Complete Genome Sequencing And Analysis Of Virulent And Attenuated Infectious Bronchitis Virus

Infectious bronchitis is an acute,highly contagious respiratory disease caused by infectious bronchitis virus.Domestic chicken is the most important but not the only host for IBV.IBV,a representative species of coronavirus gamma group,is an enveloped and nonsegmented positive-sense,single-strand RNA virus.Owing to IBV frequent mutation and recombination,resulting in the emergence of new variant strains.IBV has a large number of serotypes,but no corresponding special relationship between serotype,genotype and pathogenic type.At present,the weak phylogenetic relationship between the attenuated vaccine of the domestic and the epidemic strain,which cause them cross protection is very poor or no cross protection.This experiment by measuring the complete genome sequence of strain HQ 10 and passaged attenuated strain HQ 110,the analysis may be changed with the virulence-related genes,and to understand the genetic evolutionary relationship of strain HQ 10.According to IBV genome sequence has been released on the GenBank,to select five complete genome sequence as a reference sequence?GenBank accession Nos:NC₀01451.1?KF411041.1?FJ807652?DQ646405?AY338732?and to design IBV whole genome amplification primers.By RT-PCR and RACE techniques,strain HQ 10 and passaged attenuated strain HQ110 were amplified and subsequently cloned and sequenced.The results showed that HQ 10 and HQ110 size of the complete genome sequence is 27762 nt.Genomic structure is 5'-Cap-1a-1b-S?S1,S2?-3a,b,c?E?-M-5a,b-N-3'Poly?A?,containing 10 open reading frames,and consistent with the classical IBV genomic structure.3 'UTR and 5' UTR of strains HQ 10 and HQ110 are 504 nt and 525 nt.Gene 1 size is 19889 nt?526?20414?and contains two open reading frames.S gene?20365?23862?size is 3498 nt,and the cleavage site of S protein is HRRRR.S1?20365?21984?and S2?21985?23862?gene respectively 1620 nt and 1878 nt,respectively,encoding 540 and 625 amino acids.Gene 3?23862?24539?contains three open reading frames:3a?173 nt?,3b?192 nt?and E?333 nt?,respectively 57,63 and 110 amino acids encoded.M gene?24508?25188?size is 681 nt,encoding 226 amino acids.Gene 5?25542?25984?contains two open reading frame 5a?198 nt?and 5b?249 nt?,which encoded 65 and 82 amino acids.N gene?25927?27156?size is 1230 nt,encoding 409 amino acids.Genome sequence homology analysis of strain HQ 10 and domestic and international reference strains showed that strain HQ 10 and LX4 have the highest homology?92.4%?and strain H52 and H120 has the lowest homology?86.2%?.Phylogenetic tree analysis of the whole genome and structural gene showed that the relationship between strain HQ 10 and LX4 is close,and the relationship with strain H52,H120 and M41 is distantly related.Although complete genome sequence similarity of the passage attenuated strains HQ110 and strain HQ10 is up to 99.88%,but the virulence between them are completely different.Through the comparison of complete genome sequences of strain HQ 10 and HQ110 found before and after passage is only 32 nucleotide changes,resulting in 24 amino acid changes.Genel has 15 nucleotide changes,causing the 12 amino acid changes.S1 gene has 6 nucleotides changes,causing 5 amino acid changes.S2 gene has 3 nucleotides changes,causing 3 amino acid changes.E gene has 2 nucleotides changes,causing 1 amino acid changes.5a and N gene has each of 1 nucleotides changes,causing 1 amino acid changes respectively.There are 2 nucleotide changes in 3'UTR region.Sequence changes are mainly concentrated in the ORF1 replicase gene,S gene and 3'UTR region.