F GENE SEQUENCING AND ANALYSIS OF NEWCASTLE DISEASE VIRUS CIRCULATING IN THE FIELD
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Date
2014
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AAU, Anand
Abstract
Newcastle disease (ND) is one of the most important infectious diseases of poultry. It is distributed world wide and has the potential to cause large economic losses in the poultry industry. ND is caused by Newcastle disease virus (NDV) which is a member of the family Paramyxoviridae, genus Avulavirus and is designated Avian paramyxovirus-I (APMV-1). The primary molecular determinant for tiie NDV pathogenicity is the fusion protein cleavage site amino acid sequences. Incidences of ND in vaccinated chicken population of Anand area of Gujarat demanded a study for pathotyping and genetic characterization of the field NDV isolates. Ten allantoic fluid samples from the SPF eggs inoculated with NDV suspected tissue suspension and preserved at the Department of veterinary Microbiology were used for the RNA isolation by QIAamp viral RNA mini kit, followed by one step RT-PCR using primers A and B to amplify F gene segments of NDV. All the ten isolates yielded expected 362bp product of F gene indicating them positive for NDV. Sequencing was done by ABI PRISM automated DNA sequencer. Length of the nucleotide sequences for NDVGUJl, NDVGUJ2, NDVGUJ3, NDVGUJ4, NDVGUJ5, NDVGUJ6, NDVGUJ7, NDVGUJ8, NDVGUJ9 and NDVGUJIO were 352nt, 352nt, 363nt, 352nt, 352nt, 353nt, 352nt, 352nt, 355nt and 354nt. The deduced amino acid sequences of F gene were obtained using ExPASy proteomics. Length of amino acid sequences were 115aa, 115aa, 119aa, 115aa, 115aa, 116aa, 115aa, 115aa, 116aaand 116aa respectively. The sequence were submitted to Genbank and the obtained Accession numbers are KJ754123, KJ754124, KJ754125, KJ754126, KJ754127, KJ754128, KJ754129, KJ754130, KJ754131 and KJ754132 respectively. The nucleotide sequences and deduced amino acid sequences of F gene of ten Anand isolates were aligned with the sequences of one Indian reference sequence Bareilly (Accession No. KF727980.1) and two reference vaccine strains viz. R2B and LaSota (Accession nos. JX316216.1 and JF950510.1 respectively) using ClustalW programme. Multiple sequence alignment of Anand isolates with reference Indian Bareilly strain and two vaccine strains R2B and LaSota revealed 7 unique mutations in Anand isolates viz. G222A, T231C, T300G, T420C, A426T, C432T and A438G. The amino acid sequences of all the ten Anand isolates showed RRQKRF between 112 and 117 positions indicating them to be of virulent type. All ten Anand isolates showed two unique amino acid changes i.e. I and E respectively at positions 69 and 104 with reference Bareilly sequence which showed M and G at these positions. Anand isolates differed with both the reference vaccine strains (R2B and LaSota) at six positions viz. 69, 82, 107, 121, 124 and 145 by showing I, E, S, A, S and N in place of L, D, T, I, G and K. The phylogenetic analysis based on F gene sequences of ten Anand isolates and 18 different NDV strains/isolates from different parts of the world available in GenBank was conducted using MEGA version 6. The analysis grouped all these 28 sequences into five different clusters. Cluster I included our ten isolates and strains Chicken/Sweden/97, Stema/Astr/2755/2001, and GDI003/2010. Our isolates showed maximum genetic similarity 94.32% with genotype XIII strain (Chicken/Sweden/97) indicating our isolates to be of genotype XIII. Our ten isolates showed least similarity 75.57% with Bl vaccine strain. The study thus emphasizes that the circulating NDV in the field are continuously evolving at genetic level and the variants are continuously emerging furthering the genetic distance with the other isolates as well the vaccine strains. This explains the vaccination failures observed in field condition and demands comprehensive studies to genotype large nimiber of NDV isolates so as to update the NDV vaccines.
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VERTINARY MICROBIOLOGY, AN ANALYSIS