods and Protocols, Methods in Molecular Biology,
Humana Press, 2003; Crowther, J. R., The ELISA Guidebook (Methods
in Molecular Biology), Humana Press, 2000; and other references
described herein.
[0045] Human Rotavirus C Virus-Like Particles
[0046] Human rotavirus C virus-like particles (VLPs) are provided
according to the present invention. The term "virus-like particle"
refers to a capsid defining an internal space. The internal space
defined by the capsid is "empty" of an intact human rotavirus C
genome and the human rotavirus C VLPs of the present invention are
therefore non-replicating and incapable of causing human rotavirus
C-associated disease.
[0047] Human rotavirus C VLPs include human rotavirus C VP6 and VP7
proteins according to embodiments of the present invention. In
further embodiments of the present invention, human rotavirus C
VLPs include human rotavirus C VP2, VP6 and VP7 proteins.
[0048] Genes encoding human rotavirus C proteins VP2, VP6 and VP7
have been identified and sequenced.
[0049] Any human Group C RV VP6 protein can be included in human
rotavirus C VLPs of the present invention. Examples of human Group
C RV VP6 proteins that can be included in human rotavirus C VLPs of
the present invention include VP6 protein of human Group C strain
S-1 VP6 (SEQ ID NO: 32); Bristol strain (SEQ ID NO: 35, Accession
CAA42504); Jajeri strain (SEQ ID NO: 36, Accession AAK26534);
CMH004 strain (SEQ ID NO: 37, Accession ABR31794); V508 strain (SEQ
ID NO: 38, Accession AAX13496); China strain (SEQ ID NO: 39,
Accession BAB83829); and BCN6 strain (SEQ ID NO: 40, Accession
CAJ41549).
[0050] Human Group C RV VP6 proteins that can be included in human
GpC RV VLPs include those known by NCBI Accession numbers BAB83829,
AAK26535, AAK26534, AAX13496, AAX13492, AAX13491, CAJ41551,
CAJ41550, CAJ41549, AAW82662, AAW82661, ABD96606, ABD96605,
ABD96604, AAA47340, AAA47339, CAA42504, AAX08120, ABR31794,
YP.sub.--392512, P69481, P69483 and P69482.
[0051] Any human Group C RV VP7 protein can be included in human
rotavirus C VLPs of the present invention. Examples of human Group
C RV VP7 proteins that can be included in human GpC RV VLPs include
those known by NCRI Accession numbers BAB83828, AAX16188, AAX16187,
AAX16186, CAJ41554, CAJ41553, CAJ41552, AAW82659, AAD25388,
BAA20340, BAA20339, AAQ93808, AAQ93807, AAA47352, BAF73591,
BAF73590, BAF73589, BAF73588, BAF73587, BAD20702, BAD20701,
BAD20700, BAD20699, AAK26533, AAK26530, ABR31795, BAC53881,
BAC53880, BAC53879, BAC53878, BAC53877, BAC53876, BAC53875,
BAC53874, ABE01860, ABE01859, ABE01858, AAF33400, AAF33399,
AAF33398, AAF33397, AAF33396, AAF33395, AAF33394, AAF33393,
AAF33392, AAF33391, AAF33390, AAF33389, BAA33952, P30216, ABO25864
and 2209225A.
[0052] Any human Group C RV VP2 protein can be included in human
rotavirus C VLPs of the present invention. Examples of human Group
C RV VP2 proteins that can be included in human rotavirus C VLPs of
the present invention include VP2 protein of human Group C strain
ASP88 VP2 (SEQ ID NO: 1); and Bristol strain (SEQ ID NO: 16,
Accession CAB52753).
[0053] In addition to these VP2, VP6 and VP7 amino acid sequences,
the term VP2, VP6 or VP7 amino acid sequence encompasses variants.
In particular embodiments, a VP2, amino acid sequence included in a
VLP composition of the present invention is a variant of ASP88 VP2
(SEQ ID No. 1). In further embodiments, a VP6, amino acid sequence
included in a VLP composition of the present invention is a variant
of S-1 VP6 (SEQ ID No. 32). In further embodiments, a VP7, amino
acid sequence included in a VLP composition of the present
invention is a variant of S-1 VP7 (SEQ ID No. 34).
[0054] In another aspect, the invention provides a rotavirus-like
particle having a core VP2 structural protein of human group C RV
of strain ASP88 protein (SEQ ID NO: 1) or a fragment or variant
thereof.
[0055] In another aspect, the invention provides a rotavirus-like
particle comprising VP6 capsid protein and VP7 capsid protein, or
fragments or variants thereof, with the proviso that said particle
does not comprise an amino acid sequence set forth in (SEQ ID NO:
16; Bristol).
[0056] In another aspect, the invention provides an isolated
polypeptide comprising an amino acid sequence selected from the
group consisting of: a) an amino acid sequence having at least 98%
identity to the amino acid sequence set forth in SEQ ID NO: 1
(ASP88 VP2 amino acid sequence); b) an amino acid sequence having
at least 99% identity to the amino acid sequence set forth in SEQ
ID NO: 1 (ASP88 VP2 amino acid sequence); c) an amino acid sequence
set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); and d)
an amino acid sequence set forth in SEQ ID NO: 32 (S-1 VP6 amino
acid sequence).
[0057] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence encoding one
or more of the inventive polypeptides described herein. In certain
embodiments the invention provides an isolated nucleic acid
molecule encoding VP2 selected from the group consisting of: a) an
isolated nucleic acid molecule encoding an inventive polypeptide
having at least 98% identity to the amino acid sequence set forth
in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); b) an isolated
nucleic acid molecule encoding an inventive polypeptide having at
least 99% identity to the amino acid sequence set forth in SEQ ID
NO: 1 (ASP88 VP2 amino acid sequence); c) an isolated nucleic acid
molecule encoding an inventive polypeptide set forth in SEQ ID NO:
1 (ASP88 VP2 amino acid sequence); and d) an isolated nucleic acid
molecule encoding an inventive amino acid sequence set forth in SEQ
ID NO: 32 (S-1 VP6 amino acid sequence).
[0058] In another aspect, the invention provides an immunogenic
composition that includes a polypeptide containing at least one
amino acid sequence of:
TABLE-US-00001 (SEQ ID NO: 2) LETIIDKEVK ENKDSTKDEK LVVTEESNGD VTA,
(SEQ ID NO: 3) LETIINKEVK ENKDSMKEDK LVVTEESNGD VTT, (SEQ ID NO: 4)
TENVEEKEIK EAKEQVKDEK QVITEENVDS PKD, (SEQ ID NO: 5) KLTEIQESSA
KTYNTLFRLF TP, (SEQ ID NO: 6) NYRNSRIKCQ TYNKLFRL, (SEQ ID NO: 7)
LNVLEG MPDYIMLRDM AV, (SEQ ID NO: 8) LNVLEE MPDYIMLRDM AV, (SEQ ID
NO: 9) LNVLDE MPDYVMLRDM AV, (SEQ ID NO: 10) AAHLQLE AITVQVESQF
LAGISAAAAN EA, (SEQ ID NO: 11) LQCKLNH NSWQELVHGR NE, (SEQ ID NO:
12) LSACIVMNMH LVG, and (SEQ ID NO: 13) IPPDQMYRLR NRLRNIP;
wherein said amino sequence is an antigenic epitope recognized by
an antibody.
[0059] In another aspect, the invention provides a antibody
preparation that recognizes an amino acid sequence of:
TABLE-US-00002 (SEQ ID NO: 2) LETIIDKEVK ENKDSTKDEK LVVTEESNGD VTA,
(SEQ ID NO: 3) LETIINKEVK ENKDSMKEDK LVVTEESNGD VTT, (SEQ ID NO: 4)
TENVEEKEIK EAKEQVKDEK QVITEENVDS PKD, (SEQ ID NO: 5) KLTEIQESSA
KTYNTLFRLF TP, (SEQ ID NO: 6) NYRNSRIKCQ TYNKLFRL, (SEQ ID NO: 7)
LNVLEG MPDYIMLRDM AV, (SEQ ID NO: 8) LNVLEE MPDYIMLRDM AV, (SEQ ID
NO: 9) LNVLDE MPDYVMLRDM AV, (SEQ ID NO: 10) AAHLQLE AITVQVESQF
LAGISAAAAN EA, (SEQ ID NO: 11) LQCKLNH NSWQELVHGR NE, (SEQ ID NO:
12) LSACIVMNMH LVG, and (SEQ ID NO: 13) IPPDQMYRLR NRLRNIP.
[0060] In another aspect, the invention provides a vector
comprising the inventive nucleic acid molecules described
herein.
[0061] In another aspect, the invention provides an isolated host
cell comprising one or more of the inventive vectors described
herein.
[0062] The inventive methods and compositions are not limited to
the VP proteins and polypeptides having the amino acid sequence
described herein in detail. Where appropriate, variants, such as
homologs from other strains and groups, may be used.
[0063] As used herein, the term "variant" defines either a
naturally occurring genetic mutant of a human rotavirus C virus or
a recombinantly prepared variation of a human rotavirus C virus,
each of which contain one or more mutations in its genome compared
to a reference human rotavirus C VP2, VP6 or VP7. The term
"variant" may also refer to either a naturally occurring variation
of a given peptide or a recombinantly prepared variation of a given
peptide or protein in which one or more amino acid residues have
been modified by amino acid substitution, addition, or
deletion.
[0064] Preferred are human rotavirus C proteins having at least
95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 1, SEQ ID No.32 or
SEQ ID No. 34. Further preferred are human rotavirus C proteins
having 99% or greater identity to SEQ ID No. 1, SEQ ID No.32 or SEQ
ID No.34.
[0065] Mutations can be introduced using standard molecular biology
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. One of skill in the art will recognize that one or
more amino acid mutations can be introduced without altering the
functional properties of the human rotavirus C VP2, VP6 or VP7
proteins.
[0066] It is also recognized by one of ordinary skilled in the art
that VP protein and polypeptide variants encompass conservative
amino acid substitutions in the amino acid sequences of the VP
proteins and polypeptides set forth in detail herein. Conservative
amino acid substitutions can be made in human rotavirus C VP2, VP6
or VP7 proteins to produce human rotavirus C VP2, VP6 or VP7
protein variants. Conservative amino acid substitutions are art
recognized substitutions of one amino acid for another amino acid
having similar characteristics. For example, each amino acid may be
described as having one or more of the following characteristics:
electropositive, electronegative, aliphatic, aromatic, polar,
hydrophobic and hydrophilic. A conservative substitution is a
substitution of one amino acid having a specified structural or
functional characteristic for another amino acid having the same
characteristic. Acidic amino acids include aspartate, glutamate;
basic amino acids include histidine, lysine, arginine; aliphatic
amino acids include isoleucine, leucine and valine; aromatic amino
acids include phenylalanine, glycine, tyrosine and tryptophan;
polar amino acids include aspartate, glutamate, histidine, lysine,
asparagine, glutamine, arginine, serine, threonine and tyrosine;
and hydrophobic amino acids include alanine, cysteine,
phenylalanine, glycine, isoleucine, leucine, methionine, proline,
valine and tryptophan; and conservative substitutions include
substitution among amino acids within each group. Amino acids may
also be described in terms of relative size, alanine, cysteine,
aspartate, glycine, asparagine, proline, threonine, serine, valine,
all typically considered to be small.
[0067] Human rotavirus C VP2, VP6 or VP7 variants can include
synthetic amino acid analogs, amino acid derivatives and/or
non-standard amino acids, illustratively including, without
limitation, alpha-aminobutyric acid, citrulline, canavanine,
cyanoalanine, diaminobutyric acid, diaminopimelic acid,
dihydroxy-phenylalanine, djenkolic acid, homoarginine,
hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine,
5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and
ornithine.
[0068] In addition, as will be appreciated by one of skill in the
art, due to the degeneracy of the genetic code, more than one
nucleic acid will encode an identical protein. Thus, nucleic acids
encoding the VP proteins and polypeptides or a variant thereof are
not limited to those nucleic acids described herein in detail.
[0069] Variants of VP proteins and polypeptides having 95%, 96%,
97%, 98%, or 99% homology to the amino acid sequence described
herein in detail are operable in the described methods and
compositions. Variants of nucleic acids having 95%, 96%, 97%, 98%,
or 99% homology to the nucleotide sequence described herein in
detail are operable in the described methods and compositions.
[0070] "Homology" refers to sequence similarity or, alternatively,
sequence identity, between two or more polynucleotide sequences or
two or more polypeptide sequences.
[0071] The terms "percent identity" and "% identity", as applied to
polynucleotide sequences, refer to the percentage of identical
nucleotide matches between at least two polynucleotide sequences
aligned using a standardized algorithm. Such an algorithm may
insert, in a standardized and reproducible way, gaps in the
sequences being compared in order to optimize alignment between two
sequences, and therefore achieve a more meaningful comparison of
the two sequences.
[0072] Percent identity between polynucleotide sequences may be
determined using one or more computer algorithms or programs known
in the art or described herein. For example, percent identity can
be determined using the default parameters of the CLUSTAL V
algorithm as incorporated into the MEGALIGN version 3.12e sequence
alignment program. This program is part of the LASERGENE software
package, a suite of molecular biological analysis programs
(DNASTAR, Madison Wis.). CLUSTAL V is described in Higgins, D. G.
and P. M. Sharp (1989; CABIOS 5:151-153) and in Higgins, D. G. et
al. (1992; CABIOS 8:189-191). For pairwise alignments of
polynucleotide sequences, the default parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals
saved"=4. The "weighted" residue weight table is selected as the
default.
[0073] Alternatively, a suite of commonly used and freely available
sequence comparison algorithms which can be used is provided by the
National Center for Biotechnology Information (NCBI) Basic Local
Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J.
Mol. Biol. 215:403-410), which is available from several sources,
including the NCBI, Bethesda, Md., and on the NCBI World Wide Web
site available on the Internet. The BLAST software suite includes
various sequence analysis programs including "blastn," that is used
to align a known polynucleotide sequence with other polynucleotide
sequences from a variety of databases. Also available is a tool
called "BLAST 2 Sequences" that is used for direct pairwise
comparison of two nucleotide sequences. "BLAST 2 Sequences" can be
accessed and used interactively on the Internet via the NCBI World
Wide Web site as well. The "BLAST 2 Sequences" tool can be used for
both blastn and blastp (discussed below). BLAST programs are
commonly used with gap and other parameters set to default
settings. For example, to compare two nucleotide sequences, one may
use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (Apr.
21, 2000) set at default parameters. Such default parameters may
be, for example: Matrix:BLOSUM62; Reward for match: 1; Penalty for
mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x
drop-off: 50; Expect: 10; Word Size: 11; Filter: on.
[0074] Percent identity may be measured over the length of an
entire defined sequence, for example, as defined by a particular
SEQ ID number, or may be measured over a shorter length, for
example, over the length of a fragment taken from a larger, defined
sequence, for instance, a fragment of at least 20, at least 30, at
least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such lengths are exemplary only, and it is
understood that any fragment length supported by the sequences
shown herein, in the tables, figures, or sequence listing, may be
used to describe a length over which percentage identity may be
measured.
[0075] The phrases "percent identity" and "% identity", as applied
to polypeptide sequences, refer to the percentage of identical
residue matches between at least two polypeptide sequences aligned
using a standardized algorithm. Methods of polypeptide sequence
alignment are well known. Some alignment methods take into account
conservative amino acid substitutions. Such conservative
substitutions, explained in more detail above, generally preserve
the charge and hydrophobicity at the site of substitution, thus
preserving the structure (and therefore function) of the
polypeptide. The phrases "percent similarity" and "% similarity",
as applied to polypeptide sequences, refer to the percentage of
residue matches, including identical residue matches and
conservative substitutions, between at least two polypeptide
sequences aligned using a standardized algorithm. In contrast,
conservative substitutions are not included in the calculation of
percent identity between polypeptide sequences.
[0076] Percent identity between polypeptide sequences may be
determined using the default parameters of the CLUSTAL V algorithm
as incorporated into the MEGALIGN version 3.12e sequence alignment
program (described and referenced above). For pairwise alignments
of polypeptide sequences using CLUSTAL V, the default parameters
are set as follows: Ktuple=1, gap penalty=3, window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default
residue weight table.
[0077] Alternatively the NCBI BLAST software suite may be used. For
example, for a pairwise comparison of two polypeptide sequences,
one may use the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21,
2000) with blastp set at default parameters. Such default
parameters may be, for example: Matrix: BLOSUM62; Open Gap: 11 and
Extension Gap: 1 penalties; Gap x drop-off: 50; Expect: 10; Word
Size: 3; Filter: on.
[0078] Percent identity may be measured over the length of an
entire defined polypeptide sequence, for example, as defined by a
particular SEQ ID number, or may be measured over a shorter length,
for example, over the length of a fragment taken from a larger,
defined polypeptide sequence, for instance, a fragment of at least
15, at least 20, at least 30, at least 40, at least 50, at least 70
or at least 150 contiguous residues. Such lengths are exemplary
only, and it is understood that any fragment length supported by
the sequences shown herein, in the tables, figures or sequence
listing, may be used to describe a length over which percentage
identity may be measured.
[0079] Furthermore, fragments of the proteins and polypeptides and
variants thereof are encompassed in the methods and compositions of
the invention, so long as the fragment is operable in effecting the
relevant biological activity as understood by the ordinarily
skilled artisan. Thus, for example, fragments include fragments of
VP2 proteins and polypeptides; where the fragment is contained in a
virus-like particle when expressed in an insect cell culture along
with a VP6 and VP7 protein or polypeptide, or a variant or fragment
thereof. Thus, for example, fragments include fragments of VP6
proteins and polypeptides; where the fragment is contained in a
virus-like particle when expressed in an insect cell culture along
with a VP7 protein or polypeptide, or a variant or fragment
thereof. Thus, for example, fragments include fragments of VP7
proteins and polypeptides; where the fragment is contained in a
virus-like particle when expressed in an insect cell culture along
with a VP6 protein or polypeptide, or a variant or fragment
thereof. Fragments also encompass those fragments which effect an
immunogenic response as described herein for a VP protein,
polypeptide or a variant there.
[0080] Processes for Making VLPs
[0081] VP2 core protein and the VP6 and VP7 capsid proteins and
polypeptides (VP proteins and polypeptides) described in the
compositions and methods described herein can be generated by
recombinant methods, such as the inventive methods described
herein, or by suitable expression methods known to the ordinarily
skilled artisan where appropriate. Nucleic acid sequences encoding
the VP proteins and polypeptides are isolated as exemplified by
nucleic acid sequences described herein.
[0082] VLPs are produced using recombinant nucleic acid technology
according to embodiments of the present invention. VLP production
includes introducing a recombinant expression vector encompassing a
DNA sequence encoding human rotavirus C VP2, VP6 and/or VP7 into a
host cell.
[0083] Specific nucleic acid sequences encoding human rotavirus C
VP2, VP6 or VP7 introduced into a host cell to produce human
rotavirus C VLPs are
[0084] It is appreciated that due to the degenerate nature of the
genetic code, alternate nucleic acid sequences encode human
rotavirus C VP2, VP6 or VP7 and variants thereof, and that such
alternate nucleic acids may be included in an expression vector and
expressed to produce human rotavirus C VLPs of the present
invention.
[0085] In embodiments of the present invention, a nucleic acid
sequence which is substantially identical to SEQ ID No. 31, SEQ ID
NO: 46, or SEQ ID No. 48 encoding human rotavirus GpC VP6, is
included in an expression vector and expressed to produce human
rotavirus C VLPs of the present invention. In further embodiments
of the present invention, a nucleic acid sequence which is
substantially identical to SEQ ID No. 33 encoding human rotavirus
GpC VP7, is included in an expression vector and expressed to
produce human rotavirus C VLPs of the present invention. In further
embodiments of the present invention, a nucleic acid sequence which
is substantially identical to SEQ ID No. 18, SEQ ID No. 42 or SEQ
ID No. 43 encoding human rotavirus GpC VP2, is included in an
expression vector and expressed to produce human rotavirus C VLPs
of the present invention.
[0086] A nucleic acid sequence which is substantially identical to
SEQ ID No. 31 or SEQ ID NO: 46 is characterized as having a
complementary nucleic acid sequence capable of hybridizing to SEQ
ID No. 31, SEQ ID NO: 46, or SEQ ID No. 48 under high stringency
hybridization conditions. Similarly, a nucleic acid sequence which
is substantially identical to SEQ ID No. 33, SEQ ID No. 18, SEQ ID
No. 42 or SEQ ID No. 43, is characterized as having a complementary
nucleic acid sequence capable of hybridizing to SEQ ID No. 33 or
SEQ ID No. 18, SEQ ID No. 42 or SEQ ID No. 43, respectively, under
high stringency hybridization conditions.
[0087] The term "nucleic acid" refers to RNA or DNA molecules
having more than one nucleotide in any form including
single-stranded, double-stranded, oligonucleotide or
polynucleotide. The term "nucleotide sequence" refers to the
ordering of nucleotides in an oligonucleotide or polynucleotide in
a single-stranded form of nucleic acid.
[0088] The term "complementary" refers to Watson-Crick base pairing
between nucleotides and specifically refers to nucleotides hydrogen
bonded to one another with thymine or uracil residues linked to
adenine residues by two hydrogen bonds and cytosine and guanine
residues linked by three hydrogen bonds. In general, a nucleic acid
includes a nucleotide sequence described as having a "percent
complementarity" to a specified second nucleotide sequence. For
example, a nucleotide sequence may have 80%, 90%, or 100%
complementarity to a specified second nucleotide sequence,
indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a
sequence are complementary to the specified second nucleotide
sequence. For instance, the nucleotide sequence 3'-TCGA-5' is 100%
complementary to the nucleotide sequence 5'-AGCT-3'. Further, the
nucleotide sequence 3'-TCGA- is 100% complementary to a region of
the nucleotide sequence 5'-TTAGCTGG-3'.
[0089] The terms "hybridization" and "hybridizes" refer to pairing
and binding of complementary nucleic acids. Hybridization occurs to
varying extents between two nucleic acids depending on factors such
as the degree of complementarity of the nucleic acids, the melting
temperature, Tm, of the nucleic acids and the stringency of
hybridization conditions, as is well known in the art. The term
"stringency of hybridization conditions" refers to conditions of
temperature, ionic strength, and composition of a hybridization
medium with respect to particular common additives such as
formamide and Denhardt's solution. Determination of particular
hybridization conditions relating to a specified nucleic acid is
routine and is well known in the art, for instance, as described in
J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; and F.
M. Ausubel, Ed., Short Protocols in Molecular Biology, Current
Protocols; 5th Ed., 2002. High stringency hybridization conditions
are those which only allow hybridization of substantially
complementary nucleic acids. Typically, nucleic acids having about
85-100% complementarity are considered highly complementary and
hybridize under high stringency conditions. Intermediate stringency
conditions are exemplified by conditions under which nucleic acids
having intermediate complementarity, about 50-84% complementarity,
as well as those having a high degree of complementarity,
hybridize. In contrast, low stringency hybridization conditions are
those in which nucleic acids having a low degree of complementarity
hybridize.
[0090] The terms "specific hybridization" and "specifically
hybridizes" refer to hybridization of a particular nucleic acid to
a target nucleic acid without substantial hybridization to nucleic
acids other than the target nucleic acid in a sample.
[0091] Stringency of hybridization and washing conditions depends
on several factors, including the Tm of the probe and target and
ionic strength of the hybridization and wash conditions, as is
well-known to the skilled artisan. Hybridization and conditions to
achieve a desired hybridization stringency are described, for
example, in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F.
et al., (Eds.), Short Protocols in Molecular Biology, Wiley,
2002.
[0092] An example of high stringency hybridization conditions is
hybridization of nucleic acids over about 100 nucleotides in length
in a solution containing 6.times.SSC, 5.times.Denhardt's solution,
30% formamide, and 100 micrograms/ml denatured salmon sperm at
37.degree. C. overnight followed by washing in a solution of
0.1.times.SSC and 0.1% SDS at 60.degree. C. for 15 minutes. SSC is
0.15M NaCl/0.015M Na citrate. Denhardt's solution is 0.02% bovine
serum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone. Under highly
stringent conditions, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No.
18 will hybridize to the complement of substantially identical
targets and not to unrelated sequences.
[0093] The term "expression vector" refers to a recombinant vehicle
for introducing a DNA sequence encoding one or more human rotavirus
C proteins into a host cell where the DNA sequence is expressed to
produce the one or more human rotavirus C proteins.
[0094] In particular embodiments, a DNA sequence encoding one or
more human rotavirus C proteins includes an open reading frame
encoding the one or more human rotavirus C proteins.
[0095] In further embodiments, 5' non-coding sequence and/or 3'
non-coding sequence is present in addition to the open reading
frame encoding the one or more human rotavirus C proteins.
Preferably, where 5' non-coding sequence and/or 3' non-coding
sequence is present, it is contiguous with the DNA sequence
encoding one or more human rotavirus C proteins, 5' non-coding
sequence, if any, is positioned upstream (5') of the start codon
and 3' non-coding sequence, if any, is positioned downstream (3')
of the stop codon.
[0096] In particular embodiments, an expression vector including
SEQ ID No. 42 or a substantially identical nucleic acid sequence is
expressed to produce human rotavirus C VP2 and self-assembled VLPs
in cells containing the expression vector. SEQ ID No. 42 includes
an open reading frame encoding human rotavirus C VP2 from strain
Asp88. Optionally, 5' non-coding sequence and/or 3' non-coding
sequence of human rotavirus C VP2 from strain Asp88 is included in
the expression vector. For example, 1-36 nucleotides of 5'
non-coding sequence can be included contiguous with the 5' end of
the nucleotide sequence encoding human rotavirus C VP2 from strain
Asp88. In particular embodiments, an expression vector including
SEQ ID No. 18 or a substantially identical nucleic acid sequence is
expressed to produce human rotavirus C VP2 and self-assembled VLPs
in cells containing the expression vector. In further embodiments,
an expression vector including SEQ ID No. 43 or a substantially
identical nucleic acid sequence is expressed to produce human
rotavirus C VP2 and self-assembled VLPs in cells containing the
expression vector.
[0097] In additional embodiments, an expression vector including
SEQ ID No. 31, SEQ ID NO: 46, SEQ ID No. 48 or a substantially
identical nucleic acid sequence is expressed to produce human
rotavirus C VP6 and self-assembled VLPs in cells containing the
expression vector.
[0098] In additional embodiments, an expression vector including
SEQ ID No. 33, SEQ ID No. 45, or a substantially identical nucleic
acid sequence is expressed to produce human rotavirus C VP7 and
self-assembled VLPs in cells containing the expression vector.
[0099] In further embodiments, an expression vector including SEQ
ID No. 31, SEQ ID NO: 46, SEQ ID No. 48 or a substantially
identical nucleic acid sequence and SEQ ID No. 33, SEQ ID No. 45,
or a substantially identical nucleic acid sequence is expressed to
produce human rotavirus C VP6, human rotavirus C VP7 and
self-assembled VLPs, in cells containing the expression vector.
[0100] In further embodiments, an expression vector including SEQ
ID No. 31, SEQ ID NO: 46, SEQ ID No. 48 or a substantially
identical nucleic acid sequence, SEQ ID No. 33, SEQ ID No. 45, or a
substantially identical nucleic acid sequence and SEQ ID No. 18,
SEQ ID No. 42, SEQ ID No. 43, or a substantially identical nucleic
acid sequence is expressed to produce human rotavirus C VP6, human
rotavirus C VP7, human rotavirus C VP2 and self-assembled VLPs in
cells containing the expression vector.
[0101] In still further embodiments, a first expression vector
including SEQ ID No. 31, SEQ ID NO: 46, SEQ ID No. 48 or a
substantially identical nucleic acid sequence and a second
expression vector including SEQ ID No. 33, SEQ ID No. 45, or a
substantially identical nucleic acid sequence are both expressed to
produce human rotavirus C VP6, human rotavirus C VP7 and
self-assembled VLPs in cells containing the expression vectors.
[0102] In still further embodiments, a first expression vector
including SEQ ID No.31, SEQ ID NO: 46, SEQ ID No. 48 or a
substantially identical nucleic acid sequence, a second expression
vector including SEQ ID No. 33, SEQ ID No. 45, or a substantially
identical nucleic acid sequence and a third expression vector
including SEQ ID No. 18, SEQ ID No. 42, SEQ ID No. 43, or a
substantially identical nucleic acid sequence are expressed to
produce human rotavirus C VP6, human rotavirus C VP7, human
rotavirus C VP2, and self-assembled VLPs in cells containing the
expression vectors.
[0103] In addition to one or more DNA sequences encoding proteins
of human rotavirus C, one or more DNA sequences encoding additional
proteins can be included in an expression vector. For example, such
additional proteins include non-human rotavirus C proteins such as
reporters, including, but not limited to, beta-galactosidase, green
fluorescent protein and antibiotic resistance reporters; and
antigens.
[0104] Expression vectors are known in the art and include plasmids
and viruses, for example. An expression vector contains a DNA
molecule that includes segment encoding a polypeptide of interest
operably linked to one or more regulatory elements that provide for
transcription of the segment encoding the polypeptide of interest.
Such regulatory elements include, but are not limited to,
promoters, terminators, enhancers, origins of replication and
polyadenylation signals.
[0105] In particular embodiments, the recombinant expression vector
encodes human rotavirus C VP2 of SEQ ID No. 1, a protein having at
least 95% identity to SEQ ID No. 1, a protein encoded by SEQ ID No.
42, or a protein encoded by a nucleic acid sequence substantially
identical to SEQ ID No. 42.
[0106] In particular embodiments, the recombinant expression vector
encodes human rotavirus C VP6 of SEQ ID No. 32, a protein having at
least 95% identity to SEQ ID No. 32, a protein encoded by SEQ ID
No. 31, or a protein encoded by a nucleic acid sequence
substantially identical to SEQ ID No. 31.
[0107] In particular embodiments, the recombinant expression vector
encodes human rotavirus C VP7 of SEQ ID No. 34, a protein having at
least 95% identity to SEQ ID No. 34, a protein encoded by SEQ ID
No. 33 or SEQ ID No. 45, or a protein encoded by a nucleic acid
sequence substantially identical to SEQ ID No. 33 or SEQ ID No.
45.
[0108] In further embodiments, the recombinant expression vector
encodes human rotavirus C VP6 of SEQ ID No. 32, a protein having at
least 95% identity to SEQ ID No. 32, a protein encoded by SEQ ID
No. 31, or a protein encoded by a nucleic acid sequence
substantially identical to SEQ ID No. 31; and human rotavirus C VP7
of SEQ ID No. 34, a protein having at least 95% identity to SEQ ID
No. 34, a protein encoded by SEQ ID No. 33 or SEQ ID No. 45, or a
protein encoded by a nucleic acid sequence substantially identical
to SEQ ID No. 33 or SEQ ID No. 45.
[0109] A preferred expression vector of the present invention is a
baculovirus.
[0110] Expression of human rotavirus C VP2, VP6 and/or VP7 encoded
by a recombinant expression vector is accomplished by introduction
of the expression vector into a eukaryotic or prokaryotic host cell
expression system such as an insect cell, mammalian cell, yeast
cell, bacterial cell or any other single or multicellular organism
recognized in the art. In preferred embodiments, a eukaryotic host
cell is used. Host cells are optionally primary cells or
immortalized derivative cells. Immortalized cells are those which
can be maintained in-vitro for at least 5 replication passages.
[0111] Host cells containing the recombinant expression vector are
maintained under conditions where human rotavirus C proteins are
produced. The human rotavirus C proteins self-associate to produce
VLPs of the present invention in the host cell.
[0112] The invention provides a host cell containing a nucleic acid
sequence according to the invention. Host cells may be cultured and
maintained using known cell culture techniques such as described in
Celis, Julio, ed., 1994, Cell Biology Laboratory Handbook, Academic
Press, N.Y. Various culturing conditions for these cells, including
media formulations with regard to specific nutrients, oxygen,
tension, carbon dioxide and reduced serum levels, can be selected
and optimized by one of skill in the art.
[0113] A preferred cell line of the present invention is a
eukaryotic cell line, preferably an insect cell line, such as Sf9
or Hi5, transiently or stably expressing one or more full-length or
partial human rotavirus C proteins. Such cells can be made by
transfection (proteins or nucleic acid vectors), infection (viral
vectors) or transduction (viral vectors). The cell lines for use in
the present invention are cloned using known cell culture
techniques familiar to one skilled in the art. The cells are
cultured and expanded from a single cell using commercially
available culture media under known conditions suitable for
propagating cells.
[0114] In a preferred embodiment human rotavirus C VLPs are
produced by infection of a host cell with at least one recombinant
baculovirus encoding human rotavirus C protein(s).
[0115] It is appreciated that a single baculovirus may encode
either a single human rotavirus C protein or multiple human
rotavirus C proteins. The resulting infected cells are then
cultured under conditions whereby the encoded human rotavirus C
proteins from the respective recombinant baculoviruses are produced
and self assemble to form the capsids. The resulting human
rotavirus C VLPs are then optionally and preferably isolated.
[0116] In further preferred embodiments, the recombinant
baculovirus encodes at least human rotavirus C VP6 of SEQ ID No.
32, a protein having at least 95% identity to SEQ ID No. 32, a
protein encoded by SEQ ID No. 31, or a protein encoded by a nucleic
acid sequence substantially identical to SEQ ID No. 31, SEQ ID NO:
46 or SEQ ID No. 48; and human rotavirus C VP7 of SEQ ID No. 34, a
protein having at least 95% identity to SEQ ID No. 34, a protein
encoded by SEQ ID No. 33 or SEQ ID No. 45, or a protein encoded by
a nucleic acid sequence substantially identical to SEQ ID No. 33 or
SEQ ID No. 45.
[0117] In a further option, the recombinant baculovirus encodes
human rotavirus C VP2 of SEQ ID No. 1, a protein having at least
95% identity to SEQ ID No. 1, a protein encoded by SEQ ID No. 42,
or a protein encoded by a nucleic acid sequence substantially
identical to SEQ ID No. 42.
[0118] Any suitable baculovirus known in the art is operable in the
instant inventive process. Preferably, the baculovirus is
Autographa california nuclear polyhedrosis virus.
[0119] Processes for infecting cells with baculovirus are known in
the art. Following infection of a host cell the inventive process
proceeds by culturing the host cells under conditions such that
protein(s) produced self assemble to form VLPs.
[0120] A VLP of the present invention optionally includes a
non-human rotavirus C protein or peptide in contact with or bonded
to at least one of the human rotavirus C proteins VP2, VP6 or VP7.
Bonding of the non-human rotavirus C protein or peptide is
achieved, for example, by expression of a fusion construct
including a nucleic acid sequence encoding VP2, VP6 or VP7 and the
non-human rotavirus C protein or peptide. Thus, the non-human
rotavirus C protein or peptide is optionally a fusion protein or
peptide wherein the non-human rotavirus C protein is synthesized as
a single polypeptide chain with a human rotavirus C structural
protein.
[0121] The non-human rotavirus C protein is optionally fused with
glutathione-S-transferase (GST) for rapid isolation. A human
rotavirus C protein is also optionally fused to GST.
[0122] Chemical bonding methods are optionally used to bond a VLP
and a non-human rotavirus C protein or peptide, illustratively
including reaction using a cross-linking agent such as carbodiimide
or glutaraldehyde.
[0123] In particular embodiments, the non-human rotavirus C protein
or peptide included in the VLP includes one or more antigenic
epitopes such that the VLP serves to present the one or more
antigenic epitopes to the immune system of a subject to induce
antibody generation.
[0124] In a further option, the non-human rotavirus C protein or
peptide is a targeting moiety such as a receptor ligand or
receptor. A targeting moiety is included in the VLP to direct the
VLP to a target, such as to a particular cell type.
[0125] Human rotavirus C VLPs produced in a host cell are
optionally isolated. The term "isolated" in reference to a human
rotavirus C VLP describes a human rotavirus C VLP which is
separated from a cell in which the human rotavirus C VLP is
produced and which is substantially free of host cell components
not intended to be associated with the human rotavirus C VLP.
Generally, human rotavirus C VLPs are separated from whole cell
extracts of host cells. Numerous processes of isolating VLPs are
known in the art and are applicable to isolation of human rotavirus
C VLPs illustratively including sucrose continuous and
discontinuous gradients, cesium chloride single and multi-density
gradient centrifugation, size-exclusion chromatography, antigen
capture chromatography, affinity chromatography, or other suitable
process known in the art. An exemplary method for isolating human
rotavirus C VLPs of the present invention is described in Gillock,
ET. et al, 1997. J. Virol., 71:2857-2865.
[0126] Human rotavirus C VLPs having different compositions, that
is, different "types" of human rotavirus C VLPs are optionally
present in a composition of the present invention. For example,
human rotavirus C VLPs including human rotavirus C VP2 are
optionally included in a composition with antigen presenting human
rotavirus C VLPs including a non-human rotavirus C protein or
peptide and/or human rotavirus C VLPs containing a cargo
moiety.
[0127] In one aspect, the invention provides a method of making a
human group C rotavirus-like particle comprising: constructing a
first baculovirus vector comprising a nucleic acid molecule
comprising a sequence encoding a human group C RV VP6 capsid
protein operably linked to a baculovirus promoter that drives
expression of said protein in an insect cell; constructing a second
baculovirus vector comprising a nucleic acid molecule comprising a
sequence encoding a human group C RV VP7 capsid protein operably
linked to a baculovirus promoter that drives expression of said
protein in an insect cell; and infecting an insect cell culture
with said first and second baculovirus vector under conditions that
promote expression of the VP6- and VP7 capsid proteins.
[0128] In one embodiment of the present invention, the method
further comprises constructing a third baculovirus vector
comprising a nucleic acid molecule comprising a sequence encoding a
human group C RV VP2 core protein operably linked to a baculovirus
promoter that drives expression of said protein in an insect cell;
and infecting an insect cell culture with said first, second, and
third baculovirus vector under conditions that promote expression
of the VP6 capsid protein and VP7 capsid protein and said VP2 core
protein.
[0129] In another aspect, the invention provides a rotavirus-like
particle made by the herein described inventive method.
[0130] A virus-like particle containing a fragment of the VP
proteins described herein can be formed by any of the above
described methods for making a human group C rotavirus-like
particle, and also includes: constructing a third baculovirus
vector comprising a nucleic acid molecule comprising a sequence
encoding a core protein operably linked to a baculovirus promoter
that drives expression of said protein in an insect cell; infecting
an insect cell culture with said first, second, and third
baculovirus vector under conditions that promote expression of the
VP6- and VP7 capsid proteins, and the VP2 core protein.
[0131] In one embodiment of the present invention, the core protein
is a group C VP2 protein of ASP 88 strain.
[0132] Rotavirus particles are harvested, typically from cell
culture supernatant for inclusion in an immunogenic composition
including a vaccine composition. The rotavirus particles may be
isolated from the cell culture supernatant, for example by
filtration and/or centrifugation. The isolated rotavirus particles
are optionally lyophilized, such as for later resuspension in a
pharmaceutically acceptable carrier.
Pharmaceutical Compositions and Processes
[0133] Pharmaceutical Compositions and Processes
[0134] Vaccines and methods for their use to induce active immunity
and protection against human rotavirus C-induced illness in a
subject are provided according to the present invention.
[0135] In particular embodiments, human rotavirus C VLPs are
administered as antigens for prevention or treatment of human
rotavirus C infection such as by serving as an active vaccine
component, or by eliciting an immune response in a host organism.
Vaccine delivery may occur prior to or following human rotavirus C
infection of a host organism or patient. A vaccine optionally
contains one or more adjuvants and preservatives or other
pharmaceutically acceptable carrier.
[0136] In particular embodiments, vaccine compositions include one
or more types of human rotavirus C VLP admixed with a
pharmaceutically acceptable carrier.
[0137] The term "pharmaceutically acceptable carrier" refers to a
carrier which is substantially non-toxic to a subject and
substantially inert to the human rotavirus C VLPs included in a
vaccine composition. A pharmaceutically acceptable carrier is a
solid, liquid or gel in form and is typically sterile and pyrogen
free.
[0138] An immunogenic composition of the present invention may be
in any form suitable for administration to a subject.
[0139] An immunogenic composition is administered by any suitable
route of administration including oral and parenteral such as
intravenous, intradermal, intramuscular, intraperitoneal, mucosal,
nasal, or subcutaneous routes of administration.
[0140] For example, an immunogenic composition for parenteral
administration may be formulated as an injectable liquid including
a rotavirus and a pharmaceutically acceptable carrier. Examples of
suitable aqueous and nonaqueous carriers include water, ethanol,
polyols such as propylene glycol, polyethylene glycol, glycerol,
and the like, suitable mixtures thereof; vegetable oils such as
olive oil; and injectable organic esters such as ethyloleate.
Proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of a desirable
particle size in the case of dispersions, and/or by the use of a
surfactant, such as sodium lauryl sulfate. A stabilizer is
optionally included such as, for example, EDTA, EGTA, and an
antioxidant.
[0141] A solid dosage form for administration or for suspension in
a liquid prior to administration illustratively includes capsules,
tablets, powders, and granules. In such solid dosage forms,
rotavirus particles are admixed with at least one carrier
illustratively including a buffer such as, for example, sodium
citrate or an alkali metal phosphate illustratively including
sodium phosphates, potassium phosphates and calcium phosphates; a
filler such as, for example, starch, lactose, sucrose, glucose,
mannitol, and silicic acid; a binder such as, for example,
carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,
sucrose, and acacia; a humectant such as, for example, glycerol; a
disintegrating agent such as, for example, agar-agar, calcium
carbonate, plant starches such as potato or tapioca starch, alginic
acid, certain complex silicates, and sodium carbonate; a solution
retarder such as, for example, paraffin; an absorption accelerator
such as, for example, a quaternary ammonium compound; a wetting
agent such as, for example, cetyl alcohol, glycerol monostearate,
and a glycol; an adsorbent such as, for example, kaolin and
bentonite; a lubricant such as, for example, talc, calcium
stearate, magnesium stearate, a solid polyethylene glycol or sodium
lauryl sulfate; a preservative such as an antibacterial agent and
an antifungal agent, including for example, thimerosal, sorbic
acid, gentamycin and phenol; and a stabilizer such as, for example,
EDTA, EGTA, and an antioxidant.
[0142] Solid dosage forms optionally include a coating such as an
enteric coating. The enteric coating is typically a polymeric
material. Preferred enteric coating materials have the
characteristics of being bioerodible, gradually hydrolyzable and/or
gradually water-soluble polymers. The amount of coating material
applied to a solid dosage generally dictates the time interval
between ingestion and drug release. A coating is applied having a
thickness such that the entire coating does not dissolve in the
gastrointestinal fluids at pH below 3 associated with stomach
acids, yet dissolves above pH 3 in the small intestine environment.
It is expected that any anionic polymer exhibiting a pH-dependent
solubility profile is readily used as an enteric coating in the
practice of the present invention to achieve delivery of the active
agent to the lower gastrointestinal tract. The selection of the
specific enteric coating material depends on properties such as
resistance to disintegration in the stomach; impermeability to
gastric fluids and active agent diffusion while in the stomach;
ability to dissipate at the target intestine site; physical and
chemical stability during storage; non-toxicity; and ease of
application.
[0143] Suitable enteric coating materials illustratively include
cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl
cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl
cellulose, cellulose acetate, cellulose acetate phthalate,
cellulose acetate trimellitate, hydroxypropylmethyl cellulose
phthalate, hydroxypropylmethyl cellulose succinate and
carboxymethylcellulose sodium; acrylic acid polymers and
copolymers, preferably formed from acrylic acid, methacrylic acid,
methyl acrylate, ammonium methylacrylate, ethyl acrylate, methyl
methacrylate and/or ethyl; vinyl polymers and copolymers such as
polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate
phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl
acetate copolymers; shellac; and combinations thereof. A particular
enteric coating material includes acrylic acid polymers and
copolymers described for example U.S. Pat. No. 6,136,345.
[0144] The enteric coating optionally contains a plasticizer to
prevent the formation of pores and cracks that allow the
penetration of the gastric fluids into the solid dosage form.
Suitable plasticizers illustratively include triethyl citrate
(Citroflex 2), triacetin (glyceryl triacetate), acetyl triethyl
citrate (Citroflec A2), Carbowax 400 (polyethylene glycol 400),
diethyl phthalate, tributyl citrate, acetylated monoglycerides,
glycerol, fatty acid esters, propylene glycol, and dibutyl
phthalate. In particular, a coating composed of an anionic
carboxylic acrylic polymer typically contains approximately 10% to
25% by weight of a plasticizer, particularly dibutyl phthalate,
polyethylene glycol, triethyl citrate and triacetin. The coating
can also contain other coating excipients such as detackifiers,
antifoaming agents, lubricants (e.g., magnesium stearate), and
stabilizers (e.g. hydroxypropylcellulose, acids or bases) to
solubilize or disperse the coating material, and to improve coating
performance and the coated product.
[0145] Liquid dosage forms for oral administration include
rotavirus and a pharmaceutically acceptable carrier formulated as
an emulsion, solution, suspension, syrup, or elixir. A liquid
dosage form of a vaccine composition of the present invention may
include a wetting agent, an emulsifying agent, a suspending agent,
a sweetener, a flavoring, or a perfuming agent.
[0146] Detailed information concerning customary ingredients,
equipment and processes for preparing dosage forms is found in
Pharmaceutical Dosage Forms: Tablets, eds. H. A. Lieberman et al.,
New York: Marcel Dekker, Inc., 1989; and in L. V. Allen, Jr. et
al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,
8th Ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins,
2004; A. R. Gennaro, Remington: The Science and Practice of
Pharmacy, Lippincott Williams & Wilkins, 20th ed., 2003; and J.
G. Hardman et al., Goodman & Gilman's The Pharmacological Basis
of Therapeutics, McGraw-Hill Professional, 10th ed., 2001.
[0147] An adjuvant is optionally included in a virus composition
according to embodiments of the present invention. Adjuvants are
known in the art and illustratively include Freund's adjuvant,
aluminum hydroxide, aluminum phosphate, aluminum oxide, saponin,
dextrans such as DEAE-dextran, vegetable oils such as peanut oil,
olive oil, and/or vitamin E acetate, mineral oil, bacterial
lipopolysaccharides, peptidoglycans, and proteoglycans.
[0148] The term "subject" is used herein to refer to a human.
Non-human animals, illustratively including cows, horses, sheep,
goats, pigs, dogs, cats, birds, poultry, and rodents, are also
referred to by the term subject in particular embodiments of the
present invention.
[0149] A vaccine composition of the present invention may be in any
form suitable for administration to a subject.
[0150] A vaccine composition is administered by any suitable route
of administration including oral and parenteral such as
intravenous, intradermal, intramuscular, intraperitoneal, mucosal,
nasal, or subcutaneous routes of administration.
[0151] The phrase "therapeutically effective amount" refers to an
amount effective to induce an immunological response and prevent or
ameliorate signs or symptoms of human rotavirus C-mediated disease.
Induction of an immunological response in a subject can be
determined by any of various techniques known in the art,
illustratively including detection of anti-human rotavirus C
antibodies, measurement of anti-human rotavirus C antibody titer
and/or lymphocyte proliferation assay. Signs and symptoms of human
rotavirus C-mediated disease may be monitored to detect induction
of an immunological response to administration of a vaccine
composition of the present invention in a subject.
[0152] Administration of a vaccine composition according to a
method of the present invention includes administration of one or
more doses of a vaccine composition to a subject at one time in
particular embodiments. Alternatively, two or more doses of a
vaccine composition are administered at time intervals of
weeks--years. A suitable schedule for administration of vaccine
composition doses depends on several factors including age and
health status of the subject, type of vaccine composition used and
route of administration, for example. One of skill in the art is
able to readily determine a dose and schedule of administration to
be administered to a particular subject.
[0153] Immunogenicity of human rotavirus C VLPs is tested by any of
various assays known in the art. In a particular example, purified
human rotavirus C VLPs are administered intramuscularly to mice
with or without an adjuvant. Immunogenicity is assayed by measuring
immunoglobulin titers including IgM, IgA and/or IgG in blood
samples obtained at various times after administration.
[0154] Neutralizing antibody titers are measured by neutralization
assays known in the art, such as those generally described in Kuby,
J., Immunology, 3rd ed. W.H. Freeman and Co., New York, N.Y., 1997.
Since human rotavirus C does not grow in culture, sera from mice
injected with human rotavirus C VLPs are serially diluted two-fold
in duplicate wells and incubated with trypsin-activated porcine
rotavirus C. Activated porcine rotavirus C or serum-free MEM medium
is incubated in the absence of mouse serum and serve as positive
and negative controls, respectively. MA104 cells in MEM medium
supplemented with trypsin are added to each well. After incubation
at 37.degree. C. for 18 hours, cells are fixed with formalin.
Porcine rotavirus C antigens in the fixed MA104 cells are detected
by incubating cells with HRP-labeled rabbit IgG against human
rotavirus VLPs, and then tetramethyl benzidine. Neutralizing
antibody titer in a serum is defined as the reciprocal of the
highest dilution giving a 70% reduction in absorbance value
compared to that in the virus control.
[0155] Optionally, antibodies raised to immunogenic human rotavirus
C VLPs are administered to a subject for prevention or therapeutic
treatment relating to human rotavirus C-mediated disease.
[0156] Additional therapeutics that are optionally administered
with the vaccine composition or antibodies raised to human
rotavirus C VLPs include antivirals such as amantadine,
rimantadine, gancyclovir, acyclovir, ribavirin, penciclovir,
oseltamivir, foscarnet zidovudine (AZT), didanosine (ddI),
lamivudine (3TC), zalcitabine (ddC), stavudine (d4T), nevirapine,
delavirdine, indinavir, ritonavir, vidarabine, nelfinavir,
saquinavir, relenza, tamiflu, pleconaril, interferons; steroids and
corticosteroids such as prednisone, cortisone, fluticasone and
glucocorticoid; antibiotics; analgesics; antidiarrheals, fluid
replacement; or other treatments for rotavirus infection.
[0157] The invention also provides a pharmaceutical kit that
includes one or more receptacles containing one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0158] In a preferred embodiment, the kit contains an antibody
specific for human rotavirus C VP2, human rotavirus C VP6, human
rotavirus C VP7, the polypeptide of SEQ ID NO:1, an epitope or a
variant thereof, the polypeptide of SEQ ID NO:32, an epitope or a
variant thereof, the polypeptide of SEQ ID NO:34, an epitope or a
variant thereof, or any human rotavirus C epitope, a polypeptide or
protein of the present invention, or a nucleic acid molecule of the
invention, alone or in combination with adjuvants, antivirals,
antibiotics, analgesic, bronchodilators, or other pharmaceutically
acceptable excipients. The present invention further encompasses
kits comprising a container containing a pharmaceutical composition
of the present invention and instructions for use.
[0159] Also provided is a diagnostic kit for detecting human
rotavirus C infection that contains human rotavirus C VLPs as
reagents for the detection of human rotavirus C antibodies. It is
further appreciated that a diagnostic kit optionally includes
ancillary reagents such as buffers, solvents, a detectable label
and other reagents necessary and recognized in the art for
detection of an antibody in a biological sample.
[0160] Detection of Anti-Human Rotavirus C Antibodies
[0161] Human rotavirus C VLPs are used to detect anti-human
rotavirus C antibodies in a biological sample according to
embodiments of a process of the present invention.
[0162] The term "biological sample" refers to a sample obtained
from a biological organism, a tissue, cell, cell culture medium, or
any medium suitable for mimicking biological conditions, or from
the environment. Non-limiting examples include, saliva, gingival
secretions, cerebrospinal fluid, gastrointestinal fluid, mucous,
urogenital secretions, synovial fluid, blood, serum, plasma, urine,
cystic fluid, lymph fluid, ascites, pleural effusion, interstitial
fluid, intracellular fluid, ocular fluids, seminal fluid, mammary
secretions, and vitreal fluid, feces, and nasal secretions.
Environmental samples such as sewage or water samples can be used.
In a preferred embodiment, the sample is serum, plasma or whole
blood.
[0163] A process of detecting anti-human rotavirus C antibodies in
a biological sample according to the present invention includes
contacting a biological sample with recombinant human rotavirus C
VLPs and detecting formation of a complex between anti-human
rotavirus C antibodies present in the biological sample and the
human rotavirus C VLPs. Formation of the complex between anti-human
rotavirus C antibodies present in the biological sample and the
human rotavirus C VLPs is indicative of exposure of the subject to
human rotavirus C sufficient to activate the immune system of the
subject to produce anti-human rotavirus C antibodies. Formation of
the complex specifically indicates presence of anti-human rotavirus
C antibodies since other enteric virus antibodies, particularly
anti-human rotavirus A antibodies, do not form a complex with the
human rotavirus C VLPs.
[0164] In a preferred embodiment, human rotavirus C VLPs are used
to detect anti-human rotavirus C antibodies in a biological sample
to diagnose current and recent human rotavirus C infection in a
subject.
[0165] In a further preferred embodiment human rotavirus C VLPs are
used in a process of assessing the immune status of an individual
with respect to past or present exposure to a human rotavirus C
antigen in human rotavirus C infection susceptible organisms,
particularly in a human subject.
[0166] Detecting formation of a complex between anti-human
rotavirus C antibodies present in a biological sample and human
rotavirus C VLPs is achieved by any of various methods known in the
art, illustratively including detection of a label attached to
human rotavirus C VLPs or attached to the anti-human rotavirus C
antibodies. The term "label" or "labeled" refers to any composition
which can be used to detect, qualitatively or quantitatively, a
substance attached to the label. Suitable labels include a
fluorescent moiety, a radioisotope, a chromophore, a bioluminescent
moiety, an enzyme, a magnetic particle, an electron dense particle,
and the like. The term "label" or "labeled" is intended to
encompass direct labeling of human rotavirus C VLPs or an antibody
by coupling (i.e., physically linking) a detectable substance to
the human rotavirus C VLPs or antibody, as well as indirect
labeling of the human rotavirus C VLPs or antibody by interaction
with another reagent that is directly labeled. An example of
indirect labeling of a primary antibody includes detection of a
primary antibody using a fluorescently labeled secondary
antibody.
[0167] Labels used in detection of complex formation depend on the
detection process used. Such detection processes are incorporated
in particular assay formats illustratively including ELISA, western
blot, immunoprecipitation, immunocytochemistry, immuno-fluorescence
assay, liquid chromatography, flow cytometry, other detection
processes known in the art, or combinations thereof.
[0168] In one embodiment, an ELISA is used to detect the presence
of human rotavirus C antibodies in a biological sample.
[0169] In one configuration of an ELISA for human rotavirus C
antibodies, human rotavirus C VLPs are coated on a support such as
a microtiter plate, beads, slide, silicon chip or other solid
support such as a nitrocellulose or PVDF membrane. A biological
sample is incubated with the human rotavirus C VLPs on the support
and the presence of complex between antibodies to human rotavirus C
and human rotavirus C VLPs is detected by standard ELISA protocols.
For example, a complex between human rotavirus C VLPs and human
rotavirus C antibodies is detected by reaction of a labeled
secondary antibody with the anti-human rotavirus C antibodies and
detection of the label.
[0170] Another example of an ELISA for human rotavirus C antibodies
is a sandwich ELISA. One embodiment of a sandwich ELISA includes
depositing a binding antibody onto a solid support. The binding
antibody is optionally a non-competing antibody that recognizes
human rotavirus C VLPs. The binding antibody is incubated with
human rotavirus C VLPs. The complex is washed to remove any unbound
material and a detectable label, such as a fluorescently labeled
antibody directed to human rotavirus C VLPs, is applied. The
detectable label is detected, if present, indicating the presence
of anti-human rotavirus C antibody in the biological sample.
[0171] Further details of ELISA assays in general are found in
Crowther, J. R., The ELISA Guidebook (Methods in Molecular
Biology), Humana Press, 2000; and Wild, D., The Immunoassay
Handbook, 3rd Edition, Elsevier Science, 2005.
[0172] A human rotavirus C antibody detection kit is provided
including one or more types of human rotavirus C VLPs and ancillary
reagents for use in detecting anti-human rotavirus C antibodies in
a biological sample. Ancillary reagents are any signal producing
system materials for detection of a complex between an anti-human
rotavirus C antibody and a human rotavirus C VLP in any suitable
detection process such as ELISA, western blot, immunoprecipitation,
immunocytochemistry, immuno-fluorescence, mass spectrometry, or
other assay known in the art.
[0173] Optionally, an anti-human human rotavirus C antibody assay
kit according to embodiments of the present invention includes
human rotavirus C VLPs attached to a solid substrate. Suitable
solid substrates include, but are not limited to, microtiter
plates, chips, tubes, membranes, such as nylon or nitrocellulose
membranes, and particles, such as beads. Attachment of
protein-containing materials to solid substrates is well-known in
the art and includes, but is not limited to, adsorption.
[0174] In a preferred embodiment, a human rotavirus C antibody
detection kit of the present invention illustratively includes one
or more types of human rotavirus C VLPs; and one or more ancillary
reagents such as a high binding microtiter plate or other support,
blocking agent, washing buffer such as phosphate buffered saline, a
labeled anti-immunoglobulin antibody, and matching detection
agents, swab or other sample collection devices, control reagents
such as labeled non-competing or unlabelled reagents, control
nucleotide sequence and relevant primers and probes, and other
materials and reagents for detection. The kit optionally includes
instructions printed or in electronically accessible form and/or
customer support contact information.
[0175] Anti-immunoglobulin antibodies in a signal producing system
or otherwise are optionally labeled with a fluorophore, biotin,
peroxidase, or other enzymatic or non-enzymatic detection label. It
is appreciated that a signal producing system may employ an
unlabeled primary antibody and a labeled secondary antibody derived
from the same or a different organism. It is further appreciated
that non-antibody signal producing systems are similarly
operable.
[0176] It is further appreciated that a kit optionally includes
ancillary reagents such as buffers, solvents, a detectable label
and other reagents necessary and recognized in the art for
detection of an antibody in a biological sample.
[0177] VLPs Containing a Cargo
[0178] Optionally, the VLP contains a cargo in the internal space
defined by the VLP. In particular embodiments, a cargo moiety is a
substance to be delivered to a subject or cell. Exemplary cargo
moieties include an antigen, a nucleic acid which is not an intact
human rotavirus C genome and a therapeutic agent.
[0179] Particularly provided is a process of delivery of genetic
information whereby genetic material is encapsulated in a human
rotavirus C capsid which is then introduced into a host cell. The
genetic material is optionally DNA or RNA, or modifications
thereof. The genetic information is optionally derived from a human
rotavirus C or other viral or nonviral organism, or is
synthetic.
[0180] A cargo is incorporated in the internal space defined by a
human rotavirus C VLP by any of various methods including
introducing the cargo into a host cell such that human rotavirus C
VLPs are produced in the presence of the cargo and thereby include
the cargo in the internal space. Alternatively or additionally, a
cargo is incorporated in the internal space by incubating produced
human rotavirus C VLPs with the cargo such that the cargo enters
the internal space, e.g. by diffusion.
[0181] VLP Antibodies
[0182] Human rotavirus C VLPs are used as antigens for production
of monoclonal or polyclonal antibodies to human rotavirus C for
clinical use such as in therapy, analysis or diagnosis; or
laboratory research.
[0183] In a preferred embodiment, human rotavirus C VLPs are used
for eliciting human rotavirus C specific antibody or T cell
responses to the VP2, VP6, VP7 or any antigen included in the human
rotavirus C VLPs, in vivo (e.g., for protective or therapeutic
purposes or for providing diagnostic antibodies) and in vitro
(e.g., by phage display technology or another technique useful for
generating synthetic antibodies).
[0184] As used herein, the terms "antibody" and "antibodies" relate
to monoclonal antibodies, polyclonal antibodies, bispecific
antibodies, multispecific antibodies, human antibodies, humanized
antibodies, chimeric antibodies, camelized antibodies, single
domain antibodies, single-chain Fvs (scFv), single chain
antibodies, disulfide-linked Fvs (sdFv), and anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the invention), and epitope-binding fragments of any
of the above. In particular, antibodies include immunoglobulin
molecules and immunologically active fragments of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site.
Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD,
IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2),
or subclass.
[0185] As used herein, the term "antibody fragment" defines a
fragment of an antibody that immunospecifically binds to a human
rotavirus C virus, any epitope of the human rotavirus C virus or
human rotavirus C VLP. Antibody fragments may be generated by any
technique known to one of skill in the art. For example, Fab and
F(ab')2 fragments may be produced by proteolytic cleavage of
immunoglobulin molecules, using enzymes such as papain (to produce
Fab fragments) or pepsin (to produce F(ab') 2 fragments). F(ab') 2
fragments contain the complete light chain, and the variable
region, the CH 1 region and the hinge region of the heavy chain.
Antibody fragments are also produced by recombinant DNA
technologies. Antibody fragments may be one or more complementarity
determining regions (CDRs) of antibodies.
[0186] Human rotavirus C-specific antibodies are provided according
to the present invention which specifically bind to human rotavirus
C and do not specifically bind to other rotavirus types such as
rotavirus A, B, D, E, F and G.
[0187] A hybridoma cell line expressing monoclonal antibody raised
against human rotavirus C VLPs of the present invention
specifically binds to human rotavirus C and does not specifically
bind to other rotavirus types such as rotavirus A, B, D, E, F and
G.
[0188] An antibody raised to human rotavirus C VLPs by any of the
methods known in the art, is optionally purified by any method
known in the art for purification of an immunoglobulin molecule,
for example, by ion exchange chromatography, affinity, particularly
by affinity for the specific antigen or size exclusion;
centrifugation; differential solubility; or by any other standard
techniques for the purification of proteins. It is also appreciated
thatan inventive antibody or fragments thereof may be fused to
heterologous polypeptide sequences known in the art to facilitate
purification.
[0189] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use
chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule in which different portions of the antibody are derived
from different animal species, such as antibodies having a variable
region derived from a murine monoclonal antibody and a constant
region derived from a human immunoglobulin. Methods for producing
chimeric antibodies are known in the art. (Morrison, 1985, Science,
229:1202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397).
Humanized antibodies are antibody molecules from non-human species
that bind the desired antigen having one or more complementarity
determining regions (CDRs) from the non-human species and framework
regions from a human immunoglobulin molecule. Often, framework
residues in the human framework regions are substituted with the
corresponding residue from the CDR donor antibody to alter,
preferably improve, antigen binding. These framework substitutions
are identified by methods well known in the art, such as by
modeling of the interactions of the CDR and framework residues to
identify framework residues important for antigen binding and
sequence comparison to identify unusual framework residues at
particular positions. (U.S. Pat. No. 5,585,089; Riechmann et al.,
1988, Nature 332:323). Antibodies can be humanized using a variety
of techniques known in the art including, for example, CDR-grafting
(PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101
and 5,585,089), veneering or resurfacing (Studnicka et al., 1994,
Protein Engineering 7(6):805 814; Roguska et al., 1994, PNAS.
91:969 973), and chain shuffling (U.S. Pat. No. 5,565,332).
[0190] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. (U.S. Pat. Nos. 4,444,887 and
4,716,111).
[0191] Human antibodies are readily produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin
genes.
[0192] An inventive antibody is optionally fused or conjugated to
heterologous polypeptides may be used in vitro immunoassays and in
purification methods such as affinity chromatography. (PCT
publication Number WO 93/21232; U.S. Pat. No. 5,474,981).
[0193] An inventive antibody is optionally attached to solid
supports, which are particularly useful for immunoassays or
purification of the polypeptides of the invention or fragments,
derivatives, analogs, or variants thereof, or similar molecules
having the similar enzymatic activities as the polypeptide of the
invention. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0194] Assays for Human Rotavirus C
[0195] Anti-human rotavirus C VLP antibodies of the present
invention are used to detect human rotavirus C in a biological
sample in embodiments of the present invention.
[0196] An assay for human rotavirus C in a biological sample of the
present invention includes contacting a biological sample with an
anti-human rotavirus C antibody and detecting formation of a
complex between anti-human rotavirus C antibody and the human
rotavirus C present in the biological sample. Formation of the
complex is indicative of current infection by human rotavirus C in
a subject from which a biological sample is obtained. Formation of
the complex specifically indicates presence of human rotavirus C
since other rotavirus types such as rotavirus A, B, D, E, F and G,
do not form a complex with an anti-human rotavirus C antibody of
the present invention.
[0197] In a specific embodiment, the processes further involve
obtaining a biological sample from a subject, contacting the sample
with a compound or agent capable of detecting the presence of human
rotavirus C nucleic acid in the sample in order to confirm presence
of human rotavirus C in the sample.
[0198] In further embodiments, a control sample is assayed for
presence of human rotavirus C and/or anti-human rotavirus C
antibodies and results are compared with a test sample to ascertain
a difference in presence or amount of human rotavirus C or
anti-human rotavirus C antibodies.
[0199] In another aspect, the invention provides a method of
determining exposure of a human or animal to a group C rotavirus
comprising: contacting a biological sample of said human or animal
with the inventive rotavirus-like particle described herein, under
conditions which promote binding of antibodies in said biological
sample to said rotavirus-like particles; and detecting binding of
antibodies within the biological sample with the rotavirus-like
particles. For the purposes of determining exposure of a human or
animal to a group C rotavirus, biological sample typically is blood
and/or feces; however, biological sample also includes a sample
from other tissues; e.g. an intestinal biopsy.
[0200] The invention also encompasses kits for detecting the
presence of human rotavirus C in a test sample. The kit, for
example, includes an anti-human rotavirus C antibody and optionally
includes a reagent such as a labeled secondary antibody or agent
capable of detecting an antibody in a complex with a human
rotavirus C and, in certain embodiments, for determining the titer
in the sample.
[0201] Embodiments of inventive compositions and methods are
illustrated in the following examples. These examples are provided
for illustrative purposes and are not considered limitations on the
scope of inventive compositions and methods.
Example 1
[0202] Cloning and construction of baculovirus recombinants.
Segment 5, encoding VP6, from human group C RV strain S-1 was
amplified by RT-PCR using BMJ44 (5'-AGC-CAC-ATA-GTT-CAC-ATT-TC-3')
(SEQ ID NO: 14) and BMJ141 (5'-ATC-TCA-TTC-ACA-ATG-GAT-G-3') (SEQ
ID NO: 15) (28). Segment 8, encoding VP7, from strain S-1 was
amplified by RT-PCR using primers BMJ13
(5'-AGC-CAC-ATG-ATC-TTG-TTT-3') (SEQ ID NO: 20) and BMJ14
(5'-GGC-ATT-TAA-AAA-AGA-AGA-3') (SEQ ID NO: 21) (13, 28). Segment
2, encoding VP2, from strain ASP88 was amplified by RT-PCR using
BMJ197 (5'-TCG-AGG-ACA-AAT-CGT-CCA-AG-3') (SEQ ID NO: 22) and
BMJ180 (5'-AGC-CAC-AGA-GTT-TGA-GGT-C-3') (SEQ ID NO: 23). Cloning
and construction of recombinant baculovirus expressing S-1 VP7 was
previously described (14). DNA fragments of segment 2 and 5 were
cloned into vector pVL1393 and transfections were performed with
the Bac-N-Blue transfection kit (Gibco, Grand Island, N.Y.).
Baculovirus constructs were amplified in Spodoptera frugiperda 9
(Sf9) cell culture for 2 passages, plaque purified, and then
amplified for two more passages in Sf9 cells in serum-free HyQ
SFX-Insect media (Hyclone, Logan, Utah).
[0203] FIG. 5 provides an amino acid sequence alignment for VP2
from strain ASP88 described above (SEQ ID NO: 1); human group C VP2
strain referred to as "Bristol" with protein (SEQ ID NO: 16) has
NCBI Accession CAC 44890, version CAC 44890.1 GI: 15027005; as well
as the porcine VP2 referred to as "Cowden" (SEQ ID NO: 17).
[0204] FIG. 6 is a nucleotide sequence alignment of sequences
encoding human Group C VP-2 for inventive strain ASP88 (SEQ ID NO:
18), Cowden porcine strain (SEQ ID No. 44) and Bristol (SEQ ID NO:
19, Accession AJ303139). The start and stop codons are
underlined.
[0205] FIG. 7 is a nucleotide sequence alignment of sequences
encoding human Group C VP-6 for inventive strain S-1 relative to
conventional strains Bristol (SEQ ID NO: 25, Accession CAA42504);
Jajeri (SEQ ID NO: 26, Accession AAK26534); CMH004 (SEQ ID NO: 27,
Accession ABR31794); V508 (SEQ ID NO: 28, Accession AAX13496);
China (SEQ ID NO: 29, Accession BAB83829); and BCN6 (SEQ ID NO: 30,
Accession CAJ41549). It is noted that FIG. 7 provides the sequence
comparison in a format standard in the art wherein a "dot"
indicates identity with a reference sequence. In FIG. 7, the
reference sequence is a consensus sequence (SEQ ID No. 41).
[0206] FIG. 8 is an amino acid sequence alignment of sequences
encoding human Group C VP-6 for inventive strain S-1 (SEQ ID NO:
32) relative to conventional strains Bristol (SEQ ID NO: 34,
Accession CAA42504); Jajeri (SEQ ID NO: 35, Accession AAK26534);
CMH004 (SEQ ID NO: 36, Accession ABR31794); V508 (SEQ ID NO: 37,
Accession AAX13496); China (SEQ ID NO: 38, Accession BAB83829); and
BCN6 (SEQ ID NO: 39, Accession CAJ41549). It is noted that FIG. 8
provides the sequence comparison in a format standard in the art
wherein a "dot" indicates identity with a reference sequence. In
FIG. 8, the reference sequence is a consensus sequence (SEQ ID No.
24).
TABLE-US-00003 TABLE I Comparison of VP2 Genes of Group C
Rotaviruses Asp88 Bristol.sup.3 Cowden.sup.2 ORF 2652 2652 2652
Size (aa) 884 884 884 MW (kDa).sup.4 101.57 101.67 101.68
Nucleotide and amino acid homology Asp88 -- 97.2 83.2 Bristol 98.5
-- 82.9 Cowden 92.8 92.6 --
[0207] Table I shows results of a comparison of VP2 Genes of Group
C rotaviruses. MEGA version 4 program was used for the sequence
analysis of the VP2 genes containing a single ORF extending from nt
37-2688. Results indicate that the nucleotide sequence encoding
Asp88 VP2 has 97.2% homology to the nucleotide sequence encoding
Bristol VP2, the nucleotide sequence encoding Asp88 VP2 has 83.2%
homology to the nucleotide sequence encoding Cowden (porcine) VP2,
and the nucleotide sequence encoding Bristol VP2 has 82.9% homology
to the nucleotide sequence encoding Cowden VP2. Further, the amino
acid sequence of Asp88 VP2 has 98.5% homology to the amino acid
sequence of Bristol VP2, the amino acid sequence of Asp88 VP2 has
92.8% homology to the amino acid sequence of Cowden (porcine) VP2,
and the amino acid sequence of Bristol VP2 has 92.6% homology to
the amino acid sequence of Cowden VP2. Accession numbers of group C
rotavirus Bristol strain is AJ303139. .sup.2Cowden VP2 sequence was
resequenced. .sup.3 Bristol sequence is found in Chen, Z. et al,
2002.
Example 2
[0208] Cells and superinfections. Sf9 or High Five (Hi5) insect
cells were grown and maintained in EX-CELL 420 or 405 media (Sigma,
Lenexa, Kans.) or HyQ SFX-INSECT media in shaker flasks at
27.degree. C. Sf9 and Hi5 cells were subcultured every 3 or 4 days
at a concentration of 1.times.10.sup.6 cells/ml and
5.times.10.sup.5 cells/ml, respectively. Stationary superinfections
were performed by seeding Sf9 cells in HyQ or EX-CELL 420 and Hi5
cells in HyQ or EX-CELL 405 into a T150 flask at a concentration of
3.times.10.sup.5 cells/ml. Baculovirus constructs (rVP2, rVP6 and
rVP7) were added at a multiplicity of infection (MOI) of 1 each.
Infections were carried out without proteinase inhibitors and
infected cultures were harvested at day 5. Large scale VLP
production was performed in suspension culture by seeding Sf9 cells
in EX-CELL 420 into fernbach flasks at a concentration of
1.times.10.sup.6 cells/ml. Baculovirus recombinants were added one
day later at an MOI of 1.4 each and harvested on day 4.
Example 3
[0209] Western blot. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was carried out with 12% separating and
5% stacking gels using the Laemmli discontinuous buffer system
(16). Samples were heated at 97.degree. C. for 5 min with 10%
.beta.-mecaptoethanol prior to loading and then electrophoresed.
Proteins were transferred to a PVDF membrane in transfer buffer (25
mM Tris, 192 mM glycine, 10% methanol). After blocking with 10%
(for unpurified GpC RV proteins) blotto in PBS-T for 1-2 hrs at
room temperature or 15% (for purified GpC RV proteins) blotto in
PBS-T overnight at 4.degree. C., membranes were incubated with
porcine hyperimmune serum (1:2,000) to Cowden in 5% blotto in PBS-T
overnight at 4.degree. C. or rabbit hyperimmune serum (1:20,000) to
human GpC VLPs in 10% blotto in PBS-T for 1 h. Membranes were
washed in PBS-T, incubated with horseradish peroxidase (HRP) goat
anti-pig (1:142,000) (KPL, Gaithersburg, Md.) in 5% blotto or
HRP-goat anti rabbit (1:20,000) (Pierce, Rockford, Ill.) in 10%
blotto. GpC RV proteins were visualized with Supersignal West Femto
Maximum Sensitivity Substrate Kit (Pierce, Rockford, Ill.) by
exposing membranes to film and processing with medical film
processor SRX-101A (Konica Minolta, Jakarta, Indonesia).
Example 4
[0210] Purification of VLPs. Infected cell cultures were clarified
twice at 15,344.times.g for 30 min at 4.degree. C. with a JA-14
rotor in J2-MC centrifuge. Clarified supernatants were layered over
35% sucrose cushions and centrifuged for two hrs at 107,000.times.g
(4.degree. C.) with the SW32Ti rotor in an Optima L-80 XP
Ultracentrifuge (Beckman Coulter, Fullerton, Calif.). Pellets were
resuspended in TNC buffer (10 mM Tris pH 7.4, 140 mM NaC1, 5 mM
CaC12), re-clarified in a microcentrifuge and treated twice
sequentially with equal volume Vertrel (Miller-Stephenson, Danbury,
Conn.). Samples were centrifuged 10 min at 2,095.times.g (4.degree.
C.) with a SX4750A rotor in the Allegra X-12R tabletop centrifuge
(Beckman Coulter, Fullerton, Calif.). The aqueous layer was
overlaid on top of a CsCl solution (1.2738 g/ml) and centrifuged
17-18 hrs at 111,000.times.g (4.degree. C.) with the SW40Ti rotor.
Fractions that contained VLPs were collected, diluted in TNC, and
pelleted out by centrifugation at 107,000.times.g (4.degree. C.)
for 1 hr in the SW32Ti rotor. Particles were resuspended in Hanks
balanced salt solution (Gibco, Grand Island, N.Y.) supplemented
with 10% sorbitol.
Example 5
[0211] Electron microscopy and immunoelectron microscopy. GpC RV
VLPs were examined by electron microscopy (EM) and immunoelectron
microscopy (IEM) as previously described with modifications (19).
Briefly, 1% ammonium molybdate-1% trehalose in water (pH 6.95) was
used to provide negative contrast on specimens adsorbed to nickel
formvar-carbon filmed grids (9). Each grid was pretreated with 1%
alcian blue 8GX in water to enhance hydrophilicity and provide
cationic charges to the film surface prior to applying specimens.
IEM was done by mixing 1 .mu.l of purified GpC RV VLPs or GpA RV
RRV with 1 .mu.l of rabbit antibody to GpA or GpC RV diluted 1:500
and applying to nickel formvar-carbon coated grids. After
incubation for 0.5-1 hr, the grids were blotted with filter paper,
rinsed with 0.1 M Tris buffer supplemented with 0.4% acetylated
bovine serum albumin (BSA) (Aurion, Hatfield, Pa.), and incubated
for 30 min with goat anti-rabbit secondary antibody conjugated to 6
nm colloidal gold (1:20). Grids were rinsed twice with Tris buffer
without BSA and with deionized water, blotted, stained with
ammonium molybdate-trehalose, and viewed within an FEI Technai
BioTwin transmission electron microscope at 120 KV accelerating
voltage. Images were captured digitally with a 2K.times.2K AMT
digital camera.
Example 6
[0212] Production of antisera to GpC VLPs. Rabbits (Covance,
Denver, Pa.) were screened for the presence of GpA and C RV
antibodies by antigen capture EIA prior to immunization. Rabbit
CD94, which tested negative for GpA and C antigens, was selected
for antibody production. CD94 was injected subcutaneously with 50
.mu.g GpC VLPs formulated in Freud's complete adjuvant. Subsequent
doses formulated with Freud's incomplete adjuvant were administered
three weeks after the previous injection. Five injections in total
were administered. The first bleed was 10 days after dose two and
all subsequent bleeds were scheduled three weeks after the previous
bleed. All bleeds tested positive for GpC RV antibody and were
pooled.
Example 7
[0213] Enzyme Immunoassays (EIAs). 96-well plates were coated with
100 .mu.l supernatant from GpA RRV infected MA104 cell cultures or
GpC VLPs in recombinant baculovirus-infected Sf9 cells diluted
(1:100) in coating buffer and incubated overnight at 37.degree. C.
Plates were washed with PBS-T and then blocked with 150 .mu.l 5%
blotto for 1 hr at 37.degree. C. Plates were washed and then
incubated with 1000 of serially diluted hyperimmune serum from
rabbits CD94, CD8, and 8807A in diluent (1% blotto, 0.5%
polyoxethylene ether W1 in PBS) for 2 hrs at 37.degree. C. Rabbit
CD8 was immunized with GpA RV RRV, whereas rabbit 8807A was
naturally infected with GpA RV and also immunized with GpC RV
Cowden. Plates were washed and then incubated with HRP goat
anti-rabbit IgG (KPL, Gaithersburg, Md.) diluted (1:3,000) in
diluent for 1 hr at 37.degree. C. Plates were washed 6 times with
PBS-T and then reacted with 100 .mu.l of tetramethyl benzidine
(TMB) for 10 min. Reactions were stopped with 100 .mu.l 1N HC1 and
plates were read at Abs450. Antibody titers were defined as the
reciprocal of the highest dilution of serum giving a net optical
density (OD) value (OD with virus minus OD with blotto) above
0.1.
Example 8
[0214] Kinetics of GpC RV protein synthesis in Sf9 and Hi5 cells.
Sf9 and Hi5 cells in EX-CELL or HyQ media were infected with GpC RV
VP2, VP6, and VP7 baculovirus recombinants at an MOI of 1 each and
infected cultures were harvested on days 3, 4 and 5. Infected
cultures were clarified and analyzed by Western blot using Cowden
specific porcine hyperimmune serum to determine the expression
profiles of proteins secreted into the supernatant. FIGS. 1A and 1B
are electrophoretic gels showing the kinetics of GpC RV VP6 and VP7
expression in Sf9, FIG. 1A, or Hi5, FIG. 1B, cells in different
media. In both FIGS. 1A and 1B, lane 1, Cowden strain; lanes 2-5,
infected cultures in HyQ harvested 0, 3-5 dpi; and lanes 6-9,
infected cultures in EX-CELL harvested at 0, 3-5 dpi. GpC RV VP6
and VP7 are indicated on the right. Molecular markers 54 kDa and
37.5 kDa are indicated by arrows on the left.
[0215] Expression of GpC RV VP6 and VP7 increased with time, with
the highest levels seen at 4 or 5 days post infection (dpi) in Sf9
and Hi5 cells. Use of EX-CELL media resulted in higher rotavirus
protein yields in both cell lines. Higher protein expression was
achieved in EX-CELL media than in HyQ medium and similar levels of
protein expression were observed in Sf9 and Hi5 cells. The cell
line Sf9 and EX-CELL 420 were used in further examples of GpC RV
protein production described herein. Human GpC RV VP2 was not
detectable with the serum used.
Example 9
[0216] Self-assembly and characterization of GpC RV VLPs.
Superinfections of Sf9 cells with rVP2, rVP6 and rVP7 at an MOI of
1 each resulted in the formation of intact GpC RV VLPs that have
the structural order of typical rotavirus.
[0217] FIG. 2A is an image of an electron micrograph showing VLPs
purified from cultures of Sf9 cells in EX-CELL 420 medium that were
infected with recombinant baculoviruses encoding human rotavirus C
VP2, VP6, and VP7 at an MOI of 1 each. FIG. 2B is an image of an
electron micrograph showing VLPs purified from cultures of Sf9
cells in EX-CELL 420 medium that were infected with recombinant
baculoviruses encoding human rotavirus C VP6 and VP7 at an MOI of
1.4 each. VLPs shown in FIGS. 2A and 2B were stained with 5%
ammonium molybdate-1% trehalose. The bar in FIGS. 2A and 2B
represents 100 nm.
[0218] Sf9 cells were also superinfected with all three recombinant
viruses at various MOIs (0.1, 0.2, 1 and 2 each) in order to
optimize conditions for VLP production. EM analysis of supernatants
demonstrated better VLP formation in superinfections performed at
the higher MOIs of 1 and 2. To determine if VP2 is essential for
VLP formation, superinfections were performed with and without rVP2
and analysis was performed by EM. Because robust VLP formation was
demonstrated without rVP2, all subsequent experiments were
performed excluding this recombinant.
[0219] Biochemical composition and antigenicity of purified VP6/7
VLPs were compared with a GpA RV strain, YK-1, by SDS-PAGE and
Western blot. Images comparing major structural viral proteins from
GpA RV YK-1 and GpC VLPs by SDS-PAGE and Western blot are shown in
FIGS. 3A and 3B, respectively.
[0220] Proteins from purified YK-1 (lane 1) and VLPs (lane 2) were
separated on a 12% SDS-PAGE and stained by coomassie blue, FIG. 3A,
or analyzed by Western blot, FIG. 3B. For western blot, proteins
were transferred to a PVDF membrane and incubated with rabbit
hyperimmune serum to GpC VLPs. GpC RV VP6 and VP7 are indicated on
the right. Arrows indicate molecular weight markers 54 kDa and 37.5
kDa.
[0221] Human GpC recombinant proteins VP6 and VP7 migrate at
similar molecular weights and are present at similar ratios as seen
in GpA YK-1, indicating a proper molar ratio in assembled VLPs,
illustrate in FIG. 3A. Western blot performed with human GpC VLP
specific antibody demonstrated that these human rotavirus C VLPs
were antigenic and of good quality, illustrated in FIG. 3B.
Example 10
[0222] Antigenic reactivity of GpC RV antibody. Rabbit polyclonal
antibody was produced to purified human rotavirus C VLPs. Prior to
immunization, rabbit CD94 had no antibody to GpA or C RV and after
inoculations developed a high antibody titer to GpC RV (Table II).
In controls, rabbit CD8, immunized with RRV, gave strong positive
response to GpA RV, whereas rabbit 8807A, which was naturally
infected with GpA RV and immunized with GpC RV Cowden strain, had
similar antibody titers to GpA and GpC RV antigens. Hyperimmune
serum from rabbit CD94 was utilized to enhance EIA that employs
only porcine hyperimmune sera (8). This assay proved to
specifically detect GpC RV antigens (VLPs and Cowden) but did not
react with other control samples, such as MA104, Sf9 cells, and GpA
RV RRV.
TABLE-US-00004 TABLE II Antibody Titers in Hyperimmune Sera to GpA
and C Rotaviruses Reciprocal of IgG Titer Rotavirus Group A (RRV)
Group C (VLPs) Rabbit Antigen Pre Post Pre Post CD94 GpC VLPs
<100 <100 <100 51,200 CD8 GpA RRV ND 25,600 ND <100
8807A GpC Cowden ND 1,600 ND 3,200 & GpA Hyperimmune sera were
tested and antibody titers were determined as described in the
text. Pre = pre-immunization serum. Post = post-immunization serum.
ND = not determined.
[0223] Human GpC RV VLPs were further characterized by examining
their antigenic properties with immunoelectron microscopy using
rabbit hyperimmune sera to GpA and C RV
[0224] FIG. 4A shows GpC RV VLPs immunostained with GpC-specific
rabbit hyperimmune serum. FIG. 4B shows GpC RV VLPs immunostained
with GpA-specific rabbit hyperimmune serum. FIG. 4C shows GpA RV
immunostained with GpC-specific rabbit hyperimmune serum. FIG. 4D
shows GpA RV immunostained with GpA-specific rabbit hyperimmune
serum. GpC RV VLPs were specifically labeled with GpC-specific
hyperimmune serum and GpA RVs were heavily coated with GpA-specific
hyperimmune serum. The bar in FIGS. 4A, 4B, 4C and 4D represents
100 nm.
[0225] GpC RV antibody specifically reacted with human GpC VLPs but
not with GpA RV, RRV. Correspondingly, GpA antibody exhibited
specific reactivity with RRV and not with GpC VLPs. These results
indicate the occurrence of group-specific interactions between RV
antigen and antibody and confirm the absence of cross-reactivities
between GpA and GpC RV reagents.
TABLE-US-00005 [VP2 from human strain Asp88-amino acid sequence]
(SEQ ID NO: 1)
MISRNRRRNNQQKDIGKEKQLETIIDKEVKENKDSTKEDKLVVTEESNGDVTAVKEQSN
NINLQKNDLVKEVMNIQNQTLNTVVAENKVEIEEIVKKYIPSYNTDSLIVKKLTEIQESSA
KTYNTLFRLFTPVKSYLYDINGEKKLSTRWYWKLLKDDLPAGDYSVRQFFLSLYLNVLE
GMPDYIMLRDMAVDNPYSAEAGKIVDGKSKEILVELYQDQMTEGYIRRYMSELRHKIS
GETNTAKYPAILHPVDNELNQYFLEHQLIQPLTTRNIAELIPTQLYHDPNYVFNIDAAFLT
NSRFVPPYLTQDRIGLHDGFESIWDSKTHADYVSARRFIPDLTELVDAEKQIKEMAAHLQ
LEAITVQVESQFLAGISAAAANEAFKFIIGSVLSTRTIAVEFITSNYMSLASCMYLMTIMPS
EIFLRESLVAMRLAIINTLIYPALGLAQMHYQAGEVRTPFELAEMRVANRSIRQWLHHC
NTLQFGRQITEGIIHLRFTNDIMTGRIVNLFSTMLVALSSQPFATYPLDYKRSVQRALQLL
SNRTAQIADLTRLIVYNYTTLSACIVMNMHLVGTLTVERIQATSLTSLMMLISNKTVIPEP
SSLFSYFSSNINFLTNYNEQIDNVVAEIMAAYRLNLYQQKMLMLVTRFVSRLYIFDAPKI
PPDQMYRLRNRLRNIPVERRRADVFRIIMNNRDLIEKTSERICQGVLLSYTPMPLTYVED
VGLTNVINDTNNFQIINIEEIEKTGDYSAITNALLRDTPIILKGAIPYVTNSSVIDVLSKVDT
TVFASIVKDRDISKLKPIKFIINSDSSEYYLVHNNKWTPTTTTAVYKARSQQFDIQHSVSM
LESNLFFVVYNDLFKYIKTTTVLPINAVSYDGARIMQET [VP2 from human strain
ASP88-nucleotide sequence] (SEQ ID NO: 18)
TCGAGGACAAATCGTCCAAGATGATAAGCAGAAACAGGCGCAGAAATAAC
CAACAAAAAGATATAGGAAAAGAGAAACAATTAGAGACTATAATTGACAA
AGAAGTAAAGGAAAACAAAGATTCTACAAAAGAAGATAAGCTAGTAGTTA
CGGAAGAAAGTAATGGTGACGTCACAGCTGTTAAAGAACAATCGAATAAT
ATTAATTTACAAAAGAATGATTTGGTTAAAGAAGTCATGAATATACAGAA
TCAAACATTAAATACAGTAGTTGCTGAGAATAAAGTTGAAATAGAAGAAA
TAGTTAAAAAATACATTCCCTCATATAATACTGACAGCCTTATTGTTAAA
AAGTTAACTGAAATCCAGGAATCAAGTGCTAAAACATATAATACATTATT
CAGATTATTTACTCCAGTTAAAAGTTATTTATATGACATAAATGGTGAGA
AAAAATTATCGACTAGATGGTATTGGAAATTGCTCAAAGATGATTTACCT
GCTGGTGATTACTCAGTTAGACAATTCTTCCTGTCACTATATTTAAATGT
TTTAGAGGGAATGCCCGATTACATAATGCTTCGTGATATGGCAGTGGATA
ACCCATATTCAGCAGAAGCAGGTAAAATCGTAGATGGAAAGTCTAAAGAA
ATTTTAGTTGAACTATATCAAGACCAAATGACAGAAGGGTATATTAGAAG
ATATATGTCTGAATTAAGACATAAAATATCTGGAGAAACAAATACTGCAA
AATATCCAGCTATTCTACATCCCGTGGATAATGAGCTTAATCAATACTTT
CTTGAGCATCAGTTAATTCAACCATTAACTACAAGAAATATTGCAGAATT
GATTCCAACTCAATTATATCATGATCCAAATTACGTTTTTAATATTGATG
CAGCCTTTTTAACAAATTCAAGATTTGTTCCACCATACTTAACACAGGAT
AGGATTGGATTACATGATGGATTCGAATCAATATGGGATTCAAAAACCCA
TGCTGATTACGTTTCAGCTAGAAGATTTATACCTGATTTAACTGAACTGG
TAGATGCTGAAAAGCAAATAAAAGAAATGGCTGCACATTTACAACTAGAG
GCTATTACAGTACAGGTTGAATCACAATTTTTAGCGGGAATTAGTGCTGC
TGCAGCTAATGAAGCGTTCAAATTTATAATTGGCTCAGTTTTATCTACCA
GAACAATAGCTGTAGAATTCATAACCTCAAACTATATGTCGTTAGCATCA
TGTATGTATTTAATGACTATTATGCCATCAGAGATTTTCTTGAGAGAATC
ATTAGTTGCTATGCGATTAGCAATAATAAATACCCTTATTTATCCAGCTC
TAGGTTTAGCGCAAATGCATTATCAAGCAGGTGAAGTGAGGACCCCATTC
GAATTAGCTGAGATGCGAGTAGCTAATAGATCTATTAGACAATGGTTACA
TCATTGTAATACACTTCAATTTGGTAGACAGATAACGGAAGGGATAATTC
ATCTACGATTTACTAATGATATCATGACAGGTAGGATAGTGAACTTATTT
TCAACAATGCTAGTAGCTTTATCATCTCAGCCATTCGCTACATATCCTTT
AGACTATAAAAGATCTGTACAAAGAGCATTACAACTTTTATCAAATAGAA
CAGCCCAAATAGCAGATTTAACCAGATTAATAGTATACAATTATACTACA
TTATCTGCATGTATAGTCATGAATATGCATTTAGTAGGAACTCTTACTGT
TGAACGTATACAGGCCACTTCTCTAACTTCTTTAATGATGTTAATTTCTA
ATAAGACAGTTATTCCAGAACCATCGTCTCTTTTTTCATATTTCTCTAGT
AACATTAATTTTCTTACAAATTATAATGAGCAAATTGATAATGTGGTAGC
AGAAATAATGGCCGCATATAGATTGAATTTATATCAACAGAAAATGTTGA
TGCTCGTTACCAGGTTTGTGTCAAGGTTGTACATATTTGATGCTCCTAAA
ATACCGCCAGATCAGATGTATAGATTAAGAAACCGATTAAGAAATATTCC
AGTTGAAAGAAGAAGAGCTGATGTGTTCAGAATTATTATGAATAATAGAG
ATTTAATCGAAAAAACATCAGAACGTATATGTCAGGGTGTGTTGTTATCT
TATACACCAATGCCTTTAACTTACGTTGAAGATGTCGGGTTAACAAATGT
AATTAATGACACTAATAACTTCCAAATAATTAATATAGAAGAAATTGAGA
AGACCGGTGACTATTCAGCCATAACGAATGCATTACTTCGGGATACTCCA
ATTATATTGAAAGGTGCGATTCCATATGTTACTAACTCATCAGTAATTGA
TGTTTTATCTAAAGTGGACACCACAGTGTTCGCAAGCATCGTAAAAGATA
GGGATATTTCAAAGTTAAAACCAATAAAATTCATAATTAATTCAGATTCA
TCCGAATATTATTTAGTACACAATAATAAATGGACACCAACAACAACTAC
AGCAGTATATAAAGCTAGATCTCAGCAATTTGATATACAACATTCAGTAT
CAATGCTAGAGTCAAACTTATTTTTTGTGGTATATAATGATTTATTTAAA
TACATTAAAACCACTACAGTTCTGCCGATAAATGCTGTCTCTTATGATGG
TGCAAGAATTATGCAAGAAACATAAATGATTGTATAGTATCATCTTGTAA
CGACCTCAAACTCTGTGGCT [VP2 open reading frame from human strain
ASP88-nucleotide sequence] (SEQ ID NO: 42)
ATGATAAGCAGAAACAGGCGCAGAAATAAC
CAACAAAAAGATATAGGAAAAGAGAAACAATTAGAGACTATAATTGACAA
AGAAGTAAAGGAAAACAAAGATTCTACAAAAGAAGATAAGCTAGTAGTTA
CGGAAGAAAGTAATGGTGACGTCACAGCTGTTAAAGAACAATCGAATAAT
ATTAATTTACAAAAGAATGATTTGGTTAAAGAAGTCATGAATATACAGAA
TCAAACATTAAATACAGTAGTTGCTGAGAATAAAGTTGAAATAGAAGAAA
TAGTTAAAAAATACATTCCCTCATATAATACTGACAGCCTTATTGTTAAA
AAGTTAACTGAAATCCAGGAATCAAGTGCTAAAACATATAATACATTATT
CAGATTATTTACTCCAGTTAAAAGTTATTTATATGACATAAATGGTGAGA
AAAAATTATCGACTAGATGGTATTGGAAATTGCTCAAAGATGATTTACCT
GCTGGTGATTACTCAGTTAGACAATTCTTCCTGTCACTATATTTAAATGT
TTTAGAGGGAATGCCCGATTACATAATGCTTCGTGATATGGCAGTGGATA
ACCCATATTCAGCAGAAGCAGGTAAAATCGTAGATGGAAAGTCTAAAGAA
ATTTTAGTTGAACTATATCAAGACCAAATGACAGAAGGGTATATTAGAAG
ATATATGTCTGAATTAAGACATAAAATATCTGGAGAAACAAATACTGCAA
AATATCCAGCTATTCTACATCCCGTGGATAATGAGCTTAATCAATACTTT
CTTGAGCATCAGTTAATTCAACCATTAACTACAAGAAATATTGCAGAATT
GATTCCAACTCAATTATATCATGATCCAAATTACGTTTTTAATATTGATG
CAGCCTTTTTAACAAATTCAAGATTTGTTCCACCATACTTAACACAGGAT
AGGATTGGATTACATGATGGATTCGAATCAATATGGGATTCAAAAACCCA
TGCTGATTACGTTTCAGCTAGAAGATTTATACCTGATTTAACTGAACTGG
TAGATGCTGAAAAGCAAATAAAAGAAATGGCTGCACATTTACAACTAGAG
GCTATTACAGTACAGGTTGAATCACAATTTTTAGCGGGAATTAGTGCTGC
TGCAGCTAATGAAGCGTTCAAATTTATAATTGGCTCAGTTTTATCTACCA
GAACAATAGCTGTAGAATTCATAACCTCAAACTATATGTCGTTAGCATCA
TGTATGTATTTAATGACTATTATGCCATCAGAGATTTTCTTGAGAGAATC
ATTAGTTGCTATGCGATTAGCAATAATAAATACCCTTATTTATCCAGCTC
TAGGTTTAGCGCAAATGCATTATCAAGCAGGTGAAGTGAGGACCCCATTC
GAATTAGCTGAGATGCGAGTAGCTAATAGATCTATTAGACAATGGTTACA
TCATTGTAATACACTTCAATTTGGTAGACAGATAACGGAAGGGATAATTC
ATCTACGATTTACTAATGATATCATGACAGGTAGGATAGTGAACTTATTT
TCAACAATGCTAGTAGCTTTATCATCTCAGCCATTCGCTACATATCCTTT
AGACTATAAAAGATCTGTACAAAGAGCATTACAACTTTTATCAAATAGAA
CAGCCCAAATAGCAGATTTAACCAGATTAATAGTATACAATTATACTACA
TTATCTGCATGTATAGTCATGAATATGCATTTAGTAGGAACTCTTACTGT
TGAACGTATACAGGCCACTTCTCTAACTTCTTTAATGATGTTAATTTCTA
ATAAGACAGTTATTCCAGAACCATCGTCTCTTTTTTCATATTTCTCTAGT
AACATTAATTTTCTTACAAATTATAATGAGCAAATTGATAATGTGGTAGC
AGAAATAATGGCCGCATATAGATTGAATTTATATCAACAGAAAATGTTGA
TGCTCGTTACCAGGTTTGTGTCAAGGTTGTACATATTTGATGCTCCTAAA
ATACCGCCAGATCAGATGTATAGATTAAGAAACCGATTAAGAAATATTCC
AGTTGAAAGAAGAAGAGCTGATGTGTTCAGAATTATTATGAATAATAGAG
ATTTAATCGAAAAAACATCAGAACGTATATGTCAGGGTGTGTTGTTATCT
TATACACCAATGCCTTTAACTTACGTTGAAGATGTCGGGTTAACAAATGT
AATTAATGACACTAATAACTTCCAAATAATTAATATAGAAGAAATTGAGA
AGACCGGTGACTATTCAGCCATAACGAATGCATTACTTCGGGATACTCCA
ATTATATTGAAAGGTGCGATTCCATATGTTACTAACTCATCAGTAATTGA
TGTTTTATCTAAAGTGGACACCACAGTGTTCGCAAGCATCGTAAAAGATA
GGGATATTTCAAAGTTAAAACCAATAAAATTCATAATTAATTCAGATTCA
TCCGAATATTATTTAGTACACAATAATAAATGGACACCAACAACAACTAC
AGCAGTATATAAAGCTAGATCTCAGCAATTTGATATACAACATTCAGTAT
CAATGCTAGAGTCAAACTTATTTTTTGTGGTATATAATGATTTATTTAAA
TACATTAAAACCACTACAGTTCTGCCGATAAATGCTGTCTCTTATGATGG
TGCAAGAATTATGCAAGAAACATAA VP2 from human strain ASP88-nucleotide
sequence including 36 5' non-coding bases (SEQ ID NO: 43)
GGCTTAAAAAGATCAG TCGAGGACAAATCGTCCAAGATGATAAGCAGAAACAGGCGCAGAAATAAC
CAACAAAAAGATATAGGAAAAGAGAAACAATTAGAGACTATAATTGACAA
AGAAGTAAAGGAAAACAAAGATTCTACAAAAGAAGATAAGCTAGTAGTTA
CGGAAGAAAGTAATGGTGACGTCACAGCTGTTAAAGAACAATCGAATAAT
ATTAATTTACAAAAGAATGATTTGGTTAAAGAAGTCATGAATATACAGAA
TCAAACATTAAATACAGTAGTTGCTGAGAATAAAGTTGAAATAGAAGAAA
TAGTTAAAAAATACATTCCCTCATATAATACTGACAGCCTTATTGTTAAA
AAGTTAACTGAAATCCAGGAATCAAGTGCTAAAACATATAATACATTATT
CAGATTATTTACTCCAGTTAAAAGTTATTTATATGACATAAATGGTGAGA
AAAAATTATCGACTAGATGGTATTGGAAATTGCTCAAAGATGATTTACCT
GCTGGTGATTACTCAGTTAGACAATTCTTCCTGTCACTATATTTAAATGT
TTTAGAGGGAATGCCCGATTACATAATGCTTCGTGATATGGCAGTGGATA
ACCCATATTCAGCAGAAGCAGGTAAAATCGTAGATGGAAAGTCTAAAGAA
ATTTTAGTTGAACTATATCAAGACCAAATGACAGAAGGGTATATTAGAAG
ATATATGTCTGAATTAAGACATAAAATATCTGGAGAAACAAATACTGCAA
AATATCCAGCTATTCTACATCCCGTGGATAATGAGCTTAATCAATACTTT
CTTGAGCATCAGTTAATTCAACCATTAACTACAAGAAATATTGCAGAATT
GATTCCAACTCAATTATATCATGATCCAAATTACGTTTTTAATATTGATG
CAGCCTTTTTAACAAATTCAAGATTTGTTCCACCATACTTAACACAGGAT
AGGATTGGATTACATGATGGATTCGAATCAATATGGGATTCAAAAACCCA
TGCTGATTACGTTTCAGCTAGAAGATTTATACCTGATTTAACTGAACTGG
TAGATGCTGAAAAGCAAATAAAAGAAATGGCTGCACATTTACAACTAGAG
GCTATTACAGTACAGGTTGAATCACAATTTTTAGCGGGAATTAGTGCTGC
TGCAGCTAATGAAGCGTTCAAATTTATAATTGGCTCAGTTTTATCTACCA
GAACAATAGCTGTAGAATTCATAACCTCAAACTATATGTCGTTAGCATCA
TGTATGTATTTAATGACTATTATGCCATCAGAGATTTTCTTGAGAGAATC
ATTAGTTGCTATGCGATTAGCAATAATAAATACCCTTATTTATCCAGCTC
TAGGTTTAGCGCAAATGCATTATCAAGCAGGTGAAGTGAGGACCCCATTC
GAATTAGCTGAGATGCGAGTAGCTAATAGATCTATTAGACAATGGTTACA
TCATTGTAATACACTTCAATTTGGTAGACAGATAACGGAAGGGATAATTC
ATCTACGATTTACTAATGATATCATGACAGGTAGGATAGTGAACTTATTT
TCAACAATGCTAGTAGCTTTATCATCTCAGCCATTCGCTACATATCCTTT
AGACTATAAAAGATCTGTACAAAGAGCATTACAACTTTTATCAAATAGAA
CAGCCCAAATAGCAGATTTAACCAGATTAATAGTATACAATTATACTACA
TTATCTGCATGTATAGTCATGAATATGCATTTAGTAGGAACTCTTACTGT
TGAACGTATACAGGCCACTTCTCTAACTTCTTTAATGATGTTAATTTCTA
ATAAGACAGTTATTCCAGAACCATCGTCTCTTTTTTCATATTTCTCTAGT
AACATTAATTTTCTTACAAATTATAATGAGCAAATTGATAATGTGGTAGC
AGAAATAATGGCCGCATATAGATTGAATTTATATCAACAGAAAATGTTGA
TGCTCGTTACCAGGTTTGTGTCAAGGTTGTACATATTTGATGCTCCTAAA
ATACCGCCAGATCAGATGTATAGATTAAGAAACCGATTAAGAAATATTCC
AGTTGAAAGAAGAAGAGCTGATGTGTTCAGAATTATTATGAATAATAGAG
ATTTAATCGAAAAAACATCAGAACGTATATGTCAGGGTGTGTTGTTATCT
TATACACCAATGCCTTTAACTTACGTTGAAGATGTCGGGTTAACAAATGT
AATTAATGACACTAATAACTTCCAAATAATTAATATAGAAGAAATTGAGA
AGACCGGTGACTATTCAGCCATAACGAATGCATTACTTCGGGATACTCCA
ATTATATTGAAAGGTGCGATTCCATATGTTACTAACTCATCAGTAATTGA
TGTTTTATCTAAAGTGGACACCACAGTGTTCGCAAGCATCGTAAAAGATA
GGGATATTTCAAAGTTAAAACCAATAAAATTCATAATTAATTCAGATTCA
TCCGAATATTATTTAGTACACAATAATAAATGGACACCAACAACAACTAC
AGCAGTATATAAAGCTAGATCTCAGCAATTTGATATACAACATTCAGTAT
CAATGCTAGAGTCAAACTTATTTTTTGTGGTATATAATGATTTATTTAAA
TACATTAAAACCACTACAGTTCTGCCGATAAATGCTGTCTCTTATGATGG
TGCAAGAATTATGCAAGAAACATAAATGATTGTATAGTATCATCTTGTAA
CGACCTCAAACTCTGTGGCT [VP6 from human strain S-1-nucleotide
sequence] (SEQ ID NO: 31) * 20 * 40 * 60 * 80
ATGGATGTACTTTTTTCTATAGCGAAAACCGTGTCAGATCTTAAAGAGAAAGTTGTAGTTGGAACAATTTATAC-
TAATGT * 100 * 120 * 140 * 160
AGAAGATGTTGTACAACAGACGAATGAATTGATTAGAACTTTGAATGGAAATATTTTTCATACTGGTGGCATTG-
GAACAC * 180 * 200 * 220 * 240
AGCCTCAGAAAGAGTGGAATTTTCAGCTCCCACAATTGGGTACCACTTTATTAAATTTAGATGATAATTATGTT-
CAATCA * 260 * 280 * 300 * 320
ACTAGAGGCATAATTGATTTTTTATCATCTTTTATAGAAGCTGTATGTGATGATGAAATTGTTAGAGAAGCTTC-
AAGAAA * 340 * 360 * 380 * 400
TGGTATGCAACCTCAATCACCAGCTCTTATATTATTATCTTCATCAAAATTTAAAACAATTAATTTTAATAATA-
GTTCTC * 420 * 440 * 460 * 480
AATCTATCAAAAATTGGAATGCTCAATCAAGACGTGAGAATCCTGTATATGAGTACAAAAATCCAATGTTGTTT-
GAATAT * 500 * 520 * 540 * 560
AAAAATTCTTATATTTTACAACGCGCAAATCCACAATTTGGAAGCGTCATGGGTTTAAGATATTATACAACAAG-
TAATAT * 580 * 600 * 620 * 640
TTGTCAAATTGCAGCATTTGATTCCACCCTAGCTGAAAATGCACCAAATAATACGCAACGCTTCGTTTATAATG-
GCAGAC * 660 * 680 * 700 * 720
TAAAAAGACCCATATCAAATGTTTTAATGAAAATAGAAGCTGGTGCTCCAAATATAAGCAACCCAACTATTTTA-
CCTGAT * 740 * 760 * 780 * 800
CCTAATAATCAAACAACTTGGCTTTTTAATCCGGTACAATTAATGAATGGAACATTTACCATTGAATTCTATAA-
TAATGG * 820 * 840 * 860 * 880
TCAACTAATTGATATGGTTCGAAATATGGGAATAGTTACTGTAAGAACTTTTGATTCTTATAGAATAACAATTG-
ACATGA * 900 * 920 * 940 * 960
TTAGACCAGCTGCTATGACTCAATACGTTCAACGAATTTTTCCACAAGGTGGACCTTATCATTTTCAGGCTACA-
TATATG * 980 * 1000 * 1020 * 1040
TTAACATTAAGTATATTAGATGCTACCACAGAGTCCGTTCTATGTGATTCTCATTCAGTAGAATATTCAATAGT-
AGCAAA * 1060 * 1080 * 1100 * 1120
CGTCAGAAGAGATTCAGCAATGCCAGCTGGAACTGTTTTTCAACCGGGATTTCCATGGGAACACACACTATCCA-
ATTACA * 1140 * 1160 * 1180
CTGTTGCTCAAGAAGATAATTTAGAAAGATTATTGTTAATCGCATCTGTGAAGAGAATGGTAATG
[VP6 from human strain S-1-amino acid sequence] (SEQ ID NO: 32)
MDVLFSIAKTVSDLKEKVVVGTIYTNVEDVVQQTNELIRTLNGNIFHTGG [50]
IGTQPQKEWNFQLPQLGTTLLNLDDNYVQSTRGIIDFLSSFIEAVCDDEI [100]
VREASRNGMQPQSPALILLSSSKFKTINFNNSSQSIKNWNAQSRRENPVY [150]
EYKNPMLFEYKNSYILQRANPQFGSVMGLRYYTTSNICQIAAFDSTLAEN [200]
APNNTQRFVYNGRLKRPISNVLMKIEAGAPNISNPTILPDPNNQTTWLFN [250]
PVQLMNGTFTIEFYNNGQLIDMVRNMGIVTVRTFDSYRITIDMIRPAAMT [300]
QYVQRIFPQGGPYHFQATYMLTLSILDATTESVLCDSHSVEYSIVANVRR [350]
DSAMPAGTVFQPGFPWEHTLSNYTVAQEDNLERLLLIASVKRMVM [395] [VP7 from human
strain S-1-nucleotide sequence] (SEQ ID NO: 33)
>gi|1314237|gb|U20995.1|RGU20995 Human rotavirus group C isolate
S-1 outer capsid glycoprotein (VP7) gene, complete cds
GGCATTTAAAAAAGAAGAAGCTGTCTGACAAACTGGTCTTCTTTTTAAATGGTTTGTACAACATTGTACA
CTGTTTGCGCCATTCTCTTCATTCTTTTCATTTATATATTATTATTTAGAAAAATGTTCCACCTAATAAC
TGATACTTTAATAGTGATGCTTATTTTATCTAATTGTGTAGAGTGGTCACAAGGTCAGATGTTTATTGAT
GATATACATTATAATGGTAACGTTGAGACTATCATAAATTCTACTGATCCTTTTAATGTTGAATCTTTAT
GTATTTATTTTCCAAATGCAGTTGTAGGATCACAGGGACCAGGTAAATCCGATGGACATTTGAATGATGG
TAATTATGCACAGACTATCGCCACTTTGTTTGAAACAAAAGGATTCCCAAAAGGTTCAATAATAATTAAA
ACATATACACAGACATCAGACTTTATAAATTCAGTAGAAATGACATGCTCTTATAATATAGTTATCATTC
CTGATAGCCCAAATGATTCAGAATCTATTGAACAGATAGCAGAATGGATTTTAAATGTTTGGAGATGTGA
TGACATGAATTTGGAAATTTATACTTATGAACAAATTGGAATAAACAATTTATGGGCTGCATTTGGTAGT
GACTGTGATATATCTGTCTGTCCATTAGATACTACAAGTAATGGAATCGGATGTTCACCAGCTAGTACAG
AAACTTATGAAGTTGTATCAAATGACACCCAATTGGCCTTAATTAATGTTGTGGATAATGTTAGACATAG
AATACAGATGAACACTGCTCAATGTAAATTGAAAAATTGTATTAAGGGTGAAGCTCGACTGAATACTGCA
CTAATAAGAATTTCAACATCATCAAGTTTTGATAATTCATTGTCACCATTAAATAACGGCCAAACAACAA
GATCGTTTAAAATAAATGCAAAGAAATGGTGGACTATATTTTATACAATAATTGATTATATTAATACAAT
TGTACAATCAATGACTCCCAGACATCGGGCGATTTATCCAGAAGGGTGGATGTTGAGGTATGCGTAAACA
AGATCATGTGGCT [VP7 from human strain S-1-nucleotide sequence of
open reading frame] (SEQ ID NO: 45) ATGGTTTGTACAACATTGTACA
CTGTTTGCGCCATTCTCTTCATTCTTTTCATTTATATATTATTATTTAGAAAAATGTTCCACCTAATAAC
TGATACTTTAATAGTGATGCTTATTTTATCTAATTGTGTAGAGTGGTCACAAGGTCAGATGTTTATTGAT
GATATACATTATAATGGTAACGTTGAGACTATCATAAATTCTACTGATCCTTTTAATGTTGAATCTTTAT
GTATTTATTTTCCAAATGCAGTTGTAGGATCACAGGGACCAGGTAAATCCGATGGACATTTGAATGATGG
TAATTATGCACAGACTATCGCCACTTTGTTTGAAACAAAAGGATTCCCAAAAGGTTCAATAATAATTAAA
ACATATACACAGACATCAGACTTTATAAATTCAGTAGAAATGACATGCTCTTATAATATAGTTATCATTC
CTGATAGCCCAAATGATTCAGAATCTATTGAACAGATAGCAGAATGGATTTTAAATGTTTGGAGATGTGA
TGACATGAATTTGGAAATTTATACTTATGAACAAATTGGAATAAACAATTTATGGGCTGCATTTGGTAGT
GACTGTGATATATCTGTCTGTCCATTAGATACTACAAGTAATGGAATCGGATGTTCACCAGCTAGTACAG
AAACTTATGAAGTTGTATCAAATGACACCCAATTGGCCTTAATTAATGTTGTGGATAATGTTAGACATAG
AATACAGATGAACACTGCTCAATGTAAATTGAAAAATTGTATTAAGGGTGAAGCTCGACTGAATACTGCA
CTAATAAGAATTTCAACATCATCAAGTTTTGATAATTCATTGTCACCATTAAATAACGGCCAAACAACAA
GATCGTTTAAAATAAATGCAAAGAAATGGTGGACTATATTTTATACAATAATTGATTATATTAATACAAT
TGTACAATCAATGACTCCCAGACATCGGGCGATTTATCCAGAAGGGTGGATGTTGAGGTATGCGTAA
[VP7 from human strain S-1-amino acid sequence] (SEQ ID NO: 34)
MVCTTLYTVCAILFILFIYILLFRKMFHLITDTLIVMLILSNCVEWSQGQMFIDDIHYNG
NVETIINSTDPFNVESLCIYFPNAVVGSQGPGKSDGHLNDGNYAQTIATLFLTKGFPKGS
IIIKTYTQTSDFINSVEMTCSYNIVIIPDSPNDSESIEQIAEWILNVWRCDDMNLEIYTY
EQIGINNLWAAFGSDCDISVCPLDTTSNGIGCSPASTETYEVVSNDTQLALINVVDNVRH
RIQMNTAQCKLKNCIKGEARLNTALIRISTSSSFDNSLSPLNNGQTTRSFKINAKKWWTI
FYTIIDYINTIVQSMTPRHRAIYPEGWMLRYA [VP6 from human strain
S-1-nucleotide sequence including 5' and 3' non-coding; start and
stop codons of ORF underlined] (SEQ ID No. 48)
GGCATTTAAAATCTCATTCACAATGGATGTACTTTTTTCTATAGCGAAAACCGTGTCAGATCTTAAAGAGAAAG-
TTGTAG
TTGGAACAATTTATACTAATGTAGAAGATGTTGTACAACAGACGAATGAATTGATTAGAACTTTGAATGGAAAT-
ATTTTT
CATACTGGTGGCATTGGAACACAGCCTCAGAAAGAGTGGAATTTTCAGCTCCCACAATTGGGTACCACTTTATT-
AAATTT
AGATGATAATTATGTTCAATCAACTAGAGGCATAATTGATTTTTTATCATCTTTTATAGAAGCTGTATGTGATG-
ATGAAA
TTGTTAGAGAAGCTTCAAGAAATGGTATGCAACCTCAATCACCAGCTCTTATATTATTATCTTCATCAAAATTT-
AAAACA
ATTAATTTTAATAATAGTTCTCAATCTATCAAAAATTGGAATGCTCAATCAAGACGTGAGAATCCTGTATATGA-
GTACAA
AAATCCAATGTTGTTTGAATATAAAAATTCTTATATTTTACAACGCGCAAATCCACAATTTGGAAGCGTCATGG-
GTTTAA
GATATTATACAACAAGTAATATTTGTCAAATTGCAGCATTTGATTCCACCCTAGCTGAAAATGCACCAAATAAT-
ACGCAA
CGCTTCGTTTATAATGGCAGACTAAAAAGACCCATATCAAATGTTTTAATGAAAATAGAAGCTGGTGCTCCAAA-
TATAAG
CAACCCAACTATTTTACCTGATCCTAATAATCAAACAACTTGGCTTTTTAATCCGGTACAATTAATGAATGGAA-
CATTTA
CCATTGAATTCTATAATAATGGTCAACTAATTGATATGGTTCGAAATATGGGAATAGTTACTGTAAGAACTTTT-
GATTCT
TATAGAATAACAATTGACATGATTAGACCAGCTGCTATGACTCAATACGTTCAACGAATTTTTCCACAAGGTGG-
ACCTTA
TCATTTTCAGGCTACATATATGTTAACATTAAGTATATTAGATGCTACCACAGAGTCCGTTCTATGTGATTCTC-
ATTCAG
TAGAATATTCAATAGTAGCAAACGTCAGAAGAGATTCAGCAATGCCAGCTGGAACTGTTTTTCAACCGGGATTT-
CCATGG
GAACACACACTATCCAATTACACTGTTGCTCAAGAAGATAATTTAGAAAGATTATTGTTAATCGCATCTGTGAA-
GAGAAT
GGTAATGTAGATAAGCTAGAAGACTAAACATCTTCTATGCGGCCTACATACCATGTAGCATGAATCACGACTGG-
GTTTAG
TCCATGCTCGCATAGGGGCAAATATGCATGATATGGATGATCCCCAGAAGGATGAAATGTGAACTATGTGGCT
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Journal of Clinical Microbiology 30:2129-34. [0260] 35. Zeng, C.
Q., et al. 1998. Journal of Virology 72:201-8. [0261] 36. Zeng, C.
Q., et al. 1996. Journal of Virology 70:2736-42.
[0262] Any patents or publications mentioned in this specification
are incorporated herein by reference to the same extent as if each
individual publication is specifically and individually indicated
to be incorporated by reference.
[0263] The compositions and methods described herein are presently
representative of preferred embodiments, exemplary, and not
intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art. Such
changes and other uses can be made without departing from the scope
of the invention as set forth in the claims. All numerical ranges
described herein include all integers and decimal values within the
range and are also inclusive of the endpoints.
Sequence CWU 1
1
471884PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C
strain ASP88 VP2 protein 1Met Ile Ser Arg Asn Arg Arg Arg Asn Asn
Gln Gln Lys Asp Ile Gly1 5 10 15Lys Glu Lys Gln Leu Glu Thr Ile Ile
Asp Lys Glu Val Lys Glu Asn 20 25 30Lys Asp Ser Thr Lys Glu Asp Lys
Leu Val Val Thr Glu Glu Ser Asn 35 40 45Gly Asp Val Thr Ala Val Lys
Glu Gln Ser Asn Asn Ile Asn Leu Gln 50 55 60Lys Asn Asp Leu Val Lys
Glu Val Met Asn Ile Gln Asn Gln Thr Leu65 70 75 80Asn Thr Val Val
Ala Glu Asn Lys Val Glu Ile Glu Glu Ile Val Lys 85 90 95Lys Tyr Ile
Pro Ser Tyr Asn Thr Asp Ser Leu Ile Val Lys Lys Leu 100 105 110Thr
Glu Ile Gln Glu Ser Ser Ala Lys Thr Tyr Asn Thr Leu Phe Arg 115 120
125Leu Phe Thr Pro Val Lys Ser Tyr Leu Tyr Asp Ile Asn Gly Glu Lys
130 135 140Lys Leu Ser Thr Arg Trp Tyr Trp Lys Leu Leu Lys Asp Asp
Leu Pro145 150 155 160Ala Gly Asp Tyr Ser Val Arg Gln Phe Phe Leu
Ser Leu Tyr Leu Asn 165 170 175Val Leu Glu Gly Met Pro Asp Tyr Ile
Met Leu Arg Asp Met Ala Val 180 185 190Asp Asn Pro Tyr Ser Ala Glu
Ala Gly Lys Ile Val Asp Gly Lys Ser 195 200 205Lys Glu Ile Leu Val
Glu Leu Tyr Gln Asp Gln Met Thr Glu Gly Tyr 210 215 220Ile Arg Arg
Tyr Met Ser Glu Leu Arg His Lys Ile Ser Gly Glu Thr225 230 235
240Asn Thr Ala Lys Tyr Pro Ala Ile Leu His Pro Val Asp Asn Glu Leu
245 250 255Asn Gln Tyr Phe Leu Glu His Gln Leu Ile Gln Pro Leu Thr
Thr Arg 260 265 270Asn Ile Ala Glu Leu Ile Pro Thr Gln Leu Tyr His
Asp Pro Asn Tyr 275 280 285Val Phe Asn Ile Asp Ala Ala Phe Leu Thr
Asn Ser Arg Phe Val Pro 290 295 300Pro Tyr Leu Thr Gln Asp Arg Ile
Gly Leu His Asp Gly Phe Glu Ser305 310 315 320Ile Trp Asp Ser Lys
Thr His Ala Asp Tyr Val Ser Ala Arg Arg Phe 325 330 335Ile Pro Asp
Leu Thr Glu Leu Val Asp Ala Glu Lys Gln Ile Lys Glu 340 345 350Met
Ala Ala His Leu Gln Leu Glu Ala Ile Thr Val Gln Val Glu Ser 355 360
365Gln Phe Leu Ala Gly Ile Ser Ala Ala Ala Ala Asn Glu Ala Phe Lys
370 375 380Phe Ile Ile Gly Ser Val Leu Ser Thr Arg Thr Ile Ala Val
Glu Phe385 390 395 400Ile Thr Ser Asn Tyr Met Ser Leu Ala Ser Cys
Met Tyr Leu Met Thr 405 410 415Ile Met Pro Ser Glu Ile Phe Leu Arg
Glu Ser Leu Val Ala Met Arg 420 425 430Leu Ala Ile Ile Asn Thr Leu
Ile Tyr Pro Ala Leu Gly Leu Ala Gln 435 440 445Met His Tyr Gln Ala
Gly Glu Val Arg Thr Pro Phe Glu Leu Ala Glu 450 455 460Met Arg Val
Ala Asn Arg Ser Ile Arg Gln Trp Leu His His Cys Asn465 470 475
480Thr Leu Gln Phe Gly Arg Gln Ile Thr Glu Gly Ile Ile His Leu Arg
485 490 495Phe Thr Asn Asp Ile Met Thr Gly Arg Ile Val Asn Leu Phe
Ser Thr 500 505 510Met Leu Val Ala Leu Ser Ser Gln Pro Phe Ala Thr
Tyr Pro Leu Asp 515 520 525Tyr Lys Arg Ser Val Gln Arg Ala Leu Gln
Leu Leu Ser Asn Arg Thr 530 535 540Ala Gln Ile Ala Asp Leu Thr Arg
Leu Ile Val Tyr Asn Tyr Thr Thr545 550 555 560Leu Ser Ala Cys Ile
Val Met Asn Met His Leu Val Gly Thr Leu Thr 565 570 575Val Glu Arg
Ile Gln Ala Thr Ser Leu Thr Ser Leu Met Met Leu Ile 580 585 590Ser
Asn Lys Thr Val Ile Pro Glu Pro Ser Ser Leu Phe Ser Tyr Phe 595 600
605Ser Ser Asn Ile Asn Phe Leu Thr Asn Tyr Asn Glu Gln Ile Asp Asn
610 615 620Val Val Ala Glu Ile Met Ala Ala Tyr Arg Leu Asn Leu Tyr
Gln Gln625 630 635 640Lys Met Leu Met Leu Val Thr Arg Phe Val Ser
Arg Leu Tyr Ile Phe 645 650 655Asp Ala Pro Lys Ile Pro Pro Asp Gln
Met Tyr Arg Leu Arg Asn Arg 660 665 670Leu Arg Asn Ile Pro Val Glu
Arg Arg Arg Ala Asp Val Phe Arg Ile 675 680 685Ile Met Asn Asn Arg
Asp Leu Ile Glu Lys Thr Ser Glu Arg Ile Cys 690 695 700Gln Gly Val
Leu Leu Ser Tyr Thr Pro Met Pro Leu Thr Tyr Val Glu705 710 715
720Asp Val Gly Leu Thr Asn Val Ile Asn Asp Thr Asn Asn Phe Gln Ile
725 730 735Ile Asn Ile Glu Glu Ile Glu Lys Thr Gly Asp Tyr Ser Ala
Ile Thr 740 745 750Asn Ala Leu Leu Arg Asp Thr Pro Ile Ile Leu Lys
Gly Ala Ile Pro 755 760 765Tyr Val Thr Asn Ser Ser Val Ile Asp Val
Leu Ser Lys Val Asp Thr 770 775 780Thr Val Phe Ala Ser Ile Val Lys
Asp Arg Asp Ile Ser Lys Leu Lys785 790 795 800Pro Ile Lys Phe Ile
Ile Asn Ser Asp Ser Ser Glu Tyr Tyr Leu Val 805 810 815His Asn Asn
Lys Trp Thr Pro Thr Thr Thr Thr Ala Val Tyr Lys Ala 820 825 830Arg
Ser Gln Gln Phe Asp Ile Gln His Ser Val Ser Met Leu Glu Ser 835 840
845Asn Leu Phe Phe Val Val Tyr Asn Asp Leu Phe Lys Tyr Ile Lys Thr
850 855 860Thr Thr Val Leu Pro Ile Asn Ala Val Ser Tyr Asp Gly Ala
Arg Ile865 870 875 880Met Gln Glu Thr233PRTArtificial
sequenceAntigenic epitope of human rotavirus group C VP2 protein
2Leu Glu Thr Ile Ile Asp Lys Glu Val Lys Glu Asn Lys Asp Ser Thr1 5
10 15Lys Asp Glu Lys Leu Val Val Thr Glu Glu Ser Asn Gly Asp Val
Thr 20 25 30Ala333PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 3Leu Glu Thr Ile Ile Asn Lys Glu Val
Lys Glu Asn Lys Asp Ser Met1 5 10 15Lys Glu Asp Lys Leu Val Val Thr
Glu Glu Ser Asn Gly Asp Val Thr 20 25 30Thr433PRTArtificial
SequenceAntigenic epitope of porcine rotavirus group C VP2 protein
4Thr Glu Asn Val Glu Glu Lys Glu Ile Lys Glu Ala Lys Glu Gln Val1 5
10 15Lys Asp Glu Lys Gln Val Ile Thr Glu Glu Asn Val Asp Ser Pro
Lys 20 25 30Asp522PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 5Lys Leu Thr Glu Ile Gln Glu Ser Ser
Ala Lys Thr Tyr Asn Thr Leu1 5 10 15Phe Arg Leu Phe Thr Pro
20618PRTArtificial SequenceAntigenic epitope of porcine rotavirus
group C VP2 protein 6Asn Tyr Arg Asn Ser Arg Ile Lys Cys Gln Thr
Tyr Asn Lys Leu Phe1 5 10 15Arg Leu718PRTArtificial
SequenceAntigenic epitope of human rotavirus group C VP2 protein
7Leu Asn Val Leu Glu Gly Met Pro Asp Tyr Ile Met Leu Arg Asp Met1 5
10 15Ala Val818PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 8Leu Asn Val Leu Glu Glu Met Pro Asp
Tyr Ile Met Leu Arg Asp Met1 5 10 15Ala Val918PRTArtificial
SequenceAntigenic epitope of porcine rotavirus group C VP2 protein
9Leu Asn Val Leu Asp Glu Met Pro Asp Tyr Val Met Leu Arg Asp Met1 5
10 15Ala Val1029PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 10Ala Ala His Leu Gln Leu Glu Ala Ile
Thr Val Gln Val Glu Ser Gln1 5 10 15Phe Leu Ala Gly Ile Ser Ala Ala
Ala Ala Asn Glu Ala 20 251119PRTArtificial SequenceAntigenic
epitope of porcine rotavirus group C VP2 protein 11Leu Gln Cys Lys
Leu Asn His Asn Ser Trp Gln Glu Leu Val His Gly1 5 10 15Arg Asn
Glu1213PRTArtificial SequenceAntigenic epitope of human rotavirus
group C VP2 protein 12Leu Ser Ala Cys Ile Val Met Asn Met His Leu
Val Gly1 5 101317PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 13Ile Pro Pro Asp Gln Met Tyr Arg Leu
Arg Asn Arg Leu Arg Asn Ile1 5 10 15Pro1420DNAArtificial
Sequencesynthetic primer 14agccacatag ttcacatttc
201519DNAArtificial Sequencesynthetic primer 15atctcattca caatggatg
1916884PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C
Bristol strain VP2 protein 16Met Ile Ser Arg Asn Arg Arg Arg Asn
Asn Gln Gln Lys Asn Ile Glu1 5 10 15Lys Glu Lys Gln Leu Glu Thr Ile
Ile Asn Lys Glu Val Lys Glu Asn 20 25 30Lys Asp Ser Met Lys Glu Asp
Lys Leu Val Val Thr Glu Glu Ser Asn 35 40 45Gly Asp Val Thr Thr Ala
Lys Glu Gln Ser Asn Asn Ile Asn Leu Gln 50 55 60Lys Asn Asp Leu Val
Lys Glu Val Met Asn Ile Gln Asn Gln Thr Leu65 70 75 80Asn Thr Val
Val Thr Glu Asn Lys Val Glu Ile Glu Glu Ile Val Lys 85 90 95Lys Tyr
Ile Pro Ser Tyr Asn Thr Asp Ser Leu Ile Val Lys Lys Leu 100 105
110Thr Glu Ile Gln Glu Ser Ser Ala Lys Thr Tyr Asn Thr Leu Phe Arg
115 120 125Leu Phe Thr Pro Val Lys Ser Tyr Leu Tyr Asp Ile Asn Gly
Glu Lys 130 135 140Lys Leu Ser Thr Arg Trp Tyr Trp Lys Leu Leu Lys
Asp Asp Leu Pro145 150 155 160Ala Gly Asp Tyr Ser Val Arg Gln Phe
Phe Leu Ser Leu Tyr Leu Asn 165 170 175Val Leu Glu Glu Met Pro Asp
Tyr Ile Met Leu Arg Asp Met Ala Val 180 185 190Asp Asn Pro Tyr Ser
Ala Glu Ala Gly Lys Ile Val Asp Gly Lys Ser 195 200 205Lys Glu Ile
Leu Ile Glu Leu Tyr Gln Asp Gln Met Thr Glu Gly Tyr 210 215 220Ile
Arg Arg Tyr Met Ser Glu Leu Arg His Lys Ile Ser Gly Glu Thr225 230
235 240Asn Thr Ala Lys Tyr Pro Ala Ile Leu His Pro Val Asp Asn Glu
Leu 245 250 255Asn Gln Tyr Phe Leu Glu His Gln Leu Ile Gln Pro Leu
Thr Thr Arg 260 265 270Asn Ile Ala Glu Leu Ile Pro Thr Gln Leu Tyr
His Asp Pro Asn Tyr 275 280 285Val Phe Asn Ile Asp Ala Ala Phe Leu
Thr Asn Ser Arg Phe Val Pro 290 295 300Pro Tyr Leu Thr Gln Asp Arg
Ile Gly Leu His Asp Gly Phe Glu Ser305 310 315 320Ile Trp Asp Ser
Lys Thr His Ala Asp Tyr Val Ser Ala Arg Arg Phe 325 330 335Ile Pro
Asp Leu Thr Glu Leu Val Asp Ala Glu Lys Gln Ile Lys Glu 340 345
350Met Ala Ala His Leu Gln Leu Glu Ala Ile Thr Val Gln Val Glu Ser
355 360 365Gln Phe Leu Ala Gly Ile Ser Ala Ala Ala Ala Asn Glu Ala
Phe Lys 370 375 380Phe Ile Ile Gly Ser Val Leu Ser Thr Arg Thr Ile
Ala Val Glu Phe385 390 395 400Ile Thr Ser Asn Tyr Met Ser Leu Ala
Ser Cys Met Tyr Leu Met Thr 405 410 415Ile Met Pro Ser Glu Ile Phe
Leu Arg Glu Ser Leu Val Ala Met Gln 420 425 430Leu Ala Ile Ile Asn
Thr Leu Ile Tyr Pro Ala Leu Gly Leu Ala Gln 435 440 445Met His Tyr
Gln Ala Gly Glu Val Arg Thr Pro Phe Glu Leu Ala Glu 450 455 460Met
Gln Val Ala Asn Arg Ser Ile Arg Gln Trp Leu His His Cys Asn465 470
475 480Thr Leu Gln Phe Gly Arg Gln Ile Thr Glu Gly Ile Ile His Leu
Arg 485 490 495Phe Thr Asn Asp Ile Met Thr Gly Arg Ile Val Asn Leu
Phe Ser Thr 500 505 510Met Leu Val Ala Leu Ser Ser Gln Pro Phe Ala
Thr Tyr Pro Leu Asp 515 520 525Tyr Lys Arg Ser Val Gln Arg Ala Leu
Gln Leu Leu Ser Asn Arg Thr 530 535 540Ala Gln Ile Ala Asp Leu Thr
Arg Leu Ile Val Tyr Asn Tyr Thr Thr545 550 555 560Leu Ser Ala Cys
Ile Val Met Asn Met His Leu Val Gly Thr Leu Thr 565 570 575Val Glu
Arg Ile Gln Ala Thr Ser Leu Thr Ser Leu Met Met Leu Ile 580 585
590Ser Asn Lys Thr Val Ile Pro Glu Pro Ser Ser Leu Phe Ser Tyr Phe
595 600 605Ser Ser Asn Ile Asn Phe Leu Thr Asn Tyr Asn Glu Gln Ile
Asp Asn 610 615 620Val Val Ala Glu Ile Met Ala Ala Tyr Arg Leu Asn
Leu Tyr Gln Gln625 630 635 640Lys Met Leu Met Leu Val Thr Arg Phe
Val Ser Arg Leu Tyr Ile Phe 645 650 655Asp Ala Pro Lys Ile Pro Pro
Asp Gln Met Tyr Arg Leu Arg Asn Arg 660 665 670Leu Arg Asn Ile Pro
Val Glu Arg Arg Arg Ala Asp Val Phe Arg Ile 675 680 685Ile Met Asn
Asn Arg Asp Leu Ile Glu Lys Thr Ser Glu Arg Ile Cys 690 695 700Gln
Gly Val Leu Leu Ser Tyr Thr Pro Met Pro Leu Thr Tyr Val Glu705 710
715 720Asp Val Gly Leu Thr Asn Val Ile Asn Asp Thr Asn Asn Phe Gln
Ile 725 730 735Ile Asn Ile Glu Glu Ile Glu Lys Thr Gly Asp Tyr Ser
Ala Ile Thr 740 745 750Asn Ala Leu Leu Arg Asp Thr Pro Ile Ile Leu
Lys Gly Ala Ile Pro 755 760 765Tyr Val Thr Asn Ser Ser Val Ile Asp
Val Leu Ser Lys Val Asp Thr 770 775 780Thr Val Phe Ala Ser Ile Val
Lys Asp Arg Asp Ile Ser Lys Leu Lys785 790 795 800Pro Ile Lys Phe
Ile Ile Asn Ser Asp Ser Ser Glu Tyr Tyr Leu Val 805 810 815His Asn
Asn Lys Trp Thr Pro Thr Thr Thr Thr Ala Val Tyr Lys Ala 820 825
830Arg Ser Gln Gln Phe Asp Ile Gln His Ser Val Ser Met Leu Glu Ser
835 840 845Asn Leu Phe Phe Val Val Tyr Asn Asp Leu Phe Lys Tyr Ile
Lys Thr 850 855 860Thr Thr Val Leu Pro Ile Asn Ala Val Ser Tyr Asp
Gly Ala Arg Ile865 870 875 880Met Gln Glu Thr17884PRTPorcine
rotavirusMISC_FEATUREporcine rotavirus group C Cowden strain VP2
protein 17Met Ile Ser Arg Asn Arg Arg Arg Asn Thr Gln Gln Lys Asp
Ala Glu1 5 10 15Lys Glu Lys Gln Thr Glu Asn Val Glu Glu Lys Glu Ile
Lys Glu Ala 20 25 30Lys Glu Gln Val Lys Asp Glu Lys Gln Val Ile Thr
Glu Glu Asn Val 35 40 45Asp Ser Pro Lys Asp Val Lys Glu Gln Ser Asn
Thr Val Asn Leu Gln 50 55 60Lys Asn Asp Leu Val Lys Glu Val Ile Asn
Ile Gln Asn Gln Thr Leu65 70 75 80Asn Thr Ile Val Ala Glu Asn Lys
Val Glu Ile Glu Glu Val Val Lys 85 90 95Lys Tyr Ile Pro Ser Tyr Ser
Thr Asp Lys Leu Ile Val Lys Lys Leu 100 105 110Thr Glu Ile Gln Glu
Ser Ser Ala Lys Thr Tyr Asn Lys Leu Phe Arg 115 120 125Leu Phe Thr
Pro Val Lys Ser Tyr Leu Tyr Asp Val Asn Gly Glu Lys 130 135 140Lys
Leu Ser Thr Arg Trp Tyr Trp Lys Leu Leu Lys Asp Asp Leu Pro145 150
155 160Ala Gly Asp Tyr Ser Val Arg Gln Phe Phe Leu Ser Leu Tyr Leu
Asn 165 170 175Val Leu Asp Glu Met Pro Asp Tyr Val Met Leu Arg Asp
Met Ala Val 180 185 190Asp Asn Pro Tyr Ser Ala Glu Ala Gly Lys Ile
Val Asp Glu Lys Ser 195 200 205Lys Glu Ile Leu Val Glu Ile Tyr Gln
Asp Gln Met Thr Glu Gly Tyr 210 215
220Ile Arg Arg Tyr Met Ser Asp Leu Arg His Arg Ile Ser Gly Glu
Thr225 230 235 240Asn Thr Ala Lys Tyr Pro Ala Ile Leu His Pro Val
Asp Glu Glu Leu 245 250 255Asn Lys Tyr Phe Leu Glu His Gln Leu Ile
Gln Pro Leu Thr Thr Arg 260 265 270Asn Ile Ala Glu Leu Ile Pro Thr
Gln Leu Tyr His Asp Pro Asn Tyr 275 280 285Val Phe Asn Ile Asp Ala
Ala Phe Leu Thr Asn Ser Arg Phe Val Pro 290 295 300Pro Tyr Leu Thr
Gln Asp Arg Ile Gly Leu His Asp Gly Phe Glu Ser305 310 315 320Ile
Trp Asp Ala Lys Thr His Ala Asp Tyr Val Ser Ala Arg Arg Phe 325 330
335Val Pro Asp Leu Thr Glu Leu Val Asp Ala Glu Lys Gln Met Lys Glu
340 345 350Met Ala Ala His Leu Gln Leu Glu Ala Ile Thr Val Gln Val
Glu Ser 355 360 365Gln Phe Leu Ala Gly Ile Ser Ala Ala Ala Ala Asn
Glu Ala Phe Lys 370 375 380Phe Ile Ile Gly Thr Val Leu Ser Thr Arg
Thr Ile Ala Val Glu Phe385 390 395 400Ile Thr Ser Asn Tyr Met Ser
Leu Ala Ser Cys Met Tyr Leu Met Thr 405 410 415Ile Met Pro Ser Glu
Ile Phe Leu Arg Glu Ser Leu Val Ala Met Gln 420 425 430Leu Ala Val
Ile Asn Thr Leu Thr Tyr Pro Ala Leu Gly Leu Ala Gln 435 440 445Met
His Tyr Gln Ala Gly Glu Ile Arg Thr Pro Phe Glu Leu Ala Glu 450 455
460Met Gln Val Ala Asn Arg Pro Ile Arg Gln Trp Leu His His Cys
Asn465 470 475 480Thr Leu Gln Phe Gly Arg Gln Val Thr Glu Gly Val
Thr His Leu Arg 485 490 495Phe Thr Asn Asp Ile Met Thr Gly Arg Ile
Val Asn Leu Phe Ser Thr 500 505 510Met Leu Val Ala Leu Ser Ser Gln
Pro Phe Ala Thr Tyr Pro Leu Asp 515 520 525Tyr Lys Arg Ser Val Gln
Arg Ala Leu Gln Leu Leu Ser Asn Arg Thr 530 535 540Ala Gln Ile Ala
Asp Leu Thr Arg Leu Ile Val Tyr Asn Tyr Thr Thr545 550 555 560Leu
Ser Ala Cys Ile Val Met Asn Met His Leu Val Gly Thr Leu Thr 565 570
575Val Glu Arg Ile Gln Ala Thr Ala Leu Thr Ser Leu Ile Met Leu Ile
580 585 590Ser Asn Lys Thr Val Ile Pro Glu Pro Ser Ser Leu Phe Ser
Tyr Phe 595 600 605Ser Ser Asn Ile Asn Phe Leu Thr Asn Tyr Asn Glu
Gln Ile Asp Asn 610 615 620Val Val Ala Glu Ile Met Ala Ala Tyr Arg
Leu Asp Leu Tyr Gln Gln625 630 635 640Lys Met Leu Met Leu Val Thr
Arg Phe Val Ser Arg Leu Tyr Ile Phe 645 650 655Asp Ala Pro Lys Ile
Pro Pro Asp Gln Met Tyr Arg Leu Arg Asn Arg 660 665 670Leu Arg Asn
Ile Pro Val Glu Arg Arg Arg Ala Asp Val Phe Arg Ile 675 680 685Ile
Met Asn Asn Arg Asp Leu Ile Glu Lys Thr Ser Glu Arg Ile Cys 690 695
700Gln Gly Val Leu Leu Ser Tyr Ser Pro Met Pro Leu Thr Tyr Val
Glu705 710 715 720Asp Val Gly Leu Thr Asn Val Val Asn Asp Thr Asn
Gly Phe Gln Ile 725 730 735Ile Asn Ile Glu Glu Ile Glu Lys Thr Gly
Asp Tyr Ser Ala Ile Thr 740 745 750Asn Ala Leu Leu Arg Asp Thr Pro
Ile Ile Leu Lys Gly Ala Ile Pro 755 760 765Tyr Val Thr Asn Ser Ser
Val Ile Asp Val Leu Ser Lys Ile Asp Thr 770 775 780Thr Val Phe Ala
Ser Ile Val Lys Asp Arg Asp Ile Ser Lys Leu Lys785 790 795 800Pro
Ile Lys Phe Thr Ile Asn Ser Asp Ser Ser Glu Tyr Tyr Leu Val 805 810
815His Asn Asn Lys Trp Thr Pro Thr Thr Thr Thr Ala Val Tyr Lys Ala
820 825 830Arg Ser Gln Gln Phe Asn Ile Gln His Ser Val Ser Met Leu
Glu Ser 835 840 845Asn Leu Phe Phe Val Val Tyr Asn Asp Leu Phe Lys
Tyr Ile Lys Thr 850 855 860Thr Thr Val Leu Pro Ile Asn Ala Val Ser
Tyr Asp Gly Ala Arg Ile865 870 875 880Met Gln Glu Thr182720DNAHuman
rotavirus Cmisc_featureHuman rotavirus group C strain ASP88 VP2
nucleotide sequence 18tcgaggacaa atcgtccaag atgataagca gaaacaggcg
cagaaataac caacaaaaag 60atataggaaa agagaaacaa ttagagacta taattgacaa
agaagtaaag gaaaacaaag 120attctacaaa agaagataag ctagtagtta
cggaagaaag taatggtgac gtcacagctg 180ttaaagaaca atcgaataat
attaatttac aaaagaatga tttggttaaa gaagtcatga 240atatacagaa
tcaaacatta aatacagtag ttgctgagaa taaagttgaa atagaagaaa
300tagttaaaaa atacattccc tcatataata ctgacagcct tattgttaaa
aagttaactg 360aaatccagga atcaagtgct aaaacatata atacattatt
cagattattt actccagtta 420aaagttattt atatgacata aatggtgaga
aaaaattatc gactagatgg tattggaaat 480tgctcaaaga tgatttacct
gctggtgatt actcagttag acaattcttc ctgtcactat 540atttaaatgt
tttagaggga atgcccgatt acataatgct tcgtgatatg gcagtggata
600acccatattc agcagaagca ggtaaaatcg tagatggaaa gtctaaagaa
attttagttg 660aactatatca agaccaaatg acagaagggt atattagaag
atatatgtct gaattaagac 720ataaaatatc tggagaaaca aatactgcaa
aatatccagc tattctacat cccgtggata 780atgagcttaa tcaatacttt
cttgagcatc agttaattca accattaact acaagaaata 840ttgcagaatt
gattccaact caattatatc atgatccaaa ttacgttttt aatattgatg
900cagccttttt aacaaattca agatttgttc caccatactt aacacaggat
aggattggat 960tacatgatgg attcgaatca atatgggatt caaaaaccca
tgctgattac gtttcagcta 1020gaagatttat acctgattta actgaactgg
tagatgctga aaagcaaata aaagaaatgg 1080ctgcacattt acaactagag
gctattacag tacaggttga atcacaattt ttagcgggaa 1140ttagtgctgc
tgcagctaat gaagcgttca aatttataat tggctcagtt ttatctacca
1200gaacaatagc tgtagaattc ataacctcaa actatatgtc gttagcatca
tgtatgtatt 1260taatgactat tatgccatca gagattttct tgagagaatc
attagttgct atgcgattag 1320caataataaa tacccttatt tatccagctc
taggtttagc gcaaatgcat tatcaagcag 1380gtgaagtgag gaccccattc
gaattagctg agatgcgagt agctaataga tctattagac 1440aatggttaca
tcattgtaat acacttcaat ttggtagaca gataacggaa gggataattc
1500atctacgatt tactaatgat atcatgacag gtaggatagt gaacttattt
tcaacaatgc 1560tagtagcttt atcatctcag ccattcgcta catatccttt
agactataaa agatctgtac 1620aaagagcatt acaactttta tcaaatagaa
cagcccaaat agcagattta accagattaa 1680tagtatacaa ttatactaca
ttatctgcat gtatagtcat gaatatgcat ttagtaggaa 1740ctcttactgt
tgaacgtata caggccactt ctctaacttc tttaatgatg ttaatttcta
1800ataagacagt tattccagaa ccatcgtctc ttttttcata tttctctagt
aacattaatt 1860ttcttacaaa ttataatgag caaattgata atgtggtagc
agaaataatg gccgcatata 1920gattgaattt atatcaacag aaaatgttga
tgctcgttac caggtttgtg tcaaggttgt 1980acatatttga tgctcctaaa
ataccgccag atcagatgta tagattaaga aaccgattaa 2040gaaatattcc
agttgaaaga agaagagctg atgtgttcag aattattatg aataatagag
2100atttaatcga aaaaacatca gaacgtatat gtcagggtgt gttgttatct
tatacaccaa 2160tgcctttaac ttacgttgaa gatgtcgggt taacaaatgt
aattaatgac actaataact 2220tccaaataat taatatagaa gaaattgaga
agaccggtga ctattcagcc ataacgaatg 2280cattacttcg ggatactcca
attatattga aaggtgcgat tccatatgtt actaactcat 2340cagtaattga
tgttttatct aaagtggaca ccacagtgtt cgcaagcatc gtaaaagata
2400gggatatttc aaagttaaaa ccaataaaat tcataattaa ttcagattca
tccgaatatt 2460atttagtaca caataataaa tggacaccaa caacaactac
agcagtatat aaagctagat 2520ctcagcaatt tgatatacaa cattcagtat
caatgctaga gtcaaactta ttttttgtgg 2580tatataatga tttatttaaa
tacattaaaa ccactacagt tctgccgata aatgctgtct 2640cttatgatgg
tgcaagaatt atgcaagaaa cataaatgat tgtatagtat catcttgtaa
2700cgacctcaaa ctctgtggct 2720192736DNAHuman rotavirus
Cmisc_featureHuman rotavirus group C Bristol strain VP2 nucleotide
sequence 19ggcttaaaaa gatcagttga ggacaaatcg ttcaagatga taagcagaaa
caggcgcaga 60aataatcaac aaaaaaacat agaaaaagag aaacaattag agactataat
taacaaagaa 120gttaaggaaa acaaagattc tatgaaagaa gataagctag
tagttacaga agaaagcaat 180ggagacgtca caactgctaa agaacaatcg
aataatatta atttacaaaa gaatgatttg 240gttaaagaag tcatgaatat
acagaatcaa acattaaata cagtagttac tgagaataaa 300gttgaaatag
aagaaatagt taaaaaatac attccatcat ataatactga tagcctcatt
360gttaaaaagt taactgaaat ccaggaatca agtgctaaaa catataatac
attgtttaga 420ttatttactc cagttaaaag ttatttatat gatataaatg
gtgagaaaaa attatcgact 480agatggtatt ggaaattgct caaagatgat
ttacctgctg gtgattactc agttagacaa 540ttcttcctgt cactatattt
aaatgtttta gaggaaatgc ccgattacat aatgcttcgt 600gatatggcag
tggataaccc atattcagca gaagcaggta aaatcgtaga tggaaagtct
660aaagaaattt tgattgaact atatcaagac cagatgacag aaggatatat
tagaagatat 720atgtctgaat taagacataa aatatctgga gagacaaata
ctgcaaaata cccagctatt 780ctacatcccg tggataatga acttaatcaa
tactttcttg agcatcagtt aattcaacca 840ttaactacaa ggaacattgc
agaattgatt ccaactcaat tatatcatga tccaaattac 900gtttttaata
ttgatgcagc ctttttaaca aattcaagat ttgttccacc atacttaaca
960caggatagga ttggattaca tgatggattt gaatcaatat gggattcaaa
aactcatgct 1020gattacgttt cagctagaag atttatacct gatttaactg
aactggtgga tgctgaaaag 1080caaataaaag aaatggctgc acatttacaa
ctagaggcta ttacggtaca ggttgaatca 1140caatttttag caggaattag
tgctgctgca gctaatgaag cgtttaaatt tataattggc 1200tcagttttat
ctaccagaac aatagctgta gaattcataa cctcaaacta tatgtcacta
1260gcatcatgta tgtatttaat gactattatg ccatcagaga ttttcttaag
agaatcatta 1320gttgctatgc aattagcaat aataaatacc cttatttatc
cagctctagg tttagcgcaa 1380atgcattatc aagcaggtga agtgaggact
ccattcgaat tagctgaaat gcaagtagct 1440aatagatcta ttagacaatg
gttacatcat tgtaatacac ttcaatttgg tagacagata 1500acggaaggga
taattcatct acgatttact aatgatatca tgacaggcag gatagtgaac
1560ttattttcaa caatgttagt ggctctatca tctcagcctt tcgctacata
tcctttagac 1620tataaaagat ctgtacaaag agcgttacaa cttttatcaa
atagaacagc tcaaatagca 1680gatttaacca gattaatagt atacaattat
actacattat ctgcttgtat agttatgaat 1740atgcatttag taggaactct
tactgttgaa cgtatacaag ccacttctct aacttcttta 1800atgatgttaa
tctctaataa gacagttatt ccggaaccat cgtctctttt ttcatatttc
1860tctagtaaca ttaattttct tacaaattat aatgagcaaa ttgataatgt
ggtagcagaa 1920ataatggccg catatagatt gaatttatat caacagaaaa
tgttgatgct cgttaccaga 1980tttgtgtcaa agttatacat atttgatgct
cctaagatac caccagatca gatgtataga 2040ttaagaaacc gattaagaaa
tattccagtt gaaagaagaa gagctgacgt attcagaatt 2100attatgaata
atagagattt aatcgaaaaa acatcagaac gtatatgcca gggtgtgctg
2160ttatcttata caccaatgcc tttaacttac gttgaagatg tcgggttaac
aaatgtaatt 2220aatgacacta atagctttca aataattaat attgaagaaa
ttgagaagac cggtgactat 2280tcagctataa cgaatgcatt acttcgggat
actccaatca tattgaaagg tgcgattcca 2340tatgttacta actcatcagt
aattgatgtt ttatctaaag tggacaccac agtgttcgca 2400agcattgtaa
aagataggga catttcaaag ttaaaaccaa taaaattcat aattaattca
2460gattcatccg aatattattt agtacataat aataaatgga caccaacaac
aactacagca 2520gtatataaag ctagatctca gcaatttgat atacaacatt
cagtatcaat gctagagtca 2580aacttatttt ttgtggtata taatgattta
tttaaataca ttaaaaccac tacagttctg 2640ccgataaatg ctgtctctta
tgacggtgca agaattatgc aagaaacata aatgattgta 2700tagtatcatc
ttgtgacgac ctcaaacttt gtggct 27362018DNAArtificial
Sequencesynthetic primer 20agccacatga tcttgttt 182118DNAArtificial
Sequencesynthetic primer 21ggcatttaaa aaagaaga 182220DNAArtificial
Sequencesynthetic primer 22tcgaggacaa atcgtccaag
202319DNAArtificial Sequencesynthetic primer 23agccacagag tttgaggtc
1924395PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C VP6
consensus sequence 24Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val
Ser Asp Leu Lys Lys1 5 10 15Lys Val Val Val Gly Thr Ile Tyr Thr Asn
Val Glu Asp Val Val Gln 20 25 30Gln Thr Asn Glu Leu Ile Arg Thr Leu
Asn Gly Asn Ile Phe His Thr 35 40 45Gly Gly Ile Gly Thr Gln Pro Gln
Lys Glu Trp Asn Phe Gln Leu Pro 50 55 60Gln Leu Gly Thr Thr Leu Leu
Asn Leu Asp Asp Asn Tyr Val Gln Ser65 70 75 80Thr Arg Gly Ile Ile
Asp Phe Leu Ser Ser Phe Ile Glu Ala Val Cys 85 90 95Asp Asp Glu Ile
Val Arg Glu Ala Ser Arg Asn Gly Met Gln Pro Gln 100 105 110Ser Pro
Ala Leu Ile Leu Leu Ser Ser Ser Lys Phe Lys Thr Ile Asn 115 120
125Phe Asn Asn Ser Ser Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg
130 135 140Arg Glu Asn Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Phe
Glu Tyr145 150 155 160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro
Gln Phe Gly Ser Val 165 170 175Met Gly Leu Arg Tyr Tyr Thr Thr Ser
Asn Thr Cys Gln Ile Ala Ala 180 185 190Phe Asp Ser Thr Leu Ala Glu
Asn Ala Pro Asn Asn Thr Gln Arg Phe 195 200 205Val Tyr Asn Gly Arg
Leu Lys Arg Pro Ile Ser Asn Val Leu Met Lys 210 215 220Ile Glu Ala
Gly Ala Pro Asn Ile Ser Asn Pro Thr Ile Leu Pro Asp225 230 235
240Pro Asn Asn Gln Thr Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn
245 250 255Gly Thr Phe Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile
Asp Met 260 265 270Val Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe
Asp Ser Tyr Arg 275 280 285Ile Thr Ile Asp Met Ile Arg Pro Ala Ala
Met Thr Gln Tyr Val Gln 290 295 300Arg Ile Phe Pro Gln Gly Gly Pro
Tyr His Phe Gln Ala Thr Tyr Met305 310 315 320Leu Thr Leu Ser Ile
Leu Asp Ala Thr Thr Glu Ser Val Leu Cys Asp 325 330 335Ser His Ser
Val Glu Tyr Ser Ile Val Ala Asn Val Arg Arg Asp Ser 340 345 350Ala
Met Pro Ala Gly Thr Val Phe Gln Pro Gly Phe Pro Trp Glu His 355 360
365Thr Leu Ser Asn Tyr Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu
370 375 380Leu Leu Ile Ala Ser Val Lys Arg Met Val Met385 390
395251185DNAHuman rotavirus Cmisc_featureHuman rotavirus group C
VP6 Bristol nucleotide sequence 25atggatgtac ttttttctat agcgaaaact
gtgtcagatc ttaaaaagaa agttgtggtt 60ggaacaattt atactaatgt agaagatgtt
gtacaacaga cgaatgaatt gattagaact 120ttaaatggaa atatttttca
tactggtggc attggaacac agcctcagaa agagtggaat 180tttcagctgc
cacaattggg tacaacttta ttaaatttag atgataatta tgttcaatca
240actagaggca taatcgattt tttatcatct tttatagaag ctgtatgtga
tgatgaaatt 300gttagagaag cttcaagaaa tggtatgcaa cctcaatcac
cagctcttat attattatct 360tcatcaaaat ttaaaacaat taattttaat
aatagttctc aatctattaa aaattggaat 420gctcaatcaa gacgtgagaa
tcctgtatat gagtataaaa atccaatgtt gtttgaatat 480aaaaattcgt
atattttaca acgcgcaaat ccacaatttg gaagcgtcat gggtttaaga
540tattatacaa caagtaatac ttgtcaaatt gcagcatttg attccaccct
agctgaaaat 600gcaccaaaca atacacaacg cttcgtttat aatggcagac
taaaaagacc catatcaaac 660gttttaatga aaatagaagc tggtgctcca
aatataagca acccaactat tttacctgat 720cctaataatc aaacaacttg
gctttttaat ccggtacaat taatgaatgg aacatttacc 780attgaattct
ataataatgg tcaactaatt gatatggttc gaaatatggg aatagttact
840gtaagaactt ttgattctta tagaataaca attgacatga ttagaccagc
tgctatgact 900caatacgttc aacgaatttt tccacaaggt ggaccttatc
attttcaggc tacatatatg 960ttaacattaa gtatattaga tgctaccaca
gagtccgttc tatgtgattc tcattcagtg 1020gaatattcaa tagtagcaaa
cgttagaaga gattcagcga tgccagctgg aactgttttt 1080caaccgggat
ttccatggga acacacacta tccaattaca ctgttgctca agaagataat
1140ttagaaagat tattgttaat tgcatctgtg aagagaatgg taatg
1185261185DNAHuman rotavirus Cmisc_featureHuman rotavirus group C
VP6 Jajeri nucleotide sequence 26atggatgtac ttttttctat agcgaaaact
gtgtcagatc ttaaaaagaa agttgtagtt 60ggaacaattt atactaatgt agaagatgtt
gtacaacaga cgaatgaatt gattagaact 120ttgaatggaa atatttttta
tactggtggt attggaacac agcctcagaa agagtggaat 180tttcagctac
cacaattggg tacaacttta ttaaatttag atgacaatta tgttcaatca
240accagaggca taattgattt tttatcatct tttatagaag ctgtatgtga
tgatgaaatt 300gttagagaag cttcaagaaa tggtatgcaa cctcaatcac
cagctcttat attattatct 360tcatcaaaat ttaaaacaat taattttaat
aatagttctc aatctatcaa aaattggaat 420gctcaatcaa gacgtgagaa
tcctgtatat gagtacaaaa atccaatggt gtttgaatat 480aaaaattctt
atattttaca acgcgcaaat ccacaatttg gaagcgtcat gggtttaaga
540tattatacaa caagtaatac ttgtcaaatt gcagcatttg attccaccct
agctgaaaat 600gcaccaaaca atacgcaacg cttcgtttat aatggcagac
taaaaagacc catatcaaat 660gttttaatga aaatagaagc tggtgcccca
aatataagca acccaactat tttacctgat 720cctaataatc aaacaacttg
gctttttaat ccagtacagt taatgaatgg aacattcact 780attgaattct
ataataatgg tcaactaatt gatatggttc gaaatatggg aatagttacc
840gtaagaactt ttgattctta tagaataaca attgacataa ttagaccagc
tgctatgact 900caatacgttc aacgaatttt tccacaaggt ggaccttatc
attttcaggc tacatatatg 960ttaacattaa gtatattaga tgctactaca
gagtccgttc tatgtgattc tcattcagta 1020gaatattcaa tagtagcaaa
cgtcagaaga gattcagcga tgccagctgg
aactgttttt 1080caaccaggat ttccatggga acacacacta tccaattaca
ctgttgctca agaagataat 1140ttagaaagat tattgttaat cgcatctgtg
aagagaatgg taatg 1185271185DNAHuman rotavirus Cmisc_featureHuman
rotavirus group C VP6 CMH004 nucleotide sequence 27atggatgtac
ttttttctat agcgaaaact gtatcagatc ttaaaaagaa agttgtagtt 60ggaacaattt
atactaatgt agaagatgtt gtacaacaga cgaatgaatt gattagaact
120ttgaatggaa atatttttca tactggtggc attggaacac agcctcataa
agagtggaat 180tttcagctac cacaattagg tacaacttta ttaaatttag
atgataatta tgttcaatca 240actagaggca taattgattt tttatcatct
tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa cctcaatcac cagctcttat attattatct 360tcatcaaaat
ttaaaacaat taattttaat aatagttctc aatctatcaa aaattggaat
420gctcaatcaa gacgtgagaa tcctgtatat gagtacaaaa atccaatgtt
gtttgaatat 480aaaaattctt atattttaca acgcgcaaat ccacaatttg
gaagtgttat gggtttaaga 540tattacacaa caagtaatac ttgtcaaatt
gcagcatttg attccaccct agctgaaaat 600gcaccgaaca atacgcaacg
cttcgtttat aatggcagac taaaaagacc catatcaaat 660gttttaatga
aaatagaagc tggtgctcca aatataagca acccaactat tttacctgat
720cctaataatc aaacaacttg gctttttaat ccggtacaat taatgaatgg
aacatttacc 780attgaattct ataataatgg tcaactaatt gatatggttc
gaaatatggg aatagttact 840gtaagaactt ttgattctta tagaataaca
attgacatga ttagaccagc tgctatgacc 900caatacgttc aacgaatttt
tccacaaggt ggaccttatc attttcaggc tacatatatg 960ttaacattaa
gtatattaga tgctaccaca gaatccgttc tatgtgattc tcattcagta
1020gaatattcaa tagtagcaaa cgtcagaaga gattcagcga tgccagctgg
aactgttttt 1080caaccgggat ttccatggga acacacacta tccaattaca
ctgttgctca agaagataat 1140ttagagagat tattgttaat cgcatctgtg
aagagaatgg taatg 1185281185DNAHuman rotavirus Cmisc_featureHuman
rotavirus group C VP6 V508 nucleotide sequence 28atggatgtac
ttttttctat agcgaaaact gtgtcagatc ttaaaaagaa agttgtagtt 60ggaacaattt
ataccaatgt agaagatgtt gtacaacaga cgaatgaatt gattagaact
120ttgaatggaa atgtttttca tactggtggc attggaacac agcctcagaa
agagtggaat 180tttcaactac cacaattggg tacaacttta ttaaatttag
atgataatta tgttcaatca 240actagaggca taattgattt tttatcatct
tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa ccccaatcac cagctcttat attattatct 360tcatcaaaat
ttaaaacaat taatttgaat aatagttctc aatctatcaa aaattggaat
420gctcaatcaa gacgtgagaa tcctgtatat gagtacaaaa atccaatgtt
gtttgaatat 480aaaaattctt atattttaca acgcgcaaat ccacaatttg
gaagcgtcat gggtttaaga 540tattatacaa caagtaatac ttgtcaaatt
gcagcatttg attccaccct agctgaaaat 600gcaccaaaca atacgcaacg
cttcgtttat aatggcagac taaaaagacc catatcaaat 660gttttaatga
aaatagaagc tggtgctcca aatataagca acccaactat tttacctgat
720cctaataatc aaacaacttg gctttttaat ccagtacaat tgatgaatgg
aacattcacc 780attgagttct ataataatgg tcaactaatt gatatggttc
gaaatatggg aatagttact 840gtaagaactt ttgattctta tagaataaca
attgacatga ttagaccagc tgctatgact 900caatacgttc aacgaatttt
tccacaaggc ggaccttatc attttcaggc tacatatatg 960ttaacgttaa
gtatattaga tgctaccaca gagtccgttt tatgtgattc tcattcagta
1020gaatattcaa tagtagccaa cgtcagaaga gattcagcga tgccagctgg
aaccgttttt 1080caaccgggat ttccatggga acacacacta tccaattaca
ctgttgctca agaagataat 1140ttagaaagat tattgttaat cgcatctgtg
aagagaatgg taatg 1185291185DNAHuman rotavirus Cmisc_featureHuman
rotavirus group C VP6 China nucleotide sequence 29atggatgtac
ttttttctat agcgaaaact gtgtcagatc ttaaaaagaa agttgtagtt 60ggaacaattt
atactaatgt agaagatgtt gtacaacaga cgaatgaact gattagaact
120ttgaatggaa atatttttca tactggtggt attggaacac agccccagaa
agagtggaat 180tttcagctac cacaattggg tacaacttta ttaaatttag
atgataatta tgttcaatca 240actagaggca taattgattt tttatcatct
tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa cctcaatcac cagctcttat attattatct 360tcatcaaaat
ttaagacaat taattttaat aatagttctc aatctatcaa aaattggaat
420gctcaatcaa gacgtgagaa tcctgtatat gaatacaaaa atccaatgtt
gttggaatat 480aaaaattctt atattttaca acgcgcaatt ccacaatttg
gaagcgtcat gggtttaaga 540tattatacaa caagtaatac ttgtcaaatt
gcagcatttg attccaccct agctgaaaat 600gcaccaaaca atacgcaacg
cttcgtttat aatggcagac taaaaagacc catatcaaat 660gttttaatga
aaatagaagc tggtgctcca aatataagca acccaactat tttacctgat
720cctaataatc aaacaacttg gctttttaat ccggtacaat taatgaatgg
aacattcacc 780attgaattct ataataatgg tcaactaatt gatatggttc
gaaatatggg aatagttact 840gtaaggactt ttgattctta tagaataaca
attgacatga ttagaccagc tgctaggact 900caatacgttc aacaaatttt
tccacaaggt ggaccttatc attttcaggc tacatatatg 960ttaacattaa
gtatattaga tgctaccaca gagtccgttc tatgtgattc tcattcagta
1020gaatattcaa tagtagcaaa cgtcagaaga gattcagcga tgccagctgg
aactgttttt 1080caaccgggtt ttccatggga acacacacta tccaattaca
ctgttgctca agaagataat 1140ttagaaagat tattgttaat cgcatctgtg
aagagaatgg taatg 1185301185DNAHuman rotavirus Cmisc_featureHuman
rotavirus group C VP6 BCN6 nucleotide sequence 30atggatgtac
ttttttctat agcgaaaact gtgtcagatc ttaaaaagaa agttgtagtt 60ggaacaattt
atactaatgt agaagatgtt gtacaacaga cgaatgaatt gattagaact
120ttaaatggaa atgtttttca tactggtggc attggaacac agcctcagaa
agagtggaat 180tttcagctgc cacaattggg tacaacttta ttaaatttag
atgataatta tgttcaatca 240actagaggca taattgattt tttatcatct
tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa cctcaatcac cagctcttat attattatct 360tcatcaaagt
ttaaaacaat taattttaat aatagttctc aatccattaa aaattggaat
420gctcaatcaa gacgtgagaa tcctgtatat gagtataaaa atccaatgtt
gtttgaatat 480aaaaattcgt atattttaca acgcgcaaat ccacaatttg
gaagcgtcat gggtttaaga 540tactatacaa caagtaatac ttgtcaaatt
gcagcatttg attccacctt agctgaaaat 600gcaccaaaca atacacaacg
tttcgtttat aatggcagac taaaaagacc catatcaaac 660gttttaatga
aaattgaagc tggtgctcca aatataagca acccaactat tttacctgat
720cctaataatc aaacaacttg gctatttaat ccggtacaat taatgaatgg
aacatttacc 780attgaattct ataataatgg tcaactaatt gatatggttc
gaaatatggg aatagttact 840gtaagaactt ttgattctta tagaataaca
attgacatga ttagaccagc tgctatgact 900caatacgttc aacgaatctt
tccacaaggt ggaccttatc attttcaggc tacatatatg 960ttaacattaa
gtatattaga tgctaccaca gagtccgttc tatgtgattc tcattcagtg
1020gaatattcaa tagtagcaaa cgttagaaga gattcagcga tgccaactgg
aactgttttt 1080caaccgggat ttccatggga acacacacta tccaattaca
ctgttgctca agaagataat 1140ttagaaagat tattgttaat tgcatctgtg
aagagaatgg taatg 1185311185DNAHuman rotavirus Cmisc_featureHuman
rotavirus group C VP6 S1 nucleotide sequence 31atggatgtac
ttttttctat agcgaaaacc gtgtcagatc ttaaagagaa agttgtagtt 60ggaacaattt
atactaatgt agaagatgtt gtacaacaga cgaatgaatt gattagaact
120ttgaatggaa atatttttca tactggtggc attggaacac agcctcagaa
agagtggaat 180tttcagctcc cacaattggg taccacttta ttaaatttag
atgataatta tgttcaatca 240actagaggca taattgattt tttatcatct
tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa cctcaatcac cagctcttat attattatct 360tcatcaaaat
ttaaaacaat taattttaat aatagttctc aatctatcaa aaattggaat
420gctcaatcaa gacgtgagaa tcctgtatat gagtacaaaa atccaatgtt
gtttgaatat 480aaaaattctt atattttaca acgcgcaaat ccacaatttg
gaagcgtcat gggtttaaga 540tattatacaa caagtaatat ttgtcaaatt
gcagcatttg attccaccct agctgaaaat 600gcaccaaata atacgcaacg
cttcgtttat aatggcagac taaaaagacc catatcaaat 660gttttaatga
aaatagaagc tggtgctcca aatataagca acccaactat tttacctgat
720cctaataatc aaacaacttg gctttttaat ccggtacaat taatgaatgg
aacatttacc 780attgaattct ataataatgg tcaactaatt gatatggttc
gaaatatggg aatagttact 840gtaagaactt ttgattctta tagaataaca
attgacatga ttagaccagc tgctatgact 900caatacgttc aacgaatttt
tccacaaggt ggaccttatc attttcaggc tacatatatg 960ttaacattaa
gtatattaga tgctaccaca gagtccgttc tatgtgattc tcattcagta
1020gaatattcaa tagtagcaaa cgtcagaaga gattcagcaa tgccagctgg
aactgttttt 1080caaccgggat ttccatggga acacacacta tccaattaca
ctgttgctca agaagataat 1140ttagaaagat tattgttaat cgcatctgtg
aagagaatgg taatg 118532395PRTHuman rotavirus CMISC_FEATUREHuman
rotavirus group C VP6 S1 protein sequence 32Met Asp Val Leu Phe Ser
Ile Ala Lys Thr Val Ser Asp Leu Lys Glu1 5 10 15Lys Val Val Val Gly
Thr Ile Tyr Thr Asn Val Glu Asp Val Val Gln 20 25 30Gln Thr Asn Glu
Leu Ile Arg Thr Leu Asn Gly Asn Ile Phe His Thr 35 40 45Gly Gly Ile
Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe Gln Leu Pro 50 55 60Gln Leu
Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn Tyr Val Gln Ser65 70 75
80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser Phe Ile Glu Ala Val Cys
85 90 95Asp Asp Glu Ile Val Arg Glu Ala Ser Arg Asn Gly Met Gln Pro
Gln 100 105 110Ser Pro Ala Leu Ile Leu Leu Ser Ser Ser Lys Phe Lys
Thr Ile Asn 115 120 125Phe Asn Asn Ser Ser Gln Ser Ile Lys Asn Trp
Asn Ala Gln Ser Arg 130 135 140Arg Glu Asn Pro Val Tyr Glu Tyr Lys
Asn Pro Met Leu Phe Glu Tyr145 150 155 160Lys Asn Ser Tyr Ile Leu
Gln Arg Ala Asn Pro Gln Phe Gly Ser Val 165 170 175Met Gly Leu Arg
Tyr Tyr Thr Thr Ser Asn Ile Cys Gln Ile Ala Ala 180 185 190Phe Asp
Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn Thr Gln Arg Phe 195 200
205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile Ser Asn Val Leu Met Lys
210 215 220Ile Glu Ala Gly Ala Pro Asn Ile Ser Asn Pro Thr Ile Leu
Pro Asp225 230 235 240Pro Asn Asn Gln Thr Thr Trp Leu Phe Asn Pro
Val Gln Leu Met Asn 245 250 255Gly Thr Phe Thr Ile Glu Phe Tyr Asn
Asn Gly Gln Leu Ile Asp Met 260 265 270Val Arg Asn Met Gly Ile Val
Thr Val Arg Thr Phe Asp Ser Tyr Arg 275 280 285Ile Thr Ile Asp Met
Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln 290 295 300Arg Ile Phe
Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr Tyr Met305 310 315
320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu Ser Val Leu Cys Asp
325 330 335Ser His Ser Val Glu Tyr Ser Ile Val Ala Asn Val Arg Arg
Asp Ser 340 345 350Ala Met Pro Ala Gly Thr Val Phe Gln Pro Gly Phe
Pro Trp Glu His 355 360 365Thr Leu Ser Asn Tyr Thr Val Ala Gln Glu
Asp Asn Leu Glu Arg Leu 370 375 380Leu Leu Ile Ala Ser Val Lys Arg
Met Val Met385 390 395331063DNAHuman rotavirus Cmisc_featureHuman
rotavirus group C VP7 S1 nucleotide sequence 33ggcatttaaa
aaagaagaag ctgtctgaca aactggtctt ctttttaaat ggtttgtaca 60acattgtaca
ctgtttgcgc cattctcttc attcttttca tttatatatt attatttaga
120aaaatgttcc acctaataac tgatacttta atagtgatgc ttattttatc
taattgtgta 180gagtggtcac aaggtcagat gtttattgat gatatacatt
ataatggtaa cgttgagact 240atcataaatt ctactgatcc ttttaatgtt
gaatctttat gtatttattt tccaaatgca 300gttgtaggat cacagggacc
aggtaaatcc gatggacatt tgaatgatgg taattatgca 360cagactatcg
ccactttgtt tgaaacaaaa ggattcccaa aaggttcaat aataattaaa
420acatatacac agacatcaga ctttataaat tcagtagaaa tgacatgctc
ttataatata 480gttatcattc ctgatagccc aaatgattca gaatctattg
aacagatagc agaatggatt 540ttaaatgttt ggagatgtga tgacatgaat
ttggaaattt atacttatga acaaattgga 600ataaacaatt tatgggctgc
atttggtagt gactgtgata tatctgtctg tccattagat 660actacaagta
atggaatcgg atgttcacca gctagtacag aaacttatga agttgtatca
720aatgacaccc aattggcctt aattaatgtt gtggataatg ttagacatag
aatacagatg 780aacactgctc aatgtaaatt gaaaaattgt attaagggtg
aagctcgact gaatactgca 840ctaataagaa tttcaacatc atcaagtttt
gataattcat tgtcaccatt aaataacggc 900caaacaacaa gatcgtttaa
aataaatgca aagaaatggt ggactatatt ttatacaata 960attgattata
ttaatacaat tgtacaatca atgactccca gacatcgggc gatttatcca
1020gaagggtgga tgttgaggta tgcgtaaaca agatcatgtg gct
106334332PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C
VP7 S1 nucleotide sequence 34Met Val Cys Thr Thr Leu Tyr Thr Val
Cys Ala Ile Leu Phe Ile Leu1 5 10 15Phe Ile Tyr Ile Leu Leu Phe Arg
Lys Met Phe His Leu Ile Thr Asp 20 25 30Thr Leu Ile Val Met Leu Ile
Leu Ser Asn Cys Val Glu Trp Ser Gln 35 40 45Gly Gln Met Phe Ile Asp
Asp Ile His Tyr Asn Gly Asn Val Glu Thr 50 55 60Ile Ile Asn Ser Thr
Asp Pro Phe Asn Val Glu Ser Leu Cys Ile Tyr65 70 75 80Phe Pro Asn
Ala Val Val Gly Ser Gln Gly Pro Gly Lys Ser Asp Gly 85 90 95His Leu
Asn Asp Gly Asn Tyr Ala Gln Thr Ile Ala Thr Leu Phe Glu 100 105
110Thr Lys Gly Phe Pro Lys Gly Ser Ile Ile Ile Lys Thr Tyr Thr Gln
115 120 125Thr Ser Asp Phe Ile Asn Ser Val Glu Met Thr Cys Ser Tyr
Asn Ile 130 135 140Val Ile Ile Pro Asp Ser Pro Asn Asp Ser Glu Ser
Ile Glu Gln Ile145 150 155 160Ala Glu Trp Ile Leu Asn Val Trp Arg
Cys Asp Asp Met Asn Leu Glu 165 170 175Ile Tyr Thr Tyr Glu Gln Ile
Gly Ile Asn Asn Leu Trp Ala Ala Phe 180 185 190Gly Ser Asp Cys Asp
Ile Ser Val Cys Pro Leu Asp Thr Thr Ser Asn 195 200 205Gly Ile Gly
Cys Ser Pro Ala Ser Thr Glu Thr Tyr Glu Val Val Ser 210 215 220Asn
Asp Thr Gln Leu Ala Leu Ile Asn Val Val Asp Asn Val Arg His225 230
235 240Arg Ile Gln Met Asn Thr Ala Gln Cys Lys Leu Lys Asn Cys Ile
Lys 245 250 255Gly Glu Ala Arg Leu Asn Thr Ala Leu Ile Arg Ile Ser
Thr Ser Ser 260 265 270Ser Phe Asp Asn Ser Leu Ser Pro Leu Asn Asn
Gly Gln Thr Thr Arg 275 280 285Ser Phe Lys Ile Asn Ala Lys Lys Trp
Trp Thr Ile Phe Tyr Thr Ile 290 295 300Ile Asp Tyr Ile Asn Thr Ile
Val Gln Ser Met Thr Pro Arg His Arg305 310 315 320Ala Ile Tyr Pro
Glu Gly Trp Met Leu Arg Tyr Ala 325 33035395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 Bristol protein sequence
35Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1
5 10 15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val
Gln 20 25 30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Ile Phe
His Thr 35 40 45Gly Gly Ile Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe
Gln Leu Pro 50 55 60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn
Tyr Val Gln Ser65 70 75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser
Phe Ile Glu Ala Val Cys 85 90 95Asp Asp Glu Ile Val Arg Glu Ala Ser
Arg Asn Gly Met Gln Pro Gln 100 105 110Ser Pro Ala Leu Ile Leu Leu
Ser Ser Ser Lys Phe Lys Thr Ile Asn 115 120 125Phe Asn Asn Ser Ser
Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg 130 135 140Arg Glu Asn
Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Phe Glu Tyr145 150 155
160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile
Ala Ala 180 185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn
Thr Gln Arg Phe 195 200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile
Ser Asn Val Leu Met Lys 210 215 220Ile Glu Ala Gly Ala Pro Asn Ile
Ser Asn Pro Thr Ile Leu Pro Asp225 230 235 240Pro Asn Asn Gln Thr
Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn 245 250 255Gly Thr Phe
Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260 265 270Val
Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg 275 280
285Ile Thr Ile Asp Met Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln
290 295 300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr
Tyr Met305 310 315 320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu
Ser Val Leu Cys Asp 325 330 335Ser His Ser Val Glu Tyr Ser Ile Val
Ala Asn Val Arg Arg Asp Ser 340 345 350Ala Met Pro Ala Gly Thr Val
Phe Gln Pro Gly Phe Pro Trp Glu His 355 360 365Thr Leu Ser Asn Tyr
Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370 375 380Leu Leu Ile
Ala Ser Val Lys Arg Met Val Met385 390 39536395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 Jajeri protein sequence
36Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1
5 10 15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val
Gln 20 25
30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Ile Phe Tyr Thr
35 40 45Gly Gly Ile Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe Gln Leu
Pro 50 55 60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn Tyr Val
Gln Ser65 70 75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser Phe Ile
Glu Ala Val Cys 85 90 95Asp Asp Glu Ile Val Arg Glu Ala Ser Arg Asn
Gly Met Gln Pro Gln 100 105 110Ser Pro Ala Leu Ile Leu Leu Ser Ser
Ser Lys Phe Lys Thr Ile Asn 115 120 125Phe Asn Asn Ser Ser Gln Ser
Ile Lys Asn Trp Asn Ala Gln Ser Arg 130 135 140Arg Glu Asn Pro Val
Tyr Glu Tyr Lys Asn Pro Met Val Phe Glu Tyr145 150 155 160Lys Asn
Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val 165 170
175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile Ala Ala
180 185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn Thr Gln
Arg Phe 195 200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile Ser Asn
Val Leu Met Lys 210 215 220Ile Glu Ala Gly Ala Pro Asn Ile Ser Asn
Pro Thr Ile Leu Pro Asp225 230 235 240Pro Asn Asn Gln Thr Thr Trp
Leu Phe Asn Pro Val Gln Leu Met Asn 245 250 255Gly Thr Phe Thr Ile
Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260 265 270Val Arg Asn
Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg 275 280 285Ile
Thr Ile Asp Ile Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln 290 295
300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr Tyr
Met305 310 315 320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu Ser
Val Leu Cys Asp 325 330 335Ser His Ser Val Glu Tyr Ser Ile Val Ala
Asn Val Arg Arg Asp Ser 340 345 350Ala Met Pro Ala Gly Thr Val Phe
Gln Pro Gly Phe Pro Trp Glu His 355 360 365Thr Leu Ser Asn Tyr Thr
Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370 375 380Leu Leu Ile Ala
Ser Val Lys Arg Met Val Met385 390 39537395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 CMH004 protein sequence
37Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1
5 10 15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val
Gln 20 25 30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Ile Phe
His Thr 35 40 45Gly Gly Ile Gly Thr Gln Pro His Lys Glu Trp Asn Phe
Gln Leu Pro 50 55 60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn
Tyr Val Gln Ser65 70 75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser
Phe Ile Glu Ala Val Cys 85 90 95Asp Asp Glu Ile Val Arg Glu Ala Ser
Arg Asn Gly Met Gln Pro Gln 100 105 110Ser Pro Ala Leu Ile Leu Leu
Ser Ser Ser Lys Phe Lys Thr Ile Asn 115 120 125Phe Asn Asn Ser Ser
Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg 130 135 140Arg Glu Asn
Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Phe Glu Tyr145 150 155
160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile
Ala Ala 180 185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn
Thr Gln Arg Phe 195 200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile
Ser Asn Val Leu Met Lys 210 215 220Ile Glu Ala Gly Ala Pro Asn Ile
Ser Asn Pro Thr Ile Leu Pro Asp225 230 235 240Pro Asn Asn Gln Thr
Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn 245 250 255Gly Thr Phe
Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260 265 270Val
Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg 275 280
285Ile Thr Ile Asp Met Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln
290 295 300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr
Tyr Met305 310 315 320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu
Ser Val Leu Cys Asp 325 330 335Ser His Ser Val Glu Tyr Ser Ile Val
Ala Asn Val Arg Arg Asp Ser 340 345 350Ala Met Pro Ala Gly Thr Val
Phe Gln Pro Gly Phe Pro Trp Glu His 355 360 365Thr Leu Ser Asn Tyr
Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370 375 380Leu Leu Ile
Ala Ser Val Lys Arg Met Val Met385 390 39538395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 V508 protein sequence
38Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1
5 10 15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val
Gln 20 25 30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Val Phe
His Thr 35 40 45Gly Gly Ile Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe
Gln Leu Pro 50 55 60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn
Tyr Val Gln Ser65 70 75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser
Phe Ile Glu Ala Val Cys 85 90 95Asp Asp Glu Ile Val Arg Glu Ala Ser
Arg Asn Gly Met Gln Pro Gln 100 105 110Ser Pro Ala Leu Ile Leu Leu
Ser Ser Ser Lys Phe Lys Thr Ile Asn 115 120 125Leu Asn Asn Ser Ser
Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg 130 135 140Arg Glu Asn
Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Phe Glu Tyr145 150 155
160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile
Ala Ala 180 185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn
Thr Gln Arg Phe 195 200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile
Ser Asn Val Leu Met Lys 210 215 220Ile Glu Ala Gly Ala Pro Asn Ile
Ser Asn Pro Thr Ile Leu Pro Asp225 230 235 240Pro Asn Asn Gln Thr
Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn 245 250 255Gly Thr Phe
Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260 265 270Val
Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg 275 280
285Ile Thr Ile Asp Met Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln
290 295 300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr
Tyr Met305 310 315 320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu
Ser Val Leu Cys Asp 325 330 335Ser His Ser Val Glu Tyr Ser Ile Val
Ala Asn Val Arg Arg Asp Ser 340 345 350Ala Met Pro Ala Gly Thr Val
Phe Gln Pro Gly Phe Pro Trp Glu His 355 360 365Thr Leu Ser Asn Tyr
Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370 375 380Leu Leu Ile
Ala Ser Val Lys Arg Met Val Met385 390 39539395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 China protein sequence
39Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1
5 10 15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val
Gln 20 25 30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Ile Phe
His Thr 35 40 45Gly Gly Ile Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe
Gln Leu Pro 50 55 60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn
Tyr Val Gln Ser65 70 75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser
Phe Ile Glu Ala Val Cys 85 90 95Asp Asp Glu Ile Val Arg Glu Ala Ser
Arg Asn Gly Met Gln Pro Gln 100 105 110Ser Pro Ala Leu Ile Leu Leu
Ser Ser Ser Lys Phe Lys Thr Ile Asn 115 120 125Phe Asn Asn Ser Ser
Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg 130 135 140Arg Glu Asn
Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Leu Glu Tyr145 150 155
160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Ile Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile
Ala Ala 180 185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn
Thr Gln Arg Phe 195 200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile
Ser Asn Val Leu Met Lys 210 215 220Ile Glu Ala Gly Ala Pro Asn Ile
Ser Asn Pro Thr Ile Leu Pro Asp225 230 235 240Pro Asn Asn Gln Thr
Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn 245 250 255Gly Thr Phe
Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260 265 270Val
Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg 275 280
285Ile Thr Ile Asp Met Ile Arg Pro Ala Ala Arg Thr Gln Tyr Val Gln
290 295 300Gln Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr
Tyr Met305 310 315 320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu
Ser Val Leu Cys Asp 325 330 335Ser His Ser Val Glu Tyr Ser Ile Val
Ala Asn Val Arg Arg Asp Ser 340 345 350Ala Met Pro Ala Gly Thr Val
Phe Gln Pro Gly Phe Pro Trp Glu His 355 360 365Thr Leu Ser Asn Tyr
Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370 375 380Leu Leu Ile
Ala Ser Val Lys Arg Met Val Met385 390 39540395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 BCN6 protein sequence
40Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1
5 10 15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val
Gln 20 25 30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Val Phe
His Thr 35 40 45Gly Gly Ile Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe
Gln Leu Pro 50 55 60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn
Tyr Val Gln Ser65 70 75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser
Phe Ile Glu Ala Val Cys 85 90 95Asp Asp Glu Ile Val Arg Glu Ala Ser
Arg Asn Gly Met Gln Pro Gln 100 105 110Ser Pro Ala Leu Ile Leu Leu
Ser Ser Ser Lys Phe Lys Thr Ile Asn 115 120 125Phe Asn Asn Ser Ser
Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg 130 135 140Arg Glu Asn
Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Phe Glu Tyr145 150 155
160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile
Ala Ala 180 185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn
Thr Gln Arg Phe 195 200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile
Ser Asn Val Leu Met Lys 210 215 220Ile Glu Ala Gly Ala Pro Asn Ile
Ser Asn Pro Thr Ile Leu Pro Asp225 230 235 240Pro Asn Asn Gln Thr
Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn 245 250 255Gly Thr Phe
Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260 265 270Val
Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg 275 280
285Ile Thr Ile Asp Met Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln
290 295 300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr
Tyr Met305 310 315 320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu
Ser Val Leu Cys Asp 325 330 335Ser His Ser Val Glu Tyr Ser Ile Val
Ala Asn Val Arg Arg Asp Ser 340 345 350Ala Met Pro Thr Gly Thr Val
Phe Gln Pro Gly Phe Pro Trp Glu His 355 360 365Thr Leu Ser Asn Tyr
Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370 375 380Leu Leu Ile
Ala Ser Val Lys Arg Met Val Met385 390 395411185DNAHuman rotavirus
Cmisc_featureHuman rotavirus group C VP6 nucleotide consensus
sequence 41atggatgtac ttttttctat agcgaaaact gtgtcagatc ttaaaaagaa
agttgtagtt 60ggaacaattt atactaatgt agaagatgtt gtacaacaga cgaatgaatt
gattagaact 120ttgaatggaa atatttttca tactggtggc attggaacac
agcctcagaa agagtggaat 180tttcagctac cacaattggg tacaacttta
ttaaatttag atgataatta tgttcaatca 240actagaggca taattgattt
tttatcatct tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag
cttcaagaaa tggtatgcaa cctcaatcac cagctcttat attattatct
360tcatcaaaat ttaaaacaat taattttaat aatagttctc aatctatcaa
aaattggaat 420gctcaatcaa gacgtgagaa tcctgtatat gagtacaaaa
atccaatgtt gtttgaatat 480aaaaattctt atattttaca acgcgcaaat
ccacaatttg gaagcgtcat gggtttaaga 540tattatacaa caagtaatac
ttgtcaaatt gcagcatttg attccaccct agctgaaaat 600gcaccaaaca
atacgcaacg cttcgtttat aatggcagac taaaaagacc catatcaaat
660gttttaatga aaatagaagc tggtgctcca aatataagca acccaactat
tttacctgat 720cctaataatc aaacaacttg gctttttaat ccggtacaat
taatgaatgg aacatttacc 780attgaattct ataataatgg tcaactaatt
gatatggttc gaaatatggg aatagttact 840gtaagaactt ttgattctta
tagaataaca attgacatga ttagaccagc tgctatgact 900caatacgttc
aacgaatttt tccacaaggt ggaccttatc attttcaggc tacatatatg
960ttaacattaa gtatattaga tgctaccaca gagtccgttc tatgtgattc
tcattcagta 1020gaatattcaa tagtagcaaa cgtcagaaga gattcagcga
tgccagctgg aactgttttt 1080caaccgggat ttccatggga acacacacta
tccaattaca ctgttgctca agaagataat 1140ttagaaagat tattgttaat
cgcatctgtg aagagaatgg taatg 1185422655DNAHuman rotavirus
Cmisc_featureHuman rotavirus group C strain ASP88 VP2 S1 nucleotide
sequence of open reading frame 42atgataagca gaaacaggcg cagaaataac
caacaaaaag atataggaaa agagaaacaa 60ttagagacta taattgacaa agaagtaaag
gaaaacaaag attctacaaa agaagataag 120ctagtagtta cggaagaaag
taatggtgac gtcacagctg ttaaagaaca atcgaataat 180attaatttac
aaaagaatga tttggttaaa gaagtcatga atatacagaa tcaaacatta
240aatacagtag ttgctgagaa taaagttgaa atagaagaaa tagttaaaaa
atacattccc 300tcatataata ctgacagcct tattgttaaa aagttaactg
aaatccagga atcaagtgct 360aaaacatata atacattatt cagattattt
actccagtta aaagttattt atatgacata 420aatggtgaga aaaaattatc
gactagatgg tattggaaat tgctcaaaga tgatttacct 480gctggtgatt
actcagttag acaattcttc ctgtcactat atttaaatgt tttagaggga
540atgcccgatt acataatgct tcgtgatatg gcagtggata acccatattc
agcagaagca 600ggtaaaatcg tagatggaaa gtctaaagaa attttagttg
aactatatca agaccaaatg 660acagaagggt atattagaag atatatgtct
gaattaagac ataaaatatc tggagaaaca 720aatactgcaa aatatccagc
tattctacat cccgtggata atgagcttaa tcaatacttt 780cttgagcatc
agttaattca accattaact acaagaaata ttgcagaatt gattccaact
840caattatatc atgatccaaa ttacgttttt aatattgatg cagccttttt
aacaaattca 900agatttgttc caccatactt aacacaggat aggattggat
tacatgatgg attcgaatca 960atatgggatt caaaaaccca tgctgattac
gtttcagcta gaagatttat acctgattta 1020actgaactgg tagatgctga
aaagcaaata aaagaaatgg ctgcacattt acaactagag 1080gctattacag
tacaggttga atcacaattt ttagcgggaa ttagtgctgc tgcagctaat
1140gaagcgttca aatttataat tggctcagtt ttatctacca gaacaatagc
tgtagaattc 1200ataacctcaa actatatgtc gttagcatca tgtatgtatt
taatgactat tatgccatca 1260gagattttct tgagagaatc attagttgct
atgcgattag caataataaa tacccttatt 1320tatccagctc taggtttagc
gcaaatgcat tatcaagcag gtgaagtgag gaccccattc 1380gaattagctg
agatgcgagt agctaataga tctattagac aatggttaca tcattgtaat
1440acacttcaat ttggtagaca gataacggaa gggataattc atctacgatt
tactaatgat
1500atcatgacag gtaggatagt gaacttattt tcaacaatgc tagtagcttt
atcatctcag 1560ccattcgcta catatccttt agactataaa agatctgtac
aaagagcatt acaactttta 1620tcaaatagaa cagcccaaat agcagattta
accagattaa tagtatacaa ttatactaca 1680ttatctgcat gtatagtcat
gaatatgcat ttagtaggaa ctcttactgt tgaacgtata 1740caggccactt
ctctaacttc tttaatgatg ttaatttcta ataagacagt tattccagaa
1800ccatcgtctc ttttttcata tttctctagt aacattaatt ttcttacaaa
ttataatgag 1860caaattgata atgtggtagc agaaataatg gccgcatata
gattgaattt atatcaacag 1920aaaatgttga tgctcgttac caggtttgtg
tcaaggttgt acatatttga tgctcctaaa 1980ataccgccag atcagatgta
tagattaaga aaccgattaa gaaatattcc agttgaaaga 2040agaagagctg
atgtgttcag aattattatg aataatagag atttaatcga aaaaacatca
2100gaacgtatat gtcagggtgt gttgttatct tatacaccaa tgcctttaac
ttacgttgaa 2160gatgtcgggt taacaaatgt aattaatgac actaataact
tccaaataat taatatagaa 2220gaaattgaga agaccggtga ctattcagcc
ataacgaatg cattacttcg ggatactcca 2280attatattga aaggtgcgat
tccatatgtt actaactcat cagtaattga tgttttatct 2340aaagtggaca
ccacagtgtt cgcaagcatc gtaaaagata gggatatttc aaagttaaaa
2400ccaataaaat tcataattaa ttcagattca tccgaatatt atttagtaca
caataataaa 2460tggacaccaa caacaactac agcagtatat aaagctagat
ctcagcaatt tgatatacaa 2520cattcagtat caatgctaga gtcaaactta
ttttttgtgg tatataatga tttatttaaa 2580tacattaaaa ccactacagt
tctgccgata aatgctgtct cttatgatgg tgcaagaatt 2640atgcaagaaa cataa
2655432736DNAHuman rotavirus Cmisc_featureHuman rotavirus group C
strain ASP88 VP2 nucleotide sequence 43ggcttaaaaa gatcagtcga
ggacaaatcg tccaagatga taagcagaaa caggcgcaga 60aataaccaac aaaaagatat
aggaaaagag aaacaattag agactataat tgacaaagaa 120gtaaaggaaa
acaaagattc tacaaaagaa gataagctag tagttacgga agaaagtaat
180ggtgacgtca cagctgttaa agaacaatcg aataatatta atttacaaaa
gaatgatttg 240gttaaagaag tcatgaatat acagaatcaa acattaaata
cagtagttgc tgagaataaa 300gttgaaatag aagaaatagt taaaaaatac
attccctcat ataatactga cagccttatt 360gttaaaaagt taactgaaat
ccaggaatca agtgctaaaa catataatac attattcaga 420ttatttactc
cagttaaaag ttatttatat gacataaatg gtgagaaaaa attatcgact
480agatggtatt ggaaattgct caaagatgat ttacctgctg gtgattactc
agttagacaa 540ttcttcctgt cactatattt aaatgtttta gagggaatgc
ccgattacat aatgcttcgt 600gatatggcag tggataaccc atattcagca
gaagcaggta aaatcgtaga tggaaagtct 660aaagaaattt tagttgaact
atatcaagac caaatgacag aagggtatat tagaagatat 720atgtctgaat
taagacataa aatatctgga gaaacaaata ctgcaaaata tccagctatt
780ctacatcccg tggataatga gcttaatcaa tactttcttg agcatcagtt
aattcaacca 840ttaactacaa gaaatattgc agaattgatt ccaactcaat
tatatcatga tccaaattac 900gtttttaata ttgatgcagc ctttttaaca
aattcaagat ttgttccacc atacttaaca 960caggatagga ttggattaca
tgatggattc gaatcaatat gggattcaaa aacccatgct 1020gattacgttt
cagctagaag atttatacct gatttaactg aactggtaga tgctgaaaag
1080caaataaaag aaatggctgc acatttacaa ctagaggcta ttacagtaca
ggttgaatca 1140caatttttag cgggaattag tgctgctgca gctaatgaag
cgttcaaatt tataattggc 1200tcagttttat ctaccagaac aatagctgta
gaattcataa cctcaaacta tatgtcgtta 1260gcatcatgta tgtatttaat
gactattatg ccatcagaga ttttcttgag agaatcatta 1320gttgctatgc
gattagcaat aataaatacc cttatttatc cagctctagg tttagcgcaa
1380atgcattatc aagcaggtga agtgaggacc ccattcgaat tagctgagat
gcgagtagct 1440aatagatcta ttagacaatg gttacatcat tgtaatacac
ttcaatttgg tagacagata 1500acggaaggga taattcatct acgatttact
aatgatatca tgacaggtag gatagtgaac 1560ttattttcaa caatgctagt
agctttatca tctcagccat tcgctacata tcctttagac 1620tataaaagat
ctgtacaaag agcattacaa cttttatcaa atagaacagc ccaaatagca
1680gatttaacca gattaatagt atacaattat actacattat ctgcatgtat
agtcatgaat 1740atgcatttag taggaactct tactgttgaa cgtatacagg
ccacttctct aacttcttta 1800atgatgttaa tttctaataa gacagttatt
ccagaaccat cgtctctttt ttcatatttc 1860tctagtaaca ttaattttct
tacaaattat aatgagcaaa ttgataatgt ggtagcagaa 1920ataatggccg
catatagatt gaatttatat caacagaaaa tgttgatgct cgttaccagg
1980tttgtgtcaa ggttgtacat atttgatgct cctaaaatac cgccagatca
gatgtataga 2040ttaagaaacc gattaagaaa tattccagtt gaaagaagaa
gagctgatgt gttcagaatt 2100attatgaata atagagattt aatcgaaaaa
acatcagaac gtatatgtca gggtgtgttg 2160ttatcttata caccaatgcc
tttaacttac gttgaagatg tcgggttaac aaatgtaatt 2220aatgacacta
ataacttcca aataattaat atagaagaaa ttgagaagac cggtgactat
2280tcagccataa cgaatgcatt acttcgggat actccaatta tattgaaagg
tgcgattcca 2340tatgttacta actcatcagt aattgatgtt ttatctaaag
tggacaccac agtgttcgca 2400agcatcgtaa aagataggga tatttcaaag
ttaaaaccaa taaaattcat aattaattca 2460gattcatccg aatattattt
agtacacaat aataaatgga caccaacaac aactacagca 2520gtatataaag
ctagatctca gcaatttgat atacaacatt cagtatcaat gctagagtca
2580aacttatttt ttgtggtata taatgattta tttaaataca ttaaaaccac
tacagttctg 2640ccgataaatg ctgtctctta tgatggtgca agaattatgc
aagaaacata aatgattgta 2700tagtatcatc ttgtaacgac ctcaaactct gtggct
2736442736DNAPorcine rotavirusmisc_featurePorcine rotavirus group C
strain Cowden VP2 nucleotide sequence 44ggtttaaaaa gatcaatcga
ggacaaatcg tccaagatga taagcagaaa tagacgtaga 60aacactcaac agaaagatgc
tgaaaaggaa aagcagacag aaaatgtgga ggagaaagag 120ataaaggaag
ctaaagaaca agttaaagat gaaaagcaag tgattacaga agaaaacgtc
180gatagtccta aggatgttaa agaacaatca aacaccgtaa atctacaaaa
aaatgactta 240gttaaagaag ttataaatat ccaaaatcaa acattgaata
caatagttgc tgagaataaa 300gtggaaattg aagaagtggt taaaaagtat
attccatcat actcaactga caagctaata 360gttaaaaaat taactgaaat
tcaagaatca agtgctaaaa catacaataa attgtttaga 420ttatttacac
cggttaagag ttatctatat gatgtaaatg gagagaaaaa actatccact
480agatggtatt ggaaactact taaagatgat ctacctgctg gtgattactc
agttagacaa 540ttctttctat ctttatactt gaatgtatta gatgaaatgc
ctgattatgt tatgcttcgt 600gatatggctg tggataatcc atattcagca
gaggcaggaa aaatagtaga tgaaaaatca 660aaagaaatcc tagtagaaat
atatcaagat caaatgactg aagggtatat acggagatat 720atgtctgatt
tgagacatag aatatctggt gaaacgaata ctgctaaata tccagctatt
780ttacatcctg tagatgaaga actaaataaa tactttcttg agcaccaact
gattcaacca 840ttgactacaa gaaatatagc agaattaatt ccaactcaat
tgtatcatga tccaaattat 900gtgtttaaca ttgatgctgc atttttaaca
aactcaagat ttgttccacc gtatctaaca 960caagatagaa ttggattaca
tgatggattt gagtcaattt gggatgcaaa aacacatgct 1020gattacgttt
cagctagaag atttgtacct gatttaactg agttagttga tgctgaaaaa
1080cagatgaaag aaatggcagc acatttacag cttgaagcta ttacagtgca
agttgaatca 1140caattcttgg caggaattag tgcagcggca gctaatgaag
catttaagtt tataattggt 1200actgtgctgt caactagaac aatagcagta
gaattcatca catcaaatta tatgtcatta 1260gcgtcatgta tgtatttaat
gacgattatg ccatcagaaa tctttttgag agaatcgcta 1320gttgcaatgc
agttagcagt aataaatact cttacctatc cagctttagg attagcacaa
1380atgcattatc aggcaggtga aataagaacg ccctttgaac tagcagaaat
gcaagtagca 1440aataggccca ttaggcaatg gttgcatcat tgtaatacac
ttcaatttgg cagacaagta 1500actgaaggag taacacatct acggtttaca
aatgacatca tgacaggtag aatagttaat 1560ctcttttcaa ctatgttggt
agctttatca tctcagcctt ttgctacata tccattagat 1620tacaaaagat
ctgtccagag agcattacag cttctttcaa acaggactgc tcaaatagct
1680gatttgacta gattaatagt gtacaactat acaacattgt cagcatgcat
agtcatgaac 1740atgcatttgg ttggaacctt aactgtagaa cgtatacaag
ctacagcttt aacttcactg 1800ataatgttga tatccaataa aacggttatt
ccagaaccat catccctctt ttcatacttt 1860tctagtaata ttaatttctt
gacaaactac aatgaacaga ttgataacgt agtggctgaa 1920ataatggcag
catacagact agatctatat caacagaaaa tgctaatgct tgttactcga
1980tttgtttcac gactgtacat atttgatgct cctaagatac caccagacca
gatgtataga 2040ttaagaaata gactgaggaa cattccagtt gaaagaagaa
gagcagatgt gttcagaatc 2100attatgaata acagagatct tatagagaaa
acatcagaac gcatttgtca aggagtgtta 2160ctatcatatt caccaatgcc
attaacatat gttgaggatg ttggtttgac aaatgtggtt 2220aatgacacta
atggttttca gataataaac attgaagaaa tcgagaagac aggtgattat
2280tcagcaatta caaacgcatt actccgtgat actccaatca tactgaaggg
cgctattccg 2340tacgttacca attcatcagt aatagatgtt ctatctaaaa
tagatacaac agtgtttgcg 2400agtatcgtta aagacagaga tatttcaaaa
ttaaaaccga taaaattcac aattaattca 2460gactcatcag aatactattt
agtacacaat aataaatgga caccaacaac aacaactgct 2520gtgtacaaag
ccagatctca gcaatttaat atacaacatt cagtgtcaat gttagagtca
2580aacttgttct ttgttgtata taatgatctg tttaagtaca tcaaaacaac
tacagtatta 2640ccaatcaatg ccgtgtctta tgatggtgcg agaattatgc
aggaaacatg aactgattaa 2700ttatatcatc ttgtgatgac ctcaaactct gtggct
273645999DNAHuman rotavirus Cmisc_featureHuman rotavirus group C
VP7 S1 nucleotide sequence of open reading frame 45atggtttgta
caacattgta cactgtttgc gccattctct tcattctttt catttatata 60ttattattta
gaaaaatgtt ccacctaata actgatactt taatagtgat gcttatttta
120tctaattgtg tagagtggtc acaaggtcag atgtttattg atgatataca
ttataatggt 180aacgttgaga ctatcataaa ttctactgat ccttttaatg
ttgaatcttt atgtatttat 240tttccaaatg cagttgtagg atcacaggga
ccaggtaaat ccgatggaca tttgaatgat 300ggtaattatg cacagactat
cgccactttg tttgaaacaa aaggattccc aaaaggttca 360ataataatta
aaacatatac acagacatca gactttataa attcagtaga aatgacatgc
420tcttataata tagttatcat tcctgatagc ccaaatgatt cagaatctat
tgaacagata 480gcagaatgga ttttaaatgt ttggagatgt gatgacatga
atttggaaat ttatacttat 540gaacaaattg gaataaacaa tttatgggct
gcatttggta gtgactgtga tatatctgtc 600tgtccattag atactacaag
taatggaatc ggatgttcac cagctagtac agaaacttat 660gaagttgtat
caaatgacac ccaattggcc ttaattaatg ttgtggataa tgttagacat
720agaatacaga tgaacactgc tcaatgtaaa ttgaaaaatt gtattaaggg
tgaagctcga 780ctgaatactg cactaataag aatttcaaca tcatcaagtt
ttgataattc attgtcacca 840ttaaataacg gccaaacaac aagatcgttt
aaaataaatg caaagaaatg gtggactata 900ttttatacaa taattgatta
tattaataca attgtacaat caatgactcc cagacatcgg 960gcgatttatc
cagaagggtg gatgttgagg tatgcgtaa 999461343DNAHuman rotavirus
Cmisc_featureHuman rotavirus group C VP6 S1 nucleotide sequence
46atctcattca caatggatgt acttttttct atagcgaaaa ccgtgtcaga tcttaaagag
60aaagttgtag ttggaacaat ttatactaat gtagaagatg ttgtacaaca gacgaatgaa
120ttgattagaa ctttgaatgg aaatattttt catactggtg gcattggaac
acagcctcag 180aaagagtgga attttcagct cccacaattg ggtaccactt
tattaaattt agatgataat 240tatgttcaat caactagagg cataattgat
tttttatcat cttttataga agctgtatgt 300gatgatgaaa ttgttagaga
agcttcaaga aatggtatgc aacctcaatc accagctctt 360atattattat
cttcatcaaa atttaaaaca attaatttta ataatagttc tcaatctatc
420aaaaattgga atgctcaatc aagacgtgag aatcctgtat atgagtacaa
aaatccaatg 480ttgtttgaat ataaaaattc ttatatttta caacgcgcaa
atccacaatt tggaagcgtc 540atgggtttaa gatattatac aacaagtaat
atttgtcaaa ttgcagcatt tgattccacc 600ctagctgaaa atgcaccaaa
taatacgcaa cgcttcgttt ataatggcag actaaaaaga 660cccatatcaa
atgttttaat gaaaatagaa gctggtgctc caaatataag caacccaact
720attttacctg atcctaataa tcaaacaact tggcttttta atccggtaca
attaatgaat 780ggaacattta ccattgaatt ctataataat ggtcaactaa
ttgatatggt tcgaaatatg 840ggaatagtta ctgtaagaac ttttgattct
tatagaataa caattgacat gattagacca 900gctgctatga ctcaatacgt
tcaacgaatt tttccacaag gtggacctta tcattttcag 960gctacatata
tgttaacatt aagtatatta gatgctacca cagagtccgt tctatgtgat
1020tctcattcag tagaatattc aatagtagca aacgtcagaa gagattcagc
aatgccagct 1080ggaactgttt ttcaaccggg atttccatgg gaacacacac
tatccaatta cactgttgct 1140caagaagata atttagaaag attattgtta
atcgcatctg tgaagagaat ggtaatgtag 1200ataagctaga agactaaaca
tcttctatgc ggcctacata ccatgtagca tgaatcacga 1260ctgggtttag
tccatgctcg cataggggca aatatgcatg atatggatga tccccagaag
1320gatgaaatgt gaactatgtg gct 1343471353DNAHuman rotavirus
Cmisc_featureHuman rotavirus group C VP6 Bristol nucleotide
sequence 47ggcatttaaa atctcattca caatggatgt acttttttct atagcgaaaa
ctgtgtcaga 60tcttaaaaag aaagttgtgg ttggaacaat ttatactaat gtagaagatg
ttgtacaaca 120gacgaatgaa ttgattagaa ctttaaatgg aaatattttt
catactggtg gcattggaac 180acagcctcag aaagagtgga attttcagct
gccacaattg ggtacaactt tattaaattt 240agatgataat tatgttcaat
caactagagg cataatcgat tttttatcat cttttataga 300agctgtatgt
gatgatgaaa ttgttagaga agcttcaaga aatggtatgc aacctcaatc
360accagctctt atattattat cttcatcaaa atttaaaaca attaatttta
ataatagttc 420tcaatctatt aaaaattgga atgctcaatc aagacgtgag
aatcctgtat atgagtataa 480aaatccaatg ttgtttgaat ataaaaattc
gtatatttta caacgcgcaa atccacaatt 540tggaagcgtc atgggtttaa
gatattatac aacaagtaat acttgtcaaa ttgcagcatt 600tgattccacc
ctagctgaaa atgcaccaaa caatacacaa cgcttcgttt ataatggcag
660actaaaaaga cccatatcaa acgttttaat gaaaatagaa gctggtgctc
caaatataag 720caacccaact attttacctg atcctaataa tcaaacaact
tggcttttta atccggtaca 780attaatgaat ggaacattta ccattgaatt
ctataataat ggtcaactaa ttgatatggt 840tcgaaatatg ggaatagtta
ctgtaagaac ttttgattct tatagaataa caattgacat 900gattagacca
gctgctatga ctcaatacgt tcaacgaatt tttccacaag gtggacctta
960tcattttcag gctacatata tgttaacatt aagtatatta gatgctacca
cagagtccgt 1020tctatgtgat tctcattcag tggaatattc aatagtagca
aacgttagaa gagattcagc 1080gatgccagct ggaactgttt ttcaaccggg
atttccatgg gaacacacac tatccaatta 1140cactgttgct caagaagata
atttagaaag attattgtta attgcatctg tgaagagaat 1200ggtaatgtag
ataagctaga gggctaaaca tcttctatgc ggcctacata ccatgtagca
1260tgaatcacga ctgggtttag tccatgcttg cataggggca aatatgcatg
atatggatga 1320tccccagaag gatgaaatgt gaactatgtg gct 1353
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