Complete Genomic Cloning And Development In The Reverse Genetics System For Infectious Bursal Disease Virus

The study of viruses and their interactions with host cells and organisms has benefited greatly from the ability to engineer specific mutations into viral genomes,a technique known as reverse genetics.To develop the reverse genetics system,or so-called genetic rescue techniques for infectious bursal disease virus (IBDV),Birnaviridae family,the full-length cDNA of the double segment of the chicken IBDV was amplified and cloned by long RT-PCR in the first-step.A comparison of four purification and extraction methods of RNA from IBDV( HZ2 strain,isolated originally in Zhejiang Province)-infected chicken embryoid fibroblasts (CEF) showed that the ultracentrifugation followed by proteinase K digestion extracted dsRNA more effectively. Then reverse transcription was carried out at 50C using Superscipt II enzyme,followed by RNase H digestion. Amplification of single stranded cDNA in a single step resulted in the synthesis of the full-length segment A of 3259 bp and segment B of 2827 bp respectively. The amplified products were cloned and sequenced,identifying that it was an IBDV. This method is superior to other methods based on amplifying different parts of the genome many times,therefore the cloning procedure was simplified.Analysis of the full-length of segment A of IBDV isolatedin Zhejiang Province,including an attenuated strain HZ2,an attenuated vaccine strain JD1 and a virulent field isolate ZJ2000,revealed the identity of IBDV with two overlapping open reading frames( ORF ) flanked by 5' -and 3' -noncoding regions( NCR ) in 3259bp long. The strains shared high identity with each other at nucleotide or deduced amino acid level,and also had four unique sites H253,N279,T284,R330 which are common in other attenuated and some classic or highly virulent strains. The virulent strain ZJ2000 had several key amino acid mutations located in hypervariant region of VP2 and near the VP2-VP4 cleavage site of polypeptide,one of which is in the new identified 11 aasmall peptide. It may be related to the virulence. Sequence comparison supported that VP2 is not the sole determinant of the virulence. The highly conservation in 5' - and 3' - NCR of different strains indicated the NCR may be not responsible for the virulence. But the same conservation appeared in VPS revealed another complex relationship between VP5 and the virulence.The full-length nucleotide sequence including the 5' - and 3' -noncoding regions (NCR) of IBDV strain HZ2 and JD1 was established. The nucleotide sequence encoding VP1,an RNA-dependent RNA polymerase (RdRp),of HZ2 and JD1 were aligned with that of all other IBDV strains. Surprisingly,An in-frame start codon (ATG) is presented in the 5' -NCR upstream of usual VP1 genes which are coded by all the compared strains except an Canadian strain QC-2. According to the sequences information of other two birnaviruses including IPNV and DXV,we suppose the advanced in-frame start codons presented generally in the first ORFs of birnaviruses do not come by accident and play an important role in the initiation of virus proteins translation. The ACG may be the start codon in other IBDV strains in the same position. For IBDV,it is possible that the VP1 gene started from the first or second in-frame start codon respectively results in two different size products. The potential sites responsible for IBDV virulence were also searched and listed. The results showed that substitution at positions 4,13,256 may be the key amino acids related to the virus adaption or attenuation.The genetic tags were introduced to two segments of HZ2 strain respectively that were distinguished from the parent virus sequences by site-directed silent mutagenesis. The cDNA clone of segment A was engineered to contain two silent nucleotide changes to create a Nae I site (A to C and A to C at nt 2368 and 2371,respectively). At the same time,the 3'-NCR of coding strand of segment A was inserted a G at nt 3243 or a C at 3235. The former is expected to change the stem-loop secondary structure at the 3' terminus of segment A. The cDNA cl...