Isolation of different non-cytopathogenic bovine viral diarrhoea (BVD) viruses from cytopathogenic BVD virus stocks using reverse plaque formation method

Veterinary Microbiology, 38 (1993) 173-179 Elsevier Science Publishers B.V., Amsterdam

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Short Communication

Isolation of different non-cytopathogenic bovine viral diarrhoea (BVD) viruses from cytopathogenic BVD virus stocks using reverse plaque formation method Shigeyuki Nakamuraa, Akio Fukushob, Yoshimitsu Inoue", Hideharu Sasaki" and N o b u o Ogawa ~ INational VeterinaryAssay Laboratory, Tokyo, Japan bExotic Diseases Research Division, National Institute of Animal Health, Tokyo, Japan (Accepted 6 April 1993)

ABSTRACT Non-cytopathogenic (NCP) bovine viral diarrhoea (BVD) viruses were isolated from three cytopathogenic (CP) BVD virus stocks using the reverse plaque formation method, which was based on intrinsic interference. By means of an exaltation of Newcastle disease virus (END) test, these NCP BVD viruses were divided into two groups; END phenomenon positive (END + ) and END phenomenon negative (END-) viruses. Additionally, the END + NCP BVD viruses interfered only with CP BVD virus whereas the END- NCP BVD viruses interfered with vesicular stomatitis virus as well as CP BVD virus. Differences in antigenicity existed among the three CP strains, however, each group of parent CP BVD virus and derivative NCP BVD virus was antigenicallyindistinguishable.

Key words:Bovine viral diarrhoea virus; Reverse plaque formation; Virus isolation

INTRODUCTION

Bovine viral diarrhoea (BVD) virus, a member of the genus Pestivirus of the Flaviviridae (Francki et al., 1991 ), is an important pathogen of cattle, causing diarrhoea and mucosal disease. These are two biotypes of BVD virus, the cytopathogenic (CP) biotype and the non-cytopathogenic (NCP) biotype. The CP biotype viruses induce vacuolation and cell death in infected cell monolayers, whereas NCP biotype viruses do not cause any cytopathogenie effect-(CPE) in vitro. The CP BVD viruses can be readily titrated and Correspondence to: Shigeyuki Nakamura, National Veterinary Assay Laboratory, 1-15-1 Tokura, Kokubunji, Tokyo, 185 Japan.

0378-1135/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved.

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cloned using the conventional plaque assay, whereas NCP BVD viruses usually require indirect assay methods. In our laboratory the NCP BVD viruses have been assayed by either a homologous interference method with a CP BVD virus (Gillespie et al., 1962) or by a heterologous interference method with vesicular stomatitis virus (VSV). The reverse plaque formation (RPF) method (Fukusho et al., 1976; Laude., 1978; Itoh et al., 1983) based on homologous or heterologous interference has been used for the titration of the NCP BVD viruses. An additional assay is by the exaltation of Newcastle disease virus (END) (Kumagai et al., 1961; Inaba et al., 1963; 1968) during replication in bovine testicle (BT) cell cultures. Most NCP BVD viruses examined have been END phenomenon positive (END + ) (Inaba et al., 1963; 1968), although one END phenomenon negative (END-) strain has been reported (Itoh et al., 1984). With hog cholera virus, another Pestivirus, an END- mutant has been isolated from an END + strain (Shimizu et al., 1970). Studies on the genomic sequences of BVD viruses have identified the insertion of a cellular RNA in the genome ofa CP BVD virus strain (Collett et al., 1988; Meyers et al., 1989). It has been suggested that in persistently infected cattle, an NCP BVD virus can mutate to the CP biotype by incorporating cellular sequences in a recombination event (Meyers et al., 1991 ). However little is known about the interchange of CP and NCP biotypes in vitro. In the present study, we have isolated NCP BVD viruses from CP virus stocks using the RPF method and compared the interference patterns and antigenicity of these NCP isolates. MATERIALS AND METHODS

CP BVD viruses used in this study were the Nose (Kodama et al., 1974), NADL (Gutekunst and Malquist., 1963) and KS86(+ ) (Shimizu et al., 1989 ) strains. These viruses were propagated in BT cell cultures for the preparation of virus stocks. The Nose strain of CP BVD virus and the New Jersey serotype of VSV were used as challenge viruses for the homologous and heterologous interference methods respectively. The Miyadera strain of Newcastle disease virus (NDV) was used for END testing. BT cells were grown in Eagle's minimum essential medium (MEM) supplemented with 10% foetal bovine serum (FBS). The BT cells and FBS used were confirmed free of adventitious BVD virus by the homologous interference assay and indirect immunofluorescence tests. Neutralizing antibodies against BVD virus were not detected in the FBS by the microtitration serum neutralization test (Shimizu et al., 1989). Antisera were prepared from cattle immunized with cloned Nose or NADL strains. The modified RPF method was used for isolation of the NCP BVD viruses from the CP BVD virus stocks. Serial 10-fold dilution of the CP strains of BVD virus (0.2 ml ) were inoculated onto confluent BT cell monolayers grown

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in 60 m m plastic petri dishes. Six dishes were inoculated with each dilution. The inoculated cells were incubated at 37 C for 1 hr and then the inoeulum were aspirated. The infected cell monolayers were covered with 5 ml of methyl cellulose overlay medium consisting of 1.5% methyl cellulose in MEM containing 2% FBS. The dishes were then held at 37 C for 5 days in a CO2 incubator. Two of 6 dishes inoculated with each dilution were fixed with methanol and stained with 0.2% crystal violet solution to count the ordinary plaques produced by the CP BVD virus. The overlay medium of the remaining 4 dishes was removed by washing with warmed MEM, and reinoeulated with Nose strain of the CP BVD virus or VSV at a multiplicity of 2 plaque forming units/ cell in each dish was overlaid again with 5 ml of methyl cellulose overlay medium consisting of 1.2% methyl cellulose in MEM. After further incubation for 2 days, the cultures were fixed and stained by the method mentioned above to count the number of reverse plaques. The reverse plaque consisted of the cells infected with NCP BVD virus, which were resistant to Nose strain or VSV, standing out against the background of the disintegrated cell sheet. The reverse plaques were visualized as violet-colored colonies. The NCP BVD viruses isolated from the cells composing reverse plaques were cloned by the limiting dilution method using the procedure of interference with Nose strain or VSV. The cells composing reverse plaques were collected by trypsinization, and then the Nose strain or VSV used as a challenge virus was neutralized with antiserum against each virus. The cells were washed and suspended with MEM and then frozen and thawed three times. The supernatant was used to isolate virus. The cloning procedure by limiting dilution was repeated three times to obtain a pure clone of the isolate. RESULTS AND DISCUSSION

Small and clear conventional plaques were produced in BT cell monolayers by the three CP strains of BVD virus tested. NCP BVD viruses were detected in the CP BVD virus stocks (Nose, NADL and KS86( + ) strains) by the RPF method. Table 1 shows virus titers obtained by conventional plaque and RPF methods. The virus titers of Nose and NADL strains by the conventional plaque method were 10 to 100 times higher than those by the RPF method using Nose strain or VSV as challenge viruses. On the other hand, the virus titers of KS86 ( + ) strain obtained by these method were similar. NCP BVD viruses which interfered with the Nose strain or VSV were isolated from each of the three virus stocks. The isolated NCP BVD viruses were compared by the END method, the homologous interference method using Nose strain, and the heterologous interference method using VSV. By means of END method, these viruses were divided into two groups; an END + group and an E N D - group. These were designated as Nose-END +, NADL-END +, K S 8 6 ( + ) E N D + and Nose-

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TABLE 1 Comparative virus titration of CP BVD virus stocks by the plaque and reverse plaque formation method Strain

Virus titer (PFU/0.2 ml)

Nose NADL KS86(+ )

Plaque a

Nose-RPF b

VSV-RPF ¢

6.3× 106

8.0× 10 ~ 2.7 × 106 2.2× 106

7.0× 104 1.9× 10 ~ 1.7× 106

3 . 0 × 10 7

2.5 X 106

"Virus titer calculated from ordinary plaques which were formed by CP BVD virus. bVirus titer calculated from reverse plaques which were formed by NCP BVD virus after challenged with Nose strain. cVirus titer calculated from reverse plaques which were formed by NCP BVD virus after challenged with VSV. TABLE 2 Antigenic relationship between parent and isolated viruses Strain

Antiserum to strain Nose

Nose Nose-END + Nose-ENDNADL NADL-END + NADL-ENDKS86(+) KS86( + )END + KS86 ( + ) E N D -

4,096" 4,096 4,096

NADL 256 256 256

512 512 512

4,096 2,048 2,048

512 256 1,024

256 256 256

aNeutralizing antibody titer. The titer were expressed as the reciprocal of the highest serum dilution neutralizing the virus.

E N D - , NADL-END-, K S 8 6 ( + ) E N D - strains, respectively. BT cell cultures infected with the isolated NCP BVD viruses showed no CPE after challenge with the CP BVD virus (Nose strain). The END + NCP BVD viruses interfered with only CP BVD virus whereas the E N D - NCP BVD viruses interfered with both CP BVD virus and VSV. These results indicate that the END + NCP BVD virus shows homologous interference but the E N D - NCP BVD virus induces homologous and also heterologous interference. Antigenic properties were compared by the serum neutralization test (Table 2). Although antigenic differences existed among the three CP strains, the

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parental CP BVD virus and isolated NCP BVD virus in each group were antigenically indistinguishable. The RPF method has proved to be highly sensitive for detecting NCP strains of BVD virus (Itoh et al., 1983 ) and hog cholera virus (Fukusho et al., 1976; Laude., 1978 ). When the RPF method was performed using Nose strain as a challenge virus, reverse plaques were produced by either END+ and ENDviruses. However, the reverse plaques were produced only by END- viruses when VSV was used as a challenge virus. Therefore, by selecting a challenge virus, the RPF method described herein could be applicable for the detection of different NCP virus in CP BVD virus stocks. It is interesting to note that both END+ and END- NCP BVD viruses were isolated concomitantly from CP BVD virus stocks. After CP BVD virus stocks were purified by further successive plaque clonings, they also contained NCP BVD viruses (Data not shown). Though the three CP viruses tested were antigenically different to each other, parental CP and pairs of NCP BVD viruses isolated were antigenically identical to each other. It would appear that the mutation from the CP biotype to the NCP biotype occurs during the propagation of CP viruses in vitro. In addition this change in biotype gives rise to 2 groups of NCP viruses with distinct interference patterns. The CP BVD virus can always be isolated from mucosal disease (MD) affected animals in addition to the persisting NCP BVD virus (Brownlie et al., 1984; Bolin et al., 1985; Shimizu et al., 1989). Though field isolates of BVD virus varied antigenically, antigenic similarity was shown between CP and NCP pairs of viruses isolated from individual MD affected animals (Howard et al., 1987; Corapi et al., 1988; Shimizu et al., 1989). This observation led to the hypothesis that CP BVD virus isolated from cattle with MD arose by mutation of the persistent NCP BVD virus (Howard et al., 1987; Corapi et al., 1988). Effect of interaction of CP and NCP BVD viruses on the mechanism of induction of MD is, however, not yet clarified. If the genomic distinction between CP and NCP biotypes is to be clarified, it wilI be essential to examine homologous pairs of viruses (Brownlie., 1990). In the present study, we isolated homologous pairs of CP and NCP (END+ and END-) viruses. These pairs of viruses will be valuable for the detailed investigation of genomic sequences of BVD viruses. Further studies on the mutation of BVD viruses, as well as END+ and END- viruses, will be undertaken. ACKNOWLEDGEMENTS

We thank Dr. L.J. Gleeson (CSIRO, Australian Animal Health Laboratory) for critically reading of the manuscript, Dr. 0. Itoh (our laboratory) for his kind supply of Nose and NADL strains and Dr. M. Shimizu (National

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Institute of Animal Health) for his kind supply of KS86 ( ÷ ) strain of BVD virus.

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