key: cord-0265027-4bnhqfyf authors: Smith, D. R.; Singh, C.; Green, J.; Lueder, M. R.; Arnold, C. E.; Voegtly, L. J.; Long, K. A.; Rice, G. K.; Luquette, A.; Miner, H.; Glang, L.; Bennett, A.; Miller, R.; Malagon, F.; Cer, R. Z.; Bishop-Lilly, K. A. title: Genomic and Virological Characterization of SARS-CoV-2 Variants in a Subset of Unvaccinated and Vaccinated U.S. Military Personnel date: 2021-12-16 journal: nan DOI: 10.1101/2021.12.16.21267862 sha: 34211d0f8f261968b5aca61340e74ab10139295b doc_id: 265027 cord_uid: 4bnhqfyf The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of emerging variants, to trace the movement of variants, and to monitor effectiveness of countermeasures. Additionally, determining the amount of viable virus present in clinical samples is helpful to better understand the impact these variants have on viral shedding. In this study, we analyzed nasal swab samples collected between March 2020 and early November 2021 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries stationed worldwide as a part of the Defense Health Agency (DHA) Global Emerging Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse-transcription PCR (qRT-PCR) positive samples were characterized by next-generation sequencing and a subset was analyzed for isolation and quantification of viable virus. Not surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. military personnel beginning in July 2021 and primarily represents cases of vaccine breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in nasal swab samples from Delta variant cases when compared to cases involving other variants. Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving Delta as compared to unvaccinated personnel infected with other variants prior to the availability of approved vaccines. This study provides important insight about the genomic and virological characterization of SARS-CoV-2 isolates from a unique study population with a global presence. The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the 58 causative agent of coronavirus disease 2019 , has led to a historic global pandemic. Multiple SARS-CoV-2 genetic variants are circulating globally, some of which have mutations 60 of concern or mutations of interest that could impact transmissibility and spread, antigenicity or 61 virulence (1). The World Health Organization (WHO) classifies many of these as variants of 62 concern (VOC) or variants of interest (VOI) based on mutations that may impact SARS-CoV-2 63 countermeasures, including vaccines, therapeutics, and diagnostics. Current VOC as defined by This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (TMPRSS2) have demonstrated enhanced susceptibility and isolation of SARS-CoV-2 (11). Plaque or median tissue culture infectious dose (TCID50) assays are commonly used to quantify 91 viable virus, but new methods have been described like the S-Fuse assay that uses a cell reporter This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. formalin prior to staining with crystal violet. For the CPE assay, cells were seeded in T25 cm 2 146 flasks and each flask was infected with 0.5mL aliquots from a 1:10 dilution in Dulbecco's 147 Modified Eagle Medium (DMEM) followed by a 1 hr incubation at 37°C, 5% CO2. After 148 incubation, 5mL of DMEM supplemented with 5% heat-inactivated FBS, 1% 149 Penicillin/Streptomycin was incubated for 4 days at 37°C, 5% CO2. CPE was monitored daily. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We have sequenced over 2,300 nasal swab samples collected from March 2020 until early 160 November 2021 and of those, we produced 1,304 coding complete genomes for which we then This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 1B and D) . The "other" category for Figure 1B This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 mutations in the furin cleavage site that allow the virus to enter cells more efficiently (27, 28) . 224 Another study found that four mutations in the nucleocapsid (N) protein, which is an important 225 protein for viral replication, improve the viruses' ability to form infectious particles. These four This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 with other variants. When we analyzed our results by vaccine manufacturer, we continued to This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Our results highlight the importance of characterizing the SARS-CoV-2 genome and 269 infectious viral titers of emerging variants isolated from unvaccinated and vaccinated personnel. This work contributes to the growing body of evidence that the increased fitness of Delta is 271 primarily due to viral genetic determinants that increase infectivity or replication. This helps to This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2021. 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Viral Infection and Transmission in a large well-traced outbreak caused by the Delta SARS-CoV-2 411 variant Clinical and virological features of 413 SARS-CoV-2 variants of concern: a retrospective cohort study comparing B.1.1.7 (Alpha The Delta 417 SARS-CoV-2 variant has a higher viral load than the Beta and the historical variants in nasopharyngeal 418 samples from newly diagnosed COVID-19 patients Viral dynamics of acute SARS-421 CoV-2 infection and applications to diagnostic and public health strategies PubMed Central PMCID: PMCPMC8297933 following competing interests: JW is an employee of Quest 424 JW is an employee of Bioreference Laboratories. NDG has a consulting agreement for 425 Tempus and receives financial support from Tempus to develop SARS-CoV-2 diagnostic tests. SMK, SWO, 426 and YHG have a consulting agreement with the NBA