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Molecular Analysis Of Pathotype Specific Variations In Surface Glycoprotein Genes Of Velogenic And Lentogenic Newcastle Disease Virus Isolates

KrishiKosh

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Title Molecular Analysis Of Pathotype Specific Variations In Surface Glycoprotein Genes Of Velogenic And Lentogenic Newcastle Disease Virus Isolates
 
Creator Sanjay, Kapgate Sunil
 
Contributor Kumanan, K.
Balachandran, C.
Chandran, N. Daniel Joy
Raj, G. Dhinakar
 
Subject Newcastle disease virus
Fusion Protein Gene
Haemagglutinin-neuraminidase protein
Indirect immunofluorescence
 
Description The present study describes the whole genome sequencing of two Newcastle
disease virus isolates, their phylogenetic relationship with other global isolates, their
genome details, cloning and expression of fusion and haemagglutinin-neuraminidase
protein genes and structural analysis of these proteins. NDV-2 had an Intracerebral
pathogenicity index of 1.85 and NDV-4, 1.76. Molecular pathotyping confirmed the
velogenic nature of NDV-2 with the fusion protein cleavage site sequence (FPCS) of
^112RRQRRF^117. However, NDV-4 with the FPCS sequence of ^112GRQGRL^117, found to be
lentogenic as per OIE definition. Whole genome of NDV-2 and NDV-4 was found to be
15,186 nt long (GenBank accession numbers GU187941 and HM357251). In both the
isolates, the genome had six different genes in the order of 3’-leader-NP-P/V/W-M-F-HNL-
trailer-5’. The length of the 3’ leader and 5’ trailer of NDV-2 and NDV-4 were 55 nt and
114 nt respectively.
Four different phylogenetic trees were constructed with F gene sequence, HN gene
sequence, whole genome sequence and whole genome sequence excluding HN gene
sequence. NDV-2 was found to be phylogenetically related to an Indian isolate and two
exotic isolates namely Italien and Herts/33. All the four phylogenetic trees placed NDV-2
in genotype IV. In contrast, NDV-4 was found to be phylogenetically related to Chinese
isolates and two vaccine viruses namely, LaSota and B1. Phylogenetic analysis of NDV-4
with F gene sequence, HN gene sequence and whole genome sequence placed this isolate
in genotype II. However, phylogenetic tree with whole genome sequence excluding HN
gene sequence placed NDV-4 in genotype VII.
Predicted amino acid sequences of NP protein showed that the N-terminal of the
protein was found to be conserved. In contrast, the C terminal amino acids of NP protein
showed high variability. Predicted amino acid sequences of P protein showed variations.
Comparison of nuclear localization signals of M protein showed variations in NDV-4 and
NDV-2. HN protein of NDV-2 had 571 amino acids whereas NDV-4 had 577 amino acids.
Alignment of L gene sequences demonstrated six linear conserved domains. In cloning
experiments, RT-PCR resulted in the amplification of 1.7 kb F gene and 1.8 kb HN gene
with both the isolates. F and HN genes of NDV-2 were expressed in pTrixE Neo 1.1. those
of NDV-4 in pcDNA3.1 vector. Recombinant F and HN proteins were approximately 55
kDa and 63 kDa in size respectively.
Structural analysis of F protein revealed a trimer, with distinct head, neck and stalk
regions. HN protein contained six β-sheet motifs along with four helices. Three
dimensional locations of variable amino acids in the conformational epitopes and linear
epitopes in HN protein revealed that NDV-2 had five amino acid changes in the
conformational epitopes. NDV-4 had one amino acid change in the linear epitope.
 
Date 2016-05-20T16:51:21Z
2016-05-20T16:51:21Z
2010
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/66182
 
Language en
 
Format application/pdf
 
Publisher Tamil Nadu Veterinary and Animal Sciences University