End-point dilution-fluorescent antibody technique for cloning hog cholera virus

Hog cholera virus was cloned by incubating selected pretitrated dilutions of the virus on PK-15 cell cultures for 2 hours. After a thorough washing, the coverslip cell cultures were overlaid with medium containing 0.1% hog cholera immune serum to prevent secondary foci. Forty-eight hours later, the cultures were vigorously washed and maintenance medium containing 5% bovine fetal serum was added. When examined by the fluorescent antibody technique 18 hours later, single plaques were observed in some cultures with no evidence of secondary foci. The virus clone subsequently yielded a homogeneous population of hog cholera virus that retained the characteristics of the parent strain; pathogenicity of the virus clone in pigs was demonstrated, and specific immunofluorescence occurred in infected cell cultures stained with fluorescein isothiocyanate-labeled antibody. The method used gave reasonable assurance of the cloned virus' freedom from extraneous agents.