Adaptation of a velogenic Newcastle disease virus to vero cells: assessing the molecular changes before and after adaptation.
A velogenic Newcastle disease virus isolate was passaged 50 times in Vero cell culture and the virus was assessed for the molecular changes associated with the passaging. At every 10th passage, the virus was characterized conventionally by mean death time (MDT) analysis, intracerebral pathogenicity index (ICPI) and virus titration. At increasing passage levels, a gradual reduction in the virulence of the virus was observed. Molecular characterization of the virus included cloning and sequencing of a portion of the fusion gene (1349 bp) encompassing the fusion protein cleavage site (FPCS), which was previously amplified by reverse transcription-polymerase chain reaction. Sequence analysis revealed a total of 135 nucleotide substitutions which resulted in the change of 42 amino acids between the velogenic virus and the 50th passage virus. The predicted amino acid motif present at the cleavage site of the virulent virus was (109)SRRRRQRRFVG(119) and the corresponding region of the adapted adapted virus was (109)SGGRRQKRFIG(119). Pathogenicity studies conducted in 20-week-old seronegative birds revealed gross lesions such as petechial haemorrhages in the trachea, proventricular junction and intestines, and histopathological changes such as depletion and necrosis of the lymphocytes in thymus, spleen, bursa and caecal tonsils in the birds injected with the velogenic virus and absence of the lesions in birds injected with the adapted virus. The 50th-passage cell culture virus was back-passaged five times in susceptible chickens and subjected to virulence attribute analysis and sequence analysis of the FPCS region, with minor difference found between them.
Mohan
C Madhan
CM
Department of Animal Biotechnology, Madras Veterinary College, Chennai, India. sohinimadhan@yahoo.com
Dey
Sohini
S
Kumanan
K
K
Manohar
B Murali
BM
Nainar
A Mahalinga
AM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002645:Chickens; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003588:Cytopathogenic Effect, Viral; D008297:Male; D008969:Molecular Sequence Data; D009521:Newcastle Disease / Q000821:virology*; D009522:Newcastle disease virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D014709:Vero Cells / Q000821:virology*; D014760:Viral Fusion Proteins / Q000235:genetics; D014774:Virulence
D012367:RNA, Viral; D014760:Viral Fusion Proteins