Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2000 Mar 29;52(3):301–307. doi: 10.1016/0166-0934(94)00161-9

Optimization of in vitro growth conditions for enterotropic murine coronavirus strains

SR Compton 1,∗, DF Winograd 1, DJ Gaertner 1
PMCID: PMC7119611  PMID: 7601904

Abstract

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 106 TCID50/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.

Keywords: Murine coronavirus, Mouse hepatitis virus strain RI and Y, Enterotropism, J774A.1 cell, Plaque assay

References

  1. Barthold S.W. Mouse hepatitis virus biology and epizootiology. In: Bhatt P.N., Jacoby R.O., editors. Viral and Mycoplasmal Infections of Laboratory Rodents. Academic Press; New York: 1986. pp. 571–601. [Google Scholar]
  2. Barthold S.W. Host age and genotypic effects on enterotropic mouse hepatitis virus infection. Lab. Anim. Sci. 1987;37:36–40. [PubMed] [Google Scholar]
  3. Barthold S.W., Smith A.L., Lord P.F.S., Bhatt P.N., Jacoby R.O., Main A.J. Epizootic coronaviral typhlocolitis in suckling mice. Lab. Anim. Sci. 1982;32:376–383. [PubMed] [Google Scholar]
  4. Barthold S.W., Smith A.L., Povar M.L. Enterotropic mouse hepatitis virus infection in nude mice. Lab. Anim. Sci. 1985;35:613–618. [PubMed] [Google Scholar]
  5. Barthold S.W., Beck D.S., Smith A.L. Enterotropic coronavirus (mouse hepatitis virus) in mice: influence of host age and strain on infection and disease. Lab. Anim. Sci. 1993;43:276–284. [PubMed] [Google Scholar]
  6. Casebolt D.B., Stephensen C.B. Monoclonal antibody solution hybridization assay for detection of mouse hepatitis virus infection. J. Clin. Microbiol. 1992;30:608–612. doi: 10.1128/jcm.30.3.608-612.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Compton S.R., Barthold S.W., Smith A.L. The cellular and molecular pathogenesis of coronaviruses. Lab. Anim. Sci. 1993;43:15–28. [PubMed] [Google Scholar]
  8. Gaertner D.J., Winograd D.F., Compton S.R., Paturzo F.X., Smith A.L. Development and optimization of plaque assays for rat coronaviruses. J. Virol. Methods. 1993;43:53–64. doi: 10.1016/0166-0934(93)90089-A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Homberger F.R., Smith A.L., Barthold S.W. Detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction. J. Clin. Microbiol. 1991;29:2789–2793. doi: 10.1128/jcm.29.12.2789-2793.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kunita S., Terada E., Goto K., Kagiyama N. Sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction. Lab. Anim. Sci. 1992;42:593–598. [PubMed] [Google Scholar]
  11. Lucas A., Flintoff W., Anderson R., Percy D., Coulter M., Dales S. In vivo and in vitro models of demyelinating diseases: tropism of JHM strain of murine hepatitis virus for cells of glial origin. Cell. 1977;12:553–560. doi: 10.1016/0092-8674(77)90131-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Reed L.J., Muench H. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 1938;27:493–497. [Google Scholar]
  13. Yamada Y.K., Yabe M., Yamada A., Taguchi F. Detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection. Lab. Anim. Sci. 1993;43:285–290. [PubMed] [Google Scholar]

Articles from Journal of Virological Methods are provided here courtesy of Elsevier

RESOURCES