Plaque formation of Quesland V4 lentogenic strain of Newcastle disease virus adapted in chick embryo fibroblast cells

Abstract

Vaccination is one of the important methods for preventing and controlling of the Newcastle disease in Iran. Inactivated Newcastle disease vaccine is produced in the Razi Vaccine and Serum Research Institute, Iran, and is mainly used for vaccination against Newcastle disease. Clone vaccines are used in many countries nowadays. One of the ways for cloning a virus is propagation of the virus on cell cultures to obtain discrete plaques. In this study, monolayer chick embryo fibroblast (CEF) cell cultures were prepared by a standard method. Monolayer CEF cells were used for plaque production trial. Confluent monolayer CEF cells were in tissue culture plates and inoculated with avian paramyxovirus-1/chicken/Australia/Queensland/V4/1966 (QV4) (V4 seed of Newcastle vaccine) with 109.7 50 % egg infection dose. Various dilutions of the viruses were inoculated into monolayer CEF cell cultures that were supplemented with magnesium sulfate and trypsin and agar overlay medium. No plaques were observed on CEF tissue culture plates inoculated with V4 strain without adaptation. Eight discrete large and small plaques were obtained in the 10−5 dilution of adapted viruses (titer 3 × 107 plaque-forming units (PFU)/ml) with 2–4 mm diameter at 80 h post inoculation, and in the 10−7–10−10 dilution, there was no any plaque obtained. A dilution of 10−4 produced 28 plaques (1.75 × 108 PFU/ml) but not discrete plaque, and at 10−1–10−3 dilution, plaques were too much that they were not countable. Eight various plaques at 10−5 were collected and propagated for further investigations. The present assay is a useful tool for demonstration of discrete plaques using the adapted v4 of Newcastle disease virus within 3 days on monolayer CEF cell cultures covered with modified overlay media containing magnesium ions, diethylaminoethyl dextran, and ionoagar to ascertain the purity. This technique for plaque formation by v4 of Newcastle disease virus provides a method for establishing the purity of seed stock used in the production of vaccine.