key: cord-0737067-moqzdup7 authors: Lucas, Shantelle; Jones, Morris; Kothari, Sangita; Madlambayan, Adrian; Ngo, Christopher; Chan, Carmen; Goraichuk, Iryna V. title: A 336-Nucleotide In-Frame Deletion in ORF7a Gene of SARS-CoV-2 Identified in Genomic Surveillance by Next-Generation Sequencing date: 2022-02-14 journal: J Clin Virol DOI: 10.1016/j.jcv.2022.105105 sha: 1c40b0a998f8f8fb1bd07c5272aea86b6dc03434 doc_id: 737067 cord_uid: moqzdup7 nan ORF7a gene identified in a patient using Next-Generation Sequencing (NGS) during baseline surveillance. A nasal sample was collected on July 7 th , 2021, from a previously tested SARS-CoV- workflows (Broad Viral Genomics) on the Terra platform (app.terra.bio) (8) . Briefly, the SARS-CoV-2 genome was assembled using a reference-based assembly approach of qualityfiltered raw reads with the reference genome Wuhan-Hu-1 (NC_045512.2) (9). A total of 8,363,544 raw paired-end reads were generated. The genome consensus of the isolate, designated CA-SMCPHL-072321.3, was called from 2,071,958 SARS-CoV-2 reads using the minimap2 aligner (10) . This resulted in the mean read depth coverage of 5,066 reads. The final genome consensus was 29,548 nucleotides (nt) long and was deposited in GISAID under the accession number EPI_ISL_6159211. The genome was classified as Nextstrain clade 20I (11) from Pango lineage B.1.1.7 (12) . During the annotation of this genome, we noticed a 336-nt deletion in the gene-encoding accessory protein ORF7a (27, 753) ( Figure 1 ). Although numerous studies have been performed for some other SARS-CoV-2 viral proteins (13) (14) (15) (16) (17) , studies on putative activity and role of ORF7a is just starting to arise. It has been demonstrated that SARS-CoV-2 ORF7a protein inhibits type I interferon (IFN-1) signaling (18, 19) , interacts with CD14+ monocytes (20) , induces the nuclear factor kappa B (NF-κB) pathway (21) , and thus triggers expression of proinflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). This forms the basis of a likely mechanism through which ORF7a mediates the potentially fatal cytokine storm progression, indicating that ORF7a may be a key viral factor for clinical severity of COVID-19 (22) . Previous studies reported complete ORF7a gene loss (23, 24) , multiple length inframe and frame-shift deletions in ORF7a (25) (26) (27) (28) (29) (30) (31) , as well as large ORF7a ∆370 , ORF7a ∆227 , and ORF7a ∆392 that resulted in the fusion of ORF7a with ORF6, ORF7b, and ORF8 genes, respectively (32, 33) . However, the ORF7a ∆336 reported here is currently the longest detected in-frame deletion within the ORF7a gene, which makes the gene only 30 nt long (Figure 2 ). ORF7a deletions are found to impact SARS-CoV-2 pathogenicity (31) . In vitro viral challenge experiments demonstrated that the C-terminal truncation of ORF7a results in a replication defect, which was found to be associated with elevated IFN response to SARS-CoV-2 (31) . It was also shown that complete deletion of ORF7a reduces viral replication (34) . This suggests that strains with deletions in ORF7a are more likely to emerge in immunocompromised patients. Hence, further experiments are needed to determine the functional outcomes of different deletions. Unfortunately, a variety of deletions in the ORF7a region can be under-investigated similarly to ORF8 deletions that have been shown to often not be reported by the standard data analysis pipelines, which are frequently simply represented by stretch ambiguous bases or as gaps in the consensus sequence due to poor NGS coverage (35) . Thus, non-canonical genes are generally excluded from genomic and clinical analyses despite their importance for understanding SARS-CoV-2 evolution and replication dynamics, which have vital implications in vaccine development and control strategies for COVID-19 (6, 36, 37) . These findings highlight the necessity of submission of the raw sequencing reads in public databases in order to assess the spread of deletion strains. Clinical features of patients infected with 2019 novel coronavirus in Wuhan SARS-CoV-2: don't ignore non-canonical genes A putative new SARS-CoV protein, 3c, encoded in an ORF overlapping ORF3a Conflicting and ambiguous names of overlapping ORFs in the SARS-CoV-2 genome: A homology-based resolution Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design Non-canonical translation in RNA viruses US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Phylogenetic analysis of SARS-CoV-2 A new coronavirus associated with human respiratory disease in China Minimap2: pairwise alignment for nucleotide sequences Nextstrain: real-time tracking of pathogen evolution A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 Ubiquitination of SARS-CoV-2 ORF7a promotes antagonism of interferon response Evasion of Type I Interferon by SARS-CoV-2 Structural insight reveals SARS-CoV-2 ORF7a as an immunomodulating factor for human CD14 Activation of NF-κB and induction of proinflammatory cytokine expressions mediated by ORF7a protein of SARS-CoV-2 A Conservative Replacement in the Transmembrane Domain of SARS-CoV-2 ORF7a as a Putative Risk Factor in COVID-19 Emergence of a Severe Acute Respiratory Syndrome Coronavirus 2 Virus Variant With Novel Genomic Architecture in Hong Kong Expansion of a SARS-CoV-2 Delta variant with an 872 nt deletion encompassing Genomic surveillance of SARS-CoV-2 in Thailand reveals mixed imported populations, a local lineage expansion and a virus with truncated ORF7a Identification of eight SARS-CoV-2 ORF7a deletion variants in 2,726 clinical specimens Replication Kinetics of B.1.351 and B.1.1.7 SARS-CoV-2 Variants of Concern Including Assessment of a B.1.1.7 Mutant Carrying a Defective ORF7a Gene A deletion in SARS-CoV-2 ORF7 identified in COVID-19 outbreak in Uruguay Rapid spread of a SARS-CoV-2 Delta variant with a frameshift deletion in ORF7a An 81-Nucleotide Deletion in SARS-CoV-2 ORF7a Identified from Sentinel Surveillance in Arizona SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits immune suppression Identification of multiple large deletions in ORF7a resulting in in-frame gene fusions in clinical SARS-CoV-2 isolates Genome Sequences of Three SARS-CoV-2 ORF7a Deletion Variants Obtained from Patients in Hong Kong Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines Unconventional viral gene expression mechanisms as therapeutic targets Origin, Evolution and Stability of Overlapping Genes in Viruses: A Systematic Review We thank the nurses and staff at San Mateo County Public Health Planning and Policy for the SARS-CoV-2-positive sample -and for technical assistance.We gratefully acknowledge authors from originating and submitting laboratories of the sequences from the GISAID and GenBank databases. A table of the contributors is available in Supplementary Table S1 . The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper