Genomic sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely instituted during the coronavirus disease 2019 (COVID-19) pandemic to track the emergence and spread of new lineages. While this powerful tool can influence public health decisions and therapeutic development, not all regions of the world have had equal access to sequencing capacity, affecting surveillance. One such underrepresented region is the Caribbean islands, including the Dominican Republic (DR). To determine retrospectively what lineages were circulating in the DR in the first year of the pandemic, when there were strict travel restrictions imposed, we sequenced SARS-CoV-2 from nasal swab and saliva samples collected between July 2020 and February 2021. We investigated whether COVID-19 outbreaks were seeded by single or multiple introductions and established epidemiological linkages to other countries. Using 98 newly sequenced samples, we identified 16 SARS-CoV-2 lineages in the DR, indicating many independent introductions from diverse geographic areas. Further, we show that analyzing both globally prevalent and globally rare lineages within the DR highlights different aspects of international disease transmission.IMPORTANCEThis study uses genome sequencing of severe acute respiratory syndrome coronavirus 2 samples collected in an undersampled region of the world-the Caribbean, specifically the Dominican Republic-to make novel inferences about the dynamics of disease spread during a period of the coronavirus disease 2019 pandemic when many diverse lineages were co-circulating globally.

Pathogen sequencing during the COVID-19 pandemic has generated more whole genome sequencing data than for any other epidemic, allowing epidemiologists to monitor the transmission and evolution of SARS-CoV-2. However, large parts of the world are heavily underrepresented in sequencing efforts, including the Caribbean islands. We performed genome sequencing of SARS-CoV-2 from upper respiratory tract samples collected in Haiti during the spring of 2020. We used phylogenetic analysis to assess the pandemic dynamics in the Caribbean region and observed that the epidemic in Haiti was seeded by multiple introductions, primarily from the United States. We identified the emergence of a SARS-CoV-2 lineage (B.1.478) from Haiti that spread into North America, as well as evidence of the undocumented spread of SARS-CoV-2 within the Caribbean. We demonstrate that the genomic analysis of a relatively modest number of samples from a severely under-sampled region can provide new insight on a previously unobserved spread of a specific lineage, demonstrating the importance of geographically widespread genomic epidemiology.

Abstract Background: Children (<18 years old) were not initially considered significant sources of infection (SOIs) for SARS-CoV-2. Risk mitigation strategies were thus prioritized for adults, and vaccination was inaccessible for children until mid-2021. Emergence of novel variants led to significant increases in COVID-19 cases in both children and adults. Whether these emergence events and increased vulnerability of unvaccinated children had a synergistic effect resulting in increased caseloads in adults requires further exploration. Methods: A retrospective cohort study was conducted among 3,545 workers diagnosed with COVID-19. Case details were compiled during contact investigations. Variants of concern were identified following sequencing of biological samples collected through employer-based testing programs. Logistic regression was performed to compare the odds of having a child SOI based on the dominant variant in the workforce. Results: One-fourth (24.5%) of the cohort reported having a child in-residence; 11.2% identified a child as their SOI. In Alpha-dominant months, the odds of having a child SOI were 0.3, and the child SOI was likely older (5-17 years old). The odds of having a child SOI increased to 1.3 and 2.2 in Delta- and Omicron-dominant months, respectively. The odds of having younger child SOIs (<5 years old) were significantly higher in Omicron-dominant months. Conclusions: Children were highly likely to acquire the virus and posed a significant risk of transmission to their adult caretakers during Delta- and Omicron-dominant months. Without proper mitigation strategies in both the home and the workplace, child-associated transmission can threaten operations in the forms of staff shortages.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis work was supported in part by the Division of Intramural Research (DIR) of the NIAID/NIH### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Analysis was conducted using deidentified data that were collected for public health purposes and determined as exempted from IRB as assessed by both National Institutes of Health IRB and George Washington University IRB.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

AbstractBackgroundNirmatrelvir/ritonavir, the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease inhibitor, reduces the risk of hospitalization and death by coronavirus disease 2019 (COVID-19) but has been associated with symptomatic rebound after therapy completion.MethodsSix individuals with relapse of COVID-19 symptoms after treatment with nirmatrelvir/ritonavir, 2 individuals with rebound symptoms without prior antiviral therapy and 7 patients with acute Omicron infection (controls) were studied. Soluble biomarkers and serum SARS-CoV-2 nucleocapsid protein were measured. Nasal swabs positive for SARS-CoV-2 underwent viral isolation and targeted viral sequencing. SARS-CoV-2 anti-spike, anti–receptor-binding domain, and anti-nucleocapsid antibodies were measured. Surrogate viral neutralization tests against wild-type and Omicron spike protein, as well as T-cell stimulation assays, were performed.ResultsHigh levels of SARS-CoV-2 anti-spike immunoglobulin G (IgG) antibodies were found in all participants. Anti-nucleocapsid IgG and Omicron-specific neutralizing antibodies increased in patients with rebound. Robust SARS-CoV-2–specific T-cell responses were observed, higher in rebound compared with early acute COVID-19 patients. Inflammatory markers mostly decreased during rebound. Two patients sampled longitudinally demonstrated an increase in activated cytokine-producing CD4+ T cells against viral proteins. No characteristic resistance mutations were identified. SARS-CoV-2 was isolated by culture from 1 of 8 rebound patients; Polybrene addition increased this to 5 of 8.ConclusionsNirmatrelvir/ritonavir treatment does not impede adaptive immune responses to SARS-CoV-2. Clinical rebound corresponds to development of a robust antibody and T-cell immune response, arguing against a high risk of disease progression. The presence of infectious virus supports the need for isolation and assessment of longer treatment courses.Clinical trials registrationNCT04401436.

Although the risk of SARS-CoV-2 transmission through lung transplantation from acutely infected donors is high, the risks of virus transmission and long-term lung allograft outcomes are not as well described when using pul-monary organs from COVID-19-recovered donors. We describe successful lung transplantation for a COVID-19-related lung injury using lungs from a COVID-19-recovered donor who was retrospectively found to have detectable genomic SARS-CoV-2 RNA in the lung tissue by multiple highly sensitive assays. However, SARS-CoV-2 subgenomic RNA (sgRNA), a marker of viral replication, was not detectable in the donor respiratory tissues. One year after lung transplantation, the recipient has a good functional status, walking 1 mile several times per week without the need for supplemental oxygen and without any evidence of donor-derived SARS-CoV-2 transmission. Our findings highlight the limitations of current clinical laboratory diagnostic assays in detecting the persistence of SARS-CoV-2 RNA in the lung tissue. The persistence of SARS-CoV-2 RNA in the donor tissue did not appear to represent active viral replication via sgRNA testing and, most importantly, did not negatively impact the allograft outcome in the first year after lung transplantation. sgRNA is easily performed and may be a useful assay for assessing viral infectivity in organs from donors with a recent infection.

Clinical rebound of COVID-19 after nirmatrelvir/ritonavir treatment has been reported. We performed clinical, virologic, and immune measurements in seven patients with symptomatic rebound, six after nirmatrelvir/ritonavir treatment and one without previous treatment. There was no evidence of severe disease or impaired antibody and T-cell responses in people with rebound symptoms.

B-cell-depleting therapies may lead to prolonged disease and viral shedding in individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and this viral persistence raises concern for viral evolution. We report sequencing of early and late samples from a 335-day infection in an immunocompromised patient. The virus accumulated a unique deletion in the amino-terminal domain of the spike protein, and complete deletion of ORF7b and ORF8, the first report of its kind in an immunocompromised patient. Unique viral mutations found in this study highlight the importance of analyzing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts.

Rapid identification of newly emerging or circulating viruses is an important first step toward managing the public health response to potential outbreaks. A portable virus capture device, coupled with label-free Raman spectroscopy, holds the promise of fast detection by rapidly obtaining the Raman signature of a virus followed by a machine learning (ML) approach applied to recognize the virus based on its Raman spectrum, which is used as a fingerprint. We present such an ML approach for analyzing Raman spectra of human and avian viruses. A convolutional neural network (CNN) classifier specifically designed for spectral data achieves very high accuracy for a variety of virus type or subtype identification tasks. In particular, it achieves 99% accuracy for classifying influenza virus type A versus type B, 96% accuracy for classifying four subtypes of influenza A, 95% accuracy for differentiating enveloped and nonenveloped viruses, and 99% accuracy for differentiating avian coronavirus (infectious bronchitis virus [IBV]) from other avian viruses. Furthermore, interpretation of neural net responses in the trained CNN model using a full-gradient algorithm highlights Raman spectral ranges that are most important to virus identification. By correlating ML-selected salient Raman ranges with the signature ranges of known biomolecules and chemical functional groups—for example, amide, amino acid, and carboxylic acid—we verify that our ML model effectively recognizes the Raman signatures of proteins, lipids, and other vital functional groups present in different viruses and uses a weighted combination of these signatures to identify viruses.

• Mesa Accula is not impacted by 28881 GGG > AAC mutations, despite FDA warning alert. • Mesa Accula can detect new viral variants B.1.1.7, R.1, P. 2, B.1.526 and B. 1. 351. • Continuous evaluation of SARS-CoV-2 variants' impact on diagnostic tests is needed.

Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.

The infectious virion of hepatitis C virus (HCV) is made up of the viral nucleocapsid surrounded by an envelope that contains an ER-derived membrane bilayer, cellular lipids, and the viral E1 and E2 glycoproteins. Because the infectious HCV particle contains both protein and lipid layers, selective disruption of these layers and analysis for the presence or absence of resulting virion components can be used to study the virion assembly process. This chapter describes an experimental method to measure HCV virion envelopment, which can reveal the mechanisms of how specific viral protein-protein interactions and host factors contribute to the process of HCV envelopment.

Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.

ABSTRACT The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly. IMPORTANCE Hepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection.