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Exploring and accurately detecting new adulteration markers in sesame oil is an important measure for sesame oil adulteration monitoring. In this study, two endogenous flavors sulfurol and γ-nonalactone which can be used as potential adulteration markers were first discovered in sesame oil and accurately quantified. First, the two endogenous flavors were discovered using gas chromatography-mass spectrometry (GC-MS), and their structures were confirmed by comparing the mass spectrograms with the NIST spectral library. Then the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using direct methanol extraction pretreatment and vanillin-D3 as an internal standard was developed for rapid quantitation and application. The method was successfully validated with recoveries ranging from 88.5% to 102.2% and relative standard deviations between 2.6% and 10.5% ( n = 6). The combined method of GC-MS and LC-MS/MS was indicated to be efficient and highly sensitive for detection of sulfurol and γ-nonalactone in edible oil. Subsequently, 31 sesame oils from the market were detected, revealing that 31 samples contained the identified flavors within a relatively consistent range. However, the concentration of these flavor substances in one sample was abnormally high, indicating that there was a potential risk of adulteration. Therefore, the developed method shows good potential for quality evaluation and adulteration screening of sesame oil.

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M-K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology., Competing Interests: Declaration of interest The authors declare no competing interests.

Dysregulated host immune responses contribute to disease severity and worsened prognosis in COVID-19 infection and the underlying mechanisms are not fully understood. In this study, we observed that IL-33, a damage-associated molecular pattern molecule, is significantly increased in COVID-19 patients and in SARS-CoV-2-infected mice. Using IL-33 -/- mice, we demonstrated that IL-33 deficiency resulted in significant decreases in bodyweight loss, tissue viral burdens, and lung pathology. These improved outcomes in IL-33 -/- mice also correlated with a reduction in innate immune cell infiltrates, i.e., neutrophils, macrophages, natural killer cells, and activated T cells in inflamed lungs. Lung RNA-seq results revealed that IL-33 signaling enhances activation of inflammatory pathways, including interferon signaling, pathogen phagocytosis, macrophage activation, and cytokine/chemokine signals. Overall, these findings demonstrate that the alarmin IL-33 plays a pathogenic role in SARS-CoV-2 infection and provides new insights that will inform the development of effective therapeutic strategies for COVID-19., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. In the brain, no detectable viral RNA and minimal residential immune cell activation was observed in the surviving mice post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and gene expression levels associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent systemic T helper 1 prone cellular immune responses and strong sera neutralizing antibodies against Delta and Omicron variants months post-acute infection. Overall, our findings suggest that infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long-COVID patients and provides new insights into the role of systemic and brain residential immune factors in PASC pathogenesis., Competing Interests: The authors declare the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Singh, Adam, Aditi, Peng, Yu, Zou, Kulkarni, Kan, Jiang, Shi, Samir, Cisneros and Wang.)

Drug resistance poses a significant challenge in the development of effective therapies against SARS-CoV-2. Here, we identified two double mutations, M49K/M165V and M49K/S301P, in the 3C-like protease (3CLpro) that confer resistance to a novel non-covalent inhibitor, WU-04, which is currently in phase III clinical trials (NCT06197217). Crystallographic analysis indicates that the M49K mutation destabilizes the WU-04-binding pocket, impacting the binding of WU-04 more significantly than the binding of 3CLpro substrates. The M165V mutation directly interferes with WU-04 binding. The S301P mutation, which is far from the WU-04-binding pocket, indirectly affects WU-04 binding by restricting the rotation of 3CLpro's C-terminal tail and impeding 3CLpro dimerization. We further explored 3CLpro mutations that confer resistance to two clinically used inhibitors: ensitrelvir and nirmatrelvir, and revealed a trade-off between the catalytic activity, thermostability, and drug resistance of 3CLpro. We found that mutations at the same residue (M49) can have distinct effects on the 3CLpro inhibitors, highlighting the importance of developing multiple antiviral agents with different skeletons for fighting SARS-CoV-2. These findings enhance our understanding of SARS-CoV-2 resistance mechanisms and inform the development of effective therapeutics., (© 2024. The Author(s).)

Host anti-viral factors are essential for controlling SARS-CoV-2 infection but remain largely unknown due to the biases of previous large-scale studies toward pro-viral host factors. To fill in this knowledge gap, we perform a genome-wide CRISPR dropout screen and integrate analyses of the multi-omics data of the CRISPR screen, genome-wide association studies, single-cell RNA-Seq, and host-virus proteins or protein/RNA interactome. This study uncovers many host factors that are currently underappreciated, including the components of V-ATPases, ESCRT, and N-glycosylation pathways that modulate viral entry and/or replication. The cohesin complex is also identified as an anti-viral pathway, suggesting an important role of three-dimensional chromatin organization in mediating host-viral interaction. Furthermore, we discover another anti-viral regulator KLF5, a transcriptional factor involved in sphingolipid metabolism, which is up-regulated, and harbors genetic variations linked to COVID-19 patients with severe symptoms. Anti-viral effects of three identified candidates (DAZAP2/VTA1/KLF5) are confirmed individually. Molecular characterization of DAZAP2/VTA1/KLF5-knockout cells highlights the involvement of genes related to the coagulation system in determining the severity of COVID-19. Together, our results provide further resources for understanding the host anti-viral network during SARS-CoV-2 infection and may help develop new countermeasure strategies., (© 2024. The Author(s).)

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has provided unprecedented insight into mutations enabling immune escape. Understanding how these mutations affect the dynamics of antibody-antigen interactions is crucial to the development of broadly protective antibodies and vaccines. Here we report the characterization of a potent neutralizing antibody (N3-1) identified from a COVID-19 patient during the first disease wave. Cryogenic electron microscopy revealed a quaternary binding mode that enables direct interactions with all three receptor-binding domains of the spike protein trimer, resulting in extraordinary avidity and potent neutralization of all major variants of concern until the emergence of Omicron. Structure-based rational design of N3-1 mutants improved binding to all Omicron variants but only partially restored neutralization of the conformationally distinct Omicron BA.1. This study provides new insights into immune evasion through changes in spike protein dynamics and highlights considerations for future conformationally biased multivalent vaccine designs., (© 2023. The Author(s).)

Background: Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication., Results: Here, we use proximity ligation sequencing to identify virus-host RNA interactions for four strains of Zika virus (ZIKV) and one strain of dengue virus (DENV-1) in human cells. We find hundreds of coding and non-coding RNAs that bind to DENV and ZIKV viruses. Host RNAs tend to bind to single-stranded regions along the virus genomes according to hybridization energetics. Compared to SARS-CoV-2 interactors, ZIKV-interacting host RNAs tend to be downregulated upon virus infection. Knockdown of several short non-coding RNAs, including miR19a-3p, and 7SK RNA results in a decrease in viral replication, suggesting that they act as virus-permissive factors. In addition, the 3'UTR of DYNLT1 mRNA acts as a virus-restrictive factor by binding to the conserved dumbbell region on DENV and ZIKV 3'UTR to decrease virus replication. We also identify a conserved set of host RNAs that interacts with DENV, ZIKV, and SARS-CoV-2, suggesting that these RNAs are broadly important for RNA virus infection., Conclusions: This study demonstrates that host RNAs can impact virus replication in permissive and restrictive ways, expanding our understanding of host factors and RNA-based gene regulation during viral pathogenesis., (© 2023. The Author(s).)

The highly mutated BA.2.86, with over 30 spike protein mutations in comparison to Omicron BA.2 and XBB.1.5 variants, has raised concerns about its potential to evade COVID-19 vaccination or prior SARS-CoV-2 infection-elicited immunity. In this study, we employ a live SARS-CoV-2 neutralization assay to compare the neutralization evasion ability of BA.2.86 with other emerged SARS-CoV-2 subvariants, including BA.2-derived CH.1.1, Delta-Omicron recombinant XBC.1.6, and XBB descendants XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1 and FL.1.5.1. Our results show that BA.2.86 is less neutralization evasive than XBB sublineages. XBB descendants XBB.1.16, EG.5.1, and FL.1.5.1 continue to significantly evade neutralization induced by the parental COVID-19 mRNA vaccine and a BA.5 Bivalent booster. Notably, when compared to XBB.1.5, the more recent XBB descendants, particularly EG.5.1, display increased resistance to neutralization. Among all the tested variants, CH.1.1 exhibits the greatest neutralization evasion. In contrast, XBC.1.6 shows a slight reduction but remains comparably sensitive to neutralization when compared to BA.5. Furthermore, a recent XBB.1.5-breakthrough infection significantly enhances the breadth and potency of cross-neutralization. These findings reinforce the expectation that the upcoming XBB.1.5 mRNA vaccine would likely boost the neutralization of currently circulating variants, while also underscoring the critical importance of ongoing surveillance to monitor the evolution and immune evasion potential of SARS-CoV-2 variants.

The rapid evolution of SARS-CoV-2 Omicron sublineages mandates a better understanding of viral replication and cross-neutralization among these sublineages. Here we used K18-hACE2 mice and primary human airway cultures to examine the viral fitness and antigenic relationship among Omicron sublineages. In both K18-hACE2 mice and human airway cultures, Omicron sublineages exhibited a replication order of BA.5 ≥ BA.2 ≥ BA.2.12.1 > BA.1; no difference in body weight loss was observed among different sublineage-infected mice. The BA.1-, BA.2-, BA.2.12.1-, and BA.5-infected mice developed distinguishable cross-neutralizations against Omicron sublineages, but exhibited little neutralization against the index virus (i.e. USA-WA1/2020) or the Delta variant. Surprisingly, the BA.5-infected mice developed higher neutralization activity against heterologous BA.2 and BA.2.12.1 than that against homologous BA.5; serum neutralizing titres did not always correlate with viral replication levels in infected animals. Our results revealed a distinct antigenic cartography of Omicron sublineages and support the bivalent vaccine approach.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve after its emergence. Given its importance in viral infection and vaccine development, mutations in the viral Spike gene have been studied extensively; however, the impact of mutations outside the Spike gene are poorly understood. Here, we report that a triple deletion (ΔSGF or ΔLSG) in nonstructural protein 6 (nsp6) independently acquired in Alpha and Omicron sublineages of SARS-CoV-2 augments nsp6-mediated antagonism of type-I interferon (IFN-I) signaling. Specifically, these triple deletions enhance the ability of mutant nsp6 to suppress phosphorylation of STAT1 and STAT2. A parental SARS-CoV-2 USA-WA1/2020 strain containing the nsp6 ΔSGF deletion (ΔSGF-WA1) shows reduced susceptibility to IFN-I treatment in vitro , outcompetes the parental strain in human primary airway cultures, and increases virulence in mice; however, the ΔSGF-WA1 virus is less virulent than the Alpha variant (which has the nsp6 ΔSGF deletion and additional mutations in other genes). Analyses of host responses from ΔSGF-WA1-infected mice and primary airway cultures reveal activation of pathways indicative of a cytokine storm. These results provide evidence that mutations outside the Spike protein affect virus-host interactions and may alter pathogenesis of SARS-CoV-2 variants in humans.

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2., (© 2023. Springer Nature Limited.)

The effectiveness of early COVID-19 vaccines in reducing the severity of the disease has led to a focus on developing next-generation vaccines that can prevent infection and transmission of the virus. One promising approach involves the induction of mucosal immunity through nasal administration and a variety of mucosal vaccine candidates using different platforms are currently in development. Live-attenuated viruses, less pathogenic versions of SARS-CoV-2, have promising features as a mucosal vaccine platform and have the potential to induce hybrid immunity in individuals who have already received mRNA vaccines. This review discusses the potential benefits and considerations for the use of live-attenuated SARS-CoV-2 intranasal vaccines and highlights the authors' work in developing such a vaccine platform., Competing Interests: Declaration of Competing Interest P.-Y.S. has filed a patent on the reverse genetic system. P.-Y.S. received compensation from Pfizer for COVID-19 vaccine development. There are no competing interests for the other authors., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Chemical modifications of transcripts with a 5' cap occur in all organisms and function in many aspects of RNA metabolism. To facilitate analysis of RNA caps, we developed a systems-level mass spectrometry-based technique, CapQuant, for accurate and sensitive quantification of the cap epitranscriptome. The protocol includes the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by a 17 min chromatography-coupled tandem quadrupole mass spectrometry run for the identification and quantification of individual CNs. The total time required for the protocol can be up to 7 d. In this approach, 26 CNs can be quantified in eukaryotic poly(A)-tailed RNA, bacterial total RNA and viral RNA. This protocol can be modified to analyze other types of RNA and RNA from in vitro sources. CapQuant stands out from other methods in terms of superior specificity, sensitivity and accuracy, and it is not limited to individual caps nor does it require radiolabeling. Thanks to its unique capability of accurately and sensitively quantifying RNA caps on a systems level, CapQuant can reveal both the RNA cap landscape and the transcription start site distribution of capped RNA in a broad range of settings., (© 2023. Springer Nature Limited.)

A reliable and efficient serological test is crucial for monitoring neutralizing antibodies against SARS-CoV-2 and its variants of concern (VOCs). Here, we present an integrated research-clinical platform for a live SARS-CoV-2 neutralization assay, utilizing highly attenuated SARS-CoV-2 (Δ3678_WA1-spike). This strain contains mutations in viral transcription regulation sequences and deletion in the open-reading-frames 3, 6, 7, and 8, allowing for safe handling in biosafety level 2 (BSL-2) laboratories. Building on this backbone, we constructed a genetically stable reporter virus (mGFP Δ3678_WA1-spike) by incorporating a modified green fluorescent protein sequence (mGFP). We also constructed mGFP Δ3678_BA.5-spike and mGFP Δ3678_XBB.1.5-spike by substituting the WA1 spike with variants BA.5 and XBB.1.5 spike, respectively. All three viruses exhibit robust fluorescent signals in infected cells and neutralization titers in an optimized fluorescence reduction neutralization assay that highly correlates with a conventional plaque reduction assay. Furthermore, we established that a streamlined robot-aided Bench-to-Clinics COVID-19 Neutralization Test workflow demonstrated remarkably sensitive, specific, reproducible, and accurate characteristics, allowing the assessment of neutralization titers against SARS-CoV-2 variants within 24 h after sample receiving. Overall, our innovative approach provides a valuable avenue for large-scale testing of clinical samples against SARS-CoV-2 and VOCs at BSL-2, supporting pandemic preparedness and response strategies.

The re-emergence of Zika virus (ZIKV) remains a major public health threat that has raised worldwide attention. Accumulating evidence suggests that ZIKV can cause serious pathological changes to the human nervous system, including microcephaly in newborns. Recent studies suggest that metformin, an established treatment for diabetes may play a role in viral infection; however, little is known about the interactions between ZIKV infection and metformin administration. Using fluorescent ZIKV by flow cytometry and immunofluorescence imaging, we found that ZIKV can infect microglia in a dose-dependent manner. Metformin diminished ZIKV replication without the alteration of viral entry and phagocytosis. Our study demonstrated that metformin downregulated ZIKV-induced inflammatory response in microglia in a time- and dose-dependent manner. Our RNA-Seq and qRT-PCR analysis found that type I and III interferons (IFN), such as IFNα2, IFNβ1 and IFNλ3 were upregulated in ZIKV-infected cells by metformin treatment, accompanied with the downregulation of GBP4, OAS1, MX1 and ISG15. Together, our results suggest that metformin-mediated modulation in multiple pathways may attribute to restraining ZIKV infection in microglia, which may provide a potential tool to consider for use in unique clinical circumstances., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and adapt after its emergence in late 2019. As the causative agent of the coronavirus disease 2019 (COVID-19), the replication and pathogenesis of SARS-CoV-2 have been extensively studied by the research community for vaccine and therapeutics development. Given the importance of viral spike protein in viral infection/transmission and vaccine development, the scientific community has thus far primarily focused on studying the structure, function, and evolution of the spike protein. Other viral proteins are understudied. To fill in this knowledge gap, a few recent studies have identified nonstructural protein 6 (nsp6) as a major contributor to SARS-CoV-2 replication through the formation of replication organelles, antagonism of interferon type I (IFN-I) responses, and NLRP3 inflammasome activation (a major factor of severe disease in COVID-19 patients). Here, we review the most recent progress on the multiple roles of nsp6 in modulating SARS-CoV-2 replication and pathogenesis., Competing Interests: Declaration of competing interest X.X. and P.-Y.S. have filed a patent on the reverse genetic system. Other authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In this ongoing study, substantially increased ancestral SARS-CoV-2 neutralizing responses were observed 1 month after a third 10-µg BNT162b2 dose given to 5 to 11-year olds versus neutralizing responses post-dose 2. After dose 3, increased neutralizing responses against Omicron BA.1 and BA.4/BA.5 strains were also observed. The safety/tolerability profile was acceptable. (NCT04816643)., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society.)

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Pawlak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The coronavirus disease 2019 (COVID-19) pandemic has significantly impacted global public health safety and the economy. Multiple antiviral drugs have been developed, and some have received regulatory approval and/or authorization. The use of nutraceuticals can be beneficial for preventing and treating COVID-19 complications. AHCC is a standardized, cultured extract of an edible mushroom Lentinula edodes of the Basidiomycete family of fungi that is enriched in acylated α-1,4-glucans. Here, we evaluated the effects of the oral administration of AHCC on the host response to SARS-CoV-2 infection in two murine models, K18-hACE2 transgenic mice and immunocompetent BALB/c mice. Oral administration of AHCC every other day for one week before and one day post SARS-CoV-2 infection in both strains of mice decreased the viral load and attenuated inflammation in the lungs. AHCC treatment also significantly reduced SARS-CoV-2-induced lethality in the K18-hACE2 mice. AHCC administration enhanced the expansion of γδ T cells in the spleen and lungs before and after viral infection and promoted T helper 1-prone mucosal and systemic T cell responses in both models. In AHCC-fed BALB/c mice, SARS-CoV-2 specific IgG responses were also enhanced. In summary, AHCC supplementation enhances host resistance against mild and severe COVID-19 infection primarily via the promotion of innate and adaptive T cell immune responses in mice.

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC 50  ≈ 0.50 μM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses., Competing Interests: Declaration of competing interest S.G., K.L., A.M. and J.P.S. are employees of ATEA Pharmaceuticals, Inc. The other authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium., (© 2023 New York Academy of Sciences.)

The rapid spread and strong immune evasion of the SARS-CoV-2 Omicron subvariants has raised serious concerns for the global COVID-19 pandemic. These new variants exhibit generally reduced fusogenicity and increased endosomal entry pathway utilization compared to the ancestral D614G variant, the underlying mechanisms of which remain elusive. Here, we show that the C-terminal S1 mutations of the BA.1.1 subvariant, H655Y and T547K, critically govern the low fusogenicity of Omicron. Notably, H655Y also dictates the enhanced endosome entry pathway utilization. Mechanistically, T547K and H655Y likely stabilize the spike trimer conformation as suggested by increased molecular interactions in structural modeling and enhanced S1 shedding of their reversion mutants K547T and Y655H in viral producer cells. Importantly, the H655Y mutation also determines the low fusogenicity and enhanced dependence on the endosomal entry pathway of other Omicron subvariants, including BA.2, BA.2.12.1, BA.4/5, and BA.2.75. Together, these results uncover mechanisms governing Omicron subvariant entry and provide insights into altered Omicron tissue tropism and pathogenesis. IMPORTANCE Omicron has been shown to predominantly use the endosomal entry pathway, resulting in reduced lung tropism and reduced disease severity; however, the underlying mechanism is not fully understood. In addition, whether the most recent Omicron subvariants, including BA.5 and BA.2.75, use the same pathway as their ancestor for entry is currently not known. In this study, we show that T547K and H655Y mutations in the C terminus of the S1 subunit critically determine the enhanced dependence on the endosomal entry pathway as well as the reduced cell-cell fusion activity of Omicron BA.1, BA.1.1, and other subvariants. Further experiments and molecular modeling suggest that H655Y and K547T stabilize the spike trimer conformation, likely contributing to the decreased fusogenicity and endosomal entry. Our work uncovers novel mechanisms underlying the distinct entry pathway of Omicron subvariants and advances our understanding of their biological characteristics.

Remdesivir (GS-5734; VEKLURY) is a single diastereomer monophosphoramidate prodrug of an adenosine analog (GS-441524). Remdesivir is taken up by target cells and metabolized in multiple steps to form the active nucleoside triphosphate (GS-443902), which acts as a potent inhibitor of viral RNA-dependent RNA polymerases. Remdesivir and GS-441524 have antiviral activity against multiple RNA viruses. Here, we expand the evaluation of remdesivir's antiviral activity to members of the families Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae. Using cell-based assays, we show that remdesivir can inhibit infection of flaviviruses (such as dengue 1-4, West Nile, yellow fever, Zika viruses), picornaviruses (such as enterovirus and rhinovirus), and filoviruses (such as various Ebola, Marburg, and Sudan virus isolates, including novel geographic isolates), but is ineffective or is significantly less effective against orthomyxoviruses (influenza A and B viruses), or hepadnaviruses B, D, and E. In addition, remdesivir shows no antagonistic effect when combined with favipiravir, another broadly acting antiviral nucleoside analog, and has minimal interaction with a panel of concomitant medications. Our data further support remdesivir as a broad-spectrum antiviral agent that has the potential to address multiple unmet medical needs, including those related to antiviral pandemic preparedness., (© 2023. The Author(s).)

Background: Safe and effective vaccines against coronavirus disease 2019 (Covid-19) are urgently needed in young children., Methods: We conducted a phase 1 dose-finding study and are conducting an ongoing phase 2-3 safety, immunogenicity, and efficacy trial of the BNT162b2 vaccine in healthy children 6 months to 11 years of age. We present results for children 6 months to less than 2 years of age and those 2 to 4 years of age through the data-cutoff dates (April 29, 2022, for safety and immunogenicity and June 17, 2022, for efficacy). In the phase 2-3 trial, participants were randomly assigned (in a 2:1 ratio) to receive two 3-μg doses of BNT162b2 or placebo. On the basis of preliminary immunogenicity results, a third 3-μg dose (≥8 weeks after dose 2) was administered starting in January 2022, which coincided with the emergence of the B.1.1.529 (omicron) variant. Immune responses at 1 month after doses 2 and 3 in children 6 months to less than 2 years of age and those 2 to 4 years of age were immunologically bridged to responses after dose 2 in persons 16 to 25 years of age who received 30 μg of BNT162b2 in the pivotal trial., Results: During the phase 1 dose-finding study, two doses of BNT162b2 were administered 21 days apart to 16 children 6 months to less than 2 years of age (3-μg dose) and 48 children 2 to 4 years of age (3-μg or 10-μg dose). The 3-μg dose level was selected for the phase 2-3 trial; 1178 children 6 months to less than 2 years of age and 1835 children 2 to 4 years of age received BNT162b2, and 598 and 915, respectively, received placebo. Immunobridging success criteria for the geometric mean ratio and seroresponse at 1 month after dose 3 were met in both age groups. BNT162b2 reactogenicity events were mostly mild to moderate, with no grade 4 events. Low, similar incidences of fever were reported after receipt of BNT162b2 (7% among children 6 months to <2 years of age and 5% among those 2 to 4 years of age) and placebo (6 to 7% among children 6 months to <2 years of age and 4 to 5% among those 2 to 4 years of age). The observed overall vaccine efficacy against symptomatic Covid-19 in children 6 months to 4 years of age was 73.2% (95% confidence interval, 43.8 to 87.6) from 7 days after dose 3 (on the basis of 34 cases)., Conclusions: A three-dose primary series of 3-μg BNT162b2 was safe, immunogenic, and efficacious in children 6 months to 4 years of age. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04816643.)., (Copyright © 2023 Massachusetts Medical Society.)

Reverse genetic systems are widely used to engineer recombinant viruses with desired mutations. In response to the COVID-19 pandemic, four types of reverse genetic systems have been developed for SARS-CoV-2: (i) a full-length infectious clone that can be used to prepare recombinant SARS-CoV-2 at biosafety level 3 (BSL3), (ii) a trans-complementation system that can be used to produce single-round infectious SARS-CoV-2 at BSL2, (iii) an attenuated SARS-CoV-2 vaccine candidate (with deletions of viral accessory genes) that may be developed for veterinary use as well as for antiviral screening at BSL2, and (iv) replicon systems with deletions of viral structural genes that can be used at BSL2. Each of these genetic systems has its advantages and disadvantages that can be used to address different questions for basic and translational research. Due to the long genomic size and bacteria-toxic sequences of SARS-CoV-2, several experimental approaches have been established to rescue recombinant viruses and replicons, including (i) in vitro DNA ligation, (ii) bacterial artificial chromosome (BAC) system, (iii) yeast artificial chromosome (YAC) system, and (iv) circular polymerase extension reaction (CPER). This review summarizes the current status of SARS-CoV-2 genetic systems and their applications for studying viral replication, pathogenesis, vaccines, and therapeutics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: X.X. and P.-Y.S. have filed a patent on the reverse genetic system of SARS-CoV-2. C.K. does not have any conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages, including the BA.2-derived BA.2.75.2 and the BA.5-derived BQ.1.1 and XBB.1, have accumulated additional spike mutations that may affect vaccine effectiveness. Here we report neutralizing activities of three human serum panels collected from individuals 23-94 days after dose 4 of a parental mRNA vaccine; 14-32 days after a BA.5 bivalent booster from individuals with 2-4 previous doses of parental mRNA vaccine; or 14-32 days after a BA.5 bivalent booster from individuals with previous SARS-CoV-2 infection and 2-4 doses of parental mRNA vaccine. The results showed that a BA.5 bivalent booster elicited a high neutralizing titer against BA.4/5 measured at 14-32 days after boost; however, the BA.5 bivalent booster did not produce robust neutralization against the newly emerged BA.2.75.2, BQ.1.1 or XBB.1. Previous infection substantially enhanced the magnitude and breadth of BA.5 bivalent booster-elicited neutralization. Our data support a vaccine update strategy that future boosters should match newly emerged circulating SARS-CoV-2 variants., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, noncovalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC 50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment., Competing Interests: The authors declare the following competing financial interest(s): N.H., L.Z., W.Z., Q.T., H.Y., J.H., and Q.H. are co-inventors of patents that cover 3CLpro inhibitors in this study., (© 2023 The Authors. Published by American Chemical Society.)

Background: The emergence of immune-escape variants of severe acute respiratory syndrome coronavirus 2 warrants the use of sequence-adapted vaccines to provide protection against coronavirus disease 2019., Methods: In an ongoing phase 3 trial, adults older than 55 years who had previously received three 30-μg doses of the BNT162b2 vaccine were randomly assigned to receive 30 μg or 60 μg of BNT162b2, 30 μg or 60 μg of monovalent B.1.1.529 (omicron) BA.1-adapted BNT162b2 (monovalent BA.1), or 30 μg (15 μg of BNT162b2 + 15 μg of monovalent BA.1) or 60 μg (30 μg of BNT162b2 + 30 μg of monovalent BA.1) of BA.1-adapted BNT162b2 (bivalent BA.1). Primary objectives were to determine superiority (with respect to 50% neutralizing titer [NT 50 ] against BA.1) and noninferiority (with respect to seroresponse) of the BA.1-adapted vaccines to BNT162b2 (30 μg). A secondary objective was to determine noninferiority of bivalent BA.1 to BNT162b2 (30 μg) with respect to neutralizing activity against the ancestral strain. Exploratory analyses assessed immune responses against omicron BA.4, BA.5, and BA.2.75 subvariants., Results: A total of 1846 participants underwent randomization. At 1 month after vaccination, bivalent BA.1 (30 μg and 60 μg) and monovalent BA.1 (60 μg) showed neutralizing activity against BA.1 superior to that of BNT162b2 (30 μg), with NT 50 geometric mean ratios (GMRs) of 1.56 (95% confidence interval [CI], 1.17 to 2.08), 1.97 (95% CI, 1.45 to 2.68), and 3.15 (95% CI, 2.38 to 4.16), respectively. Bivalent BA.1 (both doses) and monovalent BA.1 (60 μg) were also noninferior to BNT162b2 (30 μg) with respect to seroresponse against BA.1; between-group differences ranged from 10.9 to 29.1 percentage points. Bivalent BA.1 (either dose) was noninferior to BNT162b2 (30 μg) with respect to neutralizing activity against the ancestral strain, with NT 50 GMRs of 0.99 (95% CI, 0.82 to 1.20) and 1.30 (95% CI, 1.07 to 1.58), respectively. BA.4-BA.5 and BA.2.75 neutralizing titers were numerically higher with 30-μg bivalent BA.1 than with 30-μg BNT162b2. The safety profile of either dose of monovalent or bivalent BA.1 was similar to that of BNT162b2 (30 μg). Adverse events were more common in the 30-μg monovalent-BA.1 (8.5%) and 60-μg bivalent-BA.1 (10.4%) groups than in the other groups (3.6 to 6.6%)., Conclusions: The candidate monovalent or bivalent omicron BA.1-adapted vaccines had a safety profile similar to that of BNT162b2 (30 μg), induced substantial neutralizing responses against ancestral and omicron BA.1 strains, and, to a lesser extent, neutralized BA.4, BA.5, and BA.2.75 strains. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04955626.)., (Copyright © 2023 Massachusetts Medical Society.)

Sirt6 activation has emerged as a promising drug target for the treatment of various human diseases, while only limited Sirt6 activators have been reported. Herein, a series of novel pyrrolo[1,2-a]quinoxaline-based derivatives have been identified as potent and selective Sirt6 activators with low cytotoxicity. Sirt6-knockdown findings have validated the on-target effects of this class of Sirt6 activators. Docking studies indicate the protonated nitrogen on the side chain of 38 forms π-cation interactions with Trp188, further stabilizing it into this extended binding pocket. New compounds 35, 36, 38, 46, 47, and 50 strongly repressed LPS-induced proinflammatory cytokine/chemokine production, while 38 also significantly suppressed SARS-CoV-2 infection with an EC 50 value of 9.3 μM. Moreover, compound 36 significantly inhibited the colony formation of cancer cells. These new molecules may serve as useful pharmacological tools or potential therapeutics against cancer, inflammation, and infectious diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the global coronavirus disease 2019 (COVID-19) pandemic, contains a unique, four amino acid (aa) "PRRA" insertion in the spike (S) protein that creates a transmembrane protease serine 2 (TMPRSS2)/furin cleavage site and enhances viral infectivity. More research into immunogenic epitopes and protective antibodies against this SARS-CoV-2 furin cleavage site is needed., Methods: Combining computational and experimental methods, we identified and characterized an immunogenic epitope overlapping the furin cleavage site that detects antibodies in COVID-19 patients and elicits strong antibody responses in immunized mice. We also identified a high-affinity monoclonal antibody from COVID-19 patient peripheral blood mononuclear cells; the antibody directly binds the furin cleavage site and protects against SARS-CoV-2 infection in a mouse model., Findings: The presence of "PRRA" amino acids in the S protein of SARS-CoV-2 not only creates a furin cleavage site but also generates an immunogenic epitope that elicits an antibody response in COVID-19 patients. An antibody against this epitope protected against SARS-CoV-2 infection in mice., Interpretation: The immunogenic epitope and protective antibody we have identified may augment our strategy in handling COVID-19 epidemic., Funding: The National Natural Science Foundation of China (82102371, 91542201, 81925025, 82073181, and 81802870), the Chinese Academy of Medical Sciences Initiative for Innovative Medicine (2021-I2M-1-047 and 2022-I2M-2-004), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2020-PT310-006, 2019XK310002, and 2018TX31001), the National Key Research and Development Project of China (2020YFC0841700), US National Institute of Health (NIH) funds grant AI158154, University of California Los Angeles (UCLA) AI and Charity Treks, and UCLA DGSOM BSCRC COVID-19 Award Program. H.Y. is supported by Natural Science Foundation of Jiangsu Province (BK20211554 andBE2022728)., Competing Interests: Declaration of interests H.Y. have filed a patent related to the antiviral activity of R3P1-B9 and R4P1-C2 antibodies. The other authors declared that no conflict of interest exists., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC 50 ) below 50 μM against mNG-SARS-CoV-2; 16 of these had EC 50 values below 10 μM and three NPs (holyrine A, alotaketal C, and bafilomycin D) had EC 50 values in the nanomolar range. We demonstrated the pan-SARS-CoV-2 activity of these three lead antivirals against SARS-CoV-2 highly transmissible Omicron subvariants (BA.5, BA.2 and BA.1) and highly pathogenic Delta VOCs in human Calu-3 lung cells. Notably, holyrine A, alotaketal C, and bafilomycin D, are potent nanomolar inhibitors of SARS-CoV-2 Omicron subvariants BA.5 and BA.2. The pan-SARS-CoV-2 activity of alotaketal C [protein kinase C (PKC) activator] and bafilomycin D (V-ATPase inhibitor) suggest that these two NPs are acting as host-directed antivirals (HDAs). Future research should explore whether PKC regulation impacts human susceptibility to and the severity of SARS-CoV-2 infection, and it should confirm the important role of human V-ATPase in the VOC lifecycle. Interestingly, we observed a synergistic action of bafilomycin D and N-0385 (a highly potent inhibitor of human TMPRSS2 protease) against Omicron subvariant BA.2 in human Calu-3 lung cells, which suggests that these two highly potent HDAs are targeting two different mechanisms of SARS-CoV-2 entry. Overall, our study provides insight into the potential of NPs with highly diverse chemical structures as valuable inspirational starting points for developing pan-SARS-CoV-2 therapeutics and for unravelling potential host factors and pathways regulating SARS-CoV-2 VOC infection including emerging omicron BA.5 subvariants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Distinct SARS-CoV-2 Omicron sublineages have evolved showing increased fitness and immune evasion than the original Omicron variant BA.1. Here, we report the neutralization activity of sera from BNT162b2 vaccinated individuals or unimmunized Omicron BA.1-infected individuals against Omicron sublineages and "Deltacron" variant (XD). BNT162b2 post-dose 3 immune sera neutralized USA-WA1/2020, Omicron BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and XD-spike SARS-CoV-2s with geometric mean titres (GMTs) of 1335, 393, 298, 315, 216, 103, and 301, respectively; thus, BA.4/5 SARS-CoV-2 spike variant showed the highest propensity to evade vaccine neutralization compared to the original Omicron variants BA.1. BA.1-convalescent sera neutralized USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and Deltacron-spike SARS-CoV-2s with GMTs of 15, 430, 110, 109, 102, 25, and 284, respectively. The unique mutation F486V in the BA.4/5 spike contributes to the increased evasion of antibody neutralization by sublineage BA.4/5. The low neutralization titres of vaccinated sera or convalescent sera from BA.1 infected individuals against the emerging and rapidly spreading Omicron BA.4/5 variants provide important results for consideration in the selection of an updated vaccine in the current Omicron wave.Trial registration: ClinicalTrials.gov; identifier: NCT04368728.

We performed an annotation of 35 mutations in the spike protein of the SARS-CoV-2 Omicron variant. Our analysis of the mutations indicates that Omicron has gained prominent immune evasion and potential for enhanced transmissibility. Previous modeling study has revealed that continued evolution in both immune evasion and enhanced transmissibility by SARS-CoV-2 would compromise vaccines as tools for the pandemic control. To combat the future variants of SARS-CoV-2, the world needs novel antiviral drugs that are effective at curb viral spreading without introducing additional selective pressure towards resistant variants.

Development of optimal SARS-CoV-2 vaccines to induce potent, long-lasting immunity and provide cross-reactive protection against emerging variants remains a high priority. Here, we report that a modified porous silicon microparticle (mPSM) adjuvant to SARS-CoV-2 receptor-binding domain (RBD) vaccine activated dendritic cells and generated more potent and durable systemic humoral and type 1 helper T (Th) cell- mediated immune responses than alum-formulated RBD following parenteral vaccination, and protected mice from SARS-CoV-2 and Beta variant challenge. Notably, mPSM facilitated the uptake of SARS-CoV-2 RBD antigens by nasal and airway epithelial cells. Parenteral and intranasal prime and boost vaccinations with mPSM-RBD elicited stronger lung resident T and B cells and IgA responses compared to parenteral vaccination alone, which led to markedly diminished viral loads and inflammation in the lung following SARS-CoV-2 Delta variant challenge. Overall, our results suggest that mPSM is effective adjuvant for SARS-CoV-2 subunit vaccine in both systemic and mucosal vaccinations., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Infections with mosquito-borne flaviviruses, such as Dengue virus, ZIKV virus, and West Nile virus, pose significant threats to public health. Flaviviruses cause up to 400 million infections each year, leading to many forms of diseases, including fatal hemorrhage, encephalitis, congenital abnormalities, and deaths. Currently, there are no clinically approved antiviral drugs for the treatment of flavivirus infections. The non-structural protein NS4B is an emerging target for drug discovery due to its multiple roles in the flaviviral life cycle. In this review, we summarize the latest knowledge on the structure and function of flavivirus NS4B, as well as the progress on antiviral compounds that target NS4B., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as the Omicron variants that are highly transmissible and immune evasive, underscores the need to develop therapeutic antibodies with broad neutralizing activities. Here, we used the LIBRA-seq technology, which identified SARS-CoV-2-specific B cells via DNA barcoding and subsequently single-cell sequenced BCRs, to identify an antibody, SW186, which could neutralize major SARS-CoV-2 variants of concern, including Beta, Delta, and Omicron, as well as SARS-CoV-1. The cryo-EM structure of SW186 bound to the receptor binding domain (RBD) of the viral spike protein showed that SW186 interacted with an epitope of the RBD that is not at the interface of its binding to the ACE2 receptor but is highly conserved among SARS coronaviruses. This epitope encompasses a glycosylation site (N343) of the viral spike protein. Administration of SW186 in mice after they were infected with SARS-CoV-2 Alpha, Beta, or Delta variants reduced the viral loads in the lung. These results demonstrated that SW186 neutralizes diverse SARS coronaviruses by binding to a conserved RBD epitope, which could serve as a target for further antibody development.