Use of Polymerase Chain Reaction for Efficient Cloning of dsRNA Segments of Infectious Bursal Disease Virus

Abstract

A method is described for efficient cloning of the large RNA segment of infectious bursal disease virus (IBDV) after reverse transcription and cDNA amplification. Complementary DNA segments of IBDV were prepared using reverse transcriptase and specific primers homologous to the conserved region at the 3' end of the IBDV sequence. The resulting cDNA segments were amplified using Taq DNA polymerase and a pair of specific primers. Three separate primer pairs were used for amplification, each yielding a cDNA fragment of the predicted size. The amplified products were directly used for cloning into a cloning vector, pCR1000. Three overlapping cDNA clones, containing the entire coding region of the large RNA segment of IBDV, were obtained. The identity of these clones was confirmed by hybridization with IBDV-specific probe, as well as by sequence analysis. By this method, approximately 3.2 kilobases of the large genome segments of two different strains of IBDV were cloned, and their complete nucleotide sequence was determined.

Genotyping of Canadian field strains of infectious bursal disease virus

“…and the reverse (IBDV-R, 5?-CAC AGC TAT CCT CCT TAT GG-3?) primers were designed to amplify a 929-base-pair fragment from nucleotides 440 to 1368, based on the Delaware E strain VP2 gene sequence (Vakharia et al, 1992). To allow a comparison of restriction fragment length polymorphism (RFLP) fragments from Canadian IBDV strains with results from previous US reports Jackwood et al, 2001), the forward (IBDV-FN, 5?-GCC CAG AGT CTA CAC CAT AAC-3?)…”

“…and the reverse (IBDV-RN, 5?-CCC GGA TTA TGT CTT TGA AG-3?) primers amplified a 743-base-pair fragment of the IBDV VP2 gene sequence from nucleotides 606 to 1348, based on the Delaware E strain VP2 gene sequence (Vakharia et al, 1992).…”

Tissue culture infectivity of different strains of infectious bursal disease virus is determined by distinct amino acids in VP2

“…D16677; K, D16678; OKYMT, D83985; and TKSMT, D84071), Bayliss et al (1990) (strains PGB98, D00868; and Cu-1, D00867) and Mundt & Müller (1995) (strain P2, X84034) and from strain D78 (V. N. Vakharia, personal communication), were compared to obtain a consensus sequence. Sequences of bursa-derived IBDV strains have been published by Vakharia et al (1992) (strain E/Del, D10065), Bayliss et al (1990) (52/70, D00869), Thiry et al (1992) (Edgar, A33255), Kibenge et al (1990) (STC, D00499), Yamaguchi et al (1996) (OKYM, D49706; and TKSM, D84072), Brown et al (1994) (DV86, Z25482; and UK661, X92760), Hudson et al (1986) (002-73, X03993), Vakharia et al (1994) (GLS, M97346), Yamaguchi et al (1997) (LukertBP, D16679), Lana et al (1992) (variant A strain, M64285), Pitcovski et al (1998) (KS, L42284) and Heine et al (1991) (variant E strain, D10065). Unpublished sequences from strains U-26 (AF091099), Miss (AF091098) and 3212 (AF091097) (H. S. Sellers, P. N. Villegas, D. J. Jackwood & B. S. Seal, unpublished results) were also included in the comparison.…”

Purification of a recombinant protein produced in a baculovirus expression system by immobilized metal affinity chromatography

“…Complementary DNA segments of E/DEL IBDV were synthesized by the method of Gubler and Hoffman13 using genomic RNA as a template (bursa derived) and a synthetic primer binding specifically to the 3' end of the VP2 gene sequence. 42 The cDNA segments were cloned into EcoRI site of pGEM-7Z+ vector using EcoRI adaptors-ligation system (Promega, WI). The complete nucleotide sequence of plasmid pEDEL-2 (E/DEL VP2 cDNA clone) was determined and submitted to EMBL Database under the accession number X 54858.…”