title: 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 plaque-reduction 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.
authors.affiliation: Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: D J
authors.initials: DJ
authors.lastname: Gaertner
authors.forename: D F
authors.initials: DF
authors.lastname: Winograd
authors.forename: S R
authors.initials: SR
authors.lastname: Compton
authors.forename: F X
authors.initials: FX
authors.lastname: Paturzo
authors.forename: A L
authors.initials: AL
authors.lastname: Smith
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D002460:Cell Line; D003332:Coronaviridae / Q000254:growth & development* / Q000276:immunology / Q000302:isolation & purification; D003333:Coronaviridae Infections / Q000276:immunology; D003637:DEAE-Dextran; D003911:Dextrans; D005347:Fibroblasts / Q000382:microbiology; D051379:Mice; D009500:Neutralization Tests; D051381:Rats / Q000276:immunology / Q000382:microbiology*; D012685:Sepharose; D013047:Specific Pathogen-Free Organisms; D014357:Trypsin; D010948:Viral Plaque Assay; D014776:Virus Cultivation
chemical_list: D000914:Antibodies, Viral; D003911:Dextrans; D012685:Sepharose; C025614:sephadex; D003637:DEAE-Dextran; D014357:Trypsin