Development of a novel real-time PCR-based strategy for simple and rapid molecular pathotyping of Newcastle disease virus
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.
KeywordsNewcastle Disease Virus Avian Influenza Virus Newcastle Disease Infectious Bronchitis Virus TaqMan Assay
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.
- 1.OIE (2011) Newcastle Disease. In: Manual of diagnostic tests and vaccines for terrestrial animals, vol 1, chap 2.3.14. pp 576–589Google Scholar
- 2.Lamb RA, Parks GD (2002) Paramyxovirinae: the viruses and their replication. In: Knipe DM, Howley PM (eds) Fields virology, vol 1. Lippincott Williams & Wilkins Inc, Philadelphia, Chapter 41, pp 1449–1496Google Scholar
- 4.Aldous EW, Mynn JK, Banks J, Alexander DJ (2003) A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol 32(3):239–256. doi: 10.1080/030794503100009783 PubMedCrossRefGoogle Scholar
- 5.Fratnik Steyer A, Rojs OZ, Krapez U, Slavec B, Barlic-Maganja D (2010) A diagnostic method based on MGB probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type 1. J Virol Methods 166(1–2):28–36. doi: 10.1016/j.jviromet.2010.02.012 PubMedCrossRefGoogle Scholar
- 16.Wang CY, Hsu CJ, Chen HJ, Chulu JL, Liu HJ (2008) Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of Newcastle disease virus. Vet Microbiol 130(1–2):28–36. doi: 10.1016/j.vetmic.2007.12.015 PubMedCrossRefGoogle Scholar
- 18.Dortmans JC, Rottier PJ, Koch G, Peeters BP (2011) Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence. J Gen Virol 92(Pt 2):336–345. doi: 10.1099/vir.0.026344-0 PubMedCrossRefGoogle Scholar
- 20.Kim LM, King DJ, Guzman H, Tesh RB, Travassos da Rosa AP, Bueno R Jr, Dennett JA, Afonso CL (2008) Biological and phylogenetic characterization of pigeon paramyxovirus serotype 1 circulating in wild North American pigeons and doves. J Clin Microbiol 46(10):3303–3310. doi: 10.1128/JCM.00644-08 PubMedCrossRefGoogle Scholar
- 29.OIE (2011) Newcastle disease. In: Terrestrial animal health code, vol 2, chapter 10.9. pp 1–12Google Scholar
- 30.Cattoli G, Fusaro A, Monne I, Molia S, Le Menach A, Maregeya B, Nchare A, Bangana I, Maina AG, Koffi JN, Thiam H, Bezeid OE, Salviato A, Nisi R, Terregino C, Capua I (2010) Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa–implications for diagnosis and control. Vet Microbiol 142(3–4):168–176. doi: 10.1016/j.vetmic.2009.09.063 PubMedCrossRefGoogle Scholar