Optimization of in vitro growth conditions for enterotropic murine coronavirus strains

Enterotropic mouse hepatitis virus (MHV) strains have been difficult to grow in cell culture. In an attempt to develop an efficient in vitro cultivation system for enterotropic MHV strains (MHV-RI and MHV-Y), 8 murine cell lines were inoculated with MHV-RI- or MHV-Y-infected infant mouse intestinal homogenates and screened for the production of cytopathic effects. MHV-RI and MHV-Y consistently produced cytopathic effects only in J774A.1 cells. Both strains produced titers of 10(6) TCID(50)/ml in subsequent passages in J774.1 cells. MHV strains -1, -3, -A59, -JHM, -S and -DVIM also produced high-titer viral stocks in J774A.1 cells. Therefore J774A.1 cells are the first cells found that support the replication of these 8 enterotropic and respiratory MHV strains. After passage in J774A.1 cells, MHV-RI and MHV-Y could infect previously non-susceptible cell lines (17C1-1, CMT-93, N18 and NCTC 1469), though cytopathic effects were often negligible with MHV-RI. MHV-Y, but not MHV-RI, grew in L2(Percy) cells. Using L2(Percy) cells, an agarose overlay and Giemsa staining, MHV-Y could be quantified by plaque assay. Infant mouse bioassays, plaque assays and cell culture infections were compared for their sensitivity in detecting MHV-Y in infected intestinal homogenates and cell supernatants.