The Pirbright Institute, Ash Road, Woking, Surrey GU24 0NF, UK.
Viruses. 2020 Sep 29;12(10):1102. doi: 10.3390/v12101102.
The infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2' cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.
传染性支气管炎病毒（IBV）可引起家禽高度接触传染性和具有重要经济意义的呼吸道疾病。在实验室中，大多数 IBV 株仅限于在原代器官培养物或鸡胚中复制，而不能在细胞培养物中复制，这使得对其基本病毒学的研究变得困难。IBV 进入细胞是由病毒粒子表面的大糖蛋白（刺突，S）蛋白介导的，S 蛋白由 S1 和 S2 亚单位组成。先前的研究表明，S2'切割位点负责 IBV Beaudette 株的扩展嗜性。本研究旨在探讨蛋白酶处理是否能扩展其他 IBV 株的嗜性。在这里，我们证明在细胞培养物中进行 IBV 繁殖时添加外源性胰蛋白酶可显著提高病毒滴度。我们使用一组表现出不同嗜性的 IBV 株，通过在存在胰蛋白酶的情况下在细胞培养物中连续传代来评估刺突切割对进入和复制的影响。可以维持复制超过连续传代，表明添加外源性蛋白酶足以克服感染的障碍。在细胞培养物中连续传代后，在 S1 和 S2 亚单位中均发现了突变。这项工作提供了一个概念验证，即外源性蛋白酶可以消除在原本不允许感染的细胞中复制 IBV 的障碍，为进一步研究难以捉摸的田间株提供了平台，并能够在体外进行可持续的疫苗生产。
