VIROLOGY 182, 562-569 (1991)

Sequence Conservation of Gene 8 between Human and Porcine Group C Rotaviruses and Its Relationship to the VP7 Gene of Group A Rotaviruses’

YUAN QlAN,* BAOMING JIANG,t LINDA J. SAIF,i’ SHIEN Y. KANG,t YOSHIRO lSHlMARU,+ YASUTAKA YAMASHITAJ MITSUAKI OSETO,” AND KIM Y. GREEN*v2

*Laboratory of infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; tFood Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster,

Ohio 4469 1; +lshimaru Pediatric Clinic, 6-5-l Sanbancho, Matsuyama, Ehime 790, Japan,’ QEhime Prefectural Institute of Public Health, 234-8 Sanban-cho. Matsuyama, Ehime 790, Japan; and “Matsuyama Prefectural Institute of Public Health,

132 Kita-Mochida-cho, Matsuyama, Ehime 790, Japan

Received December 13, 1990; accepted February 12, 199 1

cDNA libraries from porcine group (Gp) C rotavirus strain Cowden and a human Gp C rotavirus strain were gener- ated. The complete nucleotide sequence of gene 8 from the Cowden strain was determined from gene 8-specific clones and viral transcript RNA. A full-length gene 8 clone was generated from the human Gp C virus by polymerase chain reaction (PCR) using primers deduced from the 3’ and 5’ ends of the Cowden strain gene 8, and the sequence of the human Gp C gene 8 was determined from this clone and gene 8 clones in the cDNA library. The gene 8 from the Cowden or the human Gp C strain is 1063 nucleotides in length and contains a long open reading frame beginning at the 49th nucleotide from the 5’ end and terminating with a stop codon 16 bases from the 3’ end. The encoded protein contains 332 amino acids (predicted molecular weight of 37.3 kDa) with two potential N-linked glycosylation sites in the porcine strain and three in the human strain. The polypeptide products derived from in vitro translation of the transcript RNA generated from a porcine gene 8 clone containing the entire open reading frame were analogous in size with the Gp A VP7. The gene 8 of porcine and human Gp C rotaviruses exhibited considerable nucleotide and deduced amino acid sequence identity (83.8 and 88.0%, respectively). Comparison of the Gp C gene 8 protein sequence with the VP7 protein of Gp A rotavirus revealed structural similarities, although the overall amino acid identity was low (~30%). These data suggest that the gene 8 of the porcine or human Gp C rotavirus encodes a protein corresponding to the VP7 outer capsid glycoprotein of Gp A rotaviruses and that the eighth gene is highly conserved in the porcine and human Gp C strains examined in this study. o 1991 Academic Press. Inc

INTRODUCTION

Rotaviruses are etiological agents of severe diarrhea in the young of humans and many animal species (Ka- pikian and Chanock, 1990). These viruses are members of the family Reoviridae and have been as- signed to at least seven groups designated A through G (Estes and Cohen, 1989; Saif, 1990). Among them, Gp A is clearly the most important in diarrhea1 disease (Kapikian et al., 1989). The importance of “non-group A” rotaviruses in diarrhea1 disease has not been estab- lished. However, reports of severe gastroenteritis in adults and children associated with Gp B or C rotavi- ruses (Hung et a/., 1984; Beards et al., 1989; Bonsdorff and Svensson, 1988; Brown et a/., 1988; Caul et al., 1990; Chen et a/., 1988; Dimitrov et a/., 1986; Penar- anda et a/., 1989; lshimaru et al., 1990; Matsumoto et

’ The nucleotlde sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned Accession Nos. M61100 and M6 1101.

2 To whom reprint requests should be addressed.

al., 1989; Ushijima et al., 1989) suggest that these vi- ruses could emerge as important pathogens.

Gp C rotaviruses were initially detected in piglets with diarrhea (Saif et al., 1980). The Gp C viruses were identified as rotaviruses because of similarities to Gp A rotaviruses in morphology by negative stain electron microscopy and genomic structure (i.e., 11 segments of double-stranded RNA). However, the lack of sero- logic cross-reactivity with Gp A rotavirus caused them to be designated “atypical” rotaviruses or “pararota- viruses” (Bohl et a/., 1982). Limited serologic surveys have suggested that Gp C rotavirus infections are common in pigs in North America, Europe, and Austra- lia (Saif et al., 1980; Saif and Theil, 1985; Nagesha et a/., 1988). In humans, Gp C rotavirus has been asso- ciated with sporadic cases and outbreaks of diarrhea in several countries, suggesting that this group is glob- ally distributed. The unavailability of diagnostic re- agents has hampered epidemiologic studies of Gp C rotavirus infection in humans.

Hybridization studies have shown that the human and porcine Gp C rotaviruses are closely related geneti-

0042-6822/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

562

SEQUENCE CONSERVATION OF GENE 8 563

tally (Bridger et al., 1986; Qian et al., 1991). In this study, cDNA libraries of a porcine Gp C reference strain and a human Gp C rotavirus were generated in order to further investigate the molecular basis for the genetic relatedness within Gp C as well as the lack of relatedness among different rotavirus groups and to develop reagents for epidemiological studies of Gp C rotaviruses.

MATERIALS AND METHODS

Viruses. Porcine Gp C rotavirus reference strain Cowden was propagated in gnotobiotic piglets as de- scribed (Bohl eta/., 1982). Human Gp C rotavirus strain 88-220 was purified from a stool specimen collected from a child with diarrhea in Matsuyama, Japan, in 1988 (Ishimaru et a/., 1990).

Construction of cDNA libraries. Single-stranded (ss) RNA of porcine or human Gp C rotavirus was produced by in vitro transcription of purified virus particles (Qian et a/., 1991). The cDNA Synthesis Plus kit (Amersham Co.) was used for cDNA synthesis from the RNA tran- scripts of the human or porcine Gp C virus. A “random primer” included in the kit was used to initiate cDNA synthesis. In addition, transcripts of the porcine Cow- den strain were polyadenylated (Sippel, 1973) and oligo(dT) was used to initiate cDNA synthesis. Follow- ing first-strand cDNA synthesis using reverse tran- scriptase, second-strand cDNA synthesis was accom- plished using Escherichia co/i RNase and DNA poly- merase included in the kit. The double-stranded (ds) cDNA was blunt ended with T4 DNA polymerase, li- gated into the unique Smal site of vector pTZ18R (Pharmacia Co.) and used for transformation of DH-5 a! E. co/i cells (BRL). Positive clones were identified by colony hybridization using 32P-labeled RNA transcripts produced from homologous virus particles as probe (Qian et al., 1991).

Determination of gene specificity of positive cDNA clones by Northern blot hybridization. Northern blot hy- bridization was pet-formed as described (Qian et a/., 1991). Positive cDNA clones were labeled with 32P by nick translation (Maniatis et al., 1975) and used as probes against viral dsRNA. DNA fragments were gen- erated from clones of known gene specificity using PCR (GeneAmp, Perkin Elmer Cetus), radiolabeled with 32P by nick translation, and used as probes for further characterization of clones in the libraries by dot blot hybridization.

Sequencing. Dideoxynucleotide sequencing was performed on both transcript RNA and cloned DNA templates (Sanger et a/., 1977). The sequence of cloned DNA was determined using a T7 DNA polymer- ase sequencing kit (Pharmacia). Synthetic oligonucleo-

..C..T...C AA..c..... R..R.....T . . . ..G.... .G..TC.... G..T..T... 850 860 870 880 890 900

CTTATCAGAa TRTCTACRTC ATClULGTTTT GATAATTCAT TGTCACCATT AMT‘RTGGA ..A..A.... .T..A..... ,,..,,.,.. . . ..A.C..C

910 920 930 940 950 960 CRRACAACAC GATCATTTAA AATARATGCA AAhRARTGGT GGACGATATT TTACACTATR . . . . . . . ..R . . ..G..... ..G....... . . ..T..... . ..T..A...

970 980 990 loo0 1010 1020 ATT‘ATTACA TCAATACIVLT TATRCP.AACT RTGACTCCGA GACRTCGGGC CRTTTRTCCA . . . . . . ..T. .T........ .G.....G.A . . . . . . ..c. A.........

1030 1040 1050 1060 ‘MGGTTGGR TGCTGAGATb CGCGT-MC.4 RGATCATGTG GCT . . . ..G.... ..T....G.. T.........

FIG. 1. Complete nucleotide sequences of the gene 8 of Gp C porcine Cowden strain and the human 88-220 strain. The se- quences correspond to the plus strand (mRNA sense) of the dsRNA in the 5’to 3’direction. The m-frame start codon beginning at nucleo- tide 49 and the stop codon beginning at nucleotide 1045 are under- lined. Only nucleotides which differ are shown in the sequence of the human strain. The nucleotide (n) sequence of the gene 8 from the Cowden strain was determined from ssRNA transcripts (n l-l 039) and the following overlapping clones from the cDNA library: CA2-28 (n243-1063); CA4-6 (n589-1063); CAl-30 (n270-1063); and PC8- 66 (nl-1062). The sequence of the gene 8 from the human 88-220 strain was determined from the following overlapping clones: HC8F- 23 (nl-1020); HC4-47 (n42-816); HC9-76 (n42-816); HC5-39 (nl 14-982); and two full-length clones (HC8F-1 and HC8F-3).

tides approximately 18 bases long were deduced from the cDNA insert sequences and used to initiate se- quencing on viral transcript RNA in the presence of reverse transcriptase as described (Gorziglia et al., 1986). For initiation of sequencing at the 3’ end of the gene 8 transcript, an oligonucleotide (5’-AGCCACAT- GATClTG-3’) deduced from the sequence adjacent to the poly(A) tract was used.

Construction of a full-length clone of the gene 8 of porcine or human Gp C rotaviruses. A full-length gene 8 cDNA of porcine or human Gp C rotavirus was pro- duced using PCR (Larralde and Flores, 1990). Briefly,

564 QIAN ET AL.

130 110 150 160 170 180 IYIx*rxvs DNDSvcmTC SYNIWIPDS PlwSESTERI AEwILNvwnc DDnNl,DIYTY .I.....PT. ..IN...... . . . . . . . . . . &zJ...P. . . . ..E....

190 200 210 220 230 240 E**G*DNLwA Am3sDcD”s” Cm,rrnmGI GCSPASTETI EVL D LA LLNVVDNVKH ..I..N.... . . . . . ..I.. . . . . ..s... p ..“....... .T R.

250 260 270 280 290 300 RIQnNT*SCK LWCIKOEIR LNT*hIRIST ssslDNsLoP LNDGQTTRSF KINAKKWWTI . . . . . ..P.. . . . .._. ,.N....... ,..,._,.,_

310 320 330 FmfIDYINT IIPTHTPRHR ALYPEGumLR IA . . . . . . . . . .“.A...... . . . . . . . .

FIG. 2. The deduced amino acid sequences of the gene 8 proteins of Cowden porcine and 88-220 human Gp C strains. The arrow indi- cates the potential cleavage site of the predicted protein. The poten- tial N-linked glycosylation sites are boxed. Only amino acids which differ are shown in the sequence of the human strain.

an oligonucleotide (V-AGCCACATGATCTTGTT- TACGC-3’) deduced from the 3’ end of the porcine gene 8 was used to initiate cDNA synthesis from the porcine or human Gp C gene 8 transcripts in the pres- ence of reverse transcriptase prior to PCR. An addi- tional oligonucleotide (5’-GCATTTAAAAAAGAAGAA- GCTGT-3’) deduced from the 5’ end of the porcine gene 8 was used in the PCR. The amplified DNA frag- ment was purified on a 1.2% low melting point agarose gel (BRL) and chromatographed on a NACS column (BRL). The dsDNA ends were blunt ended with T4 DNA polymerase, ligated into the Smal site of the plasmid pTZ18, and transformed into DH 5 Q f. co/i cells as described above.

In vitro transcription, translation and immunoprecipi- tation. For in vitro transcription and translation experi- ments, two porcine gene 8 clones were selected. One (PC8-38) contained a cDNA insert with the correct T7 promoter orientation as well as a full open reading frame, and the other (PC8-18) contained a full open reading frame in the opposite orientation. Transcript RNA was obtained in vitro using a Riboprobe System kit (Promega) following the manufacturer’s instruction. In vitro translation was performed using a nuclease- treated rabbit reticulocyte lysate in the presence or ab- sence of canine microsomal membranes (Promega). One microgram of transcript RNA and 0.8 mCi of [35S]- methionine/ml (>800 CVmmol; 1 Ci = 37 GBq) were added to 50 ~1 of the reaction mixture. After incubation at 30” for 1 hr, the in vitro translation products were immunoprecipitated with either hyperimmune anti- serum raised in gnotobiotic piglets against the porcine Cowden strain or piglet preimmunization serum and

analyzed in a 12% polyacrylamide gel containing 0.1% SDS (Laemmli, 1970). A Cowden virus-infected MA- 104 cell lysate containing radiolabeled viral polypep- tides prepared as described previously was included in some experiments (Jiang et al., 1990). After electropho- resis, the gel was soaked in an enhancer (Enlightning, Du Pont) for 30 min, dried, and exposed to X-Omat film (Kodak) overnight.

RESULTS

Comparison of the gene 8 sequences. Several partial clones specific for the gene 8 of either porcine or hu- man Gp C rotavirus were selected by Northern blot hybridization or dot blot hybridization from the gene libraries and sequenced. Because polyadenylation was employed in the cloning of the porcine virus from transcript RNA, the sequence of the 3’ end of the por- cine gene 8 was considered to begin at the first base after the poly(A) tract. Initiating with an oligonucleotide

4

3

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4

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FIG. 3. Comparison of the hydropathicity profiles of the gene 8 protein of porcine and human Gp C rotaviruses and the VP7 glyco- protein of Gp A rotavirus by the method of Kyte and Doolittle (1982). Hydropathicity profiles of (A) the deduced gene 8 protein of Gp C porcine Cowden strain; (B) the deduced gene 8 protein of the human Gp C 88-220 strain; and (C)the VP7 glycoprotein of the Gp A porcine strain Gottfried. The nucleotide sequence of the VP7 gene of the Gottfried strain was reported previously (Gorziglia et al., 1988). Hy- drophobic areas are shown above the horizontal axes and hydro- philic areas are below.

SEQUENCE CONSERVATION OF GENE 8 565

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6i 1% 202 2&Q

Human Group C

68 135 262 2&Q

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FIG. 4. Amino acid identity between Gp C porcine and human rotavirus gene 8 proteins, and between the gene 8 protein of the Gp C Cowden strain and the VP7 protein of the Gp A porcine strain Gottfried illustrated by matrix plot analysis. (A) Plot of the human Gp C gene 8 protein vs the porcine Gp C gene 8 protein. (B) Plot of the porcine Gp C gene 8 protein vs the porcine Gp A VP7 protein. Matrix analysis was performed using

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the MacMolly computer program (Soft Gene, Berlin, Germany).

deduced from the sequence next to the poly(A) tract, the remaining nucleotide sequence of the Cowden strain gene 8 was determined using primer extension on the viral transcript RNA template and the cDNA in- serts from the clones. The sequence of the last 24 nucleotides from the 3’ end of the gene was deter- mined only from the cDNA clones. The sequence of the gene 8 of human Gp C was determined by se- quence analysis of several full-length clones generated by PCR as well as partial clones from the human Gp C gene library.

The complete nucleotide sequences of the gene 8 of the Cowden strain and the human Gp C rotavirus are shown in Fig. 1. The gene 8 of the porcine Cowden strain or the human Gp C rotavirus is 1063 nucleotides in length and contains a long open reading frame be- ginning at the 49th nucleotide from the 5’ end and ter- minating with a stop codon 16 bases from the 3’ end. For each gene, the first ATG at bases 49 through 5 1 is conserved (TAAAAG). An additional in-phase ATG is located at bases 124 through 126 in both gene 8s (AAAAAT). Neither sequence is consistent with Ko- zak’s strong initiation sequence (AXXmG) (Kozak, 1981). Beginning with the first ATG at the 49th nucleo- tide from the 5’end, the predicted protein contains 332 amino acids. The aligned sequence comparison showed that the first 79 nucleotides from the 5’ end and the last 22 nucleotides from the 3’ end are com- pletely conserved between the gene 8 of porcine and human Gp C virus. Within the coding region, nucleotide substitutions are scattered with the exception of one clustered divergent region located at nucleotides 135 through 183. The overall nucleotide identity of these two genes is 83.8%.

Comparison of the predicted gene 8 proteins. The aligned amino acid sequences deduced from the por- cine and human Gp C virus gene 8 showed extensive conservation between these two proteins (Fig. 2). The overall identity of the deduced amino acid sequences is 88.0%. The basic structural features of these two proteins appeared similar, as shown by the hydropathi- city profile of Kyte and Doolittle (1982) (Figs. 3A and 3B). Each of the two in-phase initiation codons pre- cedes a region of hydrophobic amino acids at the N- terminus of the protein. The structural similarity was also shown by the presence and location of cysteine and proline residues (1 1 cysteines and 11 prolines conserved in both strains). The amino acid identity of the porcine and human GpC gene 8 proteins is illus- trated by matrix plot analysis (Fig. 4A). In general, the amino acid substitutions were scattered, with the ex- ception of one clustered divergent region located at amino acids 31 through 45 which corresponded to the clustered nucleotide divergent region (135 through 183). This clustered amino acid divergent region is lo- cated in the second hydrophobic region. There were two conserved potential N-linked glycosylation sites (Asn-X-Ser/Thr) located at amino acid residues 67 through 69 and 225 through 227 in both gene 8 pro- teins. In the human rotavirus gene 8 protein there was an additional potential glycosylation site at amino acid residues 152 through 154.

In vitro translation and immunoprecipitation. The translation products produced in vitro from transcripts derived from porcine gene 8 clones PC8-38 (correct T7 promoter orientation) and PC8-18 (opposite orienta- tion) are shown in Fig. 5A. The lane containing the prod- uct obtained by in vitro translation of transcripts from

566

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QIAN ET

A B FIG. 5. Polyacrylamide gel electrophoresis analysis of radiolabeled

in vitro translation products from transcripts generated by in vitro transcription of the gene 8 clone of the porcine Gp C Cowden strain. Clone PC8-38 (contains nucleotides 13 to 1059) was in the correct T7 promoter orientation and clone PC8-18 (contains nucleotides 4 to 1061) was in the opposite orientation. Samples were subjected to electrophoresis in a 12% polyacrylamide gel, and the bands were visualized by fluorography. (A) 35S-labeled translation products from the reactions in the presence (with) or absence (w/o) of canine micro- somal membranes from (lane a) clone PC8-18 (w/o); (lane b) clone PC8-18 (with); (lane c) clone PC8-38 (w/o); (lane d) clone PC8-38 (with); (lane f) clone PC8-38 (w/o) precipitated with hyperimmune serum to porcine Gp C rotavirus; (lane g) clone PC8-38 (with) precipi- tated with hyperimmune serum to porcine Gp C rotavirus; (lane h) clone PC8-38 (w/o) precipitated with preimmune serum; and (lane i) clone PC8-38 (with) precipitated with preimmunization serum. ‘Y- labeled molecular weight markers (Amersham) are shown in lane e. “M” on the left indicates the molecular weight (kDa) of the molecular weight marker and on the right indicates the molecular weight of the in vitro translation product. (B) Comparison of the in vitro translation products from the PC8-38 clone with the 35S-labeled lysate of MA- 104 cells infected with the Cowden strain. “M” on the left indicates the molecular weight of the “C-labeled molecular weight markers (lane a) and on the right the molecular weight of the 33-kDa outer capsid protein from the porcine Gp C strain Cowden in an infected MA-1 04 cell lysate (lane C) identified by Jiang et al. (1990). Lane b contains the in vitro translation products from the clone PC8-38 pre- pared in the absence of canine microsomal membranes.

clone PC8-38 in the absence of canine microsomal membranes showed two bands with approximate mo- lecular weights of 34.5 and 37.0 kDa (lane c). In the presence of canine microsomal membranes in the translation reaction of clone PC8-38, three bands with approximate molecular weights of 34.5, 37, and 39 kDa were observed (lane d). No polypeptide product was detected from clone PC8-18 in the presence or absence of microsomal membranes (lanes a and b, respectively). The translation products from clone PC8-38 obtained either in the absence or in the pres- ence of canine microsomal membranes were immuno- precipitated with hyperimmune serum (lanes f and g,

AL.

respectively), whereas these products were not precipi- tated with preimmune serum (lanes h and i, respec- tively). The 34.5-kDa in vitro translation product from the clone PC8-38 obtained in the absence of micro- somal membranes was consistent in size with an outer capsid protein of 33 KDa identified in a radiolabeled lysate of MA-l 04 cells infected with the Gp C Cowden strain (Fig. 5B).

Comparison of the gene 8 protein of Gp C with the l/f7 of Gp A rotavirus. Comparison of the gene 8 pro- tein of the porcine Gp C rotavirus strain Cowden with the outer capsid glycoprotein VP7 of the porcine Gp A rotavirus strain Gottfried revealed striking similarities in structure and organization between these proteins, al- though overall amino acid identity was low (less than 30%). The hydrophobicity profile of the Gp A VP7 was similar to that of the Gp C gene 8 protein (Fig. 3C). However, the matrix plot comparing the amino acid sequence of the gene 8 protein of the Gp C porcine strain with the VP7 of Gp A porcine strain Gottfried demonstrated considerable divergence in amino acid identity (Fig. 4B). The two in-phase initiation codons, each of which precedes a hydrophobic region at the N-terminus of the gene 8 protein of the Gp C strains, are conserved in all Gp A VP7 genes examined (Estes and Cohen, 1989). A glutamine at amino acid residue 51 of the Gp AVP7 is conserved in all strains described thus far and has been identified as a cleavage site in the protein (Estes and Cohen, 1989; Stirzaker et al., 1987). This glutamine is conserved at amino acid resi- due 50 in both human and porcine gene 8 proteins (Fig. 2). The potential N-linked glycosylation sites in the gene 8 protein of the Gp C strains are also similar in location to those of the VP7 of Gp A rotavirus. In addi- tion, when the aligned amino acid sequences of the gene 8 protein of the Gp C strain Cowden and the VP7 of Gp A porcine strain Gottfried were compared, sev- eral conserved amino acid regions were observed (Fig. 6). Among these are amino acid residues 105 through 1 15 in the VP7 of Gottfried strain with amino acid resi- dues 110 through 120 of the gene 8 protein of Cowden strain; 135 through 142 in Gottfried with 140 through 147 in Cowden; 156 through 207 in Gottfried with 161 through 212 in Cowden; and 288 through 304 in Gott- fried with 293 through 309 in the Cowden strain. Of interest, all these conserved regions were located in regions of amino acid sequence in the VP7 of Gp A rotaviruses that are conserved among different sero- types (Estes and Cohen, 1989).

DISCUSSION

The gene coding assignments for Gp C rotavirus have not been established. However, the sequence analysis of individual gene segments should provide

SEQUENCE CONSERVATION OF GENE 8

166 207

ADLILNEWLCNPMDITLYYYQQTGEANKWISMGSSCTVKVCPLNTQTLGIGC

AEWILNVWFCDDMNLDIYTYEQTGIDNLWAAFGSDCDVSVCPLDTTMNGIGC 161 212

1 A B C 326

567

FIG. 6. Location of conserved amino acid regions between the Gp C Cowden gene 8 protein and the Gp A Gottfried VP7 depicted on a schematic representation of the Gp A VP7. Regions defined as divergent in the ammo acid sequence among VP7 proteins from different serotypes of Gp A rotaviruses are indicated with a solid box. The stippled line represents highly conserved amino acid sequences among Gp A VP7s. A, B, and C are antigenic sites where serotype-specific epitopes have been identified in the Gp A VP7 (Dyall-Smith eta/., 1986). The open boxes contain comparisons of amino acid sequences of the Gp A VP7 (upper line) which are conserved with amino acid sequences of the Gp C gene 8 protein (lower line). The conserved amino acrd regions between the Gp A and the Gp C proteins are numbered and identical amino acids are in bold

information relating to the encoded protein function when comparisons are made with Gp A rotaviruses (Bremont et a/., 1990). The gene segment 7, 8, or 9 of Gp A rotavirus (depending on the strain) encodes the outer capsid glycoprotein VP7 which has been identi- fied as one of the proteins of Gp A rotaviruses involved in virus neutralization (Estes and Cohen, 1989). A 39- or 37-kDa glycoprotein had previously been identified in the outer capsid of purified Gp C Cowden virus parti- cles in structural protein studies; this protein was anal- ogous in size and location to the VP7 glycoprotein of Gp A rotavirus (Bremont et al., 1988; Jiang eta/., 1990). The structure and organization of the predicted gene 8 protein of the human and porcine Gp C strains in this study resemble those of the VP7 protein of Gp A rota- virus which is consistent with a coding assignment of gene 8 for VP7 in these two Gp C strains. In addition, in vitro translation of transcripts generated from a gene 8 clone in the presence of canine microsomal mem- branes suggested that the Cowden gene 8 encodes a glycoprotein of similar size to the Gp A VP7. It is of interest that the in vitro translation products from the porcine gene 8 clone contained two bands. It is possi- ble that these two proteins resulted from initiation at the two in-frame start codons. A similar observation has been made from in vitro translation studies of the Gp A VP7 gene (Chan et a/., 1986, Stirzaker et a/., 1987). In the presence of canine microsomal mem- branes, an additional translation product from the por- cine gene 8 clone was detected with a molecular weight of 39.0 kDa, which was consistent with modifi- cation by glycosylation and with the size reported by Bremont et al. (1988) for a 39-kDa outer capsid glyco- protein identified in purified virions.

In general, among VP7s from different Gp A sero- types, the overall amino acid sequences are con- served. However, several discrete regions of variable amino acid sequence have been defined upon align- ment of the VP7 sequences from different serotypes (Gunn et al., 1985; Green et al., 1989). It is thought that these variable regions are involved in the determination of serotype specificity. Only one clustered amino acid variable region was found between the two deduced Gp C gene 8 proteins but it was located in a hydropho- bic area of the protein which, if analogous to the Gp A VP7, is not likely involved in the antigenic structure because it is removed by cleavage (Stirzaker et a/., 1987). Thus, the overall 88% homology between the human and porcine Gp C gene 8 proteins was consis- tent with shared serotype specificity. However, further studies will be required to confirm the antigenic rela- tionships among Gp C rotaviruses.

Previous studies showed the lack of genetic related- ness among rotaviruses from different groups (Bridger et al., 1986; Eiden et al,, 1986; Qian et a/., 1991; Saif and Theil, 1985; Saif, 1990). The data in this study demonstrate that although the overall nucleotide iden- tity is low, there are some structural similarities be- tween the corresponding VP7 genes of Gps A and C. Furthermore, regions of amino acid conservation be- tween the gene 8 protein of Gp C and the VP7 of Gp A rotaviruses were identified and these were located in conserved regions of the VP7 of Gp A rotaviruses. It is possible that these regions provide a common essen- tial function. For example, amino acid residues 133 to 146, which are conserved in all Gp A VP7 proteins ex- amined, were reported to share similarities with Cap-binding sites of other proteins (Estes and Cohen,

568 QIAN ET AL.

1989). A similar region of amino acid sequence was found in the gene 8 proteins of the Gp C strains in this study.

It has been proposed that human Gp C rotaviruses were originally derived from pigs through interspecies transmission (Penaranda et a/., 1989). Based upon the sequence analysis in this study, it is likely that the hu- man and porcine Gp C rotaviruses did indeed evolve from a common ancestral source. The data in this study also suggest that Gps A and C rotaviruses may have evolved from a more distant common ancestral source. Additional sequence analysis may yield further insight into evolutionary relationships.

Whether Gp C rotaviruses are only of minor impor- tance as etiologic agents of human disease and will remain so or whether they are emerging as pathogens is not clear. Molecular studies should allow the devel- opment of reagents to address a number of important epidemiological issues. In addition, molecular studies of rotaviruses that are morphologically similar but anti- genitally diverse may lead to a better understanding of mechanisms that are responsible for the important role of rotaviruses in diarrhea1 disease.

ACKNOWLEDGMENTS

We extend our appreciation to Drs. Albert Z. Kapikian and Robert M. Chanock for advice and critical review of the manuscript; to Dr. Jerry Keith for assistance with the computer analysis; to Mr. Myron Hill and Dr. Peter Collins for synthesis of primers; and to Johnna Sears for assistance in preparation of the photographs.

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BOHL, E. H., SAIF, L. J., THEIL, K. W., AGNES, A. G., and CROSS, R. F. (1982). Porcine pararotavirus: Detection, differentiation from rota- virus, and pathogenesis in gnotobiotic pigs. J. C/in. Microbial. 15, 312-319.

BONSDORFF, CH. V., and SVENSSON, L. (1988). Human serogroup C rotavirus in Finland. Stand. J. Infect. Dis. 20, 475-478.

BREMONT, M., COHEN, J., and MCCRAE, M. A. (1988). Analysis of the structural polypeptides of a porcine group C rotavirus. 1. Viral. 62, 2183-2185.

BREMONT, M., CHABANNE-VAUTHEROT, D., VANNIER, P., MCCRAE, M. A., and COHEN, J. (1990). Sequence analysis of the gene (6) encoding the major capsid protein (VP6) of group C rotavirus: Higher than expected homology to the corresponding protein from group A virus. Virology 178, 579-583.

BRIDGER, J. C., PEDLEY, S., and MCCRAE, M. A. (1986). Group C rota- viruses in humans. J. C/in. Microbial. 23, 760-763.

BROWN, D. W. G., MATHAN, M. M., MATHEW, M., MARTIN, R., BEARDS, G. M., and MATHAN, V. I. (1988). Rotavirus epidemiology in Vellore, South India: Group, subgroup, serotype, and electropherotype. 1. C/in. Microbial. 26, 241 O-2414.

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