VIROLOGY

542-547

190,

Nucleotide

(1992)

Sequence of Gene 5 Encoding the Inner Capsid Protein (VP6) of Bovine Group C Rotavirus: Comparison with Corresponding Genes of Group C, A, and B Rotaviruses HIROSHI TSUNEMITSU,+ JON R. GENTSCH,* ROGER I. GLASS,*~II KIM Y. GREEN,~ YUAN QIAN,§ AND LINDA J. SAiFt

BAOMING JIANG,*~+’

*Viral Gastroenteritis Unit, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333; “Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322; +Hokkaido Prefectural Shintoku Animal Husbandry Experiment Station, Shintoku, Hokkaido 081, Japan; BLaboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; and tFood Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 4469 1 Received

April

29,

1992;

accepted

May

27,

1992

To further study the molecular characteristics of group (gp) C rotaviruses, we produced, cloned, and sequenced cDNA to gene 5 of the Shintoku strain of bovine gp C rotavirus. The resulting clone was specific for gene 5 and was genetically related to the human and porcine gp C rotaviruses, as demonstrated by Northern blot hybridization analysis. The Shintoku gene 5 is 1352 nucleotides in length and has one open reading frame encoding a polypeptide of 395 amino acids with a predicted molecular mass of 44.5 kDa. Comparative sequence analysis indicated that: (i) the Shintoku gene 5 protein shared 88.4 to 90.6% homology with the VP6 of the human (Bristol and 88-220) and porcine (Cowden) strains of gp C rotaviruses, but only low homology with the VP6 of bovine gp A (RF) and human gp B (ADRV) rotaviruses (41.3 and 16.30/o, respectively); (ii) the predicted secondary structure was highly conserved among the gene 5 proteins of the bovine, porcine, and human gp C rotaviruses; and (iii) seven highly conserved regions were identified for the first time in the deduced primary amino acid sequences of gene 5 of gp C and gene 6 of gp A rotaviruses. However, only three of these highly conserved areas were present in the regions of VP6, where the secondary structure was predicted to be similar for the rotavirus strains examined. These three regions may contribute to common epitopes between the two groups of rotaviruses. Our results, in comparison with data for other rotaviruses, indicate that gene 5 of the bovine gp C rotavirus codes for the major inner capsid protein (VP6). o 1992 Academic PWSS, h.

Rotaviruses, one of the most common infectious agents causing severe gastroenteritis in humans and animals (7, 2), are currently classified into seven serogroups (A to G) based on the antigenic and genetic distinctness of each group (2, 3). The group (gp) C rotaviruses were originally isolated from a diarrheic pig (4) and have subsequently been associated with outbreaks of diarrhea in humans and cows (5-9). The relative severity of diarrhea associated with gp C rotaviruses compared with that of other agents, such as gp A rotaviruses, is not known: however, in humans, gp C rotaviruses have been reported to cause mild, severe, or even fatal gastroenteritis (lo- 12). The recent isolation in Japan of a gp C rotavirus (Shintoku strain) from cows with severe diarrhea indicated that gp C rotaviruses have a wider host range than was originally thought (9). Serologic analysis showed that this virus was antigenically closely related to the prot‘otype Cowden porcine gp C rotavirus, but was distantly related to gp A and B rotaviruses (9). Hybridization studies demonstrated the genetic relatedness of the Shintoku genes 5, 6, and 8 with the corre’

To whom

0042.6822/92

reprint

requests

should

$5.00

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction I” any form reserved.

sponding genes of porcine gp C rotavirus, using the Cowden cDNA probes (13, 14). In Gp A rotaviruses, the inner capsid protein, VP6, possesses group or subgroup antigens, and because it constitutes more than 50% of virion proteins, the VP6 is a target protein for diagnosis and serogrouping (I, 2). Biochemical and molecular studies have demonstrated that the VP6 of gp A rotaviruses studied to date, including human, porcine, bovine, and simian isolates, is encoded by genome segment 6 (I). Gene 6 is composed of 1356 nucleotides for all strains of gp A rotaviruses except the equine H-2 strain, which contains 1362 nucleotides (75). In addition, several conserved regions of amino acid sequences have been identified that may determine the specificity of different subgroups (15). An abundant protein was also identified in the inner capsid of a porcine gp C rotavirus that was similar to that found in gp A rotaviruses (16, 17). In contrast to the protein in gp A rotavirus, this protein in gp C rotavirus is encoded by gene 5 of the porcine (Cowden) and human (Bristol and 88-220) gp C rotaviruses (1820). Furthermore, the sequences among genes encoding VP6 of the Cowden, Bristol, and 88-220 strains

be addressed.

542

SHORT 1 GCATTTAAAATCTCATTCACA

82

156

231

m Met

COMMUNICATIONS

543

GAT GTG CTG TTC TCC ATT GCG AAA ACA GTT TCA GAG CTT AAA AAG AGA GTG GTA GTT Asp Val Leu Phe Ser Ile Ala Lys Thr Vat Ser Glu Leu Lys Lys Arg Vat Val Val 20

GGA ACA ATT TAC ACT AAT GTT GAA GAC ATA ATT CAA CAG ACT AAT GAG TTG ATT AGA ACT TTG AAT GGA AGT ACG Gly Thr Ile Tyr Thr Asn Val GLu Asp Ile Ile Gin Gin Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Ser Thr

45

TTC CAT ACT GGT GGC ATT GGC ACG CAA CCT CAG AAA GAC TGG GTT GTT CAA CTT CCT CAA CTA GGC ACG ACT CTA Phe His Thr Gly Gly Ile Gly Thr Gln Pro Gln Lys Asp Trp Val Val Gln Leu Pro Gln Leu Gly Thr Thr Leu

70

CTT AAT CTC GAT GAT AAT TAT GTG CM Leu Asn Leu Asp Asp Asn Tyr Val Gln

95

TCT GCA AGA GGT ATA ATT GAT TAT CTG CCC TCA TTT ATT GAA GCT GTA Ser Ala Arg Gly Ile Ile Asp Tyr Leu Ala Ser Phe Ile Glu Ala Val

306

TGT GAT GAT GAA ATG GTT AGA GAA GCG TCA AGA AAT GGA ATG CAA CCT CAA TCA CCA ACA TTA ATC GCA TTA GCT Cys Asp Asp Glu Met Val Arg Glu Ala Ser Arg Asn Gly Met Gln Pro Gln Ser Pro Thr Leu Ile Ala Leu Ala 120

381

TCA TCA AAA TTC AAA ACT ATT AAT TTC AAT AAT AGT TCT CAG TCT ATC AAA AAT TGG AGC GCT CAA TCT AGA CGT Ser Ser Lys Phe Lys Thr Ile Asn Phe Asn Asn Ser Ser Gln Ser Ile Lys Asn Trp Ser Ala Gln Ser Arg Arg 145

456

GAA AAT CCA GTT TAT GAA TAT AAG AAT CCA ATG GTT TTT GAA TAT AGA AAT TCA TAT ATT CTG CAT CGC GCA GAT Glu Asn Pro Val Tyr Glu Tyr Lys Asn Pro Met Val Phe Glu Tyr Arg Asn Ser Tyr Ile Leu His Arg Ala Asp 170

531

CAA CAG TTT CGA AAT GCT ATG CGA TTG AGA TAT TAT ACA ACA AGT AAT ACT TGT CAA ATT GCA GCA TTT GAT TCT Gln Gln Phe Gly Asn Ala Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gin Ile Ala Ala Phe Asp Ser 195

606

ACT ATG GCT GAG AAT GCA CCA AAT AAT ACA CAA AGG TTC ATT TAT CAT GGA AGA TTG AAG AGA CCA ATC TCT AAT Thr Met Ala Glu Asn Ala Pro Asn Asn Thr Gln Arg Phe Ile Tyr His Gly Arg Leu Lys Arg Pro Ile Ser Asn 220

681

GTG CTA ATG AAA GTT GAA CGT GGC GCT CCA AAT GTT AAT AAC CCA ACA ATA CTA CCA GAT CCA ACT AAC CAA ACT Val Leu Met Lys Val Glu Arg Gly Ala Pro Asn Val Asn Asn Pro Thr Ile Leu Pro Asp Pro Thr Asn Gln Thr 245

756

ACG TGG TTG TTT AAT CCA GTA CAA GTG ATG AAC GGA ACA TTT ACA ATT Thr Trp Leu Phe Asn Pro Val Gln Val Met Asn Gly Thr Phe Thr Ile

a31

GAT ATG GTT AGA AAT ATG GGA ATA GCA ACT GTC AGA ACT TTT GAT TCA TAT AGG ATT ACG ATA GAT ATG ATT AGA Asp Met Val Arg Asn Met Gly Ile Ala Thr Val Arg Thr Phe Asp Ser Tyr Arg Ile Thr Ile Asp Met Ile Arg 295

906

CCA GCT GCT ATG ACC CAG TAT GTA CAG CAA TTG TTC CCT GTT GGT GGA CCA TAC TCT CAT CAA GCT GCT TAT ATG Pro Ala Ala Met Thr Gln Tyr Val Gln Gln Leu Phe Pro Val Gly Gly Pro Tyr Ser His Gln Ala Ala Tyr Met 320

981

CTT ACA CTT AGT GTG CTG GAT GCC ACG ACA GAA TCT GTT TTG TGC GAC TCT CAT TCA GTC GAT TAT TCG ATA GTT Leu Thr Leu Ser Val Leu Asp Ala Thr Thr Glu Ser Val Leu Cys Asp Ser His Ser Val Asp Tyr Ser Ile Vat 345

1056

CCC AAT ACG AGA AGG CAT TCA GCG ATG CCA GCT GGA ACG GTT TTC CAA CCA GGA TTT CCA TGG GAA CAG ACG TTG Ala Asn Thr Arg Arg Asp Ser Ala Met Pro Ala Gly Thr Val Phe Gln Pro Gly Phe Pro Trp Glu Gln Thr Leu 370

1131

TCC AAC TAC ACG GTT GCT CAA GAG GAT AAT TTG GAA AGA TTG TTG CTA GTT GCA TCC GTG AAG AGA ATG GTG ATG Ser Asn Tyr Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu Leu Leu Val Ala Ser Val Lys Arg Met Val Met 395

1206 1305

TAGATAAGCTAGAGATCTAAACAATCTCTATGTGGACTACACACCATGTAGCATGATTCACGAATGGGTTTAGTCTACACTTGCGTAGGGGCAAATGCG CATGATGTAGATGATCCCCAGGAGGATGAAATGTGAACTATGTGGCT

GAG TTT TAT AAC AAT GGC CAG TTA GTT Glu Phe Tyr Asn Asn Gly Gln Leu Val 270

FIG. 1. Nucl’eotide and deduced amino acid sequences of gene 5 of the bovine gp C Shintoku strain. The nucleotide sequence corresponds to the viral plus-strand RNA. The start and stop codons are underlined. The nucleotide sequence is numbered on the left and the amino acid sequence is indicated on the right. The sequences have been assigned Accession Number M88768 by the EMBUGenBank Data Libraries.

were highly conserved (18,20,21). However, no molecular studies have been done on gp C rotaviruses from other species. Recently, the identification and adaptation to cell culture of the bovine gp C Shintoku strain has provided the opportunity to further investigate the molecular biology and the evolution of gp C rotaviruses. Moreover, because the Shintoku strain is only the second gp C rotavirus adapted to serial growth in cell culture (9), this virus may serve as another model for the detailed investigation of gp C rotaviruses. Thus, we have cloned and sequenced gene 5 of the Shintoku strain of gp C rotavirus and compared it with the VP6 genes from other porcine and human strains of gp C rotaviruses as well as from gp A and B rotaviruses. The Shintoku strain of bovine gp C rotavirus was propagated in MA1 04 cells as previously reported (9). Infected cell lysates were clarified by low-speed centrif-

ugation, extracted with trichlorotrifluoroethane, and partially purified by centrifugation through 30% sucrose cushions (17). Other gp C rotaviruses used in this study included the human (88-196) and the porcine (Cowden, NB, WH, Ah, HF, KH, and Wi) strains (13, 14). The NCDV (gp A) and ATI (gp B) strains of bovine rotaviruses were also included as controls in the hybridization assays. The in vitro transcription of the Shintoku genomic RNA was performed as previously described (22), except that partially purified viral particles were used in the assay. The transcript RNA obtained was used to make gene-5-specific cDNA by polymerase chain reaction (PCR), using two oligonucleotide primers, 3297 and 3299. Primer 3297 (5’-AGCCACATAGTTCACAlT3’) was complementary to the 3’ end of the Cowden gene 5 (plus strand), and the plus-sense primer 3299

544

SHORT

COMMUNICATIONS TABLE

COMPARISON

1

OF THE VP6 OF GP C, A, AND B ROTAVIRUSES

9P C”

Size (aa) MW (KDa) Pl

sp Ah

Shintoku

Cowden

Bristol/88-220

395 44.5 6.7

395 44.7 6.3

395 44.7 6.9 Amino

Shintoku Cowden Bristol/88-220 RF Gottfried F114 H-2 ADRV a From b From c From

90.6 -

Refs. 18 and 20. Refs. 15 and 26. Cited Ref. 27.

here

were

representative

acid

RF 397 44.8 5.5

sequence

homology

88.4 90.9

strains

Gottfried

(5’-GCATTTAAAATCTCATTC-3’) corresponded to the 5’ end of gene 5. Both primers were treated with T4 polynucleotide kinase (Bethesda Research Laboratories, Gaithersburg, MD) prior to use. The first strand cDNA was synthesized from the Shintoku transcript RNA by reverse transcription using the primer 3297. The single-stranded cDNA was then subjected to PCR amplification with Tao polymerase (Perkin-Elmer Cetus, Norwalk, CT), using both primers as described (23). The amplified full-length cDNA was treated with Klenow enzyme (BRL), purified by centrifugation through a Sephacryl S-400 spun column (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ), and ligated into the vector pTZl8R (Pharmacia) by T4 DNA ligase (BRL). The recombinant DNA was then used to transform competent Escherichia co/i (DH5cy) (BRL), and positive clones were identified by hybridization using 32P-labeled plasmid DNA probe against Shintoku dsRNA. Sequencing was conducted by the Sanger dideoxy nucleotide chain termination method (24) with [35S]deoxyadenosine 5’-[a-thioltriphosphate (NEN Research Products, Boston, MA). For cDNA clones, sequences were determined using the Sequenase (Version 2.0; United States Biochemical Co., Cleveland, OH). The viral transcript RNA was sequenced using an RNA Sequencing Kit (Boehringer-Mannheim Biochemicals, Indianapolis, IN). Five cDNA clones (BC5-58, BC5-70, BC5-77, BC578, and BC5-92) all coterminal with the 3’ end of gene 5, were sequenced, spanning nucleotides 392 to

subgroups

B”

Fl14

H2

ADRV

397 45.0 5.8

397 44.9 6.1

399 45.2 6.1

391 43.7 5.3

40.3 41.3 41.5 91.9

41.3 42.5 43.3 91.4 94.5

41.0 41.8 42.3 97.0 90.5 90.9 -

16.3 18.3 19.5 17.1 15.6 17.1 16.4

(9/o)

41.3 42.3 42.8 -

of four

w

(RF/I,

Gottfried/lI,

FI-14/l

+ II, and

H-2/nonl/lI).

1352. In order to determine the sequence of nucleotides 1 to 391, another minus-sense primer (from nucleotides 434 to 417; 5’-TlllTGATAGACTGAGAA-3’) was synthesized based on the sequence from cDNA clones. This primer and the primer 3299 were used to amplify a cDNA (-430 bp) from transcript RNA using the PCR technique and the fragment was directly sequenced. Nucleotide sequences generated from both cDNA clones and PCR products were further confirmed by the direct sequencing of viral transcript RNA. The Shintoku gene 5 is 1352 bp in length and possesses one long open reading frame (ORF) beginning at nucleotide 22 and terminating at nucleotide 1206 (Fig. 1). The AUG start codon has a purine A in position -3 and a G in position $4 and therefore is a strong initiating codon based on Kozak’s rules (25). The gene 5 sequence of the bovine Shintoku strain was the same length as those of the human Bristol and 88-220 strains (18,20) and the porcine Cowden strain reported recently (20), but was three nucleotides longer than that of the Cowden strain reported earlier (21). By comparative nucleotide sequence analysis, the Shintoku gene 5 shared 82.0% homology with both the Cowden and the Bristol gene 5, 56.5% homology with the corresponding gene of the RF bovine gp A rotavirus, and virtually no homology with the corresponding gene of the ADRV human gp B rotavirus. Furthermore, the sequences were highly conserved at both the 5’ and 3’ ends of the gene 5 of the three gp C strains (Shintoku, Cowden, and Bristol) (data not shown). These conserved 5’ and 3’ terminal sequences should permit the

SHORT

COMMUNICATIONS

545

A B C/D E

VII

---A-----L-------

_________

P-,-N-------------,-A----IN-L----T--T--

----y----L

---T-----,-m-----

______-__

R-V-N-------------,-A----,S-p----N--N--

----F----L

WLNAGSEI-V-G--YSC

-I---A--L-l

Q-EHIVQ-R-VLTTATITLLPD-ERFSFPRVITSADGA

------------mm-,, ---------------I

---YF---IL L-J

RPNNVE---LL---II

271 300 330 360 DMVRN.MGIATVRTFDSYRITIDMIRPAAMTQYVQQLFPVGGPYSHQAAYMLTLSVLDATTESVLCDSHSVDYSIVANTRRDSAMPAGTV -----.--VV-------------------------RL--Q----py--A------,---------------D------V---------------.--,V-------------------------R,--Q----HF--T------,---------------E------V-----------

NTYQARF-TIIA-N--TI-LSFQLM--PN--PA-AAL--NAQ-FEHH-TVG---RIES-VC----A-ASETMLAN-TSV-QEY-I-V-P-

361 397 FQPGFPWEQTLSNYTVAQEDNLERLLLVASVKRMVM* --------Q------------------V-------*

--------H------------------I-------* -P--MN-TDLIT--SPSR----Q-VFTV--IRS-LVK*

FIG. 2. Comparison 88-220 (C/D) strains, only one sequence is program (28). Consensus its homology with that

of the deduced amino acid sequence of the Shintoku gene 5 (A) with the VP6 sequences of the gp C Cowden (B), Bristol or and the gp A RF strain (E). The VP6 sequences of the Bristol and 88-220 strains were completely homologous (20) thus presented. Multiple sequence alignment was performed using the Genetics Computer Group (GCG) sequence analysis sequences are indicated by the character “-” and one gap was introduced into each gp C VP6 sequence to maximize of gp A rotaviruses. Seven highly conserved regions (I to VII; each with a block of 9 to 19 amino acids) are boxed

design of PCR primers which will be useful for the detection of gp C rotaviruses in clinical specimens. The ORF of the Shintoku gene 5 encodes a polypeptide of 395 amino acids, the same size as those re-

,

ported for three other gp C rotaviruses, but two to four amino acids shorter than those reported for gene 6 of gp A rotaviruses and four amino acids longer than that for gene 5 of a gp B rotavirus (Table 1). The predicted

.

I

, FIG.

method to those

I

I..

I.,

AmA

D *.

*.

I

.

.

.

.

I.

.,

2-A.+ *.

.

I.

I-.&“.

6

0,.

3. Comparison of the hydropathic plot of the Shintoku gene 5 protein (A) with those of the Cowden (B), Bristol (C), and RF (D) VP6 by the described (29). Areas above the horizontal line are hydrophobic and those below the line are hydrophilic. Seven regions corresponding identified in Fig. 2 are also indicated.

546

SHORT

B

12345

6

7

8

9

IO

COMMUNICATIONS

11

blot hybridization analysis of the Shintoku gene 5 FIG. 4. Northern probe against dsRNA from gp A, B, and C rotaviruses. Viral dsRNA was extracted from partially purified particles, resolved in 10% polyacrylamide gels, and transferred to the Nytran membranes as described (13, 14). The hybridization was performed using “P-labeled clone BC5-58 (consisted of nucleotides of 392 to 1352) as a probe (specific activity: 650 Ci/mmole) under the stringency conditions (50% formamide, 5x SSC and 42 C) (73, 14). (A) dsRNAelectropherotypes of bovine gp A (NCDV; lane l), B (ATI; lane 2) and C (Shintoku; lane 3) rotaviruses; human gp C (88-l 96; lane 4) rotavirus; and porcine gp C rotaviruses designated NB (lane 5) Ah (lane 6) Wi (lane 7) Cowden (lane 8) WH (lane 9) HF (lane 1 O), and KH (lane 1 1). (B) The corresponding autoradiogram with gene 5 indicated by an arrowhead. Lanes 8 to 11 were electrophoresed on a different gel.

molecular mass (44.5 kDa) was consistent with those for the calculated gene 5 products (44.7 kDa) of the porcine (Cowden) and the human (Bristol and 88-220) strains (18, 20), as well as with the protein identified by radiolabeling of Cowden-infected cells or by in vitro translation of the Cowden cDNA clones (17, 19). The predicted protein has a pl value of 6.7, which was similar to the values of the three other gp C strains (Table 1). In addition, the pl values of all gp C rotaviruses were higher than those of representatives from four subgroups of gp A rotaviruses as well as the gp B ADRV strain (Table 1). The functional implication of this difference in the isoelectric point among different groups of rotaviruses remains to be investigated. The deduced amino acid sequence of the Shintoku gene 5 shared more homology with the corresponding VP6 of the

Cowden (90.6%) and the Bristol/88-220 (88.4%) gp C strains than with the VP6 of gp A (41.3%) and B (16.3%) rotaviruses, further indicating the distant relationship among gp C, A, and B rotaviruses. The amino acid identities of the VP6 between other gp C and gp A or B rotaviruses, among gp A rotaviruses, or between gp A and B rotaviruses were also calculated for the comparisons (Table 1). To look for antigenically conserved regions, we aligned and compared the deduced amino acid sequence of the Shintoku gene 5 with the VP6 sequences of the Cowden, Bristol, and 88-220 gp C strains as well as with the RF gp A strain (Fig. 2). Seven regions (I to VII) were identified as highly conserved (70 to 92% homology). A total of 57 substitutions were observed among the amino acid sequences of gene 5 proteins from the four gp C strains examined. Interestingly, 21 (36.8%) of these substitutions were identical between either the two animal strains or the two human strains, indicating that there may be animal- or human-specific sequences. Furthermore, 21 (36.8%) of the 57 substitutions were conserved among the Cowden, Bristol, and 88-220 strains, whereas 12 (21.1 o/o)of the 57 changes were conserved among the bovine (Shintoku) and the human (Bristol and 88-220) strains. These data suggest that the Shintoku strain is less closely related to the two human strains than the Cowden strain, although they have a common genetic origin. The hydropathic plots of the VP6 from the three gp C strains and the RF gp A strain demonstrated that the secondary structures of the three gp C polypeptides were highly conserved as well (Fig. 3). These profiles differed in most regions from that of the corresponding plot of the RF gp A strain. Of great interest was the apparent conservation of both primary and predicted secondary structures of at least three hydrophilic regions (IV, V, and VI) between gp C and A rotaviruses (Figs. 2 and 3). These regions could contribute to potential common epitopes between distinct serogroups of rotaviruses and appear to support our previous findings of common epitopes in the VP6 of gp A and C rotaviruses, as demonstrated by the binding of crossreactive monoclonal antibodies in ELISA (30). The specificity of cDNA clones was confirmed by Northern blot hybridization (Fig. 4). Figure 4A shows an ethidium bromide-stained gel of viral dsRNA of human and animal origin. In Fig. 4B, the cDNA probe specifically recognized the genomic segment 5 of the homologous bovine (lane 3) and heterologous human (lane 4) as well as porcine (lanes 5 to 11) gp C rotaviruses. In control experiments, the probe did not react with the corresponding genes from bovine gp A (NCDV) and B (ATI) rotaviruses (lanes 1 and 2). Our results correlate

SHORT

COMMUNICATIONS

with previous reports that the Cowden cDNA probes also hybridized with the corresponding genes of homologous and heterologous gp C rotaviruses (13, 14), further confirming the genetic relatedness of gp C rotaviruses from different species. ACKNOWLEDGMENTS We extend our appreciation to Steve Monroe for assistance with the computer analysis, to Anil Parwani for help in the hybridization assay, and to John O’Connor for critical reading of the manuscript. This study was supported in part by special Research Grants Y02-AI90002-02 from the NIAID-lntra-Agency Agreement and 89-34116. 4625 from the U.S. Department of Agriculture, Cooperative State Research Service. This work was initiated by the senior author in partial fulfillment of the requirements for the Ph.D. degree from the Graduate School of the Ohio State University.

REFERENCES 1. ESTES, M. K., and COHEN, J., Microbial. Rev. 53, 41 O-449 (1989). 2. SAIF, L. J., In “Viral Diarrhea of Man and Animals” (L. J. Saif and K. W. Theil, Eds.), pp. 73-95. CRC Press, Boca Raton, FL, 1990. 3. BRIDGER, J. C., Ciba Found. Symp. 128, 5-23 (1987). 4. SAIF, L. J., BOHL, E. H., THEIL. K. W., CROSS, R. F., and HOUSE, J. A., /. C/in. Microbial. 12, 105-l 1 1 (1980). 5. DIMITROV, D. H., ESTES, M. K., RANGELOVA, S. M., SHINDAROV, L. M., MELNICK, J. L., and GRAHAM, D. Y., infect. Immun. 41, 523-526 (1983). 6. NICOLAS, J. C., COHEN, J., FORTIER, B., LOURENCO, M. H., and BRICOUT, F., \/iro/ogy 124, 181-184 (1983). 7. PE~~ARANDA, M. E., Cus~rr, W. D.. SINARACHATANANT, P., TAYPOR, D. N., LIKANONSAKUL, S., SAIF, L., and GLASS, R. I.,). infect Dis. 160, 392-397 (1989). 8. RODGER, S. M., BISHOP, R. F., and HOLMES, I. H., /. C/in. Microbial. 16, 724-726 (1982). 9. TSUNEMITSU, H., SAIF, L. J., JIANG, B. M., SHIMIZU, M., MASANOBU, H., YAMAGUCHI, T., ISHIYAMA, T., and HIRAI, T., J. C/in. Microbiol. 29, 2609-2613 (1991).

547

10. CAUL, E. O., ASHLEY, C. R., DARVILLE, J. M., and BRIDGER, J. C., 1. Med. Viral. 30, 201-205 (1990). 11. ISHIMARU, Y., NAKANO, H., OSETO, M., YAMASHITA, Y., KOBAYASHI, N., and URASAWA, S., Acta Paediafr. Jpn. 32, 523-529 (1990). 12. MATSUMOTO, K., HATANO, M., KOBAYASHI, K., HASEGAWA, A., YAMAZAKI, S.. NAKATA, S., CHIBA, S.. and KIMURA, Y., J. Infect. Dis. 160, 611-615(1989). 13. JIANG, B. M., QIAN, Y., TSUNEMITSU, H., GREEN, K. Y., and SAIF, L. J., virology 184, 433-436 (1991). 14. JIANG, B. M., TSUNEMITSU, H., QIAN, Y., GREEN, K. Y., OSETO, M., YAMASHITA, Y., and SAIF, L. J.. Arch. Viral. in press. 15. GORZIGLIA, M., HOSHINO, Y., NISHIKAWA, K., MALOY, W. L., JONES, R. W., KAPIKIAN, A. Z., and CHANOCK, R. M., /. Gen. Viral. 69, 1659-1669 (1988). 76. BREMONT, M., COHEN, J., and MCCRAE, M. A., /. Viral. 62, 21832185 (1988). 77. JIANG, B. M., SAIF, L. J., KANG, S. Y., and KIM, J. H., J. Viral. 64, 3171-3178 (1990). 18. COOKE, S. J., LAMBDEN, P. R., CAUL, E. O., and CLARKE, I. N., Virology 184, 781-785 (1991). 19. QIAN, Y., JIANG, B., SAIF, L. J., KANG, S. Y.. and GREEN, K. Y., Virology 184, 752-757 (1991). 20. QIAN, Y., JIANG, B., SAIF, L. J., KANG, S. Y., ISHIMARU, Y., YAMASHITA. Y., OSETO, M., and GREEN, K. Y., J. Gen. Viral., submitted. 21. BREMONT, M., CHABANNE-VAUTHEROT, D., VANNIER, P., MCCRAE, M. A., and COHEN, J., Viroiogy 178, 579-583 (1990). 22. JIANG, B. M., and SAIF, L. J., Arch. Viral. 124, 181-l 85 (1992). 23. LARRALDE, G., and FLORES, J., Virology 179, 469-473 (1990). 24. SANGER, F., NICKLEN, S., and COULSON, A. R., Proc. Nat/. Acad. Sci. USA 74, 5463-5467 (1977). 25. KOZAK, M.. Nucleic Acids Res. 9, 5233-5252 (1981). 26. COHEN, J., LEFEVRE, F., ESTES, M. K., and BREMONT, M., Virology 138, 178-182 (1984). 27. CHEN, G-M., WERNER-ECKERT, R., HUNG, T., and MACKOW, E. R., t’iroiogy 182, 820-829 (1991). 28. DEVEREUX, J., HAEBERLI, P., and SMITHIES, O., Nucleic Acids Res. 12, 387-395 (1984). 29. KYTE, J., and DOOLITTLE, R. F., J. MO/. Biol. 157, 105-l 32 (1982). 30. TSUNEMITSU, H., OJEH, C. K., JIANG, B., SIMKINS, R. A., WEILNAU, P. A., and SAIF, L. J., viroiogy, submitted.