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1996 Characterization of the P and G types and the VP4 and VP7 gene divergence among group A bovine rotaviruses Srinivas Mummidi Iowa State University
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Characterization of the P and G types and the VP4 and VP7 gene
divergence among group A bovine rotaviruses
by
Srinivas Muminidi
A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY
Major Immunobiology Major Professor: Dr. Prem S. Paul
Iowa State University
Ames, Iowa
1996 DMI Number; 9712581
UMI Microform 9712581 Copyright 1997, by UMI Company. All rights reserved.
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For theKtfra llege iii
This thesis is dedicated to my parents
and grandparents TABLE OF CONTENTS
ACKNOWLEDGMENTS V
ABSTRACT VI
CHAPTER L GENERAL INTRODUCTION
CHAPTER 2. LITERATURE REVIEW 6
CHAPTERS. BOVINE ROTAVIRUSES 26
CHAPTER 4. THE VP4 AND VP7 OF BOVINE ROTAVIRUS VMRI ARE ANTIGENICALLY AND GENETICALLY CLOSELY RELATED TO P-TYPE 5, G-TYPE 6 STRAINS 51
CHAPTER 5. RELATIVE FREQUENCY OF P AND G TYPES OF ROTAVIRUSES ASSOCIATED WITH CALF DIARRHEA AS DETERMINED BY NESTED REVERSE TRANSCRIPnON POLYMERASE CHAIN REACTION 78
CHAPTER 6. COMPARATIVE ANALYSIS OF THE VPS* SEQUENCES OF P[5] AND P[11] ROTAVIRUSES ISOLATED FROM CALVES WITH DIARRHEA 95
CHAPTER 7. SEQUENCE AND PHYLOGENETIC ANALYSIS OF THE VP7 GENE OF A BOVINE ROTAVIRUS WTTH G6 SUBTYPE 107
CHAPTERS. GENERAL CONCLUSIONS 112 V
ACKNOWLEDGMENTS
It gives me a great pleasure to acknowledge the inspirational guidance of my Major
Professor, Dr. Prem S. Paul, during this study. Dr. Paul has provided the motivation and the insight which allowed me to take this dissenation to its fruition. I wish to thank my present and previous POS committee members, Drs. James Roth. Chris Minion, Nolan Hartwig,
Bruce Janke, John Andrews, and Robert Benbow for their scientific input and critical evaluation of the manuscripts included in this dissertation. I would also like to thank Drs. Hank Harris and Mike Wannemuehler for their support. It would not have been possible to complete this dissertation work without the friendship and assistance of Dr. Xiang-Jin Meng, Dr. Igor Morozov, Dr. Alexei Zaberezhny, Dr. Eric Vaughn. Dr. Yanjin Zhang, Dr. Young Lyoo, Dr. Theerapol Sirinarumitr, Dr. Pomtippa Nawagitgul. Dr. Peter Akoto, Ms. Marsha Morgan, Ms. Susan Schuchaskie, Ms. Susan Hartmann, Ms. Dana McCullough, Ms. Mary
Brooks, Ms. Kelly Hicks, and Ms. Laura Pusateri. My heartfelt thanks go to my wife, Sridevi Allam, for her love and support. Finally, I would like to acknowledge the encouragement provided by my present mentors, Drs. Sunil Ahuja, Seema Ahuja, Robert
Clark, and Tony Valente. vi
ABSTRACT
Group A rotavirus is one of the most important etiological agents associated with neonatal calf diarrhea. The surface proteins. VP4 and VP7. are responsible for the induction of neutralizing antibodies to the virus, and determination of viral serotypes and genotypes.
Types based on VP4 and VP7 are referred to as P-types and G-types. respectively. A vaccine is commercially available but is not effective in many herds. We posmlated that this may be due to the incoporation of a virus in the vaccine which differs from that prevalent in the field. In this particular study, we sought to determine the relative frequency of occurrence of P and
G types associated with rotaviruses isolated from calves with diarrhea and the genetic variation associated with the VP4 and VP7 genes of bovine rotaviruses (BRV).
Initially, we characterized the VP4 and VP7 genes of the BRV strain VMRI, which showed antigenic differences with the strain incorporated in the vaccine. Our results indicated
that the VP4 of VMRI was different from that of the vaccine strain. The distribution of P and G types of rotaviruses associated with calf diarrhea was determined using multiplex reverse transcription-polymerase chain reaction. Our results confirm diat P[5]:G6 rotaviruses are the most predominant viruses associated with calf diarrhea in the field. This is the first comprehensive study on the distribution of P types
among BRV in the U.S. Nucleotide sequence analysis of the N-terminal trypsin cleavage fragment of VP4
(VPS* region) of 9 P[5] type rotaviruses and 10 P[11] type rotaviruses obtained from
diarrheic calves revealed a high degree of nucleotide homology among the field strains
indicating a low mutation rate in BRV VP4 genes. Finally, during our studies on genetic variation in the VP7 gene in BRV. we have
detected an isolate designated VMRI-29 in the feces of a diarrheic calf. Sequence analysis of
VP7 gene of VMRI-29 showed 82% nucleotide homology and 90% amino acid identity, and vii differences in the variable regions with that of G6 BRV reference strains. This is the first report of a genotypic subtype among G6 type BRV outside Japan. I
CHAPTER 1. GENERAL INTRODUCTION
Dissertation organization
This dissertation deals with the molecular epidemiology and genetic variation in the
VP4 and VP7 genes of bovine rotaviruses (BRV). The thesis consists of a general
introduction, a review of rotavirus biology, a review of BRV with emphasis on recently
published literature, four different manuscripts, and general conclusions. The general introduction describes the dissertation organization and the rationale behind this study. All
four manuscripts are included in their entirety and are formatted according to requirements of
the individual journals. The Ph.D. candidate, Srinivas Mummidi, is the senior author of all the papers included in this dissertation. The first manuscript describes the characterization of
the genes coding for immunogenic proteins of a BRV VMRI. This manuscript has been
pubUshed in Veterinary Microbiology. The work encompassing antigenic characterization of
VMRI and the Northern blot hybridization was a portion of Master's thesis submitted to the Iowa State University by Ms. Mary Brooks, who is listed as a second author on this paper.
Dr. Young Lyoo and Dr. Alexei Zaberezhny are listed as co-authors on this manuscript as
they have made contributions to the polymerase chain reaction (PGR) amplification and cloning of the VP4 and VP7 genes. The second manuscript, which has been submitted for publication, describes the relative prevalence of distribution of P and G types in BRV field
isolates in the U.S. as determined by a multiplex PGR tj^ing assay. The third manuscript
describes the sequence variation occurring in the VPS* domain of the VP4 protein of BRV
field isolates, has yet to be submitted for publication. The fourth manuscript describes the isolation, VP7 gene sequence and phylogenetic analysis of VMRI-29, which represents a G6
subtype rotavirus. Dr. Robert Holland is listed as a co-author on this paper because he was
involved with initial detection of the rotavirus infection in the calf from which VMRI-29 is
isolated and his contribution towards delineating the history of this isolate. This manuscript
has been published in Virus Genes. 2
Rationale Rotavirases are an important cause of enteric disease complex and have been shown to be associated with diarrhea in calves between 1-2 weeks of age. It has been estimated that diarrheal disease can cause up to 5-20% mortality in calves and about half a billion dollars in economic losses (Saif et al., 1994). There was a significant statistical association between the presence of rotavirus and calf diarrhea (Reynolds et al., 1986). A modified live virus vaccine is available commercially in the U.S., but the efficacy of this vaccine has been questioned by studies which have shown that the incidence of diarrhea is similar in vaccinated and unvaccinated herds (Waltoer-Toews et al., 1985). Immunization strategies to prevent rotavirus diarrhea have been developed by a number of research workers, and the present view is that passive transfer of antibodies to the calf through colostrum and milk is the most effecdve way to prevent rotavirus induced calf diarrhea (Theil, 1990). This can be effectively achieved by immunization of the pregnant dam. Several smdies have also shown that the immunfty against rotaviruses is serotype specific (Offit and Clark, 1985; Murakami et al., 1986). One reason given for vaccination failure is the absence of the most prevalent serotypes in the vaccine (Brooks and Paul, 1989; Brooks, 1989; Mummidi et al., 1992;
Parwani et al., 1992; Parwani et al., 1993; Saif et al., 1994). To develop effective vaccination strategies to control calf diarrhea, it is important to determine the extent of serotypic and genetic diversity associated with field rotavirus isolates. To understand this diversity associated with rotaviruses, my colleagues and I have cloned VP4 and VP7 genes of BRV strain VMRI, developed a mutiplex PGR based technique to distinguish BRV serotypes and genotypes in fecal samples, and sequenced the genes encoding the immunogenic proteins of rotavirus isolates from fecal samples.
Isolation and characterization of BRV VMRI with a supershort electropherotype has been previously reported (Paul et al., 1988). This vims showed antigenic differences with
NCDV-Lincoln strain (which is present in the vaccine). Also, Northern blot hybridization 3 data indicated that the VP4 coding genes between these strains were different (Brooks,
1989). Further studies using Northern blot hybridization on ceil culture adapted viruses also indicated that the strains similar to VMRI were most prevalent in the field. Therefore, we cloned, sequenced and analyzed the genes coding for the immunogenic proteins from this strain.
Preliminary studies in OUT laboratory and in other laboratories using PGR generated complementary DNA (cDNA) probes have shown that the predominant serotypes present in fecal samples are P (VP4) types 5 and 11 and G (VP7) types 6 and 10 (Mummidi et al.,
1992; Parwani et al., 1993). It is well established that G6 and GIO serotypes are the most predominant rotaviruses associated with calf diarrhea in the field (Snodgrass et al., 1990;
Mummidi et al., 1992; Parwani et al., 1992; Parwani et al., 1993). In one large scale smdy performed on 102 rotavirus positive fecal samples from diarrheic calves from U.S. and
Canada, Parwani et al. (1993) have shown that 29% of the field strains had P5 type virus,
11% had PI 1 type virus and less than 2% had PI type virus. Over 50% of the field samples could not be typed with respect to their P type (Parwani et al., 1993). This prompted us to examine the prevalence of the P and G type of rotavimses using a highly sensitive multiplex
PGR typing assay which was developed for cell culmre adapted BRV (Isegawa et al., 1993).
The results of this study are reported in the second manuscript of this thesis. Limited information is available regarding the genetic variation within a serotype of
BRV. Also no information, to our knowledge, exists about the VP4 and VP7 genes from non cell culture adapted BRV field isolates. Information on the genetic variation in the immunogenic proteins of the field isolates will help in development of effective vaccines to combat diarrheal disease in calves. The results of our analysis on the sequence variation in the VPS* genes among field isolates of BRV are reported in the third manuscript. During our studies involving the genetic variation in the VP7 encoding genes in the field isolates, we have discovered the presence of a G6 subtype vims, which we have designated VMRI-29. 4
In the fourth manuscript we report the sequence and phylogenetic analysis of the VF7 gene of this isolate.
References Brooks, M.A., 1989. Characterization of antigenic relationships among bovine rotaviruses, M.S. thesis, Iowa State University.
Brooks, M.A. and Paul, P.S., 1989. Prevalence of gene 4 types among bovine rotavirus field isolates, abstr. 80, p. 16. Proc. 70th Annu. Meet. Conf. Res. Workers in Anim. Dis., 1989.
Isegawa, Y., Nakagomi, O., Nakagomi, T., Ishida, S., Ishida, S., Uesugi, S. and Ueda, S., 1993. Determination of bovine rotavirus G and P serotypes by polymerase chain reaction. Mol. and Cell. Probes, 7: 277-284.
Mununidi, S., Zaberezhny, A., Lyoo, Y.S. and Paul, P.S., 1992. Nucleic acid probes for distinguishing bovine rotavirus P and G types, abstr. 64, p. 13. Proc. 73rd Annu. Meet. Conf. Res. Workers in Anim. Dis., 1992.
Murakami, Y.. Nishioka, N., Watanabe, T. and Kuniyasu, C., 1986. Prolonged excretion and failure of cross protection between distinct serotypes of bovine rotavirus. Vet. Microbiol., 12: 7-14.
Offit, P.A. and Clark, H.F., 1985. Matemal antibody-mediated protection against gastroenteritis due to rotavirus in new bom mice is dependent upon both serotype and titer of antibody. J. Infect. Dis., 152: 1152-1158.
Parwani, A.V., Rosen, B.I., Lucchelli, A., Hussein, H.A., Navvaro, L.E. and Saif, L.J., 1992. Characterization of field isolates of Group A bovine rotaviruses using G and P Type- specific probes, abstr. 65, p. 14. Proc. 73rd Annu. Meet. Conf. Res. Workers in Anim. Dis., 1992. 5
Parwani, A.V., Hussein, H.A., Rosen, B.I., Lucchelli, A. and Saif, L.J., 1993. Characterization of field strains of group A bovine rotaviruses by using polymerase chain reaction-generated G and P type-specific cDNA probes. J. Clin. Microbiol., 31: 2010-2015.
Paul, P.S., Lyoo, Y.S., Woode. G.N., Zheng, S., Greenberg, H.B., Matsui, S., Schwartz, K.J. and Hill, H.T., 1988. Isolation of a bovine rotavirus with a "super-short" RNA electrophoretic pattern fi:oni a calf with diarrhea. J. Clin. Microbiol., 26: 2139-2143.
Reynolds, D.J., Morgan, J.H., Chanter, N., Jones, P.W., Bridger, J.C., Debney, T.G. and Bunch, K.J., 1986. Microbiology of calf diarrhoea in southern Britain. Vet. Rec., 119: 34-39.
Saif, L.J., Rosen, B.I. and Parwani, A.V., 1994. Animal rotaviruses. In: ed. A.Z. Kapikian, Viral infections of gastrointestinal tract. Marcel-Drekker, Inc., New York, NY, pp. 289-314.
Snodgrass, D.R., Fitzgerald T., Campbell, I., Scott, F.M.M., Browning, G.F., Miller, D.L., Herring, A.J. and Greenberg, H.B., 1990. Rotavirus serotypes 6 and 10 predominate in cattle. J. Clin. Microbiol., 28: 504-507.
Theil, K.W., 1990. Group A rotaviruses . In: ed. L.J. Saif, and K.W. Theil, Viral diarrhea of man and animals. CRC press, Boca Raton, FL, pp. 35-72.
Walmer-Toews, D., Martin, S.W., Meek, A.H., McMillan, I. and Crouch, C.F., 1985. A field trial to evaluate the efficacy of a combined rotavirus-coronavirus/£yc/ienc/i/a co/i vaccine in dairy cattle. Can. J. Comp. Med., 49: 1-9. 6
CHAPTER 2. LITERATURE REVIEW
History and classiHcation
Rotaviruses are classified under the genus rotavirus of the family Reoviridae and are
named as such for their characteristic "wheel-like" appearance in negatively stained
preparations (Flewett et al., 1974). Light and Hodes (1943) reported that they were able to
reproduce enteritis in calves with filtrates from stools of diarrheic children. Later, electron
microscopic studies revealed that these viral agents in fact had rotavirus morphology (Hodes,
1977). Mebus et al. (1969) isolated a diarrheal agent from calves which was designated as
Nebraska calf diarrhea virus. They also reproduced diarrhea in calves by serially inoculating filtrates of fecal material. This agent was cultivated in fetal bovine primary cultures (Mebus et al., 1971). In humans the virus was visualized by thin section electron microscopy of duodenal biopsies by Bishop et al. (1973). Initial smdies usually referred to this agent as a
reo-like virus. Femelius et al. (1972) demonstrated that these viruses are antigenically distinct from reovirus. The double stranded nature of rotavirus genome and their similarity to
reoviruses was suggested and established in several smdies (Welch and Thompson, 1973;
Newman et al., 1975; Rodger et al., 1975). Rotaviruses have been isolated from a variety of species including swine, horses, dogs, cats, chicken and turkeys (Estes, 1983). A number of excellent reviews have appeared which cover various aspects of rotavirus biology, genetics, pathogenesis and inmiunity (Estes et al., 1983; Estes and Cohen, 1989; Bellamy
and Both, 1990; Theil, 1990; Paul and Stevenson, 1992; Paul and Lyoo, 1993; Saif et al.,
1994; Estes, 1996; Kapikian and Chanock, 1996).
Rotaviruses were initially shown to share a group specific antigen irrespective of the species they infect. Later smdies have demonstrated the presence of rotaviruses with
additional group specificities in humans and animals and these have been referred to as
atypical rotaviruses or pararotaviruses (Bohl et al., 1982). Currently, based on group specificities, rotaviruses are classified into groups A-G (McNulty et al., 1984). Groups A. 7
B, and rarely C are present in humans whereas the animal viruses are distributed among aU the groups. Using convalescent calf sera, and later monoclonal antibodies, it has been demonstrated that group A rotaviruses have "subgroup" specificities which are encoded in the
VP6 (Kapikian et al., 1981; Greenberg et al., 1982). Based on this, group A rotaviruses are classified as subgroup I and subgroup II. Also, studies indicated the existence of neutralization antigens of virus which were distinct from the group specific antigens (Kalica et al., 1981; Kapikian et al., 1981). These were found to be encoded by genes specifying the surface proteins VP4 (VP3 in earlier literature) and VP7 (Greenberg et al., 1983; Taniguchi et al., 1985; Hoshino et al., 1985; Liu et al., 1988). A binary scheme for classifying the viruses based on these neuttalization specificities was proposed (Hoshino et al., 1985; Estes and Cohen, 1989). In this scheme, the serotypes determined by VP4 are referred to as P types (for protease-sensitive) whereas the serotypes based on the VP7 are referred to as G types (for glycoprotein).
An important advance in smdies on rotavirus has been the development of cell culture adaptation methods (Babuik et al., 1977). Rotaviruses have been found to be very difficult to adapt to cell culture. The discovery that the addition of trypsin enhances the infectivity of rotaviruses has given a great impems to studying their biology. Recombinant DNA and other molecular biology approaches have provided an insight into the replication of rotaviruses and in characterization of its immunogenic proteins (Estes, 1996; Kapikian and Chanock, 1996).
Rotavirus structure The structure of rotaviruses has been studied in detail in recent years using cryoelectron microscopy and computer image processing (Prasad et al., 1988; Shaw et al.,
1993; Yeager et al., 1994). It has been proposed that the rotavirus is organized as a triple shelled particle with the inner most layer enclosing the 11 double stranded genomic RNA segments (Prasad and Chui, 1993; Shaw et al., 1993). The outer shell is composed of VP4 8 and VP7 proteins whereas the intermediate layer consists of the VP6 trimers. Both the outer and the intermediate shells show a T = 13 levo icosahedral symmetry. The inner most layer is composed mainly of the VP2 protein and this is most probably a T=I structure (Patton et al., 1995). This layer also consists of VPl and VPS proteins which form the subcore particles. The VP7 protein forms the smooth outer shell of the triple shelled virion particle whereas the VP4 forms the spike structure. There are sixty spikes protmding from the surface of each virion. The spike appears to be composed of VP4 dimers and it seems to closely interact with the VP7 and the VP6 proteins (Shaw et al., 1993). The base of the spike appears to form a hexameric structure which might have a scaffolding function (Yeager et al., 1994). Three types of channels have been described traversing the outer and intermediate shells of the rotavirion which might serve as conduits for metabolite entry and mRNA extrusion.
Rotaviral genes and proteins Nonstructural proteins and genes Five of the eleven proteins encoded by the rotavirus genome are non stmctural and are designated NSP1-NSP5. These proteins are mainly involved in rotavirus replication, assembly and maturation (reviewed by Patton et al., 1995). NSPl is a 53 kDa protein and is encoded by gene 5 segment. The N-terminus of this protein has a cysteine rich region which is thought to be involved in specific binding of viral mRNAs (Hua et al., 1994). The NSPl is a 35 kDa protein which shows similarity to bacterial proteases and metalloproteinases. NSP2 has also been demonstrated to have RNA binding activity and might be involved in
RNA replication and/or assembly (Kattoura et al., 1992; Kattoura et al., 1994). The NSP3 protein contains a basic region between amino acids 83-150 which forms a a helical structure which is present in single strand RNA binding proteins. Several other motifs have been described in this protein including heptad peptide repeats of hydrophobic residues, a leucine 9
zipper region and a region similar to the human immunodeficiency virus-1 tat protein
(Mattion et al., 1994; Patton. 1995). It has been demonstrated that NSP3 binds to the consensus sequence at the 3' end of the viral mRNAs although its exact function has not been delineated (Poncet et al., 1993). The NSP4 protein has oligomerization domains and most
likely serves as a intracellular receptor for immature rotavirion particles during the budding
process through the endoplasmic reticulum (Patton, 1995). Recent evidence indicates that
NSP4 is a viral enterotoxin and might be responsible for diarrhea in rotavirus infections (Ball
et al., 1996). The NSP5 has sites for phosphorylation and is posmlated to have a regulatory
role during viral repUcation (Patton, 1995). Several of the characteristics of the non structural genes and proteins are summarized in Table 1. Structural proteins and genes The rotavirus particle is composed of six structural proteins and the eleven double stranded genomic RNA segments (Table 2). The VPl, VP2, VP3 and VP6 proteins are
involved in RNA replication and transcription (reviewed by Patton, 1995). The VPl is a basic protein and it is found to have several motifs which are associated with RNA dependent RNA polymerases. However, it shows replicase activity only in conjunction with the VP3.
The VP3 has guanyl transferase activity and is involved in the capping of the messenger RNAs. The VP2 protein has a helix tum helix motif, an RNA binding region and a leucine zipper. It is probably involved in viral replicase activity. The VP6 protein is myristylated
and is proline rich. The VP6 protein is essential for the transcriptase activity of the rotavirus
particles, however, it alone does not possess polymerase activity. The VP6 protein carries
the rotavirus group specific antigen as well as the sub-group antigens. Based on the
serological reactivity of VP6 protein, at least 7 groups (Groups A-G) have been defined
among rotaviruses (Theil, 1990). Most of the information discussed in this review pertains
to group A rotaviruses as they have been studied the most. As this dissertation is mainly
concerned with the two outer capsid proteins VP4 and VP7, these proteins will be dealt with 10
Table 1. Features of rotavirus non-structural proteins^
Protein No. of aa (Kd) Protein Biochemical Biological [ Modifications properties properties NSPl 486-495 58.6 Slightly basic, Zn Cytoskeleton fingers at amino associated, might terminus which are determine plaque essential for size, host range binding rotavirus restriction (?) RNA "
NSP2 317 36.7 Basic, RNA Accumulation in binding, viroplasm during multimerization viral replication, essential for possible complex binding with VPl and VP3
NSP3 315 34.6 Slighdy acidic, a Associated with helical, RNA cytoskeleton, binding, heptad binds 3' end of repeat regions RNA
NSP4 175 20.2 Uncleaved 3 hydrophobic RER signal amino terminal transmembrane sequence, N- regions, Ca*"^ glycoprotein, linked binding tetramer morphogenesis glycosylation (?)
NSP5 198 21.7 Phosphorylate Slightly basic, Associated with d, 0-linked Ser-Thir rich, RNA the viroplasm. glycosylation binding regulatory role in transcription (?) ^ Modified from Patton, 1995
in detail.
The VP4 protein is encoded by die gene segment 4 in group A rotaviruses. The VP4 gene in most of the animal rotaviruses is 2362 base pairs and encodes a polypeptide of 776 amino acids (Fig. 1). As discussed previously, the VP4 protein forms the spike structure of
rotavirion and forms about 1.5% of the virion protein. A number of biological propenies are
associated with the VP4 protein including hemagglutination, plaque size, tissue tropism. 11
Table 2. Features of rotavirus structural proteins"
Protein No. of u (Kd) Protein Biochemical Biological aa Modifications properties properties VPI 1088 125 Basic, RNA Subcore protein, dependent RNA replication polymerase, ODD motif
VP2 880 102 Myristylated, RNA binding. Leu Core protein, cleaved zipper replication
VPS 835 98 Basic, guanyl Sub core protein, transferase transcription
VP4 776 87 Cleaved by Dimer, amino- Outer capsid proteases tenninal (3 strands, spike protein, carboxy terminal a virulence, helices neutralization Ag, cell attachment, plaque size VP6 397 44 Myristylated Hydrophobic, Iimer capsid trimer protein, transcription, subgroup antigen
VP7 326 37 N-linked high Bicistronic, integral Outer capsid mannose membrane glycoprotein, residues glycoprotein, two neutr^ation followed by hydrophobic amino antigen trimming terminal regions, putative calcium binding site "Modified from Mattion et al., 1994
pathogenicity, viral replication phenotype, and host range restriction (Estes and Cohen,
1989). There is an increasing evidence that it might serve as a viral receptor for cell attachment (Ludert et al., 1996). Analysis of the polypeptide stmcture indicates that the amino terminal region (-60% of the protein) forms p strands and is hydrophobic where as the carboxy terminal region forms a helices. It has been suggested that the amino terminal portion forms the head of the spike whereas the a helix between residues 494-557 might 12 84 180. 241. .247 580, I 10 ,776
Region B I I I I VPS* VPS* Fig. 1. General schematic showing the feamres of rotavirus VP4. Region B extends from residues 84-180. The trypsin cleavage sites are indicated at residues 241 and 247. The variable regions in the VPS* domain is also shown. form the stem of the spike. There are two conserved trypsin cleavage sites at positions 241 and 247 in animal rotaviruses . Following trypsin cleavage, VP4 protein is cleaved into the
N-terminal VP8* and die C-terminal VPS* fragments. The preferred trypsin cleavage site appears to be at the residue 247, at least in the rhesus rotavirus RRV (Lopez et al., 1985). In this manuscript, VP8* refers to the N terminal 241 amino acids (242 amino acids in case of
B223) whereas the VPS* refers to the C terminal S29 amino acids of the VP4. The peptide between the VP8* and VPS* fragments is referred to as the connecting peptide. The region between 384- 404 shows similarities to die fusion regions of several viral proteins (Mackow et al., 1988; Mattion et al., 1994). Between die amino acids 494 to SS4 there are several heptad repeats which might facilitate dimerization of VP4. The cysteines at positions 318,
380 and 774 are conserved among all the VP4 proteins whose sequences have been determined except in case of the strains 69M and B223. Initially group A rotaviruses were serotyped based on the cross neutralization tests using hyperimmune antisera. It was previously thought that VP7 elicited neutralizing antibodies exclusively. Subsequendy it was discovered diat monoclonal antibodies directed towards VP4 can also neutralize rotaviruses and die neutralization specificities encoded by 13
VP4 and VP7 can segregate independently (Greenberg et al., 1983; Hoshino et al., 1985;
Offit and Blavat, 1986). The discovery of two surface antigens capable of eliciting neutralizing antibodies led to the proposal of a binary system of classification akin to the one used to distinguish serotypes in the influenza viruses (Hoshino et al., 1985; Estes and
Cohen, 1989). The serotypes based on VP4 protein are referred to as P types (for protease- sensitive) whereas the serotypes based on VP7 protein are designated as G types (for glycoprotein) (Estes and Cohen, 1989). It has also been suggested that the term P-serotypes be reserved for defining antigenic relationships between VP4 protein through serological assays (for example, neutralization tests using antisera raised against baculovirus expressed VP4 or by using reassortants or by neutralizing monoclonal antibodies) and the term P- genotypes be used when the relationship between different VP4 proteins is determined through sequence analysis (Sereno and Gorziglia, 1994; Estes, 1996). A description of cuirendy described P-serotypes and P-genotypes is shown in Table 3. A more thorough discussion of P and G types which are pertinent to this dissertation appears in Chapter 3.
A number of different approaches have been used to define the antigenic and immunogenic properties of the VP4 protein. Two linear antigenic determinants have been postulated in the VP4 protein based on the neutralization activities of peptides derived from
VP8* and VP5* subunits (Arias et al., 1987; Streckert et al., 1988; Hansen et al., 1992).
The neutralization antibody inducing peptides which map to the VP8* region are derived from just upstream of the trypsin cleavage site. Larralde and Gorziglia (1992), expressed three different peptides derived from VP8* in E. coli and used them to determine the antigenically important regions. Region B which comprises amino acids 85-183 was responsible for antigenic specificity whereas regions A and C were responsible for cross- reactivity among different VP4 proteins. Another approach which has been used to determine the neutralization epitopes on VP4 is by sequence analysis of the virus variants selected in the presence of hyperimmune serum or in the presence of monoclonal antibodies. 14
Table 3. Rotavirus P-genotypes and P-serotypes^
Genotype Serotype Origin''/strain
1 6 BO/C486, Bo/NCDV, Bo/A5 2 Si/SAll 3 5 HU/HC:R3, Si/RRV, Cay^, Fe/FRV64 4 IB Hu/RV-5, Hu/DS-1, Hu/S2, Hu/L26 5 7 Bo/UK, BO/B641, Bo/61 A, Bo/V1005, Bo/VMRI, Bo/678 6 2A Hu/M37, Hu/RV-3, Hu/ST3 2B Po/Gottftied 7 9 Po/OSU, Po/TFR-41. Po/YM, Po/CRW-8 8 lA Hu/Wa, Hu/P, HU/VA70, Hu/D, Hu/WI6l 9 3 Hu/K8, Hu/PAISI 10 4 Hu/69M 11 8 BO/B223, BO/A44, Bo/KK3, Hu/116E 12 Eq/H-2, Eq/Fl-14 13 Po/MDR-13 14 Hu/HALl 166, HU/PA169 15 Ov/Lpl5 16 10 Mu/Eb, Mu/EDIM 17 Bo/993/83, Pigeon/PO-l3 18 Eq/L338 19 Po/4F 20 Mu/EHP ^Adapted from Estes, 1996.
''Species of origin indicated in abbreviations: Bo, bovine; Ca, canine; Ch, chicken; Eq, equine; Fe/feline; Hu, human; La, lapine; Mu, murine; Ov, ovine; Po, porcine; Ty, turkey 15
The variable regions which include amino acids 92-192,393. and 588 showed mutations in response to the presence of the hyperimmune antiserum (Gorziglia et al., 1990). Zhou et al.
(1994) described the presence of a neutralization epitope on VP4 which is involved in conformational change in the virus structure subsequent to antibody binding. It has also been demonstrated that the specific interactions between the VP4 and VP7 proteins might be important in determining the antigenic nature of rotaviruses (Chen et al., 1992).
There are 780 molecules of VP7 protein in a rotavirus virion and it is the most abundant protein in die outer capsid (Estes and Cohen, 1989). It comprises about 30% of the marnre virion particle and is the second most abundant protein. It has been suggested that the VP7 protein might exist as multimers but this is not conclusively proven. VP7 may be encoded by genes 7, 8, or 9 depending on the virus strain. The VP7 gene is usually 1062 base pairs (bp) long and encodes a 326 amino acid polypeptide (Fig. 2). Two in-frame initiation codons exist in the VP7 gene which are present at nucleotide positions 49 and 139
1. .6 23 33 44 124 .155 326. . 1_. 1 1 1 1 1 # K[1 H21" Ca"^"*" binding
L_J U 1 I A B C
VR-2 VR-4 VR-6 r~i n I—I r~i r~i i~in c=] • VR-l VR-3 VR-5 VR-7 VR-8 VR-9
Fig. 2. Features of rotavirus VP7. The arrow indicates the signal peptide cleavage region.
The two hydrophobic domains (HI and H2) are shown. The three antigenic regions A, B, and C extend from aa 86-101, 142-152, and 208-221, respectively. The variable regions VR-1 (9-20), VR-2 (25-32), VR-3 (37-52), VR-4 (65-76), VR-5 (87-101), VR-6 (120-132).
VR-7 (142-151), VR-8 (209-225), and VR-9 (236-243) are also indicated. 16 and make the gene potentially bicistronic. The translated VP7 protein has a signal sequence and is cleaved between residues 50 and 51 (Fig. 2). Two hydrophobic regions are present upstream of the cleavage site. The first hydrophobic region extends from amino acid residues 6 to 23 and the second hydrophobic region extends from amino acid residues 33 to
48 (Estes and Cohen, 1989). The mature VP7 protein is an integral membrane protein, although it lacks the KDEL endoplasmic reticulum retention signal. However, LPITGS, a conserved sequence between residues, might play a role in its localization (Mattion et al.,
1994). The biological role of VP7 is not very clear, but it has been reported that VP7 gene can influence the virus growth in vitro (Xu and Woode, 1994). VP7 may also alter die receptor binding specificity of rotaviruses through its interaction with VP4 (Mendez et al.,
1996). As previously mentioned serotypes based on VP7 protein are referred to as G types and these are presented in Table 4.
Nine variable regions (VR1-VR9) and several antigenic regions have been defined in the VP7 protein (Nishikawa et al., 1989). The variable regions, which are defined based on the sequence analysis of the VP7 proteins in different serotypes are VR-1 (aa 9-20), VR-2
(25-32), VR-3 (37-53), VR-4 (65-76), VR-5 (87-100), VR-6 (119-132), VR-7 (141-150),
VR-8 (208-224), and VR-9 (235-242) (Fig. 2). Only six of these regions are present in the mature protein. The antigenic regions have been identified by the sequence analysis of virus variants selected in the presence of hyperimmune antisera or monoclonal antibodies (Dyall-
Smith et al., 1986; Hoshino et al., 1994). Dyall-Smith et al., (1986) have determined the presence of three antigenic regions in the VP7 based on the sequence analysis of monoclonal antibody neutralization resistant mutants. These are antigenic regions A (aa 87-101), B (aa
141-151) and C (aa 208-224) (Dyall-Smith et al., 1986; Taniguchi et al., 1988, Nishikawa et al., 1989). Further analysis also demonstrated antigenic regions between aa 65-76 (region
D), aa 189-190 (region E), aa 235-242 (region F) and at aa 291 (Lazdins et al., 1995). Interestingly, several of the antigenic regions overlap with the variable regions in the VP7 17
Table 4. Rotavirus G serotypes^
Serotype Origin''/Strain
1 HuAVa, Hu/D, Bo/T449 2 Hu/DS-1, Hu/S2, Hu/RV-5 3 Hu/P, Hu/RV-3, Si/SAl 1, Si/RRV, Ca/K9, Eq/H-2, Fe/FRV-1, Mu/EDIM, Mu/EB, Po/MDR-13, Po/CRW-8 4 HU/ST3, HuA^A70, Po/Gottfried, Po/SB-2 5 Po/OSU, Po/MDR-13, Eq/H-1 6 Bo/NCDV, Bo/UK, Bo/B641, BoA^MRI, Bo/C486, Bo/KN-4, Hu/PA151 7 Ch/Ch2, Ty/Tyl, Pigeon/PO-13, Bo/993/83 8 Hu/69M, Hu/HALl 166, Bo/678, Bo/A5 9 HU/116E, Hu/WI61 10 BO/B223, Bo/A44, Bo/KK3, Bo/61 A, Bo/V1005, Ov/Lpl4, Hu/I321 11 Po/YM 12 Hu/L26, HU/L27 13 Eq/L338 14 Eq/F123 " Adapted from Estes, 1996.
'' Abbreviations for the species of origin from Table 3.
protein. Extensive sequence variation is also observed in the second hydrophobic domain of the VP7 and surprisingly this region appears to be main target for cytotoxic T-lymphocytes in mice (Franco et al., 1993; Estes, 1996).
Rotavirus replication cycle Only the triple layered rotavirion particles seem to initiate infection and the viral interaction with the cell seems to be mediated through VP4, although a role for VP7 has not been completely excluded (Ruggeri et al., 1991; Ludert et al., 1996). The cellular receptor 18 for rotaviruses has not been identified. However it appears that the in vivo cellular receptor for rotaviruses is a glycoconjugate and the mechanism of virus-cell interaction may be either sialic acid dependent or independent (Fukudome et al., 1989; Mendez et al., 1994). Also it has been demonstrated that the mode of cell entry of trypsin activated and non-trypsin treated rhesus rotavirus virions is different (Kaijot et al., 1988). The trypsin activated viruses seem to gain entry through the mechanism of direct penetration where as the non-trypsin treated virions seem to be taken up by endocytosis. Only the former mode of entry results in a productive infection (Kaijot et al., 1988). Following virus entry, the virion associated RNA dependent RNA polymerase is activated, resulting in the synthesis of plus-strands from minus-strand templates of the genomic double stranded RNA segments. The synthesis of the minus-strands peaks at 6-9 hours post infection whereas that of the plus strands is maximal at 9-12 hours post infection (Stacy-Phipps and Patton, 1987). The genome replication of rotaviruses is asymmetrical in that the viral plus-strand serves as a template to produce the double stranded RNA. The morphogenesis of the rotavirus particle probably involves the sequential interaction of the structural and non-structural proteins with the newly synthesized
RNA (Patton, 1995). The virion particles are transientiy enveloped as they bud from the endoplasmic reticulum and are released through host cell lysis (McNulty et al., 1976).
The aspects of rotavirus pathogenesis and hnmunity to rotaviral infections are dealt with in detail in the following chapter.
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CHAPTER 3. BOVINE ROTAVIRUSES
Several aspects of BRV have been extensively reviewed previously (Theil. 1990:
Paul and Lyoo, 1993; Saif et al.. 1994). This review mainly deals with the recent advances in genetics, epidemiology and virulence of BRV.
History
Rotaviruses are classified under the genus rotavirus of family Reoviridae (Matthews.
1979). BRV was first identified in the filtrates of fecal material from field cases of diarrheic calves, which induced diarrhea in e.xperimentally inoculated calves (Mebus. 1969).
Rotaviruses have now been reported from several mammalian and avian species and are recognized as important pathogens of medical and economical consequence (Estes. 1983).
Morphology BRV are indistinguishable from other rotaviruses by electron microscopic examination. The rotaviruses have a triple layered strucmre (double capsid particles in earlier literature) and are characterized by a well defined layers of "outer rim", "short spikes", and a
"wide hub", which gives the particles awheel-like appearance (McNulty, 1978; iVIattion et al., 1994). Besides the triple layered particle, two other types of particles are also evident during electron microscopic examination: double layered particles (single shelled particle in earlier literature) and virus cores. Only the triple layered particles are complete and infectious
(Mattion et al., 1994). The morphogenesis of BRV in cell culture and intestinal cells have been reviewed previously (McNulty, 1978; Theil. 1990).
Propagation
Several cell culture systems can support the growth of group A BRV in vitro. These include primary cell cultures derived from fetal bovine kidney, fetal bovine lung and cell lines 27 such as baby hamster kidney (BHK), Vero, embryonic bovine trachea, and human hepatocellular carcinoma (Hep G2) (reviewed by Saif et al.. 1994). The fetal monkey kidney cell line, MA-104, has been extensively used for cell culture adaptation and growth of
BRV. The discovery that trypsin enhances the infectivity of rotaviruses and the use of roller tubes for virus growth have gready simplified the adaptation of group A BRV to cell culture
(Babiuk et al., 1977). However, group B and group C BRV are extremely fastidious and there is only a single report describing the adaptation of a group C BRV to cell culture
(Tsunemitsu et al., 1991).
Physicochemical and biochemical properties
BRV are highly ubiquitous and are extremely stable in the environment. They are excreted in feces in very high numbers which can be up to 10® -10* infectious doses of virus per gram (Woode et al., 1976). BRV are stable for months at 4°C. Ijaz et al. (1994) have examined the effect of relative humidity and temperature on the survivability of aerosolized
UK BRV strain. In their study, the half-life of UK virus was 18 hours when relative humidity was maintained at about 50%. However reduced thermostability was observed when the virus was incubated at 50°C for 1 hour in the presence of MgCl, (Welch and
Thompson, 1973). The virus is resistant to extraction with lipid solvents such as ether and chloroform, indicating the absence of an envelope (Welch and Thompson, 1973). BRV are also resistant to low pH (pH 3.0) for 1 hour at room temperature. The rotavirus particle is disrupted when treated with alcohols and 95% ethanol can be used as a disinfectant for BRV
(Bishai et al., 1978). The oiple layered virus particle has a density of 1.36 g ml"' in CsCl whereas the double layered particle has a density of 1.38 g ml"' in CsCl (McNulty, 1978).
The intact BRV contains at least six structural proteins (VPl, VP2, VP3, VP4, VP6, and VP7). Several investigators reported a higher number of polypeptides associated with the BRV particle. These might be the artifacts generated due to proteolytic processing of 28 virion polypeptides, different electrophoresis conditions, or different giycosyiation patterns
(Kouvelos et al., 1984). Cohen, (1977) demonstrated the association of RNA polymerase activity with purified calf rotavirus. The VP2 protein of BRV has been shown to have RNA binding activity (Boyle and Holmes, 1986).
Genome and genetics The core of the BRV particle consists of 11 double stranded genomic RNA segments in addition to VPl, VP2, and VPS proteins. The genomic RNA constimtes about 12% of die rotavirus particle. The total molecular mass of the genome is 12.35 X 10® daltons. The size of individual segments ranges from 0.4 X 10® to 2.25 X 10® daltons (Rixon et al., 1984).
The RNA segments of BRV can be visualized after deproteination of virus particles and analysis by polyacrylamide gel electrophoresis (PAGE). The pattern produced by the migration of the RNA segments through polyacrylamide gels has been referred to as
"electropherotype". Group A BRV are characterized by 4-2-3-2 electropherotype pattem, whereas group B viruses have a 4-2-2-3 pattem and group C have a 4-3-2-2 pattem (Theil,
1990). The electropherotype of group A rotaviruses can be "long", "short", or "super-short" based on the migration pattem. In general, the migration pattem of group A BRV is long (Theil, 1990). However, there have been reports of short and super-short migration patterns among these viruses (Paul et al., 1988; Theil et al., 1988; Allen et al., 1989; Parwani et al.,
1995; Chinasagaram et al., 1995). The supershort migration pattem in the VMRI strain is due to the rearrangement in the 11th genomic segment (Paul et al., 1988; Matsui et al.,
1990). The functional assignment of the different segments of the rotaviruses has been determined through reassortant studies and in vitro translation. The genes encoding all the stmctural and nonstructural proteins from BRV have been sequenced except for the guanyl transferase, VPS (Saif et al., 1994). Hybridization of rotaviral genomic RNA from several 29
strains with labeled in vitro transcribed probes indicates that the group A BRV share a high
degree of homology in several gene segments and can be classified as a single genogroup
(Matsuda and Nakagomi, 1989; Munmiidi et al., 1996a). At least two different gene 5 segments are present among group A BRV (Lu et al., 1994; Xu et al., 1994; Mummidi et al.,
1996a). The nucleotide sequences of genes 6 and 10 of bovine group C rotaviruses have
been reported (Jiang et al., 1993).
Incidence and detection of BRV Several methods have been described to determine the presence of rotaviruses in feces
including electron microscopy, enzyme-linked immunosorbent assay (ELISA), latex
agglutination, and PAGE of rotavirus RNA. Electron microscopy is highly specific for
rotavirus detection (Theil, 1990). ELISA is the most frequently used method to detect the
presence of group A BRV in feces. But wide spread use of group A based ELISAs
precludes the detection of other groups of rotaviruses. Detection of rotavirus nucleic acid in
PAGE gels has been extensively used and can detect the presence of non group A
rotaviruses. However, confirmation of the group should ideally be performed using
serological assays. Hammami et al. (1990) reported that concentrated PAGE was highly
specific for identification of BRV from feces. In their study 19% of 77 fecal samples diarrheic calves were positive for rotavirus. The prevalence rate of BRV infections has been
variously estimated between 30-83% (Theil, 1990; Behymer et al., 1991). A survey of 450
neonatal calves indicated that the overall prevalence of rotavirus in calves from Ohio, USA, was 16.4% (Lucchelli et al., 1992). In a closed dairy herd, 14.6% of the stool samples
tested were positive for rotavirus (Ishizaki et al., 1995). Chinasagaram et al. (1995) reported that by using reverse transcription-polymerase chain reaction (RT-PCR) about 90% of the fecal samples from clinical and non-clinical cases were positive for group A rotaviruses
whereas about 70% of these samples were positive for group B rotaviruses. A high 30 prevalence of antibodies (47-56%) to group C rotaviruses was observed in adult cattle in the
U.S. and Japan (Tsunemitsu et al., 1992a).
Antigenic relationstiips and serotypes Most of the group A BRV can be classified as sub-group I based on their reactivity with sub-group specific monoclonal antibodies (Theil and McClosky, 1989). The first well characterized virus among BRV is the NCDV-Lincoln strain. A strain which showed some antigenic differences from the NCDV strain was subsequendy isolated from U.K. (Woode et al., 1974; Woode et al., 1983). Further studies have indicated diat a different rotavirus serot3^e is also present in calf diarrhea cases (Murakami et al., 1983; Woode et al., 1983;
Snodgrass et al., 1984; Ojeh et al., 1984; Briissow et al., 1987). Based on the serological reactivity of these viruses they have been classified as a distinct serotype (serotype 10)
(Snodgrass et al., 1990; Matsuda et al., 1990). Sequence analysis indicated that the VP7 gene of G6 viruses differs from that of the GIO type viruses (EUeman et al., 1983; Glass et al., 1985; Taniguchi et al., 1990; Xu et al., 1991). Snodgrass et al. (1990) and Taniguchi et al. (1991) reported the isolation of G8 serotype rotaviruses from cattle. Recendy, NCDV- Cody strain has been assigned to G8 serotype based on serological and sequence analysis
(Lu et al., 1995; Chang et al., 1995). A serotype 1 BRV, T449, was isolated from Argentina
(Blackball et al., 1992). Briissow et al. (1992) reported the isolation of 993/83, a G7 serotype BRV. There is also evidence for the existence of G2, G3, and Gil rotaviruses in cattle (Hussein et al., 1993). However, these isolates have not been fully characterized yet.
Matsuda et al. (1993) reported that the BRV KN-4 showed two way cross neutralization with both G6 and GIO rotaviruses. Further analysis indicated that KN-4 had P type 11. Only sequence analysis could confirm the G6 serotype assignment of KN-4 strain (Matsuda et al..
1993). Recendy we have reported the VP7 sequence analysis of VMRI-29, which showed a high degree of homology with that of KN-4 (Mummidi et al., 1996b). The VP7 gene of this 31 isolate had a low nucleotide homology (81%) with the reference G6 strains. However, amino acid sequence analysis indicated that it is a G6 type virus. Similar subtypes or variants have also been reported from other laboratories. Briissow et al. 1990 reported that V1005 might be a G10 subtype virus.
Although the VP7 gene sequence of UK was similar to that of NCDV, its VP4 gene sequence differed from that of NCDV (Kantharidis et al., 1988). This confirmed the presence of a second bovine VP4 type. Sequencmg of several BRV strains confirmed worldwide distribution of viruses with UK like VP4 (Hardy et al., 1991; Taniguchi et al.,
1993; Mummidi et al., 1996a). Northern blot hybridization was used to determine the relationships between the strains NQDV, VMRI and B223 indicated that the VP4 genes between these strains might be different (Brooks and Paul, 1989; Brooks, 1989). It was also indicated in another study that there are antigenic differences between NCDV and B641 VP4 proteins (Zheng et al., 1989). Subsequent smdies using genetic reassortants and sequence analysis confirmed the presence of 3 different VP4 types among BRV (Matsuda et al., 1990;
Hardy et al., 1992). VP4 sequence analysis of the 993/83 isolate indicated that it is closely related to pigeon rotavirus (Isegawa et al., 1994; Rohwedder et al., 1995). Even though serological tests indicated that V1005 might represent a distinct P type, sequence analysis indicated that in fact it is a P5 type virus (Snodgrass et al., 1992; Briissow et al., 1994). There have been only a few reports regarding the isolation of non group A rotaviruses in cattle. Voderfecht et al. (1986) reported the detection of a group B rotavirus which is associated with formation of epithelial syncytia in experimentally inoculated calves. The group C BRV strain, Shintoku, is antigenically distinguishable from Cowden strain, which is a group C porcine rotavirus strain (Tsunemitsu et al., 1992b). This indicates serological diversity exists among group C rotaviruses. 32
Epidemiology The mode of transmission of rotaviruses is through the fecal-orai route. It has also been suggested that transmission can occur through aerosols (Ijaz et al., 1994). Morbidity in case of rotavirus infections can be as high as 80-90% in unexposed herds. Mortality in rotavirus infected herds is generally low unless additional enteric pathogens are present. BRV have a world wide distribution (Saif et al., 1994). Several methods have been used to study the rotavirus epidemiology including ELISA, electropherotype, presence of serum neutralization antibodies. Northern blot hybridization, dot blot hybridization and PGR. In most cases, a single electropherotype has been found to be associated with a rotavirus infected herd (Theil and McCloskey, 1989; Lucchelli et al., 1992; Ishizaki et al., 1995). The observed electropherotypes were also different from that of the vaccine strain (Theil and
McCloskey, 1989; Lucchelli et al., 1992; Chinasagaram et al., 1995). Ishizaki et al. (1995) reported the persistence of a single electropherotype in a closed dairy herd. The beef and dairy cattle which are subjected to different management practices show differences in incidence of rotavirus infections and the distribution of group A BRV serotypes. Dairy herds show a more complex distribution of serotypes as compared to the beef herds (Bellinzoni et al., 1989a). The G serotype distribution among field rotavirus isolates in different countries has been studied extensively. The distribution of the G6 type rotaviruses in dairy and beef herds has been examined in Argentina using ELISA (Bellinzoni et al., 1989a). In their study, about 85% of the isolates from beef herds (n=72) were classified as serotype 6 viruses and the rest were classified as non serotype 6 or untypable viruses. However, only 44% of the 43 fecal samples from dairy herds were classified as G6 type viruses. The rest were either non serotype 6 viruses or untypable. Snodgrass et al. (1990) examined the serotype distribution among cattle in U.K. using Mab based ELISAs for Gl, G2, G3, G5, G6 and PI 1 types. About 66% of the 162 samples were typed as G6 viruses; about 31% of the 33 samples remained untypable (Snodgrass et al., 1990). In a German smdy, serum neutralizing antibodies to G6 serotype were most predominant in different age groups of cattle followed by antibodies to G8 and GIO serotype viruses (Briissow et al., 1990). We and others have determined by using PGR generated cDNA probes that P5:G6 isolates are the most commonly occurring rotaviruses associated with calf rotaviral diarrhea in the U.S.
(Mummidi et al., 1992; Parwani et al., 1992). G type distribution among 102 BRV field samples from the U.S. and Canada was determined using a dot blot hybridization technique
(Parwani et al., 1993). In this smdy 36% were G6 viruses, 13% were GIO viruses, and 3% were G8 type. About 25% of the samples were untypable. In a follow-up study, the same group reported that 22% of the G6, G8 and GlO-negative samples can be classified as Gl,
G2, G3 and G4 types using cDNA probes (Hussein et al., 1993). A monoclonal antibody based ELIS A was also used to determine the prevalence of other G types associated with calf rotaviruses (Hurtado et al., 1995). In this study, about 7% of 134 G6 and GlO-negative viruses from the U.S. were positive for Gl, G2 and G3 type rotaviruses. This implies that there are additional rotavirus G-serotypes associated with calf diarrhea.
Information on P serotype distribution among BRV field isolates is limited due to the lack of serological reagents. Using cDNA probes specific for different P types Parwani et al.
(1993) reported that 2% of the samples were PI, 20% were P5 and 9% were PI 1 type (n=93). The rest were classified as untypable vimses. However using a multiplex PGR typing technique we have observed that most of the group A rotaviruses associated with calf diarrhea are typable with respect to their P type (Mummidi and Paul, submitted).
Several laboratories also examined the P and G types associated with cell culture adapted BRV. A semi-nested PGR assay was used to determine the P and G types of cell culture adapted BRV isolated in Japan (Isegawa et al., 1993; Suzuki et al., 1993). It was found in this study that P5:G6 viruses were the most predominant group (42.5%). PI I:G6 viruses formed the next largest group (17.5%). A similar survey was undertaken on 34 cell 34 culture adapted BRV of German origin (Briissow et al., 1994). P5:G6 viruses comprised
56% of the isolates whereas P5:GI0 were 32%. Surprisingly no PI 1 viruses were identified in this smdy. However, these figures may not reflect the situation in the field as there are differences among serotypes in their ability to grow in cell culture (Snodgrass et al., 1990).
Virulence and pathogenesis
It has been generally accepted that there is an age dependent resistance to rotavirus induced gastroenteritis in animals. However a recent study indicates that rotaviruses can cause disease in cattle irrespective of their age (Bridger, 1994). Recent smdies also indicated that there are differences in virulence between different rotavirus strains (Torres-Medina et al., 1985; Bridger et al., 1992). It has been proposed that low virulent strains of BRV cause disease in 2-week old calves whereas the virulent strains cause disease in calves irrespective of age (Bridger, 1994). Virulent strains of BRV are reported to spread more extensively and preferentially colonize the proximal portion of the small intestine (Bridger et al., 1992; Hall et al., 1993). Infection with virulent strains also cause severe damage to enterocytes and villi
(Hall et al., 1993). It has been speculated that VP4 might be responsible for virus spread in
vivo and the subsequent damage to intestinal epithelium, as it determines the virus spread in
vitro. However recent evidence indicates rotavirus virulence is multigenic and other viral factors might be related to pathogenesis (Broome et al., 1993). Simultaneous infection of calves with enterotoxigenic E. coli and rotavirus results in extensive intestinal damage and severe clinical iUness (Moon et al., 1978; Tzipori et al., 1983; Torres-Medina, 1984).
Several host and microbial factors might influence the severity of disease in dually infected calves including among others the amount of feed, dose of E. coli, and strains of E. coli and
rotavirus used for infection (Runnels et al., 1986). Most of the studies on the histological changes in intestine following BRV infection
indicate that infection is initiated in the proximal region of the small intestine and then 35 progresses more distally. However, there is inconclusive evidence regarding the region in small intestine which is mostly affected following the infection (Carpio et al., 1981; Torres-
Medina, 1984). The infection with BRV leads to villous attophy due to destruction of enterocytes (Mebus et al., 1971). This leads to the replacement of the villous epithelium with immature cuboidal cells. The immature cells lack the fiiU complement of Na^-K"^ ATPase and disaccharidases which results in hypertonicity in the large intestine due to accumulation of lactose (Middleton, 1978). Diarrhea in BRV infected calves develops within 18-24 hours.
The ensuing symptoms ui the affected calf are due to the loss of fluids and electrolytes. The symptoms persist until the redevelopment of mature villous epithelium.
Immunity, prevention and control Only a limited amount of information is available on the immune response of calves after exposure to rotavirus infection. Serum and fecal IgM and IgG antibodies developed after calves are challenged with rotavirus (Saif, 1987). Neonatal calves develop intestinal
IgA after oral inoculation of rotaviruses (Vonderfecht and Osbum, 1982). IgGl was the predominant serum antibody 2 to 3 weeks after infection. Local antibodies in the gut play an important role in die determining the protection to rotavirus infections (Woode et al., 1975).
Besser et al. (1988) indicated that the luminal antibody in the intestine of calves is derived by transudation from the circulating passive antibody, and this antibody can prevent rotavirus induced diarrhea. However, the role of neutralizing antibody in protection of calves against rotavirus infections has been questioned in one particular study (Bridger and Oldham, 1987).
An increase of bovine CT)8'^ cells and BoWCl'^y/S lymphocytes in the epithelium and lamina propria of small intestine was reported after infection of calves with rotavirus (Parsons et al..
1993). This is suggestive of involvement of these subsets of T lymphocytes in the immune response to rotavirus. Also, CD8* depleted calves showed an increase of rotavirus shedding. 36
The CDS"^ cells are thought to be involved in limiting primary rotavirus infection (Oldham et al., 1993).
BRV are ubiquitous in distribution, and it is impractical to eliminate them from the environment. However good management practices can help in controlling the exposure of calves to BRV. Several factors can determine the severity of rotavirus induced enteritis including the virulence of BRV, age of the calf, amount of virus to which the animal is exposed, immune status of the animal, and interaction with other pathogens. Basically two strategies have been developed to control rotavirus diarrhea in calves. One approach is oral vaccination of calves with modified live BRV. However the presence of maternal antibody in calves might neutralize the virus thus rendering the vaccine ineffective. Although this approach was successful under experimental conditions, oral vaccination of calves in endemically infected herds was found not to be protective (De Leeuw et al., 1980). A more successful method has been to enhance the colostral and milk antibody levels by dam vaccination. Vaccination of pregnant cows with inactivated or modified live vaccine was shown to be effective in decreased incidence of rotavirus associated diarrhea in calves
(Snodgrass, 1986; Castracci et al., 1987; Bellinzoni et al., 1989b; Castrucci et al., 1993). It has been suggested that intramuscular and intramammary injection of modified live vaccine into cows leads to better colostral and milk antibody levels (Saif et al., 1983). Colostrum derived from vaccinated cows was also shown to be effective in controlling calf diarrhea
(Castrucci et al., 1984; Castrucci et al., 1989). Due to the serotypic differences among the
BRV infecting cattle, several studies have addressed the stimulation of heterotypic antibodies by single serotype immunization (Snodgrass et al., 1984; Murakami et al., 1986; Bridger and
Oldham, 1987). Failure of cross protection between different serotypes in experimentally inoculated calves was observed by Murakami et al. (1986). Snodgrass et al. (1984), observed that immunization of cows with a single rotavirus serotype leads to the development of serotype specific neuuralizing antibodies and neutralizing antibodies to heterotypic strains 37 to which previous exposure has occurred. However, development of cross-neutralizing antibodies was observed in adult cows after immunization with a single serotype (Brussow et al., 1988). Woode et al. (1983) smdied cross protection between two G6 type viruses
NCDV and B641, and GIO vims B223 in gnotofaiotic calves. NCDV failed to protect the calves challenged with B223 and also surprisingly calves inoculated with B641. In a follow up study, B223 was found to induce both homologous and heterologous protection (Woode et al., 1987). Xu et al. (1993) further dissected the immune response to NCDV and B223 in gnotobiotic calves. It was suggested in this study that B223 induced a cross reactive immune response to several serotypes whereas NCDV induced a narrow immune response. Data from this smdy also indicated that the protective immune response to G6 viruses might be directed against VP4 (Xu et al., 1993). In contrast, the immunodominant immune response in the case of B223 is directed towards VP7 (Xu and Woode, 1993). Snodgrass et al. (1991) found that the development of heterotypic antibodies during primary rotavims infection is modest. But in vaccinated adult cows, die immune response to heterologous virus strains was more pronounced (Snodgrass et al., 1991). A subunit vaccine consisting of E. coli expressed VPS* domain from c486 was used to immunize cattle and was found to be effective in enhancing neutralization antibody titers in colostrum and milk. However, lower neutralization titers were induced against PI I type viruses (Lee et al., 1995).
Concluding remarks
During the past two and a half decades tremendous progress has been made in understanding rotavirus induced neonatal diarrhea in calves especially in terms of pathogenesis, epidemiology, and immunity. It is clear from recent smdies that two major VP4 types (P[5] and P[l 1]) and two major VP7 types (G6 and GIO) of group A rotaviruses are most likely associated with rotavirus induced diarrhea in calves. However, the role of group B and group C rotaviruses in the etiology of calf diarrhea is not clear. Another area of 38 concern is the induction of heterotypic immunity using a single serotype vaccination. Is a multivalent vaccine incorporating two or more strains necessary for induction of protective immunity? This question has not been answered satisfactorily to date. Recent advances in the development of vaccines including the use of virus like particles to immunize cattle should allow the incorporation of several protective antigens from multiple serotypes to provide a broad spectrum of immunity.
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CHAPTER 4. THE VP4 AND VP7 OF BOVINE ROTAVIRUS VMRI ARE ANTIGENICALLY AND GENETICALLY CLOSELY RELATED TO P-TYPE 5, G-TYPE 6 STRAINS
A Paper Published By Veterinary Microbiology
Srinivas Mummidi, Mary A. Brooks, Prem S. Paul.
Young S. Lyoo, and Alexei D. Zaberezhny
Abstract We have previously reported the isolation of a bovine rotavirus, designated VMRI, with a super-short electropherotype. We have characterized this strain further as it has shown antigenic differences with the prototype G6 strain NCDV-Lincoln. In diis communication, we report the antigenic, and molecular characterization and the nucleotide sequence of ±e VP4 and VP7 genes of this strain. Virus neutralization tests indicated 2- to 13- fold differences in the titers between NCDV-Lincoln, B64I and VMRI strains. Northern blot hybridization results indicated a degree of heterogeneity in the VP4 gene of these strains which can be detected under conditions of high stringency. The VP4 and VP7 genes of the
VMRI strain were cloned and sequenced and compared with the published sequences of other bovine rotavirus strains. The VP4 gene of VMRI had a high degree of homology with tiiat of
UK and B641 strains but differed significantiy from that of both NCDV-Lincoln and B223 strains. Sequence analysis of the VP7 gene of VMRI and other strains indicated a high degree of conservation, and the amino acid identity between the different strains was 96%.
Sequence information regarding these strains and field isolates will assist in the generation of effective vaccination strategies for control of neonatal calf diarrhea. 52
1. Introduction
Rotaviruses are a major cause of diarrhea in the young of many species, including cattle. They are classified under the family Reoviridae, and are characterized by the presence of 11 double stranded (ds) genomic RNA segments and a triple layered protein shell enclosing the genome (Estes and Cohen, 1989; Paul and Lyoo, 1993; Mattion et al., 1994).
The outer capsid is composed of two proteins, VP4 and VP7, both of which are responsible for eliciting neutralizing antibodies. The VP4 protein is encoded by gene 4 whereas die VP7 protein is found to be synthesized by gene 7,8 or 9 depending on the virus strain. The VP4 is an 88 kDa protease-sensitive protein usually composed of 776 amino acids, and is cleaved into two fragments designated VPS* and VPS* on treatment with trypsin. The VPS* fragment has been shown to specify both serotype and cross reactive epitopes whereas VPS* was shown to specify inter-serotype specific epitopes. VP7 has been considered to play a major role in the humoral immune response to rotaviruses, though recent smdies emphasized the protective role of VP4 protein in rotaviral infections (Ward et al., 1990; Ward et al.,
1993). The presence of two surface proteins responsible for neutralization has led to a binary system of classification of rotaviruses with serotypes based on VP4 designated as P types and those based on VP7 referred to as G types (Estes and Cohen, 1989). Classification into
G and P types is based on the neutralization test, but other procedures, such as nucleic acid hybridization, PCR and nucleotide sequence analysis have been used to predict the serotype
(Flores et al., 1990; Gouvea et al., 1990; Green et al., 1988). A universal VP4 typing system is currendy not available because of the paucity of appropriate reagents. However, such a typing system has been established for some human rotavirus strains using recombinant baculovirus expressed VP4 proteins (Gorziglia et al., 1990). At least 18 P types and 14 G types have been identified among rotaviruses (Dunn et al.. 1994; Sereno and 53
Gorziglia, 1994). Sereno and Gorziglia (1994) also describe die presence of 15 P-genotypes based on the amino acid sequence of the VP4 protein of various strains.
There are three main P types in cattle, the prototypic strains being NCDV-Lincoln CP- type 1), UK and B641 (P-type 5), and B223 (P-type 11) (Paul and Lyoo, 1993; Saif et al..
1994). Nucleic acid hybridization indicates a large proportion of bovine rotavirus field isolates are untypable with respect to P type (Mummidi et al., 1992; Parwani et al., 1992;
Parwani et al., 1993). Although Snodgrass et al. (1992) predicted the presence of another P- type existing among bovine rotaviruses through reassortant studies, as represented by
V1005, a recent study indicated that V1005 is in fact a P5 type virus (Brussow et al.. 1994). Recendy, Isegawa et al. (1994) sequenced the VP4 gene of a bovine rotavirus isolate from Germany, 993/83, which indicated that it might represent a new P-type. Two main G types have been identified in cattle, G6 type represented by NCDV-
Lincoln and GIO type represented by B223. Several groups have also idendfied Gl, G2, G3, G8 and Gl 1 types among cattle using virus neutralization, nucleic acid hybridization, and sequence analysis (Taniguchi et al., 1991; Blackball et al., 1992; Hussein et al., 1993).
Snodgrass et al. (1990) found through serological analyses that G6 and GIO types are predominant among the rotavirus isolates associated with calf diarrhea. This was also confirmed by dot blot hybridization of RNA extracted from fecal samples with type specific probes (Parwani et al., 1993). The different G and P types occurring in bovine rotaviruses along with the representative strains are summarized in Table 1. In order to gain a better understanding of antigenic and genetic diversity among bovine rotaviruses, we have initiated smdies to isolate and characterize the bovine rotavirus isolates.
In an earlier report, we described the isolation and preliminary characterization of a bovine rotavirus with a "super-short" RNA electropherotype (Paul et al.. 1988). Nucleotide sequence analysis of the gene 11 equivalent of the VMRI strain indicated that the 3' untranslated region of the gene contained a duplication of a region derived from the coding 54
Table I. The P and G serotypes of selected bovine rotavirus strains
Strain P-type G-type Origin Reference NCDV-Lincoln^ I 6 United States Mebus et al.. 1969 c486 I 6 Canada Babuik et al., 1977 UK'' 5 6 United Kingdom Woode et al., 1974 B64I 5 6 United States Woode et al., 1983 B-60 5 6 Australia Huang et al., 1992 VMRI 5 6 United States Paul et al., 1988 B223" 11 10 United States Woode et al., 1983 Cr 11 10 United States Parwani et al., 1993 A44 11 10 Thailand Taniguchi et al., 1991 KK-3 11 10 Japan Murakami et al., 1983 B-H 11 10 Australia Huang et al., 1992 KN-4 11 6 Japan Murakami et al., 1983 6IA 5 10 Thailand Taniguchi et al., 1991 T449 9 1 Argentina Blackball et al., 1992 J2538 1 8 United Kingdom Snodgrass et al., 1992 A5 1 8 Thailand Taniguchi et al.. 1991 V1005 5 10 Germany Brussow et al., 1987 Briissow et al., 1994 993/83" 16 7 Germany Brussow et al., 1992
^indicates type strains
portion resulting in its altered migration pattern (Matsui et al., 1990). The aim of this particular smdy was molecular characterization of VP4 and VP7 genes of VMRI strain of rotavirus and comparative analyses of VP4 and VP7 genes of different strains representing
P5 and 06 type among bovine rotaviruses. To our knowledge, this is the second U.S. strain of P5:G6 serotype in which the VP4 and VP7 genes have been characterized. Analysis of amino acid sequences of VP4 and VP7 proteins of P5:G6 bovine rotavirus strains assumes 55
importance as nucleic acid hybridization indicates that strains with this combination of the P and G type are the most common rotaviruses associated with gastroenteritis in calves in the
U.S. (Mummidi et al., 1992; Parwani et al., 1992; Parwani et al., 1993).
2. Materials and Methods
Virus cultivation and purification
The bovine rotavirus strain VMRI was isolated in our laboratory from a diarrheic calf (Paul et al., 1988). The sources of the different virus strains used in this study were described previously (Zaberezhny et al., 1994). The virus was grown on the MA104 cell line following activation by trypsin at a concentration of 10 |ig/ml as described previously
(Paul et al., 1988). The vims was grown on a large scale and concentrated by centrifugation at 72,000 X g for 2 1/2 hours. The viral pellet was treated widi one-fourth volume of
trichlorotrifluroethane (Genetron-113, Johnson Matthey Electronics, MA) and then
centrifiiged at 480 X g for 20 minutes. The supernatant was layered on a 30% sucrose cushion and centrifuged at 72,000 X g for 2 1/2 hours. The virus pellet was then resuspended in 50 mM Tris-HCI and 5 mM EDTA and stored at -20 C. Further purification,
if necessary, was performed using isopycnic gradient centrifugation in a CsCl gradient. Gradients were prepared with CsCl solutions with densities of 1.2 g/ml and 1.4 g/ml containing 5 and 10% glycerol, respectively, using a linear gradient maker. The partially
purified virus was layered onto the gradient and centrifuged at 112,000 X g for 2 hours.
Visible bands containing double shelled capsids, single shelled capsids, and empty capsids
were collected and diluted in 50 mM Tris HCl (pH 8.0) and pelleted at 112,000 X g for 1
hour. The pellet was resuspended in 50 mM Tris-HCI (pH 8.0) and stored in aliquots at -70°C. 56
Virus neutralization test
Antisera preparation and viral neutralization were performed as previously described
(Paul et al., 1988). For the viral neutralization test, 200 fluorescent focus forming units (ffu) of the virus was mixed with equal volumes of diluted antisera and incubated for 1 hour at
37 C. The virus-serum mixture was inoculated into each of 4 wells (0.2 ml/well) of an
MA 104 cell monolayer in a 96-well plate. At 1 1/2 hours post infection, the inoculum was replaced with the cell culture medium containing 0.5 mg/ml trypsin. At 20 to 24 hours post infection, cells were fixed with methanol and stained for rotavirus antigen by an indirect immunofluorescence test. The reported neutralization titer was the reciprocal of the highest dilution of the serum that neutralized 50% or more of the virus.
In vitro transcription
In vitro transcription was performed as described with slight modifications (Flores et al., 1982). Briefly, the transcription was performed using the partially purified virions at 42 C for 6 hours in the presence of 8 mM ATP; 2.5 mM GTP, UTP, and CTP; 70 mM Tris- base 0.5 mM S-Adenosylmethionine; 15 mM magnesium acetate; 260 mM sodium acetate and lOU of RNase inhibitor. RNA was then precipitated by adding 2M LiCl and incubating overnight at 4 C. The pellet was washed with 100% ethanol followed by 70% ethanol, briefly dried under vacuum, and then resuspended in water. This RNA was utilized for cDNA synthesis. For generating labeled probes for the Northern blot hybridization we used a similar procedure as outlined above except that in the transcription reaction GTP was
32 reduced to 0.5 mM and 125-250 ^Ci of Pa -GTP (Amersham, Arlington Heights, IL) was included and the reaction was carried out in the presence of 0.1% bentonite. The labeled probe was precipitated as described above.
Polyacrylamide gel electrophoresis (PAGE) and Northern blot hybridization
Genomic ds RNA (400 ng) was extracted from partially purified bovine rotavirus strains NCDV-Lincoln. VMRI, B641 and B223 and separated by PAGE. Electrophoresis of 57
RNA segments was performed in 0.75 mm thick polyacryiamide slab gels with 10% resolving and 3.5% stacking gels. Electrophoresis was performed at a constant current of 10 mA for 15 hours at 4 C. Gels were stained with ethidium bromide to visualize the RNA bands and photographed. The dsRNA was electrophoretically transferred to nylon membrane (Zeta Probe, Bio-Rad laboratories, Richmond. CA) according to manufacturer's instructions with the following modifications. The dsRNA was partially denatured in 0.1 M
NaOH for 5 minutes, neutralized and transferred to nylon membrane at 80V overnight. The
a membranes were air dried and then baked for two hours at 80 C. The membranes were
0 prehybridized for 2 hours at 52 C in a solution containing 2.5 X SSC, 4 X Denhardt's solution, 100 mM Tris-HCl (pH 8.0), 2 mM EDTA, 7% SDS, 200 |j.g/ml salmon testes DNA, and 50% deionized formamide. Hybridization was then performed using denatured ^"P-labeled RNA probe. Following hybridization, membranes were washed four times at room temperature in 1 X SSC with 0.1% SDS, twice in 2 X SSC with 0.1% SDS and twice in 1 X SSC at 52 C. The filters were kept moist and exposed to X-OMAT film (Eastman
Kodak Co., Rochester, NY).
Reverse transcription and polymerase chain reaction (RT-PCR) and molecular cloning of
VP4 and VP7 genes
First strand cDNA was synthesized using avian myeloblastosis reverse u^scriptase (Invitrogen, San Diego, CA) with VP4 and VP7 gene-specific primers corresponding to the
3' terminus sequence. The primers used for the cDNA synthesis were (5')GGTCAG-
ATCCTCCAGAAGCTG(3') for the VP4 gene and (5')GGTCACATCATACTAAT-
CCTA(3') for the VP7 gene. The first strand cDNA was amplified in a PCR in the presence of 0.2 mM of each dNTP; 1 mM of each primer; 1.5 mM MgCU; 50 mM KCl; 20 mM Tris
HCl, pH 8.0; and 2.5U of Taq DNA polymerase (Boehringer Mannheim, Indianapolis. IN) in a 100 |il volume. The reaction was overlaid with 50 ^ll of mineral oil and carried out in a
temperature cycler (Coy, Troy, MI). The 5' end primers used for amplification were 58
(5')GCTATAAAATG-GCTTCGCTC(3') for the VP4 gene and (5')GGCnTAAAAGAG-
AGAATTTC(3') for the VP7 gene. The 3' end primers were the same used to prime the
9 cDNA synthesis. The PGR amplification conditions for VP4 gene were 94 C for 1 1/2
minutes, 48 C for 2 1/2 minutes, and 72 C for 3 minutes for the first cycle followed by 30
o 9 o cycles of a profile consisting of 94 C for 2 minutes, 48 C for 2 minutes, and 72 C for 2
minutes. The last cycle consisted of an extension period of 10 minutes. The PGR
a 0 amplification conditions used for VP7 gene were 92 G for 1 minute, 48 G for 45 seconds,
and 72 G for 1.5 minutes for 30 cycles. The amplified products were purified from the reaction using glass milk procedure (Bio 101. La Jolla, GA). DNA was resuspended in
water and was blunt ended and phosphorylated using lOU each of DNA polymerase and T4 polynucleotide kinase in the presence of 50 mM Tris-HGl, pH 7.5, 10 mM MgGU, 1 mM
DTT, 20 mM dNTPs and 1 mM rATP. The reaction was terminated after 30 minutes and ±e DNA was run through an agarose gel. The specific band was cut out, purified and was then
ligated to Sma I cut Bluescript SK + vector (Stratagene, La Jolla, GA). The ligation mix was used to transform competent DH5a cells by the GaGli method (Sambrook et al., 1989).
Ampicillin resistant colonies containing the recombinant plasmids were scored for inserts by
blue-white selection and the presence of the insert was confirmed by restriction analysis of isolated plasmid DNA.
Nucleotide sequence analysis of VMRJ VP4 and VP7 genes
Three clones of VMRIVP4 gene were completely sequenced using universal.
reverse, and gene-specific oligonucleotides. Sequencing was performed by Sanger's
dideoxy chain termination method using an automated fluorescent sequencer (Applied
Biosystems, Foster Gity, GA). The nucleotide sequence of VMRI VP7 gene was determined by directly sequencing the PGR product. Sequence analysis was performed using MacVector
(International Biotechnologies, New Haven. GT) and GeneWorks (IntelliGenetics, Inc.,
Mountain View, GA ) software. The nucleotide sequences of VMRI VP4 and VP7 genes 59
have been deposited with the genbank and have been assigned accession numbers U53923 and U53924, respectively.
3. Results
Antigenic relatedness of VMRI, B641 and NCDV-Lincoln strains
Cross-neutralization tests with hyperimmune antisera demonstrated that NCDV-
Lincohi, VMRI, and B641 were closely related antigenically and possibly belonged to the same serotype (Table 2). However, 2- to 13- fold differences in the neutralization titers of antisera were observed between NCDV-Lincoln, VMRI, and B641 which suggested that some antigenic differences existed between these viral strains. These strains have been shown to be antigenically distinct from B223 (Paul et al., 1988; Woode et al., 1987).
Table 2. Antigenic relationships among bovine rotavirus strains by virus neutralization^.
Neutralization titers (ratios) of antisera to bovine rotavims strains Virus NCDV-Lincoln VMRI B641 NCDV-Lincoln 32, 000 (1.00) 15, 885 (4.30) 8,038 (1.45) VMRI 16, 000 (2.00) 68, 086 (1.00) 22, 600 (0.50) B64I 12, 708 (2.50) 31, 770 (2.14) 11, 721 (1.00)
^Data represent the reciprocal dilution giving 50% reduction in fluorescing foci. The ratio of the neutralization titer of serum against the homologous vims to that for the heterologous virus is given in the parentheses.
Gene segment homology among bovine rotaviruses
Genetic relatedness among the four bovine rotavirus strains was determined by RNA-
RNA hybridization using Northern blot (Fig. 1). The radiolabeled ssRNA probes of NCDV-
Lincoln and VMRI were hybridized to RNA of NCDV-Lincoln, VMRI. B641. and B223. 0 Fig. I. Northern blot hybridization of ds RNA preparations at 52 C and 50% formamide.
Panel A: UV light photography of gels after ethidium bromide staining. Panel B: Autoradiogram after hybridization to the NCDV-Lincoln probe. Panel C: Autoradiogram after hybridization to VMRI probe. NCDV-Lincoln (lane 1), VMRI (lane 2), B641 (lane 3) and
B223 (lane 4). Segments l-Il of rotavirus RNA are denoted at the left. 7,8,9|
10
11 62
Using the relatively high stringent conditions which allow a base pair mismatch of 21%,
NCDV-Lincoln probe hybridized strongly to all segments of homologous NCDV-Lincoln
RNA and all heterologous gene segments except gene segment 4 of VMRI and B641 (Fig.
I). NCDV-Lincoln probe hybridized to heterologous B223 gene segments with the exception of genes 4, 5, and 9. VMRI probe hybridized to all segments of VMRI, B641, and NCDV-Lincoln except gene segment 4 of NCDV-Lincoln. As observed with the NCDV-
Lincoln probe, the VMRI probe hybridized to 8 segments of B223 with no apparent
hybridization to gene segments 4,5, and 9.
Sequence analysis of VMRI VP4 gene
The VMRI gene 4 was 2362 base pairs long and had an open reading frame of 776
amino acids. The open reading frame extended from nucleotide position 10 to the nucleotide position 2328 and codes for a protein of 86.26 kDa with a pl of 4.89. It had three potential trypsin cleavage sites at amino acid residues 241,246, and 247. The deduced amino acid sequence also revealed the presence of conserved cysteines at positions 216, 318,380, and
774. The conserved cysteine at position 203, which is present in most other rotavirus strains, is absent in the VP4 gene of P5 type strains. Nucleotide sequence comparison with the other published VP4 genes of P5 type showed a high degree of homology (Kantharidis et al., 1988; Hardy et al., 1991; Taniguchi et al., 1993). The nucleotide sequence of VP4 gene of
VMRI shares 91%, 93% and 94% homology with that of UK, 61A, and B641, respectively.
The deduced VP4 protein sequence had 94%, 95%, and 96% amino acid identities with that
of B641, UK, and 61A, respectively. The sequence relationships between different strains
belonging to the P5 serotype are summarized in Table 3. The alignment of VMRI VP4 amino
acid sequence with that of other P5 type strains is shown in Fig. 2. The VPS* region, which
is the N-terminal portion of cleaved VP4, shared 95-98% amino acid identity with other P5
type su-ains in this region. In the hypervariable region between the amino acids 71-204, 63
Table 3. Nucleotide homologies and amino acid identities of VP4 among bovine
rotaviruses belonging to P5 serotype^
Virus strain UK B641 61A VMRI LfK XX 9i 95 95" B641 92 XX 94 94 61A 93 92 XX 96 VMRI 91 94 93 XX
'^e nucleotide homologies are indicated on the bottom half of the diagonal
and the amino acid homologies are indicated in italics on the upper half.
VMRI VP4 shared 98% identity with VP4 of the surain 61A whereas with the VP4 proteins of B641 and UK it shared 92-93% identity.
Sequence analysis of VMRI VP7 gene
The VMRI VP7 gene appears to be 1062 base pairs long; the uncertainty regarding the length is due to die 3' end primer which we have used to amplify the VP7 gene. The recombinant clones as well as the PGR product were 1063 base pairs in length which was one nucleotide longer than what has been reported for VP7 gene for most other rotavirus strains. The gene had three open reading frames and the predicted VP7 protein was 326 amino acids in length. The VP7 gene of VMRI shared 93,95, and 96% nucleotide homology with that of NCDV-Lincoln, UK, and 3641, respectively (Glass et al., 1985;
EUeman et al., 1983; Hardy et al., 1991). The predicted protein showed a 96% amino acid identity with that of other G6 VP7 proteins (Table 4). The alignment of the VMRI VP7 protein with that of other strains is shown in Fig. 3. As is the case with other VP7 proteins the start codon at position 49 precedes the first hydrophobic region which extends from residues 6 to 23 (Estes and Gohen. 1989). Twenty-nine amino acids downstream of this. 64
VMRI VP4 MASLIYRQLLANSYAVDLSDEIQSVGSEKNQRVTVNPGPFAQTGYAPVNWGPGEVNDSTWQPVLOGPYQPASFD 75 UK VP4 K C P.. 75 B641 VP4 0 R P.. 75 61A VP4 P 75 VMRI VP4 LPVGNHMLiAPTqgqvwBqm»aiBMviLHPovTifTMinifugCTavLvaianm'AMMV«iim«ao iso OK VP4 a ^ .M V.. .V iso B641 VP4 R V ISO 61A VP4 V...V ISO VMRI VP4 .TaiMicTom»rrioerBimicimnmTO8n?Bvm5DErasRSQESAcrEYiNNGLP 22S UK VP4 225 B641 VP4 • •:• ~ 225 61A VP4 225
VMRI VP4 PIQNTRNVVPVAISSBS1££S5V0AMEDIWSKTSLHKQIQYN1U)IIIRFRF0KSIIKSGGLA7KMAEISFXAAH 300 UK VP4 300 8641 VP4 300 61A VP4 jfn PF r. K G 300
VMRI VP4 YQYNYMKDGEEVTAHTTCSVNGVNDFSFNGGSLPTDFAISRYEVIKENSYVYVDYWDDSC3TFRNMVYVRSLAANL 375 UK VP4 R - A 375 B641 VP4 IS H A 375 61A VP4 TP - - T A - ^ 375
VMRI VP4 NDVMCSGGDYSFALPAGQWPVMKGCAATLHTAGVTLSTQFTDYVSLNSLRFRFRIAVEEPSFTITRTRVSKLYGL 450 UK VP4 450 B641 VP4 450 6U VP4 450
VMRI VP4 PAANPNGGREYYEVAGRFSLISLVPSNDDYQAPIMNSVTVRQDLERRLNELREEFNNLSQEIAVSQLIDLAMLPL 525 UK VP4 525 B641 VP4 525 61A VP4 525
VMRI VP4 DMFSMFSGIEGTVKAPKSHATNVMRKFKSSXLASSVSMLTDSLSDAASSIARSTSlRSISSTASAiailZSCQICO £00 UK VP4 s A 600 B641 VP4 A I S.. 600 61A VP4 .A. .N...... VS •«• 600
VMRI VP4 AVMB9*TISSQVSQISGKIJlIJCEITTQTEGMNn3DISAAVLKAKIDRSICIVDPHALPDVITEASEKFIRNRAYRV 675 OK VP4 R Q 675 B641 VP4 S N K 675 61A VP4 Q..F A 675
VMRI VP4 IDGDEAFEACTDGRFFAlfKVETLEEMPSNIEKFADLVTHSPVISAIIDFKTLKNIJIDNYGITREQAFNI.LRSNPK 750 UK VP4 F.M N D.. 750 B641 VP4 S FDM R 750 61A VP4 F.MG N 750
VMRI VP4 VLRGFIDPNNPIIKNRIEQLIMQCRL 776 OK VP4 Q 776 B641 VP4 M.Q 776
Fig. 2. The alignment of the predicted VP4 protein with other P5 type VP4 proteins. The three variable regions as defined by Mattion et al. (1994) are shown as shaded areas. The residues of the "region B" is indicated in bold. The connecting peptide is underlined. 65
Table 4. Nucleotide homologies and amino acid identities of VP7 among bovine rotavirus strains belonging to G6 serotype^
Virus strain UK B641 NCDV- B-60 VMRI Lincohi UK XX 97 97 96 96 B641 96 XX 96 95 96 NCDV-Lincoln 96 94 XX 96 96 B-60 94 92 92 XX 94 VMRI 95 96 93 91 XX
^e nucleotide homologies are indicated on the bottom half of the diagonal and the amino
acid homologies are indicated in italics on the upper half.
two mediionines occur in tandem in front of the second hydrophobic region, a feature it shares with NCDV-Lincoln and B-60 VP7 proteins. The second hydrophobic region extends from residues 33 to 48. The conserved glutamine residue at position 51 in the group A
rotavims VP7 proteins was also present in the VMRIVP7. There are three potential glycosylation sites occurring at positions 69. 238, and 318. The VP7 of VMRI strain shares
the glycosylation site at position 69 with B641, UK, and B-60 strains. This particular glycosylation site is absent in the NCDV-Lincoln strain.
4. Discussion
The overall goal of our studies is to better characterize the field isolates of bovine
rotaviruses associated with gastroenteritis with calves. A ioiowledge of genetic variation of
the VP4 and VP7 genes in the field will allow us to better define the vaccination strategies to
control rotaviral diarrhea in calves. Several groups have studied the relative contribution of
VP4 and VP7 in conferring innmunity to rotaviral infections (Ward et al.. 1990; Ward et al.. VR-1 VR-2 VR-3 VMRI VP7 MYGIEYTTllLISLT?SITLI.miLKSlTWlMPYIIYRIW.IAVli:lA'Jl!«INAQNYGVNLPITGSMDfrAYiU
VMRI VP7 DYVNQIIQTMSKRSRSLNSSAFYYRV 326 NCDV VP? 326 UK VP7 326 B641 VP? 326 fl-60 VP? K S 326
Fig. 3. The alignment of the predicted VP7 protein of VMRI with that of other G6 strains. The variable regions as defined by
Nishikawa et al. (1989) are shaded. The residues in bold indicate the different antigenic regions as defined by Dyall-Smith et al.
(1986). Note that the antigenic regions A, B, C overlap with VR-5, VR-7, and VR-8, respectively. 67
1993: Xu et al., 1993). Ward et al. (1993), showed that in natural rotavirus infections in infants the major neutralization response was directed to VP4. Xu et al. (1993) reported that the virus strain involved may determine the immunodominant neutralizing antigens during the primary immune response in rotaviral infections. A follow up study emphasized the importance of VP4 protein in providing heterologous immunity (Xu and Woode, 1993).
Sequence analyses of the reference strains and field isolates would potentially indicate the amount of antigenic drift occurring in nature as a result of mechanisms such as immune selection. In this study, we have presented andgenic and genetic characterisdcs of the VMRI strain. The first evidence regarding the antigenic namre of VMRI strain was through the cross-neutralization test which showed that VMRI strain was closely related to B641 and less closely to NCDV-Lincoln. Northern blot hybridization indicated the VP4 genes of VMRI and B641 might be more closely related to each other than to the VP4 genes of NCDV- Lincoln or B223 and was confirmed by subsequent sequence analysis of the VMRI VP4 gene. Gorziglia et al. (1990) suggested that the strains which share more than 89% of amino acid identity in their VP4s can be classified as die same P-type as that determined by neutralization test. Thus VMRI can be classified as a P-type 5 rotavims. In the VPS* region, the VMRI strain showed 95% amino acid identity with B641 and UK and 98% identity with 61A. Larralde and Gorziglia (1992) demonstrated that a region between amino acid 84-180 ("peptide B") in the VPS* is involved in serotype specificity. They also defined regions A (amino acids 1-102) and C (amino acids 150-251) which showed serotype cross reactivity. In region B between amino acids 84 to 180 the degree of identity is 93% with
UK, 95% with B641. and 98% with 61A (Sereno and Gorziglia. 1994). In 61A strain, similar amino acids were substituted at corresponding positions. When compared to B641. 3 significant amino acid changes were present in this region. These substimtions are at residues 140 (Leu-»Phe), 141 (Phe-^Val), and 162 (Ile-^Thr). It is unclear at the present 68
time if these changes can play a significant role in the immune response to rotaviral infections. Further sequence analysis studies of field isolates are needed as the region B was found to be immunodominant to regions A and C (Larralde and Gorziglia, 1992).
It is interesting to note that Cys-203 is absent in all P5 strains studied to date. Many of the animal rotavirus strains including NCDV-Lincoln and c486 have a consei-ved Cys-203, but most human strains lack this residue. A phylogenetic analysis of the VP4 polypeptides of several rotaviral strains by Patton et al. (1993) indicated that all species with Cys-203 clustered together and might have evolved from a common ancestor. Strains which lack Cys- 203. may have originated from and are more closely related to an ancestral gene which subsequentiy evolved into the various animal rotaviral strains which have Cys-203 and the human rotaviral strains. The lack of Cys-203 in VP4 of the P5 strains indicates that only a single disulfide bond between Cys-318 and Cys-380 is present in the VP4 proteins of these strains which may affect the tertiary structure of the protein (Patton et al.. 1993). We also analyzed the connecting peptide which comprises the amino acids between the residues 241 and 247 in the VP4 protein. All P5 type strains had Arg-241 which is conserved in all group A rotavirus strains. Residue 244 is glutamic acid in VMRIVP4 whereas it is lysine in all other P5-type VP4 sequences. The lysine present at residue 244 in the UK, B641 and 61A viruses creates a potential trypsin cleavage site and these viruses share diis characteristic with the VP4 of the strain K8 (P-type 9). In the sttains UK, B641 and 61A an arginine occurs at position 246 instead of the usual conserved 247 position found in most group A animal rotaviruses (Fig. 2). The VMRI VP4 is unique in that it shares Arg-
246 with other P5 type strains and also has the conserved Arg-247. The presence of an extra cleavage site due to the presence of lysine in the connecting peptide has been observed in the human rotavirus strains Wa, P and VA70 (Estes and Cohen, 1989). Thus, there are three potential trypsin cleavage sites in strains VMRI, UK and 61A whereas B641 has four such sites in the connecting peptide. The presence of an extra cleavage does not seem to confer 69
any selective growth advantage to the virus in vitro (Huang et al., 1993). Although previous studies indicated that the presence of an extra cleavage site correlated with virulence, the evidence seems to be inconclusive (Estes and Cohen, 1989; Broome et al., 1993; Burke et al., 1994). All of the P5-type strains also had the conserved Arg-231 which is posmlated to be a potential trypsin cleavage site.
The VP4 protein of VMRI has 34 prolines and most of these are conserved between all the P5 type strains. Sequencing of VP4 genes from several group A rotavirus strains indicated there are 19 proline residues which are highly conserved among them (Estes and Cohen, 1989). However, all the VP4 proteins of the P5 type strains sequenced to date lack the proline residues at positions 76 and 669. The strains VMRI, UK and B 641 are unique in that they have a proline in their connecting peptide at position 245. However in the strain 61A, a glutamic acid replaces the proline at this position. The significance of a proline residue in the cormecting peptide is not known. Sequence analysis of the VP7 proteins from several rotavirus strains has revealed
nine regions which show sequence variability (VR-1 to VR-9) (Green et al., 1987). Six of these are present in the mamre protein. Although a high degree of amino acid conservation occurs in these regions among the G-type 6 strains, the VMRI strain showed several amino acid changes which localize to the variable regions. These include residues 12 (Phe-»Ser),
73 (Ser-^Asn), and 221 (Thr-+Pro). Sequencing of neutralizing escape mutants of G1 and
G3 serotypes indicated the presence of three antigenic regions in the VP7 protein: Region A
(87-101), Region B (143-152), and Region C (208-221) (Dyall-Smith et ai., 1986). Three
more regions have been recendy defined using G2 and G3 viruses: Region D (65-76),
Region E (189-190) and Region F (235-245) (Kirkwood et al., 1993). All of these regions
seem to correspond to the regions of sequence variability found in the mature VP? protein. A
very high degree of homology is found in the A-F regions among the different G6 strains
(91-100% homology). These relationships are illustrated in Fig. 3. Two changes in the 70
VMRIVP7 amino acid sequence, at residues 73 and 221 in the variable regions also map to the antigenic regions D and C, respectively. Comparison of G6 VP7 with that of GIG strains revealed homologies of 43%, 60%, 57%. 50%, 50%, and 91% in regions A, B, C, D, E, and F, respectively. The overall homology of VP7 between 06 and GIO strains is up to
80% at the amino acid level. It is of interest to note that amino acid 69 is aspartic acid in the strain NCDV-Lincoln but is asparagine in the other G6 strains and this might lead to a different glycosylation pattern in these strains. Since glycosylation can alter the antigenicity of a protein, further smdies are needed to smdy the degree of amino acid conservation at this residue in several field isolates. It is to be noted that NCDV-Lincoln is the vaccine strain currently being used in the field in the United States. Another point of interest is the lack of cross hybridization between the gene segment 5 of NCDV-Lincoln/VMRI and gene segment 5 of B223. Xu et al. (1994) reported that gene 5 segments of UK and 3223 strains show 75% nucleotide homology and an amino acid identity of 71%. However, RF (P1:G6) and
UK strains shared 89% nucleotide homology and 95% amino acid identity. These results seem to be consistent with our Northern blot hybridization data. Further smdies on the sequence diversity of gene 5 segment from different bovine rotavirus strains are warranted as recent smdies have indicated that gene 5 in mice can confer a selective growth advantage in vivo (Broome et al., 1993). Overall we have found a high degree of conservation in VP4 and VP7 of bovine rotaviruses of the P5;06 serotype. However, a certain degree of variation between different strains is evident in the 'region B' and the connecting peptide of
VP4. Additional sequence analysis of field isolates should reveal the degree of genetic
variation occurring within the P5:G6 serotype and other serotypes causing rotaviral diarrhea
in calves. This information may result in more effective vaccination strategies for prevention and control of rotavirus infection. 71
Acknowledgments
We would like to thank Drs. J. Lundy and H. Hills for their help in nucleotide sequence analysis and Drs. J. Roth, C. Minion, R. Benbow and J. Andrews for their suggestions and critical review of this manuscript. This work was supported in part by grants from the Animal Health Care Division of the Biotechnology Research and
Development Corporation and the Iowa Livestock Health Advisory Council, State of Iowa.
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Zaberezhny, A.D., Lyoo, Y.S. and Paul, P.S., 1994. Prevalence of P types among porcine rotaviruses using subgenomic VP4 gene probes. Vet. Microbiol., 39: 97-110. 78
CHAPTER 5. RELATIVE FREQUENCY OF P AND G TYPES OF ROTAVIRUSES ASSOCIATED WITH CALF DIARRHEA AS DETERMINED BY NESTED REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION
A Paper Submitted to Journal of Clinical Microbiology
Srinivas Mummidi and Prem S. Paul
Group A rotaviruses are important agents of diarrhea in neonatal animals including calves. They are classified into different serotypes based on the specificities of the outer capsid proteins, VP4 (P serotypes and P genotypes) and VP7 (G-serotypes). Three P genotypes, P[l], P[5] and
P[ll], and tw^o G serotypes, G6 and GIO, are mainly associated with rotaviruses isolated from calf diarrhea cases. Previous studies using serological and hybridization techniques have indicated that G6 type viruses are the predominant viruses associated with calf diarrhea. However, the distribution of P genotypes in the field is relatively unclear. In this study, we report the distribution of P types and G types of rotaviruses associated with calf diarrhea using a multiplex PCR system. Our results confirm that
P[5]:G6 rotaviruses were the predominant viruses associated with calf diarrhea in the field in United States. In addition these studies indicate that
(i) most of the field isolates in our study were typable with respect to their P
type, (ii) a large number of calves in the study were dually infected with strains which differ in their P and/or G types (iii) about 15% of the samples 79 were not tvpable with respect to their G type, and (iv) the present vaccine strain in United States (P[1]:G6) is very rare in the field. The results from this study provide the basis for development of multivalent vaccines for control of calf diarrhea.
Rotaviruses are an important cause of enteric disease in neonatal animals including calves (Paul and Lyoo, 1993; Saif et al., 1994; Kapikian, 1996). They are classified under the family Reoviridae and are characterized by the presence of a triple shelled virus structure and 11 double stranded RNA segments (Mattion et al., 1994). The two outer surface proteins, VP4 and VP7. are responsible for eliciting neutralization antibodies against the virus. A binary system is used to classify group A rotaviruses based on the antigenic specificities of VP4 gene (P types) and VP7 gene (G types) (Estes and Cohen, 1989). Recently it has been proposed that the term P serotypes should be used for VP4 specificities of rotaviruses determined by using serological assays and P genotypes be used when the relationship between VP4 proteins is established through sequence analysis and by hybridization (Estes, 1996). There are mainly 3 P genotypes (P[l], P[5], and P[11]) and 3
G serotypes (G6, G8, GIO) recognized in cattle (Paul and Lyoo. 1993; Saif et al., 1994).
Previous studies using northern blot hybridization, dot blot hybridization with PGR generated cDNA probes, and liquid hybridization indicated that P[5]: G6 viruses are the most commonly occurring vimses in the field (Brooks and Paul, 1989; Brooks, 1989; Mummidi et. al., 1992; Parwani et al., 1992; Redmond et al., 1992; Parwani et al., 1993). This has also been demonstrated in cell culture adapted viruses and with a limited number of fecal samples using multiplex PGR techniques (Isegawa et al., 1993; Suzuki et al., 1993; Briissow et al., 1994; Gouvea et al., 1994a; Gouvea et al., 1994b). However, using PGR generated cDNA probes in dot blot hybridization, a majority of the field samples were found to be untypable with respect to their P type (Mummidi et al.. 1992; Parwani et al., 1992; Parwani 80 et al., 1993). Thus the distribution of P types among rotaviruses associated with calf diarrhea is not clear (Parwani et al., 1993). PGR is about 5,000 times more sensitive in the detection of rotavirus nucleic acid when compared to hybridization techniques (Xu et al.,
1990; Kapikian, 1996). We have adapted the multiplex PGR assay developed by Isegawa et al. (1993) for determining the P and G types of BRV from calf fecal samples. In this paper we report the relative prevalence of P and G types among rotaviruses in fecal samples and intestinal tissues from diarrheic calves in the United States.
MATERIALS AND METHODS Virus strains and Rotavirus field samples. The origin and cultivation of the different rotavirus sttains used in this study have been previously described (Zaberezhny et al., 1994; Mummidi et al., 1996). Rotavirus positive fecal samples, intestinal contents or intestinal tissues have been obtained from veterinary diagnostic laboratories in Iowa, Michigan, Wyoming, Galifomia, Pennsylvania and North Garolina representing midwest, western and eastern regions of the United States. All the samples were positive for the presence of rotaviruses by one or more of these techniques: ELISA, electron microscopy, latex agglutination, and polyacrylamide gel electrophoresis of rotavirus RNA.
Purification of rotavirus RNA. Extraction of fecal samples was performed according to previously described technique with some modifications (Xu et al., 1990; Gentsch et al.,
1992). Approximately 200 milligrams or 0.2 ml of feces was mixed with equal volume of
0.1 M sodium acetate buffer with 1% SDS and sonicated for three minutes. The homogenized fecal sample was extracted with equal quantities of phenol-chloroform (pH 6.2) and centrifuged at 12 000 x g for 10 min. The double stranded RNA was purified from the supernatant by using RNaid kit according to manufacturer's instmctions (Bio 101, La Jolla.
GA). RNA was eluted from the glass powder in DEPG treated water. In the case of the 81 intestinal tissues, RNA was isolated by using guanidium isothiocyanate procedure (RNA isolation kit, Stratagene. La Jolla, CA).
Reverse transcription and polymerase chain reaction. The purified double stranded RNA was denatured by heating it at 95°C for 3 minutes in the presence of 7%
DMSO and then by quick chilling on ice. Complementary DNA was synthesized in the presence of VP4 specific primer (nt 1909 to 1930, caaccgcagctgatatatcatc) or VP? specific primer (nt 1042 to 1062, ggtcacatcatacattgctaa) by using a cDNA cycle kit (Invitrogen, San Diego, CA) according to manufacturer's instructions. Two microliters of the cDNA was used for PCR amplification. PCR was performed in a 50 td reaction in the presence of 0.2 mM of each dNTP, 20 pM of each primer, Taq extender buffer, 2.5 U of Taq extender (Stratagene, La Jolla, CA) and 2.5 U of Taq polymerase (Boehringer-Mannheim,
IndianapoUs, Indiana). Non specific amplification of cDNA at lower temperatures was prevented by using a hot start technique with wax beads. The sequences of the common upper primers for the cDNA amplificadon of the VP4 and VP7 genes are ttcattattgggacgattcaca (nt 1067-1088), and ggctttaaaaga(c)gagaatttc (nt I to 21), respectively.
The temperatures for thermal cycling for the first PCR were 2 cycles of a profile consisting of 92°C for 2 min, 45°C for 1 min and 72°C for 3 min followed by 30 cycles of 92°C for 1 min,
45''C for 45 sec, and 72°C for 1.5 min. Negative controls were run concurrently along with all sets of PCRs to determine the presence of any laboratory contamination. The amplification products of the first PCR were detected after agarose gel electrophoresis and staining with ethidium bromide. The reaction product was then diluted 1; 100 in water and used to determine the type of the virus. The type specific primers used for second PCR were ttaaattcatctcttagttctc (P[l]), ggccgcatcggataaagagtcc (P[5]), tgcctcataatattgttggtct (P[l I]), ggtatcagctatttcgtttgat (G6), and aacgttctagtatttgtggtct (GIO). The reaction consisted of 30 cycles of a profile with 92°C for 45 sec, 50°C for 45 sec. and 72°C for 45 sec. The products 82 of the second PGR were electrophoresed on a 1.5% agarose gel and detected after staining with ethidium bromide.
Specificity of the PGR. The specificity of the P typing reaction was tested using the porcine rotavirus strains, OSU and Gottfried and simian rotavirus strain, EIRV. VP7 genes from several reference rotavirus strains [Wa (Gl), DS-l (G2), EIRV (G3), Gottfried (G4),
OSU (G5), and NCDV-Cody (G8)] were amplified and tested by using multiplex PGR as described above. Amplification products of correct size were not detected after the second
PGR which indicated that ±ese typing assays are specific.
RESULTS The VP4 common primers used in this study amplified a 864 bp fragment from P[l],
P[5], and P[11] type viruses (Isegawa et al., 1993). In the typing reaction, a mixture of VP4 type specific primers resulted in the amplification of a 460 bp product with a P[l] type virus, a 659 bp product with a P[5] type virus, and a 332 bp product with a P[11] type virus (Fig.
1). In a slight modification of G typing method described by Isegawa et al. (1993), we used primers to amplify the entire VP7 gene from the field isolates and then used G6 and GIO specific primers in a second PGR reaction. The second PGR resulted in the ampUfication of a 336 bp product with a G6 virus and a 692 bp product with a GIO vims (Fig. 2). The P typing and G typing reactions were highly specific for the detection of P[l], P[5], and P[11] viruses, and G6 and GIO viruses, respectively, in the panel of reference strains that we tested for specificity. In this study, we have tested the relative prevalence of P[l], P[5], and P[11], and
G6, and GIO types in 102 rotavirus positive fecal samples and intestinal tissues from diarrheic calves. Table 1 summarizes the distribution of P and G types among different field samples isolated from several states in the U. S. We successfully deteraiined the P type of Fig 1. Determination of P type of the reference and field strains of bovine rotaviruses. Lanes
1,2 and 3 show the amplification products from the bovine rotavirus strains, NCDV (P[l]), VMRI (P[5]), and B223 (P[l 1]) after the nested PGR. The amplification products from field isolates are shown in lanes 4 (P[5]) and 5 (P[I I]). The two bands in the lane 6 indicate the presence of mixture of P[5] and P[11] types. M 1 2 3 4 5 Fig 2. Determination of G type of the reference and field strains of bovine rotaviruses. Lanes 1 and 2 show the amplification products from the bovine rotavirus strains, VMRI (G6), and B223 (GIO) after the nested PGR. The amplification products from field isolates are shown in lanes 3 (G6) and 4 (GIO). The two bands in the lane 5 indicate the presence of a mixture of G6 and GIO serotypes. M 1 2 3 4 87
Table 1. P and G types of bovine rotaviruses isolated from different states in the U.S.
Number of isolates typed Type lA MI WY GA PA NG Total P5:G6 7 9 8 I 30
P5:G10 I - 1 - - 2
P5:G? 4 1 - 2 - 7
PII:G6 1 2 1 - I 5
Pll:G10 5 1 2 - 3 11
P11:G? 2 - 1 I - - 4
Pl+ll G6+10 - I - - - - 1
P5+1I G6 5 2 - 6 3 - 16
P5+11 GIO 2 2 - 4 - 1 9
P5+11 G6+10 2 1 1 5 I - 10
P5+II G? 2 - 1 - 1 - 4
P?:G6 - - 1 - - - 1
P?:G? - - - - 2 - 2 Total 31 19 16 16 14 6 102 the rotaviruses from a majority (98%) of the samples. In some instances, we did not detect the PGR product after first amplification. However, the second PGR resulted in amplification of the correct size bands. For VP7 type determination, we were unable to assign the G type in 16.7% of the samples. P[5] type viruses were detected in about 76.5% of the samples tested (Table 2). Another interesting observation was that we detected bands corresponding to both P[5] and P[11] type viruses in a large number of samples (38.2%).
However, only 25% of the samples with mixed P[5] and P[11] types also had mixed G6 and
GIO bands. P[11] viruses were detected in 19.6% of the samples tested. Only one of the
102 samples tested had an amplified band corresponding to the P[l] type. 88
DISCUSSION
Both surface proteins VP4 and VP7 are important for the host immune response to rotaviruses. The immunodominant protein responsible for protection might differ from one strain to another (Xu et ai., 1993). It is important to determine the distribution of different P types and G types of bovine rotaviruses associated with calf diarrhea because this information can lead to development of better control measures for its prevention. Several studies previously addressed the problem of the distribution of the VP4 and VP7 types in the field using monoclonal antibody based ELIS As and PGR generated cDNA probes (Bellinzoni et al., 1989; Snodgrass et al., 1990; Redmond et al., 1992; Mummidi et al., 1992; Parwani et
Table 2. Relative frequencies of P and G types among bovine rotavirus field isolates
Number of isolates typed P or G type G6 GIO G6+10 G? Total
P5 30 2 - 7 39
Pll 5 11 - 4 20
Pl+Pll - - 1 - 1 P5+P11 16 9 10 4 39
P? 1 - - 2 3 Total 52 22 11 17 102 al., 1993; Hussein et al., 1993). The serological tests and tests based on nucleic acid hybridization indicate that G6 and GIO viruses are the predominant G serotypes associated with calf rotaviruses (Brooks and Paul, 1989; Snodgrass et al., 1990; Mummidi et al., 1992;
Parwani et al., 1992; Parwani et al., 1993). The simation regarding the distribution of P types is not clear. Using nucleic acid hybridization, it was observed that P[5] rotaviruses are the most frequently associated with calf diarrhea. However, a majority of the field samples remained untypable with respect to P type as determined by dot blot hybridization lest
(Mummidi et al., 1992; Parwani et al., 1992; Parwani et al., 1993). In this study we have 89 determined the relative frequency of P and G types of group A bovine rotaviruses associated with calf diarrhea using a highly sensitive multiplex PGR assay.
Our results confirm that P[5]:G6 type viruses are the most frequently associated group A BRV with calf diarrhea. Our P typing results are different from those results obtained using dot blot hybridization technique by us (Mununidi et al., 1992) and by Parwani et al. (1993). In this study, we were able to type more than 98% of the field samples with respect to their P type as opposed to approximately 40% obtained using dot blot hybridization. The reasons which might have contributed to this discrepancy might be (i) the multiplex PGR technique is several thousand fold more sensitive than dot blot hybridization, (ii) the probes used for P-typing were derived from the hypervariable region of the VP4 gene and differences in diis region might have contributed to low level signal during hybridization, and (iii) low amount of rotavirus RNA in some samples. In our study, 40.2% of the calves were singly infected with either P[5]:G6 or P[11]:G10 viruses and 6.9% were singly infected with P[5]:G10 or P[11]:G6 viruses. About 39.2% of the samples were dually infected with two different strains as indicated by the presence of two different P types and/or G types. The unusually large number of samples which showed bands specific for both P[5] and P[11] viruses is an unprecedented observation. Surprisingly however, we could only amplify a single band either corresponding to G6 or GIO type viruses from a majority of these samples. This indicates that these calves might be infected with viruses which share a conunon G type but differ in their P type. About 25% of the P[5] and P[11] positive samples were also positive for G6 and GIO types. We could not determine the G types of rotaviruses in about 17% of the samples we tested. This is in agreement with previously reported results from other laboratories (Snodgrass et al., 1990; Parwani et al.. 1993; Hussein et al.. 1993).
Several other G types have been found to be associated with calf diarrhea including Gl. G2.
G3, G7. G8, and Gil viruses (Snodgrass et al.. 1990; Briissow et al.. 1992; Blackball et al., 1992; Parwani et al., 1993; Hussein et al., 1993) which would have remained undetected 90 in our study. Further studies are underway in our laboratory to characterize the untypable viruses. We have also observed differences in the relative frequency of the various rotavirus strains among the selected states. For example, in California, 93% of the samples were found to be dually infected with two strains. This might reflect differences among the states with respect to management practices.
Although we cannot rule out the detection of other P types from cattle in future, our smdy indicated the relative frequency of different rotavirus P types associated with calf diarrhea. Ideally, the determination of P and G serotypes should be performed using serological tests such as monoclonal antibody based ELISAs. However nucleic acid hybridization and PGR based assays have served as alternative tests for determining rotavirus serotypes (Xu et al., 1990; Gouvea et al., 1990; Parwani et al., 1993). These sunrogate tests can play an important role in determination of P types because the serological reagents for characterizing VP4s are not widely available and because of the relative instability of the VP4 protein in B223 like viruses. Several authors have previously indicated that the presently available commercial vaccine in United States may be ineffective due to the presence of P[l] type virus in the vaccine (Brooks and Paul, 1989; Brooks. 1989; Mummidi et al.. 1992;
Parwani et al., 1992; Parwani et al., 1993). The extensive distribution of P[5] and P[11] type viruses detected in this study warrants the development of multivalent vaccines for the control of calf diarrhea.
ACKNOWLEDGMENTS
We thank Drs. C. Minion, N. Hartwig, B. Janke. J. Roth, and S. Winiarczyk for critically reading the manuscript. Dr. S. Goel, Dr. G. Erickson, Dr. R. Holland. Dr. P.
Blanchard. Dr. S. Kapil, Dr. C. Heame. Mr. B. Stoffregen. Ms. Beverly Fowles and Ms.
S. Hartmann for supplying rotavirus positive samples, and Dr. Gary Polking for synthesis of 91 oligonucleotide primers. This work was supported by a grant from Iowa Livestock Health
Advisory Council.
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2. Blackball, J., R. Bellinzoni, N. Mattion, M. K. Estes, J. L. La Torre, and G. Magnusson. 1992. A bovine rotavirus serotype I: Serologic characterization of the virus and nucleotide sequence determination of the stmctural glycoprotein VP7 gene. Virology. 189:833-837.
3. Brooks M. A., and P. S. Paul. 1989. Prevalence of gene 4 types among bovine rotavirus field isolates, abstr. 80, p. 16, Proc. 70th Annu. Meet. Conf. Res. Workers in Ani. Dis., 1989.
4. Brooks, M. A. 1989. Characterization of antigenic relationships among bovine rotaviruses, M.S. Thesis, Iowa State University.
5. Briissow, H., O. Nakagomi, G. Gena, and W. Eichhorn. 1992. Isolation of an avian-like group A rotavirus from a calf with diarrhea. J. Clin. Microbiol. 30:67-73.
6. Briissow, H., A. Rohwedder, O. Nakagomi, J. Sidoti, and W. Eichchorn. 1994. Bovine rotavirus V1005 a P5, not a P12, type like all viruses in a German survey. J. Clin. Microbiol. 32:2876-2879.
7. Estes M. K. 1996. Rotaviruses and their replication, p. 1625-1655. B.N. Fields, D.M. Knipe, P.M. Howley (ed.). Fields Virology, 3th edition. Lippincott- Raven Publishers. Philadelphia. 92
8. Estes M. K., and J. Cohen. 1989. Rotavirus gene structure and function. Microbiol. Rev. 53:410-449
9. Gentsch, J. R., R. I. Glass, P. Woods, V. Gouvea, M. Gorziglia, J. Flores, B. K. Das, and M. K. Bhan. 1992. Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol. 30:1365- 1373
10. Gouvea V., R. I. Glass, P. Woods, K. Taniguchi, H. F. Clark, B. Forrester, and Z. Fang. 1990. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J. Clin. Microbiol. 28:276- 282
11. Gouvea V., N. Santos, and M. C. Timenetsky. 1994a. Identification of bovine and porcine rotavirus G types by PCR. J. Clin. Microbiol. 32:1338-1340
12. Gouvea V., N. Santos, and M. C. Timenetsky. 1994b. VP4 typing of bovine and porcine group A rotavirus by PCR. J. Clin. Microbiol. 32:1333-1337
13. Hussein, H. A., A. V. Parwani, B. I. Rosen, A. Lucchelli, and L. J. Saif. 1993. Detection of rotavirus serotypes Gl. G2, G3, and G11 in feces of diarrheic calves by using polymerase chain reaction-derived cDNA probes. J. Clin. Microbiol. 31:2491-2496.
14. Isegawa Y., O. Nakagomi, T. Nakagomi, S. Ishida, S. Uesugi, and S . Ueda. 1993. Determination of bovine rotavirus G and P serotypes by polymerase chain reaction. Mol. and Cell. Probes. 7:277-284
15. Kapikian A. Z., and R. M. Chanock. 1996. Rotaviruses, p. 1657-1708. In B.N. Fields, D.M. Knipe, P.M. Howley (ed.). Fields Virology, 3rd edition. Lippincott-Raven Publishers, Philadelphia.
16. Mattion N. M., J. Cohen, and M. K. Estes. 1994. The rotavirus proteins, p. 169-249. In A.Z. Kapikian (ed.). Viral infections of the gastrointestinal tract. 2nd edition. Marcel Dekker. Inc.. New York. 93
17. Mummidi, S., A. Zaberezhny, Y. S. Lyoo, and P. S. Paul. 1992. Nucleic acid probes for distinguishing bovine rotavirus P and G types, abstr. 64, p. 13. In Proc. 73rd Annu. Meet. Conf. Res. Workers in Animal Dis., 1992.
18. Mummidi, S., M. A. Brooks, P. S. Paul, Y. S. Lyoo, and P. S. Paul. 1996. The VP4 and VP7 of bovine rotavirus VMRI are antigenically and genetically closely related to P-type 5, G-type 6 strains. Vet. Microbiol. 51:241-255.
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CHAPTER 6. COMPARATIVE ANALYSIS OF THE VPS* SEQUENCES
OF P[5] AND P[11] ROTAVIRUSES ISOLATED FROM CALVES WITH
DIARRHEA
A Paper To Be Submitted To Virus Genes
Srinivas Mummidi and Prem S. Paul
Abstract. In this study we have determined the nucleotide sequences of the VPS* domain of the VP4 gene of 9 P[5] type rotaviruses and 10 P[11] type rotaviruses obtained from diarrheic calves and compared them to the cell culture adapted P[5] and P[11] type surains. The sequence of VPS* was determined from a 1.93 Kb piece of die VP4 gene directly amplified by RT-PCR from the fecal samples. The P[5] type isolates showed 90-98 % nucleotide homology and 93-100 % amino acid identity with that of the cell culture adapted strains and 95-100% nucleotide homology and 97-100% amino acid identity among themselves. Comparison of the VPS* nucleotide sequences of P[11] type field isolates with that of the P[11] type cell culture adapted viruses revealed 94-97 % homology. The deduced amino acid sequences indicated 95-9S % identity between the P[11] field isolates and P[11] cell culture adapted viruses. The P[l 1] field isolates themselves showed extensive sequence conservation at both nucleotide (94-99 %) and amino acid (97-100 %) levels. This information should be useful in developing more effective vaccines for the control of rotavirus induced diarrhea in calves.
Group A rotavimses are one of the leading etiological agents associated with calf diarrhea (I). An ideal vaccine for its control should incorporate strains which circulate most frequently in the cattle population. The major rotavirus neutralization antigens are VP4 and
VP7. and they determine the serotype of the virus. There are two major VP4 genotypes 96
(P[5] and P[11]) and two major VP7 serotypes (G6 and GIO) associated with calf diarrhea
(2, 3,4, 5, Mummidi and Paul, submitted for publication). We have also observed, using multiplex PGR typing assays, that in many cases rotavirus positive diarrheic calves are dually infected with strains differing in their P type (Mummidi and Paul, submitted for publication).
There is very limited sequence information available on the VP4 genes from bovine rotavirus strains, and no information is available, to our knowledge, from non-cell culture adapted bovine rotaviruses. In this paper, we report the nucleotide and amino acid sequence analysis of the N-terminal proteolytic cleavage fragment (VP8*) of the VP4 gene of P[5] and P[11] type rotaviruses. The serotype specific epitopes are mostly localized to the VPS* domains and hence we postulated that this is the region that is most likely to show genetic variation (6. 7).
The history of the different P[5]-type and P[1 l]-type cell culture adapted bovine rotaviruses has been previously described (8, 9, 10, 11, 12). Rotavirus positive fecal samples from diarrheic calves were obtained from different regions in the U.S. (midwest, eastern, and western regions). The P types of the rotaviruses in this smdy were determined either by using VP4 specific cDNA probes or by a multiplex PGR technique (3, Mummidi and Paul, submitted). For sequencing the VPS* domain, a 1.93 Kb fragment from VP4 was amplified by reverse transcription and polymerase chain reaction. The double stranded RNA from approximately 200 ^1 or 0.2 g of fecal sample was purified by a glass matrix method
(Bio 101, La JoUa, GA). The purified double stranded RNA was denatured by heating it at
95°G for 3 minutes in the presence of 7% DMSO and then by quick chilling on ice.
Gomplementary DNA was synthesized in the presence of VP4 specific primer (nt 1909 to
1930, caaccgcag-ctgatatatcatc) by using a cDNA cycle kit (Invitrogen. San Diego. GA) according to manufacturer's instructions. Two microliters of the cDNA was used for PGR amplification. PGR was performed in a 50 al reaction in the presence of 0.2 mM of each dNTP. 20 pM of each primer. Taq extender buffer. 2.5 U of Taq extender (Stratagene. La 97
Jolla, CA) and 2.5 U of Taq polymerase (Boehringer-Mannheim, Indianapolis, IN) in a thermal cycler (Coy, Troy, MI). Non specific amplification of cDNA at lower temperatures was prevented by using a hot start technique with wax beads. The sequence of the upper primer for the PGR amplification is ggctttataatggcttcgcta (nt 1 to 21). The temperatures for thermal cycling for the PGR were 2 cycles of a profile consisting of 92°G for 2 min, 45°G for
1 min and 72°G for 5 min followed by 30 cycles of 92°G for I min, 45°C for 45 sec, and
72°C for 2.5 min. Negative controls without DNA were run concurrendy along with all sets of PCRs to determine the presence of any laboratory contamination. The amplified fragments were purified by Geneclean (Bio 101, La Jolla, GA), and were sequenced by a modified Sanger's dideoxy method using an automated fluorescent sequencer (ABI. Foster City, CA).
Analysis of the sequences was performed by using GeneWorks (IntelliGenetics, Inc., Mountain View, GA). The nucleotide sequences of the VP4 genes of the cell culture adapted strains, UK, B641, 61A , VMRI, B223, KK3, and A44 have been described previously (12,
13, 14, 15, 16). The P[5] type field strains showed 90-98 % nucleotide homology in the VPS* region with the cell culmre adapted viruses (Table 1). The least amount of nucleotide homology was with that of the UK strain (90-92 %) and the highest degree of homology was with VMRI strain (97-98 %). Sequence analysis indicated that there is a nucleotide homology of 95-100 % among P[5] type field strains. The predicted VP8* domains of P[5] type field strains showed 93-100 % amino acid identity with that of cell culture adapted vimses whereas they showed 97-100 % amino acid identity among themselves. All the P[5] type field strains were found to lack Cys-203 as in case of the cell culture adapted viruses (Fig. 1). The Cys-203 is conserved among several human and rotavirus strains (17). The sequence of the connecting peptide between the VP8* and VP5* domains in all the field strains was RSIEPRK (Fig. 1).
No information is available on the antigenic regions and neutralization epitopes of the VP4 of
P[5] type viruses, so it becomes imperative to interpret the sequence data in terms of what is 98
Table 1. Nucleotide homologies^ and amino acid identities'' of the VPS* region of the P[5] type field isolates with that of the ceil culture adapted viruses. Virus UK B641 6IA VMRI 6 11 19 30 55 184 204 206 UK*-' XX 91 92 91 92 92 92 91 91 90 91 91 90
B641' 91 XX 92 94 94 94 94 93 94 93 93 94 94
61A' 95 94 XX 95 95 95 95 94 95 94 94 94 93
VMRT 95 95 98 XX 98 98 98 97 98 97 98 98 98
5 95 95 99 100 XX 100 99 98 98 97 97 98 97 6 95 95 98 100 100 XX 99 98 98 97 97 98 97 11 95 95 98 100 99 100 XX 98 98 97 97 98 97 19 93 93 97 98 98 98 98 XX 96 95 97 97 96
30 94 95 98 100 99 100 99 98 XX 98 97 98 98 55 94 94 98 99 98 99 98 97 98 XX 96 97 98 184 94 93 97 98 98 98 98 97 98 97 XX 97 97 204 94 94 98 99 99 99 99 98 99 98 98 XX 97 206 93 94 97 98 98 98 98 97 98 98 97 98 XX ^ Nucleotide homologies are shown in the upper diagonal half in italics '' Amino acid identities are shown in the lower diagonal half Cell culture adapted P[5] type strains known in general for VP4 of group A rotaviruses. Sequence analysis of several rotavirus strains has indicated the presence of a hyper-variable region between amino acid residues 71-
204 (6). The importance of this region was also established by analyzing virus mutants selected in the presence of hyperimmune semm or monoclonal antibodies, which indicated specific amino acid changes (18, 19). Larralde and Gorziglia (1992), have shown that the antigenic region B (aa 84-180) in the VPS* is responsible for serotype specificity (20). In the region B. the P[5] field strains had an amino acid identity of 90-94 % with UK. 92-96 % with B641, 95-99% with 61A and 96-100% with VMRI. 99
UK VPS* MASLIYRQLL ANSYAVNLSD EIQSVGSGKN QKVTVNPGPF AQTGVrai"7NW 50 VMRI VPS' D E 50 B641 VPS- D E D R 50 61A VPS* D E 50 5 VPS* D E 50 6 VP8* D E 50 11 VPS* D E 50 19 VPS* T.D E 50 30 VPS* D E 50 55 VPS* D E 50 184 VPS* D E 50 204 VPS* D E 50 206 VPS* D E 50
UK VPS* GPGEVNDSTV VQPVLDGPYQ PAPFDLPVCSI WMLLAPTRPG ^;VVEGTDNSG 100 VMRI VPS* S G 100 B641 VPS* R G 100 61A VPS* P S G 100 5 VPS* S G 100 6 VPS* S G 100 11 VPS* S G 100 19 VPS* H S IG 100 30 VPS* S G 100 55 VPS* S G 100 1S4 VPS' S A 100 204 VPS* S G 100 206 VPS* S G 100
UK VPS* RWLSVILIEP GVaSETRTm MFGSSKQWV SNVSDTKWKF VEMVKTAVDG 150 VMRI WS* T T L. ..A M 150 B641 VPS* T T L L F..M 150 61A VPS* T T M 150 5 VPS* T T L M 150 6 VPS* T T L. ..A M 150 11 VPS* T T L. ..A M 150 19 VPS* T T L. ..A M 150 30 VPS* T T N...L. ..A M 150 55 VPS* T T S. ..A M 150 184 VPS* T T R.L. ..A M 150 204 VPS* S.TA T L. ..A M 150 206 VPS* T T S. ..A M 150
Fig 1. Multiple alignment of the predicted amino acid sequences of the VPS* domain of
P[5] type field isolates with that of the cell culture adapted vimses. Also shown is the connecting peptide (aa 241-247). 100
UK VPS * DY2iEWGTLLS DTKLYC301KY GRRLFIYEGE TPNATTKGYF rTNYASAEVR 200 VMRI VPS* E IV V... 200 B641 VPS* I.I E R IV...T.V... 200 61A VPS* E V IV V... 200 5 VPS* E IV V... 200 6 VPS* E IV V... 200 11 VPS* E V v... 200 19 VPS* E IV V... 200 30 VPS* E IV V... 200 55 VPS* P E IV V... 200 1S4 VPS* E IV V... 200 204 VPS* E IV V... 200 206 VPS* QT R. .E IV V... 200
UK VPS* PYSDFYIISR SQESACTEYI NNGLPPIQNT RNWPVAISS RSIKPRE 247 VMRX VPS* E..R 247 B641 VPS* L K 247 61A VPS* Q.. 247 5 VPS* E..K 247 6 VPS* E..K 247 11 VPS* E..K 247 19 VPS* G E..K 247 30 VPS* E..K 247 55 VPS* L E..K 247 1S4 VPS* L E..K 247 204 VPS* E..K 247 206 VPS* E..K 247
Fig 1. (continued)
Analysis of nucleotide sequence in the VPS* region of P[11] type field strains indicated that they shared 94-97 % homology with that of the cell culture adapted viruses, B223, BCK3 and A44 (Table 2). The field strains shared 94-99 % nucleotide homology among themselves. The predicted VPS* domains of field strains showed amino acid identity of 95-98 % with that of the cell culture adapted strains and amino acid identity of 97-100 % among themselves. The P[11] type field strains shared the conserved cysteine at position
217 with the cell culture adapted strains (Fig. 2). However, they lacked the cysteine at position SO. where a tyrosine replaces the cysteine residue present in B223 and A44 strains.
More interestingly, in P[ 11 ] field isolates, there is a C-»A transversion at nucleotide position lOl
Table 2. Nucleotide homologies^ and amino acid identities'' of the VPS* region of P[I I] type field isolates with that of the cell culture adapted viruses.
Virus B223 KK3 A44 29 59 68 77 106 175 177 197 200 1768 B223'' XX 95 96 97 97 97 95 96 97 97 98 97 97
KKS" 97 XX 95 94 93 94 95 96 94 94 94 94 94
A44' 97 95 XX 94 94 94 95 96 94 94 95 94 94
29 98 96 95 XX 98 98 95 95 98 98 99 98 98
59 98 96 95 99 XX 99 94 95 99 99 99 98 98
68 98 96 95 99 99 XX 95 95 99 99 99 98 98
77 98 98 95 98 98 98 XX 98 95 95 95 94 95
106 97 97 95 97 97 97 99 XX 95 95 95 94 95 175 98 97 95 99 99 99 98 97 XX 99 99 98 98
177 98 96 95 99 99 99 98 97 99 XX 99 98 98
197 98 97 95 99 100 99 98 98 100 100 XX 99 99
200 98 96 95 98 98 98 98 97 98 98 98 XX 98
1768 98 96 95 98 99 98 98 97 99 99 99 98 XX ^ Nucleotide homologies are shown in the upper diagonal half in italics ^ Amino acid identities are shown in the lower diagonal half Cell culture adapted P[ 11] type strains
586 resulting in the change of His-193 residue in cell culture adapted strains to Asn in field strains (Fig. 2). The significance of this particular amino acid change is not known. There are 11 prolines which are highly conserved between the field strains and the cell culture adapted strains in the VPS* region. All of the P[11] field strains, except 177, share the three arginine residues in the connecting peptide with the cell culture adapted viruses. In 177, the arginine at residue 145 is replaced by a glycine. The region B of P[11] type field strains had amino acid identity of 98-100 % with that of B223, and KK3 and amino acid identity of 94-
96% with that of A44. 102
B223 VPS' MASLIYRQLL YNSYSVDLSD EITNIGAEKK ENVTVQLGEF AQSQYAPVSW 50 KK3 VPS* Q 50 A44 VPS* I.G 50 29 VPS* 50 59 VPS* 50 68 VPS* 50 77 VPS* 50 106 VPS* 50 175 VPS* 50 177 VPS* 50 197 VPS* 50 200 VPS* 50 176S VPS* 50
B223 VPS* GSGETLSOIV EEQTLDGPYA PDSSNLPSNC WYLVNPSNDG WFSVTDNST 100 KK3 VPS* P T Y 100 A44 VPS* W 100 29 VPS* S Y 100 59 VPS* S Y 100 68 VPS* S Y 100 77 VPS* T Y 100 106 VPS* T Y 100 175 VPS* S Y 100 177 VPS* S P.Y 100 197 VPS* S Y 100 200 VPS* Y 100 1768 VPS* S Y 100
B223 VPS* FWMFTYLVLP NTAQTMVTVN VMNETVNISI DNSGSTYREV DYHCTSSTQA 150 KK3 VPS* 150 A44 VPS* E P..S ISO 29 VPS* 150 59 VPS* 150 68 VPS* N 150 77 VPS* L, 150 106 VPS* L V 150 175 VPS* 150 177 VPS* 150 197 VPS* 150 200 VPS* S 150 1768 VPS* C 150
Fig 2. Multiple alignment of the predicted amino acid sequences of the VPS* domain of P[11] type field strains with that of the cell culture adapted P[11] strains. Also shown is
the connecting peptide (aa 242-248). 103
B223 VPS* YGSRNYLNTA HKLQAYRSDG DC3IISNYWGA ErTQGDLHVGT YSHE«/PNAVI 200 KK3 VPS* 200 A44 VPS* 200 29 VPS* N T.. 200 59 VPS* N 200 6S VPS- N 200 77 VPS* N 200 106 VPS* N 200 175 VPS* N 200 177 VPS* N 200 197 VP8* N 200 200 VPS* G N 200 176S VPS* N N 200
B223 VPS* NLNADFYVIP DSQQETCTEY IRGGLPAMQT TTYVTPISVA IRSQRIAR 248 KK3 VPS* S I T V. 24S A44 VPS* I V v.. 24S 29 VPS* K E 248 59 VPS* S K 248 68 VPS* K E v.. 248 77 VPS* 1 248 106 VPS* I A 248 175 VPS* F K v.. 248 177 VPS* K GV.. 248 197 VPS* K v.. 248 200 VPS* .1 K v.. 248 1768 VPS* K v.. 248
Fig. 2 (continued)
It is evident from the sequence analysis that the differences between P[5] type field isolates and P[5] type cell culture adapted viruses are more prominent than differences between P[11] type field isolates and P[1I] type cell culture adapted strains. The VP8* field strains show a lesser degree of nucleotide homology and amino acid identity with UK and
B641 than they show with that of 61A and VMRI. We do not understand at this point if the differences which we have observed between the field isolates and UK surain are because of a gradual genetic drift occurring temporally or due to the changes that have occurred due to cell culture adaptation. The latter theory seems to be unlikely as the field isolates showed high degree of homologies with cell culture adapted strains VMRI and 61 A. Both the P[5] type and P[11] type field isolates seem to share a high degree of homology in the VP4 gene among themselves, irrespective of their state of origin in the U.S. As VP4 is an imponant 104 determinant of immunity against rotavirus infection, it is surprising that there is very limited genetic variation occurring within a P type.
Very few studies have been published comparing the VPS* regions of different VP4 alleles among human or animal rotaviruses (21, 22). The Au-1 VP4 alleles derived firom bodi human and feline rotaviruses shared 93.8 to 99.7 % nucleotide homology and 95.5-100
% amino acid identity (21). Similarly a high degree of amino acid identity was observed among VPS* sequences of M37-like viruses (22). Our studies also reveal comparable results with regard to UK-like (P[5]) and B223-like (P[l 1]) alleles. VPS* domain of VP4 is a major region involved in the serotype specific neutralization of rotaviruses (7). The neuuralizing antibodies to VP4 are predominant in convalescent antisera and is immunodominant in conferring protection in children emphasizing its important role in immune response to rotaviruses (23). This study provides for the first time, the information on the sequence variation in the VPS* region among P[5] and P[11] rotaviruses isolated from fecal samples obtained from diarrheic calves. This knowledge should be useful in development of
recombinant vaccines for the control of rotavirus induced neonatal calf diarrhea.
References
1. Paul P.S., and Lyoo Y.S., Vet Microbiol 37, 299-317, 1993.
2. Snodgrass D.R., Fitzgerald T., Campbell I., Scott F.M.M., Browning G.F., Miller D.L., Herring A.J., and Greenberg H.B., J Clin Microbiol 28, 504-507, 1989.
3. Mummidi S.. Zaberezhny A., Lyoo Y.S., and Paul P.S., abstr 64, p 13, Proc 73rd Annu Meet Conf Res Workers in Anim Dis. 1992.
4. Parwani A.V., Rosen B.I., Lucchelli A., Hussein H.A., Navvaro L.E., and Saif L.J.. abstr 65. p 14. Proc 73rd Annu Meet Conf Res Workers in Anim Dis. 1992. 105
5. Parwani A.V., Hussein H.A.. Rosen B.I., Lucciielli A., and Saif L.J., J Clin Microbiol SI, 2010-2015. 1993.
6. Estes M.K.. and Cohen J., Microbiol Rev 53, 410-449. 1989.
7. Kapikian A.Z., and Chanock R.M., In Fields Virology, 3rd edition. Ed Fields B,N.. BCnipe D.M., and Howley P.M., Lippincott-Raven Publishers. Philadelphia, pp 1657-1708, 1996.
8. Woode G.N., Bridger J.C., Hall G.A., and Dennis M.J., Res Vet Sci 16. 102-105, 1974.
9. Woode G.N., Kelso N.E., Simpson T.F., Gaul S.K., Evans L.E., and Babuik L., J Clin Microbiol 18, 358-364, 1983.
10. Paul F.S.. Lyoo Y.S., Woode G.N., Zheng S., Greenberg H.B., Matsui S., Schwartz K.J., and Hill H.T., J Clin Microbiol 26, 2139-2143, 1988.
11. Taniguchi K., Urasawa T., Pongsuwanna Y., Choonthanom M., Jayavasu C., and Urasawa, S., J Gen Virol 72, 2929-2937, 1991.
12. Mummidi S., Brooks M.A., Paul P.S., Lyoo Y.S., and Zaberezhny A.. Vet Microbiol 51, 241-255. 1996.
13. Kantharidis P., Dyall-Smith M.L., Tregear G.W., and Holmes I.H., Virology 166. 308-315, 1988.
14. Hardy M.E., Woode G.N., Xu Z., and Gorziglia M.. J Virol 65, 5535-5538. 1991.
15. Hardy M.E., Gorziglia M., and Woode G.N., Virology 191, 291-300. 1992.
16. Taniguchi K., Urasawa T., and Urasawa S., J Gen Virol 74, 1215-1221. 1993.
17. Patton J.T.. Hua J., and Mansell E.A., J Virol 67, 4848-4855. 1993. 106
18. Mackow E.R.. Shaw R.D., Matsui S.M., Vo P.T.. Dang M.N.. and Greenberg H.B., Proc Natl Acad Sci USA 85. 645-649, L988.
19. Kirkwood C.D.. Bishop R.F.. and Couison B.S.. Arch Virol 141. 587-600, 1996.
20. Larralde G.. and Gorziglia M., J Virol 66, 7438-7443, 1992.
21. Nakagomi O., Isegawa Y., Ueda S., Gema G.. Sarasini A.. Kaga E., Nakagomi T.. and Flores J., J Gen Virol 74, 1709-1713, 1993.
22. Santos N., Gouvea V., Timenetsky M.C., Clark H.F.. Riepenhoff-Talty M.. and Garbarg-Chenon A.. J Gen Virol 75, 1775-1780. 1994.
23. Ward R.L., McNeal M.M., Sander D.S., Greenberg H.B.. and Bernstein D.I.. J Virol 67, 464-468, 1993. 107
CHAPTER 7. SEQUENCE AND PHYLOGENETIC ANALYSIS OF THE VP7 GENE OF A BOVINE ROTAVIRUS WITH G6 SUBTYPE
A Paper Published by Virus Genes
Srinivas Mummidi, Prem S. Paul, and Robert E. Holland
Abstract. A bovine rotavirus, designated VMRI-29, was detected in the feces of a diarrheic calf. Sequence analysis of the surface glycoprotein VP7 gene showed 81 to 82% nucleotide homology and 90% amino acid identity with G6 bovine rotavirus reference strains. In contrast, this isolate had high nucleotide (97%) and amino acid identity (98%) with the Japanese bovine rotavirus strain KN-4. The consensus amino acid sequence of VMRI-29 and KN-4 in the variable regions of the VP7 protein was also different from the reference strains. We found the VR-3 region to be the most divergent region between VMRI- 29 and the reference strains. The implications of these findings are discussed with relation to
rotavirus evolution and the existence of a "genotypic" subtype within the 06 rotaviruses.
Group A rotaviruses are a predominant cause of rotaviral diarrhea in the young of several species including man and cattle (1,2). The outer capsid of group A rotaviruses is composed of two proteins VP4 and VP7. As both VP4 and VP7 elicit neutralizing antibodies during the immune response to rotaviral infection, a binary system of classification has been
proposed with serotypes based on VP4 designated as P types and those based on VP7
referred to as G types (1). Two major G types predominate in cattle with NCDV being the
prototype G6 strain whereas B223 serves as the prototype GIO strain (2). Recently G6 and
GIO strains have been isolated from humans and their VP7 genes characterized (3.4.5). 108
Studies with variants selected with monoclonal antibodies have revealed several regions in the VP7 protein which are thought to be important in neutralization (6,7). We have initiated smdies to examine the extent of variation in the VP7 gene among bovine rotavirus isolates and report here the detection and sequence data on the VP7 gene of a bovine rotavirus isolate which appears to be a G6 genotypic subtype.
The rotavirus VMRI-29 was detected in the feces of a 3-week; old diarrheic calf at a
Michigan farm in 1991. The herd has a history of sporadic outbreaks of diarrhea and the calves are routinely vaccinated at birth with a modified live virus vaccine containing NCDV- Lincoln strain with a P type 1 and G type 6. We have amplified the VP7 gene from this isolate by RT-PCR and analyzed its sequence. The nucleotide sequence of the VP7 gene of VMRI-29 indicated that it had a 81-82% homology with that of different bovine G6 strains and a 75% homology with B223. In contrast, it showed 97% homology with the VP7 gene of KN-4, a strain isolated from Japan (8). The predicted VP7 polypeptide of VMRI-29 also had a high degree of identity with that of strain ECN-4. The VP7 proteins of VMRI-29 and BCN-4 shared 98% identity whereas they shared 90 to 91% identity with the VP7 proteins of the previously described bovine G6 strains. Nine discrete variable regions designated VR-l through VR-9 and three antigenic regions have been reported based on the comparative sequence analysis of VP7 proteins from several rotavirus strains and by sequence analysis of virus variants selected in the presence of antisera (9,6). The predicted VP7 polypeptide of VMRI-29 differed from the reference G6 strains at several amino acid residues which localize to the variable and antigenic regions.
Several differences between VMRI-29 and the reference G6 strains were also evident in the
H2 signal peptide region and the VR-3 region which extended from positions 33 to 48 and 37 to 53 of VP7 protein, respectively (Fig. 1). This region of VP7 protein also has been shown to contain an immunodominant cytotoxic T cell epitope (10). The H2 signal peptide and the
VR-3 region were highly conserved between VMRI-29 and KN-4 VP7 proteins. Other 109 amino acid differences were localized to VR-8 region and antigenic region C (Fig. 1). We constructed phylogenetic trees to determine the evolutionary relationships between the VP7 sequences of VMRI-29. KN-4 and the published VP7 sequences of several rotavirus strains using the PAUP program (Swofford. Illinois Natural History Survey, Champaign. Illinois,
U.S.A.). The minimal length tree obtained from the VP7 protein sequences indicated that several of the standard G6 strains formed a single cluster, whereas VMRI-29. KN-4. PA151 and PA 169 formed a distinct cluster.
Rotavirus strains Variable Regions VR-3 VR-8 37 53 208 224 VMRI-29 FLFVWLMAIVTSAQNY STTTPDTFETVATAEKL KN-4 ...N...... NCDV-Lincoln ..LI..IL.TIIN B641 ..LI.AIL.TIMN PA151 ...... W..IAN....
Fig. 1. Comparison of deduced amino acid sequences of VP7 gene of VMRI-29 in the
variable regions VR3 and VR8 with that of other G6 type rotaviruses.
Previous studies have shown that the VP? proteins of rotaviruses belonging to a
particular serotype and isolated from a single animal species share amino acid identities of
95% or higher and there is a very high degree of species specific conservation in the variable
regions of the protein (9). However, our results indicate that there is genetic variability in the
VP7 gene among G6 type rotaviruses. This genetic variation should be taken into
consideration for rational vaccine design for prevention of rotavirus diarrhea as this
variability is mainly localized to the antigenically important regions of VP7. This acquires 110
further importance as previous studies have indicated that KN-4 which has VP7 like VMRI-
29 is not neutralized by G6 or GIG specific monoclonal antibodies (8). Based on the
phylogenetic tree it can be speculated that diere is a conmion ancestral gene from which
VMRI-29, PA 151. and PA169 VP7 genes might have evolved and genetic reassortment
might have played a role in the evolution of human G6 type viruses. Further serological and
molecular epidemiological studies are needed to determine the distribution and assess the
biological significance of two G6 genotypic subtypes among bovine rotavirus field isolates.
References
1. Estes M.K., and Cohen J.. Microbiol Rev 53, 410-449. 1989.
2. Paul P.S., and Lyoo Y.S., Vet Microbiol 37, 299-317. 1993.
3. Dunn S.J., Greenberg H.B., Ward R.L., Nakagomi O., Bums J.W., Vo P.T., Pax K.A., Das M., Gowda K., and Rao C.D., J Clin Microbiol 31, 165-169, 1993.
4. Geraa G., Sarasini A., Parea M., Arista S., Miranda P., Briissow H., Hoshino Y., and Rores J., J Clin Microbiol 30, 9-16, 1992.
5. Gema G., Steele A., Hoshino Y.. Serene M., Garcia D., Sarasini A., and Flores J.. J Gen Virol 75, 1781-1784, 1994.
6. Dyall-Smith M.L., Lazdins I., Tregear G.W.. and Holmes I.H., Proc Nad Acad Sci USA 83, 3465-3468, 1986.
7. Kirkwood C., Masendycz P.J.. and Coulson B.S.. Virology 196. 79-88, 1993.
8. Matsuda Y.. Isegawa Y.. Woode G.N., Zheng S., Kaga E.. Nakagomi T., Ueda S.. and Nakagomi O., J Clin Microbiol 31, 354-358 1993.
9. Nishikawa K., Hoshino Y.. Taniguchi K.. Green K.Y.. Greenberg H.B., Kapikian A.Z.. Chanock R.M.. and Gorziglia M.. Virology 171. 503-515. 1989. Ill
10. Franco M.A., Prieio I.. Labbe M.. Ponset D.. Borras-Cuesta F.. and Cohen J., J Gen Virol 74. 2579-2586. 1993.
Appendix
UK
52.
NCDV KN-4
Circle diagram indicating the phylogenetic relationships of the VP7 proteins from different
G6 type viruses of animal and human origin. The tree was consuructed using PAUP program
using "exhaustive' search option. The amino acid sequence of the VP7 protein of T449
(bovine G type 1 strain) was used as an outgroup. 112
CHAPTER 8. GENERAL CONCLUSIONS
Rotaviruses are one of the most important viral agents associated with neonatal calf diarrhea, and control of diarrhea in dairy and beef herds remains an important goal to attain.
Research in the past decade has revealed an extreme complexity in the serotypic distribution among group A rotaviruses. At present, there are about 19 VP4 genotypes and 14 VP7 serotypes recognized among group A rotaviruses from all species (Estes, 1996). Some of the VP4 genotypes might even comprise two or more VP4 serotypes (Estes, 1996). Genetic reassortment can potentially result in any combination of VP4 and VP7 resulting in another level of complexity. VP4 and VP7 interactions might give rise to unique epitopes (Chen et al., 1992). Another fascinating aspect of rotavirus epidemiology is the potential for cross- species transmission because of their ubiquitous nature. One of the extreme examples is the BRV 993/83, which has avian-like VP4 and VP7 and is potentially a reassortant from rotaviruses infecting two different classes of vertebrates (Brussow et al., 1992). My colleagues and I were interested in tiie extent of genetic variation existing among BRV and we undertook a series of studies to analyze the VP4 and VP7 genes of BRV, the distribution of VP4 and VP7 types, and the variation within VP4 and VP7 genes. This information is pertinent as several reports indicated that the presentiy available vaccine against calf diarrhea in the field is ineffective. In the first manuscript of this dissertation, my colleagues and I describe the characterization of VP4 and VP7 genes of a BRV strain designated VMRI. This strain has a
"super-short" migration pattern which distinguishes it from many of the BRV field isolates which have a "long" electropherotype pattern (Paul et al., 1988). Initial characterization indicated that this virus is antigenically distinguishable from NCDV. which is the virus strain present in the vaccine. This observation was further strengthened when northern blot hybridization indicated that the VP4 gene of VMRI failed to hybridize with that of NCDV
under high stringency conditions (Brooks, 1989). This prompted us to clone and sequence 113 the VP4 and VP7 genes of VMRI strain. The sequence information indicated that the VP4 gene of VMRI is different from that of NCDV and is more closely related to that of UK and
B641. Also this smdy confirms the earlier observation of the presence of a "bovine" genogroup where in all the strains studied by northern blot hybridization shared most of the their genes (Matsuda and Nakagomi, 1989).
In the second manuscript we report the prevalence of different P and G types associated with rotaviruses isolated from diarrheic calves. We initially developed a dot blot hybridization technique for distinguishing P and G types of BRV. For this we used cDNA probes amplified by RT-PCR from the hyper-variable regions of the VP4 genes of BRV and the VP7 genes to detect viral genomic RNA extracted from feces under high stringency conditions (Mummidi et al., 1992). However, this technique had several drawbacks including insufficient RNA in the samples and low hybridization signals. We have modified a multiplex RT-PCR technique developed for typing cell culture adapted BRV to type rotavimses from fecal samples from diarrheic calves. The typing assays were specific for detection of the P and G types of rotavirus strains associated with calf diarrhea. The results from this study confirmed the previous observation from our laboratory and others that P[5]: G6 viruses are the most predominant group associated with calf diarrhea. However, the observations resulting from this smdy are much more comprehensive than previously reported. The P type of only 40% of the field samples could be determined using dot-blot hybridization (Mummidi et al., 1992; Parwani et al., 1992; Parwani et al., 1993). Using the multiplex RT-PCR assay we were able to determine P types of 98% of the rotavirus isolates from fecal samples. Although, it is possible that the samples that were tested in the previous studies did have isolates with unique P types, this was unlikely because, to our knowledge, only 4 P types have been so far associated with BRV. P[l] type seems to occur very infrequently in the field and there is only a single report describing the existence of P[ 16] 114
type. However, we cannot completely rule out the association of additional P types associated with calf diarrhea.
Another interesting observation arising out of this study is approximately 40% of the
rotavirus positive samples that we have tested had both P[5] and P[11] types associated with
them. This is an unprecedented observation, as previous studies did not report such an extensive occurrence of P[ll] type viruses in the field. An alternative explanation for the above observation is that the primers used in this study might be recognizing strains in which
VP4 gene has undergone a recombination event. Such strains would be expected to have sequences derived from the VP4 genes of both P[5] and P[ 11 ] type strains. However, this type of recombinational event has not been reported in the VP4 gene of rotaviruses. We have also observed that 75% of the samples exhibiting dual infection with P[5] or P[11] had only a single G type associated with them. This probably indicates that the VP4 gene reassortment is much more frequent than that of VP7 gene and also P[11]: G6 viruses occur much more frequently than previously reported. However, the former observation should be confirmed
in a more systematic smdy which addresses die reassortment of rotaviral RNA segments in
vivo. We, as well as others, have observed that P[5] genotype tends to segregate preferentially, though not exclusively, with the 06 type. We do not understand the reasons
for this preferential segregation and whether it confers any biological advantages to the virus. We have also observed regional variation in the distribution of P and G types, which may
reflect the differences in management practices and also might be influenced by the nature of
the herd (dairy or beef).
It has to be noted that the multiplex PGR assay is only a surrogate for serological
assays to determine the P type of a rotavirus. Recent advances in expression of BRV VP4 in
insect cell systems might allow development of serological reagents to determine P type of a
virus directly (Lyoo et al.. unpublished data). This approach may itself have disadvantages
as it is known that the VP4 in B223-like viruses is unstable, at least during certain 115 purification protocols (Chen and Ramig, 1992). We have limited this study to the detection of G6 and GIO type rotaviruses as they appear to be the predominant among BRV
(Snodgrass et al.. 1990). Rotaviruses with six other G types also appear to be involved in the etiology of calf diarrhea (Blackball et al., 1992; Briissow et al.. 1992; Parwani et al..
1993; Hussein et al., 1993). Only a single strain belonging to the atypical G types has been well characterized in the U. S. (NCDV-Cody, G8) (Lu et al.. 1995). Future studies should focus on cell culture adaptation and characterization of rotaviruses associated with calf diarrhea which appear to have an atypical G type.
We believe the epidemiological data obtained in this smdy wiU be useful in development of effective vaccines to control neonatal calf diarrhea. It has been suggested that maternal vaccination during late pregnancy to enhance the colostral antibodies is probably the most effective way to protect the calves from rotavirus induced diarrhea. As some studies indicate heterotypic antibody response during primary rotavirus infection is limited (Snodgrass et al., 1991), a multivalent vaccine may be an effective way to control group A rotavirus induced diarrhea. An interesting question arises upon the smdy of distribution of P and G types among BRV: there seem to be a limited number of VP4 types associated with calf diarrhea, whereas the number of VP7 types appear to be considerably larger. Are there some host related or virus associated factors which lead to this preponderance of VP7 types? Is it because VP4 is conferring a degree of host specificity and limits the amount of interspecies transmission? In this context, it is interesting to note that asymptomatic human strains isolated in India have a
B223-Iike VP4 (Das et al., 1993). These conflicting questions need to be addressed in a future study.
In the third manuscript, we describe the sequence analysis of the VPS* region of
BRV isolates after amplifying it directly from the fecal samples. This is the first report in which the VPS* sequences from non-cell culture adapted viruses belonging to P[5] and P[11] 116 types have been studied. Sequence analysis shows a limited degree of genetic variation when the VPS* regions of the cell culture adapted and that of the non cell culture adapted P[5] viruses were compared. The VPS* regions of the P[5] and P[11] type viruses in the fecal samples appear to be essentially homogenous. This smdy further confirms the previous observation that mutation rate of rotaviruses is lower than that observed in the single stranded
RNA virus families like orthomyxoviruses, coronaviriuses. picomaviruses, and retroviruses
(Flores et al., 1988).
In the fourth manuscript, we describe the isolation and the VP7 sequence analysis of a rotavirus field isolate which we have designated as VMRI-29. This isolate was detected during the smdy of the geneuc variation in the VP7 genes of rotavirus isolates associated with calf diarrhea. In general, we have observed that the rotavims field isolates belonging to a single serotype exhibit 96% to 97% nucleotide homology and a 96% aa identity to that of reference strains. This has also been observed in case of VP7 genes of rotaviruses recovered from asymptomatic infants (Flores et al., 1988). The VP7 gene of VMRI-29 exhibited a nucleotide homology of 81-83% with that of the standard G6 strains, only marginally higher than the 75% homology it exhibited with that of B223, which is a G10 virus. Twenty five percent of the differences in the nucleotide sequence of VMRI-29 VP7 are localized to the first and second codons resulting in 34 amino acid differences in the polypeptide sequence with that of NCDV-Lincoln strain, indicating a long period of divergence from the standard strains. In contrast, VMRI, a standard G6 strain shows 13 amino acid changes when compared to that of NCDV-Lincoln. Most of the changes that were observed in case of
VMRI-29 were localized to VR-3 and VR-S regions of the VP7. The VR-3 region has been determined to be important in the generation of rotavirus specific cytotoxic T-lymphocyte activity in mice and the VR-8 overlaps with antigenic region C, which is the most immunodominant of all the antigenic regions in the VP7. and this probably indicates the role of selective immunological pressure in the evolution of VMRI-29 VP7. 117
We have also studied the relationship of VMRI-29 VP7 with that of the recently sequenced human G6 type viruses (Gema et al., 1994). Phylogenetic trees constructed by parsimony analysis from the predicted polypeptide sequences of the VP7 proteins of VMRI-
29, PA151 (human G6 type rotavirus) and the standard strains indicate that the VP7s of
PA 151 and VMRI-29 cluster together. This might be indicative of a common ancestral gene for the VP7 genes of VMRI-29 and PA 151 and a possible role of inter-species transmission in the evoluuon of rotaviral strains. The VP7 gene of VMRI-29 is unique in that it is one of the first examples of a VP7 gene sharing more homology with that of a strain originating from a heterologous species, than with that of the strains from the homologous host within a serotype. However, we have observed several differences between VMRI-29 and PA 151 in the variable regions and especially the antigenic region C. It has been reported that several amino acids in the antigenic regions B and C are highly conserved between PA 151 and PA169 and strain P, a G3 type strain (Gema et al.. 1994). Based on these observations, it has been hypothesized that rotaviruses might occur in a "serotypic continuum rather than by discrete serotypes" (Gema et al., 1994). A comparative analysis of the VP7 protein sequences of VMRI-29, KN-4, PA 151 and the reference G6 strains supports this hypothesis. VMRI-29 isolate was found to have a nucleotide homology of 97% and amino acid identity of 98% with the BRV isolate FCN-4, which was isolated in Japan (Matsuda et al., 1993). This indicates that the strains with VMRI-29 like VP7 might have a broader distribution than recognized till now. Some of the questions which can be addressed in the future with respect to the smdies carried out in the present dissertation are: 1. Defining the antigenic regions and neutralization epitopes in the VP4 and VP7 proteins of
VMRI and B223 strains. 2. Identification and characterization of vimses with atypical VP4 and VP7 t}'pes associated with rotavirus induced calf diarrhea. 118
3. Can dam vaccination with a multivalent vaccine protect calves against rotavirus induced diarrhea under field conditions?
4. Are there other unidentified genetic and antigenic VP4 and VP7 subtypes or variants occurring in the field? What is the mechanism of their evolution? How important are they in
the etiology, pathogenesis and epidemiology of rotavirus induced diarrhea in calves?
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