Molecular Epidemiology Of The Infectious Bronchitis Virus Isolated In China Between 1995 And 2004

Twenty avian infectious bronchitis (IB) viruses (IBV) were isolated from outbreaks in chickens in 11 provinces of China between 1995 and 2004. The Biological characteristics of 20 IBV isolates were analyzed. Structural protein (Spike glycoprotein, Membrane glycoprotein, Nucleocapsid protein, Envelope protein) genes and nonstructural protein genes (3a, 3b, 5a, 5b) in the 3' end of genome were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and then were cloned, sequenced and compared with IBV reference strains. The phylogenetic trees were also constructed based on the nucleotide sequences of the structural protein genes and those of the nonstructural protein genes in order to trace the source of domestic IBV isolates, to determine IBV genotypes exist in China, and the variation, the sequence identity, the phylogenetic relationships between Chinese isolates and reference strains.Typical signs including dwarfing, stunting, curling and death of embryo were observed in the third to seventh passages when each of the twenty Chinese isolates was inoculated into 9-day-old to 11-day-old specific-pathogen-free (SPF) chicken embryos. Diagnoses based on electron microscopy examination performed on allantoic fluids of different passages showed all twenty isolates had typical coronavirus morphology (Virions enveloped, slightly pleomorphic, spherical, 80nm-120nm in diameter. Surface projections of envelope distinct, club-shaped, spaced widely apart and dispersed evenly over all the surface.). No other agents such as Newcastle disease virus were detected. The above results elementarily suggested that the twenty viruses isolated in this study were avian infectious bronchitis viruses.Partial genome sequence of 3' end analysis showed that the 20 viruses had the S-3-M-5-N genes order that was typical of avian coronaviruses. A total of 6752 bp~6828 bp were found in a region beginning from the start codon of ORF S gene to the stop codon ORF of N gene of the 20 IBV isolates. The length of ORF3a, ORF5a, ORF5b and ORF of N genes were composed of 174 bp, 198 bp, 249 bp and 1230 bp respectively except CK/CH/LSD/031, ORF3a of which contained 144 bp. But the length of ORF of S genes were composed of 3477 bp~3510 bp, ORF3b were composed of 189 bp~219 bp, ORF3c were composed of 309 bp~330 bp, ORF of M genes were composed of 672 bp~681 bp. Deletion, insertion and mutation were found in the genome sequences of 20 isolates compared with reference strains which maybe one of the reasons lead to variation of genome of IBV isolates in China.The sequence identity and the phylogenetic relationships between the genes derived from the 20 isolates and the reference strains indicated that IBV isolates of China were branched into several genetic clusters. Serotypic evolution in IBV is associated primarily with the sequences of S1 glycoprotein and the genetic diversity of IBV is mainly monitored on analysis of S1 gene because the S1 subunit of spike glycoprotein of IBV is responsible for inducing neutralizing and serotype-specific antibodies in chickens and mutations in the antigenically important spike glycoprotein S1 subunit leads to theemergence and proliferation of variant serotypes associated with disease outbreaks. Based on the phylogenetic tree of nucleotide sequences of SI genes, IBV isolates in China were classified into eight Genotypes. Genotypes I, Genotype II, Genotype V and Genotype IX formed indigenous genotypes of China and 18 IBV isolates in this study were included in the four genotypes. IBVs in same genotypes showed more than 90% sequence similarities except BJ, SC021202 and CK/CH/LDL/04II isolates. IBV isolates in genotype III were 4/91 serotype. Genotype IV consisted of eight Chinese isolates that showed close relationship with Korean IBV isolates. Massachusetts serotype was present in China in 1990s and was in a separate genotype (VI) and most of IBV vaccines used in China belonged to this genotype. Two isolates, HN99 and CK/CH/LHN/001, which might be a reisolation of vaccine strains, clustered into genotype VII. Those results suggested most of IBV isolates in China formed indigenous genotypes and higher nucleotide sequence identity were found among them. Furthermore, low nucleotide sequence identity between the genes of IBV isolates of China and vaccine strains used in China also indicated that IBV vaccine strains and field isolates belonged to different genotypes, which may be responsible for frequent outbreaks of IB in vaccinated flocks in China. Hence, developing vaccines from local strains is necessary for IBV control in China.The comparison and phylogenetic analysis of gene sequences indicated that gene recombinations were occurring within China isolates. Phylogenetic analysis of SI genes and genes 3 showed that CK/CH/LLN/98I and CK/CH/LSD/03I shared as high as 96.9% and 96.7% nucleotide identities of SI protein gene with isolate QXIBV and nucleotide identities of gene 3 was 91.3% between CK/CH/LLN/98I and CK/CH/LHLJ/99, 92.6% between CK/CH/LSD/03I and U/CH/LDT3/03. By phylogenetic analysis of M genes, genes 5 and N genes, isolate CK/CH/LLN/98I, together with Ark DPI and Jilin vaccine isolates, belonged to an evolutionary cluster and shared higher nucleotide and deduced amino acid identities (>94.6%). Isolate CK/CH/LSD/03I, which shared more than 97.3% nucleotide identities of M gene, gene 5 and N gene with H52 vaccine, and Mass type IBVs belonged to the same evolutionary cluster. The analysis above showed that recombinations between field isolates and vaccine strains occured during the evolution of CK/CH/LLN/98I and CK/CH/LSD/03I. In conclusion, recombinations were another main reason leading to the variation of genome of IBV isolates in China.Due to virulence recovery and the influence of selective pressure from environment, IB live vaccine strains may spread in flocks and new serotype IBVs or IBV variations may emerge. Although Mass-type vaccines were commonly used in China, Chinese Mass-type strains were also isolated. Phylogenetic relationships indicated that Mass type IBV strains in China formed a genotype together with HI20, H52, and D41 vaccine strains and they shared a high degree of identities (95.2%~99.9%) of of SI gene sequence. In addition, the CK/CH/LHN/001 strain shared more than 99.0% similarity of each gene from S gene to N gene with Jilin vaccine strains. Take together, the emergence of new IBV strains might have a relationship to the application of attenuated vaccines. Theses isolates may have come from the vaccine strains by point mutation due to immunologic pressures caused by the widespread use of live vaccines, although the possibility that some of them were reisolations of vaccine strains cannot beexcluded.Some study indicated that the prevalence of IBV showed geographical characteristic. In our study, Korea strain K069-01, belong to Korea genotype III which was the main genotype in Korea, showed 90% gene sequence similarities with China genotype IV isolates. In addition, the N gene of Korea isolates K514-03 and K1255-03 had a close phylogenetic relationship with China IBV isolates (genotype VD). In conclusion, Korean isolates had a closer relationship with China ones. This may owe to the increased trade of agricultural products including poultry between two countries.