| Infectious bronchitis (IB) is an acute, highly contagious viral infectious disease caused byinfectious bronchitis virus (IBV). IBV is a gammacoronavirus of the subfamily Coronavirinae，familyCoronaviridae，Order Nidovirales. To explore the mechanism of attenuation of pathogenic IBV strain,provide highlights for design of new vaccines and study of pathogenesis, a IBV strain ck/CH/LDL/97Iwas used as a model virus for investigating the difference of complete genomic sequence betweenparent pathogenic IBV ck/CH/LDL/97I P5(P5) and embryo-passaged attenuated ck/CH/LDL/97I PI15(P115), virus replication in vivo and in vitro, levels of specific antibody, and the expression profiles ofcytokine and mediator gene after inoculated with P5or PI15，respectively.Sequences of27,680and27,682nucleotides were obtained from ck/CH/LDL/97I P5and P115,respectively. The genome organization was as follows:5’-UTR-gene1(ORFla, lb)-S-gene3(ORFs3a,3b, E)-M-gene5(ORFs5a,5b)-N-UTR-3’. Based on sequence analysis, ck/CH/LDL/97I wasgenetically closely related to US IBV strains, suggesting a common oirgin or an epidemiological linkbetween ck/CH/LDL/97I and US strains. By comparing the P115with its parental virus P5, no sequencechanges were observed in nsp5, nsp6, nsp7, nsp8nsplO, ORF3b or0RF5a after115passages. Changeswere observed in nsp4，nsp9，nsp11/12, nsp14, nsp15，nspl6and ORF3a, but these did not result inamino acid substitutions and/or showed variations because of amplification of specific viralsubpopulations. Two and three nucleotide changes were observed, respectively, in the5’-and3’-UTRsof ck/CH/LDL/97I PI15，compared to the parent strain P5. Most nucleotide mutations were found in theS region, with the most in S2gene between P5and PI15，and some of the nucleotide mutations resultedin amino acid substitutions. Among the nine nsps in the ORF1region which showed nucleotidedifferences between the pathogenic P5and attenuated PI15viruses, nsp3had the most nucleotidechanges. In addition, P115had two nucleotide insertions in the intergenic untranslated region betweenM gene and gene5.In the current study, the growth characteristics of IBV strains P5and PI15in embryonated eggswere compared by real-time RT-PCR to determine the amounts of virus in allantoic lfuid collected fromembryonated eggs inoculated with each virus over a period of36h. P5and PI15exhibited similargrowth profiles in eggs, with peak viral growth at24h post-inoculation. Moreover, the replicationcapacity of P5and PI15were assessed by detecting IBV-derived RNA in9tissues using real-timeRT-PCR. The result showed that most tissues had detectable viral RNA from days4-21post-inoculationwith P5and P115, respectively. However, no RNA was detected in some tissues. Remarkably, chickensinoculated with P5showed higher viral genomic RNA loads than those inoculated with PI15from days7-21, except for trachea, lung and Harderian gland tissues from chickens in the P115-inoculated groupon day4，which showed higher replication of viral genomic RNA than those in the P5-inoculated group.Most of the tissues were positive for virus recovery on days4and7after inoculation with P5or PI15.In addition, chickens inoculated with P5virus had higher virus recovery rates than those inoculated withP115virus. The replication capacity of attenuated P115was found to be reduced in several tissues according to virus recovery and real-time RT-PCR, compared to P5-inoculated chickens. ELISA testshowed that chickens inoculated with P5had higher antibody titers than those inoculated with PI15，implying slightly decreased antigenicity of IBV after serial embryo passages. We also compared the SIgene in re-isolated viruses from chickens inoculated with pathogenic P5and attenuated PI15. The resultshowed that subpopulations of viruses were found in the tissues of both P5-and P115-inoculatedchickens, as indicated by variations in the SI genes.Lastly, to investigate the relationship between IBV with different virulence and host immuneresponse, the expression profiles of15cytokine and mediator genes in tissue samples of Harderiangland, thymus, cecal tonsil, bursa of Fabricius and spleen was measured by real-time PCR. The resultsshowed dysregulation in the expression of several cytokine and mediator genes in five types of immunetissues infected with either virulent IBV P5or low-virulent P115strains. Both P5and P115inoculationcaused an increase in IFN-a expression in spleen, indicating that IFN-a induction was an importantinnate immune response to IBV. Both spleen tissues harvested from PI15-and P5-innoculated birdsexpressed high cMGF levels at ifve time points, although cMGF expression was greater in the P5groupthan the PI15group, the cMGF up-regulation was presumably driven by IBV proliferation. |
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