Characterization of SARS-CoV-2 ORF6 deletion variants detected in a nosocomial cluster during routine genomic surveillance, Lyon, France

During routine molecular surveillance of SARS-CoV-2 performed at the National Reference Center of Respiratory Viruses (Lyon, France) (<i>n</i> = 229 sequences collected February–April 2020), two frameshifting deletions were detected in the open reading frame 6, at the same position (27267). While a 26-nucleotide deletion variant (D26) was only found in one nasopharyngeal sample in March 2020, the 34-nucleotide deletion (D34) was found within a single geriatric hospital unit in 5/9 patients and one health care worker in April 2020. Phylogeny analysis strongly suggested a nosocomial transmission of D34, with potential fecal transmission, as also identified in a stool sample. No difference in disease severity was observed between patients hospitalized in the geriatric unit infected with WT or D34. <i>In vitro</i> D26 and D34 characterization revealed comparable replication kinetics with the wild-type (WT), but differential host immune responses. While interferon-stimulated genes were similarly upregulated after infection with WT and ORF6 variants, the latter specifically induced overexpression of 9 genes coding for inflammatory cytokines in the NF-kB pathway, including <i>CCL2/MCP1</i>, <i>PTX3</i>, and <i>TNFα</i>, for which high plasma levels have been associated with severe COVID-19. Our findings emphasize the need to monitor the occurrence of ORF6 deletions and assess their impact on the host immune response.

本研究在法国里昂国家呼吸道病毒参考中心开展严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）常规分子监测，对2020年2月至4月收集的229条病毒序列进行分析。研究人员在开放阅读框6（open reading frame 6, ORF6）的同一位置（27267）检出2种移码缺失变异：其中26核苷酸缺失变异株（D26）仅在2020年3月的1份鼻咽拭子样本中被检出；34核苷酸缺失变异株（D34）则于2020年4月在某老年病院单元内的9名患者中的5名及1名医护人员中被检出。系统发育分析强烈提示D34存在医院内传播，且存在经粪便传播的可能性——该变异株亦在1份粪便样本中被检出。感染野生型（wild-type, WT）或D34的老年病房住院患者之间，疾病严重程度无显著差异。体外（in vitro）对D26与D34的特性表征显示，二者与野生型毒株的复制动力学特征相近，但宿主免疫应答存在差异。尽管感染野生型及ORF6变异株后，干扰素刺激基因的上调水平相似，但后者可特异性诱导NF-κB通路中9种炎性细胞因子编码基因的过表达，包括CCL2/MCP1、PTX3及TNFα，上述细胞因子的血浆高表达已被证实与重症新型冠状病毒肺炎（COVID-19）密切相关。本研究结果强调，需加强对ORF6缺失变异株的监测，并评估其对宿主免疫应答的影响。