ABSTRACT
The complete genome sequence of a virulent Newcastle disease virus (vNDV) strain isolated from an exotic parakeet (Melopsittacus undulatus) is described here. The virulent strain parakeet/Pak/R-Pindi/SFR-16/2016 was isolated from a bird reared as a pet in the province of Punjab in the northern region of Pakistan in 2016. Phylogenetic analysis classified the isolate as a member of NDV class II, subgenotype VIIi, in genotype VII.
GENOME ANNOUNCEMENT
Newcastle disease (ND) is the one of the most economically important viral diseases affecting many species of birds. Newcastle disease is a devastating disease of poultry worldwide due to high mortality in commercial young chickens (1). Newcastle disease virus (NDV) is classified as an avian paramyxovirus type 1 (AMPV-1), belonging to the genus Avulavirus, family Paramyxoviridae, order Mononegavirales (2). NDV is an enveloped virus having a negative-sense, nonsegmented, and single-stranded RNA, with a genome length of 15.2 kb (3, 4). The NDV genome encodes six transcriptional units, nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large protein (L), with an additional V protein that is expressed by RNA editing of P mRNA (5).
An NDV isolate designated parakeet/Pak/R-Pindi/SFR-16/2016 (SFR-16) was identified and classified as a member of the newly emerging subgenotype VIIi in poultry production facilities and pet birds in Pakistan. Viruses classified in this subgenotype are widely spread through Asia, the Middle East, eastern European countries, like Turkey, Georgia, Bulgaria, and recently in Indian peafowl causing outbreaks of virulent NDV (vNDV) with high mortality in poultry flocks, suggesting the existence of a fifth panzootic (6–8). Nonsignificant changes in the complete genome sequences of ND viruses recently reported in chickens, ducks and parakeets in Pakistan revealed apparent spill-over of the viruses among them (7).
Oropharyngeal and cloacal swabs, along with blood samples, were collected from healthy parakeets and inoculated into 9- to 10-day-old (NDV-specific-antibody-free) chicken eggs. A hemagglutination assay (HA) was performed, and NDV was suspected by a positive HA test result (9). The positive HA allantoic fluid was used for viral RNA extraction using TRIzol LS (Invitrogen, USA), according to the manufacturer’s protocol. The first-strand cDNA (cDNA) was synthesized using the Thermo Scientific reverse transcriptase PCR (RT-PCR) kit using random hexamer (Thermo Scientific, USA), according to the manufacturer’s recommendations. The complete genome was sequenced as described previously (3), and the BioEdit software was used for the sequence analysis (10).
Sequence analysis showed that the complete genome length of the SFR-16 isolate was 15,192 nucleotides (nt). The SFR-16 strain has the highest homology (>99%) to the sequences of previously isolated ND viruses from duck (AW-123) and chicken (SFR-611) (GenBank accession numbers KU845252 and KM670337, respectively). The amino acid sequence identities of the NP, P, M, HN, F, and L proteins between SFR-16 and AW-123 strains are 99.38%, 100%, 99.17%, 99.82%, 99.27%, and 100%, respectively. SFR-16 was classified as velogenic NDV, with an intracerebral pathogenicity index (ICPI) of 1.70 in day-old specific-NDV antibody-free chicks; the result is consistent with the polybasic amino acid motif and a polyethylene at position 117 (112RRQKR↓F117) at the fusion protein cleavage site, which is typical for virulent NDV isolates (9). The current scenario of the highly related viral transmission in various pet birds in Pakistan highlights the importance of an active surveillance program.
Accession number(s).
The complete genome sequence of NDV strain parakeet/Pak/R-Pindi/SFR-16/2016 has been deposited in GenBank under the accession number KX791183.
ACKNOWLEDGMENTS
We thank Claudio L. Afonso (Newcastle Disease Lead Scientist: SEPRL, Athens, GA, USA) for his guidance and generous support in active surveillance program for Newcastle disease in Pakistan. This is part of Collaborative Research between South East Poultry Research Laboratory (SEPRL), Athens, GA, and Quality Operations Lab (QOL), University of Veterinary and Animal Sciences, Lahore, Pakistan.
The funding for this work was supported by the U.S. Department of Agriculture (agreement 58-0210-3-009) to Shafqat Fatima Rehmani and Abdul Wajid for Quality Operation Lab of the University of Veterinary and Animal Sciences, Lahore, Pakistan.
Footnotes
Citation Wajid A, Basharat A, Khan TA, Wasim M, Rehmani SF. 2017. Complete genome sequence of a velogenic Newcastle disease virus strain isolated from a clinically healthy exotic parakeet (Melopsittacus undulatus) in Pakistan. Genome Announc 5:e01581-16. https://doi.org/10.1128/genomeA.01581-16.
REFERENCES
- 1.Rehmani SF, Wajid A, Bibi T, Nazir B, Mukhtar N, Hussain A, Lone NA, Yaqub T, Afonso CL. 2015. Presence of virulent Newcastle disease virus in vaccinated chickens in farms in Pakistan. J Clin Microbiol 53:1715–1718. doi: 10.1128/JCM.02818-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Afonso CL, Amarasinghe GK, Bányai K, Bào Y, Basler CF, Bavari S, Bejerman N, Blasdell KR, Briand FX, Briese T, Bukreyev A, Calisher CH, Chandran K, Chéng J, Clawson AN, Collins PL, Dietzgen RG, Dolnik O, Domier LL, Dürrwald R, Dye JM, Easton AJ, Ebihara H, Farkas SL, Freitas-Astúa J, Formenty P, Fouchier RA, Fù Y, Ghedin E, Goodin MM, Hewson R, Horie M, Hyndman TH, Jiāng D, Kitajima EW, Kobinger GP, Kondo H, Kurath G, Lamb RA, Lenardon S, Leroy EM, Li CX, Lin XD, Liu L, Longdon B, Marton S, Maisner A, Muhlberger E, Netesov SV, Nowotny N. 2016. Taxonomy of the order Mononegavirales: update 2016. Arch Virol 161:2351–2360. doi: 10.1007/s00705-016-2880-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Wajid A, Wasim M, Rehmani SF, Bibi T, Ahmed N, Afonso CL. 2015. Complete genome sequence of a recent panzootic virulent Newcastle disease virus from Pakistan. Genome Announc 3(3):e00658-15. doi: 10.1128/genomeA.00658-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Dimitrov KM, Ramey AM, Qiu X, Bahl J, Afonso CL. 2016. Temporal, geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus). Infect Genet Evol 39:22–34. doi: 10.1016/j.meegid.2016.01.008. [DOI] [PubMed] [Google Scholar]
- 5.Miller PJ, Decanini EL, Afonso CL. 2010. Newcastle disease: evolution of genotypes and the related diagnostic challenges. Infect Genet Evol 10:26–35. doi: 10.1016/j.meegid.2009.09.012. [DOI] [PubMed] [Google Scholar]
- 6.Miller PJ, Haddas R, Simanov L, Lublin A, Rehmani SF, Wajid A, Bibi T, Khan TA, Yaqub T, Setiyaningsih S, Afonso CL. 2015. Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features. Infect Genet Evol 29:216–229. doi: 10.1016/j.meegid.2014.10.032. [DOI] [PubMed] [Google Scholar]
- 7.Wajid A, Rehmani SF, Wasim M, Basharat A, Bibi T, Arif S, Dimitrov KM, Afonso CL. 2016. Complete genome sequence of a virulent Newcastle disease virus strain isolated from a clinically healthy duck (Anas platyrhynchos domesticus) in Pakistan. Genome Announc 4(4):e00730-16. doi: 10.1128/genomeA.00730-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Fuller C, Löndt B, Dimitrov KM, Lewis N, van Boheemen S, Fouchier R, Coven F, Goujgoulova G, Haddas R, Brown I. 15 December 2015. An epizootiological report of the re-emergence and spread of a lineage of virulent Newcastle disease virus into eastern Europe. Transbound Emerg Dis doi: 10.1111/tbed.12455. [DOI] [PubMed] [Google Scholar]
- 9.World Organisation for Animals Health (OIE) 2012. Newcastle disease, p 555–574. A manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees), 7th ed, vol 1, part 2. Biological standards commission World Organisation for Animal Health, Paris, France. [Google Scholar]
- 10.Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. J Nucleic Acids S41:95–98. [Google Scholar]