The S2 Subunit of Infectious Bronchitis Virus Beaudette Is a Determinant of Cellular Tropism.
The S2 Subunit of Infectious Bronchitis Virus Beaudette Is a Determinant of Cellular Tropism.
复制标题
DOI：
10.1128/jvi.01044-18
复制
发表时间：
2018-10-01
影响因子：
5.4
通讯作者：
Britton P
中科院分区：
文献类型：
--
作者：
Bickerton E;Maier HJ;Stevenson-Leggett P;Armesto M;Britton P
尽管存在许多不同的疫苗，传染性支气管炎仍然是全球家禽业的一个主要问题。目前，包括减毒活疫苗和灭活疫苗在内的IBV疫苗都是在有胚胎的鸡蛋上生长的，由于大多数IBV菌株不能在培养细胞中生长，这是一个既繁琐又昂贵的过程。IBV的反向遗传系统为生产合理设计和更有效的疫苗创造了机会。IBV Beaudette具有在Vero细胞上生长的额外倾向，这一观察结果也引发了用培养细胞而不是用胚胎卵生产IBV疫苗的可能性。本研究确定了IBV Beaudette S糖蛋白参与扩展细胞趋向性决定的区域。这一信息将有助于合理设计可在Vero细胞上生长的下一代IBV疫苗。禽γ冠状病毒传染性支气管炎病毒（IBV）的刺突(S)糖蛋白由两个亚基（S1和S2）组成，在体内具有毒力作用，并在体外负责细胞趋向性。我们之前已经证明，用强毒性M41-CK株的相应区域替换IBV无毒Beaudette株的S糖蛋白外结构域，产生重组病毒BeauR-M41(S)，具有M41-CK的体外细胞亲和性。IBV Beaudette株能够在鸡原代肾细胞和Vero细胞中复制，而IBV M41-CK株只能在原代细胞中复制。为了研究IBV S在Vero细胞中负责生长的区域，我们在Beaudette基因组背景下产生了一系列表达嵌合S糖蛋白的重组IBV，其中包括来自Beaudette和M41-CK S基因序列的区域。Beaudette S的S2亚基，而不是S1亚基，被发现赋予了Vero细胞生长的能力。将beaudette特异性氨基酸的各种组合引入M41的S2亚基，以确定在Vero细胞中生长时赋予趋向性的最低要求。IBV在Vero细胞中生长和产生感染性子代病毒的能力随后被缩小到仅围绕S2 '切割位点的3个氨基酸。相反，将3个与Beaudette相关的氨基酸与M41中的相应氨基酸交换，就足以阻止Vero细胞中的Beaudette生长。尽管存在许多不同的疫苗，传染性支气管炎仍然是全球家禽业的一个主要问题。目前，包括减毒活疫苗和灭活疫苗在内的IBV疫苗都是在有胚胎的鸡蛋上生长的，由于大多数IBV菌株不能在培养细胞中生长，这是一个既繁琐又昂贵的过程。IBV的反向遗传系统为生产合理设计和更有效的疫苗创造了机会。IBV Beaudette具有在Vero细胞上生长的额外倾向，这一观察结果也引发了用培养细胞而不是用胚胎卵生产IBV疫苗的可能性。本研究确定了IBV Beaudette S糖蛋白参与扩展细胞趋向性决定的区域。这一信息将有助于合理设计可在Vero细胞上生长的下一代IBV疫苗。
Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells. The spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulence in vivo, and is responsible for cellular tropism in vitro. We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the in vitro cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2′ cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells. IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.