Comparative Study On The Relationship Between The Virulence And Gene Sequence Of Attenuated IBDV Strains And Passage Strains In Chickens

Infectious bursal disease (IBD) is an acute and highly contagious viral disease of young chickenscaused by Infectious bursal disease virus (IBDV). The epidemic of very virulent IBDV strainsin the late1980s, make the cause of the virulence variation of IBDV become the focus of research. Butso far, the virulence factor of IBDV was not clear. In order to provide more molecular basis for thevirulence of IBDV, the attenuated IBDV strains were serially passaged in specific-pathogen-free (SPF)chickens in this study, and the genome sequences of the attenuated IBDV strains and the passage strainsin chickens were amplified and analyzed. At the same time, the IBDV E2strain was passaged4successive times in chicken embryo, and the the genome sequences of the E2strain and the passagestrains in chicken embryo were amplified and analyzed. The main results in this study are as follows:(1) Two attenuated IBDV strains were passaged five or six times in SPF chickens. Both attenuatedstrains increased in virulence during the passage in susceptible chickens as evidenced by the decrease inbursa/body weight ratios and B:B index.(2) The genome sequences of the attenuated IBDV strains and the passage strains in chickens weresegmentally amplified by RT-PCR and molecular clone technology. A direct sequence alignmentanalysis results showed that the genome B segment were conservative with no nucleotide mutationduring the passage in chickens; however, there were5nucleotide mutations in genome A segment,which resulted in3amino acid substitutions at position80(from R to G)、125(from P to S) and130(from C to R) in VP5，an amino acid substitution at position253(from H to Q or N) in VP2and anamino acid substitution at position778(from V to M) in VP3. Further homologous analysisdemonstrated that the mutation of amino acid at position253in VP2made the strains after passage inchickens have a higher identity with the classical strains and the very virulent strains, and a loweridentity with the attenuated strains; Those results suggested that the mutation of amino acid at253sitein VP2may be one of the molecular mechanism of IBDV virulence enhancement during the passage inchickens. The3-D structure prediction of VP2revealed that amino acid residue at position253exposedat the surface of VP2projection domain, demostrating the amino acid mutation from H to Q or N at thissite may improve its hydrophilicity, which may facilitate the interaction between IBDV and the cellreceptor in the host, so as to increase the pathogenicity of IBDV.(3) Sequence analysis of the IBDV E2strain and its passage strains in chicken embryo showed thatthere were some unique amino acids variation in VP2between the classical strains and the attenuatedstrains:253(Q�'H)、279(D�'N)、284(A�'T)、330(S�'R), which further elucidated the molecularmechanism of IBDV virulence attenuation during the passage in chicken embryo.