Vol. 64, No. 1
JOURNAL OF VIROLOGY, Jan. 1990, p. 411-413
0022-538X/90/010411-03$02.00/0 Copyright © 1990, American Society for Microbiology
Lack of Cosegregation of the Subgroup II Antigens and 6 in Porcine Rotaviruses
on
Genes 2
LENNART SVENSSON, 12* LUIS PADILLA-NORIEGA, 12'3 KOKI TANIGUCHI,4 AND HARRY B. GREENBERG' 2 Department of Medicine and Medical Microbiology, Stanford University School of Medicine, Stanford, California 943051; Palo Alto Veterans Administration Medical Center, Palo Alto, California 943042*; Unidad de Investigacion Clinica en Enfermedades Infecciosas y Parasitarias, Instituto Mexicano del Seguro Social, Mexico City 04000, Mexico3; and
Department of Hygiene, Sapporo Medical College, Chuo-Ku, Sapporo, Japan4 Received 12 June 1989/Accepted 13 September 1989
The rotavirus subgroup I and II specificities associated with gene 2 and 6 products (vp2 and vp6, respectively) were shown not to cosegregate in a number of porcine rotavirus strains. The porcine OSU rotavirus strain and OSU-vp7-like strains were all found to possess a subgroup H-specific region on vp2 and a subgroup I-specific region on vp6. Of interest is the observation that the subgroup II-specific epitope on vp2 appears to be present only in human and porcine rotavirus strains, suggesting a possible human-pig ancestral lineage for gene 2.
vp2 is present exclusively in human and porcine rotavirus strains. The rotavirus strains analyzed in the present study are shown in Table 1. For immunologic characterization, radioimmune precipitation assay, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) assay were used. Radioimmune precipitation assays and ELISAs were performed essentially as described previously (9, 11). IF studies were performed with cover slips containing MA-104 cells that were infected with trypsin-activated rotavirus. After a 30-min incubation with MAbs, the immune reactivity was visualized with an antimouse biotin-streptavidin fluorescein system from Amersham Corp. Four MAbs were used in the study. A previously undescribed vp2 group A-specific MAb designated 6E8 was isolated from a mouse immunized with Wa rotavirus. This MAb reacts with all group A rotaviruses tested. vp6 SG Iand II-specific MAbs (255/60 and 631/9, respectively), previously produced and characterized by Greenberg et al. (4), and a vp2 SG II-specific MAb, YO-60, produced and characterized by Taniguchi et al. (14), were also used. The specificity of YO-60 for human vp6 SG II strains was confirmed before the study by analyzing more than 200 human rotavirus strains, and a complete correlation with SG II strains was seen. The analysis of the SG II vp2 reactivity patterns of rotaviruses derived from various animal species is summarized in Table 1. For comparison, the vp6 SG I and II specificities are also shown. For the strains (J1515, 572, 566, 7325, 451, 7360, and EE TC16) that had not previously been assigned a vp6 subgroup classification, a ratio (SG I/SG II or SG II/SG I) of >3 was used (10). The vp2 group-reactive MAb 6E8 reacted with all human and animal strains except for a variety of isolates from chickens and turkeys. As expected, the SG II vp6 MAb 631/9 reacted with the human strains Wa and WI 61, the porcine strain Gottfried, and J1515 (a Gottfried-vp7-like porcine strain). All other strains except those isolated from avian species reacted with the vp6directed SG I MAb 255/60. The SG II vp2-specific MAb YO-60 reacted with all human SG II strains but not with the human SG I strains DS-1 and 69M. More interestingly, YO-60 also reacted with all nine porcine rotavirus strains analyzed, independent of vp6 subgroup specificity. Several
Rotavirus subgroup (SG) I and II specificity has been localized to epitopes or regions on both vp2 and vp6 (4, 14). Genetic and immunologic characterizations have shown that the subgroup specificities of vp2 and vp6 are encoded by genes 2 and 6, respectively (2, 8). Greenberg et al. (4) used hybridoma technology to produce vp6 SG I- and II-specific monoclonal antibodies (MAbs). These antibodies have been used successfully to determine the subgroups of animal and human strains (4, 12). The use of these and other subgroupspecific MAbs has also shown that subgroup specificity is more complex than originally thought. Examples of this antigenic complexity include the isolation of human (10) and animal rotaviruses that do not react with SG I or II MAbs (2, 4) and the detection of a virus strain that bears both subgroup specificities on vp6 (6). However, the great majority of the serogroup A rotaviruses studied to date have been classified into one of the two subgroups. The majority of human strains appear to be SG II viruses, while all typed animal strains, except for one rabbit (13) and three pig (6) rotavirus strains, belong to SG I. The recent observations that vp2 contains an antigenic domain which, in virtually all human strains tested to date (14), cosegregates with the SG II-specific domain on vp6 and that serotype-specific epitopes reside on two different proteins, vp4 and vp7 (1, 5, 7), make antigenic classification even more complex. The realization that both type and subgroup antigenic specificities are encoded by more than one gene begs the question of how frequently separate genes coding for proteins with type or subgroup specificity cosegregate in the field. In a recent study, it was reported that genes 4 and 9, which encode type-specific antigens vp4 and vp7, respectively, segregate independently in nature (7). In this paper, we present evidence that the subgroup specificities of rotavirus also segregate independently in nature. In a previous study with human strains, the SG II specificities on vp2 and vp6 had been found to cosegregate (14). In the current study, we observed that in wild-type porcine strains, the two specificities are clearly not linked and can segregate independently, presumably by gene reassortment that occurs in the field. Furthermore, we show that among several animal rotavirus strains, a unique and highly conserved SG II-specific epitope *
on
Corresponding author. 411
412
J. VIROL.
NOTES
TABLE 1. Reactivity of human and animal rotaviruses with various subgroup-specific MAbs as demonstrated by IF and ELISA
A B C
D
Assay (optical density at 450 nm)b for: Rotavirus strain
Wa
WI 61 Price DS-1 69M Gottfried J1515 OSU 572 566 7325 451 7360 EE TC16 SA-11 RRV
vp2 with
Source'
SrcaMAb: Human Human Human Human Human
Pig Pig Pig Pig Pig Pig Pig Pig Pig Vervet monkey Rhesus monkey SA-11/OSU 66d SA-11/OSU 86d SA-11/OSU 43d Calf NCDV Calf UK Lamb Lamb Foal Foal 82/309 F Rabbit TC 1039 Cat K9 1037 Dog Ch2 Chicken Tyl Turkey AR NA-1 Bird AR 2 Bird AR 1294 Bird AR 428 Bird AR B Bird
vp6 with MAb:
6E8
YO-60
631/9
255/60
+ (0.850)
+ (0.835) + (0.472) + (0.405) - (0.007) - (0.017) + (0.626) + (0.895) + (0.912) + (0.412) + (0.426) + (0.668) + (0.547) + (0.776) + (0.646) - (0.045) - (0.039) + (0.493) - (0.051) + (0.532) - (0.036) - (0.025) - (0.041) - (0.036) - (0.058) - (0.048) - (0.006) - (0.020) - (0.016) - (0.012) - (0.038) - (0.018) - (0.010) - (0.041)
+ + + +
-
+C
_
+ (0.399) + (0.451) + (0.339) + (0.279)
+ (0.657) + (0.845) + (0.454) + (0.391) + (0.329) + (0.522) + (0.534) + (0.661) + (0.599) + (0.512) + (0.788) + (0.538) + (0.714) + (0.701) + (1.205) + (0.625) + (0.531) + (0.712) + (0.693) + (0.813) + (0.625) - (0.009) - (0.015) - (0.007) - (0.027) - (0.008) - (0.015) - (0.026)
-
-
-
3vp4 p3V p3
vp>
469k
+ +
+ +C +C +C +C +C +C
-
+ + +
-
-
492k
-
446k
Vp60.
*
S
430k
+ + + + + + + + +
FIG. 1. Immunoprecipitation of a [35S]methionine-labeled porcine (OSU strain) rotavirus lysate with SG I- and II-specific MAbs. Lane A, OSU-infected cell lysate; lane B, OSU-infected cell lysate and SG II vp6-specific MAb 631/9; lane C, OSU-infected cell lysate and SG I vp6-specific MAb 255/60; lane D, OSU-infected cell lysate and SG II vp2-specific MAb YO-60. Molecular mass markers (Amersham) are shown on the right as follows: phosphorylase b, 92 kilodaltons; bovine serum albumin, 69 kilodaltons; ovalbumin, 46 kilodaltons; carbonic anhydrase, 30 kilodaltons.
-
-
-
-
a Strains J1515 and EE TC16 were donated by Linda Saif (Ohio State University); strains 572, 566, 7325, 451, and 7360 were donated by Prem Paul (Iowa State University); the avian strains were donated by K. Nagaraga (University of Minnesota); and the lamb and foal strains were donated by Graham Beards. b + and - signify IF and/or ELISA result. Optical densities are mean values of three ELISAs. Cutoff value, 0.1. c Subgroup designation was established by using a SG I/SG II or SG II/ SG I ratio of >3 (10). d Reassortant: 66, SA-11 genes 1, 3, 5, and 10; 86, SA-11 genes 1, 2, 3, and 5; and 43, SA-11 genes 3, 5, 8, and 9.
porcine strains, including OSU, contained a SG I-reactive epitope on vp6 but a SG II region on vp2. This reactivity is in clear contrast to the vp6 SG I human strains DS-1 and 69M, which were not recognized by the vp2 SG II-specific MAb YO-60. Radioimmune precipitation and IF assays showed that the OSU rotavirus strain has two different subgroup specificities located on different proteins (Fig. 1; Table 1). The SG I specificity is located on vp6, as demonstrated by reactivity with MAb 255/60, and the SG II reactivity is located on vp2. To determine if the SG II reactivity of the OSU strain cosegregated with gene 2 or gene 6, reassortment libraries were constructed from MA-104 cells coinfected with OSU and SA-11 (serotype 3, subgroup I). Each gene-reassortant strain was plaque purified three times before the RNA profile was analyzed (data not shown). RNA profile, ELISA, and IF studies of the reassortants confirmed that the reactivity of
YO-60 was linked to the vp2 encoded by gene 2 (2, 8, 14) of the OSU strain (Table 1). Hoshino et al. (6), after analyzing 49 rotavirus strains with six different vp6-specific MAbs, suggested that human rotavirus may have two ancestral lineages: one to human SG I, vervet monkey, and cow rotavirus strains, and the other to OI human and pig rotavirus strains. The SG II human-pig SG ancestral lineage is further supported by our vp2 characterization, which shows that only pig and human rotavirus strains bear SG II-specific antigenic regions on vp2. Gorziglia et al. (3) compared the amino acid sequence of the vp6 of porcine rotavirus Gottfried strain with that of human rotavirus Wa strain vp6 and found 98% homology. At present, no such comparative vp2 amino acid sequence is available for these strains. Such information will be valuable for further studies of the evolution of group A rotaviruses. We predict a high degree of conservation of the vp2 amino acid sequence between human and porcine strains. The antigenic similarity between pig and human rotaviruses with respect to vp2, vp6, and vp7 shows that the pig-human ancestral lineage is worth further attention. Furthermore, the demonstration that subgroup genes as well as genes encoding neutralization antigens do not cosegregate emphasizes again the importance of carefully defining a serologic classification scheme for rotavirus. Since these studies clearly demonstrate that the antigenic epitope on vp2 defined by MAb YO-60 does not invariably cosegregate with the SG II domain on vp6, it is not reasonable to continue to label this epitope a SG II-specific region. Perhaps the subgroup label should be exclusively reserved for antigens on vp6, while antigenic regions on other proteins could be described with
NOTES
VOL. 64, 1990
the protein designation followed by an antigen designation (such as vp2a, vp2b, etc.). We are grateful to Graham Beards (Regional Virus Laboratory, Birmingham Hospital, Birmingham, United Kingdom), Linda Saif (Ohio State University, Columbus), Prem Paul (Iowa State University, Ames), and K. Nagaraga (University of Minnesota, Minneapolis) for providing various rotavirus strains. This work was supported by grants K88-16R-8508 and K8816F-8490-01 from the Swedish Medical Research Council.
7.
LITERATURE CITED Bastardo, J. W., J. L. McKimm-Breschkin, S. Sonza, L. D. Mercer, and I. H. Holmes. 1981. Preparation and characterization of antisera to electrophoretically purified SAl virus polypeptides. Infect. Immun. 34:641-647. Estes, M. K., E. L. Palmer, and J. F. Obijeski. 1983. Rotaviruses: a review. Curr. Top. Microbiol. Immunol. 105:123-184. Gorziglia, M., Y. Hoshino, K. Nishikawa, W. L. Maloy, R. W. Jones, A. Z. Kapikian, and R. M. Chanock. 1988. Comparative sequence analysis of the genomic segment 6 of four rotaviruses each with a different subgroup specificity. J. Gen. Virol. 69: 1659-1669. Greenberg, H., V. McAuliffe, J. Valdesuso, R. Wyatt, J. Flores, A. Kalica, Y. Hoshino, and N. Singh. 1983. Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies. Infect. Immun. 39:91-99. Greenberg, H. B., J. Valdesuso, K. van Wyke, K. Midthun, M. Walsh, V. McAuliffe, R. G. Wyatt, A. R. Kalica, J. Flores, and Y. Hoshino. 1983. Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus. J. Virol. 47:267-275. Hoshino, Y., M. Gorziglia, J. Valdesuso, J. Askaa, R. I. Glass,
9.
1.
2. 3.
4.
5.
6.
8.
10.
11.
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and A. Z. Kapikian. 1987. An equine rotavirus (FI-14 strain) which bears both subgroup I and subgroup II specificities on its vp6. Virology 157:488-496. Hoshino, Y., M. M. Sereno, K. Midthun, J. Flores, A. Z. Kapikian, and R. M. Chanock. 1985. Independent segregation of two antigenic specificities (vp3 and vp7) involved in neutralization of rotavirus infectivity. Proc. Natl. Acad. Sci. USA 82: 8701-8704. Kalica, A. R., H. B. Greenberg, R. G. Wyatt, J. Flores, M. M. Sereno, A. Z. Kapikian, and R. M. Chanock. 1981. Genes of human (strain Wa) and bovine (strain UK) rotaviruses that code for neutralization and subgroup antigens. Virology 112:385-390. Shaw, R. D., D. L. Stoner-Ma, M. K. Estes, and H. B. Greenberg. 1985. Specific enzyme-linked immunoassay for rotavirus serotypes 1 and 3. J. Clin. Microbiol. 22:286-291. Svensson, L., L. Grahnquist, C.-A. Pettersson, M. Grandien, G. Stintzing, and H. B. Greenberg. 1988. Detection of human rotaviruses which do not react with subgroup I- and II-specific monoclonal antibodies. J. Clin. Microbiol. 26:1238-1240. Svensson, L., H. Sheshberadaran, S. Vene, E. Norrby, and G. Wadell. 1987. Serum antibody responses to individual viral polypeptides in human rotavirus infections. J. Gen. Virol.
68:643-651. 12. Svensson, L., I. Uhnoo, M. Grandien, and G. Wadell. 1986. Molecular epidemiology of rotavirus infection in Uppsala, Sweden 1981. Disappearance of a predominant electropherotype. J. Med. Virol. 18:101-111. 13. Tanaka, T. N., M. E. Conner, D. Y. Graham, and M. K. Estes. 1988. Molecular characterization of three rabbit rotavirus strains. Arch. Virol. 98:253-265. 14. Taniguchi, K., T. Urasawa, and S. Urasawa. 1986. Reactivity patterns to human rotavirus strains of a monoclonal antibody against vp2, a component of the inner capsid of rotavirus. Arch. Virol. 87:135-141.
JOURNAL OF VIROLOGY, Jan. 1990, p. 411-413
0022-538X/90/010411-03$02.00/0 Copyright © 1990, American Society for Microbiology
Lack of Cosegregation of the Subgroup II Antigens and 6 in Porcine Rotaviruses
on
Genes 2
LENNART SVENSSON, 12* LUIS PADILLA-NORIEGA, 12'3 KOKI TANIGUCHI,4 AND HARRY B. GREENBERG' 2 Department of Medicine and Medical Microbiology, Stanford University School of Medicine, Stanford, California 943051; Palo Alto Veterans Administration Medical Center, Palo Alto, California 943042*; Unidad de Investigacion Clinica en Enfermedades Infecciosas y Parasitarias, Instituto Mexicano del Seguro Social, Mexico City 04000, Mexico3; and
Department of Hygiene, Sapporo Medical College, Chuo-Ku, Sapporo, Japan4 Received 12 June 1989/Accepted 13 September 1989
The rotavirus subgroup I and II specificities associated with gene 2 and 6 products (vp2 and vp6, respectively) were shown not to cosegregate in a number of porcine rotavirus strains. The porcine OSU rotavirus strain and OSU-vp7-like strains were all found to possess a subgroup H-specific region on vp2 and a subgroup I-specific region on vp6. Of interest is the observation that the subgroup II-specific epitope on vp2 appears to be present only in human and porcine rotavirus strains, suggesting a possible human-pig ancestral lineage for gene 2.
vp2 is present exclusively in human and porcine rotavirus strains. The rotavirus strains analyzed in the present study are shown in Table 1. For immunologic characterization, radioimmune precipitation assay, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) assay were used. Radioimmune precipitation assays and ELISAs were performed essentially as described previously (9, 11). IF studies were performed with cover slips containing MA-104 cells that were infected with trypsin-activated rotavirus. After a 30-min incubation with MAbs, the immune reactivity was visualized with an antimouse biotin-streptavidin fluorescein system from Amersham Corp. Four MAbs were used in the study. A previously undescribed vp2 group A-specific MAb designated 6E8 was isolated from a mouse immunized with Wa rotavirus. This MAb reacts with all group A rotaviruses tested. vp6 SG Iand II-specific MAbs (255/60 and 631/9, respectively), previously produced and characterized by Greenberg et al. (4), and a vp2 SG II-specific MAb, YO-60, produced and characterized by Taniguchi et al. (14), were also used. The specificity of YO-60 for human vp6 SG II strains was confirmed before the study by analyzing more than 200 human rotavirus strains, and a complete correlation with SG II strains was seen. The analysis of the SG II vp2 reactivity patterns of rotaviruses derived from various animal species is summarized in Table 1. For comparison, the vp6 SG I and II specificities are also shown. For the strains (J1515, 572, 566, 7325, 451, 7360, and EE TC16) that had not previously been assigned a vp6 subgroup classification, a ratio (SG I/SG II or SG II/SG I) of >3 was used (10). The vp2 group-reactive MAb 6E8 reacted with all human and animal strains except for a variety of isolates from chickens and turkeys. As expected, the SG II vp6 MAb 631/9 reacted with the human strains Wa and WI 61, the porcine strain Gottfried, and J1515 (a Gottfried-vp7-like porcine strain). All other strains except those isolated from avian species reacted with the vp6directed SG I MAb 255/60. The SG II vp2-specific MAb YO-60 reacted with all human SG II strains but not with the human SG I strains DS-1 and 69M. More interestingly, YO-60 also reacted with all nine porcine rotavirus strains analyzed, independent of vp6 subgroup specificity. Several
Rotavirus subgroup (SG) I and II specificity has been localized to epitopes or regions on both vp2 and vp6 (4, 14). Genetic and immunologic characterizations have shown that the subgroup specificities of vp2 and vp6 are encoded by genes 2 and 6, respectively (2, 8). Greenberg et al. (4) used hybridoma technology to produce vp6 SG I- and II-specific monoclonal antibodies (MAbs). These antibodies have been used successfully to determine the subgroups of animal and human strains (4, 12). The use of these and other subgroupspecific MAbs has also shown that subgroup specificity is more complex than originally thought. Examples of this antigenic complexity include the isolation of human (10) and animal rotaviruses that do not react with SG I or II MAbs (2, 4) and the detection of a virus strain that bears both subgroup specificities on vp6 (6). However, the great majority of the serogroup A rotaviruses studied to date have been classified into one of the two subgroups. The majority of human strains appear to be SG II viruses, while all typed animal strains, except for one rabbit (13) and three pig (6) rotavirus strains, belong to SG I. The recent observations that vp2 contains an antigenic domain which, in virtually all human strains tested to date (14), cosegregates with the SG II-specific domain on vp6 and that serotype-specific epitopes reside on two different proteins, vp4 and vp7 (1, 5, 7), make antigenic classification even more complex. The realization that both type and subgroup antigenic specificities are encoded by more than one gene begs the question of how frequently separate genes coding for proteins with type or subgroup specificity cosegregate in the field. In a recent study, it was reported that genes 4 and 9, which encode type-specific antigens vp4 and vp7, respectively, segregate independently in nature (7). In this paper, we present evidence that the subgroup specificities of rotavirus also segregate independently in nature. In a previous study with human strains, the SG II specificities on vp2 and vp6 had been found to cosegregate (14). In the current study, we observed that in wild-type porcine strains, the two specificities are clearly not linked and can segregate independently, presumably by gene reassortment that occurs in the field. Furthermore, we show that among several animal rotavirus strains, a unique and highly conserved SG II-specific epitope *
on
Corresponding author. 411
412
J. VIROL.
NOTES
TABLE 1. Reactivity of human and animal rotaviruses with various subgroup-specific MAbs as demonstrated by IF and ELISA
A B C
D
Assay (optical density at 450 nm)b for: Rotavirus strain
Wa
WI 61 Price DS-1 69M Gottfried J1515 OSU 572 566 7325 451 7360 EE TC16 SA-11 RRV
vp2 with
Source'
SrcaMAb: Human Human Human Human Human
Pig Pig Pig Pig Pig Pig Pig Pig Pig Vervet monkey Rhesus monkey SA-11/OSU 66d SA-11/OSU 86d SA-11/OSU 43d Calf NCDV Calf UK Lamb Lamb Foal Foal 82/309 F Rabbit TC 1039 Cat K9 1037 Dog Ch2 Chicken Tyl Turkey AR NA-1 Bird AR 2 Bird AR 1294 Bird AR 428 Bird AR B Bird
vp6 with MAb:
6E8
YO-60
631/9
255/60
+ (0.850)
+ (0.835) + (0.472) + (0.405) - (0.007) - (0.017) + (0.626) + (0.895) + (0.912) + (0.412) + (0.426) + (0.668) + (0.547) + (0.776) + (0.646) - (0.045) - (0.039) + (0.493) - (0.051) + (0.532) - (0.036) - (0.025) - (0.041) - (0.036) - (0.058) - (0.048) - (0.006) - (0.020) - (0.016) - (0.012) - (0.038) - (0.018) - (0.010) - (0.041)
+ + + +
-
+C
_
+ (0.399) + (0.451) + (0.339) + (0.279)
+ (0.657) + (0.845) + (0.454) + (0.391) + (0.329) + (0.522) + (0.534) + (0.661) + (0.599) + (0.512) + (0.788) + (0.538) + (0.714) + (0.701) + (1.205) + (0.625) + (0.531) + (0.712) + (0.693) + (0.813) + (0.625) - (0.009) - (0.015) - (0.007) - (0.027) - (0.008) - (0.015) - (0.026)
-
-
-
3vp4 p3V p3
vp>
469k
+ +
+ +C +C +C +C +C +C
-
+ + +
-
-
492k
-
446k
Vp60.
*
S
430k
+ + + + + + + + +
FIG. 1. Immunoprecipitation of a [35S]methionine-labeled porcine (OSU strain) rotavirus lysate with SG I- and II-specific MAbs. Lane A, OSU-infected cell lysate; lane B, OSU-infected cell lysate and SG II vp6-specific MAb 631/9; lane C, OSU-infected cell lysate and SG I vp6-specific MAb 255/60; lane D, OSU-infected cell lysate and SG II vp2-specific MAb YO-60. Molecular mass markers (Amersham) are shown on the right as follows: phosphorylase b, 92 kilodaltons; bovine serum albumin, 69 kilodaltons; ovalbumin, 46 kilodaltons; carbonic anhydrase, 30 kilodaltons.
-
-
-
-
a Strains J1515 and EE TC16 were donated by Linda Saif (Ohio State University); strains 572, 566, 7325, 451, and 7360 were donated by Prem Paul (Iowa State University); the avian strains were donated by K. Nagaraga (University of Minnesota); and the lamb and foal strains were donated by Graham Beards. b + and - signify IF and/or ELISA result. Optical densities are mean values of three ELISAs. Cutoff value, 0.1. c Subgroup designation was established by using a SG I/SG II or SG II/ SG I ratio of >3 (10). d Reassortant: 66, SA-11 genes 1, 3, 5, and 10; 86, SA-11 genes 1, 2, 3, and 5; and 43, SA-11 genes 3, 5, 8, and 9.
porcine strains, including OSU, contained a SG I-reactive epitope on vp6 but a SG II region on vp2. This reactivity is in clear contrast to the vp6 SG I human strains DS-1 and 69M, which were not recognized by the vp2 SG II-specific MAb YO-60. Radioimmune precipitation and IF assays showed that the OSU rotavirus strain has two different subgroup specificities located on different proteins (Fig. 1; Table 1). The SG I specificity is located on vp6, as demonstrated by reactivity with MAb 255/60, and the SG II reactivity is located on vp2. To determine if the SG II reactivity of the OSU strain cosegregated with gene 2 or gene 6, reassortment libraries were constructed from MA-104 cells coinfected with OSU and SA-11 (serotype 3, subgroup I). Each gene-reassortant strain was plaque purified three times before the RNA profile was analyzed (data not shown). RNA profile, ELISA, and IF studies of the reassortants confirmed that the reactivity of
YO-60 was linked to the vp2 encoded by gene 2 (2, 8, 14) of the OSU strain (Table 1). Hoshino et al. (6), after analyzing 49 rotavirus strains with six different vp6-specific MAbs, suggested that human rotavirus may have two ancestral lineages: one to human SG I, vervet monkey, and cow rotavirus strains, and the other to OI human and pig rotavirus strains. The SG II human-pig SG ancestral lineage is further supported by our vp2 characterization, which shows that only pig and human rotavirus strains bear SG II-specific antigenic regions on vp2. Gorziglia et al. (3) compared the amino acid sequence of the vp6 of porcine rotavirus Gottfried strain with that of human rotavirus Wa strain vp6 and found 98% homology. At present, no such comparative vp2 amino acid sequence is available for these strains. Such information will be valuable for further studies of the evolution of group A rotaviruses. We predict a high degree of conservation of the vp2 amino acid sequence between human and porcine strains. The antigenic similarity between pig and human rotaviruses with respect to vp2, vp6, and vp7 shows that the pig-human ancestral lineage is worth further attention. Furthermore, the demonstration that subgroup genes as well as genes encoding neutralization antigens do not cosegregate emphasizes again the importance of carefully defining a serologic classification scheme for rotavirus. Since these studies clearly demonstrate that the antigenic epitope on vp2 defined by MAb YO-60 does not invariably cosegregate with the SG II domain on vp6, it is not reasonable to continue to label this epitope a SG II-specific region. Perhaps the subgroup label should be exclusively reserved for antigens on vp6, while antigenic regions on other proteins could be described with
NOTES
VOL. 64, 1990
the protein designation followed by an antigen designation (such as vp2a, vp2b, etc.). We are grateful to Graham Beards (Regional Virus Laboratory, Birmingham Hospital, Birmingham, United Kingdom), Linda Saif (Ohio State University, Columbus), Prem Paul (Iowa State University, Ames), and K. Nagaraga (University of Minnesota, Minneapolis) for providing various rotavirus strains. This work was supported by grants K88-16R-8508 and K8816F-8490-01 from the Swedish Medical Research Council.
7.
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