key: cord-281410-y558a5jf authors: Akashi, H.; Inaba, Y.; Miura, Y.; Sato, K.; Tokuhisa, S.; Asagi, M.; Hayashi, Y. title: Propagation of the Kakegawa strain of bovine coronavirus in suckling mice, rats and hamsters date: 1981 journal: Arch Virol DOI: 10.1007/bf01314841 sha: doc_id: 281410 cord_uid: y558a5jf The Kakegawa strain of bovine coronavirus was easily propagated in suckling mice. Infected animals died with nervous symptoms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. The 3rd passage viral material from infected mice evoked the same disease in suckling mice, rats and hamsters inoculated by the intracerebral or by the subcutaneous route. Viruses recovered from mice, rats and hamsters could be clearly differentiated from mouse hepatitis virus strain 2 by the neutralization test. W'ith 1 Figure Accepted November 5, 1980 Summary The Kakegawa strain of bovine coronavirus was easily propagated in suckling mice. Infected animals died with nervous symptoms, and serial passage was readily accomplished by intraeerebral inoculation with brain emulsions. The 3rd passage viral material from infected mice evoked the same disease in suckling mice, rats and hamsters inoculated by the intracerebral or by the subcutaneous route. Viruses recovered from mice, rats and hamsters could be clearly differentiated from mouse hepatitis virus strain 2 by the neutralization test. A bovine coronavirus (BCV) has been recognized as one of the causative agents of diarrhea in calves (8) . l~ecently the Kakagawa strain isolated from the feces of a cow with cpizootic diarrhea has been identified as BCV (1, 9) . However, the study of bovine diarrheal disease produced by coronavirus has been greatly hampered by difficulty in isolating virus in cell cultures and the lack of experimental hosts other than cattle. Recently many members of the eorona~drus family" have been found to grow in suckling mice (5--7). The present paper describes briefly our recent observation that the Kakegawa strain of BCV readily propagates in suckling mice, rats and hamsters. The Kakegawa strain of BCV used for mouse inoculation was at, the 10th passage level in primary bovine kidney cell cultures. Conventionally reared oneday-old mice (strain ddY), rats and Syrian hamsters were each inoculated with 0.01-ml of the viral materials by the intracerebral (ic) route and with 0.02-ml by the subcutaneous (s.c.) route. Virus recovery and infectivity assay were carried out. by inoculation into cell cultures of BEK-1 ecll, derived from bovine embryonic kidney (3). The a:nti-Kakegawa strain immune serum having a homologous neutralizing antibody titer of 4096 was prepared in specific germ-free rabbits with virus grown in BEK-1 cells as described previously (1), and the anti-mouse hepatitis virus (MHV) strain 2 rabbit immune serum having a neutralizing antibody titer of about 10,000 by 50 per cent plaque reduction neutralization test was kindly supplied by Dr. K. Fujiwara, University of Tokyo, Japan. Neutralization (NT) test was carried out by the method described previously (1) . Viruses recovered from the brains of affected animals containing 200 TCIDs0 were mixed with an equal volume of two-fold serum dilutions. Each mixture was assayed for infectivity by using two tubes of BEK-1 cell cultures per dilution. The antibody titer was expressed as the reciprocal of the highest serum dilution that showed no cytopathic changes in at least, one of the two tubes. Fig. 1 . Four-day-old mouse 3 days after intraeerebral inoculation with the 3rd mouse brain passaged Kakegawa strain (left) and an uninoeulated 4-day-old mouse (right) Four litters (10 to 12/litter) of suckling mice were inoculated intracerebrally with infected tissue culture fluid containing 106-5 TCID50/ml. At daily intervals, four animals were sacrificed and their brains taken. The inoculated animals developed an illness 4 days post-inoculation (p. i.) and began to die at 5 days p. i. The affected animals became anemic and showed mild neural symptoms such as weak limbs and sluggish staggering gait. The virus titers of infected brains rose at 2 days p. i. with a subsequent gradual rise to a maximum of 10 s.~ TCIDs0/g at 5 days p. i. Serial passage in suckling mice was readily accomplished by the i. c. route with 10 per cent, (w/v) brain emulsions (Fig. 1) . The incubation period fell to 2 to 3 days by the 3rd passage. Uninoculated mice and mice inoculated with normal brain emulsions remained healthy. Virus at the 3rd passage level in suckling mice containing 105.2 TCIDs0/ml also produced a clinical illness with the same symptoms described above. It caused death in all animals when ineoulated into one litter of suckling mice (10/litter) by the s.c. route, and into rats (9 or 12/litter) and hamsters (7 or 8/litter) by the i.e. or s.c. route. Table 1 Neutralizing antibody titer The results of one way cross NT test are summarized in Table 1 . The homologous NT titers were much higher than the heterologous liters. These findings showed that the viruses recovered from the brains of affected animals were antigenieally the same as the cell culture passaged virus and could be clearly differentiated from the MttV strain 2. A histological study of affected animals showed inflammation in the brains and vacuolar degeneration of the intestinal villi only in the case of s.c. inoculation. However, there was no evidence of inflammation in the liver and other organs (Dr. 3/[. Kubo, personal communiea, tion). KAYE et al. (4) reported that they had adapted the American strain of calf diarrhea coronavirus to suckling mouse brain. On ~he other hand, DEA et al. (2) reported that the American strain and their new isolates of calf diarrhea coronavirus were not virulent for suckling mice, hamsters and guinea pigs. Our present results support Kaye's findings. NT tests and histological studies have ruled out contamination with MHV. Further studies, however, wil] be necessary to investigate the immunological and pa~thological differences between BCV and MI-IV. Properties of a coronavirus isolated from a cow with epizootic diarrhea Physicochemical and biological properties of neonatal calf diarrhea eoronaviruses isolated in Quebec and comparison with the Nebraska calf eoronavirus Replication of bovine coronavirus in cell line BEN-i culture Calf diarrhoea coronavirus Antigenic relationship between human eoronavirus strain OC 43 and hemagglutinating encephalomyelitis virus strain 67N of swine: Antibody responses in human and animal sera Coronaviruses: A comparative review Feline Infectious Peritonitis Virus. II. Propagation in suckling mouse brain Neonatal calf diarrhea: Purification and electron microscopy of a coronavirus-Iike agent Epizootic diarrhoea of adult cattle associated with a coronavirus-like agent Authors' address: Dr. H. AKAS~I, National Institute o~' Animal ttealth A-1011 Wien. Fiir den Text~eil verantwortlieh: Dr. Wilhelm Schwabl, M61kerbastei 5, A-1011 WiSh. Fiir den Anzeigenteil verantwortlieh: NIag. Bruno Schweder, M61kerbastei 5, A-1011 Wien