Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells

Veterinary Microbiology, 8 (1983) 135--139 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

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PRIMARY ISOLATION OF CYTOPATHIC BOVINE ROTAVIRUSES ON F E T A L R H E S U S M O N K E Y K I D N E Y C E L L S

YOSUKE MURAKAMI, NOBUYOSHI NISHIOKA, YUJI HASHIGUCHI and CHIKARA KUNIYASU Hokkaido Branch Laboratory, National Institute of Animal Health, Hitsujigaoka 4, Toyohira-ku, Sapporo, Hokkaido 061-01 (Japan) (Accepted 26 October 1982)

ABSTRACT

Murakami, Y., Nishioka, N., Hashiguchi, Y. and Kuniyasu, C., 1983. Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells. Vet. Microbiol., 8: 135--139. Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.

INTRODUCTION I t is quite difficult to isolate a n d cultivate r o t a v i r u s e s o n cell cultures, even t h o u g h an e x t r e m e l y large n u m b e r o f virus particles are p r e s e n t in t h e i n f e c t e d feces ( M c N u l t y , 1 9 7 8 ) . A great deal o f e f f o r t has b e e n m a d e t o cultivate r o t a v i r u s e s in cell c u l t u r e s ( M a t s u n o et al., 1 9 7 7 ; A l m e i d a et al., 1 9 7 8 ; B a r n e t t et al., 1 9 7 9 ; Clark et al., 1 9 7 9 ; S c h o u b et al., 1 9 7 9 ; S m i t h et al., 1 9 7 9 ; G r a h a m a n d Estes, 1 9 8 0 ; W y a t t et al., 1 9 8 0 ) . R e c e n t l y several r o t a v i r u s e s derived f r o m d i f f e r e n t species have b e e n successfully isolated a n d / o r c u l t i v a t e d o n cell c u l t u r e s in t h e p r e s e n c e o f t r y p s i n ( B a b i u k et al., 1 9 7 7 ; Theil et al., 1 9 7 7 ; M c N u l t y et al., 1 9 7 9 ; W y a t t et al., 1 9 8 0 ; F u k u s h o et al., 1 9 8 1 ; H o s h i n o et al., 1 9 8 1 ) . A l m o s t all o f t h e successful results f o l l o w e d serial passages o f t h e virus s a m p l e s o n cell c u l t u r e s or s u s c e p t i b l e e x p e r i m e n t a l animals. N o r e p o r t has b e e n p u b l i s h e d c o n c e r n i n g p r i m a r y isolation o f c y t o p a t h i c r o t a v i r u s e s o n cell c u l t u r e s f r o m fecal samples, exc e p t o n e case o f feline r o t a v i r u s ( H o s h i n o et al., 1 9 8 1 ) . T h e p r e s e n t p a p e r

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© 1983 Elsevier Science Publishers B.V.

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describes the primary isolation of cytopathic bovine rotaviruses on MA104 cells from fecal samples of clinically affected calves. MATERIALS AND METHODS

The fecal samples used in this study were obtained from 51 calves from 22 herds affected by acute diarrhea over the period from April 1978 to May 1980 in Japan. A 10% suspension of the feces was prepared in phosphatebuffered saline (PBS, pH 7.2) supplemented with 1000 U of penicillin, 500 /~g of streptomycin and 2.5 ~g of Fungizone (Squibb, U.S.A.) per ml. The fecal suspension was clarified by centrifugation at 1500 g for 20 min and the supernatant fluid subsequently centrifuged at 15000 g for 30 min. The supernatant fluid was finally filtrated through a 450 membrane filter (Toyo Roshi, Japan). The continuous line of fetal rhesus m o n k e y kidney (MA104) cells used was grown in Eagle's minimum essential medium with Earle's salt containing 10% of calf serum, 10% of tryptose phosphate broth, 0.15% of sodium bicarbonate, and 100 ~g of kanamycin and 2.5 ~g of Fungizone per ml. The maintenance medium (MM) for the cells after inoculation was of the same composition as the growth medium; it contained 1 ug per ml of trypsin (Type 1, Sigma Chemical, U.S.A.) and 0.11% of bovine serum albumin instead of calf serum. The fecal extract was treated with 10 pg per ml of trypsin at 37°C for 30 min prior to inoculation into the MA104 cells. After removal of the growth medium, confluent tube cultures of MA104 cells i l l × 100 mm in size) were washed twice with 2 ml of Earle's balanced salt solution (Earle's BSS). The trypsin-treated fecal extracts were diluted from 10 ° to 10 -4 with MM and then 0.1 ml of each dilution inoculated into each of eight tube cultures. The inoculum was removed after adsorption at 37°C for 1 h. Next, the culture was washed once with Earle's BSS, and then 0.5 ml of MM were added. The inoculated cultures were incubated in a roller apparatus at 37 ° C. After incubation for 24--48 h, the cells were collected from the cultures by vigorous shaking and examined for virus antigen by immunofluorescence (IF). Fresh MM was applied to these cultures, and incubation was continued. When the cells showed cytopathic change, the culture tubes were frozen and thawed three times and centrifuged at 1500 g for 10 min. The supernatants were passaged more than three times on the MA104 cells by the terminal dilution method. The cytopathic agents were identified by IF and electron microscopy (EM). The cultures that failed to show the virus antigen by IF at primary isolation were further passaged three times by the m e t h o d described above. Antiserum used for IF was prepared against the Lincoln strain of bovine rotavirus (Mebus et al., 1971) in specific pathogen-free guinea-pigs (Nippon Inst. Biol., Japan) which had been shown to be free of rotavirus antibody by the complement fixation test against the Lincoln strain. The procedures of

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purification and immunization for the virus have been described (Murakami et al., 1981). Direct IF for detection of the virus antigen on infected cells was conducted by using the antiserum conjugated with fluorescein. Conjugation of the antiserum and staining were carried out according to the technique described by Kawamura (1969). The specimens were examined by a Nikon F l u o p h o t fluorescence microscope. Demonstration of virus particles by EM was carried out as follows. The fecal extract and infected culture fluid, partially purified by a series of low-speed centrifugations, were further centrifuged at 100 000 g for 2 h. The pellets were taken up in 0.1 ml of distilled water. A drop of this fluid was mixed with an equal a m o u n t of 2% phosphotungstic acid (pH 6.0), and the mixture placed on a 400-mesh collodion carbon-coated grid. The excess fluid was removed and the specimen was examined by a Hitachi HS-9 EM. RESULTS

A total of 51 fecal samples obtained from 22 herds was examined for bovine rotaviruses. Virus isolation on MA104 cells was compared with the detection of virus particles by EM in the fecal samples and the detection of virus antigens in the cells by IF within 24--48 h after inoculation. The results are given in Table I. Rotavirus particles were demonstrated in 10 fecal samples from 6 herds by EM. The rotavirus antigens were detected in 14 fecal samples from 7 herds by IF and included the samples positive by EM. On the other hand, within 7 days after inoculation, cytopathic bovine rotaviruses were isolated from all of the 14 fecal samples that were positive by IF on the MA104 cells. The infected cells became granular or rounding, and then detached from the glass surface. The cytopathic change resulted in the destruction of the entire monolayer. Their infective titers were less than 103 TCIDs0 per ml. The isolates were passaged three to five times on MA104 cells in the presence of trypsin by the terminal dilution method, and then TABLE I Primary isolation of bovine rotavirus from field cases, as compared with detection of virus antigens by immunofluorescence and particles by electron microscopy in feces Total fecal samples examined a

Positivefecal samples examined by Electron microscopy

Immunofluorescence

Primary isolation

51 (22) 5

10 (6)

14 (7)

14 (7)

a Fecal samples were derived from diarrheic calves. b Numbers in parentheses indicate the number of herds from which the samples were obtained.

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their identification as rotaviruses was confirmed by EM and IF. Though the fecal samples that failed to react with IF at the initial inoculation were given three further passages, no bovine rotaviruses were isolated from any of them. DISCUSSION Recent reports have described the isolation and cultivation of rotaviruses derived from several animal species involving bovine on cell cultures (Babiuk et al., 1977; Theft et al., 1977; McNulty et al., 1979; Wyatt et al., 1980; Fukusho et al., 1981; Hoshino et al., 1981). In almost all cases, the successful results depended on serial passages of the cell cultures in the presence of trypsin and by monitoring for IF antigen (Babiuk et al., 1977; Theft et al., 1977; McNulty et al., 1979; Fukusho et al., 1981), or on the serial passage of affected feces in gnotobiotic animals prior to adaptation to the cell cultures (Wyatt et al., 1980). In the present study, we obtained the primary isolation and serial cultivation of cytopathic bovine rotaviruses from all of the fecal samples that were positive by both IF and EM. The m e t h o d described in this study is highly sensitive for the isolation of bovine rotaviruses from affected feces on cell cultures. It is assumed that our success largely depends on the presence of trypsin in the fecal samples and the culture media in combination with rolling cultivation as described by Fukusho et al. (1981). Although we were able to demonstrate numerous virus particles in the feces, the infective titers of the isolates at the initial cultivation (less than 10 a TCIDs0 per ml) were much lower than the titer of detectable virus in negatively stained specimens examined by EM (107--109 particles per ml; Hsiung et al., 1979). It is possible that this phenomenon may correlate with our observation that single-shelled particles were predominant in the affected feces of this study rather than double-shelled particles (data not shown), since the double-shelled particles are believed to be mainly associated with infectivity (Flewett and Woode, 1978). The methods described in this study appear to be suitable for the isolation, propagation and identification of bovine rotaviruses in the feces of calves with acute diarrhea.

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