Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation (preprint)

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

Positive selection underlies repeated knockout of ORF8 in SARS-CoV-2 evolution (preprint)

Knockout of the ORF8 protein has repeatedly spread through the global viral population during SARS-CoV-2 evolution. Here we use both regional and global pathogen sequencing to explore the selection pressures underlying its loss. In Washington State, we identified transmission clusters with ORF8 knockout throughout SARS-CoV-2 evolution, not just on novel, high fitness viral backbones. Indeed, ORF8 is truncated more frequently and knockouts circulate for longer than for any other gene. Using a global phylogeny, we find evidence of positive selection to explain this phenomenon: nonsense mutations resulting in shortened protein products occur more frequently and are associated with faster clade growth rates than synonymous mutations in ORF8. Loss of ORF8 is also associated with reduced clinical severity, highlighting the diverse clinical impacts of SARS-CoV-2 evolution.

Quantifying how single dose Ad26.COV2.S vaccine efficacy depends on Spike sequence features (preprint)

It is of interest to pinpoint SARS-CoV-2 sequence features defining vaccine resistance. In the ENSEMBLE randomized, placebo-controlled phase 3 trial, estimated single-dose Ad26.COV2.S vaccine efficacy (VE) was 56% against moderate to severe–critical COVID-19. SARS-CoV-2 Spike sequences were measured from 484 vaccine and 1,067 placebo recipients who acquired COVID-19 during the trial. In Latin America, where Spike diversity was greatest, VE was significantly lower against Lambda than against Reference and against all non-Lambda variants [family-wise error rate (FWER) p < 0.05]. VE also differed by residue match vs. mismatch to the vaccine-strain residue at 16 amino acid positions (4 FWER p < 0.05; 12 q-value ≤ 0.20). VE significantly decreased with physicochemical-weighted Hamming distance to the vaccine-strain sequence for Spike, receptor-binding domain, N-terminal domain, and S1 (FWER p < 0.001); differed (FWER ≤ 0.05) by distance to the vaccine strain measured by 9 different antibody-epitope escape scores and by 4 NTD neutralization-impacting features; and decreased (p = 0.011) with neutralization resistance level to vaccine recipient sera. VE against severe–critical COVID-19 was stable across most sequence features but lower against viruses with greatest distances. These results help map antigenic specificity of in vivo vaccine protection.

Sensitivity of Rapid Antigen Tests Against SARS-CoV-2 Omicron and Delta Variants (preprint)

Rapid Antigen Tests (RAT) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of eight commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR Cycle threshold (CT) value (a proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using CT value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Local-Scale phylodynamics reveal differential community impact of SARS-CoV-2 in metropolitan US county (preprint)

SARS-CoV-2 transmission is largely driven by heterogeneous dynamics at a local scale, leaving local health departments to design interventions with limited information. We analyzed SARS-CoV-2 genomes sampled between February 2020 and March 2022 jointly with epidemiological and cell phone mobility data to investigate fine scale spatiotemporal SARS-CoV-2 transmission dynamics in King County, Washington, a diverse, metropolitan US county. We applied an approximate structured coalescent approach to model transmission within and between North King County and South King County alongside the rate of outside introductions into the county. Our phylodynamic analyses reveal that following stay-at-home orders, the epidemic trajectories of North and South King County began to diverge. We find that South King County consistently had more reported and estimated cases, COVID-19 hospitalizations, and longer persistence of local viral transmission when compared to North King County, where viral importations from outside drove a larger proportion of new cases. Using mobility and demographic data, we also find that South King County experienced a more modest and less sustained reduction in mobility following stay-at-home orders than North King County, while also bearing more socioeconomic inequities that might contribute to a disproportionate burden of SARS-CoV-2 transmission. Overall, our findings suggest a role for local-scale phylodynamics in understanding the heterogeneous transmission landscape. One Sentence SummaryAnalysis of SARS-CoV-2 genomes in King County, Washington show that diverse areas in the same metropolitan region can have different epidemic dynamics.

Paxlovid-like nirmatrelvir/ritonavir fails to block SARS-CoV-2 transmission in ferrets (preprint)

Despite the continued spread of SARS-CoV-2 and emergence of variants of concern (VOC) that are capable of escaping preexisting immunity, therapeutic options are underutilized. In addition to preventing severe disease in high-risk patients, antivirals may contribute to interrupting transmission chains. The FDA has granted emergency use authorizations for two oral drugs, molnupiravir and paxlovid. Initial clinical trials suggested an efficacy advantage of paxlovid, giving it a standard-of-care-like status in the United States. However, recent retrospective clinical studies suggested a more comparable efficacy of both drugs in preventing complicated disease and case-fatalities in older adults. For a direct efficacy comparison under controlled conditions, we assessed potency of both drugs against SARS-CoV-2 in two relevant animal models; the Roborovski dwarf hamster model for severe COVID-19 in high-risk patients and the ferret model of upper respiratory tract disease and transmission. After infection of dwarf hamsters with VOC omicron, paxlovid and molnupiravir were efficacious in mitigating severe disease and preventing death. However, a pharmacokinetics-confirmed human equivalent dose of paxlovid did not significantly reduce shed SARS-CoV-2 titers in ferrets and failed to block virus transmission to untreated direct-contact ferrets, whereas transmission was fully suppressed in a group of animals treated with a human-equivalent dose of molnupiravir. Prophylactic administration of molnupiravir to uninfected ferrets in direct contact with infected animals blocked productive SARS-CoV-2 transmission, whereas all contacts treated with prophylactic paxlovid became infected. These data confirm retrospective reports of similar therapeutic benefit of both drugs for older adults, and reveal that treatment with molnupiravir, but not paxlovid, may be suitable to reduce the risk of SARS-CoV-2 transmission.

Genomic analysis of early spread of monkeypox virus in Washington State (preprint)

Genomic analysis of the monkeypox virus outbreak in Washington State using 109 viral genome sequences collected from July-August 2022 shows low overall genetic diversity, multiple introductions into the state with ongoing community transmission, and potential for coinfection of an individual by multiple strains. Biography Dr. Roychoudhury is Acting Instructor and Director of COVID-19 NGS in the Department of Laboratory Medicine and Pathology at the University of Washington and Associate in the Vaccine and Infectious Disease Division at the Fred Hutchinson Cancer Research Center. Her primary research interests are pathogen genomics and mathematical models of viral evolution and host-pathogen interactions.

Safety, Immunogenicity and Efficacy of NVX-CoV2373 in Adolescents in PREVENT-19: A Randomized, Phase 3 Trial (preprint)

BACKGROUND Over 20% of cases and 0.4% of deaths from Covid-19 occur in children. Following demonstration of safety and efficacy of the adjuvanted, recombinant spike protein vaccine NVX-CoV2373 in adults, the PREVENT-19 trial enrolled adolescents. METHODS Safety, immunogenicity, and efficacy of NVX-CoV2373 were evaluated in adolescents aged 12 to <18 years in an expansion of PREVENT-19, a phase 3, randomized, observer-blinded, placebo-controlled trial in the United States. Participants were randomized 2:1 to two doses of NVX-CoV2373 or placebo 21 days apart, and followed for a median of 2 months after second vaccination. Primary end points were serologic non-inferiority of neutralizing antibody (NA) responses compared with young adults (18 to <26 years) in PREVENT-19, protective efficacy against laboratory-confirmed Covid-19, and assessment of reactogenicity/safety. RESULTS Among 2,247 participants randomized between April-June 2021, 1,491 were allocated to NVX-CoV2373 and 756 to placebo. Post-vaccination, the ratio of NA geometric mean titers in adolescents compared to young adults was 1.5 (95% confidence interval [CI] 1.3 to 1.7). Twenty Covid-19 cases (all mild) occurred: 6 among NVX-CoV2373 and 14 among placebo recipients (vaccine efficacy [VE]: 79.5%, 95% CI, 46.8 to 92.1). All sequenced viral genomes (11/20) were identified as Delta variant (Delta variant VE: 82.0% [95% CI: 32.4 to 95.2]). Reactogenicity was largely mild-to-moderate, transient, and more frequent in NVX-CoV2373 recipients and after the second dose. Serious adverse events were rare and evenly distributed between treatments. CONCLUSIONS NVX-CoV2373 was safe, immunogenic, and efficacious in the prevention of Covid-19 and those cases caused by the Delta variant in adolescents. (Funded by the Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority and the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health; PREVENT-19 ClinicalTrials.gov number, NCT04611802).

Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike (preprint)

The SARS-CoV-2 spike protein is the target of neutralizing antibodies and the immunogen used in all currently approved vaccines. The global spread of the virus has resulted in emergence of lineages which are of concern for the effectiveness of immunotherapies and vaccines based on the early Wuhan isolate. Here we describe two SARS-CoV-2 isolates with large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis showed that the deletions result in complete reshaping of the antigenic surface of the NTD supersite. The remodeling of the NTD affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite for both spike variants. A unique escape mechanism with high antigenic impact observed in the {Delta}N135 variant was based on the loss of the Cys15-Cys136 disulfide due to the P9L-mediated shift of the signal peptide cleavage site and deletion of residues 136-144. Although the observed large loop and disulfide deletions are rare, similar modifications became independently established in several other lineages, highlighting the possibility of a general escape mechanism via the NTD supersite. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.

Evidence for SARS-CoV-2 Delta and Omicron co-infections and recombination (preprint)

Between November 2021 and February 2022, SARS-CoV-2 Delta and Omicron variants co-circulated in the United States, allowing for co-infections and possible recombination events. We sequenced 29,719 positive samples during this period and analyzed the presence and fraction of reads supporting mutations specific to either the Delta or Omicron variant. Our sequencing protocol uses hybridization capture and is thus less subject to artifacts observed in amplicon-based approaches that may lead to spurious signals for recombinants. We identified 20 co-infections, one of which displayed evidence of a low recombinant viral population. We also identified two independent cases of infection by a Delta-Omicron recombinant virus, where 100% of the viral RNA came from one clonal recombinant. In both cases, the 5'-end of the viral genome was from the Delta genome, and the 3'-end from Omicron, though the breakpoints were different. Delta-Omicron recombinant viruses were rare, and there is currently no evidence that the two Delta-Omicron recombinant viruses identified are more transmissible between hosts compared to the circulating Omicron lineages.

Narrow transmission bottlenecks and limited within-host viral diversity during a SARS-CoV-2 outbreak on a fishing boat (preprint)

The long-term evolution of viruses is ultimately due to viral mutants that arise within infected individuals and transmit to other individuals. Here we use deep sequencing to investigate the transmission of viral genetic variation among individuals during a SARS-CoV-2 outbreak that infected the vast majority of crew members on a fishing boat. We deep-sequenced nasal swabs to characterize the within-host viral population of infected crew members, using experimental duplicates and strict computational filters to ensure accurate variant calling. We find that within-host viral diversity is low in infected crew members. The mutations that did fix in some crew members during the outbreak are not observed at detectable frequencies in any of the sampled crew members in which they are not fixed, suggesting viral evolution involves occasional fixation of low-frequency mutations during transmission rather than persistent maintenance of within-host viral diversity. Overall, our results show that strong transmission bottlenecks dominate viral evolution even during a superspreading event with a very high attack rate.

Rapid and Accurate Identification of SARS-CoV-2 Omicron Variants Using Droplet Digital PCR (RT-ddPCR) (preprint)

Background Mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in the recently described Omicron variant. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals. Objective To develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment. Study Design Using three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant. Results Mutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen. Conclusion We describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.

A method for variant agnostic detection of SARS-CoV-2, rapid monitoring of circulating variants, detection of mutations of biological significance, and early detection of emergent variants such as Omicron (preprint)

The rapid emergence of new SARS-CoV-2 variants raises a number of public health questions including the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to better understand patterns of transmission and possible load on healthcare resources. Next-Generation Sequencing (NGS) is the primary method for detecting and tracing the emergence of new variants, but it is expensive, and it can take weeks before sequence data is available in public repositories. Here, we describe a Polymerase Chain Reaction (PCR)-based genotyping approach that is significantly less expensive, accelerates reporting on SARS-CoV-2 variants, and can be implemented in any testing lab performing PCR. Specific Single Nucleotide Polymorphisms (SNPs) and indels are identified that have high positive percent agreement (PPA) and negative percent agreement (NPA) compared to NGS for the major genotypes that circulated in 2021. Using a 48-marker panel, testing on 1,128 retrospective samples yielded a PPA and NPA in the 96.3 to 100% and 99.2 to 100% range, respectively, for the top 10 most prevalent lineages. The effect on PPA and NPA of reducing the number of panel markers was also explored. In addition, with the emergence of Omicron, we also developed an Omicron genotyping panel that distinguishes the Delta and Omicron variants using four (4) highly specific SNPs. Data from testing demonstrates the capability to use the panel to rapidly track the growing prevalence of the Omicron variant in the United States in December 2021.

De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: A case report (preprint)

SARS-CoV-2 remdesivir resistance mutations have been generated in vitro but have not been reported in patients receiving treatment with the antiviral agent. We present a case of an immunocompromised patient with acquired B-cell deficiency who developed an indolent, protracted course of SARS-CoV-2 infection. Remdesivir therapy alleviated symptoms and produced a transient virologic response, but her course was complicated by recrudescence of high-grade viral shedding. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase which was not present in pre-treatment specimens. In vitro experiments demonstrated that the mutation conferred a ~6-fold increase in remdesivir IC50 but resulted in a fitness cost in the absence of remdesivir. Sustained clinical and virologic response was achieved after treatment with casirivimab-imdevimab. Although the fitness cost observed in vitro may limit the risk posed by E802D, this case illustrates the importance of monitoring for remdesivir resistance and the potential benefit of combinatorial therapies in immunocompromised patients with SARS-CoV-2 infection.

Clinical performance characteristics of the Swift Normalase Amplicon Panel for sensitive recovery of SARS-CoV-2 genomes (preprint)

Amplicon-based sequencing methods have been central in characterizing the diversity, transmission and evolution of SARS-CoV-2, but need to be rigorously assessed for clinical utility. Here, we validated the Swift Biosciences SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens with a 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR reaction. Breadth of genome recovery was evaluated across a range of Ct values (11.3 - 36.7, median 21.6). Out of 428 positive samples, 406 (94.9%) generated genomes with < 10% Ns, with a mean genome coverage of 13,545X/SD 8,382X. No genomes were recovered from PCR-negative specimens (n = 30), or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared to whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.

Variants in nucleocapsid protein and endoRNase are found to associate with severe COVID-19 in a case-control study in Washington State, USA (preprint)

SARS-CoV-2 is spreading worldwide with continuously evolving variants, some of which occur in the Spike protein and appear to increase the viral transmissibility. However, variants that cause severe COVID-19 or lead to other breakthroughs have not been well characterized. To discover such viral variants, we assembled a cohort of 683 COVID-19 patients; 388 inpatients (“cases”) and 295 outpatients (“controls”) from April to August 2020 using electronically captured COVID test request forms and sequenced their viral genomes. To improve the analytic power, we accessed 7,137 viral sequences in Washington State to filter out viral single nucleotide variants (SNVs) that did not have significant expansions over the collection period. Applying this filter led to the identification of 53 SNVs that were statistically significant, of which 13 SNVs each had 3 or more variant copies in the discovery cohort. Correlating these selected SNVs with case/control status, eight SNVs were found to significantly associate with inpatient status (q-values<0.01). Using temporal synchrony, we identified a four SNV-haplotype (t19839-g28881-g28882-g28883) which was significantly associated with case/control status (Fisher’s exact p=2.84*10 −11 ) that appeared in April 2020, peaked in June, and persisted into January 2021. This association was replicated (OR=5.46, p-value=4.71*10 −12 ) in an independent cohort of 964 COVID-19 patients (June 1, 2020 to March 31, 2021). The haplotype included a synonymous change N73N in endoRNase, and three non-synonymous changes coding residues R203K, R203S and G204R in the nucleocapsid protein. This discovery points to the potential functional role of the nucleocapsid protein in triggering “cytokine storms” and severe COVID-19 that led to hospitalization. The study further emphasizes a need for tracking and analyzing viral sequences in correlations with clinical status.

Associations between SARS-CoV-2 variants and risk of COVID-19 hospitalization among confirmed cases in Washington State: a retrospective cohort study (preprint)

BackgroundThe COVID-19 pandemic is now dominated by variant lineages; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the risk of hospitalization following infection with nine variants of concern or interest (VOC/VOI). MethodsOur study includes individuals with positive SARS-CoV-2 RT-PCR in the Washington Disease Reporting System and with available viral genome data, from December 1, 2020 to July 30, 2021. The main analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for the risk of hospitalization following infection with a VOC/VOI, adjusting for age, sex, and vaccination status. FindingsOf the 27,814 cases, 23,170 (83.3%) were sequenced through sentinel surveillance, of which 726 (3.1%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.17, 95% CI 2.15-4.67), Beta (HR: 2.97, 95% CI 1.65-5.35), Delta (HR: 2.30, 95% CI 1.69-3.15), and Alpha (HR 1.59, 95% CI 1.26-1.99) compared to infections with an ancestral lineage. Following VOC infection, unvaccinated patients show a similar higher hospitalization risk, while vaccinated patients show no significant difference in risk, both when compared to unvaccinated, ancestral lineage cases. InterpretationInfection with a VOC results in a higher hospitalization risk, with an active vaccination attenuating that risk. Our findings support promoting hospital preparedness, vaccination, and robust genomic surveillance.

Trajectory of viral load in a prospective population-based cohort with incident SARS-CoV-2 G614 infection (preprint)

ImportanceSARS-CoV-2 viral trajectory has not been well-characterized in documented incident infections. These data will inform SARS-CoV-2 natural history, transmission dynamics, prevention practices, and therapeutic development. ObjectiveTo prospectively characterize early SARS-CoV-2 viral shedding in persons with incident infection. DesignProspective cohort study. SettingSecondary data analysis from a multicenter study in the U.S. ParticipantsThe samples derived from a randomized controlled trial of 829 community-based asymptomatic participants recently exposed (<96 hours) to persons with SARS-CoV-2. Participants collected daily mid-turbinate swabs for SARS-CoV-2 detection by polymerase-chain-reaction and symptom diaries for 14-days. Persons with negative swab for SARS-CoV-2 at baseline who developed infection during the study were included in the analysis. ExposureLaboratory-confirmed SARS-CoV-2 infection. Main outcomes and measuresThe observed SARS-CoV-2 viral shedding characteristics were summarized and shedding trajectories were examined using a piece-wise linear mixed-effects modeling. Whole viral genome sequencing was performed on samples with cycle threshold (Ct)<34. ResultsNinety-seven persons (57% women, median age 37-years) developed incident infections during 14-days of follow-up. Two-hundred fifteen sequenced samples were assigned to 15 lineages that belonged to the G614 variant. Forty-two (43%), 18(19%), and 31(32%) participants had viral shedding for 1 day, 2-6 days, and [≥]7 days, with median peak viral load Ct of 38.5, 36.7, and 18.3, respectively. Six (6%) participants had 1-6 days of observed viral shedding with censored duration. The peak average viral load was observed on day 3 of viral shedding. The average Ct value was lower, indicating higher viral load, in persons reporting COVID-19 symptoms than asymptomatic. Using the statistical model, the median time from shedding onset to peak viral load was 1.4 days followed by a median of 9.7 days before clearance. Conclusions and RelevanceIncident SARS-CoV-2 G614 infection resulted in a rapid viral load peak followed by slower decay and positive correlation between peak viral load and shedding duration; duration of shedding was heterogeneous. This longitudinal evaluation of the SARS-CoV-2 G614 variant with frequent molecular testing may serve as a reference for comparing emergent viral lineages to inform clinical trial designs and public health strategies to contain the spread of the virus. KEY POINTSO_ST_ABSQuestionC_ST_ABSWhat are the early SARS-CoV-2 G614 viral shedding characteristics in persons with incident infection? FindingsIn this prospective cohort of 97 community-based participants who collected daily mid-turbinate swabs for SARS-CoV-2 detection after recent exposure to SARS-CoV-2, viral trajectory was characterized by a rapid peak followed by slower decay. Peak viral load correlated positively with symptoms. The duration of shedding was heterogeneous. MeaningA detailed description of the SARS-CoV-2 G614 viral shedding trajectory serves as baseline for comparison to new viral variants of concern and inform models for the planning of clinical trials and transmission dynamics to end this pandemic.

Towards Discovering SARS-CoV-2 Variants of High Consequence Based on Both Surveillance and Electronically Captured Health Data: First Year Experience in Washington State (January 2020-2021) (preprint)

Background: SARS-CoV-2 is continuously evolving with the emergence of variants of interest (VOI) or with variants of concern (VOC). While Variants of High Consequence (VOHC) are well defined, no such variants have been formally documented. Here we propose an integrated strategy and application towards discovering VOHC. Methods: We utilized 7,137 viral sequences collected from COVID-19 cases in Washington State from January 19, 2020 to January 31, 2021, to identify genome-wide viral single nucleotide variants (SNVs). Utilizing a non-parametric regression model, we selected a subset of SNVs that had significant and substantial expansions over the collection period. Further, using unsupervised learning, we identified multiple SNVs forming haplotypes. To evaluate their clinical relevance, we assembled a discovery cohort of COVID-19 cases (388 inpatients and 295 outpatients) to identify SNVs and haplotypes associated with hospitalization status, a proxy for disease severity. A logistic regression model was used to assess associations of SNVs with hospitalization status in the discovery cohort. These results were validated on an independent cohort of 964 genome sequences derived from COVID-19 cases in Washington State from June 1, 2020 to March 31, 2021. Finding: The analysis of the 7,137 sequences led to identification of 107 SNVs that were statistically significant (false positive error rate q-value <0.01) and substantial expansions (maximum value of locally averaged proportions, Pmax>0.10). Forty-one SNVs were considered urgent, because their SNV proportions persisted or expanded above 10% in January 2021, the last month of the current investigation period. Correlating with clinical data, eight SNVs were found to significantly associate with inpatient status (p-values<0.001). By their synchronized dynamics, two SNVs were haplotyped and the mutant haplotype (c15933t-g16968t) was observed among patients in the discovery cohort (Fisher’s exact p=1.53*10-10), and this association was validated in the validation cohort (OR=5.38, p=10-9). Similarly, a haplotype with 4 SNVs (t19839c-g28881a-g28882a-g28883c) was observed only among inpatients (p=1.53*10-10) in the discovery cohort. Discovered haplotypic association was validated in the independent validation cohort (OR=3.69, p-value=3.44*10-10) and was further validated after adjusting for sex, age and collection time (OR=5.46, p-value=4.71*10-12). Interpretation: The mutant haplotype t19839c-g28881a-g28882a-g28883c emerged in April 2020, remained undetected over eight months, and has now begun to re-emerge. Because of its strong association with hospitalization status and re-emergence, this mutant haplotype may be a candidate variant for VOHC, pending further investigation of a) its clinical association with the disease severity, b) asymptomatic transmissibility and/or c) immune evasion to approved vaccines. While preliminary, this result indicates the importance to conduct purpose-driven clinical follow up studies to discover and validate candidate variants for VOHC. Also of interest is the mutant haplotype c15933t-g16968t which expanded in May 2020 but subsided by October 2020. Due to its association with hospitalization, we recommend continued monitoring for re-emergence of this variant and further assessment of viral phenotype.Funding: National Institutes of Health grant R01-GM129325 National Institutes of Health/National Institute of Allergy and Infectious Diseases grant UM1 AI068635Declaration of Interest: The authors declare that they have no competing interests.Ethical Approval: This study was approved by the Human Subject Review Committee at Fred Hutchinson Cancer Research Center (IRB#6007-2043) and by the University of Washington Institutional Review Board (STUDY00000408).

Towards Discovering SARS-CoV-2 Variants of High Consequence Based on Both Surveillance and Electronically Captured Health Data: First Year Experience in Washington State (January 2020-2021) (preprint)

Background: SARS-CoV-2 is continuously evolving with the emergence of variants of interest (VOI) or with variants of concern (VOC). While Variants of High Consequence (VOHC) are well defined, no such variants have been formally documented. Here we propose an integrated strategy and application towards discovering VOHC.Methods: We utilized 7,137 viral sequences collected from COVID-19 cases in Washington State from January 19, 2020 to January 31, 2021, to identify genome-wide viral single nucleotide variants (SNVs). Utilizing a non-parametric regression model, we selected a subset of SNVs that had significant and substantial expansions over the collection period. Further, using unsupervised learning, we identified multiple SNVs forming haplotypes. To evaluate their clinical relevance, we assembled a discovery cohort of COVID-19 cases (388 inpatients and 295 outpatients) to identify SNVs and haplotypes associated with hospitalization status, a proxy for disease severity. A logistic regression model was used to assess associations of SNVs with hospitalization status in the discovery cohort. These results were validated on an independent cohort of 964 genome sequences derived from COVID-19 cases in Washington State from June 1, 2020 to March 31, 2021.Finding: The analysis of the 7,137 sequences led to identification of 107 SNVs that were statistically significant (false positive error rate q-value <0.01) and substantial expansions (maximum value of locally averaged proportions, Pmax>0.10). Forty-one SNVs were considered urgent, because their SNV proportions persisted or expanded above 10% in January 2021, the last month of the current investigation period. Correlating with clinical data, eight SNVs were found to significantly associate with inpatient status (p-values<0.001). By their synchronized dynamics, two SNVs were haplotyped and the mutant haplotype (c15933t-g16968t) was observed among patients in the discovery cohort (Fisher’s exact p=1.53*10-10), and this association was validated in the validation cohort (OR=5.38, p=10-9). Similarly, a haplotype with 4 SNVs (t19839c-g28881a-g28882a-g28883c) was observed only among inpatients (p=1.53*10-10) in the discovery cohort. Discovered haplotypic association was validated in the independent validation cohort (OR=3.69, p-value=3.44*10-10) and was further validated after adjusting for sex, age and collection time (OR=5.46, p-value=4.71*10-12). Interpretation: The mutant haplotype t19839c-g28881a-g28882a-g28883c emerged in April 2020, remained undetected over eight months, and has now begun to re-emerge. Because of its strong association with hospitalization status and re-emergence, this mutant haplotype may be a candidate variant for VOHC, pending further investigation of a) its clinical association with the disease severity, b) asymptomatic transmissibility and/or c) immune evasion to approved vaccines. While preliminary, this result indicates the importance to conduct purpose-driven clinical follow up studies to discover and validate candidate variants for VOHC. Also of interest is the mutant haplotype c15933t-g16968t which expanded in May 2020 but subsided by October 2020. Due to its association with hospitalization, we recommend continued monitoring for re-emergence of this variant and further assessment of viral phenotype.Funding Information: National Institutes of Health grant R01-GM129325. National Institutes of Health/National Institute of Allergy and Infectious Diseases grant UM1 AI068635Declaration of Interests: None to declare. Ethics Approval Statement: This study was approved by the Human Subject Review Committee at Fred Hutchinson Cancer Research Center (IRB#6007-2043) and by the University of Washington Institutional Review Board (STUDY00000408).