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Arch Virol (1991) 118:269-277

© Springer-Verlag 1991 Printed in Austria

Comparison of human and porcine group C rotaviruses by Northern blot hybridization analysis

Brief Report Y. Qian 1, L. J. Saif 2, A. Z. Kapikian 1, S. Y. Kang2, B. Jiang 2, Y. Ishimaru3, Y. Yamashita 4, M. Oseto 4, and K. Y. Green 1 1Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, and 2Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, U.S.A. 3Ishimaru Pediatric Clinic, Ehime, and 4Ehime Prefecture Institute of Public Health, Ehime, Japan Accepted December 18, 1990

Summary. The genetic relationship between human and porcine Gp C rotaviruses and between Gp C and Gp A or B rotaviruses was examined by Northern blot hybridization. Cross-hybridization studies using radiolabeled ssRNA transcript probes demonstrated that the human and porcine Gp C rotaviruses shared a high degree of nucleotide sequence homology in most of the eleven gene segments; the greatest sequence divergence was observed in gene 7. Neither the human nor the porcine Gp C probe hybridized strongly with gene segments from Gp A reference strains or a Gp B bovine rotavirus. These data indicate that genetically, porcine and human Gp C rotaviruses are closely related, whereas they are quite distinct from Gp A or B suggesting that porcine and human Gp C rotaviruses may have evolved from a common ancestral source.

Rotaviruses are important etiological agents of severe diarrhea in the young of humans and many animal species I17]. These viruses are members of the family Reoviridae and have been assigned to at least seven groups designated A through G [11, 25]. Group (Gp) A, B and C rotaviruses have been found in both humans and animals, whereas Gps D, E, F and G have been found only in animals [5, 25]. Rotaviruses in these various groups share many structural similarities such as morphologic appearance by negative stain electron microscopy, and a genome containing 11 double-stranded (ds) RNA segments. However, there are major

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differences in antigenicity, genetic homology, host range and clinical relevance among groups. Molecular biologic studies of these groups may provide insight into the mechanisms responsible for these differences. Of the three groups which infect humans, Gp A is clearly the most important in the etiology of diarrheal disease and, thus, efforts are in progress to develop a vaccine [18]. However, it is not known whether other groups will emerge as important pathogens when effective vaccines for Gp A are used extensively. Until recently, the role of "non-group A" rotaviruses in human diarrheal disease was considered insignificant [6]. However, with the occurrence of large outbreaks of severe diarrhea associated with Gp B rotaviruses, predominantly in adults in several provinces of China [13], there has been increased interest in the study of these agents. Although similar Gp B outbreaks have not yet been reported outside China, recent descriptions of the association of Gp C rotaviruses with sporadic cases and a few outbreaks of diarrhea in several countries indicate that the latter group is globally distributed, and perhaps may emerge as an important new pathogen [1, 3, 7-9, 22]. For example, Gp C rotavirus outbreaks have been reported in several different areas of Japan [14, 20, 28]: in Matsuyama since 1988, Gp C rotavirus has been the most frequently identified virus in stool specimens in ill children 4 to 7 years of age [14]; human Gp C rotavirus infection was also reported in Tokyo in 1987 and 1988 during a 7 year survey [28]; and in Fukui, a large outbreak occurred among schoolchildren and their teachers at seven elementary schools in 1988 [20]. In addition, one fatal case of gastroenteritis associated with Gp C rotavirus has recently been reported in England [8]. Gp C rotaviruses have been detected in humans and pigs [-5, 23, 25]. The human and porcine group C rotaviruses are related to each other both antigenically and genetically [-6, 22, 25]. However, antigenic differences were observed with certain serologic assays such as immune electron microscopy (IEM) and immunoftuorescence [22]. Genetic analysis has been limited to genomic profile analysis ("electropherotyping"), terminal fingerprint analysis of selected genome segments and dot blot hybridization of entire viral RNA [6]. Thus, in order to further define the genetic relationship between human and porcine Gp C rotaviruses at the level of individual genes, two G p C human rotavirus samples and a single reference Gp C porcine strain were studied by Northern blot hybridization. Human GpC rotavirus samples (88-315 and 88-220) used in this study were collected in Matsuyama, Japan in 1988 [14]. Stool specimens containing these viruses were shipped on dry ice from Japan and stored at - 7 0 °C. Porcine Gp C rotavirus reference strain Cowden was propagated in gnotobiotic piglets and rotavirus positive stool specimens were used [2]. Human G p A reference strains Wa, DS-1, P, ST3, 69M and WI61 that represent serotypes 1, 2, 3, 4, 8, and 9, respectively, were propagated in MA-104 cells in the presence of trypsin (1.0 pg/ml). Bovine Gp B rotavirus, AT1 strain, was propagated in calves and rotavirus positive stool specimens were used [25].

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Virus particles were concentrated from either 10% (v/v) stool material suspended in suspension buffer [16] or from tissue culture material by genetron (1,1,2-trichloro-l,2,2-trifluoroethane; J. T. Baker Chemical Co., Phillipsburg, N J) extraction followed by centrifugation through a 6 ml 30% sucrose cushion at 120,000 x g for 90 min. To purify virus particles for in vitro transcription, the pellet from the sucrose cushion was dissolved in suspension buffer, layered onto a 40-50% CsC1 gradient and centrifuged overnight at 220,000 x g. Visible bands were collected and pelleted through suspension buffer at 220,000 x g for 2.5 h. For h u m a n Gp C samples, the CsC1 fractions (1.0 ml/each fraction) were also collected and pelleted through suspension buffer as described above. The pellet was suspended in 50 m M Tris-HC1, p H 8.0 and stored at - 7 0 °C. The h u m a n Gp C virus particles were visualized by negative stain (3% PTA, pH 7.2) electron microscopy in the third fraction from the top, which contained a visible band, and were indistinguishable morphologically from Gp A rotaviruses. Double-stranded R N A was obtained by extracting the purified virus with an equal

Fig. 1. Northern blot analysis of human and porcine Gp C rotaviruses using 32p-labelled ssRNA transcripts from porcine Gp C strain Cowden as probe. A Viral dsRNAs resolved by etectrophoresis on a 10% polyacrylamide gel and visualized by ethidium bromide staining. B Autoradiogram from Northern blot hybridization in which viral RNAs corresponding to A were electrotransferred to a nylon membrane and probed with 32P-labelled ssRNA transcripts from Cowden. Hybridization was performed at 42 °C and the film was exposed 48h. a Wa (GpA); b 69M (apA); c ATI (apB); d 88-315 (GpC); e 88-220 (GpC); f Cowden (Gp C)

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volume of phenol/chloroform (1 : 1) followed by precipitation in ethanol. The d s R N A was analyzed by electrophoresis using a 10% polyacrylamide gel [19] and visualized by ethidium bromide staining. The gel was then soaked in 0.1 M N a O H for 20 min followed by electrotransfer of the R N A to a nylon membrane (Nytran; Schleicher & Schuell Inc. U.S.A., Keene, NH) as described [27]. 32p-labelled ssRNA probes were prepared by in vitro transcription of virus particles as described [12] with incubation at 42 °C overnight. Transcription of the Gp C viruses was preceded by thermal shock at 55 °C for 1 min as reported [15]. The reaction mixture was extracted with an equal volume of phenolchloroform (1 : 1), and the ssRNA was precipitated in 2 M LiC1 at 4 °C overnight [21]. The specific activity of each ssRNA probe was 5 x 105cpm/gg and 5 x 106 cpm/ml were used in each hybridization.

Fig. 2. Northern blot hybridization analysis of human and porcine Gp C rotaviruses using 32P-labelled ssRNA transcripts from a human Gp C strain as probe. A Viral dsRNAs resolved by etectrophoresis on a 10% polyacrylamide get and visualized by ethidium bromide staining. B Autoradiogram from Northern blot hybridization in which RNAs corresponding to A were electrotransferred to a nylon membrane and probed with 32p-labelled ssRNA transcripts from No. 88-220. Hybridization was performed at 42 °C and the film was exposed 48h. a Wa (GpA); b DS-1 (apA); c P (GpA); d ST3 (6pA); e ATI (GpB); f 88-220 (Gp C); g Cowden (Gp C); h 69 M (Gp A); i W161 (Gp A)

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Hybridization was performed at 42 °C or 52 °C for 16 to 20 h in hybridization buffer consisting of 50% formamide, 2.5 x Denhardt's solution (1 x Denhardt's contains 0.02% polyvinylpyrrolidone,0.02% bovine serum albumin and 0.02% Ficoll 400), 0.1% SDS, 5 x SSC (1 x SSC contains 150mM NaC1, 15 mM sodium citrate, pH 7.0), and 100 gg/ml denatured salmon sperm DNA. Following the hybridization, membranes were washed twice in 2 >< SSC with 0.1% SDS and two more times in 0.1 x SSC with 0.1% SDS. Each wash was performed at room temperature for 20 min. The membranes were then airdried and exposed to Kodak X-Omat AR film for 24 to 48 h. The hybridization conditions described here were similar to those described by Street et al. in which it was predicted that no more than 8% sequence mismatch was allowed for RNA-DNA hybridization to occur at 52 °C [-26]. In hybridization experiments at 42 °C, the percent mismatch allowed would be predictably higher. However, similar genetic relationships were observed in our experiments at either temperature. The human and porcine Gp C rotaviruses used in this study showed the characteristic RNA electropherotype profile ofGp C rotavirus which is 4 : 2 : 3 : 2 [23, 25]. This differs from the RNA patterns of Gp A (4 : 2 : 3 : 211 and Gp B (4: 2 : 2 : 3) viruses (Fig. 1). Two bands in the gene 11 region of the porcine Cowden strain were observed in certain dsRNA preparations extracted from virus particles following in vitro transcription (Figs. 2 and 3). These two bands did not appear in dsRNA preparations prepared from virus particles purified directly from either stool or tissue culture material (data not shown). In contrast to the porcine Gp C Cowden strain (Fig21 A, lane f), gene segments 3 and 4 as well as segments 8 and 9 from the human Gp C specimens (Fig. 1 A, lanes d and e) migrated tightly together, which correlated with patterns observed from other human Gp C rotavirus samples 1-22]. An overall high degree of sequence homology between the genome of human and porcine Gp C rotaviruses is illustrated in the Northern blot hybridization (Figs. 1 and 2). When 32p-labelled ssRNA transcripts from the porcine strain Cowden were used as probe, all dsRNA gene segments of the homologous Cowden strain hybridized (Fig. 1 B, lane f). Gene segments of the human Gp C samples hybridized with the Cowden strain probe also (Fig. 1 B, lanes d and e). However, the intensity of the signal of the porcine probe with the gene 7 of each of the human Gp C samples was lower which suggests divergence between the porcine and human Gp C viruses in this gene. With genes 3 and 4 or 8 and 9, it was not clear whether each single gene hybridized with its corresponding gene in the porcine Gp C probe because these gene pairs in human Gp C rotavirus migrated closely together and resolution was not possible. A similar result was obtained when the 32p-labelled ssRNA transcripts of the human Gp C sample 88-220 were used as probe. All gene segments of the homologous human GpC virus sample 88-220 hybridized with the probe (Fig. 2 B, lane f). Most of the genes from porcine strain Cowden hybridized with the human Gp C probe as well (Fig. 2 B, lane g). In addition, the electropherotype

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of the porcine strain in the Northern blot was useful in demonstrating that each of porcine gene segments 3, 4, 8, and 9 hybridized with the human Gp C probe, although the signals with genes 3 and 4 were weaker (Fig. 2 B, laneg). Again, the signal of gene 7 was weak in this hybridization. Whether the divergence in gene 7 between human and porcine Gp C strains is related to species differences remains to be determined. The specificity of the probes used in this study for Gp C rotaviruses was shown by the lack of hybridization to either Gp A or B rotavirus strains. Thus, the ssRNA transcript probes described here should be useful in epidemiologic studies using dsRNA from test specimens in hybridization analyses. However, weak or faint signals were detected on the original autoradiogram against several Gp A rotavirus gene segments with both human or porcine Gp C probes after prolonged exposure of the film. Of interest, signals from the gene 1 of Wa, 69 M and W161 were observed after overnight exposure. In previous Northern blot hybridization studies to investigate the genetic relatedness of human and animal Gp B rotaviruses, weak hybridization signals were also detected with a Gp B probe against the gene 1 of both Gps A and C [10]. Recently, the gene encoding

Fig. 3. Northern blot hybridizationanalysis of Gp A and Gp C rotaviruses using 32p-labelled ssRNA transcripts from Gp A strain Wa as probe. A Viral dsRNAs resolved by electrophoresis on a 10% polyacrylamide gel and visualized by ethidium bromide staining. B Autoradiogram from Northern blot hybridizationin which RNAs correspondingto A were electrotransferred to a nylon membrane and probed with 32p-labelledssRNA transcripts from Wa. Hybridization was performed at 42 °C and the film was exposed 24h. a Wa (GpA); b Cowden (Gp C); c 88-220 (Gp C)

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the VP 6 of the porcine Cp C Cowden strain has been sequenced and was shown to be 55% homologous with the G p A gene6 [4]. Whether these data taken together indicate functional homology among different rotavirus groups in proteins encoded by these genes remains to be determined. To confirm the lack of homology in the genomic segments between Gp A and C rotaviruses, 32p-labelled ssRNA transcripts of Gp A strain Wa were used as a probe against Gp C viruses Cowden and 88-220. Although the homologous hybridization was strong against the dsRNA genomic segments of Wa (Fig. 3 B, lane a), a signal against any gene segment of the Gp C viruses could not be detected (Fig. 3 B, lanes b and c) even after prolonged exposure. Our observations indicate that human Gp C rotavirus strains 88-315, 88-220 and porcine group C rotavirus reference strain Cowden share strong homology among the majority of corresponding genes. Genes 3 and 4 of human and porcine Gp C rotaviruses appear to be less related, and gene 7 is apparently the most divergent. Sequence data will give further information. It is possible that human Gp C rotaviruses may have originated in pigs with subsequent transmission to humans [22] or vice versa. Nevertheless, the data from this study suggest that human and porcine Gp C rotaviruses evolved from a common ancestor. The strong genetic relatedness between human and porcine Gp C rotaviruses indicates that pigs infected with the porcine Gp C Cowden strain will be a good experimental animal model for the study of human Gp C rotavirus infection and pathogenesis. The recent adaptation of the Cowden strain to growth in a cell line [24] should facilitate these studies.

Acknowledgements We extend our appreciation to Dr. Robert M. Chanock for advice and critical review of the manuscript; to Johnna Sears for excellentassistance in preparation of the photographs. The followingGp A rotavirus strains were gifts to our laboratory; ST 3 (from J. Banatvala, St. Thomas Hospital, London, England); 69 M (from S. Matsuno, Central Virus Diagnostic Laboratory, National Institute of Health, Tokyo, Japan) and W161 (from H F. Clark, Children's Hospital, Philadelphia, Pennsylvania).

References 1. Beards GM, Desselberger U, Flewett TH (1989) Temporal and geographical distributions of human rotavirus serotypes, 1983-1988. J Clin Microbiol 27:2827-~2833 2. Bohl EH, Saif LJ, Theil KW, Agnes AG, Cross RF (1982) Porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesisin gnotobiotic pigs. J Clin Microbiol 15:312-319 3. Bonsdorf CHV, Svensson L (1988) Human serogroup C rotavirus in Finland. Scand J Infect Dis 20:475-478 4. BremontM, Chabanne-VautherotD, VannierP, McCrae MA, Cohen J (1990) Sequence analysis of the gene (6) encoding the major capsid protein (VP 6) of group C rotavirus: higher than expected homology to the corresponding protein from group A virus. Virology 178:57%583

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5. Bridger JC (1987) Novel rotavirus in animals and man. In: Novel diarrhea viruses. Ciba Found Symp 128:5-23 6. Bridger JC, Pedley S, McCrae MA (1986) Group C rotaviruses in humans. J Clin Microbiol 23:760-763 7. Brown DWG, Mathan MM, Mathew M, Martin R, Beards GM, Mathan VI (1988) Rotavirus epidemiology in Vellore, South India: group, subgroup, serotype, and electropherotype. J Clin Microbiol 26:2410-2414 8. Caul EO, Ashley CR, Darville JM, Bridger JC (1990) Group C rotavirus associated with fatal enteritis in a family outbreak. J Med ViroI 30:201-205 9. Chen G, Fan R, Guo X, Hung T (1988) Group C rotavirus found in sporadic diarrhea in China. J Exp Clin Virol 2:1-3 (In Chinese with English abstract) 10. Eiden J, Vonderfecht S, Theil K, Tortes-Medina A, Yolken RH (1986) Genetic and antigenic relatedness of human and animal strains of antigenically distinct rotaviruses. J Infect Dis 154:972-982 11. Estes MK, Cohen J (1989) Rotavirus gene structure and function. Microbiol Rev 53: 410-499 12. Flores J, Myslinski J, Kalica AR, Greenberg HB, Wyatt RG, Kapikian AZ, Chanock RM (1982) In vitro transcription of two human rotaviruses. J Virol 43:1032-1037 13. Hung T, Wang C, Fang Z, Chou Z, Chang X, Liong X, Chen G, Yao H, Chao T, Ye W, Den S, Chang W (1984) Waterborne outbreak of rotavirus diarrhea in adults in China caused by a novel rotavirus. Lancet i: 1139-1142 14. Ishimaru Y, Nakano S, Nakano H, Oseto M, Yamashita Y (t990) Group C rotavirusassociated gastroenteritis. Arch Pediatr Jap (in press) 15. Jashes M, Sandino AM, Faundez G, Avendano LF, Spencer E (1986) In vitro transcription of human pararotavirus. J Virol 57:183-190 16. Jiang BM, Saif LJ, Kang SY, IZdm JH (1990) Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus. J Virol 64: 3171-3178 17. Kapikian AZ, Chanock RM (1990) In: Fields BN et al (eds) Virology, 2nd edn. Raven Press, New York, pp 1353-1404 18. Kapikian AZ, Flores J, Midthun K, Hoshino Y, Green KY, Gorziglia M, Nishikawa K, Chanock RM, Potash LP, Perez-Schael L (1989) Strategies for the development of a rotavirus vaccine against infantile diarrhea with an update on clinical trials of rotavirus vaccines. Adv Exp Med Biol 257:67-89 19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 20. Matsumoto K, Hatano M, Kobayashi K, Hasegawa A, Yamazaki S, Nakata S, Chiba S, Kimura Y (1989) An outbreak of gastroenteritis associated with acute rotaviral infection in schoolchildren. J Infect Dis 160:611-615 21. Midthun K, Valdesuso J, Hoshino Y, Flores J, Kapikian AZ, Chanock RM (1987) Analysis by RNA-RNA hybridization assay of intertypic rotaviruses suggests that gene reassortment occurs in vivo. J Clin Microbiol 25:295-300 22. Penaranda ME, Cubitt WD, Sinarachatanant P, Taylor DN, Likanonsakul S, Saif L, Glass RI (1989) Group C rotavirus infections in patients with diarrhea in Thailand, Nepal, and England. J Infect Dis 160:392-397 23. Sail L J, Theil KW (1985) Antigenically distinct rotaviruses of human and animal origin. In: Tzipori S (ed) Infectious diarrhoea in the young: strategies for control in humans and animals. Elsevier, Amsterdam, pp 208-214 24. Saif LJ, Terrett LA, Miller KL, Gross RF (1988) Serial propagation of porcine group C rotavirus (pararotavirus) in a continuous cell line and characterization of the passaged virus. J Clin Microbiol 26:1277-1282

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25. Saif LJ (1990) Nongroup A rotaviruses. In: Saif LJ, Theil KW (eds) Viral diarrheas of man and animals. CRC Press, Boca Raton, pp 73-95 26. Street JE, Croxson MC, Chadderton WF, Bellamy AR (1982) Sequence diversity of human rotavirus strains investigated by Northern blot hybridization analysis. J Virol 43:369-378 27. Tanaka TN, Conner ME, Graham DY, Estes MK (1988) Molecular characterization of three rabbit rotavirus strains. Arch Virol 98:253-265 28. Ushijima H, Honma H, Mukoyama A, Shinozaki T, Fujita Y, Kobayashi M, Ohseto M, Morikawa S, Kitamura T (1989) Detection of group C rotavirus in Tokyo. J Med Virol 27:299-303 Authors' address: K. Y. Green, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, U.S.A. Received December 1, 1990

Comparison of human and porcine group C rotaviruses by northern blot hybridization analysis.

The genetic relationship between human and porcine Gp C rotaviruses and between Gp C and Gp A or B rotaviruses was examined by Northern blot hybridiza...
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