Comparison of Subgenomic and Total RNA in SARS-CoV-2-Challenged Rhesus Macaques
Comparison of Subgenomic and Total RNA in SARS-CoV-2-Challenged Rhesus Macaques
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DOI：
10.1128/jvi.02370-20
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发表时间：
2021-04-01
影响因子：
5.4
通讯作者：
Barouch, Dan H.
中科院分区：
文献类型：
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作者：
Dagotto, Gabriel;Mercado, Noe B.;Barouch, Dan H.
呼吸道病毒攻击研究涉及攻击病毒的管理和采样，以评估在相同的解剖位置的保护。因此，很难区分主动复制病毒和输入挑战病毒。对于SARS-CoV-2，对主动复制的病毒进行特异性监测对于研究疫苗、单克隆抗体和抗病毒药物的保护和治疗效果至关重要。我们采用了SARS-CoV-2亚基因组RNA (sgRNA) RT-PCR检测来区分生产感染与灭活或中和病毒。亚基因组rna是在细胞进入后产生的，并且很难与成熟病毒体结合，因此可能为病毒的主动复制提供标记。我们发现，在被感染的细胞裂解物中，包膜(E) sgRNA被RNA酶降解，而基因组RNA （gRNA）受到保护，可能是由于被包装成病毒粒子。为了研究sgRNA测定在体内区分输入攻击病毒和主动复制病毒的能力，我们在恢复期恒河猴和实验性SARS-CoV-2攻击后接受抗体治疗的恒河猴中比较了E sgRNA测定与标准核蛋白(N)或E总RNA (gRNA和sgRNA) RNA。在这两项研究中，E sgRNA检测结果均为阴性，表明其具有保护作用，而N和E总RNA检测结果均为阳性。这些数据表明sgRNA在预防性和治疗性SARS-CoV-2研究中监测主动复制病毒的潜在效用。制定针对SARS-CoV-2病毒的治疗和预防对策是公共卫生的优先事项。在挑战研究中，从相同的解剖位置传递和采样呼吸道病毒。因此，区分主动复制病毒和输入挑战病毒是很重要的。检测SARS-CoV-2病毒最常用的方法是靶向核衣壳总RNA的逆转录PCR (RT-PCR)，它不能区分中和的输入病毒和复制病毒。在这项研究中，我们评估了SARS-CoV-2亚基因组RNA作为恒河猴复制病毒的潜在措施。
Respiratory virus challenge studies involve administration of the challenge virus and sampling to assess for protection in the same anatomical locations. It can therefore be difficult to differentiate actively replicating virus from input challenge virus. 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.IMPORTANCE Developing therapeutic and prophylactic countermeasures for the SARS-CoV-2 virus is a public health priority. During challenge studies, respiratory viruses are delivered and sampled from the same anatomical location. It is therefore important to distinguish actively replicating virus from input challenge virus. The most common assay for detecting SARS-CoV-2 virus, reverse transcription PCR (RT-PCR) targeting nucleocapsid total RNA, cannot distinguish neutralized input virus from replicating virus. In this study, we assess SARS-CoV-2 subgenomic RNA as a potential measure of replicating virus in rhesus macaques.