Comparison of Subgenomic and Total RNA in SARS-CoV-2 Challenged Rhesus Macaques

For SARS-CoV-2, specific monitoring of actively replicating virus is critical for investigating the protective and therapeutic efficacy of vaccines, monoclonal antibodies, and antiviral drugs. We adapted a SARS-CoV-2 subgenomic RNA (sgRNA) RT-PCR assay to differentiate productive infection from inactivated or neutralized virus. Subgenomic RNAs are generated after cell entry and are poorly incorporated into mature virions, and thus may provide a marker for actively replicating virus. We show envelope (E) sgRNA was degraded by RNase in infected cell lysates, while genomic RNA (gRNA) was protected, presumably due to packaging into virions. To investigate the capacity of the sgRNA assay to distinguish input challenge virus from actively replicating virus in vivo, we compared the E sgRNA assay to a standard nucleoprotein (N) or E total (both gRNA and sgRNA) RNA in convalescent rhesus macaques and in antibody-treated rhesus macaques after experimental SARS-CoV-2 challenge. In both studies, the E sgRNA assay was negative, suggesting protective efficacy, whereas the N and E total RNA assays remained positive. These data suggest the potential utility of sgRNA to monitor actively replicating virus in prophylactic and therapeutic SARS-CoV-2 studies.

针对新型冠状病毒（SARS-CoV-2），对其活跃复制病毒进行特异性监测，对于评估疫苗、单克隆抗体及抗病毒药物的防护与治疗效果至关重要。本研究对新型冠状病毒（SARS-CoV-2）亚基因组RNA（subgenomic RNA，sgRNA）逆转录聚合酶链反应（Reverse Transcription-Polymerase Chain Reaction，RT-PCR）检测方法进行优化，以区分产毒感染与灭活或中和病毒。亚基因组RNA于病毒入侵细胞后生成，且极少整合进入成熟病毒粒子，因此可作为活跃复制病毒的检测标志物。本研究发现，感染细胞裂解液中的包膜蛋白（E）亚基因组RNA可被核糖核酸酶（RNase）降解，而基因组RNA（genomic RNA，gRNA）则可免受降解，推测其原因为基因组RNA被包装进入了病毒粒子。为探究sgRNA检测方法在体内区分输入性攻毒病毒与活跃复制病毒的能力，本研究在经实验性新型冠状病毒（SARS-CoV-2）攻毒的恢复期恒河猴与抗体处理恒河猴中，将包膜蛋白（E）sgRNA检测方法与标准核蛋白（N）或E总RNA（涵盖基因组RNA与亚基因组RNA）检测方法进行了对比。两项研究中，包膜蛋白（E）sgRNA检测均呈阴性，提示受试对象具备防护效果，而核蛋白（N）与E总RNA检测结果仍为阳性。上述结果表明，sgRNA检测有望用于新型冠状病毒（SARS-CoV-2）预防与治疗性研究中活跃复制病毒的监测。