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Regeneration of a Recombinant Infectious Bursal Disease Virus Having Four Amino Acid Substitutions in Vp2 by Reverse Genetics

M Noor
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

C Lüken
Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, 04103 Leipzig, Germany

PM Das
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

MR Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

H Müller
Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, 04103 Leipzig, Germany

Abstract/ Executive Summary:

Infectious bursal disease virus (IBDV), a virus with a double-stranded, bi-segmented RNA genome, is an economically important pathogen of chickens. Recent understanding of the molecular biology of IBDV has implicated several amino acid residues in the capsid protein VP2 in pathogenicity and tissue culture adaptation. In the present study a recombinant strain of IBDV having four mutations in VP2 (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) has been generated using reverse genetics. Desired mutations were introduced in the VP2 gene of the cloned cDNA of genome segment A of a very virulent (vv) IBDV by site-directed mutagenesis. Capped RNA transcribed in vitro from cloned cDNA of the modified segment A and wild type segment B was co-transfected into chicken embryo fibroblast (CEF) cell culture. The recombinant virus, designated as BD- 3tcC, was rescued from the transfected cell culture and characterized in vitro. BD-3tcC retained all the four desired mutations and replicated with titres only slightly lower than those of CEF cell-culture-adapted wild-type IBDV. This recombinant strain can be used in future studies for understanding the biological significance of these four amino acid residues in VP2.

Keywords: Bursal disease, Virus, Amino acid, VP2, Reverse genetics, Genetics

Location: Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

To identify the Infectious bursal disease virus (IBDV) strain (designated as BD-3tcC), which had the four amino acid substitutions in VP2, as originally suggested to be associated with tissue culture adaptation of IBDV.

Methodology:

Previously constructed full-length cDNA clones of segment A and segment B of a wild type vvIBDV (BD-3wt) in pUC19 vector (pVL561 & pVL547) and that of a modified segment A of a recombinant IBDV strain BD-3tc (pVL565) [4, 5] were used as starting materials. All the cloned cDNA had the T₇ promoter at the 5' end. BD-3tc had already two amino acid substitutions at positions 253 (Gln→His) and 284 (Ala→Thr) of VP2 gene. In the present study two additional amino acid substitutions at position 279 (Asp→Asn) and 330 (Ser→Arg) were introduced sequentially. The selected sites for mutagenesis with expected amino acid changes. First the mutagenic primer “IBDV-D279N” and a flanking primer “INCO-DC#3” were used on the pVL565 (BD-3tc) template to synthesize a 405 bp megaprimer having the desired mutation (Asp279Asn). Then the synthesized megaprimer and the other flanking primer “INCO-DC#4” were used on the same template to amplify a 677 bp VP2 gene fragment having the desired mutation. The amplified PCR product was cloned in the pCR-2.1 plasmid vector by the TA cloning method and designated as pBAU5. Full-length cloned cDNA of the modified segment A (pVL913) and the wild-type vvIBDV segment B (pVL547) were used. In brief, cDNA corresponding to genome segments A and B were transcribed in vitro using T7 polymerase by run-off transcription method from the plasmids linearised with an appropriate enzyme. The capped RNA corresponding to segments A and B were co-transfected in chicken embryo fibroblast cells in the presence of a liposome. The expression of viral proteins in transfected cells was confirmed by immuno-fluorescence assay using a monoclonal anti-VP2 antibody (1/A6) 24 h after transfection. Confluent CEF cell monolayers grown in Petri dishes were infected at a multiplicity of infection (m.o.i.) of 0.5. After incubation for 1 h at 38ºC the supernatant was removed, the cells were washed twice with PBS, and fresh medium was added. At 0, 4, 8, 16, 24 and 48 hr after infection, cell culture supernatants from two Petri dishes each were collected and centrifuged at low speed (400g) for 10 min and the clarified supernatant was preserved at -20°C to determine extracellular virus concentration. Then 2 ml fresh medium was added to the dishes. After 3 cycles of freezing and thawing the cell lysate was centrifuged at 3,000g for 10 min, the supernatant was collected and frozen at -20ºC to determine intracellular virus concentration. Virus titres in the culture supernatant and cell lysate preparation at each time point were determined by plaque assay.

Source of Information: The Bangladesh Veterinarian (2014) 31(1): 12 - 19

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The recombinant IBDV strain BD-3tcC, in the backbone of very virulent genotype and having four defined mutations in the outer capsid protein VP2, would be a valuable asset for future investigation on the biological significance of these mutations. The protocol outlined in this paper could also be used to adapt available vaccine viruses to the current field situation through site-directed mutagenesis and reverse genetics to overcome the problems of vaccination failure due to antigenic drift in field isolates.

Knowledge Management: Journal