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Authors: Kumar, Balvinder
Advisor: Prasad, Gaya
Title: Nucleic acid based identification and molecular characterization of group A rotavirus causing diarrhea in buffalo calves
Publisher: LUVAS
Language: en
Type: Thesis
Agrotags: null
Keywords: Buffalo, Group A Rotavirus, VP4 & VP7 genes, Genomic diversity, RT- PCR , PAGE
Abstract: Group A bovine rotaviruses play an important role in causing gastroenteritis and calf mortality leading to significant economic losses to farmers in India. Due to segmented nature of the RNA genome and wide host range, vast genetic and antigenic diversity exists amongst different isolates of rotavirus. Molecular characterization of group A rotavirus in terms of nucleotide sequencing of locally prevalent rotavirus strains circulating in buffalo population would eventually help in formulation of comprehensive disease diagnosis and control strategies. To address these concerns, the present study was undertaken to characterize buffalo group A rotavirus circulating in north India. A total of 455 faecal samples from buffalo calves from organized buffalo farms in north India, were screened and 33 (7.25%), 21 (4.61%) and 15 (3.3%) were xlii found positive by ELISA, PAGE and RT-PCR, respectively. All the samples revealed long electropherotypes by RNA-PAGE. VP7 and VP4 genes were amplified as evidenced by an expected PCR product of 1011bp (VP7), 864bp (VP4). All positive samples were also isolated in MA 104 cell line and tested by RT-PCR. Typing by RT PCR was done using specific primers representative of VP4 and VP7 genotypes. Genotyping by nested PCR typed G6, G10 and P[11] genotypes but VP4 genes of 11 isolates remained untyped by PCR. Amplified VP4 and VP7 PCR amplicons were cloned and screened by touch PCR. The positive recombinant bacterial colonies were subjected to sequencing. The sequences were analysed using BlastN and aligned by Clustal X. The percent identity and distance matrices of the partial VP4 and VP7 gene sequences with representatives of prototype G and P genotypes were generated by MEGA version 4. Phylogenetic trees were constructed by analyzing nucleotide and deduced amino acid sequences by MEGA 4 software. By phylogenic and evolutionary analysis the untyped VP4 gene/protein sequences were classified as P[3] genotypes. Phylogenetic analysis also confirmed genotyping results obtained by nested PCR for G6, G10 and P[11] genotypes. In this study 73.3% of the samples were G10P[3] and 26.7% were G6P[11]. The amino acid sequences deduced from partial VP4 and VP7 gene sequences were aligned with amino acid sequences of established strains and compared. It has been concluded that G6P[11] and G10P[3] Group A rotaviruses are circulating in buffalo herds of organized farms in north India. Unusual reassortants G10P[3] of Group A rotaviruses isolated from buffalo calves shows new genomic constellations and indicate interspecies reassortment. The information may be of great use while devising vaccination strategy.
Subject: Animal Biotechnology
Theme: Nucleic acid based identification and molecular characterization of group A rotavirus causing diarrhea in buffalo calves
These Type: Ph.D
Issue Date: 2008
Appears in Collections:Thesis

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