DETECTION AND STRAIN VARIATION OF FIELD INFECTIOUS BURSAL DISEASE VIRUS (IBDV) BY RT - PCR - RELP PROFILES
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Date
2004
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AAU, Anand
Abstract
Infectious bursal, or Gumboro, disease is an acute, highly infectious viral disease of immature chickens, caused by Infectious bursal disease virus (IBDV) belonging to Avibirnavirus genus of Birnaviridae family. The disease causes considerable morbidity and mortality mainly by immunosuppression. Emergence of very virulent IBDV (vvIBDV) strains in different parts of the world including India, during the last couple of decades, has demanded further research efforts in understanding the added complexicity of the disease process and the means to characterize the genetic variation, so that proper control measures can be taken. The present study was aimed at screening IBD suspected field bursal samples by Agar gel immunodiffusion (AGID), followed by genetic variation study by molecular techniques i.e. Reverse transcription-polymerase chain reaction / restriction fragment length polymorphism (RT-PCR/RFLP). The genetic similarities among the IBDVs were estimated by phylogenetic relationship obtained by PopGene software analysis and Network analysis. Processing of the 37 field bursal samples by AGID revealed IBDV in 23 (62.16 %) samples. These samples and two live IBD vaccines were used for viral RNA extraction by Tri ReagentĀ® method, which yielded RNA of good quality and quantity, and produced targeted amplicons of approximately 643 bp by primer P1 & P2 specific for hypervariable region of IBDV VP2 gene, for 31 (83.78%) field samples and both the vaccines. RT-PCR products from 31 positive bursal and two vaccine samples were further digested with four restriction enzymes (RE) viz. Msp-I, Sac-I, Stu-I and Taq-I for RFLP analysis, which yielded two, one and zero restriction sites and eight different haplotypes (A, B, C, D, E, F, G & H). None of the samples was digested by Sac-I. Majority of the samples behaved identically to the individual RE, as 24, 28 and 28 field samples showed single RE site for Msp-I, Stu-I and Taq-I respectively. Both the vaccine samples behaved similar to the majority of the samples, except for Stu-I by showing an unique additional restriction site placing them exclusively in haplotype D. Locationwise, haplotype F was found at maximum four locations, and strainwise, haplotype A was only found in a layer strain (BV-300). Phylogenetic analysis was done with PopGene software, which clustered the 31 field IBDVs and two vaccine strains into six molecular groups (designated as IG1, IG2, IG3, IG4, IG5 and V), based on RE profiles among the IBDVs. These molecular groups were compared for the genetic similarities among them, as well as with 14 international IBDV strains (seven classical; two variant; three classical vaccine and two vvIBDV strains) taken in silico from GeneBank database and digested similarly with the same four REs by DNAsis software (Pharmacia, Uppsala, Sweden). Two field samples falling in molecular group IG5 differed maximally from majority of the field and vaccine samples, by possessing an additional Msp-I site. Taq-I digestion revealed a new restriction site in majority of the samples possibly indicating very virulent nature of the field IBDVs. Different degrees of similarity observed among the field samples, suggested circulation of variant IBDVs in the field. Network analysis further emphasized the heterogenicity among the field and vaccine IBDV strains to the reference IBDV strains taken from GeneBank. IG5 showed maximum seven and five mutations from the central cluster of the reference classical IBDV strains and vaccine strains (included in the study) respectively. IG4 showed only one mutation with V group and was thus maximally similar than the other molecular groups. Among the 14 reference strains, both the wIBDV (849VB and T1/TW) and variant (Variant A and MTA) strains showed higher number of mutations from the central cluster.
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VERTINARY MICROBIOLOGY, A STUDY