Isolation and Characterization of an Equine Rotavirus

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1983, p /83/ $02.OO/O Copyright C 1983, American Society for Microbiology Vol. 18, No. 3 Isolation and Characterization of an Equine Rotavirus YASUTAKA HOSHINO,* RICHARD G. WYATT, HARRY B. GREENBERG, ANTHONY R. KALICA, JORGE FLORES, AND ALBERT Z. KAPIKIAN Laboratory ofinfectious Diseases, National Institute ofallergy and Infectious Diseases, Bethesda, Maryland Received 24 March 1983/Accepted 15 June 1983 A rotavirus, designated as the H-1 strain, was isolated from a diarrheic foal in primary African green monkey kidney cells and MA104 cells. This cell cultureadapted strain hemagglutinated erythrocytes of human group 0, rhesus monkeys, guinea pigs, and sheep. It was found to be similar, if not identical, to porcine rotaviruses (strains OSU, EE, and A-580) by plaque reduction neutralization and hemagglutination inhibition tests, and, in addition, it was found to belong to subgroup 1. This equine rotavirus has an RNA electrophoretic migration pattern which was distinct from those of the three strains of porcine rotavirus. The serological relationship established by plaque reduction neutralization and hemagglutination inhibition tests between the equine (H-1) and porcine (OSU, EE, and A-580) rotaviruses is an example of a rotavirus of the same serotype being isolated from different species. The H-1 strain was distinct from four human rotavirus serotypes (Wa, DS-1, P, and St. Thomas 4) as well as from bovine rotavirus NCDV, simian rotavirus MMU18006, and canine rotavirus CU-1 by plaque reduction neutralization tests. This equine isolate (H-1) was found to be related antigenically to canine CU-1 and bovine NCDV rotaviruses in a one-way fashion by hemagglutination inhibition tests. Rotaviruses are well established as important etiological agents of acute gastroenteritis in various mammalian and avian species, including newborn horses. In 1975, Flewett et al. (8) reported scours in newborn foals in northern Humberside, England, and detected rotaviruses in feces from foals by electron microscopy. In 1976, Kanitz (C. L. Kanitz, Proc. Annu. Conv. Am. Assoc. Equine Practnr., Dallas, Tex., 1976, p ) successfully reproduced a neonatal foal diarrhea syndrome experimentally and identified a rotavirus as the causative agent. Woode et al. (43) reported that foal rotaviruses were indistinguishable from those from children, calves, piglets, and mice morphologically by electron microscopy and antigenically by complement fixation, fluorescent antibody tests, and immune electron microscopy. In 1977, Thouless et al. (40) reported that foal rotaviruses were neutralized by at least a fourfold greater dilution of homologous (foal) antiserum than by heterologous (human, calf, piglet, lamb, mouse, and rabbit) serum, as determined by fluorescent focus neutralization tests. In 1978 from Australia (37, 41) and from the United States (7), in 1979 from New Zealand (6), and in 1982 from Ireland (36), visualization by electron microscopy of rotaviruses in feces from diarrheic foals was 585 reported. In 1979, Smith and Tzipori (35) reported that foal rotaviruses have an RNA electropherotype that is distinct from those of calf, pig, mouse, deer, and human rotaviruses. Rodger et al. (33), in 1980, reported that different equine rotavirus isolates showed variations in the electrophoretic mobiities of some double-stranded (ds) RNA segments. Subsequently, the serological evidence by complement fixation and fluorescent antibody tests of widespread infection of horses with rotaviruses was reported from Japan (23, 38) and from the United States (5, 32). In 1981, Imagawa et al. (22) successfully propagated a foal rotavirus in MA104 cells and demonstrated that their isolate was distinct from bovine (NCDV) rotavirus by using tube neutralization tests. To develop effective rotavirus vaccines for both human and veterinary medicine, it is important to elucidate the serotypic diversity of these agents. It is of particular interest that simian, canine, and certain equine rotaviruses have recently been shown to be similar, if not identical, to the third human rotavirus serotype (19a-21, 44). This report describes the isolation, propagation, and partial characterization of an equine rotavirus, as well as a serological comparison of this agent with human, simian, canine, porcine, and bovine rotaviruses.

2 586 HOSHINO ET AL. MATERIALS AND METHODS Preparation of fecal inoculum. A 20% clarified fecal suspension of diarrhea from a foal at a racing stable at Newmarket, England, was kindly supplied by T. H. Flewett under U.S. Department of Agriculture permit 6328 (46). Cell culture. An established cell line of fetal rhesus monkey kidney (MA104) was used for rotavirus isolation and for plaque reduction neutralization (PRN) tests. Primary African green monkey kidney (AGMK) cells were used for reisolation of rotavirus. Growth medium was Eagle minimal essential medium, with 8 to 9% fetal calf serum or calf serum and antibiotics. Maintenance medium was the same but did not contain serum. Isolation and propagation of equine rotavirus in cultured cells. Confluent monolayer cultures (culture tubes, 16 by 125 mm) were washed three times with Leibovitz L-15 medium or Eagle minimal essentidl medium and were inoculated with 0.1 ml of fecal suspension (pretreated for 60 min at 37 C with trypsin at a final concentration of 5,u g/ml). After a 1-h adsorption at 37 C, cell cultures were washed once with maintenance medium, fed with 1.0 ml of maintenance medium supplemented with 0.25,ug of trypsin per ml, and incubated on a roller apparatus at 37 C. At 1 week after infection if cytopathic effects (CPE) were not observed or at the time the cells showed 50 to 80% CPE, the culture tubes were frozen and thawed quickly three times, and cell lysates were inoculated into fresh cell cultures as described above. Subgroup antigen assay. The immune adherence hemagglutination assay (IAHA) (27), enzyme-linked immunosorbent assay (ELISA) (10), or both were employed to subgroup equine rotavirus. Human rotavirus strains DS-1 (subgroup 1) and Wa (subgroup 2) were used as positive controls. Solid-phase radioimmunoassays (RIA) were performed as described previously (10). Viruses. The following rotaviruses were used as reference strains: porcine rotaviruses OSU, EE, A-580 (furnished by E. H. Bohl), and P-1 (furnished by J. G. Lecce); bovine rotavirus NCDV (furnished by C. A. Mebus); rhesus monkey rotavirus MMU18006 (furnished by N. J. Schmidt); canine rotavirus CU-1; and human rotaviruses Wa, DS-1, P, and St. Thomas 4. The St. Thomas 4 strain belongs to a newly defined fourth serotype (45) and was used in the form of a reassortant virus between a temperature-sensitive mutant of a cultivatable bovine rotavirus (UK strain) and a human rotavirus (St. Thomas 4) (13). All rotaviruses were triply plaque purified before use. Antisera. All hyperimmune antisera used in PRN and hemagglutination inhibition (HI) tests were prepared in guinea pigs as described previously (11, 44). Preparation of ds RNA and polyacrylamide gel electrophoresis. ds RNAs of equine and porcine rotaviruses were extracted from the infected MA104 cell lysates by a previously described method (26). Segmented ds RNA was run overnight in a 12% polyacrylamide slab gel with 3.5% stacking gel at 15 to 20 ma of constant current by the method of Laemmli (30), except that sodium dodecyl sulfate was omitted from all solutions. Ethidium bromide staining and photography of RNA under UV light were performed as reported previously (26). J. CLIN. MICROBIOL. PRN tests. PRN tests were performed as described previously (21, 44). Neutralizing antibody titers were expressed as a reciprocal of the calculated serum dilution causing a 60% reduction in plaque counts. If the difference between homologous and heterologous reciprocal neutralizing titers was 20-fold, two viruses were considered to be different antigenically and, thus, serotypically distinct (21, 44). HA and HI tests. The hemagglutinating antigens were prepared from the infected MA104 cell lysates extracted with trichlorotrifluoroethane (Genetron 113, Allied Chemical Co.) followed by sedimentation through a 40% sucrose cushion by ultracentrifugation. The pellets were reconstituted in phosphate-buffered saline (ph 7.4) in 1/30 of the original volume of cell lysates and were then employed for hemagglutination (HA) and HI tests. The erythrocytes of human group 0, rhesus monkeys, sheep, and guinea pigs were used for HA at 0.5% concentration in phosphate-buffered saline supplemented with 0.3% bovine serum albumin. The HI titer of the hyperimmune serum (twofold dilutions) was expressed as the reciprocal of the highest dilution of serum which completely inhibited hemagglutination. RESULTS Isolation and propagation of rotavirus in cultured cells. CPE were not observed during the first two passages of strain H-1 in either the MA104 cells or the reisolation attempts in primary AGMK cells. The first sign of CPE was detected 3 days postinfection at passage 3 in both cell types. CPE consisted of increased cell granularity, obscure cell boundaries, cell rounding, and eventual cell detachment from the glass walls; CPE were consistently observed thereafter Ṡubgroup antigen assay. The rotavirus in the original fecal material from a diarrheic foal was found to belong to subgroup 2 by using RIA (10). However, the same fecal material was not sub using ELISA (Table 1), and the tissue culture-adapted equine rotavirus (H-1) grown in either AGMK or MA104 cells was shown to belong to subgroup 1 by using ELISA (Table 1) and IAHA (Table 2). In attempting to reconcile these conflicting data, subgroup testing of the original feces was reattempted by using RIA. Common monoclonal antibodies detected rotavirus common antigen(s); however, both subgroup 1 and 2 monoclones failed to detect subgroup-specific antigen(s) on repeated RIA tests (Table 3). Strains EE, A-580, and P-1 of porcine rotavirus were found to belong to subgroup 1 (Table 1). HA and HI tests. The partially purified H-1 strain of equine rotavirus hemagglutinated erythrocytes of human group 0 (titer, 1:32), rhesus monkeys (1:32), guinea pigs (1:4), and sheep (1:4). Since the hemagglutinating pattern of human group 0 erythrocytes was firmer and

3 VOL. 18, 1983 TABLE 1. Subgroup specificity of equine (H-1), and porcine (OSU, EE, A-580, and P-1) rotaviruses determined by using ELISA with monoclonal antibodies Optical densitya with a single dilution of indicated Rotavirus monoclone Subgroup Sub- Sub- Comgroup lb group 2' mond Human (DS-1) Human (Wa) Original foal Not subfeces (H-1) MA104-grown equine (H-1) Porcine (OSU) Porcine (EE) Porcine 2% (A-580) Porcine (P-1) nm. Sum of optical density of two wells x 100 at 400 b Monoclone 255/60 with subgroup 1 specificity. Monoclone 631/9 with subgroup 2 specificity. d Mixture of monoclones 952/224, 260/9, 260/12, 631/ 16, 631/24, and 255/18, designed as a broadly reactive reagent. better than that of the rhesus monkeys, human group 0 erythrocytes were used for the HI test. The H-1 strain of equine rotavirus and the OSU strain of porcine rotavirus were found to be similar, if not identical, by the HI test (Table 4). The CU-1 strain of canine rotavirus and the TABLE 2. Subgroup specificity of equine (H-1) and porcine (OSU, EE, and A-580) rotaviruses determined by IAHA Reciprocal of IAHA antigen titer of gnotobiotic calf postinfection antiserum Rotavirus to indicated rotavirus Subgroup Human sub- Human subgroup 1 group 2 (DS-1) (Fh) Human 16 <2 1 (DS-1) Human (Wa) < Original foal <2 <2 Not subfeces (H-1) MA104-grown 32 <2 1 equine (H-1) Porcine <2 <2 Not sub- (OSU) Porcine (EE) 64 <2 1 Porcine 32 <2 1 (A-580) EQUINE ROTAVIRUS 587 TABLE 3. Subgroup specificity of equine (H-1) rotavirus as determined by RIA Reciprocal of RIA antigen titer with a single dilution of Rotavirus indicated monoclones Subgroup Sub- Sub-bComn group ia group 2b Commonc Human (DS-1) Human (Wa) 1, ,153 1, Original foal Not subfeces (H-1) a Monoclone 255/60 with subgroup 1 specificity. b Monoclone 631/9 with subgroup 2 specificity. c Mixture of monoclones 952/224, 260/9, 260/12, 631/ 16, 631/24, and 255/18, designed as a broadly reactive reagent. NCDV strain of bovine rotavirus were related to the H-1 strain in a one-way fashion, and rhesus rotavirus was distinct from equine H-1. The canine and simian viruses were similar, if not identical, by HI test. Antigenic relationships between equine and other mammalian rotaviruses. Table 5 summarizes the results of the antigenic relationships between equine and other mammalian rotaviruses studied by PRN tests. The equine (H-1) and porcine (OSU) rotaviruses were found to be similar, if not identical, by this reciprocal crossneutralization test. It is striking that the equine (H-1) rotavirus was distinct from the four serotypes of human rotavirus (Wa, DS-1, P, and St. Thomas 4), as well as from rhesus (MMU18006), canine (CU-1), and bovine (NCDV) rotaviruses by the PRN test. Antigenic relationship of equine and three strains of porcine rotaviruses. To further study the close antigenic relationship between this isolate of equine rotavirus and porcine rotaviruses, two additional porcine rotavirus strains (EE and A-580) were examined by using PRN tests. Table 6 shows a two-way antigenic relationship between the H-1 strain of equine rotavirus and OSU, EE, and A-580, which together represent a single serotype of porcine rotavirus. Polyacrylamide gel electrophoresis of segmented viral ds RNAs. Since equine rotavirus was found to be closely related antigenically to porcine rotaviruses and since there is always a possibility of cross-contamination in the laboratory, electrophoretic migration patterns of ds RNAs of equine and porcine rotaviruses were compared. The H-1 strain of equine rotavirus has an electropherotype of ds RNA distinct from those of OSU, EE, and A-580 of porcine rotaviruses (Fig. 1). The H-1 strain was also shown to be distinct from porcine rotavirus P-1 in ds RNA electrophoretic migration pattern.

4 588 HOSHINO ET AL. J. CLIN. MICROBIOL. TABLE 4. Comparison of equine (H-1), porcine (OSU), simian (MMU18006), canine (CU-1), and bovine (NCDV) rotaviruses by HI tests Reciprocal of HI titer of hyperimmune guinea pig antiserum to indicated rotavirus Rotavirus Equine Porcine Simian Canine Bovine (H-1) (OSU) (MMU18006) (CU-1) (NCDV) Equine (H-1) 5,120a 10, ,120 Porcine (OSU) 5,120 40, ,560 Simian (MMU18006) ,480 2, Canine (CU-1) 1,280 2,560 20,480 5, Bovine (NCDV) ,960 a Homologous values are shown in boldface type. DISCUSSION The results of the present study demonstrated by PRN tests that equine rotavirus strain H-1 is distinct from four human rotavirus serotypes (Wa, DS-1, P, and St. Thomas 4) as well as being distinct from bovine (NCDV), rhesus monkey (MMU18006), and canine (CU-1) rotaviruses, based on the criterion of 20-fold or greater differences in antibody titer. This isolate (H-1) of equine rotavirus was found to be indistinguishable from one serotype of porcine rotavirus (OSU, EE, and A-580) by PRN and HI tests. However, it differs in the electrophoretic mobility of certain ds RNA segments with each of these porcine rotavirus strains (Fig. 1). It is not surprising that the rotaviruses from two different animal species belong to the same serotype, since it has been well established that simian (MMU18006 and SAii), canine (CU-1, A79-10, and LSU75C-36), some equine (H-2), and some human (M, P, B, 14, and 15) rotaviruses belong to the third human rotavirus serotype (19a-21, TABLE 5. Rotavirus 44, 45); HI tests have revealed broader crossreactivity among rotaviruses examined in this study than PRN tests. For example, although the H-1 strain of equine rotavirus was shown to be serotypically distinct from canine (CU-1) and bovine (NCDV) rotaviruses by PRN tests (Table 5), the former was found to be related to the latter in a one-way fashion by HI tests (Table 4). Kalica et al. (24) have recently reported by analyzing 16 simian (MMU18006) x bovine (UK) reassortants that rotaviral HA and neutralization are functions encoded by two different gene products: gene 4 encodes viral hemagglutinin, and gene 8 or 9 encodes a major neutralizing protein. More recently, Greenberg et al. (12) have demonstrated by analyzing a series of monoclonal antibodies directed against two surface proteins of simian rotavirus (MMU18006) that both viral HA and neutralization properties can be mediated by antibodies to gene 4 as well as to gene 8 or 9 products. They have also shown that both viral hemagglutinin and a major neu- Antigenic relationship between equine rotavirus and porcine, bovine, simian, canine, and human rotaviruses by PRN tests with hyperimmune antisera Reciprocal of 60% plaque reduction neutralizing titer of hyperimmune antiserum to indicated rotavirus Equine Porcine Bovine Rhesus Canine Human Human Human Human (St. (H-1) (OSU) (NCDV) (MMU18006) (CU-1) (DS-1) (Wa) (P) Thomas 4) Equine (H-1) 46,816a 82,687a 3,524a 689a Equine (H-1) 68,343b e -50b <80b <80b Equine (H-1) 65,785c c <80c Porcine (OSU) 21,777a 90,442a Bovine (NCDV) 840a - 81,920a Rhesus (MMU18006) 230a - - _=81,920a Canine (CU-1) 1,766b - 77,607b Human (DS-1) <80b 30,465b - Human (Wa) 982b - 65,478b Human (P) <80c - 32,482c Human (St. <80c - 24,827c Thomas 4d) a,b,c This is a composite table of values derived from three different PRN tests, a to c. Homologous values are shown in boldface type. d Human-bovine reassortant rotavirus. ' Not tested.

5 VOL. 18, 1983 TABLE 6. Comparison of equine rotavirus (H-1) and porcine rotaviruses (strains OSU, EE, and A- 580) by PRN testsa with hyperimmune antiserum Reciprocal of 60% plaque reduction neutralizing titer of hyperimmune Rotavirus antiserum to indicated rotavirus Equine Porcine Porcine Porcine (H-1) (OSU) (EE) (A-580) Equine (H-1) 53,484b 83,665 61,635 27,136 Porcine (OSU) 20,345 85,492 25,259 19,880 Porcine (EE) 11,890 82,785 21,199 9,577 Porcine (A-580) 13,932 86,473 40,416 15,964 a All values are from the same test. b Homologous values are shown in boldface type. tralizing protein exhibit distinct type specificity. For example, simian (MMU18006) and canine (CU-1) rotaviruses have been shown to be indistinguishable from each other by PRN and HI tests using monoclonal antibodies directed against gene 8 or 9 product; however, a crossreactivity between the two has not been detected by PRN and HI tests using monoclonal antibodies directed against gene 4 product (12). 2 - Therefore, although the qualitative nature and quantitative distribution of two biologically distinct antibodies (those directed against gene 4 products and gene 8 or 9 products as demonosu osu H 1-1 H EQUINE ROTAVIRUS 589 strated in a monoclonal system) in polyclonal hyperimmune antiserum employed in this study are not known, it is conceivable that the antigenic relationships among rotaviruses studied by PRN tests would be different from those examined by HI tests as shown in the present study. A variety of human, animal, and avian rotaviruses are currently being studied for serotypic diversity in our laboratory. In 1981, Kapikian et al. (27) proposed the first unified antigenic classification of human and other mammalian rotaviruses by demonstrating by IAHA and ELISA the existence of at least two distinct antigenic specificities designated subgroups 1 and 2. Moreover, Kalica et al. (25) demonstrated that subgroup antigens were encoded by the rotavirus gene 6 product (the major inner capsid protein). Subsequently, Greenberg et al. (12) confirmed and extended the previous findings by using 10 separate monoclonal antibodies directed against the protein product of gene 6. Bastardo et al. (1), using simian rotavirus SAl1, and Killen and Dimmock (29), using bovine rotavirus UK, reported independently that the monospecific hyperimmune antiserum prepared to gene 6 products of each rotavirus had a low level of neutralizing activity against homologous viruses. In addition to its practical applications described previously (10, 27), subgroup specificity plays an important role in the studies of genetics and natural history of rotaviruses. A-580 EE EE H-1 H--I A I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ j FIG. 1. Comparison of RNA electrophoretic migration patterns of H-1 strain of equine and three strains (OSU, EE, and A-580) of porcine rotaviruses. RNAs from H-1 were coelectrophoresed with OSU, EE, or A-580 as indicated. Migration was from top to bottom, and genome segments are numbered in descending order of molecular weight.

6 590 HOSHINO ET AL. For example, we have recently demonstrated for the first time the existence of two rotaviruses from one animal species which belong to the same serotype but differ in subgroup specificity (Hoshino et al. manuscript in preparation). Further studies on its biological nature and genetic significance need to be done. The equine rotavirus (H-1) in the original fecal material has been found on two separate occasions to be a subgroup 2 virus by using RIA with monoclonal antibodies (10); however, the cultivatable virus was found to be a subgroup 1 virus. As noted earlier, however, on repeated RIA testing of the original fecal material by using RIA with the same monoclonal antibodies, a subgroup determination could not be made (Table 3). A rotavirus reisolated from the original foal feces in primary AGMK cells was also a subgroup 1 virus. It is possible that the virus in the original feces was a mixture of subgroup 2 (predominantly) and subgroup 1 subpopulations, and susceptible cell substrates (MA104 and AGMK cells) selected the subgroup 1 subpopulation preferentially over the subgroup 2 subpopulation during virus multiplication. Antigenic heterogeneity of the various constituent virus particles comprising a virus strain has been well established for certain enteroviruses (2), myxoviruses (3, 4), alphaviruses (14, 15), and rhabdoviruses (42). Also, host-induced antigenic variation has been reported previously for animal viruses such as variola (19), influenza A (18), ECHO-6 (28), western equine encephalomyelitis (31), eastern equine encephalomyelitis (17), Semliki Forest, chikungunya, O'nyoun-nyong (T. B. Stim and J. R. Henderson, Bacteriol. Proc., p. 182, 1968), and Getah (16). Very recently Sabara et al. (34) reported the coexistence of electrophoretically distinct bovine rotavirus subpopulations within one animal by the use of high-resolution gels. The existence of a subgroup 1 (39) as well as subgroup 2 (J. H. Gillespie, Cornell University, Ithaca, N.Y., personal communication) equine rotaviruses obtained from a different source than the H-1 strain has been reported recently. In cross-protection studies performed by Tzipori and Walker (41) in 1978, apparent crossprotection between pig and foal rotaviruses in pigs was observed. This cross-protection between pig and foal rotaviruses can now possibly be explained since certain porcine and equine rotaviruses belong to the same serotype. However, it has been reported that there is a serotype of porcine rotavirus distinct from the OSU strain (E. H. Bohl, Ohio Agricultural and Development Center, Wooster, personal communication), and this strain does not cross-react with the H-1 strain of equine rotavirus (Hoshino et al., manuscript in preparation). Moreover, we J. CLIN. MICROBIOL. have recently reported the existence of a second equine rotavirus serotype which was found to be distinct from the H-1 strain not only in serotypic specificity but also in subgroup specificity (19a). Gaul et al. (9) have recently concluded that the cross-protection between rotaviruses is type specific, and potential vaccines should include serotypes which cause disease in the homologous animal species. If the H-1 strain of equine rotavirus is found to be of low virulence or can be further attenuated, and if the above findings by Gaul et al. are confirmed further, the H-1 strain may become a potential vaccine for use not only in foals but also in piglets. ACKNOWLEDGMENTS We thank Robert M. Chanock for valuable advice and criticism. 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