Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, highmannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.Rotaviruses (RV) are the leading viral etiologic agents of severe pediatric gastroenteritis worldwide, affecting nearly all children before the age of 5, with 2 million cases resulting in 444,000 deaths annually (33,34,40). RV nonstructural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum (ER) transmembrane glycoprotein due to the protein's high-mannose glycosylation and its critical function as an intracellular receptor for the translocation of subviral particles into the ER during virion morphogenesis (2,5,14). However, the identification of NSP4 and NSP4 amino acids (aa) 114 to 135 (NSP4 114-135 ) as enterotoxic and the redistribution of RVencoded proteins upon NSP4 silencing led to a reevaluation of NSP4 function(s) and subcellular localization(s) (4, 31).A cleaved NSP4 fragment, aa 112 to 175, is secreted from RV-infected epithelial cells, indicating that some portion of NSP4 traffics from the ER to the plasma membrane (PM) (65). The colocalization of NSP4 114-135 and the extracellular matrix proteins laminin-3 and fibronectin at the basement membrane of small-intestinal epithelia from RV strain EDIM-infected mouse pups also supports NSP4 transport to the PM during host infection (8). While both findings demonstrat...
Introduction The coronavirus disease 2019 (COVID-19) pandemic is a global public health emergency causing a disparate burden of death and disability around the world. The molecular characteristics of the virus that predict better or worse outcome are largely still being discovered. Methods We downloaded 155,958 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from GISAID. Of these genomes, 3,637 samples included useable metadata on patient outcomes. Using this subset, we evaluated whether SARS-CoV-2 viral genomic variants improved prediction of reported severity beyond age and region. First, we established whether including genomic variants as model features meaningfully increased predictive power of our model. Next, we evaluated specific variants in order to determine the magnitude of association with severity and the frequency of these variants among SARS-CoV-2 genomes. Results Logistic regression models that included viral genomic variants outperformed other models (AUC = 0.91 as compared with 0.68 for age and gender alone; p < 0.001). Among individual variants, we found 17 single nucleotide variants in SARS-CoV-2 have more than two-fold greater odds of being associated with higher severity and 67 variants associated with ≤0.5 times the odds of severity. The median frequency of associated variants was 0.15% (interquartile range 0.09%-0.45%). Altogether 85% of genomes had at least one variant associated with patient outcome. Conclusion Numerous SARS-CoV-2 variants have two-fold or greater association with odds of mild or severe outcome and collectively, these variants are common. In addition to comprehensive mitigation efforts, public health measures should be prioritized to control the more severe manifestations of COVID-19 and the transmission chains linked to these severe cases. Lay Summary This study explores which, if any, SARS-CoV-2 viral genomic variants are associated with mild or severe COVID-19 patient outcomes. Our results suggest that there are common genomic variants in SARS-CoV-2 that are more often associated with negative patient outcomes, which may impact downstream public health measures.
BackgroundRotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types.MethodsTransport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab)2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls.ResultsOnly full-length (FL), endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells.ConclusionsThe intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study.
Clinicians frequently use influenza rapid antigen tests for diagnostic testing. We tested nasal wash samples from 1 April to 7 June 2009 from 1538 patients using the QuickVue Influenza A+B (Quidel) rapid influenza antigen test and compared the results with real-time reverse transcription polymerase chain reaction (rRT-PCR) assay (gold standard). The prevalence of 2009 pandemic influenza A (pH1N1) was 1.98%, seasonal influenza type A .87%, and seasonal influenza type B 2.07%. The sensitivity and specificity of the rapid test for pH1N1 was 20% (95% CI, 8-39) and 99% (95% CI, 98-99), for seasonal influenza type A 15% (95% CI, 2-45) and 99% (95% CI, 98-99), and for influenza type B was 31% (95% CI, 9-61) and 99% (95% CI, 98-99.7). Rapid influenza antigen tests were of limited use at a time when the prevalence of pH1N1 and seasonal influenza in the United States was low. Clinicians should instead rely on clinical impression and laboratory diagnosis by rRT-PCR.
The Armed Forces Health Surveillance Center’s Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) supports and oversees surveillance for emerging infectious diseases, including respiratory diseases, of importance to the U.S. Department of Defense (DoD). AFHSC-GEIS accomplishes this mission by providing funding and oversight to a global network of partners for respiratory disease surveillance. This report details the system’s surveillance activities during 2009, with a focus on efforts in responding to the novel H1N1 Influenza A (A/H1N1) pandemic and contributions to global public health. Active surveillance networks established by AFHSC-GEIS partners resulted in the initial detection of novel A/H1N1 influenza in the U.S. and several other countries, and viruses isolated from these activities were used as seed strains for the 2009 pandemic influenza vaccine. Partners also provided diagnostic laboratory training and capacity building to host nations to assist with the novel A/H1N1 pandemic global response, adapted a Food and Drug Administration-approved assay for use on a ruggedized polymerase chain reaction platform for diagnosing novel A/H1N1 in remote settings, and provided estimates of seasonal vaccine effectiveness against novel A/H1N1 illness. Regular reporting of the system’s worldwide surveillance findings to the global public health community enabled leaders to make informed decisions on disease mitigation measures and controls for the 2009 A/H1N1 influenza pandemic. AFHSC-GEIS’s support of a global network contributes to DoD’s force health protection, while supporting global public health.
Genomic surveillance empowers agile responses to SARS-CoV-2 by enabling scientists and public health analysts to issue recommendations aimed at slowing transmission, prioritizing contact tracing, and building a robust genomic sequencing surveillance strategy. Since the start of the pandemic, real time RT-PCR diagnostic testing from upper respiratory specimens, such as nasopharyngeal (NP) swabs, has been the standard. Moreover, respiratory samples in viral transport media are the ideal specimen for SARS-CoV-2 whole-genome sequencing (WGS). In early 2021, many clinicians transitioned to antigen-based SARS-CoV-2 detection tests, which use anterior nasal swabs for SARS-CoV-2 antigen detection. Despite this shift in testing methods, the need for whole-genome sequence surveillance remains. Thus, we developed a workflow for whole-genome sequencing with antigen test-derived swabs as an input rather than nasopharyngeal swabs. In this study, we use excess clinical specimens processed using the BinaxNOW™ COVID-19 Ag Card. We demonstrate that whole-genome sequencing from antigen tests is feasible and yields similar results from RT-PCR-based assays utilizing a swab in viral transport media.
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