Complete Genome Sequence Analysis And Pathogencity Of IBDV-FJ02 Strain

Infectious bursal discase(IBD),caused by infectious bursal discase virus(IBDV),is an infectious disease that is acute and highly contagious to chicks.The antigenic and virulent variations of IBDV usually induce immune failure,thus causing serious economic losses to the poultry industry.The IBDV-FJ02 strain previously isolated by our group is one of the prevalent strains in local region.In order to study the regulation of genetic evolutionary for strain and virulence variation,this experiment,eight pairs of overlapping primers were designed basing on the IBDV genome sequences published in NCBI website,and the complete genomic sequences of IBDV-FJ02 isolate was determined DNA sequencing.The obtained sequences were spliced and then used to analyze the homology,phylogenetic relationship and characteristic amino acid sites of the two segmented genes(A and B segment)in comparison with IBDV reference strains using the biology software.Finally,the pathogenicity of IBDV-FJ02 strain is a preliminary study by SPF chick embryos and animal inoculation tests,with clear genetic evolutionary regulation of strain and clinical disease,This study will be helpful to understand the genetic evolution and clinical pathogenesis of IBDV-FJ02 strain,and provide theoretical references for the prevention and treatment of prevalent IBD in local region.The results are as follows:1.The genomic sequencing results of IBDV-FJ02 strain showed that the length of fragment A gene was 3257 bp,including two open reading frames(ORF),of which ORF2(450 bp)encoded VP5 protein,and ORF1(3039 bp)encoded VP2-4-3 polyprotein;the length of the fragment B gene was 2823 bp,which contained a complete ORF(2640 bp),encoding VP1 protein.2.The results of homology,phylogenetic relationship and characteristic amino acid sites analysis of IBDV-FJ02 strain showed that:(1)the segment A gene of IBDV-FJ02 strain had the highest nucleotide homology of 95.7%with that of American variant-E IBDV strain;segment A VP2 gene-based phylogenetic tree analysis also showed that the IBDV-FJ02 strain located at the same branch with American variant-E IBDV strain;and the characteristic amino acids of 213N,222T,249K,318D and 323E in the VP2-4-3 polyprotein of IBDV-FJ02 strain were all identical to those of variant IBDV strains;(2)the segment B gene of IBDV-FJ02 strain had the highest nucleotide homology of 96.5%with that of American classical IM strain;and segment B-based phylogenetic tree analysis showed that IBDV-FJ02 strain located at a relatively individual branch;the conservative amino acids of the VP1 protein encoded by segment B gene were identical to the classical strains and the variant strains except for 147D and 506K.3.The results of pathogenicity study of IBDV-FJ02 strain showed that:(1)7-day-old chick embryos were inoculated with 0.2 mL of IBDV-FJ02 strain,and chick embryos began to die after 181 h,and all died at 337 h.The dead embryos displayed the characteristics of embryo shrink,edema and hemorrhage,splenomegaly,liver hemorrhage and speckled necrosis,chick embryos died at late-infection period also appeared liver atrophy,pale myocardium with boiled like lesions;(2)SPF chicks were inoculated with 0.2 mL of IBDV-FJ02 strain and then subjected to cohabitation infection ?Chicks in the inoculation group and cohabitation infection group only displayed subclinical symptoms,necropsy inspection showed that obvious splenomegaly and rapid atrophy but no inflammations of bursa were visible in both groups,which were similar to the pathogenic characteristics of standard variant IBDV strains;in addition,breast muscle and leg muscle bleeding were observed in individual chicks;?According to organization pathological observation,we found that IBDV-FJ02 strain caused different degrees of lymphocytes necrosis in major immune organs in infected SPF chicks,in which the damage of the bursa of Fabricius lymphocytes is more serious,and the histopathological changes of the thymus and spleen are relatively mild;?Fluorescence quantitative PCR was used to detect IBDV infection,and results showed that the cohabitation infection group was IBDV positive at 3?21 days after challenge,and the duration of IBDV infection in the major immune organs of the inoculation group was different,the virus lasted longer in bursa and thymus,which was positively detectable at 21 d and 17 d after challenge,respectively,comparatively,the duration of IBDV infection in spleen and cecal tonsils were relatively short,which was undetectable at 14 d after challenge.In summary,our results suggest that IBDV-FJ02 strain is more close to the standard variant IBDV strains basing on genomic analysis,distinctive amino acids analysis as well as study of clinical pathogenic characteristics,while there are still some differences in pathogenicity.