Plaque formation by Newcastle virus strain V4 on cell culture and characterization with RT-PCR

Cloned vaccines are used in many countries nowadays. One of the ways for cloning a virus is propagation of the virus on cell culture to obtain discrete different plaques in order to study their morphology and genetics. In this study monolayer Madin-Darby Canine Kidney (MDCK) cell cultures were prepared by standard method. Various dilutions of the viruses were inoculated into monolayer MDCK cell cultures that were supplemented with magnesium sulfate and trypsin, and over laid with agar medium. The viruses could reproduce on these cells and caused cytopathic effect and plaques. At 10-6 virus dilution, 6 various shape and size discrete plaques were obtained and inoculated into allantoic fluid 9-11 days embryonated eggs. After 48 hrs, the allantoic fluids contain plaques were harvested and their RNA extracted. Cleavage site of fusion protein, with RT-PCR test was performed and the PCR products were purified and sequenced. The sequences of nucleotides and amino acids for each plaque were compared with those of the registered strain at gene bank as well as with each other. Molecular studies showed that all plaques are lentogenic strain of Newcastle disease virus and has about 97% to 99% homology with the strain V4 in the gene bank. The aim of this study is produce clear plaque by V4 strain of NDV on MDCK cell line and studies the molecular variations among them.