A. Mahalinga Nainar

A simplified objective method for quantification of peste des petits ruminants virus or neutralizing antibody

A simplified and standardized assay based on haemagglutination of infected culture supernatants was developed to detect peste des petits ruminants (PPR) virus growth in vero cells and to quantify PPR neutralizing antibody. Virus titres estimated by visual reading of cytopathic effects as a criterion were compared with those estimated by haemagglutination of infected supernatants and no statistically significant differences were seen between them. During quantification of PPR antibodies, the titres based on haemagglutination of supernatants on day 5 post-infection had a high sensitivity, specificity and agreement in qualititative comparison with those determined by visual examination of cytopathic effects. In quantitative comparison, the correlation coefficient between serum neutralization titres estimated by visual examination of cytopathic effects or haemagglutination of supernatants was 0.96, when haemagglutination was done on day 5 post-infection. The virus and serum neutralization titres can thus be estimated objectively using the haemagglutination of supernatants as criterion to measure endpoints. The haemagglutination-inhibition titres also correlated well with serum neutralization titres with a coefficient of 0.78. Thus the haemagglutination-inhibition test appears to be a suitable alternative to the serum neutralization test for quantification of PPR neutralizing antibody.

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 109SRRRRQRRFVG119 and the corresponding region of the adapted adapted virus was 109SGGRRQKRFIG119. 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.