| Avian infectious bronchitis virus (IBV), a member of the genus Gammacoronavirus of the family Coronaviridae, causes avian infectious bronchitis (IB), an acute, highly contagious respiratory disease in chickens. IB is of major economic importance because it is a cause of poor weight gain and feed efficiency and reduced egg production and egg quality. Live or inactivated vaccines containing strains of IBV from multiple serotypes are routinely used to protect chickens against IB infection in commercial settings. In nature, IBVs can evolve rapidly to give rise to new antigenic variants, which have complicated and increased the cost of attempts to prevent the disease by immunization. The aims of this study were to elucidate the predominant genotypes circulating in China in recent years and define their phylogenetic relationships, as well as to develop a new recombinant viral vaccine effective against the current epidemic genotypes using Marek’s disease virus (MDV) as a vector.1. Epidemiological surveillance and phylogenetic analysis of IBVs circulating in China between2009and2011A total of54IBV field strains were isolated from chicken flocks suspected of infection with IB in8provinces of China between2009and2011. Of the54strains collected,52were isolated from broiler chickens and2were isolated from egg-laying chickens. Frequent high mortality (25/52flocks) caused by coinfection with IBV and H9subtype avian influenza viruses (AIV) have been observed in broiler chicken flocks in this study. The multiple infection samples also exhibited more severe pathogenicity in inoculated embryos. This phenomenon suggested that there is potential synergism between IBV and H9subtype AIV.The S1genes of all isolates were cloned and sequenced, revealing4different S1genes, including1611-,1617-,1620-and1626-nucleotide isoforms. This demonstrated that nucleotide insertion or deletion is common in the S1gene. The sequences of S1genes from all isolates and57reference strains representing different genotypes recently circulating in China and surrounding countries or areas were aligned, and a phylogenetic tree was constructed. The results revealed that all of the isolates could be grouped into7genotypes:QX (42isolates). HN-08(5isolates), LSC7991(2isolates). TW-I (2isolates), Mass (1isolate),793/B (1isolate) and CH Ⅲ (1isolate). The QX type has been the predominant genotype recently circulating in China, and strains of this type could be further subdivided into Cluster Ⅰ (21isolates) and Cluster Ⅱ (21isolates). The classical QX-type reference strain QXIBV is in Cluster Ⅰ, and all Cluster Ⅱ strains were found in samples obtained prior tosince2010.To more clearly describe the distribution of the different genotypes in China,296isolates between2009and2011with complete S1genes published in GenBank were combined for further phylogenetic analysis. These results revealed that all350isolates could be grouped into19genotypes, including11known genotypes and8novel genotypes. The11known genotypes were QX (219isolates), HN-08(26isolates), LSC/991(22isolates), LDT3/03(12isolates), TW-I (12isolates), CH III (11isolates), Mass (10isolates), LDL/971(3isolates), CH VI (3isolates), TW-II (2isolates) and793/B (1isolate). The8novel genotypes were tentatively named undetermined genotypes I-VIII. These results strongly suggested that IBV epidemiology in China is very complex and shows distinct regional distribution characteristics; interestingly, most of the genotypes circulating in surrounding countries and areas are not found in China.To clarify the phylogenetic relationships between the different IBV genotypes circulating in China, the S1gene sequences of some strains in different genotypes were chosen for recombination analysis with RDP3.31software. The results revealed that recombination between S1genes is a common phenomenon. A series of recombination incidents not only accelerate genotype differentiation but also lead to the emergence of new genotypes. According to the results of recombinant analysis, we were able to conclude that all current epidemic genotypes in China originated from6parental genotypes:QX, LSC/99I,793/B, LDL/97I, TW-Ⅰ and TW-Ⅱ. We further concluded that all newly emerged genotypes were variants generated by one or more homologous recombination events between those6prototype strains.2. Protective efficacy of Mass type H120live vaccine against some genotypes of IBVFour IBV strains representing three predominant genotypes were selected to evaluate the protective efficacy offered by Mass type H120live vaccine according to the results of our phylogenetic analysis. The corresponding genotypes were QX type Cluster1(AH/2009/I), QX type Cluster Ⅱ (JS/2010/12). LSC/99I type (JS/2009/5) and HN08type (JS/2010/6), respectively. In this experiment,3-day-old young SPF chickens were vaccinated with HI20live vaccine and challenged21d post-vaccination with4field isolates and another virulent Mass type IBV reference strain M41. The morbidity, mortality and virus shedding from the trachea and kidney of challenged chickens were recorded for each group. The results showed that the protective efficacy was different against every virus. The vaccine conferred complete protection against the homologous Mass type M41strain, and the chickens showed low shedding rates post-challenge. Although the vaccination also conferred clinical protection against the QX-like strains AH/2009/1and JS/2010/12. high rates of virus shedding were detected in the tracheae and kidneys of challenged chickens. The vaccination did not provide good protection against strains JS/2009/5and JS/2010/6, and the challenged chickens showed serious clinical signs of infection and high rates of virus shedding. All of these results suggested that the Mass type vaccine cannot provide complete protection against new epidemic genotypes of IBVs isolated in China, and it is therefore urgent to develop new vaccines to control this disease.3. Construction of a recombinant Marek’s disease virus expressing the QX-type IBV S1proteinIn this study, we constructed a recombinant CVI988/Rispens, a strain of type Ⅰ MDV, that expresses the S1protein of the QX type IBV strain CK/CH/JS/06Ⅱ. The IRS-US intergenic region of the MDV genome was used as the gene insertion site in which the LTR promoter from the reticuloendotheliosis virus (REV) and S1gene were incorporated into the MDV genome. The transfer vector pUP-LTR-EGFP-S1-DOWN and MDV genomic DNA were co-transfected into chicken embryo fibroblast cells (CEF) to generate the recombinant virus rMDV-EGFP-S1by homologous recombination. The fluorescent plaques formed by recombinant virus were picked and purified by repeated passages in CEFs. Then, the EGFP gene was knocked out by Cre-mediated recombination, and the rendered recombinant virus containing the LTR promoter and the S1gene was designated rMDV-S1. The recombinant virus was identified through PCR and an indirect immunofluorescence antibody assay (IFA), and the results showed that the S1gene was correctly inserted into the MDV genome and was stably expressed. The rMDV-S1virus was genetically stable after30sequential passages in CEFs.In the experiment evaluating the protective efficacy of rMDV vaccination against IBV,1-day-old SPF chickens were immunized with5×103PFU rMDV-S1per bird and challenged with QX type virulent IBV strain CK/CH/JS/06Ⅱ. The morbidity and mortality were30%and5%, respectively, compared to100%and30%in the control group, a significant difference (p <0.05), as determined by chi-squared(χ2) test, in the virus shedding test, there was a significant difference in the number of tracheal swabs that tested positive for the virus between the rMDV-S1-vaccinated group and the control group (p<0.05). The virus detection rate from the kidneys of the immunized chickens was lower than that in the control group. There was a highly significant difference (p<0.01) in the number of kidneys that tested positive for the virus between the vaccinated and control groups. All of these results demonstrated that rMDV-S1is an effective and promising recombinant vaccine for the prevention of QX-like IBV infection.In an experiment evaluating the protective efficacy of rMDV vaccination against MDV, the rMDV-Sl showed similar protective efficacy against virulent MDV RB1B and its parent virus CVI988/Rispens. This result showed that the rMDV-Sl can serve as a vaccine for the concomitant immunoprophylaxis of IB and MD. |
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