Growth characteristics and protein profiles of prototype and wild-type rat coronavirus isolates grown in a cloned subline of mouse fibroblasts (L2p.176 cells)

Abstract

Rat coronaviruses (RCVs) infect laboratory rats and confound biomedical research results. In vitro systems developed so far have limited the growth in knowledge about RCVs by not permitting generation of plaque-cloned virus stocks, reliable isolation of RCVs from rat tissues, or growth of high titered stocks of all isolates. Due to the fact that less than 20% of L2(Percy) cells were becoming infected, sublines were produced and selected for maximal growth of RCVs. Screening of 238 cell sublines yielded L2p.176 cells which were highly susceptible to all RCVs tested; however, susceptibility declined after 30 passages in vitro. Low-passaged L2p.176 cells were used to isolate virus from natural outbreaks and to propagate individual RCV plaques into high titered stocks. Proteins from six RCV isolates were immunoblotted using polyclonal rat and mouse antibodies to sialodacryoadenitis virus and polyclonal monospecific rabbit and goat antibodies against the peplomer (S) and nucleocapsid (N) proteins of mouse hepatitis virus (MHV). Proteins of two prototype, one Japanese and three wild type RCVs were examined and found to be similar to those of MHV, although the exact sizes and ratios of protein forms were unique for most RCV isolates. This study reports the development of a continuous cell line which reliably supports RCVs opening an opportunity for further in vivo studies of the biology of these agents. As a first step in the characterization of RCVs, we have shown that RCV proteins are very similar to those of MHV.

SDAV, the Rat Coronavirus—How Much Do We Know about It in the Light of Potential Zoonoses

“…As currently known, SDAV in vitro propagation is possible in established cell lines, such as murine fibroblasts L-2(Percy) cells, subclone L2P-41.a, LBC cells, RCV-9, and in primary rat kidney cell cultures (RKCC). In other frequently used cell lines (VERO, Hep-2, NTCT 1469, BHK-21), infection is not possible [ 15 , 28 , 70 , 101 , 102 , 103 , 104 ]. For purification and plaque assay, L-2cells, L2P-41.a, and LBC are often used [ 100 , 101 ].…”

“…In other frequently used cell lines (VERO, Hep-2, NTCT 1469, BHK-21), infection is not possible [ 15 , 28 , 70 , 101 , 102 , 103 , 104 ]. For purification and plaque assay, L-2cells, L2P-41.a, and LBC are often used [ 100 , 101 ]. The virus samples can be stored at −60 °C for 7 years, but according to research by Jacoby et al (1975), they can become less infectious when stored at −20 °C [ 11 , 105 ].…”

Viral Disease

“…RCV replicates in vitro in primary rat kidney cells, forming multinucleate syncytia typical of coronaviral infection (Bhatt et al, 1972). Virus can be detected antigenically in infected cells within 12 h and cytopathic effects can develop within 24 h. Mouse L2 fibroblasts and sublines, as well as rat LBC cells and intestinal RCV-9 cells, can also be infected (Gaertner et al, 1996a;Hirano et al, 1986;Ohsawa et al, 1996;Percy et al, 1989Percy et al, , 1990b, and L2 sublines and LBC cells can also be used for plaque assays and plaque purification (Gaertner et al, 1993b).…”

“…RCV spreads primarily by direct contact with infected rats or by aerosol, and RCV remains infectious for at least 2 days when dried on plastic surfaces (Gaertner et al, 1996a). Experimental studies have demonstrated the ease of horizontal transmission of RCV (La Regina et al, 1992), but there is no evidence for intrauterine transmission.…”