Department of Veterinary Medicine

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Author: search.filters.author.Kumar, Sachin

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    Molecular characterization and complete genome sequence of avian paramyxovirus type 4 prototype strain duck/Hong Kong/D3/75
    (Springer Nature, 2008-10-20) Nayak, Baibaswata; Kumar, Sachin; Collins, Peter L; Samal, Siba K
    Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world. All APMVs, except avian metapneumovirus, are classified in the genus Avulavirus of the family Paramyxoviridae. At present, the APMVs of genus Avulavirus are divided into nine serological types (APMV 1–9). Newcastle disease virus represents APMV-1 and is the most characterized among all APMV types. Very little is known about the molecular characteristics and pathogenicity of APMV 2–9. As a first step towards understanding the molecular genetics and pathogenicity of APMV-4, we have sequenced the complete genome of APMV-4 strain duck/Hong Kong/D3/75 and determined its pathogenicity in embryonated chicken eggs. The genome of APMV-4 is 15,054 nucleotides (nt) in length, which is consistent with the "rule of six". The genome contains six non-overlapping genes in the order 3'-N-P/V-M-F-HN-L-5'. The genes are flanked on either side by highly conserved transcription start and stop signals and have intergenic sequences varying in length from 9 to 42 nt. The genome contains a 55 nt leader region at 3' end. The 5' trailer region is 17 nt, which is the shortest in the family Paramyxoviridae. Analysis of mRNAs transcribed from the P gene showed that 35% of the transcripts were edited by insertion of one non-templated G residue at an editing site leading to production of V mRNAs. No message was detected that contained insertion of two non-templated G residues, indicating that the W mRNAs are inefficiently produced in APMV-4 infected cells. The cleavage site of the F protein (DIPQR↓F) does not conform to the preferred cleavage site of the ubiquitous intracellular protease furin. However, exogenous proteases were not required for the growth of APMV-4 in cell culture, indicating that the cleavage does not depend on a furin site. Phylogenic analysis of the nucleotide sequences of viruses of all five genera of the family Paramyxoviridae showed that APMV-4 is more closely related to the APMVs than to other paramyxoviruses, reinforcing the classification of all APMVs in the genus Avulavirus of the family Paramyxoviridae.
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    Sequence analysis of fusion protein gene of Newcastle disease virus isolated from outbreaks in Egypt during 2006
    (2011-05-18) Mohamed, Mahmoud HA; Kumar, Sachin; Paldurai, Anandan; Samal, Siba K
    Background: Newcastle disease virus represents APMV-1 and is the most characterized among all APMV types. The F protein cleavage site sequence is a well-characterized determinant of NDV pathogenicity in chickens. In this study, the sequences of fusion protein (F) gene of three Newcastle disease virus (NDV) strains isolated from outbreak in chickens in the Al-Sharkia province of Egypt in 2006 were determined. Findings: The viral genomic RNAs were extracted from the infective allantoic fluid and F gene is amplified using primer sets designed from the available sequences of NDV strains from GenBank. The pathogenicity of NDV strains was determined by three internationally recognized tests mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index. The phylogenetic analysis showed that the Egypt isolates are closely related with the genotype II of class II NDV strains. Conclusions: The sequences of the F genes of the 2006 Egypt isolates are closely related to that of the 2005 Egypt isolate from the same province suggesting that these strains are probably circulating in the vaccinated bird population in Egypt until development of an outbreak.
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    CHARACTERIZATION AND DEVELOPMENT OF REVERSE GENETICS SYSTEM FOR AVIAN PARAMYXOVIRUS TYPE-3 AND ITS EVALUATION AS A LIVE VIRAL VECTOR
    (2010) Kumar, Sachin; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Avian Paramyxovirus (APMV) serotype 3 is one of the nine serotypes of APMV that infect a variety of avian species around the world. In chickens and turkeys, APMV-3 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-3, the complete genome sequences of prototype strain Netherlands and strain Wisconsin were determined. The genome length of APMV-3 strain Netherlands is 16,272 and for strain Wisconsin is 16,181 nucleotides (nt). Each genome consists of six non-overlapping genes in the order 3'N-P/V/W-M-F-HN-L5' similar to most of APMVs. Comparison of the APMV-3 strain Wisconsin nt and the aggregate predicted amino acid (aa) sequences with those of APMV-3 strain Netherlands revealed 67 and 78%, identity, respectively. The phylogenetic and serological analyses of APMV-3 strains Netherlands and Wisconsin indicated the existence of two subgroups within the same serotype. Both the strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenesis of APMV-3, a reverse genetics system for strain Netherlands was established in which infectious recombinant APMV-3 was recovered from a cloned APMV-3 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to parental virus. A recombinant APMV-3 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of recombinant APMV-3 expressing Newcastle disease virus (NDV) F and HN proteins demonstrated that the F protein plays a major role in protection against virulent NDV challenge. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-3 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-3 would be of considerable utility for introducing defined mutations into the genome of this virus and developing a vaccine vector for animal and human pathogens.