Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunoprecipitation captured transmembrane protease/ serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.Viruses exit from infected cells embedded with the energy required to enter new host cells. When viruses encounter new host cells, energy stored within metastable virus surface proteins is dissipated through protein refoldings and used to open the viruses and allow viral genomes to access the cell. This conversion from high-energy metastable to low-energy end stages is spatially and temporally regulated by a variety of triggers that are incorporated into the surface proteins. Depending on the virus, one or a combination of cell receptor bindings, protonations in the endosome, disulfide reductions, and proteolytic cleavages triggers viral protein refolding and opening. Insights into these activating conditions have advanced our understanding of virus-host interactions and have revealed new approaches for antiviral therapeutics.These activating virus entry events can be further dissected through research with the human CoVs (HCoVs). The HCoVs are notable pathogens (27, 48), with one of them accounting for severe acute respiratory syndrome (SARS) (12, 24). Evolution of the CoVs in their protruding surface or spike (S) proteins can change virus-activating conditions and permit zoonoses (30, 40) and virulence changes. Unraveling S protein activations is therefore central to understanding HCoV tropism, ecology, and pathogenesis.The S proteins include cell receptor-binding domains (RBDs) and virus-cell membrane fusion domains. Like other class I viral fusion proteins, the HCoV spikes require proteolytic priming to be activated (7). Notably, the majority of pathogenic HCoVs exit producer cells with unprimed S proteins (2, 34) and thus rely on target cell proteases for activation. Therefore, the HCoV cell entry factors on target cells include virus-binding agents (cell receptors) and also virus protein-cleaving agents (cell proteases).SARS-CoV binds to its ectopeptidase receptor, angiotensin-converting enzyme 2 (ACE2), with very high affinit...

had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Coronavirus-cell entry programs involve virus-cell membrane fusions mediated by viral spike (S) proteins. Coronavirus S proteins acquire membrane fusion competence by receptor interactions, proteolysis, and acidification in endosomes. This review describes our current understanding of the S proteins, their interactions with and their responses to these entry triggers. We focus on receptors and proteases in prompting entry and highlight the type II transmembrane serine proteases (TTSPs) known to activate several virus fusion proteins. These and other proteases are essential cofactors permitting coronavirus infection, conceivably being in proximity to cell-surface receptors and thus poised to split entering spike proteins into the fragments that refold to mediate membrane fusion. The review concludes by noting how understanding of coronavirus entry informs antiviral therapies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 180 million people since the onset of the pandemic. Despite similar viral load and infectivity rates between children and adults, children rarely develop severe illness. Differences in the host response to the virus at the primary infection site are among the mechanisms proposed to account for this disparity. Our objective was to investigate the host response to SARS-CoV-2 in the nasal mucosa in children and adults and compare it with the host response to respiratory syncytial virus (RSV) and influenza virus. We analyzed clinical outcomes and gene expression in the nasal mucosa of 36 children with SARS-CoV-2, 24 children with RSV, 9 children with influenza virus, 16 adults with SARS-CoV-2, and 7 healthy pediatric and 13 healthy adult controls. In both children and adults, infection with SARS-CoV-2 led to an IFN response in the nasal mucosa. The magnitude of the IFN response correlated with the abundance of viral reads, not the severity of illness, and was comparable between children and adults infected with SARS-CoV-2 and children with severe RSV infection. Expression of ACE2 and TMPRSS2 did not correlate with age or presence of viral infection. SARS-CoV-2–infected adults had increased expression of genes involved in neutrophil activation and T-cell receptor signaling pathways compared with SARS-CoV-2–infected children, despite similar severity of illness and viral reads. Age-related differences in the immune response to SARS-CoV-2 may place adults at increased risk of developing severe illness.

RationaleDespite similar viral load and infectivity rates between children and adults infected with SARS-CoV-2, children rarely develop severe illness. Differences in the host response to the virus at the primary infection site are among the proposed mechanisms.ObjectivesTo investigate the host response to SARS-CoV-2, respiratory syncytial virus (RSV), and influenza virus (IV) in the nasal mucosa in children and adults.MethodsClinical outcomes and gene expression in the nasal mucosa were analyzed in 36 children hospitalized with SARS-CoV-2 infection, 24 children with RSV infection, 9 children with IV infection, 16 adults with mild to moderate SARS-CoV-2 infection, and 7 healthy pediatric and 13 healthy adult controls.ResultsIn both children and adults, infection with SARS-CoV-2 leads to an interferon response in the nasal mucosa. The magnitude of the interferon response correlated with the abundance of viral reads and was comparable between symptomatic children and adults infected with SARS-CoV-2 and symptomatic children infected with RSV and IV. Cell type deconvolution identified an increased abundance of immune cells in the samples from children and adults with a viral infection. Expression of ACE2 and TMPRSS2 – key entry factors for SARS-CoV-2 – did not correlate with age or presence or absence of viral infection.ConclusionsOur findings support the hypothesis that differences in the immune response to SARS-CoV-2 determine disease severity, independent of viral load and interferon response at the primary infection primary site.

Background Recent COVID-19 surges are attributed to emergence of more transmissible SARS-CoV-2 variants of concern (VOCs). The relative severity of VOCs in children is unknown. Methods We performed a single-center retrospective cohort study of children ≤18 years old diagnosed with COVID-19 from October 2020–February 2022 and whose SARS-CoV-2 isolate underwent Illumina sequencing. We measured the frequency of five markers of COVID-19 severity. Logistic regression models were fitted to estimate the odds of each severity marker with each VOC. Results Among 714 children, 471 (66.0%) were infected with a VOC: 96 (13.4%) alpha, 38 (5.3%) gamma, 119 (16.7%) delta, and 215 (30.1%) omicron. High-risk medical conditions and increasing age were independently associated with COVID-19 severity. After adjusting for age, race, ethnicity, high-risk medical conditions, and COVID-19 community incidence, neither alpha, delta, nor omicron was associated with severe COVID-19. Gamma was independently associated with hospitalization (OR 6.7, 95% CI 2.0–22.1); pharmacologic treatment (OR 5.7, 95% CI 1.2–26.8); respiratory support (OR 11.9, 95% CI 2.7–62.4); and severe disease per the WHO Clinical Progression Scale (OR 11.7, 95% CI 2.1–90.5). Upon subgroup analyses, omicron was independently associated with ICU admission and severe disease per the WHO Clinical Progression Scale in children without SARS-CoV-2 immunization or prior COVID-19 infection. Conclusions Compared to non-VOC COVID-19, the gamma VOC was independently associated with increased COVID-19 severity, as was omicron in children without SARS-CoV-2 immunization or prior COVID-19 infection. SARS-CoV-2 vaccination and prior COVID-19 prevented severe outcomes during the omicron surge.

Background Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool. Methods In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at −80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing. Findings The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at −80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at −80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2. Interpretation This is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic. Funding National Institute of Allergy and Infect...

Tuberculosis remains a prevalent disease worldwide, with approximately 9 million cases diagnosed annually. The emergence of multidrug-resistant tuberculosis has proven to be a challenging international public health issue. In the United States, however, the incidence of tuberculosis has been decreasing since 1992. There were just over 9,500 reported cases in 2013, and almost 500 of those were in children younger than age 15 years. Foreign-born persons are a high-risk group and account for 65% of new cases annually. Other high-risk groups include ethnic minorities, HIV-infected patients, and people living in low-socioeconomic urban areas.