VIROLOGY
185,853-856
(1991)
Sequence
of the VP7 Gene of Chicken K~zuo
Epidemiology
Section,
Rotavirus
Ch2 Strain of Serotype
7 Rotavirus’
NISHIKAWA, YASUTAKA HOSHINO, AND MARIO GORZIGLIA* Laboratory of Infectious Diseases, National National Institutes of Health, Bethesda, Received
July 17, 199 1; accepted
Institute Maryland August
of Allergy 20892
and Infectious
Diseases,
19, 199 1
The gene that encodes the VP7 protein of the Ch2 strain of group A chicken rotavirus (serotype G7) was sequenced, and its deduced amino acid sequence analyzed. The gene is 1067 bp in length and contains a single long open reading frame of 987 nt, capable of encoding a protein of 329 amino acids. Amino acid homology of the chicken rotavirus VP7 to the corresponding protein of rotaviruses representing each of the other twelve G serotypes ranged from 58 to 63%. In addition, the VP7 protein of the Ch2 strain contains a unique amino acid sequence in variable regions VR5 (A), VR7 (B), or VR8 (C), which is consistent with previous studies indicating that these three regions are involved in G serotype specificity. The VP7 protein had amino acid homology of 19 or 33% with that of groups B (ADRV) or C (Cowden) rotavirus strains, respectively. Attempts to produce reassortants in vivo or in vitro between the Ch2 strain and strains belonging to group A or C rotavirus were unsuccessful. The low level of nucleotide identity in the 5’ end noncoding region of the VP7 gene between the Ch2 and the group A or C rOtaVirUSeS may account for this failure. o 1x11 Academic press, Inc.
In this communication, we report the nucleotide sequence of the VP7 gene of the avian rotavirus Ch2 strain, which is classified as belonging to serotype G7. The nucleotide sequence and its deduced amino acid sequence were compared with those of the VP7 gene of selected human and animal strains belonging to the other twelve G serotypes. Avian rotaviruses have been isolated from diarrheic chickens, turkey poults, and other avian species in various parts of the world (7 I). Two avian rotaviruses (from turkey or chicken) have been characterized antigenitally (72, 73). Both avian rotaviruses possess a group antigen common to all group A rotaviruses as demonstrated by fluorescent-antibody assay or complement fixation assay (72). However, VPG-specific monoclonal antibodies which react with all group A animal rotaviruses fail to recognize avian rotaviruses. In addition, the avian rotaviruses belong to neither subgroup I nor II (74). By reciprocal cross-neutralization assay between the avian Ch2 rotavirus and the twelve rotavirus strains, the Ch2 strain is shown to be distinct from each of the other twelve G serotypes (5). The Ch2 strain of chicken rotavirus was originally isolated from the intestinal contents of a chicken with diarrhea in Northern Ireland (72). In the present study an established cell line of fetal rhesus monkey kidney cells, MA104, was used for virus propagation. Viral mRNAs were produced from single capsid particles as described previously (73, and the total mRNA was hybridized to a series of primers 18 nt in length. The sequence of the Ch2 VP7 gene transcript was determined by the dideoxy-chain termination method with
Rotaviruses are members of the Reoviridae family; they contain two outer capsid proteins, VP4 and VP7, that each separately elicit neutralizing antibodies (7-3) and that are associated with serotype specificity, the VP4 with the P serotype and the VP7 with the G serotype. In the group AVP4 serotype system three distinct P serotypes and one subtype were established by neutralization with antiserum to baculovirus recombinantexpressed VP4 protein of human rotavirus strains KU, DS-1, or 1076 (4). In the VP7 serotype system 13 distinct serotypes were established by neutralization among human, animal, and avian strains (5). The nucleic acid sequence of the VP7 gene has been determined for all but one of the thirteen G serotypes, the exception being the Ch2 strain representing G7. The nucleotide sequence comparison among different VP7 genes has allowed the recognition of serotype-specific sequences (6, 7). Recently, these discrete or variable regions have been amplified by PCR and used as radioactive probes to identify the serotype of rotavirus isolates from field studies (8-70). It has been suggested that such methodology along with the sequencing of the VP7 gene may facilitate the serotyping of new isolates. This may be important in countries where the importation of certain animal rotavirus strains is restricted and, therefore, cross-neutralization tests cannot be performed. ’ The nucleotide sequence data reported in this paper submitted to the EMBL nucleotide sequence database been assigned the Accession No. X56784. ’ To whom request for reprints should be addressed.
have been and
have
a53
0042-6822/91 $3.00 Copyright (0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
854 GGCATTTAAMCAGAAATTCTCGTTTCTCACCGACGACTAGTCTGTTM
ATG TAT AGT ACT AAA TGT ACT MC Met Tyr Ser Thr Lys Cys Thr Am
ATC TTC TGT ACA CTA TTT TTA CTC GTT TTA GM Ile Phe Cys Thr Leu Phe Leu LQU Val Leu Glu TCT TCT AAA TGT TCA GCA CAG MC Ser Ser Lys Cys Ser Ala Gin Asn A
COMMUNICATIONS TTT TTC CTT GAG ATA ATA TTC TAT GTT Phe Phe Leu Glu Ile Ile Phe Tyr Val
AAG ATG TCT AAA CTA CTT AGC TGG ATT GTT ATT GTT TGC TTA TTT GTA TTC GCC ATT Lys Met Ser Lys Leu Leu Ser Trp Ile Val Ile Val Cys Leu Phe Val Phe Ala Ile
TAT GGA ATT MC Tyr Gly Ile Am
GTA CCT ATT ACC Val Pro Ile Thr
GGT TCA ATG GAT GTA GTG CTT GCG MT TCG ACA CM Gly Ser Met Asp Val Val Leu Ala(ApnmlGln
GAT CM Asp Gin
101 17 191 47 281 77
ATT GGC CTA ACG TCG ACA TTG TGT ATT TAT TAC CCA AAA GCT GCA GAT ACT GM Ile Gly Leu Thr Ser Thr Leu Cys Ile Tyr Tyr Pro Lys Ala Ala Asp Thr Glu
ATT GCA GAT CCA GAG TGG AAG GCG ACA GTG ACG CM Ile Ala Asp Pro Glu Trp Lys Ala Thr Val Thr Gln
371 107
TTA CTA TTA ACA AAG GGA TGG CCC ACA ACG TCT GTG TAT TTA MT Leu Leu Lm Thr Lys Gly Trp Pro Thr Thr Ser Val Tyr Leu Am
GAC TTG GTG ACA Asp Leu Val Thr
TGT GAT TAC MT Cys Asp Tyr Am TGT MT Cys Am
ATT GTA TTA GCA CAT TAC ACG MC Ile Val LQU Ala His Tyr Thr Asn
GTT TGT CCA CTA MT Cys Pro Leu Asn
ACT CAG ACA Thr Gln Thr
ACA
461 137
GAT GTG GCG TTG GAC ATA TCT GAG TTG GCC GAG TTT TTG CTT TAT GAA Asp Val Ala Leu Asp Ile Ser Glu Leu Ala Glu Phe Leu Leu Tyr Glu
TGG TTA Trp Leu
551 167
ATT AAG Lys
641 197
AAA Lys
TTG Leu
731 227
TGC ATA CGA CTT AAC CCT Cys Ile Arq Leu Asn Pro
821 257
CCG ATG GTA ATA CCA AAG GTG TCG CGT ATG Pro Met Val Ile Pro Lys Val Ser Arg Met
911 287
TTA GGA ATA GGA TGT CM Leu Gly Ile Gly Cys Gln
GCT ATA ATT GAC GTA GTA GAC GGA GTG MT Ala Ile Ile Asp Val Val Asp Gly Val Am
Thr
TTG TAT Leu Tyr
TAT CM Tyr Gln
CCA ATG GAC GTA ACA CTA TAT TAT TAT CAG CAG ACG AGT GAG CCT MT Pro Met Asp Val Thr Leu Tyr Tyr Tyr Gln Gln Thr Ser Glu Pro Am
Val
AGA GM Arg Glu
GAT CCT AAA Asp Pro Lys
GM Glu
MT Am
GTT GCA ATA ATT CM Val Ala Ile Ile Gln
CGT ATG MT Arg Met Asn
TGG MG Trp Lys
AAA Lys
ACT ACT MT Thr Thr Asn
CAT AAA GTA GAT TAT ACA His Lys Val Asp Tyr Thr
MA Lys
ACT GAT ACA TTT GM Thr Asp Thr Phe Glu
GTT GCG ACG TGT AAA Val Ala Thr Cys Lys
GTG TTT TAT ACA ATA GTT GAC TAT ATA MT Val Phe Tyr Thr Ile Val Asp Tyr Ile Am
AGG TCA CTT GAT GTT TCA TCG TAT TAC TAC AGA GTA TAGACTCAAGGATAAAGGTTTGATGTGACC Arg Ser Leu Asp Val Ser Ser Tyr Tyr Tyr Arg Val
FIG. 1. Complete nucleotide correspond to the plus strand indicates the potential clevage and VR8(C) are underlined.
TGG ATA GCC ATG GGT ACT MT Trp Ile Ala Met Gly Thr(AsnfIle
GTT GGA GGG CCG GAA GTG CTT GAC ATA TCT GAG MT Val Gly Gly Pro Glu Val Leu Asp Ile Ser Glu Am
TGG TGG CM Trp Trp Gin
TTT TCA MT Phe Ser Asn
TGC ACA
ATT TTG ACG ATG TCT GM Ile Leu Thr Met Ser'Glu
ATA MC Ile Asn
MT Asn
ACC ATA ATT ACG ACA ATG TCT MG Thr Ile Ile Thr Thr Met Ser Lys
1001 317
1067 329
sequence of the strain Ch2 chicken rotavirus VP7 gene and the deduced amino acid sequence. (mRNA sense) of the dsRNA. The nucleotide sequence was determined from ssRNA transcripts, site of the predicted protein. The potential N-linked glycosilation sites are boxed. Variable regions
reverse transcriptase as described elsewhere (15). The sequence of 30 nt near the 3’ end was determined using double-stranded RNA. The Ch2 VP7 gene sequence is 1067 nt in length and includes a long open reading frame (ORF) of 987 nt capable of encoding a protein of 329 amino acids, Fig. 1. The ORF starts at nt 5 1 and ends at nt 1038 with a UAG termination codon. When compared with other rotaviruses, the VP7 of strain Ch2 has an insertion of 3 amino acids in the long hydrophobic region at the NH2terminus (amino acids 1 to 53). A second in-frame AUG is found at amino acid position 30, similar to that found in other rotavirus strains. Two potential N-linked glycosylation sites are located at amino acids 72 (corresponding to 69 in others G serotypes) and 193; among the other twelve G serotypes, a second potential N-linked glycosylation site is found only in serotype G2 (at position 190) (6). The overall amino acid sequence homology between the VP7 gene of the Ch2 strain and the VP7 gene of each of the other twelve G serotypes (strains are referenced in Fig. 2) showed a smaller degree of
CGT TCA Arg Sar
The sequence The arrowhead VRS(A), VR7(B),
amino acid identity (58 to 63%) than that observed among the other twelve G serotypes (72 to 82%). Comparison of the VP7 of the Ch2 strain with the VP7 of a representative strain of porcine group C or group B rotavirus (16, 17) showed an amino acid homology of 33 and 19%, respectively, suggesting that the VP7 gene of the Ch2 strain is more closely related to the group A rotaviruses. Comparative analysis of the VP7 gene from different rotavirus serotypes has identified nine variable regions which are divergent among the different serotypes but which are highly conserved within the same serotype (6, 7). These regions are located at or adjacent to hydrophilic amino acid domains that are thought to contain neutralization epitopes (18). Based on analysis of neutralization escape mutants selected with anti-VP7 neutralizing antibodies, three of these regions designated A, B, and C orVR5 (aa 87-l 00) VR7 (aa 142-l 52) and VR8 (aa 208-224) respectively, appear to play a major role in the delineation of VP7 serotype (18-20). The overall sequence divergence in these three variable regions, between the Ch2 strain and the other twelve G
SHORT Rotavirus Strains
COMMUNICATIONS
VR5 (4
Serotype
VR7 (W
83
Ch2
7
Wa DS-1
1 2 3 4 5 6 8 9 10 11 12 13
E CaJ NCDV 69M WI61 8223 \rM L26 L338 Cowden
(GpC)
855 VRB (Cl
145
KAADTEI ADPEWKA? a7 TES- Q- N- GDAEKN- - - D- T E- A- - - N- NSS _ _ p _ Q- S - T - _ NE-A-----TK-TETE-SN_ - -TVEEm _ _ - - SSAES- O- S-TTER- - - N- N- HEA- Q- - - DKAES- Q- G-TNEVVSLNDSGSQGPGKT-
101 - DS EN- D- D - D-l-J- DTS- D- D- N-
GYLNDG
AHYTNDVALD? 142 MKDQSLE-‘M” MKD- TSE-A MKDATLQ-M IRFVSGEE--MKDGNLQ- M MKDSTQE- M MKNANEE-M MK-DSTEE--M MR-NSSLK--M MK-DGNLQ--M VQQ- SL- -v VKSTELQ- 140 CSNI VI I P-‘:
211 QTTNTDTFEI LTMnS* zm - - - -VS- - MI AE? K- - DVN- - - VASL- - D-A- - EVATA -----A---TVADS- - DI NO- TVANA LI--P----TVATM L- - D-T- - EVATA T----A---EVAA-----G---EVATA L--DPT---EVASA T--DVA---EVANA L- - D- E- - - EVATL 204 217 D- - MNGI GCSPAST
FIG. 2. Comparison of the deduced amino acid sequence of the VP7 gene of strain Ch2 in variable regions VR5(A) (aa 87 to 101) VR7(B) (aa 142 to 152) and VR8 (aa 208 to 22 1) to those of other G serotypes. References for amino acid sequences of the VP7 of Wa (24) DS-1 and ST3 (6) RRV (i), OSU (26), NCDV (26) 69M and WI61 (27) 8223 (28) YM (29) L26 (30) L338 (6) and gene 8 of GpC Cowden strain (76).
serotypes, is consistent with the distinct serotype of the Ch2 strain (Fig. 2). It is of interest that the VR5(A), VR7(B), and VR8(C) of each of the thirteen G serotypes contain 1, 2, or 3 conserved amino acids, respectively. These conserved amino acids may play a role in maintaining the conformation of their respective VP7 epitopes. Alignment of nucleotide sequences indicates that the 3’ end noncoding region of the Ch2 strain lacks six nucleotides and the overall nucleotide homology in this region between the Ch2 strain and the other twelve G serotypes is 53 to 70%. The 5’end noncoding region of the VP7 gene of the Ch2 strain contains two extra nucleotides compared to each of the other twelve group A G serotypes (strains referenced in Fig. 2) as well as the group C rotavirus strain Cowden, Fig. 3. The overall nucleotide homology in this region among the twelve G Rotavirus Strain Ch2 Wa DS-1 RRV ST3 cm NCDV 69M WI61 8223 w L26 L336 ADRV (OpB) c,,,,&,,(GpC)
serotype strains, excluding G7, is 92 to 99%. In contrast, only 56 to 62% nucleotide homology was found in this region between the Ch2 strain and the other twelve G serotypes. In addition this region of the VP7 gene of the Cowden strain exhibits a similar level of divergence from the corresponding region of each of the group A rotaviruses analyzed in this study, i.e., 36% lack of homology. Thus, it appears that there is a dimorphism at the 5’ end of the VP7 gene of group A rotaviruses and that these alternate forms differ significantly from the corresponding region of group C or B rotaviruses. Attempts to generate reassortants in vitro between the Ch2 strain and various human (strains Wa and DSl), porcine (strain SB-1 A), rhesus monkey (strain MMU 18006) or bovine (strain UK) group A rotaviruses were unsuccessful (unpublished observations). Chicken ro-
Serotype 7 1 2 3 4 5 6 a 9 10 ,l
,2 ,3
1 IO 20 GGCATTTAAAACAGAAATTCTCGTTTCTCACCGACGACTAGTCTGTTAAAATG ___* _____ --G---G-A.T-COCTGG’mm-. _____ --AC--G-A-T--CG-CTGG’-TAG--GT---CTCT--TT--’-___= ____--GC--G-A-T--CG--TGG’-TAG--GA---CTCC--TT---___= _______ G--s G-AT- - CG- TGG’ ___= _____ --G---G-A-T--CGACTGG*-TAT--GA--TT------* _-----GC--G-A-T--CG--TOG’ ___* _____ --GC--G-A-T--CG--TGG’-TAO--GT--GT---CTCC--TT ___* -____ --G---G-A-T--CGC-TGG’-TA---OT---GT---CTCC--TT mm_* ------GC--G-AAT-.CG--TGG’-TAG--GT---CTCC--TT __________ G---G-A-T--CGACTGG’-TNG--GA---CTCC--TT---___= ----.-G- - - G-A-T- Co- TGG’ ___* ____-__ G---G-A-T--CG--TGG’-TAG--GA---CTCC--TT
so
40
so
TA-
- - GT-
- - CTCC-
- TT-
- - -
-TAG-
- GA-
- - CTCC-
- TT-
- - -
-TAG--GT---CTCC--TT---____ ---_ ----TAG-
- GT-
_ - CTCC-
- TT-
- - ----
GGCAA--m--m___-em_____
A ----
GAAGCT--C-GA--AACTG-T--*.--T
FIG. 3. Comparison of the nucleotide sequence of the 5’end noncoding the gene 9 of GpB ADRV strain (17), and the GpC Cowden strain (strains each VP7 gene of group A rotaviruses and Cowden strain are indicated homology with the VP7 nucleotide sequence of the Ch2 strain.
-----
region of the VP7 gene of strain Ch2 and those of other G serotypes. referenced in legend to Fig. 2). The two single nucleotide deletions in by an asterisk and are arbitrarily located to maximize the nucleotide
SHORT
COMMUNICATIONS
taviruses, but not human or other animal rotaviruses, have been successfully passaged serially in lo- to 12day-old embryonated hen’s eggs inoculated by the intraamniotic route. Attempts to reassort group A human or animal rotavirus strains with the avian strain were not successful in this in viva system. The failure to generate reassortants between avian and human or animal rotaviruses was unexpected, because reassortants among various group A rotaviruses can be generated readily in vitro or in vivo. It should be noted that it has not been possible to generate reassortants between group A porcine (strain OSU) and group C porcine (strain Cowden) rotaviruses (unpublished observation) or between group A and group B rotaviruses (2 1). The divergence of the nucleotide sequence at the 5’ end noncoding region of the Ch2 VP7 gene, and the corresponding region of other group A rotaviruses, may be responsible for our failure to generate reassortants. It has been suggested that this region contains signals for transcription, replication, protein-RNA interactions, and assembly of the viral genome segments (22,23). Perhaps the rotavirus polymerase complexes fail to recognize the divergent sequence of the VP7 5’ end noncoding region of heterologous rotaviruses. Solubilization and purification of these enzymes may clarify whether the VP7 5’end noncoding region of the homologous but not the heterologous group of rotaviruses is recognized in a specific manner. ACKNOWLEDGMENTS We extend our appreciation to Drs. Albert Z. Kapikian and Robert M. Chanock for advice and critical review of the manuscript, to Mr. Myron Hill and Dr. Peter Collins for synthesis of primers, and to Mr. Todd J. Heishman for editorial assistance.
REFERENCES 1. HOSHINO, Y., SERENO, M. M., MIDTHUN, K., FLORES, J., KAPIKIAN, A. Z., and CHANOCK, R. M., Proc. Nat/. Acad. Sci. USA 82, 8701-8704 (1985). 2. OFFIT, P. A., CLARK, H. F., and BLAVAT, G., J. Viral. 60, 491-496 (1986). 3. KAPIKIAN, A. Z., and CHANOCK, R. M., In “Virology” (B. N. Fields, D. M. Knipe, R. M. Chanock. M. S. Hirsch, J. L. Melnick, T. P. Monath, and B. Roizman, Eds.), 2nd ed., pp. 1353-1404. Raven Press, New York, 1990. 4. GORZIGLIA, M., LARRALDE, G., KAPIKIAN, A. Z., and CHANOCK, R. M., Proc. Nat/. Acad. Sci. USA 87, 7155-7159 (1990). 5. BROWNING, G. F., CHALMERS, R. M.. FITZGERALD, T. A., and SNODGRASS, D. R../. Viral. 72, 1059-1064 (1991).
6. GREEN, K. Y., MIDTHUN, K., GORZIGLIA, M., HOSHINO, Y., KAPIKIAN, A. Z., and CHANOCK, R. M., Virology 161, 153-l 59 (1987). 7. 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). 8. GOUVEA, V., GLASS, R. I., WOODS, P., TANIGUCHI, K.. CLARK, H. F., FORRESTER, B., and FANG, Z.-Y., 1. C/in. Microbial. 28, 276282 (1990). 9. ROHWEDDER, A., and WERCHAU, H., “Abstracts of theVlll International Congress on Virology,” pp. 25-26. Berlin, 1990. 10. FLORES, J., SEARS, J., PEREZ-SCHAEL, I., WHITE, L., GARCIA, D., ~NATA, C., and KAPIKIAN, A. Z.. J. Viral. 64, 4021-4024 (1990). 11. YASON, C. V., and SCHAT, K. A., Avian Dis. 29,499-508 (1985). 12. MCNULTY, M. S., ALLAN, G. M., TODD, D., and MCFERRAN. J. B., Arch. Viral. 61, 13-21 (1979). 13. MCNULPI, M. S., ALLAN, G. M., TODD, D., MCFERRAN, J. B., and MCCRACKEN, R. M., J. Gen. Viral. 55, 405-413 (1981). 14. THEIL, K. W., and MCCLOSKEY, C. M., J. C/in. Microbial. 27, 2846-2848 (1989). 15. GORZIGLIA, M., HOSHINO, Y., BUCKLER-WHITE, A., BLUMENTALS, I., GLASS, R. I., FLORES, J., KAPIKIAN, A. Z., and CHANOCK, R. M., Proc. Nat/. Acad. Sci. USA 83, 7039-7043 (1986). 16. QIAN. Y., JIANG, B., SAIF, L. J., SHIEN, Y. K.. ISHIMARU, Y., YAMASHITA, Y., OSETO, M., and GREEN, K. Y., Virology 182,562-569 (1991). 17. CHEN, G.-M., HUNG, T., and MACKOW, E. R., i/iro/ogy 178, 3113 15 (1990). 18. DYALL-SMITH, M. L., LAZDINS, I., TREGEAR, G. W., and HOLMES, I. H., Proc. Nat/. Acad. Sci. USA 83, 3465-3468 (1986). 19. MACKOW, E. R., SHAW, R. D., MATSUI, S. M., Vo, P. T., BENFIELD, D. A., and GREENBERG, H. B., Virology 165, 511-517 (1988). 20. TANIGUCHI, K., HOSHINO, Y., NISHIKAWA, K., GREEN, K. Y., MALOY, W. L., MORITA, Y., URASAWA, S.. KAPIKIAN, A. Z., CHANOCK, R. M., and GORZIGLIA, M., I. Viral. 62, 1870-1874 (1988). 21. YOLKEN, R., ARANGO-JARAMILLO, S.. EIDEN, J.. and VONDERFECHT, S., 1. Infect. Dis. 158, 1120-l 123 (1988). 22. ESTES, M. K.. In “Virology” (B. N. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsch, J. L. Melnick, T. P. Monath, and B. Roizman, Eds.), 2nd ed., pp. 1329-1452. Raven Press, New York, 1990. 23. ESTES, M. K., and COHEN, J., /. Microbial. Rev. 53, 41 O-449 (1989). 24. RICHARDSON, M. A., IWAMOTO. A., IKEGAMI, N , NOMOTO, A., and FURUICHI, Y., J. Viral. 51, 860-862 (1984). 25. GORZIGLIA, M., AGUIRRE, Y., HOSHINO, Y., ESPARZA, J., BLUMENTALS, I., ASKAA, J., THOMPSON, M., GLASS, R. I., KAPIKIAN, A. Z.. and CHANOCK, R. M., J. Gen. Vkol. 67, 2445-2454 (1986). 26. GLASS, R. I., KEITH, J., NAKAGOMI, O., NAKAGOMI, T., ASKAA, J., KAPIKIAN, A. Z., CHANOCK, R. M., and FLORES, J., Virology 141, 292-298 (1985). 27. GREEN, K. Y., HOSHINO, Y., and IKEGAMI, N., Virology 168, 429433 (1989). 28. Xu, L.. HARBOUR, D., and MCCRAE, M. A., /. Gen. Viral. 72, 177180 (1991). 29. RUIZ, A. M., LOPEZ, I., LOPEZ, S., ESPEJO, R. T., and ARIAS, C. F., J. Viral. 162, 4331-4336 (1988). 30. TANIGUCHI. K., URASAWA, T., KOBAYASHI, N., GORZIGLIA, M., and URASAWA, S., /. Viral. 64, 5640-5644 (1990).
185,853-856
(1991)
Sequence
of the VP7 Gene of Chicken K~zuo
Epidemiology
Section,
Rotavirus
Ch2 Strain of Serotype
7 Rotavirus’
NISHIKAWA, YASUTAKA HOSHINO, AND MARIO GORZIGLIA* Laboratory of Infectious Diseases, National National Institutes of Health, Bethesda, Received
July 17, 199 1; accepted
Institute Maryland August
of Allergy 20892
and Infectious
Diseases,
19, 199 1
The gene that encodes the VP7 protein of the Ch2 strain of group A chicken rotavirus (serotype G7) was sequenced, and its deduced amino acid sequence analyzed. The gene is 1067 bp in length and contains a single long open reading frame of 987 nt, capable of encoding a protein of 329 amino acids. Amino acid homology of the chicken rotavirus VP7 to the corresponding protein of rotaviruses representing each of the other twelve G serotypes ranged from 58 to 63%. In addition, the VP7 protein of the Ch2 strain contains a unique amino acid sequence in variable regions VR5 (A), VR7 (B), or VR8 (C), which is consistent with previous studies indicating that these three regions are involved in G serotype specificity. The VP7 protein had amino acid homology of 19 or 33% with that of groups B (ADRV) or C (Cowden) rotavirus strains, respectively. Attempts to produce reassortants in vivo or in vitro between the Ch2 strain and strains belonging to group A or C rotavirus were unsuccessful. The low level of nucleotide identity in the 5’ end noncoding region of the VP7 gene between the Ch2 and the group A or C rOtaVirUSeS may account for this failure. o 1x11 Academic press, Inc.
In this communication, we report the nucleotide sequence of the VP7 gene of the avian rotavirus Ch2 strain, which is classified as belonging to serotype G7. The nucleotide sequence and its deduced amino acid sequence were compared with those of the VP7 gene of selected human and animal strains belonging to the other twelve G serotypes. Avian rotaviruses have been isolated from diarrheic chickens, turkey poults, and other avian species in various parts of the world (7 I). Two avian rotaviruses (from turkey or chicken) have been characterized antigenitally (72, 73). Both avian rotaviruses possess a group antigen common to all group A rotaviruses as demonstrated by fluorescent-antibody assay or complement fixation assay (72). However, VPG-specific monoclonal antibodies which react with all group A animal rotaviruses fail to recognize avian rotaviruses. In addition, the avian rotaviruses belong to neither subgroup I nor II (74). By reciprocal cross-neutralization assay between the avian Ch2 rotavirus and the twelve rotavirus strains, the Ch2 strain is shown to be distinct from each of the other twelve G serotypes (5). The Ch2 strain of chicken rotavirus was originally isolated from the intestinal contents of a chicken with diarrhea in Northern Ireland (72). In the present study an established cell line of fetal rhesus monkey kidney cells, MA104, was used for virus propagation. Viral mRNAs were produced from single capsid particles as described previously (73, and the total mRNA was hybridized to a series of primers 18 nt in length. The sequence of the Ch2 VP7 gene transcript was determined by the dideoxy-chain termination method with
Rotaviruses are members of the Reoviridae family; they contain two outer capsid proteins, VP4 and VP7, that each separately elicit neutralizing antibodies (7-3) and that are associated with serotype specificity, the VP4 with the P serotype and the VP7 with the G serotype. In the group AVP4 serotype system three distinct P serotypes and one subtype were established by neutralization with antiserum to baculovirus recombinantexpressed VP4 protein of human rotavirus strains KU, DS-1, or 1076 (4). In the VP7 serotype system 13 distinct serotypes were established by neutralization among human, animal, and avian strains (5). The nucleic acid sequence of the VP7 gene has been determined for all but one of the thirteen G serotypes, the exception being the Ch2 strain representing G7. The nucleotide sequence comparison among different VP7 genes has allowed the recognition of serotype-specific sequences (6, 7). Recently, these discrete or variable regions have been amplified by PCR and used as radioactive probes to identify the serotype of rotavirus isolates from field studies (8-70). It has been suggested that such methodology along with the sequencing of the VP7 gene may facilitate the serotyping of new isolates. This may be important in countries where the importation of certain animal rotavirus strains is restricted and, therefore, cross-neutralization tests cannot be performed. ’ The nucleotide sequence data reported in this paper submitted to the EMBL nucleotide sequence database been assigned the Accession No. X56784. ’ To whom request for reprints should be addressed.
have been and
have
a53
0042-6822/91 $3.00 Copyright (0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
854 GGCATTTAAMCAGAAATTCTCGTTTCTCACCGACGACTAGTCTGTTM
ATG TAT AGT ACT AAA TGT ACT MC Met Tyr Ser Thr Lys Cys Thr Am
ATC TTC TGT ACA CTA TTT TTA CTC GTT TTA GM Ile Phe Cys Thr Leu Phe Leu LQU Val Leu Glu TCT TCT AAA TGT TCA GCA CAG MC Ser Ser Lys Cys Ser Ala Gin Asn A
COMMUNICATIONS TTT TTC CTT GAG ATA ATA TTC TAT GTT Phe Phe Leu Glu Ile Ile Phe Tyr Val
AAG ATG TCT AAA CTA CTT AGC TGG ATT GTT ATT GTT TGC TTA TTT GTA TTC GCC ATT Lys Met Ser Lys Leu Leu Ser Trp Ile Val Ile Val Cys Leu Phe Val Phe Ala Ile
TAT GGA ATT MC Tyr Gly Ile Am
GTA CCT ATT ACC Val Pro Ile Thr
GGT TCA ATG GAT GTA GTG CTT GCG MT TCG ACA CM Gly Ser Met Asp Val Val Leu Ala(ApnmlGln
GAT CM Asp Gin
101 17 191 47 281 77
ATT GGC CTA ACG TCG ACA TTG TGT ATT TAT TAC CCA AAA GCT GCA GAT ACT GM Ile Gly Leu Thr Ser Thr Leu Cys Ile Tyr Tyr Pro Lys Ala Ala Asp Thr Glu
ATT GCA GAT CCA GAG TGG AAG GCG ACA GTG ACG CM Ile Ala Asp Pro Glu Trp Lys Ala Thr Val Thr Gln
371 107
TTA CTA TTA ACA AAG GGA TGG CCC ACA ACG TCT GTG TAT TTA MT Leu Leu Lm Thr Lys Gly Trp Pro Thr Thr Ser Val Tyr Leu Am
GAC TTG GTG ACA Asp Leu Val Thr
TGT GAT TAC MT Cys Asp Tyr Am TGT MT Cys Am
ATT GTA TTA GCA CAT TAC ACG MC Ile Val LQU Ala His Tyr Thr Asn
GTT TGT CCA CTA MT Cys Pro Leu Asn
ACT CAG ACA Thr Gln Thr
ACA
461 137
GAT GTG GCG TTG GAC ATA TCT GAG TTG GCC GAG TTT TTG CTT TAT GAA Asp Val Ala Leu Asp Ile Ser Glu Leu Ala Glu Phe Leu Leu Tyr Glu
TGG TTA Trp Leu
551 167
ATT AAG Lys
641 197
AAA Lys
TTG Leu
731 227
TGC ATA CGA CTT AAC CCT Cys Ile Arq Leu Asn Pro
821 257
CCG ATG GTA ATA CCA AAG GTG TCG CGT ATG Pro Met Val Ile Pro Lys Val Ser Arg Met
911 287
TTA GGA ATA GGA TGT CM Leu Gly Ile Gly Cys Gln
GCT ATA ATT GAC GTA GTA GAC GGA GTG MT Ala Ile Ile Asp Val Val Asp Gly Val Am
Thr
TTG TAT Leu Tyr
TAT CM Tyr Gln
CCA ATG GAC GTA ACA CTA TAT TAT TAT CAG CAG ACG AGT GAG CCT MT Pro Met Asp Val Thr Leu Tyr Tyr Tyr Gln Gln Thr Ser Glu Pro Am
Val
AGA GM Arg Glu
GAT CCT AAA Asp Pro Lys
GM Glu
MT Am
GTT GCA ATA ATT CM Val Ala Ile Ile Gln
CGT ATG MT Arg Met Asn
TGG MG Trp Lys
AAA Lys
ACT ACT MT Thr Thr Asn
CAT AAA GTA GAT TAT ACA His Lys Val Asp Tyr Thr
MA Lys
ACT GAT ACA TTT GM Thr Asp Thr Phe Glu
GTT GCG ACG TGT AAA Val Ala Thr Cys Lys
GTG TTT TAT ACA ATA GTT GAC TAT ATA MT Val Phe Tyr Thr Ile Val Asp Tyr Ile Am
AGG TCA CTT GAT GTT TCA TCG TAT TAC TAC AGA GTA TAGACTCAAGGATAAAGGTTTGATGTGACC Arg Ser Leu Asp Val Ser Ser Tyr Tyr Tyr Arg Val
FIG. 1. Complete nucleotide correspond to the plus strand indicates the potential clevage and VR8(C) are underlined.
TGG ATA GCC ATG GGT ACT MT Trp Ile Ala Met Gly Thr(AsnfIle
GTT GGA GGG CCG GAA GTG CTT GAC ATA TCT GAG MT Val Gly Gly Pro Glu Val Leu Asp Ile Ser Glu Am
TGG TGG CM Trp Trp Gin
TTT TCA MT Phe Ser Asn
TGC ACA
ATT TTG ACG ATG TCT GM Ile Leu Thr Met Ser'Glu
ATA MC Ile Asn
MT Asn
ACC ATA ATT ACG ACA ATG TCT MG Thr Ile Ile Thr Thr Met Ser Lys
1001 317
1067 329
sequence of the strain Ch2 chicken rotavirus VP7 gene and the deduced amino acid sequence. (mRNA sense) of the dsRNA. The nucleotide sequence was determined from ssRNA transcripts, site of the predicted protein. The potential N-linked glycosilation sites are boxed. Variable regions
reverse transcriptase as described elsewhere (15). The sequence of 30 nt near the 3’ end was determined using double-stranded RNA. The Ch2 VP7 gene sequence is 1067 nt in length and includes a long open reading frame (ORF) of 987 nt capable of encoding a protein of 329 amino acids, Fig. 1. The ORF starts at nt 5 1 and ends at nt 1038 with a UAG termination codon. When compared with other rotaviruses, the VP7 of strain Ch2 has an insertion of 3 amino acids in the long hydrophobic region at the NH2terminus (amino acids 1 to 53). A second in-frame AUG is found at amino acid position 30, similar to that found in other rotavirus strains. Two potential N-linked glycosylation sites are located at amino acids 72 (corresponding to 69 in others G serotypes) and 193; among the other twelve G serotypes, a second potential N-linked glycosylation site is found only in serotype G2 (at position 190) (6). The overall amino acid sequence homology between the VP7 gene of the Ch2 strain and the VP7 gene of each of the other twelve G serotypes (strains are referenced in Fig. 2) showed a smaller degree of
CGT TCA Arg Sar
The sequence The arrowhead VRS(A), VR7(B),
amino acid identity (58 to 63%) than that observed among the other twelve G serotypes (72 to 82%). Comparison of the VP7 of the Ch2 strain with the VP7 of a representative strain of porcine group C or group B rotavirus (16, 17) showed an amino acid homology of 33 and 19%, respectively, suggesting that the VP7 gene of the Ch2 strain is more closely related to the group A rotaviruses. Comparative analysis of the VP7 gene from different rotavirus serotypes has identified nine variable regions which are divergent among the different serotypes but which are highly conserved within the same serotype (6, 7). These regions are located at or adjacent to hydrophilic amino acid domains that are thought to contain neutralization epitopes (18). Based on analysis of neutralization escape mutants selected with anti-VP7 neutralizing antibodies, three of these regions designated A, B, and C orVR5 (aa 87-l 00) VR7 (aa 142-l 52) and VR8 (aa 208-224) respectively, appear to play a major role in the delineation of VP7 serotype (18-20). The overall sequence divergence in these three variable regions, between the Ch2 strain and the other twelve G
SHORT Rotavirus Strains
COMMUNICATIONS
VR5 (4
Serotype
VR7 (W
83
Ch2
7
Wa DS-1
1 2 3 4 5 6 8 9 10 11 12 13
E CaJ NCDV 69M WI61 8223 \rM L26 L338 Cowden
(GpC)
855 VRB (Cl
145
KAADTEI ADPEWKA? a7 TES- Q- N- GDAEKN- - - D- T E- A- - - N- NSS _ _ p _ Q- S - T - _ NE-A-----TK-TETE-SN_ - -TVEEm _ _ - - SSAES- O- S-TTER- - - N- N- HEA- Q- - - DKAES- Q- G-TNEVVSLNDSGSQGPGKT-
101 - DS EN- D- D - D-l-J- DTS- D- D- N-
GYLNDG
AHYTNDVALD? 142 MKDQSLE-‘M” MKD- TSE-A MKDATLQ-M IRFVSGEE--MKDGNLQ- M MKDSTQE- M MKNANEE-M MK-DSTEE--M MR-NSSLK--M MK-DGNLQ--M VQQ- SL- -v VKSTELQ- 140 CSNI VI I P-‘:
211 QTTNTDTFEI LTMnS* zm - - - -VS- - MI AE? K- - DVN- - - VASL- - D-A- - EVATA -----A---TVADS- - DI NO- TVANA LI--P----TVATM L- - D-T- - EVATA T----A---EVAA-----G---EVATA L--DPT---EVASA T--DVA---EVANA L- - D- E- - - EVATL 204 217 D- - MNGI GCSPAST
FIG. 2. Comparison of the deduced amino acid sequence of the VP7 gene of strain Ch2 in variable regions VR5(A) (aa 87 to 101) VR7(B) (aa 142 to 152) and VR8 (aa 208 to 22 1) to those of other G serotypes. References for amino acid sequences of the VP7 of Wa (24) DS-1 and ST3 (6) RRV (i), OSU (26), NCDV (26) 69M and WI61 (27) 8223 (28) YM (29) L26 (30) L338 (6) and gene 8 of GpC Cowden strain (76).
serotypes, is consistent with the distinct serotype of the Ch2 strain (Fig. 2). It is of interest that the VR5(A), VR7(B), and VR8(C) of each of the thirteen G serotypes contain 1, 2, or 3 conserved amino acids, respectively. These conserved amino acids may play a role in maintaining the conformation of their respective VP7 epitopes. Alignment of nucleotide sequences indicates that the 3’ end noncoding region of the Ch2 strain lacks six nucleotides and the overall nucleotide homology in this region between the Ch2 strain and the other twelve G serotypes is 53 to 70%. The 5’end noncoding region of the VP7 gene of the Ch2 strain contains two extra nucleotides compared to each of the other twelve group A G serotypes (strains referenced in Fig. 2) as well as the group C rotavirus strain Cowden, Fig. 3. The overall nucleotide homology in this region among the twelve G Rotavirus Strain Ch2 Wa DS-1 RRV ST3 cm NCDV 69M WI61 8223 w L26 L336 ADRV (OpB) c,,,,&,,(GpC)
serotype strains, excluding G7, is 92 to 99%. In contrast, only 56 to 62% nucleotide homology was found in this region between the Ch2 strain and the other twelve G serotypes. In addition this region of the VP7 gene of the Cowden strain exhibits a similar level of divergence from the corresponding region of each of the group A rotaviruses analyzed in this study, i.e., 36% lack of homology. Thus, it appears that there is a dimorphism at the 5’ end of the VP7 gene of group A rotaviruses and that these alternate forms differ significantly from the corresponding region of group C or B rotaviruses. Attempts to generate reassortants in vitro between the Ch2 strain and various human (strains Wa and DSl), porcine (strain SB-1 A), rhesus monkey (strain MMU 18006) or bovine (strain UK) group A rotaviruses were unsuccessful (unpublished observations). Chicken ro-
Serotype 7 1 2 3 4 5 6 a 9 10 ,l
,2 ,3
1 IO 20 GGCATTTAAAACAGAAATTCTCGTTTCTCACCGACGACTAGTCTGTTAAAATG ___* _____ --G---G-A.T-COCTGG’mm-. _____ --AC--G-A-T--CG-CTGG’-TAG--GT---CTCT--TT--’-___= ____--GC--G-A-T--CG--TGG’-TAG--GA---CTCC--TT---___= _______ G--s G-AT- - CG- TGG’ ___= _____ --G---G-A-T--CGACTGG*-TAT--GA--TT------* _-----GC--G-A-T--CG--TOG’ ___* _____ --GC--G-A-T--CG--TGG’-TAO--GT--GT---CTCC--TT ___* -____ --G---G-A-T--CGC-TGG’-TA---OT---GT---CTCC--TT mm_* ------GC--G-AAT-.CG--TGG’-TAG--GT---CTCC--TT __________ G---G-A-T--CGACTGG’-TNG--GA---CTCC--TT---___= ----.-G- - - G-A-T- Co- TGG’ ___* ____-__ G---G-A-T--CG--TGG’-TAG--GA---CTCC--TT
so
40
so
TA-
- - GT-
- - CTCC-
- TT-
- - -
-TAG-
- GA-
- - CTCC-
- TT-
- - -
-TAG--GT---CTCC--TT---____ ---_ ----TAG-
- GT-
_ - CTCC-
- TT-
- - ----
GGCAA--m--m___-em_____
A ----
GAAGCT--C-GA--AACTG-T--*.--T
FIG. 3. Comparison of the nucleotide sequence of the 5’end noncoding the gene 9 of GpB ADRV strain (17), and the GpC Cowden strain (strains each VP7 gene of group A rotaviruses and Cowden strain are indicated homology with the VP7 nucleotide sequence of the Ch2 strain.
-----
region of the VP7 gene of strain Ch2 and those of other G serotypes. referenced in legend to Fig. 2). The two single nucleotide deletions in by an asterisk and are arbitrarily located to maximize the nucleotide
SHORT
COMMUNICATIONS
taviruses, but not human or other animal rotaviruses, have been successfully passaged serially in lo- to 12day-old embryonated hen’s eggs inoculated by the intraamniotic route. Attempts to reassort group A human or animal rotavirus strains with the avian strain were not successful in this in viva system. The failure to generate reassortants between avian and human or animal rotaviruses was unexpected, because reassortants among various group A rotaviruses can be generated readily in vitro or in vivo. It should be noted that it has not been possible to generate reassortants between group A porcine (strain OSU) and group C porcine (strain Cowden) rotaviruses (unpublished observation) or between group A and group B rotaviruses (2 1). The divergence of the nucleotide sequence at the 5’ end noncoding region of the Ch2 VP7 gene, and the corresponding region of other group A rotaviruses, may be responsible for our failure to generate reassortants. It has been suggested that this region contains signals for transcription, replication, protein-RNA interactions, and assembly of the viral genome segments (22,23). Perhaps the rotavirus polymerase complexes fail to recognize the divergent sequence of the VP7 5’ end noncoding region of heterologous rotaviruses. Solubilization and purification of these enzymes may clarify whether the VP7 5’end noncoding region of the homologous but not the heterologous group of rotaviruses is recognized in a specific manner. ACKNOWLEDGMENTS We extend our appreciation to Drs. Albert Z. Kapikian and Robert M. Chanock for advice and critical review of the manuscript, to Mr. Myron Hill and Dr. Peter Collins for synthesis of primers, and to Mr. Todd J. Heishman for editorial assistance.
REFERENCES 1. HOSHINO, Y., SERENO, M. M., MIDTHUN, K., FLORES, J., KAPIKIAN, A. Z., and CHANOCK, R. M., Proc. Nat/. Acad. Sci. USA 82, 8701-8704 (1985). 2. OFFIT, P. A., CLARK, H. F., and BLAVAT, G., J. Viral. 60, 491-496 (1986). 3. KAPIKIAN, A. Z., and CHANOCK, R. M., In “Virology” (B. N. Fields, D. M. Knipe, R. M. Chanock. M. S. Hirsch, J. L. Melnick, T. P. Monath, and B. Roizman, Eds.), 2nd ed., pp. 1353-1404. Raven Press, New York, 1990. 4. GORZIGLIA, M., LARRALDE, G., KAPIKIAN, A. Z., and CHANOCK, R. M., Proc. Nat/. Acad. Sci. USA 87, 7155-7159 (1990). 5. BROWNING, G. F., CHALMERS, R. M.. FITZGERALD, T. A., and SNODGRASS, D. R../. Viral. 72, 1059-1064 (1991).
6. GREEN, K. Y., MIDTHUN, K., GORZIGLIA, M., HOSHINO, Y., KAPIKIAN, A. Z., and CHANOCK, R. M., Virology 161, 153-l 59 (1987). 7. 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). 8. GOUVEA, V., GLASS, R. I., WOODS, P., TANIGUCHI, K.. CLARK, H. F., FORRESTER, B., and FANG, Z.-Y., 1. C/in. Microbial. 28, 276282 (1990). 9. ROHWEDDER, A., and WERCHAU, H., “Abstracts of theVlll International Congress on Virology,” pp. 25-26. Berlin, 1990. 10. FLORES, J., SEARS, J., PEREZ-SCHAEL, I., WHITE, L., GARCIA, D., ~NATA, C., and KAPIKIAN, A. Z.. J. Viral. 64, 4021-4024 (1990). 11. YASON, C. V., and SCHAT, K. A., Avian Dis. 29,499-508 (1985). 12. MCNULTY, M. S., ALLAN, G. M., TODD, D., and MCFERRAN. J. B., Arch. Viral. 61, 13-21 (1979). 13. MCNULPI, M. S., ALLAN, G. M., TODD, D., MCFERRAN, J. B., and MCCRACKEN, R. M., J. Gen. Viral. 55, 405-413 (1981). 14. THEIL, K. W., and MCCLOSKEY, C. M., J. C/in. Microbial. 27, 2846-2848 (1989). 15. GORZIGLIA, M., HOSHINO, Y., BUCKLER-WHITE, A., BLUMENTALS, I., GLASS, R. I., FLORES, J., KAPIKIAN, A. Z., and CHANOCK, R. M., Proc. Nat/. Acad. Sci. USA 83, 7039-7043 (1986). 16. QIAN. Y., JIANG, B., SAIF, L. J., SHIEN, Y. K.. ISHIMARU, Y., YAMASHITA, Y., OSETO, M., and GREEN, K. Y., Virology 182,562-569 (1991). 17. CHEN, G.-M., HUNG, T., and MACKOW, E. R., i/iro/ogy 178, 3113 15 (1990). 18. DYALL-SMITH, M. L., LAZDINS, I., TREGEAR, G. W., and HOLMES, I. H., Proc. Nat/. Acad. Sci. USA 83, 3465-3468 (1986). 19. MACKOW, E. R., SHAW, R. D., MATSUI, S. M., Vo, P. T., BENFIELD, D. A., and GREENBERG, H. B., Virology 165, 511-517 (1988). 20. TANIGUCHI, K., HOSHINO, Y., NISHIKAWA, K., GREEN, K. Y., MALOY, W. L., MORITA, Y., URASAWA, S.. KAPIKIAN, A. Z., CHANOCK, R. M., and GORZIGLIA, M., I. Viral. 62, 1870-1874 (1988). 21. YOLKEN, R., ARANGO-JARAMILLO, S.. EIDEN, J.. and VONDERFECHT, S., 1. Infect. Dis. 158, 1120-l 123 (1988). 22. ESTES, M. K.. In “Virology” (B. N. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsch, J. L. Melnick, T. P. Monath, and B. Roizman, Eds.), 2nd ed., pp. 1329-1452. Raven Press, New York, 1990. 23. ESTES, M. K., and COHEN, J., /. Microbial. Rev. 53, 41 O-449 (1989). 24. RICHARDSON, M. A., IWAMOTO. A., IKEGAMI, N , NOMOTO, A., and FURUICHI, Y., J. Viral. 51, 860-862 (1984). 25. GORZIGLIA, M., AGUIRRE, Y., HOSHINO, Y., ESPARZA, J., BLUMENTALS, I., ASKAA, J., THOMPSON, M., GLASS, R. I., KAPIKIAN, A. Z.. and CHANOCK, R. M., J. Gen. Vkol. 67, 2445-2454 (1986). 26. GLASS, R. I., KEITH, J., NAKAGOMI, O., NAKAGOMI, T., ASKAA, J., KAPIKIAN, A. Z., CHANOCK, R. M., and FLORES, J., Virology 141, 292-298 (1985). 27. GREEN, K. Y., HOSHINO, Y., and IKEGAMI, N., Virology 168, 429433 (1989). 28. Xu, L.. HARBOUR, D., and MCCRAE, M. A., /. Gen. Viral. 72, 177180 (1991). 29. RUIZ, A. M., LOPEZ, I., LOPEZ, S., ESPEJO, R. T., and ARIAS, C. F., J. Viral. 162, 4331-4336 (1988). 30. TANIGUCHI. K., URASAWA, T., KOBAYASHI, N., GORZIGLIA, M., and URASAWA, S., /. Viral. 64, 5640-5644 (1990).
