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Differentiation of Infectious Bursal Disease Virus (IBDV) Genome Segment B of Very Virulent and Classical Lineage by Rt-pcr Amplification and Restriction Enzyme Analysis

M. R. Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

S. Rahman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M. Noor E. H. Chowdhury
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

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

Abstract/ Executive Summary:

Molecular characterization of IBDV usually relies on the analysis of segment A of the bi-segmented, double-stranded RNA genome. Although segments B of classical and very virulent IBDVs differ significantly, re-assortment of genome segments does occur, and molecular epidemiological studies demand the analysis of both segments. An RT-PCR and restriction enzyme analysis for molecular discrimination between genome segment B of classical and very virulent IBDVs is described. Tested on eight IBDV strains/isolates, the protocol successfully identified very virulent and classical IBDVs as well as a segment reassortant. This approach is a valuable tool for molecular epidemiological studies on IBDV.

Keywords: Cell-Culture-Adapted, IBDV

Location: Department of Immunology, University of Strathclyde,Glasgow, UK

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Diseases

Objectives:

To Differentiation of infectious bursal disease virus (IBDV) genome segment B of very virulent and classical lineage by RT-PCR amplification and restriction enzyme analysis

Methodology:

A total of eight IBDV strains or isolates were used in this study: five very virulent isolates, including BD 1/99, BD 2/99, BD 3/99 (Bangladesh), VB849 (Belgium), Gx (China), and three classical virulent strains, F52/70 (UK), Cu-1wt (Germany) and D-78 (a vaccine virus, Intervet). Lyophilized preparations of VB849, Gx and F52/70 were a gift from Dr. N. Eterradossi, ANSAS, Ploufragan, France. Total RNA was isolated using a kit (RNAgents Total RNA Isolation System, Promega Corporation, USA) and subjected to RT-PCR for segment A and segment B. A previously reported primer set (INCO-DC # 3F: 50 -AAC AGC CAA CAT CAA CG-30; INCO-DC # 4R: 50-GCT CGA AGT TGC TCA CCC-30) was used to amplify a 677-bp fragment from the hypervariable region of the VP2 gene in segment A. For segment B the following primers were newly designed: B-Univ-F: 50-AAT GAG GAG TAT GAG ACC GA-30; B-Univ-R: 50-CCT TCT CTA GGT CAA TTG AGT ACC-30. These amplified a 1051-bp fragment (nucleotides 319-1369) from the VP1 gene in segment B. A one-tube RT-PCR reaction was performed on a thermocycler using the Superscript One-step RT-PCR System (Invitrogen, USA) following the manufacturer’s instructions with some modifications. RTPCR for both segments A and B were run simultaneously using the same reaction profile. Briefly, the first reaction mixture (20µl), containing 13µl nuclease-free water, 1µl of each primer and 5µl RNA template in a 0.2-ml PCR tube, was placed on the thermocycler, heated at 95C for 5 min for initial denaturation of double-stranded RNA and transferred to ice immediately. Then, a 30-µl aliquot of a mastermix containing 25 µl of 29 reaction mixture (buffer and dNTPs), 1µl enzyme mix (reverse transcriptase and polymerase), 0.5 µl RNaseOut (RNase inhibitor) and 3.5µl nuclease-free water was added to each tube, mixed gently with the pipette tip and returned to the thermocycler. The reaction profile consisted of reverse transcription for 30 min at 50C, followed by activation of Taq polymerase for 2 min at 94⁰C, then 30 cycles of denaturation (at 94⁰C for 30 sec), annealing (at 55⁰C for 30 sec) and elongation (at 68⁰C for 1 min + 1 sec/cycle after the 10th cycle), and final elongation at 68C for 7 min. A portion of the RT-PCR product (7 µl) was analyzed by electrophoresis in a 1.2% agarose gel containing ethidium bromide (5 lg/ml). The RT-PCR product amplified from segment A was digested with SacI and SspI, and the product from segment B was digested with KpnI. Possible cleavage of the cDNA with the restriction enzymes was analysed by electrophoresis on a 2% agarose gel. Nucleotide sequences of the coding regions of the polyprotein gene of segment A and the VP1 gene of segment B of 35 strains of IBDV were downloaded from GenBank. The selected strains included the well-studied IBDV strains of known pathotype as well as the strains that have been reported to be segment reassortants. The nucleotide sequences were subjected to multiple alignment analysis using the Clustal W method in the MegAlign module of DNAStar software.

Source of Information: Archives of Virology, Official Journal of the Virology, Division of the International Union of Microbiological Societies, ISSN 0304-8608, Volume 157, Number 2, Arch Virol (2012) 157:333-336,

Article Link (Online Journal): DOI 10.1007/s00705-011-1159-9

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the present study, only eight isolates/strains were subjected to segment B RT-PCR followed by restriction enzyme analysis with KpnI. The presence or absence of this restriction site in the segment B nucleotide sequence of all the 35 IBDV strains was examined theoretically from the alignment report. It was observed that only the 11 IBDV strains that have a very-virulent-type segment B also possess a KpnI restriction site, GGTACC, at nucleotide position 777-782. The remaining 24 strains having a classical-type segment B do not have the KpnI restriction site at the corresponding position, as the nucleotide ‘A’ at position 780 is substituted by ‘G’. It may, therefore, be concluded that the present RT-PCR for segment B of IBDV coupled with restriction enzyme analysis with KpnI could successfully distinguish IBDV strains having segment B of classical and very virulent lineage. This method could be used as an important tool in molecular epidemiological studies on IBDV.

Knowledge Management: Journal