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Veterinary Research Communications

, Volume 31, Issue 3, pp 371–383 | Cite as

Adaptation of a Velogenic Newcastle Disease Virus to Vero Cells: Assessing the Molecular Changes Before and After Adaptation

  • C. Madhan Mohan
  • Sohini Dey
  • K. Kumanan
  • B. Murali Manohar
  • A. Mahalinga Nainar
Article

Abstract

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.

Keywords

Newcastle disease virus Vero cells fusion gene sequencing studies pathogenicity testing 

Abbreviations

AAF

amnioallantoic fluid

CPE(s)

cytopathic effect(s)

dNTPs

deoxynucleotide triphosphate

ECE

embryonated chicken eggs

FPCS

fusion protein cleavage site

HA

haemagglutination

HI

haemagglutination inhibition

ICPI

intracerebral pathogenicity index

MDT

mean death time

MuMLV

Moloney murine leukemia virus

ND

Newcastle disease

NDV

Newcastle disease virus

p.i.

post inoculation

RT-PCR

reverse transcription-polymerase chain reaction

TCID50

tissue culture infective dose 50%

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Copyright information

© Springer Science + Business Media, Inc. 2007

Authors and Affiliations

  • C. Madhan Mohan
    • 1
    • 2
  • Sohini Dey
    • 1
    • 2
  • K. Kumanan
    • 1
  • B. Murali Manohar
    • 1
  • A. Mahalinga Nainar
    • 1
  1. 1.Department of Animal BiotechnologyMadras Veterinary CollegeChennai
  2. 2.Division of Veterinary BiotechnologyIndian Veterinary Research InstitiuteBareillyIndia

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