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The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72–8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates. [ABSTRACT FROM AUTHOR]

We report a silver nanoparticle (AgNP) sensor array to detect SARS-CoV-2 viral particles utilizing five peptide receptors to produce a colorimetric response. We show that the interaction of the virus particles with the peptides interferes with the shift in plasmonic absorbance and hinders the formation of snowflake-like fractal assemblies between the AgNPs and the bridging peptides. The limit of detection in water of the sensor array was 500,000 viral copies/mL. We demonstrate the capabilities of the sensor array to distinguish between SARS-CoV-2 (Beta, Delta, and Omicron variants) from the influenza virus at the 99% confidence level through linear discriminant analysis. The sensor array was also able to discriminate three out of five negative exhaled breath condensate samples from five positive SARS-CoV-2 EBC samples. [ABSTRACT FROM AUTHOR]

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major health problem worldwide. Due to the fast emergence of SARS-CoV-2 variants, understanding the molecular mechanisms of viral pathogenesis and developing novel inhibitors are essential and urgent. Here, we investigated the potential roles of N6,2′-O-dimethyladenosine (m6Am), one of the most abundant modifications of eukaryotic messenger ribonucleic acid (mRNAs), in SARS-CoV-2 infection of human cells. Using genome-wide m6Am-exo-seq, RNA sequencing analysis, and Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, we demonstrate that phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1), a cap-specific adenine N6 -methyltransferase, plays a major role in facilitating infection of primary human lung epithelial cells and cell lines by SARS-CoV-2, variants of concern, and other coronaviruses. We show that PCIF1 promotes infection by sustaining expression of the coronavirus receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) via m6Am-dependent mRNA stabilization. In PCIF1- depleted cells, both ACE2/TMPRSS2 expression and viral infection are rescued by re-expression of wild-type, but not catalytically inactive, PCIF1. These findings suggest a role for PCIF1 and cap m6Am in regulating SARS-CoV-2 susceptibility and identify a potential therapeutic target for prevention of infection. [ABSTRACT FROM AUTHOR]

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary. [ABSTRACT FROM AUTHOR]

The breakdown and release of hyaluronan (HA) from the extracellular matrix has been hypothesized to act as an endogenous signal of injury. To test this hypothesis, we generated mice that conditionally overexpressed human hyaluronidase 1 (HYAL1). Mice expressing HYAL1 in skin either during early development or by inducible transient expression exhibited extensive HA degradation, yet displayed no evidence of spontaneous inflammation. Further, HYAL1 expression activated migration and promoted loss of DCs from the skin. We subsequently determined that induction of HYAL1 expression prior to topical antigen application resulted in a lack of an antigenic response due to the depletion of DCs from the skin. In contrast, induction of HYAL1 expression concurrent with antigen exposure accelerated allergic sensitization. Administration of HA tetrasaccharides, before or simultaneously with antigen application, recapitulated phenotypes observed in HYAL1-expressing animals, suggesting that the generation of small HA fragments, rather than the loss of large HA molecules, promotes DC migration and subsequent modification of allergic responses. Furthermore, mice lacking TLR4 did not exhibit HA-associated phenotypes, indicating that TLR4 mediates these responses. This study provides direct evidence that HA breakdown controls the capacity of the skin to present antigen. These events may influence DC function in injury or disease and have potential to be exploited therapeutically for modification of allergic responses., Introduction The early inflammatory response to tissue injury has been proposed to involve recognition of components of damaged cells by pattern recognition receptors, such as TLRs (1). These dead cell [...]

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants. Author summary: Next generation SARS-CoV-2 vaccines need to address the transmission of new virus variants that continue to threaten global health and recovery. This may require new perspectives in immunogen design as well as immune response engineering. Here we applied these ideas in a two-step proof of concept study. First, we designed conformationally-constrained immunogens expressing key residues at the RBM: ACE2 interface to mimic their stereochemical orientation in the native Spike protein. Second, we initiated the process of B cell immunity (priming) using plasmid DNA injected intra-spleen to exploit the spatial organization of a secondary lymphoid organ. Mice given a single booster immunization with intact Spike protein developed a rapid memory antibody response against RBD and Spike proteins. Importantly, immune sera neutralized authentic WA1 virus and the B.1.351/Beta, B.1.617.2/Delta, and BA.1/Omicron variants of concern. Our findings demonstrate that immunogens built on structure selection and lymphoid organ targeting are a powerful way to focus the antibody response to a conserved site of vulnerability shared between wildtype virus and variants of concern. [ABSTRACT FROM AUTHOR]

We isolated a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2 variant from a person with coronavirus disease 2019 recrudescence after nirmatrelvir/ritonavir treatment. Antiviral sensitivity and neutralizing antibody testing were performed with both parental SARS-CoV-2 and multiple variants of concern. We found that neither nirmatrelvir resistance nor absence of neutralizing immunity was a likely cause of the recrudescence. [ABSTRACT FROM AUTHOR]

Group B Streptococcus (GBS) is classified into nine serotypes that vary in capsular polysaccharide (CPS) architecture but share in common the presence of a terminal sialic acid (Sia) residue. This position and linkage of GBS Sia closely resembles that of cell surface glycans found abundantly on human cells. CD33-related Siglecs (CD33rSiglecs) are a family of Sia-binding lectins expressed on host leukocytes that engage host Sia-capped glycans and send signals that dampen inflammatory gene activation. We hypothesized that GBS evolved to display CPS Sia as a form of molecular mimicry limiting the activation of an effective innate immune response. In this study, we applied a panel of immunologic and cell-based assays to demonstrate that GBS of several serotypes interacts in a Sia- and serotype-specific manner with certain human CD33rSiglecs, including hSiglec-9 and hSiglec-5 expressed on neutrophils and monocytes. Modification of GBS CPS Sia by O acetylation has recently been recognized, and we further show that the degree of O acetylation can markedly affect the interaction between GBS and hSiglec-5, -7, and -9. Thus, production of Sia-capped bacterial polysaccharide capsules that mimic human cell surface glycans in order to engage CD33rSiglecs may be an example of a previously unrecognized bacterial mechanism of leukocyte manipulation.

The main protease (Mpro) and papain‐like protease (PLpro) play critical roles in SARS‐CoV‐2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS‐CoV‐2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual‐color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro. 3MBP5‐activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid‐transfected HEK 293T cells, and SARS‐CoV‐2‐infected TMPRSS2‐Vero cells. Results from the dual‐color probe first verified the simultaneous detection and intracellular distribution of SARS‐CoV‐2 Mpro and PLpro. This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8–43) and a short cytoplasmic helix (residues 53–60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6–18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein. Author summary: The novel coronavirus SARS-CoV-2, the causative agent of the world-wide pandemic of COVID-19, has become one of the greatest threats to human health. While rapid progress has been made in the development of vaccines, drug discovery has lagged, partly due to the lack of atomic-resolution structures of the free and drug-bound forms of the viral proteins. The SARS-CoV-2 envelope (E) protein, with its multiple activities that contribute to viral replication, is widely regarded as a potential target for COVID-19 treatment. As structural information is essential for drug discovery, we established an efficient sample preparation system for biochemical and structural studies of intact full-length SARS-CoV-2 E protein and characterized its structure and dynamics. We also characterized the interactions of amilorides with specific E protein residues and correlated this with their antiviral activity during viral replication. The binding affinity of the amilorides to E protein correlated with their antiviral potency, suggesting that E protein is indeed the likely target of their antiviral activity. We found that residue asparagine15 plays an important role in maintaining the conformation of the amiloride binding site, providing molecular guidance for the design of inhibitors targeting E protein. [ABSTRACT FROM AUTHOR]

Coccidioides immitis and C. posadasii are dimorphic fungi that transform from mycelia with internal arthroconidia in the soil to a tissue form known as a spherule in mammals. This process can be recapitulated in vitro by increasing the temperature, CO2 and changing other culture conditions. In this study, we have analyzed changes in gene expression in mycelia and young and mature spherules. Genes that were highly upregulated in young spherules include a spherule surface protein and iron and copper membrane transporters. Genes that are unique to Coccidioides spp. are also overrepresented in this group, suggesting that they may be important for spherule differentiation. Enriched GO terms in young spherule upregulated genes include oxidation-reduction, response to stress and membrane proteins. Downregulated genes are enriched for transcription factors, especially helix–loop–helix and C2H2 type zinc finger domain-containing proteins, which is consistent with the dramatic change in transcriptional profile. Almost all genes that are upregulated in young spherules remain upregulated in mature spherules, but a small number of genes are differentially expressed in those two stages of spherule development. Mature spherules express more Hsp31 and amylase and less tyrosinase than young spherules. Some expression of transposons was detected and most of the differentially expressed transposons were upregulated in spherules. [ABSTRACT FROM AUTHOR]

An 83‐year‐old female had asymptomatic SARS‐CoV‐2 infection while taking ruxolitinib. She remained RT‐PCR positive for viral RNA for >120 days, and Pegylated interferon for 4 weeks led to viral RNA clearance. The observations support combination therapy of ruxolitinib + interferon for COVID‐19. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID‐19) is caused by SARS‐CoV‐2 and has spread across the globe. SARS‐CoV‐2 is a highly infectious virus with no vaccine or antiviral therapy available to control the pandemic; therefore, it is crucial to understand the mechanisms of viral pathogenesis and the host immune responses to SARS‐CoV‐2. SARS‐CoV‐2 is a new member of the betacoronavirus genus like other closely related viruses including SARS‐CoV and Middle East respiratory syndrome coronavirus (MERS‐CoV). Both SARS‐CoV and MERS‐CoV have caused serious outbreaks and epidemics in the past eighteen years. Here, we report that one of the interferon‐stimulated genes (ISGs), cholesterol 25‐hydroxylase (CH25H), is induced by SARS‐CoV‐2 infection in vitro and in COVID‐19‐infected patients. CH25H converts cholesterol to 25‐hydrocholesterol (25HC) and 25HC shows broad anti‐coronavirus activity by blocking membrane fusion. Furthermore, 25HC inhibits USA‐WA1/2020 SARS‐CoV‐2 infection in lung epithelial cells and viral entry in human lung organoids. Mechanistically, 25HC inhibits viral membrane fusion by activating the ER‐localized acyl‐CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane. Altogether, our results shed light on a potentially broad antiviral mechanism by 25HC through depleting accessible cholesterol on the plasma membrane to suppress virus–cell fusion. Since 25HC is a natural product with no known toxicity at effective concentrations, it provides a potential therapeutic candidate for COVID‐19 and emerging viral diseases in the future. Synopsis: 25‐Hydroxycholesterol, produced by the interferon‐stimulated protein CH25H, inhibits the entry of SARS‐CoV‐2, SARS‐CoV, and MERS‐CoV. Mechanistically, 25HC activates the ER‐localized enzyme ACAT, whose activity depletes accessible cholesterol on the plasma membrane and restricts viral membrane fusion. An interferon‐stimulated gene, CH25H is induced in SARS‐CoV-2 infection in vitro and in vivo.25HC exhibits broad anti‐coronavirus activity by blocking membrane fusion.25HC activates ACAT to induce internalization of accessible cholesterol on the plasma membrane and restricts viral fusion.25HC inhibits USA‐WA1/2020 SARS‐CoV-2 infection in lung epithelial cells and virus entry in human lung organoids. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus linked to devastating neurologic diseases. Immune responses to flaviviruses may be pathogenic or protective. Our understanding of human immune responses to ZIKV in vivo remains limited. Therefore, we performed a longitudinal molecular and phenotypic characterization of innate and adaptive immune responses during an acute ZIKV infection. We found that innate immune transcriptional and genomic responses were both cell type- and time-dependent. While interferon stimulated gene induction was common to all innate immune cells, the upregulation of important inflammatory cytokine genes was primarily limited to monocyte subsets. Additionally, genomic analysis revealed substantial chromatin remodeling at sites containing cell-type specific transcription factor binding motifs that may explain the observed changes in gene expression. In this dengue virus-experienced individual, adaptive immune responses were rapidly mobilized with T cell transcriptional activity and ZIKV neutralizing antibody responses peaking 6 days after the onset of symptoms. Collectively this study characterizes the development and resolution of an in vivo human immune response to acute ZIKV infection in an individual with pre-existing flavivirus immunity. [ABSTRACT FROM AUTHOR]

Summary Interferon-regulatory factors (IRFs) are a family of transcription factors (TFs) that translate viral recognition into antiviral responses, including type I interferon (IFN) production. Dengue virus (DENV) and other clinically important flaviviruses are suppressed by type I IFN. While mice lacking the type I IFN receptor ( Ifnar1 −/− ) succumb to DENV infection, we found that mice deficient in three transcription factors controlling type I IFN production ( Irf3 −/− Irf5 −/− Irf7 −/− triple knockout [TKO]) survive DENV challenge. DENV infection of TKO mice resulted in minimal type I IFN production but a robust type II IFN (IFN-γ) response. Using loss-of-function approaches for various molecules, we demonstrate that the IRF-3-, IRF-5-, IRF-7-independent pathway predominantly utilizes IFN-γ and, to a lesser degree, type I IFNs. This pathway signals via IRF-1 to stimulate interleukin-12 (IL-12) production and IFN-γ response. These results reveal a key antiviral role for IRF-1 by activating both type I and II IFN responses during DENV infection. [ABSTRACT FROM AUTHOR]

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/ eIF2/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity. [ABSTRACT FROM AUTHOR]

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N 6 -methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3′ end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment. [Display omitted] • SARS-CoV-2 virus genome contains m6A modifications enriched in the 3′ end region of RNA • RIG-I binds to SARS-CoV-2 viral RNAs, which is inhibited by m6A RNA modifications • METLL3 regulates SARS-CoV-2 RNA m6A levels and activation of inflammation pathways • METTL3 is reduced and inflammatory genes are induced in severe COVID-19 patients Li et al. show that SARS-CoV-2 RNA contains N6 -methylation of adenosine (m6A) modifications enriched at the 3′ end that play roles in evading host immune responses to viral infection. Host cell m6A methyltransferase METTL3 adds m6A modifications in SARS-CoV-2 RNA, leading to decreased RIG-I binding and subsequently dampening the sensing and activation of innate immune responses. [ABSTRACT FROM AUTHOR]

Significant barriers to the diagnosis of latent and acute SARS-CoV-2 infection continue to hamper population-based screening efforts required to contain the COVID-19 pandemic in the absence of widely available antiviral therapeutics or vaccines. We report an aptamer-based SARS-CoV-2 salivary antigen assay employing only low-cost reagents ($3.20/test) and an off-the-shelf glucometer. The test was engineered around a glucometer as it is quantitative, easy to use, and the most prevalent piece of diagnostic equipment globally, making the test highly scalable with an infrastructure that is already in place. Furthermore, many glucometers connect to smartphones, providing an opportunity to integrate with contact tracing apps, medical providers, and electronic health records. In clinical testing, the developed assay detected SARS-CoV-2 infection in patient saliva across a range of viral loads - as benchmarked by RT-qPCR - within 1 h, with 100% sensitivity (positive percent agreement) and distinguished infected specimens from off-target antigens in uninfected controls with 100% specificity (negative percent agreement). We propose that this approach provides an inexpensive, rapid, and accurate diagnostic for distributed screening of SARS-CoV-2 infection at scale. • Detection of SARS-CoV-2 antigen in human saliva using an off-the-shelf glucometer. • Aptamer-based assay with engineered affinity and enzymatic amplification. • Widely prevalent read out device (glucometer) with low-cost reagents ($3.20/test). • Picomolar limit-of-detection with minimal cross-reactivity to other viruses. • Validated against clinical samples with 100% concordance to RT-qPCR. [ABSTRACT FROM AUTHOR]

All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specificCmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gcpreferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens. [ABSTRACT FROM AUTHOR]

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) was enhanced by CTP and Mg2+, the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac2 followed by CMP activation of Neu5Ac or activation of Neu5,9Ac2 followed by de-O-acetylation of CMP-Neu5,9Ac2. Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity. [ABSTRACT FROM AUTHOR]

Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%–27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in ∼40%–60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2. • Measuring immunity to SARS-CoV-2 is key for understanding COVID-19 and vaccine development • Epitope pools detect CD4+ and CD8+ T cells in 100% and 70% of convalescent COVID patients • T cell responses are focused not only on spike but also on M, N, and other ORFs • T cell reactivity to SARS-CoV-2 epitopes is also detected in non-exposed individuals An analysis of immune cell responses to SARS-CoV-2 from recovered patients identifies the regions of the virus that is targeted and also reveals cross-reactivity with other common circulating coronaviruses [ABSTRACT FROM AUTHOR]

The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic., Competing Interests: Competing interests statement:C.C. is an inventor on a patent titled ‘‘Cancer diagnostics, therapeutics, and drug discovery associated with macropinocytosis,’’ Pub. No.: US 2018/0335420 A1.

Diagnostic approaches that combine the high sensitivity and specificity of laboratory-based digital detection with the ease of use and affordability of point-of-care (POC) technologies could revolutionize disease diagnostics. This is especially true in infectious disease diagnostics, where rapid and accurate pathogen detection is critical to curbing the spread of disease. We have pioneered an innovative label-free digital detection platform that utilizes Interferometric Reflectance Imaging Sensor (IRIS) technology. IRIS leverages light interference from an optically transparent thin film, eliminating the need for complex optical resonances to enhance the signal by harnessing light interference and the power of signal averaging in shot-noise-limited operation to achieve virtually unlimited sensitivity. In our latest work, we have further improved our previous 'Single-Particle' IRIS (SP-IRIS) technology by allowing the construction of the optical signature of target nanoparticles (whole virus) from a single image. This new platform, 'Pixel-Diversity' IRIS (PD-IRIS), eliminated the need for z-scan acquisition, required in SP-IRIS, a time-consuming and expensive process, and made our technology more applicable to POC settings. Using PD-IRIS, we quantitatively detected the Monkeypox virus (MPXV), the etiological agent for Monkeypox (Mpox) infection. MPXV was captured by anti-A29 monoclonal antibody (mAb 69-126-3) on Protein G spots on the sensor chips and were detected at a limit-of-detection (LOD) - of 200 PFU/ml (~3.3 attomolar). PD-IRIS was superior to the laboratory-based ELISA (LOD - 1800 PFU/mL) used as a comparator. The specificity of PD-IRIS in MPXV detection was demonstrated using Herpes simplex virus, type 1 (HSV-1), and Cowpox virus (CPXV). This work establishes the effectiveness of PD-IRIS and opens possibilities for its advancement in clinical diagnostics of Mpox at POC. Moreover, PD-IRIS is a modular technology that can be adapted for the multiplex detection of pathogens for which high-affinity ligands are available that can bind their surface antigens to capture them on the sensor surface.