GENOMIC AND PHYLOGENETIC INSIGHTS INTO VELOGENIC NEWCASTLE DISEASE VIRUS GENOTYPE VII OUTBREAKS IN GENOTYPE II-VACCINATED POULTRY FLOCKS OF SOUTHERN PAKISTAN

• Dr. Shaheer I. Nawrouz

  Department of Veterinary Microbiology, Sindh Agricultural Biotechnology Institute, Tandojam, Pakistan

  Author

Newcastle Disease (ND), caused by Newcastle Disease Virus (NDV), remains a significant threat to the global poultry industry, particularly in endemic regions like Pakistan. Despite widespread vaccination programs, outbreaks of velogenic NDV continue to occur, raising concerns about vaccine efficacy and the circulation of novel or antigenically distinct viral strains. This study aimed to conduct a molecular characterization and phylogenetic analysis of velogenic NDV genotype VII strains isolated from recent outbreaks in commercial poultry flocks in the southern region of Pakistan, which were previously vaccinated with genotype II-based vaccines. Through reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of key virulence-determining genes, the isolated strains were confirmed as velogenic NDV and assigned to Genotype VII, specifically sub-genotypes VIIh and VIIi. Phylogenetic analysis revealed that these strains clustered closely with other Genotype VII viruses circulating in the region and globally, suggesting ongoing evolution and potential transboundary movement. The findings highlight a significant antigenic mismatch between the circulating Genotype VII strains and the conventionally used Genotype II vaccines, contributing to vaccine failures and persistent outbreaks. This research underscores the critical need for updated vaccine strategies, potentially incorporating Genotype VII-specific strains, and enhanced surveillance to mitigate the economic impact of ND in Pakistan's poultry sector.

[1] S. A. Ghalib, R. Kanwal, A. Zehra et al., “Review of the distribution, status and conservation of wildlife of Sindh,” Canadian Journal of Pure and Applied Sciences, vol. 12, no. 2, pp. 4519–4533, 2018.

[2] S. Sultan, N. M. I. Eldamarany, M. W. Abdelazeem, and H. A. Fahmy, “Active surveillance and genetic characterization of prevalent velogenic Newcastle disease and highly pathogenic avian influenza H5N8 viruses among migratory wild birds in Southern Egypt during 2015–2018,” Food and Environmental Virology, vol. 14, no. 3, pp. 280–294, 2022.

[3] R. E. Dizaji, A. Ghalyanchilangeroudi, M. V. Marandi et al., “Complete genome sequence of a subgenotype XXI.1.1 pigeon paramyxovirus type 1 virus (PPMV-1) isolated from Iran in 2018 and phylogenetic analysis of a possible novel, but unassigned, PPMV-1 group isolated in 2014,” Comparative Immunology, Microbiology and Infectious Diseases, vol. 73, Article ID 101565, 2020.

[4] Y.-P. Liu, F. Lee, M.-C. Cheng, C.-Y. Chang, C.-J. Chiou, and H.-J. Tsai, “Genetic diversity of avian paramyxoviruses isolated from wild birds and domestic poultry in Taiwan between 2009 and 2020,” Journal of Veterinary Medical Science, vol. 84, no. 3, pp. 378–389, 2022.

[5] K. Karamendin and A. Kydyrmanov, “Cormorants as a potentially important reservoir and carrier of Newcastle disease virus on the Asian continent,” Frontiers in Veterinary Science, vol. 8, Article ID 648091, 2021.

[6] C. S. Ross, S. Mahmood, P. Skinner et al., “JMM profile: Avian paramyxovirus type-1 and Newcastle disease: a highly infectious vaccine-preventable viral disease of poultry with low zoonotic potential,” Journal of Medical Microbiology, vol. 71, no. 8, Article ID 001489, 2022.

[7] Y. Zhang, W. Wang, Y. Li et al., “A pigeon paramyxovirus type 1 isolated from racing pigeon as an inactivated vaccine candidate provides effective protection,” Poultry Science, vol. 101, no. 10, Article ID 102097, 2022.

[8] T. Zhan, L. Xiaolong, D. He et al., “Phylogenetic analysis and pathogenicity assessment of pigeon paramyxovirus Type 1 circulating in China during 2007–2019,” Transboundary and Emerging Diseases, vol. 69, no. 4, pp. 2076–2088, 2022.

[9] R. Esmaeelzadeh-Dizaji, A. Molouki, H. Hosseini et al., “Molecular characterization of a pigeon paramyxovirus Type 1 virus isolated from Eurasian collared doves in Iran, 2017,” Journal of Veterinary Science, vol. 23, no. 3, 2022.

[10] P. L. M. Msoffe, G. H. Chiwanga, C. J. Cardona, P. J. Miller, and D. L. Suarez, “Isolation and characterization of Newcastle disease virus from live bird markets in Tanzania,” Avian Diseases, vol. 63, no. 4, pp. 634–640, 2019.

[11] B. Rima, A. Balkema-Buschmann, W. G. Dundon et al., “ICTV Virus Taxonomy Profile: Paramyxoviridae,” Journal of General Virology, vol. 100, no. 12, pp. 1593-1594, 2019.

[12] E. B. Mngumi, F. N. Mpenda, and J. Buza, “Epidemiology of Newcastle disease in poultry in Africa: systematic review and meta-analysis,” Tropical Animal Health and Production, vol. 54, no. 4, p. 214, 2022.

[13] J. H. Khorajiya, B. P. Joshi, R. A. Mathakiya, K. S. Prajapati, and S. H. Sipai, “Economic impact of genotype-XIII Newcastle disease virus infection on commercial vaccinated layer farms in India,” International Journal of Livestock Research, vol. 8, no. 5, pp. 280–288, 2018.

[14] I. Capua and D. J. Alexander, Eds., Avian Influenza and Newcastle Disease: A Field and Laboratory Manual, Springer Science & Business Media, 2009.

[15] J. H. Kuhn, Y. I. Wolf, M. Krupovic et al., “Classify viruses — the gain is worth the pain,” Nature, vol. 566, no. 7744, pp. 318–320, 2019.

[16] B. S. Seal, D. J. King, and H. S. Sellers, “The avian response to Newcastle disease virus,” Developmental & Comparative Immunology, vol. 24, no. 2–3, pp. 257–268, 2000.

[17] S. L. Butt, T. L. Taylor, J. D. Volkening et al., “Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing,” Virology Journal, vol. 15, no. 1, p. 179, 2018.

[18] M. Nooruzzaman, L. R. Barman, T. T. Mumu, E. H. Chowdhury, K. M. Dimitrov, and M. R. Islam, “A pigeon-derived subgenotype XXI. 1.2 Newcastle disease virus from Bangladesh induces high mortality in chickens,” Viruses, vol. 13, no. 8, p. 1520, 2021.

[19] A. P. da Silva, E. J. Aston, G. H. Chiwanga et al., “Molecular characterization of Newcastle disease viruses isolated from chickens in Tanzania and Ghana,” Viruses, vol. 12, no. 9, p. 916, 2020.

[20] D. L. Suarez, P. J. Miller, G. Koch, E. Mundt, and S. Rautenschlein, “Newcastle disease, other avian paramyxoviruses, and avian metapneumovirus infections,” Diseases of Poultry, vol. 109–166, 2020.

[21] K. M. Dimitrov, C. Abolnik, C. L. Afonso et al., “Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus,” Infection, Genetics and Evolution, vol. 74, Article ID 103917, 2019.

[22] L. M. Kim, D. J. King, P. E. Curry et al., “Phylogenetic diversity among low-virulence Newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates,” Journal of Virology, vol. 81, no. 22, pp. 12641–12653, 2007.

[23] S. C. Courtney, L. Susta, D. Gomez et al., “Highly divergent virulent isolates of Newcastle disease virus from the Dominican Republic are members of a new genotype that may have evolved unnoticed for over 2 decades,” Journal of Clinical Microbiology, vol. 51, no. 2, pp. 508–517, 2013.

[24] D. G. Diel, L. H. A. da Silva, H. Liu, Z. Wang, P. J. Miller, and C. L. Afonso, “Genetic diversity of avian paramyxovirus type 1: proposal for a unified nomenclature and classification system of Newcastle disease virus genotypes,” Infection, Genetics and Evolution, vol. 12, no. 8, pp. 1770–1779, 2012.

[25] A. A. Eid, A. Hussein, O. Hassanin et al., “Newcastle disease genotype VII prevalence in poultry and wild birds in Egypt,” Viruses, vol. 14, no. 10, p. 2244, 2022.

[26] M. Getabalew, T. Alemneh, D. Akeberegn, D. Getahun, and D. Zewdie, “Epidemiology, diagnosis & prevention of Newcastle disease in poultry,” American Journal of Biomedical Science and Research, vol. 3, no. 1, pp. 50–59, 2019.

[27] D. J. Alexander, “Newcastle disease and other avian paramyxovirus,” Revue Scientifique et Technique-Office International des Epizooties, vol. 19, no. 2, pp. 443–462, 2000.

[28] S. S. Ewies, A. Ali, S. M. Tamam, and H. M. Madbouly, “Molecular characterization of Newcastle disease virus (genotype VII) from broiler chickens in Egypt,” Beni-Suef University Journal of Basic and Applied Sciences, vol. 6, no. 3, pp. 232–237, 2017.

[29] T. Toyoda, T. Sakaguchi, K. Imai et al., “Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus,” Virology, vol. 158, no. 1, pp. 242–247, 1987.

[30] R. L. Glickman, R. J. Syddall, R. M. Iorio, J. P. Sheehan, and M. A. Bratt, “Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus,” Journal of Virology, vol. 62, no. 1, pp. 354–356, 1988.

[31] T. G. Morrison, “Structure and function of a paramyxovirus fusion protein,” Biochimica et Biophysica Acta (BBA)-Biomembranes, vol. 1614, no. 1, pp. 73–84, 2003.

[32] D. Leeuw, S. Olav, G. Koch, L. Hartog, N. Ravenshorst, and B. P. H. Peeters, “Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin–neuraminidase protein,” Journal of General Virology, vol. 86, no. 6, pp. 1759–1769, 2005.

[33] P. J. Miller, E. L. Decanini, and C. L. Afonso, “Newcastle disease: evolution of genotypes and the related diagnostic challenges,” Infection, Genetics and Evolution, vol. 10, no. 1, pp. 26–35, 2010.

[34] A. Panda, S. Elankumaran, S. Krishnamurthy, Z. Huang, and S. K. Samal, “Loss of N-linked glycosylation from the hemagglutinin-neuraminidase protein alters virulence of Newcastle disease virus,” Journal of Virology, vol. 78, no. 10, pp. 4965–4975, 2004.

[35] O. F. Maminiaina, P. Gil, F.-X. Briand et al., “Newcastle disease virus in Madagascar: identification of an original genotype possibly deriving from a died out ancestor of genotype IV,” PLoS One, vol. 5, no. 11, Article ID e13987, 2010.

[36] M. Munir, M. Z. Shabbir, T. Yaqub et al., “Complete genome sequence of a velogenic neurotropic avian paramyxovirus 1 isolated from peacocks (Pavo cristatus) in a wildlife park in Pakistan,” Genome Announcement, vol. 86, no. 23, Article ID 13114, p. 13113, 2012.

[37] N. Siddique, K. Naeem, M. A. Abbas et al., “Sequence and phylogenetic analysis of virulent Newcastle disease virus isolates from Pakistan during 2009–2013 reveals circulation of new sub genotype,” Virology, vol. 444, no. 1–2, pp. 37–40, 2013.

[38] S. F. Rehmani, A. Wajid, T. Bibi et al., “Presence of virulent Newcastle disease virus in vaccinated chickens in farms in Pakistan,” Journal of Clinical Microbiology, vol. 53, no. 5, pp. 1715–1718, 2015.

[39] S. F. Rehmani, “Newcastle disease vaccination: a comparison of vaccines and routes of administration in Pakistan,” Preventive Veterinary Medicine, vol. 25, no. 3–4, pp. 241–248, 1996.

[40] World Organization for Animal Health (WOAH), WOAH-Terrestrial Animal Health Code Twenty-third edition, WOAH, 2014.

[41] World Organization for Animal Health (WOAH), Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: Mammals, Birds and Bees, Office International des Epizooties, Paris, 2004.

[42] OIE Terrestrial Manual, “Chapter 2.3.14. Newcastle disease,” in Manual of diagnostic tests and vaccines for terrestrial animals, pp. 1–19, World Organization for Animal Health (OIE), Paris, 2012.

[43] J. C. Pedersen, Procedure for determining mean death time for Newcastle disease virus isolates, National Veterinary Services Laboratories Testing Protocol, Ames, Iowa, USA, 2011.

[44] E. W. Aldous, J. K. Mynn, J. Banks, and D. J. Alexander, “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 Pathology, vol. 32, no. 3, pp. 237–255, 2003.

[45] K. Tamura, G. Stecher, and S. Kumar, “MEGA11: molecular evolutionary genetics analysis Version 11,” Molecular Biology and Evolution, vol. 38, no. 7, pp. 3022–3027, 2021.

[46] K. Tamura and M. Nei, “Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees,” Molecular Biology and Evolution, vol. 10, no. 3, pp. 512–526, 1993.

[47] World Organization for Animal Health (WAH), “Newcastle disease,” in Manual of standards for diagnostic tests and vaccines, pp. 221–232, OIE, Paris, 4th edition, 2000.

[48] R. A. Lamb and D. J. F. V. Kolakofsky, “Paramyxoviridae: the viruses and their replication,” in Fields virology, Lippincott-Raven Press, 1996.

[49] J. A. Kattenbelt, M. P. Stevens, and A. R. Gould, “Sequence variation in the Newcastle disease virus genome,” Virus Research, vol. 116, no. 1–2, pp. 168–184, 2006.

[50] N. F. Habib-ur-Rehman, G. Abbas, G. Naheed et al., “Surveillance of poultry diseases in Punjab province, Pakistan; special reference to Newcastle disease,” Research Journal for Veterinary Practitioners, vol. 1, pp. 1–4, 2013.

[51] A. Ballagi-Pordany, E. Wehmann, J. Herczeg, S. Belak, and B. Lomniczi, “Identification and grouping of Newcastle disease virus strains by restriction site analysis of a region from the F gene,” Archives of Virology, vol. 141, no. 2, pp. 243–261, 1996.

[52] T. A. Khan, C. A. Rue, S. F. Rehmani et al., “Phylogenetic and biological characterization of Newcastle disease virus isolates from Pakistan,” Journal of Clinical Microbiology, vol. 48, no. 5, pp. 1892–1894, 2010.

[53] A. Bogoyavlenskiy, V. Berezin, A. Prilipov et al., “Newcastle disease outbreaks in Kazakhstan and Kyrgyzstan during 1998, 2000, 2001, 2003, 2004, and 2005 were caused by viruses of the genotypes VIIb and VIId,” Virus Genes, vol. 39, no. 1, pp. 94–101, 2009.

[54] Z. Wang, H. Liu, X. Jiangtao et al., “Genotyping of Newcastle disease viruses isolated from 2002 to 2004 in China,” Annals of the New York Academy of Sciences, vol. 1081, no. 1, pp. 228–239, 2006.

[55] C. J. Snoeck, M. F. Ducatez, A. A. Owoade et al., “Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms,” Archives of Virology, vol. 154, no. 1, pp. 47–54, 2009.

[56] S. Xiao, A. Paldurai, B. Nayak et al., “Complete genome sequences of Newcastle disease virus strains circulating in chicken populations of Indonesia,” Genome Announcement, vol. 86, no. 10, pp. 5969-5970, 2012.

[57] M. Farooq, U. Saliha, M. Munir, and Q. M. Khan, “Biological and genotypic characterization of the Newcastle disease virus isolated from disease outbreaks in commercial poultry farms in northern Punjab, Pakistan,” Virology Reports, vol. 3-4, pp. 30–39, 2014.

[58] P. J. Miller, R. Haddas, L. Simanov et al., “Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features,” Infection, Genetics and Evolution, vol. 29, pp. 216–229, 2015.

[59] S. Umar, “Retracted article: emergence of new sub-genotypes of Newcastle disease virus in Pakistan,” World's Poultry Science Journal, vol. 73, no. 3, pp. 567–580, 2017.

[60] WWF, Detail ecological assisment of fauna including limnology studies at Khanjar Lake, WWF Report, 2008.

[61] WWF, Ecological assessment of fauna at Manchhar Lake, District Dadu, Sindh Baseline Survey 2010 – 2011, WWF Survey, 2011.

[62] A. Begum, A. Zubair, S. E. Hussain, and A. R. Khan, “Waterbirds diversity and conservation at hub dam Lasbella, Karachi, Pakistan,” African Journal of Science and Research, vol. 2, no. 3, pp. 22–26, 2013.

[63] S. A. Ghalib, M. Zaheer Khan, S. M. Ahmed, A. Begum, B. Hussain, and W. Ahmed, “Study of the wildlife of Jhimpir wind corridor, district Thatta, Sindh and development of bird monitoring strategy in the area,” African Journal of Science and Research, vol. 3, no. 6, pp. 1–9, 2014.

[64] M. M. Radwan, S. F. Darwish, I. M. El-Sabagh, A. A. El-Sanousi, and M. A. Shalaby, “Isolation and molecular characterization of Newcastle disease virus genotypes II and VIId in Egypt between 2011 and 2012,” Virus Genes, vol. 47, no. 2, pp. 311–316, 2013.

[65] S. Gaurav, P. Deka, S. Das et al., “Isolation of genotype VII avian orthoavulavirus serotype 1 from barn owl from North-east India,” Avian Pathology, vol. 51, no. 1, pp. 45–50, 2022.

[66] D. J. Alexander, E. W. Aldous, and C. M. Fuller, “The long view: a selective review of 40 years of Newcastle disease research,” Avian Pathology, vol. 41, no. 4, pp. 329–335, 2012.

[67] L. Susta, M. E. B. Jones, G. Cattoli et al., “Pathologic characterization of genotypes XIV and XVII Newcastle disease viruses and efficacy of classical vaccination on specific pathogen-free birds,” Veterinary Pathology, vol. 52, no. 1, pp. 120–131, 2015.

[68] F. Perozo, R. Marcano, and C. L. Afonso, “Biological and phylogenetic characterization of a genotype VII Newcastle disease virus from Venezuela: efficacy of field vaccination,” Journal of Clinical Microbiology, vol. 50, no. 4, pp. 1204–1208, 2012.

[69] P. J. Miller, L. Mia Kim, H. S. Ip, and C. L. Afonso, “Evolutionary dynamics of Newcastle disease virus,” Virology, vol. 391, no. 1, pp. 64–72, 2009.

[70] D. G. Diel, L. Susta, S. C. Garcia et al., “Complete genome and clinicopathological characterization of a virulent Newcastle disease virus isolate from South America,” Journal of Clinical Microbiology, vol. 50, no. 2, pp. 378–387, 2012.

[71] S.-H. Cho, S.-J. Kim, and H.-J. Kwon, “Genomic sequence of an antigenic variant Newcastle disease virus isolated in Korea,” Virus Genes, vol. 35, no. 2, pp. 293–302, 2007.

[72] K.-S. Choi, S.-J. Kye, J.-Y. Kim et al., “Molecular epidemiology of Newcastle disease viruses in Vietnam,” Tropical Animal Health and Production, vol. 46, no. 1, pp. 271–277, 2014.

[73] E. Naggar, F. Rania, M. A. Rohaim et al., “Biological characterization of wild-bird-origin avian avulavirus 1 and efficacy of currently applied vaccines against potential infection in commercial poultry,” Archives of Virology, vol. 163, no. 10, pp. 2743–2755, 2018.

[74] P. J. Miller, D. J. King, C. L. Afonso, and D. L. Suarez, “Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge,” Vaccine, vol. 25, no. 41, pp. 7238–7246, 2007.

[75] P. J. Miller, C. L. Afonso, J. El Attrache et al., “Effects of Newcastle disease virus vaccine antibodies on the shedding and transmission of challenge viruses,” Developmental & Comparative Immunology, vol. 41, no. 4, pp. 505–513, 2013.

[76] J. Liu, J. Zhu, H. Xu et al., “Effects of the HN antigenic difference between the vaccine strain and the challenge strain of Newcastle disease virus on virus shedding and transmission,” Viruses, vol. 9, no. 8, p. 225, 2017.

[77] E. Shahar, R. Haddas, D. Goldenberg et al., “Newcastle disease virus: is an updated attenuated vaccine needed?,” Avian Pathology, vol. 47, no. 5, pp. 467–478, 2018.

[78] D. R. Kapczynski, C. L. Afonso, and P. J. Miller, “Immune responses of poultry to Newcastle disease virus,” Developmental & Comparative Immunology, vol. 41, no. 3, pp. 447–453, 2013.

[79] Y. Ji, T. Liu, Y. du et al., “A novel genotype VII Newcastle disease virus vaccine candidate generated by mutation in the L and F genes confers improved protection in chickens,” Veterinary Microbiology, vol. 216, pp. 99–106, 2018.

[80] M. A. Ayoub, W. K. Elfeil, D. El Boraey, H. Hammam, and M. A. Nossair, “Evaluation of some vaccination programs in protection of experimentally challenged broiler chicken against Newcastle disease virus,” American Journal of Animal and Veterinary Sciences, vol. 14, no. 3, pp. 197–206, 2019.

[81] WOAH Terrestrial Manual, Chapter 3.3.14. – Newcastle disease (infection with Newcastle disease virus), Newcastle Disease (Infection With Newcastle Disease Virus), 2021.

[82] D. J. Alexander, Y. M. Saif, H. J. Barnes et al., Diseases of poultry chapter (Newcastle disease, other avian paramyxoviruses, and pneumovirus infections), Iowa State University Press, Ames, Iowa, 2003.

[83] Y. Ji, T. Liu, Y. Jia et al., “Two single mutations in the fusion protein of Newcastle disease virus confer hemagglutinin-neuraminidase independent fusion promotion and attenuate the pathogenicity in chickens,” Virology, vol. 509, pp. 146–151, 2017.

[84] S.-H. Cho, H.-J. Kwon, T.-E. Kim et al., “Characterization of a recombinant Newcastle disease virus vaccine strain,” Clinical and Vaccine Immunology, vol. 15, no. 10, pp. 1572–1579, 2008.

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