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Restriction Enzyme Analysis of Tissue Culture-adapted Velogenic Newcastle Disease Virus

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Abstract

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.

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Abbreviations

AAF:

amnioallantoic fluid

CEF:

chicken embryo fibroblasts

CPE:

cytopathic effect(s)

dNTPs:

deoxynucleotide triphosphate

ECE:

embryonated chicken egg

HA:

haemagglutination

ICPI:

intracerebral pathogenicity index

MDT:

mean death time

MMLV:

Moloney murine leukaemia virus

ND:

Newcastle disease

NDV:

Newcastle disease virus

PI:

post-inoculation

RT-PCR:

reverse transcription–polymerase chain reaction

TCID50:

tissue culture infective dose 50%

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Authors and Affiliations

  1. National Biotechnology Centre, Indian Veterinary Research Institute, Izatnagar, 243 122, India

    C. Madhan Mohan & Sohini Dey

  2. Department of Veterinary Microbiology, Madras Veterinary College, Chennai, 600 007, India

    K. Kumanan

Authors
  1. C. Madhan Mohan
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  2. Sohini Dey
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  3. K. Kumanan
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Correspondence to C. Madhan Mohan.

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Mohan, C.M., Dey, S. & Kumanan, K. Restriction Enzyme Analysis of Tissue Culture-adapted Velogenic Newcastle Disease Virus. Vet Res Commun 30, 455–466 (2006). https://doi.org/10.1007/s11259-006-3295-3

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  • DOI: https://doi.org/10.1007/s11259-006-3295-3

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