Development and optimization of plaque assays for rat coronaviruses

Abstract

Plaque assays under Sephadex or agarose overlays are described for rat coronaviruses (RCVs) grown in L2 mouse fibroblasts. A plaque assay using Sephadex was simple; however, viable plaques could not be collected for propagation, and fixation was necessary before evaluation. Plaque formation under agarose was optimized using diethylaminoethyl-dextran (DEAE-D) in the pre-treatment and absorption media and trypsin added to the absorption media and agarose overlay. The use of DEAE-D alone, trypsin alone or trypsin combined with DEAE-D significantly increased plaque numbers and visibility. Plaque numbers were highest when pre-treatment media contained DEAE-D, absorption media contained DEAE-D and trypsin, and the agarose overlay contained trypsin. The assay was useful for plaque isolation and quantification of sialodacryoadenitis virus (SDA), Parker's rat coronavirus (PRCV) and other coronavirus isolates from rats and its specificity was demonstrated by plaquereduction neutralization testing. These methods will facilitate production of cloned virus stocks for study of RCV biology and virus quantification for in vitro and in vivo studies of RCVs.

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

“…Two L2(Percy) sublines, L2 41.a and L2 29.a were obtained from K. Holmes (Bethesda, MD, USA). Cells were maintained as previously described (Gaertner et al, 1993). Cells were tested for MycopIasma spp.…”

“…Three wild-type isolates of RCV were studied. RCV-WT was isolated from outbred Sprague Dawley (SD) rats immediately after receipt from a commercial facility housing rats endemically infected with RCV (Paturzo, 1987;Gaertner et al, 1993). Lung homogenates were inoculated into mouse pups to produce mouse brain suspensions and into L2(Percy) cells to produce the plaques used in these studies.…”

“…For virus titrations, three or more replicate 2.0 cm 2 wells of cells were exposed to 0.3 ml of inoculum per well for each virus dilution. Agarose overlays were prepared as previously described (Gaertner et al, 1993). Plates were fixed at 48 h with 10% buffered formalin and were stained with Giemsa, plaques were counted and titers were expressed as PFU per ml (Lorenz and Bogel, 1973).…”

“…Plates were fixed at 48 h with 10% buffered formalin and were stained with Giemsa, plaques were counted and titers were expressed as PFU per ml (Lorenz and Bogel, 1973). Individual plaques were collected, stored as previously described (Gaertner et al, 1993) and were inoculated into cells growing in 25 cm 2 flasks in attempts to produce plaquecloned stocks. Flasks were observed daily, harvested 48 or 72 h after inoculation and virus in infected cell lysates was quantified.…”

“…Rat coronaviruses (RCVs) are agents which commonly infect laboratory rats. RCV infections stocks of prototype RCVs in the Percy subline of mouse fibroblast L2 cells (Percy et al, 1989(Percy et al, , 1990 and improved virus quantification and yields in L2(Percy) cells treated with dietheylaminoethyl dextran (DEAE-D) and trypsin (Gaertner et al, 1991(Gaertner et al, , 1993. These improvements in culture techniques have facilitated the production of high titered stocks of previously cultivated RCV isolates and production of individual RCV plaques for further study, but when individual plaques were put back into L2(Percy) cells, the resulting stocks had only low titers (Gaertner, unpublished).…”