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Figure.  Sequence Alignment Between the 7 SARS-CoV-2 Strains and Wuhan Strain With Amino Acid Changes
Sequence Alignment Between the 7 SARS-CoV-2 Strains and Wuhan Strain With Amino Acid Changes
Table.  Characteristics of SARS-CoV-2 Strains Detected in Asymptomatic Individuals From Long-term Care Facility
Characteristics of SARS-CoV-2 Strains Detected in Asymptomatic Individuals From Long-term Care Facility
1.
Korber  B, Fischer  WM, Gnanakaran  S,  et al  Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus.   Cell. 2020;182(4):812-827.e819. doi:10.1016/j.cell.2020.06.043PubMedGoogle ScholarCrossref
2.
Benton  DJ, Wrobel  AG, Xu  P,  et al.  Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion.   Nature. 2020;588(7837):327-330. doi:10.1038/s41586-020-2772-0PubMedGoogle ScholarCrossref
3.
Yanes-Lane  M, Winters  N, Fregonese  F,  et al.  Proportion of asymptomatic infection among COVID-19 positive persons and their transmission potential: a systematic review and meta-analysis.   PLoS One. 2020;15(11):e0241536. doi:10.1371/journal.pone.0241536PubMedGoogle Scholar
4.
Li  J, Wang  H, Mao  L,  et al.  Rapid genomic characterization of SARS-CoV-2 viruses from clinical specimens using nanopore sequencing.   Sci Rep. 2020;10(1):17492. doi:10.1038/s41598-020-74656-yPubMedGoogle ScholarCrossref
5.
Yan  R, Zhang  Y, Li  Y, Xia  L, Guo  Y, Zhou  Q.  Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2.   Science. 2020;367(6485):1444-1448. doi:10.1126/science.abb2762PubMedGoogle ScholarCrossref
6.
Jiang  HW, Zhang  HN, Meng  QF,  et al.  SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70.   Cell Mol Immunol. 2020;17(9):998-1000. doi:10.1038/s41423-020-0514-8PubMedGoogle ScholarCrossref
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    Research Letter
    Infectious Diseases
    April 9, 2021

    Genome Analysis for Sequence Variants in SARS-CoV-2 Among Asymptomatic Individuals in a Long-term Care Facility

    Author Affiliations
    • 1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha
    • 2Nebraska Public Health Laboratory, University of Nebraska Medical Center, Omaha
    • 3Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center (UNMC), Omaha,
    JAMA Netw Open. 2021;4(4):e217939. doi:10.1001/jamanetworkopen.2021.7939
    Introduction

    Studies have suggested that sequence variants in the genome of SARS-CoV-2 may affect infectivity, transmission, and pathogenicity of the virus.1-3 In this study, genome analysis was performed on SARS-CoV-2 RNA recovered from 7 individuals in a long-term care facility who were asymptomatic at time of screening.

    Methods

    This cohort study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline. As per 45 CFR 46.102(I), this study did not require University of Nebraska Medical Center institutional review board approval because it supported the COVID-19 public health response. No informed patient consent was required because the individuals were deidentified and institutional review board approval was not required.

    After contact with a health care worker positive for SARS-CoV-2, 20 female residents in a long-term care facility were screened for SARS-CoV-2 symptoms and a nasopharyngeal swab was collected. At the time of screening, all the residents were asymptomatic with 7 subsequently identified as positive for SARS-CoV-2 by a commercial polymerase chain reaction (PCR) assay. A second specimen was collected from all 7 individuals, deidentified, and retested using the US Centers for Disease Control and Prevention nCOV PCR assay. Whole genome sequencing (WGS) was performed using the MinION platform.4 After uploading consensus genomes to the GISAID database, Nextclade version 0.9.0 was used for quality checks and data analysis (Table). Data analysis was performed from September to December 2020.

    Results

    The 7 asymptomatic female individuals with PCR positive results, whose ages ranged from 75 to 92 years, had PCR cycle threshold values that ranged from 17 to 27 (Table), reflecting high viral loads. During the 30 days following confirmation of COVID-19 infection, 5 individuals remained asymptomatic while 2 individuals developed lethargy and cognitive decline with no respiratory symptoms and both died after 2- and 4-weeks, respectively. The study group had less than 3 nucleotide substitutions among strains but 13 to 15 substitutions when compared with Wuhan-Hu-1 reference strain (GenBank NC045512), grouping them into clade 20C. The S:D614G variant within the spike (S) protein was present in all 7 genomes. The other variants resulted in 7 or 8 amino acid (AA) differences at the protein level (Figure). Two of the nonconserved amino acid changes were estimated to have structural changes in the S protein (S:K854N) or the nonstructural open reading frame 9b (ORF9b:N36Y) with potential association with changes in protein function.

    Discussion

    Previous studies have demonstrated that sequence variations in the S protein may increase the infectivity and pathogenicity of SARS-CoV-2.1,2 The S:D614G variant has been associated with increased infectivity and transmissibility of SARS-CoV-2.1 Studies hypothesized that S:D614 within the first S protein subunit forms a key salt bridge with lysine at 854 in the second spike subunit when in the closed formation.2 Theoretically, S:D614G removes this important bridge resulting in a structural change estimated to enhance the ability of S protein to bind to angiotensin-converting enzyme 2 receptors (ACE2R).2,5 S:K854N has not been previously identified to our knowledge. In these 7 strains, a substitution for the positively charged lysine with asparagine, a polar AA, may restore the key salt bridge between S:D614G and S:K854N. This is estimated to result in a closed structural form and reduction of S protein binding ability to ACE2R, which may in turn result in a reduction in the virus ability to establish infection.

    ORF9b antagonizes type I and type III interferons, which could reduce the cell’s ability to restrict viral replication.6 ORF9b variants may reduce pathogenicity.6 The ORF9b:N36Y variant has not been studied in vitro but it could introduce a structural change affecting the virus’ ability to antagonize interferons. Taken together, the reduced ability of the virus to propagate and to suppress interferon may explain why these individuals aged 75 years and older were asymptomatic and 5 of the 7 recovered without illness.

    A limitation of this study is the small sample size, but surveillance for sequence variations is continuing to address this issue. In vitro studies are needed to determine whether the estimated outcomes of the variants described here associated with functional changes in cells with reductions in pathogenicity of the virus.

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    Article Information

    Accepted for Publication: March 6, 2021.

    Published: April 9, 2021. doi:10.1001/jamanetworkopen.2021.7939

    Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2021 Abdalhamid B et al. JAMA Network Open.

    Corresponding Author: Baha Abdalhamid, MD, PhD, Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, NE 68198-5900 (babdalhamid@unmc.edu).

    Author Contributions: Dr Hinrichs had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

    Concept and design: Abdalhamid, Iwen, Hinrichs.

    Acquisition, analysis, or interpretation of data: All authors.

    Drafting of the manuscript: Abdalhamid, Iwen, Wiley, Hinrichs.

    Critical revision of the manuscript for important intellectual content: Abdalhamid, Iwen, Pratt, Hinrichs.

    Obtained funding: Hinrichs.

    Administrative, technical, or material support: Abdalhamid, Iwen, Hinrichs.

    Supervision: Hinrichs.

    Conflict of Interest Disclosures: Dr Hinrichs reported being a member of the Department of Health and Human Services Clinical Laboratory Improvement Advisory Committee. No other disclosures were reported.

    Additional Contributions: We thank Chris R. Bilder, PhD, for consultation and advice on statistics. Dr Bilder was not compensated.

    References
    1.
    Korber  B, Fischer  WM, Gnanakaran  S,  et al  Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus.   Cell. 2020;182(4):812-827.e819. doi:10.1016/j.cell.2020.06.043PubMedGoogle ScholarCrossref
    2.
    Benton  DJ, Wrobel  AG, Xu  P,  et al.  Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion.   Nature. 2020;588(7837):327-330. doi:10.1038/s41586-020-2772-0PubMedGoogle ScholarCrossref
    3.
    Yanes-Lane  M, Winters  N, Fregonese  F,  et al.  Proportion of asymptomatic infection among COVID-19 positive persons and their transmission potential: a systematic review and meta-analysis.   PLoS One. 2020;15(11):e0241536. doi:10.1371/journal.pone.0241536PubMedGoogle Scholar
    4.
    Li  J, Wang  H, Mao  L,  et al.  Rapid genomic characterization of SARS-CoV-2 viruses from clinical specimens using nanopore sequencing.   Sci Rep. 2020;10(1):17492. doi:10.1038/s41598-020-74656-yPubMedGoogle ScholarCrossref
    5.
    Yan  R, Zhang  Y, Li  Y, Xia  L, Guo  Y, Zhou  Q.  Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2.   Science. 2020;367(6485):1444-1448. doi:10.1126/science.abb2762PubMedGoogle ScholarCrossref
    6.
    Jiang  HW, Zhang  HN, Meng  QF,  et al.  SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70.   Cell Mol Immunol. 2020;17(9):998-1000. doi:10.1038/s41423-020-0514-8PubMedGoogle ScholarCrossref
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