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Development of a novel real-time PCR-based strategy for simple and rapid molecular pathotyping of Newcastle disease virus

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Abstract

A novel real-time PCR strategy was applied to simultaneously detect and to discriminate low-pathogenic lentogenic and virulent meso/velogenic Newcastle disease virus (NDV). The pathotyping is achieved by a three-step semi-nested PCR. A pre-amplification of the cleavage site (CS) region of the F gene is followed by a two-level duplex real-time PCR directly targeting the CS, combining detection and pathotyping in a single tube. A wide range of NDV isolates spanning all genotypes were successfully detected and pathotyped. Clinical samples from outbreaks in Sweden in 2010 that were positive by the novel PCR method were also successfully pathotyped. The method is time-saving, reduces labour and costs and provides opportunities for rapid diagnosis at remote locations and in the field. Since the same strategy was also recently applied to avian influenza virus pathotyping, it shows promise of finding broad utility in diagnostics of infectious diseases caused by different RNA viruses in various hosts.

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Acknowledgments

This work was supported by the Award of Excellence of the Swedish University of Agricultural Sciences (2007) granted to SB, the EU grant FLUTEST (SSPE-CT-2007-044429), a grant from the Swedish Civil Contingencies Agency (2:4 Krisberedskap) and a grant in the framework of the EU-project AniBioThreat (Grant Agreement: Home/2009/ISEC/AG/191) with the financial support from the Prevention of and Fight against Crime Programme of the European Union, European Commission – Directorate General Home Affairs. This publication reflects the views only of the author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. Dr. Béla Lomniczi (VMRI, Budapest, Hungary) is thanked for kindly providing many NDV isolates used in developing this assay and for constructive criticism and advice. We thank Drs. István Kiss and Siamak Zohari for useful discussions. Ulla Berglöf is thanked for expert technical assistance. The critical reading and suggestions of Dr. László Zsák (USDA; ARS, Southeast Poultry Research Laboratory, Athens, GA, USA) are highly appreciated.

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Authors and Affiliations

  1. Department of Virology, Immunobiology and Parasitology, National Veterinary Institute (SVA), Ulls väg 2B, SE-751 89, Uppsala, Sweden

    Alia Yacoub, Mikael Leijon, Karin Ullman & Sándor Belák

  2. School of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland, BT9 7BL, UK

    Michael J. McMenamy & Gordon Allan

  3. Veterinary Sciences Division, Agri-Food and Biosciences Institute, Belfast, Northern Ireland, BT4 3SD, UK

    John McKillen

  4. Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden

    Sándor Belák

  5. The Joint R&D Division in Virology of SVA and SLU, The OIE Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine, Ulls väg 2B, SE-751 89, Uppsala, Sweden

    Alia Yacoub, Mikael Leijon & Sándor Belák

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  1. Alia Yacoub
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  2. Mikael Leijon
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Correspondence to Alia Yacoub.

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A. Yacoub, M. Leijon and M. J. McMenamy contributed equally and share first authorship.

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Yacoub, A., Leijon, M., McMenamy, M.J. et al. Development of a novel real-time PCR-based strategy for simple and rapid molecular pathotyping of Newcastle disease virus. Arch Virol 157, 833–844 (2012). https://doi.org/10.1007/s00705-012-1231-0

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  • DOI: https://doi.org/10.1007/s00705-012-1231-0

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