Distinctive evolution of alveolar T cell responses is associated with clinical outcomes in unvaccinated patients with SARS-CoV-2 pneumonia.

The evolution of T cell molecular signatures in the distal lung of patients with severe pneumonia is understudied. Here, we analyzed T cell subsets in longitudinal bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia, including unvaccinated patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or with respiratory failure not linked to pneumonia. In patients with SARS-CoV-2 pneumonia, activation of interferon signaling pathways, low activation of the NF-κB pathway and preferential targeting of spike and nucleocapsid proteins early after intubation were associated with favorable outcomes, whereas loss of interferon signaling, activation of NF-κB-driven programs and specificity for the ORF1ab complex late in disease were associated with mortality. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize individuals who recover, whereas responses against nonstructural proteins and activation of NF-κB are associated with poor outcomes.

Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA.

We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP.

Africa-specific human genetic variation near CHD1L associates with HIV-1 load.

HIV-1 remains a global health crisis1, highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa2, we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV3. We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log10-transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair4. Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Social/Sexual Networks of People Newly Diagnosed with HIV in Ibadan, Nigeria.

Young men who have sex with men (YMSM) in Nigeria are ten times more likely to be living with HIV-1 than other young men. Due to stigma and criminalization of same-sex sexual behavior, YMSM sexual networks are likely to overlap with those of the general population, leading to a generalized HIV-1 epidemic. Due to limited research on social/sexual network dynamics related to HIV-1 in Nigeria, our study focused on YMSM and sought to assess the feasibility and acceptability of collecting social and sexual network data in Network Canvas from individuals newly diagnosed with HIV-1 in Ibadan, Nigeria. The Network Canvas software was piloted at three sites in Ibadan, Nigeria to collect social/sexual network data from 151 individuals newly diagnosed with HIV-1. Our study sample included 37.7% YMSM; participants reported a mean of 2.6 social alters and 2.6 sexual alters. From the 151 egos and 634 alters, 85 potential unique individuals (194 total) were identified; 65 egos/alters were collapsed into 25 unique individuals. Our success collecting network data from individuals newly diagnosed with HIV-1 in Ibadan demonstrates clear feasibility and acceptability of the approach and the use of Network Canvas to capture and manage these data.

Rab11-FIP1C Is Dispensable for HIV-1 Replication in Primary CD4+ T Cells, but Its Role Is Cell Type Dependent in Immortalized Human T-Cell Lines.

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines-Jurkat E6.1, SupT1, and H9-and in primary human CD4+ T cells. FIP1C knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, FIP1C knockout caused a cell density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (Y795W) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. IMPORTANCE The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types-human T-cell lines and primary CD4+ T cells-we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.

Resurgence of SARS-CoV-2 Delta after Omicron variant superinfection in an immunocompromised pediatric patient.

BACKGROUND: Persistent SARS-CoV-2 infection in immunocompromised hosts is thought to contribute to viral evolution by facilitating long-term natural selection and viral recombination in cases of viral co-infection or superinfection. However, there are limited data on the longitudinal intra-host population dynamics of SARS-CoV-2 co-infection/superinfection, especially in pediatric populations. Here, we report a case of Delta-Omicron superinfection in a hospitalized, immunocompromised pediatric patient. METHODS: We conducted Illumina whole genome sequencing (WGS) for longitudinal specimens to investigate intra-host dynamics of SARS-CoV-2 strains. Topoisomerase PCR cloning of Spike open-reading frame and Sanger sequencing of samples was performed for four specimens to validate the findings. Analysis of publicly available SARS-CoV-2 sequence data was performed to investigate the co-circulation and persistence of SARS-CoV-2 variants. RESULTS: Results of WGS indicate the patient was initially infected with the SARS-CoV-2 Delta variant before developing a SARS-CoV-2 Omicron variant superinfection, which became predominant. Shortly thereafter, viral loads decreased below the level of detection before resurgence of the original Delta variant with no residual trace of Omicron. After 54 days of persistent infection, the patient tested negative for SARS-CoV-2 but ultimately succumbed to a COVID-19-related death. Despite protracted treatment with remdesivir, no antiviral resistance mutations emerged. These results indicate a unique case of persistent SARS-CoV-2 infection with the Delta variant interposed by a transient superinfection with the Omicron variant. Analysis of publicly available sequence data suggests the persistence and ongoing evolution of Delta subvariants despite the global predominance of Omicron, potentially indicative of continued transmission in an unknown population or niche. CONCLUSION: A better understanding of SARS-CoV-2 intra-host population dynamics, persistence, and evolution during co-infections and/or superinfections will be required to continue optimizing patient care and to better predict the emergence of new variants of concern.

Rapid and Sensitive Detection of Antigen from SARS-CoV-2 Variants of Concern by a Multivalent Minibinder-Functionalized Nanomechanical Sensor.

New platforms for the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are urgently needed. Here we report the development of a nanomechanical sensor based on the deflection of a microcantilever capable of detecting the SARS-CoV-2 spike (S) glycoprotein antigen using computationally designed multivalent minibinders immobilized on a microcantilever surface. The sensor exhibits rapid (<5 min) detection of the target antigens down to concentrations of 0.05 ng/mL (362 fM) and is more than an order of magnitude more sensitive than an antibody-based cantilever sensor. Validation of the sensor with clinical samples from 33 patients, including 9 patients infected with the Omicron (BA.1) variant observed detection of antigen from nasopharyngeal swabs with cycle threshold (Ct) values as high as 39, suggesting a limit of detection similar to that of the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate the use of minibinders and nanomechanical sensors for the rapid and sensitive detection of SARS-CoV-2 and potentially other disease markers.

Incidence and Clinical Characteristics of Severe Acute Respiratory Syndrome Coronavirus 2 Infection After Vaccination in Children.

The objective of this single-center cohort study was to characterize the frequency, clinical characteristics, and molecular epidemiology of pediatric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection after vaccination. Between May 15, 2021, and January 1, 2022, 171 children experienced SARS-CoV-2 infection postvaccination, 146 (86%) following the Omicron variant predominance. Outcomes were generally mild and comparable before and after Omicron predominance.

De novo emergence of SARS-CoV-2 spike mutations in immunosuppressed patients.

BACKGROUND: The continuing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with decreased susceptibility to neutralizing antibodies is of clinical importance. Several spike mutations associated with immune escape have evolved independently in association with different variants of concern (VOCs). How and when these mutations arise is still unclear. We hypothesized that such mutations might arise in the context of persistent viral replication in immunosuppressed hosts. METHODS: Nasopharyngeal specimens were collected longitudinally from two immunosuppressed patients with persistent SARS-CoV-2 infection. Plasma was collected from these same patients late in disease course. SARS-CoV-2 whole genome sequencing was performed to assess the emergence and frequency of mutations over time. Select Spike mutations were assessed for their impact on viral entry and antibody neutralization in vitro. RESULTS: Our sequencing results revealed the intrahost emergence of spike mutations that are associated with circulating VOCs in both immunosuppressed patients (del241-243 and E484Q in one patient, and E484K in the other). These mutations decreased antibody-mediated neutralization of pseudotyped virus particles in cell culture, but also decreased efficiency of spike-mediated cell entry. CONCLUSIONS: These observations demonstrate the de novo emergence of SARS-CoV-2 spike mutations with enhanced immune evasion in immunosuppressed patients with persistent infection. These data suggest one potential mechanism for the evolution of VOCs and emphasize the importance of continued efforts to develop antiviral drugs for suppression of viral replication in hospitalized settings.

Screening Students and Staff for Asymptomatic Coronavirus Disease 2019 in Chicago Schools.

OBJECTIVES: To assess rates of asymptomatic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) positivity in K-8 schools with risk mitigation procedures in place, and to evaluate SARS-CoV-2 transmission in school and household contacts of these positive individuals. STUDY DESIGN: In this prospective observational study, screening testing for SARS-CoV-2 was performed by oropharyngeal swabbing and polymerase chain reaction (PCR) analysis in students and staff at K-8 private schools in high-risk Chicago ZIP codes. New coronavirus disease 2019 (COVID-19) diagnoses or symptoms among participants, household contacts, and nonparticipants in each school were queried. RESULTS: Among 11 K-8 private schools across 8 Chicago ZIP codes, 468 participants (346 students, 122 staff members) underwent screening testing. At the first school, 17 participants (36%) tested positive, but epidemiologic investigation suggested against in-school transmission. Only 5 participants in the subsequent 10 schools tested positive for an overall 4.7% positivity rate (1.2% excluding school 1). All but 1 positive test among in-person students had high PCR cycle threshold values, suggesting very low SARS-CoV-2 viral loads. In all schools, no additional students, staff, or household contacts reported new diagnoses or symptoms of COVID-19 during the 2 weeks following screening testing. CONCLUSIONS: We identified infrequent asymptomatic COVID-19 in schools in high-risk Chicago communities and did not identify transmission among school staff, students, or their household contacts. These data suggest that COVID-19 mitigation procedures, including masking and physical distancing, are effective in preventing transmission of COVID-19 in schools. These results may inform future strategies for screening testing in K-8 schools.

Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.

COVID-19 outbreak and genomic investigation in an inpatient behavioral health unit.

Background: Inpatient behavioral health units (BHUs) had unique challenges in implementing interventions to mitigate coronavirus disease 2019 (COVID-19) transmission, in part due to socialization in BHU settings. The objective of this study was to identify the transmission routes and the efficacy of the mitigation strategies employed during a COVID-19 outbreak in an inpatient BHU during the Omicron surge from December 2021 to January 2022. Methods: An outbreak investigation was performed after identifying 2 COVID-19-positive BHU inpatients on December 16 and 20, 2021. Mitigation measures involved weekly point prevalence testing for all inpatients, healthcare workers (HCWs), and staff, followed by infection prevention mitigation measures and molecular surveillance. Whole-genome sequencing on a subset of COVID-19-positive individuals was performed to identify the outbreak source. Finally, an outbreak control sustainability plan was formulated for future BHU outbreak resurgences. Results: We identified 35 HCWs and 8 inpatients who tested positive in the BHU between December 16, 2021, and January 17, 2022. We generated severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomes from 15 HCWs and all inpatients. Phylogenetic analyses revealed 3 distinct but genetically related clusters: (1) an HCW and inpatient outbreak likely initiated by staff, (2) an HCW and inpatient outbreak likely initiated by an inpatient visitor, and (3) an HCW-only cluster initiated by staff. Conclusions: Distinct transmission clusters are consistent with multiple, independent SARS-CoV-2 introductions with further inpatient transmission occurring in communal settings. The implemented outbreak control plan comprised of enhanced personal protective equipment requirements, limited socialization, and molecular surveillance likely minimized disruptions to patient care as a model for future pandemics.

Evaluation of the analytical performance of the 15-minute point-of-care DASH™ SARS-CoV-2 RT-qPCR test.

Accurate and timely diagnosis for COVID-19 diagnosis allows highly effective antiviral medications to be prescribed. The DASH™ Rapid PCR System is a sample-to-answer point-of-care platform combining state-of-the-art PCR kinetics with sequence specific hybridization. The platform's first assay, the DASH™ SARS-CoV-2/S test for anterior nares direct swab specimens, received FDA Emergency Use Authorization in March 2022 for point-of-care use. Here we report the analytical characteristics of the assay including limit of detection, dynamic range, and robustness of SARS-CoV-2 variant detection. The limit of detection was determined by testing swabs contrived with one hundred copies of wild type or Omicron BA.5 virus and detecting 20/20 and 19/20, respectively. The dynamic range was assessed with contrived swabs containing 102-106 copies; the log-linear relationship between Cq and copy input was plotted, and the qPCR efficiency calculated from the slope of the line was 101.4%. Detection of seven SARS-CoV-2 variants was demonstrated.

Deviations in RSV epidemiological patterns and population structures in the United States following the COVID-19 pandemic.

Respiratory Syncytial Virus (RSV) is a leading cause of acute respiratory tract infection, with the greatest impact on infants, immunocompromised individuals, and older adults. RSV prevalence decreased substantially in the United States (US) following the implementation of COVID-19-related non-pharmaceutical interventions but later rebounded with abnormal seasonality. The biological and epidemiological factors underlying this altered behavior remain poorly defined. In this retrospective cohort study from 2009 to 2023 in Chicago, Illinois, US, we examined RSV epidemiology, clinical severity, and genetic diversity. We found that changes in RSV diagnostic platforms drove increased detections in outpatient settings post-2020 and that hospitalized adults infected with RSV-A were at higher risk of intensive care admission than those with RSV-B. While population structures of RSV-A remained unchanged, RSV-B exhibited a genetic shift into geographically distinct clusters. Mutations in the antigenic regions of the fusion protein suggest convergent evolution with potential implications for vaccine and therapeutic development.

Genomic characterization of SARS-CoV-2 in Guinea, West Africa.

SARS-CoV-2 has claimed several million lives since its emergence in late 2019. The ongoing evolution of the virus has resulted in the periodic emergence of new viral variants with distinct fitness advantages, including enhanced transmission and immune escape. While several SARS-CoV-2 variants of concern trace their origins back to the African continent-including Beta, Eta, and Omicron-most countries in Africa remain under-sampled in global genomic surveillance efforts. In an effort to begin filling these knowledge gaps, we conducted retrospective viral genomic surveillance in Guinea from October 2020 to August 2021. We found that SARS-CoV-2 clades 20A, 20B, and 20C dominated throughout 2020 until the coincident emergence of the Alpha and Eta variants of concern in January 2021. The Alpha variant remained dominant throughout early 2021 until the arrival of the Delta variant in July. Surprisingly, despite the small sample size of our study, we also found the persistence of the early SARS-CoV-2 clade 19B as late as April 2021. Together, these data help fill in our understanding of the SARS-CoV-2 population dynamics in West Africa early in the COVID-19 pandemic.

Evolution of SARS-CoV-2 in the murine central nervous system drives viral diversification.

Severe coronavirus disease 2019 and post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral divergence were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titres and decreased morbidity compared with the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but dissemination from the CNS to the lung required the intact FCS. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Integration of individualized and population-level molecular epidemiology data to model COVID-19 outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility and immune escape have emerged periodically throughout the coronavirus disease 2019 (COVID-19) pandemic, but the impact of these variants on disease severity has remained unclear. In this single-center, retrospective cohort study, we examined the association between SARS-CoV-2 clade and patient outcome over a two-year period in Chicago, Illinois. Between March 2020 and March 2022, 14,252 residual diagnostic specimens were collected from SARS-CoV-2-positive inpatients and outpatients alongside linked clinical and demographic metadata, of which 2,114 were processed for viral whole-genome sequencing. When controlling for patient demographics and vaccination status, several viral clades were associated with risk for hospitalization, but this association was negated by the inclusion of population-level confounders, including case count, sampling bias, and shifting standards of care. These data highlight the importance of integrating non-virological factors into disease severity and outcome models for the accurate assessment of patient risk.