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.

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Introduction: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Methods: Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. Results: In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. Discussion: We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.

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.

Activity and inhibition of the SARS-CoV-2 Omicron nsp13 R392C variant using RNA duplex unwinding assays.

SARS-CoV-2 nsp13 helicase is an essential enzyme for viral replication and a promising target for antiviral drug development. This study compares the double-stranded RNA (dsRNA) unwinding activity of nsp13 and the Omicron nsp13R392C variant, which is predominant in currently circulating lineages. Using in vitro gel- and fluorescence-based assays, we found that both nsp13 and nsp13R392C have dsRNA unwinding activity with equivalent kinetics. Furthermore, the R392C mutation had no effect on the efficiency of the nsp13-specific helicase inhibitor SSYA10-001. We additionally confirmed the activity of several other helicase inhibitors against nsp13, including punicalagin that inhibited dsRNA unwinding at nanomolar concentrations. Overall, this study reveals the utility of using dsRNA unwinding assays to screen small molecules for antiviral activity against nsp13 and the Omicron nsp13R392C variant. Continual monitoring of newly emergent variants will be essential for considering resistance profiles of lead compounds as they are advanced towards next-generation therapeutic development.

Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps.

Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.

TGF-ß blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirm the latency reversal properties of in vivo TGF-ß blockade, decrease viral reservoirs and stimulate immune responses. Treatment of eight female, SIV-infected macaques on ART with four 2-weeks cycles of galunisertib leads to viral reactivation as indicated by plasma viral load and immunoPET/CT with a 64Cu-DOTA-F(ab')2-p7D3-probe. Post-galunisertib, lymph nodes, gut and PBMC exhibit lower cell-associated (CA-)SIV DNA and lower intact pro-virus (PBMC). Galunisertib does not lead to systemic increase in inflammatory cytokines. High-dimensional cytometry, bulk, and single-cell (sc)RNAseq reveal a galunisertib-driven shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrate that galunisertib, a clinical stage TGF-ß inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity.

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.

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.

SARS-CoV-2 genomics and impact on clinical care for COVID-19.

The emergence and worldwide spread of SARS-CoV-2 during the COVID-19 pandemic necessitated the adaptation and rapid deployment of viral WGS and analysis techniques that had been previously applied on a more limited basis to other viral pathogens, such as HIV and influenza viruses. The need for WGS was driven in part by the low mutation rate of SARS-CoV-2, which necessitated measuring variation along the entire genome sequence to effectively differentiate lineages and characterize viral evolution. Several WGS approaches designed to maximize throughput and accuracy were quickly adopted by surveillance labs around the world. These broad-based SARS-CoV-2 genomic sequencing efforts revealed ongoing evolution of the virus, highlighted by the successive emergence of new viral variants throughout the course of the pandemic. These genomic insights were instrumental in characterizing the effects of viral mutations on transmissibility, immune escape and viral tropism, which in turn helped guide public health policy, the use of monoclonal antibody therapeutics and vaccine development strategies. As the use of direct-acting antivirals for the treatment of COVID-19 became more widespread, the potential for emergence of antiviral resistance has driven ongoing efforts to delineate resistance mutations and to monitor global sequence databases for their emergence. Given the critical role of viral genomics in the international effort to combat the COVID-19 pandemic, coordinated efforts should be made to expand global genomic surveillance capacity and infrastructure towards the anticipation and prevention of future pandemics.

TGF-ß blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-ß blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on suppressive ART were treated with 4 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption(ATI). One macaque did not rebound, while the remaining rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with the 64Cu-DOTA-F(ab')2-p7D3-probe. Half to 1 Log decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk and single-cell RNAseq revealed a shift toward an effector phenotype in T and NK cells. In summary, we demonstrated that galunisertib, a clinical stage TGFß inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity.

SARS-CoV-2 Bottlenecks and Tissue-Specific Adaptation in the Central Nervous System.

Severe COVID-19 and post-acute sequelae of 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 diversity 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 titers and decreased morbidity compared to 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 this required reversion of the FCS deletion. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization with possible implications for the treatment of neuroinvasive COVID-19.

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.

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors.

Antiviral therapeutics to treat SARS-CoV-2 are needed to diminish the morbidity of the ongoing COVID-19 pandemic. A well-precedented drug target is the main viral protease (MPro ), which is targeted by an approved drug and by several investigational drugs. Emerging viral resistance has made new inhibitor chemotypes more pressing. Adopting a structure-based approach, we docked 1.2 billion non-covalent lead-like molecules and a new library of 6.5 million electrophiles against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 and 20 µM, respectively. Several series were optimized, resulting in low micromolar inhibitors. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. While the new chemotypes may aid further optimization of MPro inhibitors for SARS-CoV-2, the modest success rate also reveals weaknesses in our approach for challenging targets like MPro versus other targets where it has been more successful, and versus other structure-based techniques against MPro itself.

Temporal and spatial characterization of HIV/SIV infection at anorectal mucosa using rhesus macaque rectal challenge model.

The study described herein is a continuation of our work in which we developed a methodology to identify small foci of transduced cells following rectal challenge of rhesus macaques with a non-replicative luciferase reporter virus. In the current study, the wild-type virus was added to the inoculation mix and twelve rhesus macaques were necropsied 2-4 days after the rectal challenge to study the changes in infected cell phenotype as the infection progressed. Relying on luciferase reporter we noted that both anus and rectum tissues are susceptible to the virus as early as 48h after the challenge. Small regions of the tissue containing luciferase-positive foci were further analyzed microscopically and were found to also contain cells infected by wild-type virus. Phenotypic analysis of the Env and Gag positive cells in these tissues revealed the virus can infect diverse cell populations, including but not limited to Th17 T cells, non Th17 T cells, immature dendritic cells, and myeloid-like cells. The proportions of the infected cell types, however, did not vary much during the first four days of infection when anus and rectum tissues were examined together. Nonetheless, when the same data was analyzed on a tissue-specific basis, we found significant changes in infected cell phenotypes over the course of infection. For anal tissue, a statistically significant increase in infection was observed for Th17 T cells and myeloid-like cells, while in the rectum, the non-Th17 T cells showed the biggest temporal increase, also of statistical significance.

Altered RSV Epidemiology and Genetic Diversity Following the COVID-19 Pandemic.

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

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

Pathogen clearance and resolution of inflammation in patients with pneumonia require an effective local T cell response. Nevertheless, local T cell activation may drive lung injury, particularly during prolonged episodes of respiratory failure characteristic of severe SARS-CoV-2 pneumonia. While T cell responses in the peripheral blood are well described, the evolution of T cell phenotypes and molecular signatures in the distal lung of patients with severe pneumonia caused by SARS-CoV-2 or other pathogens is understudied. Accordingly, we serially obtained 432 bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia and respiratory failure, including 74 unvaccinated patients with COVID-19, and performed flow cytometry, transcriptional, and T cell receptor profiling on sorted CD8+ and CD4+ T cell subsets. In patients with COVID-19 but not pneumonia secondary to other pathogens, we found that early and persistent enrichment in CD8+ and CD4+ T cell subsets correlated with survival to hospital discharge. Activation of interferon signaling pathways early after intubation for COVID-19 was associated with favorable outcomes, while activation of NF-κB-driven programs late in disease was associated with poor outcomes. Patients with SARS-CoV-2 pneumonia whose alveolar T cells preferentially targeted the Spike and Nucleocapsid proteins tended to experience more favorable outcomes than patients whose T cells predominantly targeted the ORF1ab polyprotein complex. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize patients who recover, yet these responses progress to NF-κB activation against non-structural proteins in patients who go on to experience poor clinical outcomes.

ViralVar: A Web Tool for Multilevel Visualization of SARS-CoV-2 Genomes.

The unprecedented growth of publicly available SARS-CoV-2 genome sequence data has increased the demand for effective and accessible SARS-CoV-2 data analysis and visualization tools. The majority of the currently available tools either require computational expertise to deploy them or limit user input to preselected subsets of SARS-CoV-2 genomes. To address these limitations, we developed ViralVar, a publicly available, point-and-click webtool that gives users the freedom to investigate and visualize user-selected subsets of SARS-CoV-2 genomes obtained from the GISAID public database. ViralVar has two primary features that enable: (1) the visualization of the spatiotemporal dynamics of SARS-CoV-2 lineages and (2) a structural/functional analysis of genomic mutations. As proof-of-principle, ViralVar was used to explore the evolution of the SARS-CoV-2 pandemic in the USA in pediatric, adult, and elderly populations (n > 1.7 million genomes). Whereas the spatiotemporal dynamics of the variants did not differ between these age groups, several USA-specific sublineages arose relative to the rest of the world. Our development and utilization of ViralVar to provide insights on the evolution of SARS-CoV-2 in the USA demonstrates the importance of developing accessible tools to facilitate and accelerate the large-scale surveillance of circulating pathogens.

Challenges and Opportunities for Global Genomic Surveillance Strategies in the COVID-19 Era.

Global SARS-CoV-2 genomic surveillance efforts have provided critical data on the ongoing evolution of the virus to inform best practices in clinical care and public health throughout the pandemic. Impactful genomic surveillance strategies generally follow a multi-disciplinary pipeline involving clinical sample collection, viral genotyping, metadata linkage, data reporting, and public health responses. Unfortunately, current limitations in each of these steps have compromised the overall effectiveness of these strategies. Biases from convenience-based sampling methods can obfuscate the true distribution of circulating variants. The lack of standardization in genotyping strategies and bioinformatic expertise can create bottlenecks in data processing and complicate interpretation. Limitations and inconsistencies in clinical and demographic data collection and sharing can slow the compilation and limit the utility of comprehensive datasets. This likewise can complicate data reporting, restricting the availability of timely data. Finally, gaps and delays in the implementation of genomic surveillance data in the public health sphere can prevent officials from formulating effective mitigation strategies to prevent outbreaks. In this review, we outline current SARS-CoV-2 global genomic surveillance methods and assess roadblocks at each step of the pipeline to identify potential solutions. Evaluating the current obstacles that impede effective surveillance can improve both global coordination efforts and pandemic preparedness for future outbreaks.