DEVELOPMENT AND OPTIMIZATION OF PLAQUE ASSAYS FOR RAT CORONAVIRUSES

Plaque assays under Sephadex or agarose overlays are described for rat coronaviruses (RCVs) grown in L2 mouse fibroblasts. A plaque assay us ing Sephadex was simple; however, viable plaques could not be collecte d for propagation, and fixation was necessary before evaluation. Plaqu e formation under agarose was optimized using diethylaminoethyl-dextra n (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-t reatment media contained DEAE-D, absorption media contained DEAE-D and trypsin, and the agarose overlay contained trypsin. The assay was use ful for plaque isolation and quantification of sialodacryoadenitis vir us (SDA), Parker's rat coronavirus (PRCV) and other coronavirus isolat es from rats and its specificity was demonstrated by plaque-reduction neutralization testing. These methods will facilitate production of cl oned virus stocks for study of RCV biology and virus quantification fo r in vitro and in vivo studies of RCVs.