key: cord-267155-3rhdxzj3 authors: Sato, K.; Inaba, Y.; Matumoto, M. title: Serological relation between calf diarrhea coronavirus and hemagglutinating encephalomyelitis virus date: 1980 journal: Arch Virol DOI: 10.1007/bf01314983 sha: doc_id: 267155 cord_uid: 3rhdxzj3 Neutralizing (NT) and hemagglutination-inhibiting (HI) antibodies to calf diarrhea coronavirus (CDCV) and hemagglutinating encephalomyelitis virus of swine (HEV) (strain 67 N) were detected in high proportions of normal adult cattle and pigs in Japan. Since comparison of NT and HI titers in the serum samples suggested an antigenic difference between the viruses, cross NT and HI tests of these viruses were carried out with antisera raised in rabbits. The homologous NT titers were markedly higher than the heterologous titers. In HI tests essentially the same results were obtained. These findings indicate the presence of a marked difference in antigenic make-up between CDCV and HEV. NT and HI tests can clearly differentiate the viruses, although there is some cross reaction. Neutralizing (NT) and hcmagglutination-inhibiting (HI) antibodies to calf diarrhea, coronavirus (CDCV) and hemagglutinating encephalomyelitis virus of swine (HEV) (strain 67N) were detected in high proportions of normal adult cattle and pigs in Japan. Since comparison of NT and H I titers in the serum samples suggested an antigenic difference between the viruses, cross NT and H I tests of these viruses were carried out with autisera raised in rabbits. The homologous NT titers were markedly higher than the heterologous titers. In H I tests essentially the same results were obtained. These findings indicate the presence of a marked difference in antigenic make-up between CDCV and HEV. NT and H I tests can clearly differentiate the viruses, Mthough there is some cross reaction. $ Coronaviruses have been reported to be divided into two serological groups by the aid of the fluorescent antibody technique (4). Calf diarrhea coronavirus (CDCV) and hemagglutinating encephalomyelitis virus (HEV) 67N of swine were classified in one group, together with mouse hepatitis virus type 3 and human coronavirus 0C43. The other group comprized feline infections peritonitis virus, transmissible gastroenteritis virus of swine, canine coronavirus and human coronavirus 229E. Viruses in each group were antigenically related to each other to varying degrees, but were antigenieally unrelated to eoronaviruses of the second group. During the studies on CDCV and H E V we happened to test some normal bovine and swine sera for neutralizing (NT) and hcmagglutination-inhibiting (HI) antibodies to these viruses and obtained data suggestive of a marked antigenic difference between the viruses. Accordingly we conducted a direct comparison between the viruses by NT and H I tests using antisera raised in rabbits. CDCV passaged in bovine embryonic kidney cells (2) was kindly supplied b y Dr. C. A. Mebus, University of Nebraska, U.S.A. I n our l a b o r a t o r y the virus was shown to readily replicate and induce cytopathie effect in cultures of a continuous cell line, BEK-1, derived from bovine embryonic kidney, thus providing a sensitive, practical assay m e t h o d and a satisfactory source of the virus (1). I n the present s t u d y virus grown in BEK-1 cells was used. Strain 67N of H E V (3) was kindly supplied b y Dr. K. Hirai, Gifu University, J a p a n , and was grown in p r i m a r y swine k i d n e y cultures. NT tests were carried out b y the serum dilution m e t h o d using tube cultures of B E K -i cells with CDCV and those of p r i m a r y swine kidney with H E V . Virusserum mixtures were incubated at 37 ° C for 1 hour before inoculation into 2 tube cultures per serum dilution. The virus dose per tube was 100 TCID50. The antibody titer was expressed as the reciprocal of the highest serum dilution which shouted neutralization in at least one of the 2 tubes. An a n t i b o d y titer of 2 or higher was t a k e n as positive. H I tests were carried out b y the microtiter m e t h o d (5, 6) . Infectious culture fluid was used as H A antigen. F o r H I tests the serum was inactivated at 56 ° C for 30 minutes and t r e a t e d with Kaolin to remove non-specific inhibitors and with packed chicken erythrocytes to remove antibodies to the erythrocytes. The antigenserum mixtures were incubated at room t e m p e r a t u r e for 1 hour, mixed with chicken e r y t h r o e y t e suspension and incubated at room t e m p e r a t u r e for 1 hour. The H I a n t i b o d y titer was expressed as the reciprocal of the highest serum dilution showing complete H I and titers of 2 or higher were taken as positive. Serum samples from 10 normal adult cattle and 59 pigs were tested for NT and H I antibodies to CDCV and HEV. Many of the bovine and porcine serum samples contained N T and H I antibodies to these viruses, indicating dissemination of the viruses or antigenically related viruses in these animals. F u r t h e r m o r e , the bovine sera had m a r k e d l y higher N T titers to CDCV t h a n H E V , while the porcine sera showed similar N T titers against b o t h viruses; NT titer ratio, ttEV/CDCV, ranged from 1:64 to 1:4096 with bovine sera and from 1:1/4 to 1 : 16 with porcine sera. Representative results are shown in Table 1 . H I titers of bovine sera were Table 1 2 512 5t2 20 40 3 512 32 10 40 4 t28 i28 l0 40 5 128 128 40 40 also higher to CDCV than HEV, although they were low; HI titer ratio, HEV/ CDCV, ranged from 1:2 to 1:16. HI titers of porcine sera were very low to both viruses. HI titers of representative sera are also shown in Table i . These results suggested that there are antigenic differences between these two viruses. Therefore, we conducted a direct comparison between CDCV and HEV by cross NT and I-II tests. The antisera used were prepared in specific pathogen free rabbits with virus grown in cultured cells and concentrated by ultracentrifugation (i). The animals received an intravenous dose of the virus suspension, followed at 3 and 6 weeks intervals, by two intramuscular doses of the virus suspension mixed with Freund's complete adjuvant, and sera were obtained 3 weeks after the last dose. The results are summarized in Table 2 . The homologous NT titers were much higher than the heterologous titers. In HI tests similar results were also obtained. All the serum samples obtained before the immunization were invariably negative in these tests. These findings show the presence of a marked difference in the antigenic makeup between these two viruses. NT and I-II tests can clearly differentiate the viruses, although there is some cross reaction. Replication of bovine eoronavirus in cell line B E K -1 culture Neonatal calf diarrhea : propagation, attenuation and characteristics of a coronavirus-like agent Characteristics of a eoronavirus (strain 67N) of pigs Antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species Characteristics of a coronavirus causing vomition and wasting in pigs Hemagglutination by calf diarrhea coronavirus