key: cord-310291-z79x349o authors: Holland, LaRinda A.; Kaelin, Emily A.; Maqsood, Rabia; Estifanos, Bereket; Wu, Lily I.; Varsani, Arvind; Halden, Rolf U.; Hogue, Brenda G.; Scotch, Matthew; Lim, Efrem S. title: An 81-Nucleotide Deletion in SARS-CoV-2 ORF7a Identified from Sentinel Surveillance in Arizona (January to March 2020) date: 2020-07-01 journal: J Virol DOI: 10.1128/jvi.00711-20 sha: doc_id: 310291 cord_uid: z79x349o On January 26 2020, the first Coronavirus Disease 2019 (COVID-19) case was reported in Arizona (3rd case in the US) (1).…. O n 26 January 2020, the first coronavirus disease 2019 (COVID- 19) case was reported in Arizona (third case in the United States) (1) . Here, we report on early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sentinel surveillance in Tempe, Arizona. Genomic characterization identified an isolate encoding a 27-amino-acid in-frame deletion in accessory protein ORF7a, the ortholog of SARS-CoV immune antagonist ORF7a/X4. In anticipation of COVID-19 spreading in Arizona, we initiated a surveillance effort for the local emergence of SARS-CoV-2 starting 24 January 2020. We leveraged an ongoing influenza surveillance project at Arizona State University (ASU) Health Services in Tempe, Arizona. Individuals presenting with respiratory symptoms (ILI) were tested for influenza A and B viruses (Alere BinaxNOW). Subsequently, we tested influenzanegative nasopharyngeal (NP) swabs for SARS-CoV-2. We extracted total nucleic acid using the bioMérieux eMAG automated platform and performed real-time quantitative reverse transcription-PCR (qRT-PCR) assays specific for SARS-CoV-2 N and E genes (2, 3). Out of 382 NP swabs collected from 24 January to 25 March 2020, we detected SARS-CoV-2 in 5 swabs from 16 to 19 March (Fig. 1) . This corresponds to a prevalence of 1.31%. Given the estimated 1-to 14-day incubation period for COVID-19, the spike in cases might be related to university spring break holiday travel (8 to 15 March), as previously seen in other outbreaks (4, 5) . To understand the evolutionary relationships and characterize the SARS-CoV-2 genomes, we performed next-generation sequencing (NGS; Illumina NextSeq, 2ϫ76) directly on specimen RNA, thereby avoiding cell culture passage and potentially associated mutations. This generated an NGS data set of 20.7 to 22.7 million paired-end reads per sample. We mapped quality-filtered reads to a reference SARS-CoV-2 genome (MN908947) using BBMap (version 39.64) to generate three full-length genomes: AZ-ASU2922 (376ϫ coverage), AZ-ASU2923 (50ϫ), and AZ-ASU2936 (879ϫ) (Geneious prime, version 2020.0.5). We aligned a total of 222 SARS-CoV-2 genome sequences comprising at least 5 representative sequences from phylogenetic lineages defined by Rambaut et al. (6) . We performed phylogenetic reconstruction with BEAST (version 1.10.4; strict molecular clock, HKY ϩ ⌫ nucleotide substitution, exponential growth for coalescent model) (7-10). The ASU sequences were phylogenetically distinct, indicating independent transmissions ( Fig. 2A) . Like SARS-CoV, the SARS-CoV-2 genome carries multiple open reading frames in the 3= region. We found that the SARS-CoV-2 AZ-ASU2923 genome has an 81-nucleotide (nt) deletion in the ORF7a gene, resulting in a 27-amino-acid in-frame deletion (Fig. 2B) . The SARS-CoV ORF7a ortholog is a viral antagonist of host restriction factor BST-2/ 3) using the MAFFT v7.450 plugin, and the 5= and 3= untranslated regions (Ͻ300 nt each) were trimmed. We initiated two independent runs of 500M sampling every 50K steps and used Tracer v1.7.1 (18) to check for convergence and that all effective sample size (ESS) values for our statistics were Ͼ200, LogCombiner (7) to combine the models with a 10% burn-in, and TreeAnnotator (7) to produce an MCC tree. We used FigTree, v1.4.4 (19) , to edit the tree and color the tips based on lineages (6) and pangolin (20) Tetherin and induces apoptosis (11) (12) (13) (14) . Based on the SARS-CoV ORF7a structure (15) , the 27-amino-acid deletion in SARS-CoV-2 ORF7a maps to the putative signal peptide (partial) and first two beta strands. To validate the deletion, we performed RT-PCR using primers spanning the region and verified the amplicons by Sanger sequencing (Fig. 2C) . Similar deletions in SARS-CoV-2 genomes are emerging, notably in the ORF8 gene (16) , that may reduce virus fitness (17) . Hence, further experiments are needed to determine the functional consequences of the ORF7a deletion. Collectively, although global NGS efforts indicate that SARS-CoV-2 genomes are relatively stable, dynamic mutations can be selected in symptomatic individuals. Initial public health response and interim clinical guidance for the 2019 novel coronavirus outbreak-United States CDC. 2020. 2019-Novel coronavirus (2019-nCoV) real-time rRT-PCR panel primers and probes Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application A spatial analysis of the spread of mumps: the importance of college students and their spring-break-associated travel A dynamic nomenclature for SARS-CoV-2 to assist genomic epidemiology Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol 4:vey016 Dating of the human-ape splitting by a molecular clock of mitochondrial DNA Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods GENIE: estimating demographic history from molecular phylogenies Severe acute respiratory syndrome coronavirus ORF7a inhibits bone marrow stromal antigen 2 virion tethering through a novel mechanism of glycosylation interference SARS coronavirus 7a protein blocks cell cycle progression at G0/G1 phase via the cyclin D3/pRb pathway Overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspasedependent pathway Severe acute respiratory syndrome coronavirus gene 7 products contribute to virus-induced apoptosis Structure and intracellular targeting of the SARS-coronavirus Orf7a accessory protein Discovery of a 382-nt deletion during the early evolution of SARS-CoV-2 Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission Posterior summarization in bayesian phylogenetics using Tracer 1.7 We thank the nurses and staff at ASU Health Services, Arizona Department of Health Services, for a SARS-CoV-2-positive sample (AZ_4811) for qRT-PCR assay validation experiments, Nicholas Mellor and the ASU Genomics Facility for technical assistance, and the authors and originating and submitting laboratories of the sequences from the GISAID's EpiCoV database. A complete acknowledgments table for sequences obtained from this database is available at https://www.dropbox.com/ s/aiybuatgxjunuga/GISAID_CoV2020_Acknowledgements.xlsx?dlϭ0.