Restriction enzyme analysis of tissue culture-adapted velogenic Newcastle disease virus.
A velogenic Newcastle disease virus isolate typed to belong to group C1 by monoclonal antibody typing was adapted 50 times in chicken embryo fibroblast cell culture and 60 times in Vero cells. At every 10th passage the virus was characterized on the basis of mean death time, intracerebral pathogenicity indices and viral titration studies. A gradual reduction in the virulence of the virus was noted as the passage number increased. RT-PCR of a 254 bp region of the fusion gene encompassing the fusion protein cleavage site was carried out for the virulent as well as cell culture-adapted viruses at every 10th passage level. The amplicons were subsequently digested with three restriction enzymes, viz. AluI, HaeIII and PstI. It was found out that there was difference in banding patterns between the virulent and adapted viruses, indicating nucleotide substitutions in the virulent virus when it was sequentially passaged onto cell culture systems.
Mohan
C Madhan
CM
National Biotechnology Centre, Indian Veterinary Research Institute, Izatnagar, 243 122, India.
Dey
Sohini
S
Kumanan
K
K
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002645:Chickens; D002522:Chlorocebus aethiops; D005347:Fibroblasts / Q000166:cytology / Q000821:virology*; D008969:Molecular Sequence Data; D009522:Newcastle disease virus / Q000235:genetics* / Q000254:growth & development*; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping / Q000379:methods*; D014709:Vero Cells; D014779:Virus Replication