Abstract

Infectious bronchitis virus (IBV) is a member of genus gamma-coronavirus in the family Coronaviridae, causing serious economic losses to the poultry industry. Reverse genetics is a common technique to study the biological characteristics of viruses. So far, there is no BAC reverse genetic system available for rescue of IBV infectious clone. In the present study, a new strategy for the construction of IBV infectious cDNA clone was established. The full-length genomic cDNA of IBV vaccine strain H120 was constructed in pBAC vector from four IBV fragment subcloning vectors by homologous recombination, which contained the CMV promoter at the 5′ end and the hepatitis D virus ribozyme (HDVR) sequence and bovine growth hormone polyadenylation (BGH) sequence after the polyA tail at the 3′ end of the full-length cDNA. Subsequently, using the same technique, another plasmid pBAC-H120/SCS1 was also constructed, in which S1 gene from IBV H120 strain was replaced with that of a virulent SC021202 strain. Recombinant virus rH120 and rH120/SCS1 were rescued by transfecting the plasmids into BHK cells and passaged in embryonated chicken eggs. Finally, the pathogenicity of both the recombinant virus strains rH120 and rH120/SCS1 was evaluated in SPF chickens. The results showed that the chimeric rH120/SCS1 strain was not pathogenic compared with the wild-type IBV SC021202 strain and the chickens inoculated with rH120/SCS1 could resist challenge infection by IBV SC021202. Taken together, our results indicate that BAC reverse genetic system could be used to rescue IBV in vitro and IBV S1 protein alone might not be the key factor for IBV pathogenicity.

Key points

• BAC vector was used to construct IBV full-length cDNA by homologous recombination.

• Based on four subcloning vectors, a recombinant chimeric IBV H120/SCS1 was constructed and rescued.

• Pathogenicity of H120/SCS1 was similar to that of H120, but different to that of SC021202.

中文翻译：

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带有强毒分离株嵌合S1基因的传染性支气管炎病毒疫苗株的构建及其致病性分析。

摘要

传染性支气管炎病毒（IBV）是属伽马冠状病毒在家庭成员冠状，给家禽业造成严重的经济损失。逆向遗传学是研究病毒生物学特性的常用技术。迄今为止，尚无可用于拯救IBV传染性克隆的BAC反向遗传系统。在本研究中，建立了一种构建IBV感染性cDNA克隆的新策略。通过同源重组，在pBAC载体中，由四个IBV片段亚克隆载体通过重组重组构建IBV疫苗株H120的全长基因组cDNA，该重组子在5'端含有CMV启动子，并带有D型肝炎病毒核酶（HDVR）序列和牛生长激素。全长cDNA 3'末端的polyA尾部之后的多腺苷酸化（BGH）序列。随后，使用相同的技术，还构建了另一个质粒pBAC-H120 / SCS1，其中将来自IBV H120菌株的S1基因替换为有毒的SC021202菌株。通过将质粒转染到BHK细胞中，将重组病毒rH120和rH120 / SCS1拯救出来，并在胚胎鸡蛋中传代。最后，在SPF鸡中评估了重组病毒株rH120和rH120 / SCS1的致病性。结果表明，与野生型IBV SC021202菌株相比，rH120 / SCS1嵌合菌株没有致病性，接种了rH120 / SCS1的鸡可以抵抗IBV SC021202的攻击感染。综上所述，我们的结果表明BAC反向遗传系统可用于体外拯救IBV，而单独IBV S1蛋白可能不是IBV致病性的关键因素。通过将质粒转染到BHK细胞中，将重组病毒rH120和rH120 / SCS1拯救出来，并在胚胎鸡蛋中传代。最后，在SPF鸡中评估了重组病毒株rH120和rH120 / SCS1的致病性。结果表明，与野生型IBV SC021202菌株相比，rH120 / SCS1嵌合菌株没有致病性，接种了rH120 / SCS1的鸡可以抵抗IBV SC021202的攻击感染。两者合计，我们的结果表明BAC反向遗传系统可用于体外拯救IBV，而单独IBV S1蛋白可能不是IBV致病性的关键因素。通过将质粒转染到BHK细胞中，将重组病毒rH120和rH120 / SCS1拯救出来，并在胚胎鸡蛋中传代。最后，在SPF鸡中评估了重组病毒株rH120和rH120 / SCS1的致病性。结果表明，与野生型IBV SC021202菌株相比，rH120 / SCS1嵌合菌株没有致病性，接种了rH120 / SCS1的鸡可以抵抗IBV SC021202的攻击感染。两者合计，我们的结果表明BAC反向遗传系统可用于体外拯救IBV，而单独IBV S1蛋白可能不是IBV致病性的关键因素。结果表明，与野生型IBV SC021202菌株相比，rH120 / SCS1嵌合菌株没有致病性，接种了rH120 / SCS1的鸡可以抵抗IBV SC021202的攻击感染。两者合计，我们的结果表明BAC反向遗传系统可用于体外拯救IBV，而单独IBV S1蛋白可能不是IBV致病性的关键因素。结果表明，与野生型IBV SC021202菌株相比，rH120 / SCS1嵌合菌株没有致病性，接种了rH120 / SCS1的鸡可以抵抗IBV SC021202的攻击感染。两者合计，我们的结果表明BAC反向遗传系统可用于体外拯救IBV，而单独IBV S1蛋白可能不是IBV致病性的关键因素。

关键点

•BAC载体用于通过同源重组构建IBV全长cDNA。

•基于四个亚克隆载体，构建并拯救了重组嵌合IBV H120 / SCS1。

•H120 / SCS1的致病性与H120相似，但与SC021202不同。