Isolation of infectious bursal disease virus (IBDV) in cell culture, detection and characterization by RT-PCR / SSCP analysis
Gaba, Amit (2004) Isolation of infectious bursal disease virus (IBDV) in cell culture, detection and characterization by RT-PCR / SSCP analysis. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.
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Infectious bursal disease (IBD), an economically important infectious viral disease of poultry is caused by Infectious bursal disease virus (IBDV) belonging to Avibirnavirus genus of Birnaviridae family. The disease causes considerable morbidity and mortality mainly by immunosuppression. The re-emergence of IBD in variant or highly virulent form in different parts of the world including India during the last couple of decades, have demanded further research efforts in understanding the added complexity of the disease process and the means to diagnose and control it. At present, the disease is controlled by the combined use of live virus and inactivated oil emulsion vaccines. But these vaccines are not always safe as they may not contain the required immunogens present in the variant strains prevailing in that area. Study of the virus at molecular level is therefore an essential prerequisite for formulating a suitable vaccine, particularly with local isolates recovered from field cases. In view of this, IBDV isolation in cell culture is essential and decisive for virus identification, genetic studies and molecular characterization of the virus. The present study was aimed at isolation of field IBDVs by cell culture technique using BGM-70 cell line, detection of IBDV in cell culture by RT-PCR and characterization of the isolated IBDVs by SSCP analysis to study any possible nucleotide sequence variation among the field and vaccine viruses. Six bursal samples were collected from the IBD vaccinated birds showing enlarged and haemorrhagic bursae. By AGID, all the six samples collected were found positive for IBDV antigen. BGM-70 cell monolayers inoculated with the bursal homogenates of two representative bursal samples (BF1 and BF2) found positive for IBDV antigen by AGID, resulted in isolation of the virus. Along with the field isolates, vaccine virus (Vac) was also propagated in BGM-70 cell culture. BF1 and BF2 isolates were adapted to cell culture after five successive passages. Distinct CPE characterized by rounding and clumping of cells was first observed by 72 hours PI at third and fourth passage level for BF2 and BF1, respectively. At fifth passage, increased rounding and clumping of cells, extensive cytoplasmic granularity, marked detachment of cells from surface monolayer and cellular degeneration leading to formation of plaques were recorded by 96 hours PI. At sixth passage level, CPE was characterized by degenerative changes involving more than 80 percent cells and extensive detachment of cells at 96 hours PI.Using live IBDV vaccine (Vac) as inoculum, rounding and clumping of cells, vacoulation and detachment of very few cells was observed at 48 hours PI during first passage itself. More than 80 percent cells were detached from the monolayer and extensive cellular degeneration was observed at 96 hours PI. CPE produced by Vac isolate was similar to that produced by BF1 and BF2 isolates in BGM-70 monolayer. Cell culture supernatant and infected monolayers at each passage level were used for viral RNA extraction using Trizol reagent. RT-PCR performed for viral RNA extracted from cell culture supernatant and infected monolayer of the isolates BF1 and BF2 at sixth passage level and Vac isolate at first passage level, using P1 and P2 pair of primers specific to hypervariable region of VP2 gene resulted in generation of a targeted amplicon of 643 bp, thus, confirming the growth of IBDV in cell culture.Rest all RNA samples extracted from cell culture supernatant and infected monolayer obtained from earlier passages as well as that from the control monolayer inoculated with known negative bursal sample did not produce the targeted amplification. On SSCP analysis, RT-PCR amplified products of both isolates BF1 and BF2 as well as vaccine virus revealed similar migration pattern and produced three bands each, two of single stranded DNA and one of double stranded DNA, suggesting absence of any nucleotide sequence variation among them with respect to targeted 643 bp segment of VP2 hypervariable region.
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