Antigenic differentiation of mouse hepatitis viruses by neutralization test.

Abstract

Mouse hepatitis viruses (MHV) are members of the coronavirus group, which contain a single-stranded polyadenylated RNA genome (13, 18,20). The discovery of cell lines suitable for MHV replication and the development of assay systems have made it possible to investigate the mode of replication and other important characteristics of MHV (6,21,24). As a result, some information has become available on the molecular aspects of MHV multiplication in cultured cells (13, 17, 18,24). MHV is also known to cause hepatitis and demyelinating disease in mice. Variation in pathogenicity and organ tropism among many strains has been shown to occur (8, 16). In addition, studies of recent isolates of MHV from athymic nude mice dying with wasting disease (9) or from suckling mice with severe diarrhea (5, 10, 12) revealed some new aspects of the pathogenicity of MHV in mice (10, 11). However, despite extensive studies of MHV multiplication and many pathological examinations, little is known about the serological differences among MHV strains (5,24), though cross-reactivity of some strains of MHV with other coronaviruses has been demonstrated (16). In this communication, we report serological differences among six established and six freshly isolated strains of MHV, obtained by the neutralization test (NT) which is the only currently available method for the differentiation of closely related MHV strains. The MHV strains MHV-l (4), MHV-2 (15), MHV-3 (2), JHM (1), MHV-S (19), and MHV-A59 (14) were kindly supplied by Dr. ].e. Parker, Microbiological Associates, Inc., Bethesda, Md. MHV-A, MHV-U, MHV-Nu66 (9), MHV-K (11), and MHV-Nu67 were isolated from the livers of nude mice with hepatitis, and MHV-D was isolated from the intestines of suckling mice with severe diarrhea (10). These viruses were propagated in DBT cells (6) which were grown in Eagle's minimal essential medium (EMEM, Nissui, Tokyo) supplemented with 10% tryptose phosphate broth (TPB, Difco, Detroit, Mich.) and 8% calf serum. The virus stock used in the NT was prepared as described previously (22). Antisera to MHV-3, JHM, and MHV-S were produced in rabbits by injection of purified viruses emulsified with Freund's complete adjuvant, as previously reported (23). For NT, a modification of the method of Dulbecco (3) was employed, which was more convenient than the original method and more sensitive than the serum