Adaptation Of Infectious Bursal Disease Virus Attenuated Strains In Brutal B Cells And Isolation And Identification Of HeYD Strain

Infectious bursal disease virus (IBDV) infecteing in chicken B lymphocytes is a double-stranded RNA virus. Classical IBDV strains can infect chickens 3 to 6 weeks of aged, and cause immune deficiency, thus cause other pathogenic infections and high mortality. The very virulent strain of IBDV (vvIBDV) is able to be directly lethal to chickens, make huge economic losses in poultry industry. And IBDV also decreased the role of other vaccines significantly.IBDV is a double-stranded,double-segment RNA virus which is no envelop, belongs to family rirnaviridae, of the genus Avirirnavirus. IBDV particle is a diameter of about 60nm icosahedron, and there are 5 viral proteins encoded by IBDV, such as VP1, VP2, VP3, VP4 and VP5. VP2 is the major structural protein and the most abundant in IBDV, which significantly impacts on viral virulence and cell tropism of IBDV. As VP2 is the main antigen of IBDV, it can make the body to produce protective antibodies against IBDV virus in terms of virus prevention. The VP2 hypervariable region, there are a toxic amino acids characteristics sits and a high degree possibilities of variability.In this experiment, two strain TAD and HN3 preserved in Laboratory of Immunology, Henan Academy of Agricultural Sciences and adapted DT40 or CEF strains isolated by subculture. Hypervariable region of VP2 protein gene amplificated by RT-PCR,cloned, sequenced, and compared the results with some strains represented a number of different virulence strains each other for comparison. The results show that there are some differences bewteen the original strain sequence and the adapted strain which possiblly made by some changes occurred in the course that a virus adapt to the cell by subculture.As the role of VP2 is significant and the variability of its hypervariable region is high, the VP2 hypervariable region nucleotide sequence and amino acid sequences of original strains and cell adapted strains were compared to the analysis, for furtherly understanding variations of the virus in the adapt process and founding those amino acid residues which have important implications on the viral cell tropism. In this study, using MEGA 3.1 nucleotide and amino acid building Sequence phylogenetic tree by NJ (Neighbor Joining) method, VP2 hypervariable region gene of four strains with different virulence selected from the GenBanks and the original strains and their adapted strain have been done in phylogenetic analysis. Using MegAlin of DNAstar, two different cells adapted and the original attenuated strains are compared, for understanding of the changes frequency and mutation point of nucleotide and amino acid sequences and the nucleotide and amino acid in the process to adapt. The results show that there are minor differences in the VP2 hypervariable region gene between the DT40 cell adapted strains and the original strain, and differences in nucleotide sequence of not more than 1%, difference in amino acid sequence is not more than 10%, while on the VP2 hypervariable region sequences of the CEF adapted strains did not change. Compared the original strain with DT40 cells adapted strains,there are only four nucleotide substitution loci, and three amino acid replacement. The substitution sites in adapted strains are not the key to the virulence of IBDV sites, and replacing amino acid is not some special amino acid such as proline, so the impact on the protein structure is not evident. And the VP2 hypervariable region of CEF adapted strains, nucleotide sequence and amino acid sequences are not changed, there may be changes in other VP2 sites or other gene causeing adaptation.For further studying the cellular adaptation mechanisms in different toxic strains of IBDV, new strain HeYD suspected very virulent strain in Henan were isolated, cloned, analysed and aligned with hipervariable region sequence of other reference strains. Sequence analysis showed that there are typical very virulent strain characteristics in the VP2 hypervariable region amino acid in isolated IBDV. phylogenetic tree constructed with sequence of. VP2 hypervariable region, show that the relationship of HeYD and domestic very virulent strains is closer than the relationship it and classic domestic strains.