Rowe C L, Baker S C, Nathan M J, Fleming J O
Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153, USA.
J Virol. 1997 Apr;71(4):2959-69. doi: 10.1128/JVI.71.4.2959-2969.1997.
High-frequency RNA recombination has been proposed as an important mechanism for generating viral deletion variants of murine coronavirus. Indeed, a number of variants with deletions in the spike glycoprotein have been isolated from persistently infected animals. However, the significance of generating and potentially accumulating deletion variants in the persisting viral RNA population is unclear. To study this issue, we evaluated the evolution of spike variants by examining the population of spike RNA sequences detected in the brains and spinal cords of mice inoculated with coronavirus and sacrificed at 4, 42, or 100 days postinoculation. We focused on the S1 hypervariable region since previous investigators had shown that this region is subject to recombination and deletion. RNA isolated from the brains or spinal cords of infected mice was rescued by reverse transcription-PCR, and the amplified products were cloned and used in differential colony hybridizations to identify individual isolates with deletions. We found that 11 of 20 persistently infected mice harbored spike deletion variants (SDVs), indicating that deletions are common but not required for persistent infection. To determine if a specific type of SDV accumulated during persistence, we sequenced 106 of the deletion isolates. We identified 23 distinct patterns of SDVs, including 5 double-deletion variants. Furthermore, we found that each mouse harbored distinct variants in its central nervous system (CNS), suggesting that SDVs are generated during viral replication in the CNS. Interestingly, mice with the most severe and persisting neurological disease harbored the most prevalent and diverse quasispecies of SDVs. Overall, these findings illustrate the complexity of the population of persisting viral RNAs which may contribute to chronic disease.
高频RNA重组被认为是产生鼠冠状病毒病毒缺失变异体的重要机制。事实上，已经从持续感染的动物中分离出了许多刺突糖蛋白存在缺失的变异体。然而，在持续存在的病毒RNA群体中产生并可能积累缺失变异体的意义尚不清楚。为了研究这个问题，我们通过检查接种冠状病毒并在接种后4天、42天或100天处死的小鼠脑和脊髓中检测到的刺突RNA序列群体，评估了刺突变异体的进化情况。我们聚焦于S1高变区，因为先前的研究人员已经表明该区域易发生重组和缺失。通过逆转录PCR从感染小鼠的脑或脊髓中分离出RNA，扩增产物被克隆并用于差异菌落杂交以鉴定具有缺失的单个分离株。我们发现20只持续感染的小鼠中有11只携带刺突缺失变异体（SDV），这表明缺失很常见，但不是持续感染所必需的。为了确定在持续感染过程中是否积累了特定类型的SDV，我们对106个缺失分离株进行了测序。我们鉴定出23种不同的SDV模式，包括5种双缺失变异体。此外，我们发现每只小鼠在其中枢神经系统（CNS）中都携带不同的变异体，这表明SDV是在CNS中的病毒复制过程中产生的。有趣的是，患有最严重和持续神经疾病的小鼠携带最普遍和最多样化的SDV准种。总体而言，这些发现说明了持续存在的病毒RNA群体的复杂性，这可能导致慢性疾病。
