Skip to main content
SpringerLink
Log in

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

  • Published:
Veterinary Research Communications Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

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%

References

  • Alexander, D.J., 2001. Newcastle disease. British Poultry Science, 42, 5–22

    PubMed  CAS  Google Scholar 

  • Alexander, D.J. 1997. Newcastle disease and other paramyxoviruses. In: Diseases of Poultry, 10th ed (Iowa State University Press, Ames, IA), 541–570

    Google Scholar 

  • Alexander, D.J. and Parsons, G., 1984. Avian paramyxovirus type I infection of racing pigeons: pathogenicity experiments in pigeons and chickens. The Veterinary Record, 114, 466–469

    PubMed  CAS  Google Scholar 

  • Allan, W.H., Lancaster, J.E. and Toth, B., 1978. Newcastle disease vaccines, their production and use. FAO Animal Production and Health Series No. 10 (FAO, Rome)

  • Ballagi Pordany, A., Wehmann, E., Herczeg, J., Belak, S. and Lominiczi, B., 1996. Identification and grouping of Newcastle disease virus strains by restriction site analysis of a region from the F gene. Archives of Virology, 141, 243–261

    Article  PubMed  CAS  Google Scholar 

  • Collins, M.S., Bashiruddin, J.B. and Alexander, D.J., 1993. Deduced aminoacid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity. Archives of Virology, 128, 363–370

    Article  PubMed  CAS  Google Scholar 

  • Collins, M.S., Strong, I. and Alexander, D.J., 1994. Evaluation of the molecular basis of the pathogenicity of the variant Newcastle disease viruses termed pigeon PMV-1 viruses. Archives of Virology, 134, 403–411

    Article  PubMed  CAS  Google Scholar 

  • Collins, M.S., Strong, I. and Alexander, D.J., 1996. Pathogenicity and phylogenetic evaluation of the variant Newcastle disease viruses termed pigeon PMV-1 viruses based on the nucleotide sequence of the fusion protein gene. Archives of Virology, 141, 635–647

    Article  PubMed  CAS  Google Scholar 

  • Chomczynski, P. and Saachi, N., 1987. Single step method for RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry, 162, 156–159

    Article  PubMed  CAS  Google Scholar 

  • Fujii, Y., Sakaguchi, T., Kiyotani, K. and Yoshida, T., 1999. Comparison of substrate specificities against the fusion glycoproteins of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin like proteases. Microbiology and Immunology, 43, 133–140

    PubMed  CAS  Google Scholar 

  • Gould, A.R., Kattenbelt, J.A., Selleck, P., Hansson, E., Della-Porta, A. and Westbury, H.A., 2001. Virulent Newcastle disease in Australia: molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998–2000. Virus Research, 77, 51–60

    Article  PubMed  CAS  Google Scholar 

  • Hamid, H., Campbell, R.S.F. and Parede, L., 1991. Studies of the pathology of velogenic Newcastle disease: virus infection in non-immune and immune birds. Avian Pathology, 20, 561–575

    PubMed  CAS  Google Scholar 

  • Heuschelle, W.P. and Easterday, B.C., 1970. Local immunity and persistence of virus in the trachea of chickens following infection with Newcastle disease virus. Journal of Infectious Diseases, 121, 486–496

    Google Scholar 

  • Huang, Z., Panda, A., Elankumaran, S., Govindarajan, D., Rockemann, D.D. and Samal, S.K., 2004. The haemagglutinin neuraminidase protein of Newcastle disease virus determines tropism and virulence. Journal of Virology, 78, 4176–4184.

    Article  PubMed  CAS  Google Scholar 

  • Jungherr, E.L., Tyzzer, E.E., Brandly, C.A. and Moses, H.E., 1946. The comparative pathology of Fowl plague and Newcastle disease. American Journal of Veterinary Research, 7, 250–288

    Google Scholar 

  • Kommers, G.D., King, D.J., Seal, B.S., Carmichael, K.P. and Brown, C.C., 2002. Pathogenesis of six pigeon-origin isolates of Newcastle disease virus for domestic chickens. Veterinary Pathology, 39, 353–362

    Article  PubMed  CAS  Google Scholar 

  • Koonin, E.V. and Dolja, V.V., 1993. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Critical Reviews in Biochemistry and Molecular Biology, 28, 375–430

    PubMed  CAS  Google Scholar 

  • Kumanan, K. and Venkatesan, R.A., 1994. Adaptation of Newcastle disease virus strains to different cell culture. Indian Journal of Comparitive Microbiology, Immunology and Infectious Diseases, 15, 39–41

    Google Scholar 

  • Kumanan, K., Elankumaran, S., Vijayarani., K., Palaniswamy, K.S., Padmanabhan, V.D., Manvell, R.J. and Alexander, D.J., 1992. Characterization of Newcastle disease virus isolated in India. Journal of Veterinary Medicine B, 39, 383–387

    Article  PubMed  CAS  Google Scholar 

  • Kumanan, K., Vijayarani, K. and Padmanabhan, V.D., 1993. Adaptation of a velogenic Newcastle disease virus violate to Vero cells. The Indian Veterinary Journal, 70, 993–995

    Google Scholar 

  • Madhan Mohan, C., Dey, S. and Kumanan, K., 2005. Molecular changes of the fusion protein gene of chicken embryo fibroblast adapted velogenic Newcastle disease virus: effect on its pathogenicity. Avian Diseases, 49, 56–62.

    Article  PubMed  Google Scholar 

  • Mayo, M.A., 2002. A summary of the changes recently approved by the ICTV. Archives of Virology, 147, 1655–1656

    Article  PubMed  CAS  Google Scholar 

  • Mebatison, T., Verstegen, S., de Vaan, L.T., Romer Oberdorfer, A. and Schrier, C.C., 2001. A recombinant Newcastle disease virus with low level V protein expression is immunogenic and lacks pathogenicity for chicken embryos. Journal of Virology, 75, 420–428

    Article  Google Scholar 

  • Nagai, Y., 1995. Virus activation by host proteinases. A pivotal role in the spread of infection, tissue tropism and pathogenicity. Microbiology and Immunology, 39, 1–9

    PubMed  CAS  Google Scholar 

  • Nagai, Y., Shimokata, K., Yoshida, T., Hamaguchi, M., Iinuma, M., Maeno, K., Matsumoto, T., Klenk, H.D. and Rott, R. 1979. The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins. Journal of General Virology, 48, 263–272

    Article  Google Scholar 

  • Nagai, Y., Hamaguchi, M. and Toyoda, T., 1989. Molecular biology of Newcastle disease virus. Progress in Veterinary Microbiology and Immunology, 5, 16–64

    PubMed  CAS  Google Scholar 

  • Oberdorfer, A. and Werner, O., 1998. Newcastle disease virus detection and characterization by PCR of recent German isolates differing in pathogenicity. Avian Pathology, 27, 237–243

    PubMed  CAS  Google Scholar 

  • Parede, L. and Young, P.L., 1990. The pathogenesis of velogenic Newcastle disease virus infection of chickens of differential ages and different levels of immunity. Avian Diseases, 34, 803–808

    Article  PubMed  CAS  Google Scholar 

  • Park, M.S., Shaw, M.L., Munoz Jordan, J., Cros, J.F., Nakaya, T., Bouvier, N., Palase, P., Garcia Sastre, A. and Basler, C.F., 2003. Newcastle disease virus based assay demonstrates interferon antagonistic activity for the NDV V protein and the Nipah virus V, W and C proteins. Journal of Virology, 77, 1501–1511

    Article  PubMed  CAS  Google Scholar 

  • Russell, P.H., Samson, A.C.R. and Alexander, D.J., 1990. Newcastle disease virus variations. Applied Virology Research, 2, 177–195

    Article  Google Scholar 

  • Sambrook, J. and Russell, D.W., 2001. Molecular Cloning—A Laboratory Manual, 3rd edn (Cold Spring Harbour Laboratory Press, New York), 1.84–1.87.

    Google Scholar 

  • Seal, B.S., King, D.J. and Benett, J.D., 1995. Characterization of Newcastle disease virus isolates by reverse transcription PCR coupled to direct nucleotide sequencing and development of sequence database for pathotype prediction and molecular epidemiological analysis. Journal of Clinical Microbiology, 33, 2624–2630.

    PubMed  CAS  Google Scholar 

  • Slosaris, M., Levy, B., Katz, E., Levy, R. and Rones, Z.Z., 1989. Elevated virulence of Newcastle disease virus strains following serial passages in kidney cells in vitro. Avian Diseases, 33, 248–253.

    Article  PubMed  CAS  Google Scholar 

  • Takimoto, T., Taylor, G.L., Connaris, H.C., Crennell, S.J. and Portner, A., 2002. A role of the haemagglutinin neuraminidase protein in the mechanism of paramyxo cell membrane fusion. Journal of Virology, 76, 13028–13033.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. C. Madhan Mohan & Sohini Dey

    Present address: Division of Veterinary Biotechnology, Indian Veterinary Research Institiute, Bareilly, India

Authors and Affiliations

  1. Department of Animal Biotechnology, Madras Veterinary College, Chennai

    C. Madhan Mohan, Sohini Dey, K. Kumanan, B. Murali Manohar & A. Mahalinga Nainar

Authors
  1. C. Madhan Mohan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sohini Dey
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. Kumanan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. B. Murali Manohar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. Mahalinga Nainar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. Madhan Mohan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mohan, C.M., Dey, S., Kumanan, K. et al. Adaptation of a Velogenic Newcastle Disease Virus to Vero Cells: Assessing the Molecular Changes Before and After Adaptation. Vet Res Commun 31, 371–383 (2007). https://doi.org/10.1007/s11259-006-3502-2

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11259-006-3502-2

Keywords

Search

Navigation