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) (n = 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. In vitro 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 CCL2/MCP1, PTX3, and TNFα, 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.本研究基于法国里昂呼吸道病毒国家参考中心开展的SARS-CoV-2常规分子监测工作，该项目于2020年2月至4月期间共收集229条病毒序列。研究人员在开放阅读框6（open reading frame 6，ORF6）的27267号位点，检测到两种移码缺失突变。2020年3月，仅在1份鼻咽拭子样本中检出26核苷酸缺失突变株（D26）；而2020年4月，在某老年医院病区的9名患者中的5名以及1名医护人员体内均检出34核苷酸缺失突变株（D34）。系统发育分析强烈提示D34存在院内传播，且存在经粪便传播的可能性，该线索也在1份粪便样本中得到佐证。感染野生型毒株（wild-type，WT）与D34的老年病区住院患者，其疾病严重程度无显著差异。体外实验对D26与D34的特性分析显示，二者与野生型毒株的复制动力学特征无显著差异，但可引发不同的宿主免疫应答。尽管感染野生型毒株与ORF6突变株后，干扰素刺激基因的上调水平相似，但ORF6突变株可特异性诱导核因子κB（NF-κB）通路中9种炎症细胞因子编码基因的过度表达，其中包括CCL2/MCP1、PTX3以及TNFα——上述细胞因子的血浆高表达水平已被证实与重症COVID-19相关。本研究结果提示，亟需加强对ORF6缺失突变株的监测，并评估其对宿主免疫应答的影响。