The Phylodynamic nature of Newcastle disease virus isolated

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The Phylodynamic nature of Newcastle disease virus isolated from Malaysia during 2004-2011 Dilan A Satharasinghe 1,3, Sheau W Tan 1, Swee K Yeap 1, Muhammad Munir 4, Aini Ideris 1,2 and Abdul R Omar 1, 2 1 Institute of Bioscience, 2 Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia, 3 Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, 20400 Peradeniya, Sri Lanka 4 The Pirbright Institute, Ash Road, Pirbright, Surrey GU 24 ONF, United Kingdom ABSTRACT Newcastle disease virus (NDV) strains MB 128/4, MB76/5, IBS002/11 and IBS005/11 obtained from Newcastle disease (ND) outbreaks during 2004 to 2011 from Malaysia were characterized based on Intracerebral Pathogenicity Index (ICPI) and complete genome sequencing. Sequence analysis of the F protein cleavage site of the NDV isolates resembled virulent virus sequence of 112R/K-R-Q-R/K-R↓F117. According to the phylogenetic analysis of partial and full F gene of all the isolates belong to genotype VIIa (lineage 5a). Pairwise sequence comparison analysis (PASC) of the partial F gene sequence of the Malaysian NDV isolates with other NDV genotypes indicated that the studied NDV isolates have the highest genetic distance against genotype II viruses. In addition, PASC analysis also indicated that the recent NDV isolates, IBS002 and IBS005 which were isolated in 2011 have a higher genetic distance (20.16%) compared to NDV isolated in 2004 and 2005 (MB128-4 and MB76-5) (18.84%) when compared with genotype II viruses. This presentation discusses further the molecular characterization of the studied NDV isolates. 1. Introduction Newcastle disease virus (NDV) or Avian Paramyxovirus type I (APMV-I) has been a major threat to the poultry industry. Biosecurity and vaccination of poultry flocks against NDV are considered as effective methods of controlling outbreaks of velogenic NDV. Vaccination is performed by using strains belong to the genotype II of class II APMV-I. 3. Material and Methods Intracerebral pathogenicity index (ICPI) was calculated by using SPF day one chicks. Viral RNA was extracted from allantoic fluid and complete genome of the virus samples were subjected to Nextera XT library preparation kit. Libraries were quality controlled by using bio-analyzer and qPCR Generated libraries were sequenced by using Miseq Illumina platform. CLC genomics workbench software was used to assemble the viral genomes. Phylogenetic and PASC analysis was conducted using MEGA6 software. 2. Objectives To apply NGS technology to determine the whole genome sequence of NDV genotype strains isolated from 2004 to 2011 in Malaysia. To determine the molecular characterization and evolution of sequenced Malaysian NDV isolates. 4. Results NDV Isolates p-Distance (%) Lineages Lineage 1 Lineage 2 Lineage 3 Lineage 4 Lineage 5 MB128/04 16.26 18.84 12.58 10.91 7.59 MB76/05 IBS002/11 18.52 20.16 15.35 14.19 11.58 IBS005/11 NDV isolate F cleavage site ICPI MB128/04 112RRQKR↓F117 1.68 MB76/05 1.84 IBS002/11 112RRRKR↓F117 1.76 IBS005/11 1.71 NDV Isolates p-Distance (%) Sub lineages 5a 5b 5c 5d 5e 5f 5g 5h 5i MB128/04 3.78 7.77 6.26 6.49 6.84 14.21 13.67 16.44 MB76/05 IBS002/11 8.49 12.43 9.12 9.97 11.33 18.23 17.69 20.46 16.49 IBS005/11 5. Discussion and Conclusion According to the ICPI value and F protein cleavage site motif all NDV isolates were identified as velogenic. Based on phylogenetic and PSCA analysis all Malaysian NDV isolates were characterized under lineage 5a (genotype VIIa). The studied NDV isolates have the highest genetic distance against lineage II (genotype II) , the lineage which includes most of the current vaccine strains. Over the period of time all the NDV isolates demonstated an increasing genetic distance to all the lineages. 6. Acknowledgement This study was supported by IBS HICOE grant, Ministry of Education, Malaysia. 7. References Aldous, E.W., Mynn, J.K., Banks, J. & Alexander, D.J. (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 Pathology, 32, 239-256. Panda, A., Huang, Z., Elankumaran, S., Rockemann, D. D., & Samal, S. K. (2004). Role of fusion protein cleavage site in the virulence of Newcastle disease virus. Microbial Pathogenesis, 36(1), 1–10. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.