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Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe 2+ and peroxidated lipids. Notably, the Fe 2+ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.

Fetal immune cell functions during congenital infections are poorly understood. Zika virus (ZIKV) can vertically transmit from mother to fetus, causing nervous system infection and congenital ZIKV syndrome (CZS). We identified differential functional roles for fetal monocyte/macrophage cell types and microglia in ZIKV dissemination versus clearance using mouse models. Trafficking of ZIKV-infected primitive macrophages from the yolk sac allowed initial fetal virus inoculation, while recruited monocytes promoted non-productive neuroinflammation. Conversely, brain-resident differentiated microglia were protective, limiting infection and neuronal death. Single-cell RNA sequencing identified transcriptional profiles linked to the protective versus detrimental contributions of mononuclear phagocyte subsets. In human brain organoids, microglia also promoted neuroprotective transcriptional changes and infection clearance. Thus, microglia are protective before birth, contrasting with the disease-enhancing roles of primitive macrophages and monocytes. Differential modulation of myeloid cell phenotypes by genetically divergent ZIKVs underscores the potential of immune cells to regulate diverse outcomes during fetal infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Global cyclical outbreaks of human enterovirus infections has positioned human enterovirus A71 (EV-A71) as a neurotropic virus of clinical importance. However, there remains a scarcity of internationally approved antivirals and vaccines., Methods: In pursuit of repurposing drugs for combating human enteroviruses, we employed a comprehensive pharmacophore- and molecular docking-based virtual screen targeting EV-A71 capsid protein VP1-4, 3C protease, and 3D polymerase proteins. Among 15 shortlisted ligand candidates, we dissected the inhibitory mechanism of Tanomastat in cell-based studies and evaluated its in vivo efficacy in an EV-A71-infected murine model., Findings: We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D in vitro. Time-course studies suggested that Tanomastat predominantly disrupts early process(es) of the EV-A71 replication cycle. Mechanistically, live virus particle tracking and docking predictions revealed that Tanomastat specifically impedes viral capsid dissociation, potentially via VP1 hydrophobic pocket binding. Bypassing its inhibition on entry stages, we utilized EV-A71 replication-competent, 3D pol replication-defective, and bicistronic IRES reporter replicons to show that Tanomastat also inhibits viral RNA replication, but not viral IRES translation. We further showed that orally administered Tanomastat achieved 85% protective therapeutic effect and alleviated clinical symptoms in EV-A71-infected neonatal mice., Interpretation: Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application., Funding: MOE Tier 2 grants (MOE-T2EP30221-0005, R571-000-068-592, R571-000-076-515, R571-000-074-733) and A∗STARBiomedical Research Council (BMRC)., Competing Interests: Declaration of interests HF received grants from Synthetic Biology Translational Research Program, School of Medicine, National University of Singapore., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive., Methods: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein's GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A's host interacting partners during infection., Results: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A's involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A's top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection., Conclusions: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins., (© 2024. The Author(s).)

Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, we sequenced the C-prM-E genes from 45 DENV 2015 strains, and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Sequence analysis comparing different DENV-2 genotypes and Cosmopolitan DENV-2 sequences prior to 2015 showed a clade replacement event in the DENV-2 Cosmopolitan genotype. Additionally, a major substitution C-A314G (K73R) was found in the capsid region which may have contributed to the clade replacement event. Reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells compared to wildtype virus, as well as a decrease in NS1 production in BHK-21-infected cells. After a series of passaging, the C-A314G (K73R) mutation reverted to wildtype and was thus considered to be unstable. Next generation sequencing (NGS) of three sera collected from a single DENV2-infected patient at 1-, 2-, and 5-days post-admission was employed to examine the genetic diversity over-time and mutations that may work in conjunction with C-A314G (K73R). Results showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged, and a single non-synonymous NS4A-L115I mutation was identified. Therefore, we have identified a persistent mutation C-A314G (K73R) in all of the DENV-2 isolates, and during the course of an infection, a single new non-synonymous mutation in the NS4A region appears in the virus population within a single host. The C-A314G (K73R) thus may have played a role in the DENV-2 2015 outbreak while the NS4A-L115I may be advantageous during DENV infection within the host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tsai 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.)

Detection of cytosolic nucleic acids by pattern recognition receptors, including STING and RIG-I, leads to the activation of multiple signalling pathways that culminate in the production of type I interferons (IFNs) which are vital for host survival during virus infection. In addition to protective immune modulatory functions, type I IFNs are also associated with autoimmune diseases. Hence, it is important to elucidate the mechanisms that govern their expression. In this study, we identified a critical regulatory function of the DUSP4 phosphatase in innate immune signalling. We found that DUSP4 regulates the activation of TBK1 and ERK1/2 in a signalling complex containing DUSP4, TBK1, ERK1/2 and IRF3 to regulate the production of type I IFNs. Mice deficient in DUSP4 were more resistant to infections by both RNA and DNA viruses but more susceptible to malaria parasites. Therefore, our study establishes DUSP4 as a regulator of nucleic acid sensor signalling and sheds light on an important facet of the type I IFN regulatory system., (© 2024. The Author(s).)

Viruses are a key component of the colon microbiome, but the relationship between virome and colorectal cancer (CRC) remains poorly understood. We seek to identify alterations in the viral community that is characteristic of CRC and examine if they persist after surgery. Forty-nine fecal samples from 25 non-cancer (NC) individuals and 12 CRC patients, before and 6-months after surgery, were collected for metagenomic analysis. The fecal virome of CRC patients demonstrated an increased network connectivity as compared to NC individuals. Co-exclusion of influential viruses to bacterial species associated with healthy gut status was observed in CRC, suggesting an altered virome induced a change in the healthy gut bacteriome. Network analysis revealed lower connectivity within the virome and trans-kingdom interactions in NC. After surgery, the number of strong correlations decreased for trans-kingdom and within the bacteria and virome networks, indicating lower connectivity within the microbiome. Some co-occurrence patterns between dominant viruses and bacteria were also lost after surgery, suggesting a possible return to the healthy state of gut microbiome. Microbial signatures characteristic of CRC include an altered virome besides an altered bacterial composition. Elevated viral correlations and network connectivity were observed in CRC patients relative to healthy individuals, alongside distinct changes in the cross-kingdom correlation network unique to CRC patients. Some patterns of dysbiosis persist after surgery. Future studies should seek to verify if dysbiosis truly persists after surgery in a larger sample size with microbiome data collected at various time points after surgery to explore if there is field-change in the remaining colon, as well as to examine if persistent dysbiosis correlates with patient outcomes., (© 2024. The Author(s).)

Background: Chikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications., Methods: In the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors., Results: CHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood-brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedes albopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice., Conclusions: These findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections., (© 2024. The Author(s).)

The infectious clone has been constructed for years via various mechanisms using reverse genetics of viral RNA into cDNA. The mechanism of construction has evolved to DNA-launch plasmids which simplify infectious clone manipulation and expression in mammalian cells. Infectious clones have enormously allowed manipulation of the enterovirus genome to discover antivirals, viral replication mechanisms, and functions of essential viral proteins. Here we will be discussing methods for the production of DNA-launch human enterovirus infectious clones and viral genome engineered with a fluorescent reporter gene., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

With approximately 3.8 billion people at risk of infection in tropical and sub-tropical regions, Dengue ranks among the top ten threats worldwide. Despite the potential for severe disease manifestation and the economic burden it places on endemic countries, there is a lack of approved antiviral agents to effectively treat the infection. Flavonoids, including baicalein, have garnered attention for their antimicrobial properties. In this study, we took a rational and iterative approach to develop a series of baicalein derivatives with improved antiviral activity against Dengue virus (DENV). Compound 11064 emerged as a promising lead candidate, exhibiting antiviral activity against the four DENV serotypes and representative strains of Zika virus (ZIKV) in vitro, with attractive selectivity indices. Mechanistic studies revealed that Compound 11064 did not prevent DENV attachment at the cell surface, nor viral RNA synthesis and viral protein translation. Instead, the drug was found to impair the post-receptor binding entry steps (endocytosis and/or uncoating), as well as the late stage of DENV infection cycle, including virus assembly/maturation and/or exocytosis. The inability to raise DENV resistant mutants, combined with significant antiviral activity against an unrelated RNA virus (Enterovirus-A71) suggested that Compound 11064 targets the host rather than a viral protein, further supporting its broad-spectrum antiviral potential. Overall, Compound 11064 represents a promising antiviral candidate for the treatment of Dengue and Zika., 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 The Author(s). Published by Elsevier B.V. All rights reserved.)

The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that unmodified red blood cell-derived extracellular vesicles (RBCEVs) can inhibit SARS-CoV-2 infection in a phosphatidylserine (PS) dependent manner. Using T cell immunoglobulin mucin domain-1 (TIM-1) as an example, we demonstrate that PS receptors on cells can significantly increase the adsorption and infection of authentic and pseudotyped SARS-CoV-2 viruses. RBCEVs competitively inhibit this interaction and block TIM-1-mediated viral entry into cells. We further extend the therapeutic efficacy of this antiviral treatment by loading antisense oligonucleotides (ASOs) designed to target conserved regions of key SARS-CoV-2 genes into RBCEVs. We establish that ASO-loaded RBCEVs are efficiently taken up by cells in vitro and in vivo to suppress SARS-CoV-2 replication. Our findings indicate that this RBCEV-based SARS-CoV-2 therapeutic displays promise as a potential treatment capable of inhibiting SARS-CoV-2 entry and replication.

The fight against hand, foot, and mouth disease (HFMD) remains an arduous challenge without existing point-of-care (POC) diagnostic platforms for accurate diagnosis and prompt case quarantine. Hence, the purpose of this salivary biomarker discovery study is to set the fundamentals for the realization of POC diagnostics for HFMD. Whole salivary proteome profiling was performed on the saliva obtained from children with HFMD and healthy children, using a reductive dimethylation chemical labeling method coupled with high-resolution mass spectrometry-based quantitative proteomics technology. We identified 19 upregulated (fold change = 1.5-5.8) and 51 downregulated proteins (fold change = 0.1-0.6) in the saliva samples of HFMD patients in comparison to that of healthy volunteers. Four upregulated protein candidates were selected for dot blot-based validation assay, based on novelty as biomarkers and exclusions in oral diseases and cancers. Salivary legumain was validated in the Singapore (n = 43 healthy, 28 HFMD cases) and Taiwan (n = 60 healthy, 47 HFMD cases) cohorts with an area under the receiver operating characteristic curve of 0.7583 and 0.8028, respectively. This study demonstrates the feasibility of a broad-spectrum HFMD POC diagnostic test based on legumain, a virus-specific host systemic signature, in saliva., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Zika virus (ZIKV) is a mosquito-borne virus that has re-emerged as a significant threat to global health in the recent decade. Whilst infections are primarily asymptomatic, the virus has been associated with the manifestation of severe neurological complications. At present, there is still a lack of approved antivirals for ZIKV infections. In this study, chelerythrine chloride, a benzophenanthridine alkaloid, was identified from a mid-throughput screen conducted on a 502-compound natural products library to be a novel and potent inhibitor of ZIKV infection in both in-vitro and in-vivo assays. Subsequent downstream studies demonstrated that the compound inhibits a post-entry step of the viral replication cycle and is capable of disrupting viral RNA synthesis and protein expression. The successful generation and sequencing of a ZIKV resistant mutant revealed that a single S61T mutation on the viral NS4B allowed ZIKV to overcome chelerythrine chloride inhibition. Further investigation revealed that chelerythrine chloride could directly inhibit ZIKV protein synthesis, and that the NS4B-S61T mutation confers resistance to this inhibition. This study has established chelerythrine chloride as a potential candidate for further development as a therapeutic agent against ZIKV infection., 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.)

Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chin 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.)

Over the years, the hand, foot and mouth disease (HFMD) has sparked epidemics across many countries which mainly affected young children. While symptoms are usually mild, severe complications may arise, and some even lead to death. Such concerns, coupled with the lack of approved vaccines and antivirals to date, create an urgency in the identification of safe therapeutics against HFMD. The disease is mainly transmitted by enteroviruses like enterovirus A71 (EV-A71). Essential for enterovirus replication is the host protein, PI4KB. In this study, we investigate the antiviral efficacy of a novel PI4KB inhibitor, CUR-N399. We found that CUR-N399 displayed broad-spectrum antiviral activity against picornaviruses in cell culture models. Using a suckling mouse model of lethal EV-A71 infection, CUR-N399 was found to be well-tolerated, promote survival and reduce viral titre in mice organs. Together, these support the discovery of CUR-N399 as an antiviral against EV-A71 and potentially other closely related viruses., Competing Interests: Declaration of competing interest CUR-N399 is covered in patent/patent applications owned by Curovir AB (J.W. and G.A.). D.H.J.C., T.Y., Y.H.W and J.J.H.C. declare no potential conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice., Methods: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model., Findings: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model., Interpretation: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections., Funding: Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4., Competing Interests: Declaration of interests CTK, RCHL, TY, JJHC, and WLC have filed patents and provisional applications based on this work., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Dengue virus (DENV) is the cause of dengue fever, infecting 390 million people worldwide per year. It is transmitted to humans through the bites of mosquitoes and could potentially develop severe symptoms. In spite of the rising social and economic impact inflicted by the disease on the global population, a conspicuous lack of efficacious therapeutics against DENV still persists. In this study, catechin, a natural polyphenol compound, was evaluated as a DENV infection inhibitor in vitro. Through time-course studies, catechin was shown to inhibit a post-entry stage of the DENV replication cycle. Further investigation revealed its role in affecting viral protein translation. Catechin inhibited the replication of all four DENV serotypes and chikungunya virus (CHIKV). Together, these results demonstrate the ability of catechin to inhibit DENV replication, hinting at its potential to be used as a starting scaffold for further development of antivirals against DENV infection.

Objectives: As the world transitions to COVID-19 endemicity, studies focusing on aerosol shedding of highly transmissible SARS-CoV-2 variants of concern (VOCs) are vital for the calibration of infection control measures against VOCs that are likely to circulate seasonally. This follow-up Gesundheit-II aerosol sampling study aims to compare the aerosol shedding patterns of Omicron VOC samples with pre-Omicron variants analyzed in our previous study., Design: Coarse and fine aerosol samples from 47 patients infected with SARS-CoV-2 were collected during various respiratory activities (passive breathing, talking, and singing) and analyzed using reverse transcription-quantitative polymerase chain reaction and virus culture., Results: Compared with patients infected with pre-Omicron variants, comparable SARS-CoV-2 RNA copy numbers were detectable in aerosol samples of patients infected with Omicron despite being fully vaccinated. Patients infected with Omicron also showed a slight increase in viral aerosol shedding during breathing activities and were more likely to have persistent aerosol shedding beyond 7 days after disease onset., Conclusion: This follow-up study reaffirms the aerosol shedding properties of Omicron and should guide continued layering of public health interventions even in highly vaccinated populations., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In Taiwan, coronavirus disease 2019 (COVID-19) involving the delta variant occurred after that involving the alpha variant in 2021. In this study, we aimed to analyze the Delta variant. A total of 318 patients in Taiwan infected with delta variants were identified. The case fatality rate (CFR) of patients infected with delta variants was 0.94% in Taiwan compared with that of those infected with alpha variants (5.95%). The possible reasons for the low CFR might be hybrid immunity due to infection and rapid promotion of the COVID-19 vaccination program during the alpha variant outbreak. We identified three 21J delta variants. Two long gene deletions were detected in these severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) isolates: ORF7aΔ91 in KMUH-8 and SpikeΔ30 in KMUH-9. Protein structure prediction indicates that ORF7aΔ91 results in malfunction of NS7a as an interferon antagonist and that SpikeΔ30 results in a truncated spike protein (N679-A688del), resulting in a lower infection rate compared with the delta variant without these deletions. The impact of these two deletions on SARS-CoV-2-associated pathogenesis deserves further investigation. Delta variants still exist in many regions in the omicron era, and the backbone of the delta variant genome possibly spread worldwide in the form of delta-omicron hybrids (deltacron; e.g., XBC.1 and XAY.2), which casts a potential threat to public health. Our study further highlighted the importance of more understanding of the delta variants., (© 2023 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Background: The mosquito Aedes aegypti transmits two of the most serious mosquito-borne viruses, dengue virus (DENV) and Zika virus (ZIKV), which results in significant human morbidity and mortality worldwide. The quickly shifting landscapes of DENV and ZIKV endemicity worldwide raise concerns that their co-circulation through the Ae. aegypti mosquito vector could greatly exacerbate the disease burden in humans. Recent reports have indicated an increase in the number of co-infection cases in expanding co-endemic regions; however, the impact of co-infection on viral infection and the detailed molecular mechanisms remain to be defined., Methods: C6/36 (Aedes albopictus) cells were cultured in Dulbecco's modified Eagle medium/Mitsuhashi and Maramorosch Insect Medium (DMEM/MM) (1:1) containing 2% heat-inactivated fetal bovine serum and 1× penicillin/streptomycin solution. For virus propagation, the cells were infected with either DENV serotype 2 (DENV2) strain 16681 or ZIKV isolate Thailand/1610acTw (MF692778.1). Mosquitoes (Ae. aegypti UGAL [University of Georgia Laboratory]/Rockefeller strain) were orally infected with DENV2 and ZIKV through infectious blood-feeding., Results: We first examined viral replication activity in cells infected simultaneously, or sequentially, with DENV and ZIKV, and found interspecies binding of viral genomic transcripts to the non-structural protein 5 (NS5). When we challenged Ae. aegypti mosquitos with both DENV2 and ZIKV sequentially to probe similar interactions, virus production and vector susceptibility to infection were significantly enhanced., Conclusions: Our results suggest that DENV2 and ZIKV simultaneously establishing infection in the Ae. aegypti mosquito vector may augment one another during replication. The data also implicate the homologous NS5 protein as a key intersection between the flaviviruses in co-infection, highlighting it as a potential target for vector control., (© 2023. The Author(s).)

Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available., (© 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Hand, foot and mouth disease (HFMD) is a major public health problem among children in the Asia-Pacific region. The optimal specimen for HFMD virological diagnosis remains unclear. Enterovirus A71 (EV-A71) neutralizing antibody titres detected in paired sera were considered the reference standard for calculating the sensitivity, specificity, positive and negative predictive value of throat swabs, rectal swabs, stool, blood samples and cerebrospinal fluid (CSF) by RT-PCR or ELISA assay. In this study, clinical samples from 276 HFMD patients were collected for analysing the sensitivity of different kind of specimens. Our results showed that stool had the highest sensitivity (88%, 95% CI: 74%-96%) and agreement with the reference standard (91%). The order of diagnostic yield for EV-A71 infection was stool sample ​≥ ​rectal swab ​> ​throat swab ​> ​blood sample ​> ​CSF sample, and using a combination of clinical samples improved sensitivity for enterovirus detection. The sensitivity of ELISA for IgM antibody detection in sterile-site specimens was significantly higher than that of RT-PCR (serum/plasma: 62% vs. 2%, CSF: 47% vs. 0%) (P ​< ​0.002). In conclusion, our results suggest that stool has the highest diagnostic yield for EV-A71-infected HFMD. If stool is unavailable, rectal swabs can be collected to achieve a similar diagnostic yield. Otherwise, throat swabs may be useful in detecting positive samples. Although IgM in blood or CSF is diagnostically accurate, it lacks sensitivity, missing 40%-50% of cases. The higher proportion of severe cases and shorter interval between onset and sampling contributed to the increase in congruency between clinical testing and the serological reference standard., Competing Interests: Conflict of interest Hongjie Yu received investigator-initiated research funding from Sanofi Pasteur, GlaxoSmithKline, and Yichang HEC Changjiang Pharmaceutical Company and Shanghai Roche Pharmaceutical Company, none of which is related to HFMD and enteroviruses. All other authors report no competing interests., (Copyright © 2022 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

The omicron variants of SARS-CoV-2 have substantial ability to escape infection- and vaccine-elicited antibody immunity. Here, we investigated the extent of such escape in nine convalescent patients infected with the wild-type SARS-CoV-2 during the first wave of the pandemic. Among the total of 476 monoclonal antibodies (mAbs) isolated from peripheral memory B cells, we identified seven mAbs with broad neutralizing activity to all variants tested, including various omicron subvariants. Biochemical and structural analysis indicated the majority of these mAbs bound to the receptor-binding domain, mimicked the receptor ACE2 and were able to accommodate or inadvertently improve recognition of omicron substitutions. Passive delivery of representative antibodies protected K18-hACE2 mice from infection with omicron and beta SARS-CoV-2. A deeper understanding of how the memory B cells that produce these antibodies could be selectively boosted or recalled can augment antibody immunity against SARS-CoV-2 variants., (© 2023. The Author(s).)

Dengue and Zika viruses are mosquito-borne flaviviruses burdening millions every year with hemorrhagic fever and neurological symptoms. Baicalein was previously reported as a potential anti-flaviviral candidate and halogenation of flavones and flavanones potentiated their antiviral efficacies. Here, we reported that a chemically modified 8-bromobaicalein effectively inhibited all dengue serotypes and Zika viruses at 0.66-0.88 micromolar in cell-based system. The compound bound to dengue serotype 2 conserved pocket and inhibited the dengue RdRp activity with 6.93 fold more than the original baicalein. Moreover, the compound was mildly toxic against infant and adult C57BL/6 mice despite administering continuously for 7 days. Therefore, the 8-bromobaicalein should be investigated further in pharmacokinetics and efficacy in an animal model., (© 2023. The Author(s).)

The COVID-19 pandemic has brought about unprecedented medical and healthcare challenges worldwide. With the continual emergence and spread of new COVID-19 variants, four drug compound libraries were interrogated for their antiviral activities against SARS-CoV-2. Here, we show that the drug screen has resulted in 121 promising anti-SARS-CoV-2 compounds, of which seven were further shortlisted for hit validation: citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate. In particular, the active form of vitamin D, calcitriol, exhibits strong potency against SARS-CoV-2 on cell-based assays and is shown to work by modulating the vitamin D receptor pathway to increase antimicrobial peptide cathelicidin expression. However, the weight, survival rate, physiological conditions, histological scoring, and virus titre between SARS-CoV-2 infected K18-hACE2 mice pre-treated or post-treated with calcitriol were negligible, indicating that the differential effects of calcitriol may be due to differences in vitamin D metabolism in mice and warrants future investigation using other animal models.

Dengue is a mosquito-borne flavivirus that causes 21,000 deaths annually. Depsides and depsidones of lichens have previously been reported to be antimicrobials. In this study, our objective was to identify lichen-derived depsides and depsidones as dengue virus inhibitors. The 18 depsides and depsidones of Usnea baileyi , Usnea aciculifera , Parmotrema dilatatum , and Parmotrema tsavoense were tested against dengue virus serotype 2. Two depsides and one depsidone inhibited dengue virus serotype 2 without any apparent cytotoxicity. Diffractaic acid, barbatic acid, and Parmosidone C were three active compounds further characterized for their efficacies (EC 50 ), cytotoxicities (CC 50 ), and selectivity index (SI; CC 50 /EC 50 ). Their EC 50 (SI) values were 2.43 ± 0.19 (20.59), 0.91 ± 0.15 (13.33), and 17.42 ± 3.21 (8.95) μM, respectively. Diffractaic acid showed the highest selectivity index, and similar efficacies were also found in dengue serotypes 1-4, Zika, and chikungunya viruses. Cell-based studies revealed that the target was mainly in the late stage with replication and the formation of infectious particles. This report highlights that a lichen-derived diffractaic acid could become a mosquito-borne antiviral lead as its selectivity indices ranged from 8.07 to 20.59 with a proposed target at viral replication.

Background and Objectives: The high transmissibility of SARS-CoV-2 has exposed weaknesses in our infection control and detection measures, particularly in healthcare settings. Aerial sampling has evolved from passive impact filters to active sampling using negative pressure to expose culture substrate for virus detection. We evaluated the effectiveness of an active air sampling device as a potential surveillance system in detecting hospital pathogens, for augmenting containment measures to prevent nosocomial transmission, using SARS-CoV-2 as a surrogate., Methods: We conducted air sampling in a hospital environment using the AerosolSense TM air sampling device and compared it with surface swabs for their capacity to detect SARS-CoV-2., Results: When combined with RT-qPCR detection, we found the device provided consistent SARS-CoV-2 detection, compared to surface sampling, in as little as 2 h of sampling time. The device also showed that it can identify minute quantities of SARS-CoV-2 in designated "clean areas" and through a N95 mask, indicating good surveillance capacity and sensitivity of the device in hospital settings., Conclusion: Active air sampling was shown to be a sensitive surveillance system in healthcare settings. Findings from this study can also be applied in an organism agnostic manner for surveillance in the hospital, improving our ability to contain and prevent nosocomial outbreaks., Competing Interests: PT reports receiving grants from Roche, Arcturus, Johnson and Johnson, Sanofi Pasteur, and personal fees from AJ Biologicals, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tan, Ang, Tay, Somani, Ng, Peng, Chu, Tambyah and Allen.)

As SARS-CoV-2 Omicron and other variants of concern (VOCs) continue spreading worldwide, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with potency against diverse VOCs including Omicron subvariants BA.1, BA.2 and BA.4/5, SARS-CoV-1, and major sarbecoviruses. Crystal structure analysis of one representative nanobody, 3-2A2-4, discovers a highly conserved epitope located between the cryptic and the outer face of the receptor binding domain (RBD), distinctive from the receptor ACE2 binding site. Cryo-EM and biochemical evaluation reveal that 3-2A2-4 interferes structural alteration of RBD required for ACE2 binding. Passive delivery of 3-2A2-4 protects K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. Identification of these unique nanobodies will inform the development of next generation antibody therapies and design of pan-sarbecovirus vaccines., (© 2022. The Author(s).)

The on-going COVID-19 pandemic has given rise to SARS-CoV-2 clades and variants with differing levels of symptoms and severity. To this end, we aim to systematically elucidate the changes in the pathogenesis as SARS-CoV-2 evolved from ancestral to the recent Omicron VOC, on their mechanisms (e.g. cytokine storm) resulting in tissue damage, using the established K18-hACE2 murine model. We reported that among the SARS-CoV-2 viruses tested, infection profiles were initially similar between viruses from early clades but started to differ greatly starting from VOC Delta, where the trend continues in Omicron. VOCs Delta and Omicron both accumulated a significant number of mutations, and when compared to VOCs Alpha, Beta, and earlier predecessors, showed reduced neurotropism and less apparent gene expression in cytokine storm associated pathways. They were shown to leverage on other pathways to cause tissue damage (or lack of in the case of Omicron). Our study highlighted the importance of elucidating the response profiles of individual SARS-CoV-2 iterations, as their propensity of severe infection via pathways like cytokine storm changes as more variant evolves. This will then affect the overall threat assessment of each variant as well as the use of immunomodulatory treatments as management of severe infections of each variant., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aw, Mok, Wong, Chen, Mak, Lin, Lye, Tan and Chu.)

Zika virus (ZIKV) is an arbovirus belonging to the flavivirus genus and is transmitted in Aedes mosquito vectors. Since its discovery in humans in 1952 in Uganda, ZIKV has been responsible for many outbreaks in South America, Africa, and Asia. Patients infected with ZIKV are usually asymptomatic; mild symptoms include fever, joint and muscle pain, and fatigue. However, severe infections may have neurological implications, such as Guillain-Barré syndrome and fetal microcephaly. To date, there are no existing approved therapeutic drugs or vaccines against ZIKV infections; treatments mainly target the symptoms of infection. Preventive measures against mosquito breeding are the main strategy for limiting the spread of the virus. Antiviral drug research for the treatment of ZIKV infection has been rapidly developing, with many drug candidates emerging from drug repurposing studies, and compound screening. In particular, several studies have demonstrated the potential of natural products as antivirals for ZIKV infection. Hence, this paper will review recent advances in natural products in ZIKV antiviral drug discovery., (© 2022 The Authors. Medicinal Research Reviews published by Wiley Periodicals LLC.)

SETDB1 is a key regulator of lineage-specific genes and endogenous retroviral elements (ERVs) through its deposition of repressive H3K9me3 mark. Apart from its H3K9me3 regulatory role, SETDB1 has seldom been studied in terms of its other potential regulatory roles. To investigate this, a genomic survey of SETDB1 binding in mouse embryonic stem cells across multiple libraries was conducted, leading to the unexpected discovery of regions bereft of common repressive histone marks (H3K9me3, H3K27me3). These regions were enriched with the CTCF motif that is often associated with the topological regulator Cohesin. Further profiling of these non-H3K9me3 regions led to the discovery of a cluster of non-repeat loci that were co-bound by SETDB1 and Cohesin. These regions, which we named DiSCs (domains involving SETDB1 and Cohesin) were seen to be proximal to the gene promoters involved in embryonic stem cell pluripotency and lineage development. Importantly, it was found that SETDB1-Cohesin co-regulate target gene expression and genome topology at these DiSCs. Depletion of SETDB1 led to localized dysregulation of Cohesin binding thereby locally disrupting topological structures. Dysregulated gene expression trends revealed the importance of this cluster in ES cell maintenance as well as at gene 'islands' that drive differentiation to other lineages. The 'unearthing' of the DiSCs thus unravels a unique topological and transcriptional axis of control regulated chiefly by SETDB1., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hand, foot, and mouth disease (HFMD), which is mainly caused by coxsackievirus A16 (CVA16) or enterovirus A71 (EV-A71), poses a serious threat to children's health. However, the long-term dynamics of the neutralizing Ab (NAb) response and ideal paired-serum sampling time for serological diagnosis of CVA16-infected HFMD patients were unclear. In this study, 336 CVA16 and 253 EV-A71 PCR-positive HFMD inpatients were enrolled and provided 452 and 495 sera, respectively, for NAb detection. Random-intercept modeling with B-spline was conducted to characterize NAb response kinetics. The NAb titer of CVA16 infection patients was estimated to increase from negative (2.1, 95% confidence interval [CI]: 1.4-3.3) on the day of onset to a peak of 304.8 (95% CI: 233.4-398.3) on day 21 and then remained >64 until 26 mo after onset. However, the NAb response level of EV-A71-infected HFMD patients was much higher than that of CVA16-infected HFMD patients throughout. The geometric mean titer was significantly higher in severe EV-A71-infected patients than in mild patients, with a 2.0-fold (95% CI: 1.4-3.2) increase. When a 4-fold rise in titer was used as the criterion for serological diagnosis of CVA16 and EV-A71 infection, acute-phase serum needs to be collected at 0-5 d, and the corresponding convalescent serum should be respectively collected at 17.4 (95% CI: 9.6-27.4) and 24.4 d (95% CI: 15.3-38.3) after onset, respectively. In conclusion, both CVA16 and EV-A71 infection induce a persistent humoral immune response but have different NAb response levels and paired-serum sampling times for serological diagnosis. Clinical severity can affect the anti-EV-A71 NAb response., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) inactivation is an important step toward enhanced biosafety in testing facilities and affords a reduction in the biocontainment level necessary for handling virus-positive biological specimens. Virus inactivation methods commonly employ heat, detergents, or combinations thereof. In this work, we address the dearth of information on the efficacy of SARS-CoV-2 inactivation procedures in plasma and their downstream impact on immunoassays. We evaluated the effects of heat (56 °C for 30 min), detergent (1-5% Triton X-100), and solvent-detergent (SD) combinations [0.3-1% tri- n -butyl phosphate (TNBP) and 1-2% Triton X-100] on 19 immunoassays across different assay formats. Treatments are deemed immunoassay-compatible when the average and range of percentage recovery (treated concentration relative to untreated concentration) lie between 90-110 and 80-120%, respectively. We show that SD treatment (0.3% TNBP/1% Triton-X100) is compatible with more than half of the downstream immunoassays tested and is effective in reducing SARS-CoV-2 infectivity in plasma to below detectable levels in plaque assays. This facile method offers enhanced safety for laboratory workers handling biological specimens in clinical and research settings., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Due to the nature of RNA viruses, their high mutation rates produce a population of closely related but genetically diverse viruses, termed quasispecies. To determine the role of quasispecies in DENV disease severity, 22 isolates (10 from mild cases, 12 from fatal cases) were obtained, amplified, and sequenced with Next Generation Sequencing using the Illumina MiSeq platform. Using variation calling, unique wildtype nucleotide positions were selected and analyzed for variant nucleotides between mild and fatal cases. The analysis of variant nucleotides between mild and fatal cases showed 6 positions with a significant difference of p  < 0.05 with 1 position in the structural region, and 5 positions in the non-structural (NS) regions. All variations were found to have a higher percentage in fatal cases. To further investigate the genetic changes that affect the virus's properties, reverse genetics (rg) viruses containing substitutions with the variations were generated and viral growth properties were examined. We found that the virus variant rgNS5-T7812G (G81G) had higher replication rates in both Baby hamster kidney cells (BHK-21) and Vero cells while rgNS5-C9420A (A617A) had a higher replication rate only in BHK-21 cells compared to wildtype virus. Both variants were considered temperature sensitive whereby the viral titers of the variants were relatively lower at 39°C, but was higher at 35 and 37°C. Additionally, the variants were thermally stable compared to wildtype at temperatures of 29, 37, and 39°C. In conclusion, viral quasispecies found in isolates from the 2015 DENV epidemic, resulted in variations with significant difference between mild and fatal cases. These variations, NS5-T7812G (G81G) and NS5-C9420A (A617A), affect viral properties which may play a role in the virulence of DENV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cheng, Huang, Chin, Hung, Tsai, Chu, Chao and Wang.)

Striking number of mutations found in the spike protein of recently emerged SARS-CoV-2 Omicron subvariants BA.1, BA.2, BA.3 and BA.4/5 has raised serious concerns regarding the escape from current antibody therapies and vaccine protection. Here, we conducted comprehensive analysis on the extent of two major Omicron lineages BA.1/BA.1.1 and BA.2 to escape neutralization from the therapeutic antibodies approved by the regulatory authorities and convalescent plasma from SARS-CoV-2 patients infected during initial wave of pandemic in early 2020. We showed that Omicron BA.1/BA.1.1 were the most resistant in both magnitude and breadth against antibodies and convalescent plasma, followed by Beta, BA.2, Gamma, Delta and Alpha. While the majority of therapeutic antibodies lost binding and neutralization to Omicron variants, BRII combo (BRII-196 + BRII-198), S309, and AZ combo (COV2-2196 + COV2-2130) maintained neutralization despite of reduction due to either conserved epitope or combinational effect between the two designated antibodies. A single intraperitoneal injection of BRII combo as a prophylactic treatment protected animals from Omicron infection. Treated animals manifested normal body weight, survived infection up to 14 days, undetectable levels of infectious viruses in the lungs, and reduced lung pathology compared to the controls. Analyzing ACE2 from diverse host species showed that Omicron variants acquired ability to use mouse ACE2 for entry. These results demonstrate major antigenic shifts and potentially broadening the host range of two major Omicron lineages BA.1/BA.1.1 and BA.2, posing serious challenges to current antibody therapies and vaccine protection as well as increasing danger of spillover into the wildlife., Competing Interests: The authors have filed patent applications on antibodies BRII-196 and BRII-198 described in the manuscript. LZ, QZ, and XS are shareholders of TSB Therapeutics., (Copyright © 2022 Wang, Zhang, Zhang, Aw, Chen, Wong, Hong, Ju, Shi, Ding, Zhang, Chu and Zhang.)

Background: Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading events suggest that aerosols play an important role in driving the coronavirus disease 2019 (COVID-19) pandemic. To better understand how airborne SARS-CoV-2 transmission occurs, we sought to determine viral loads within coarse (>5 μm) and fine (≤5 μm) respiratory aerosols produced when breathing, talking, and singing., Methods: Using a G-II exhaled breath collector, we measured viral RNA in coarse and fine respiratory aerosols emitted by COVID-19 patients during 30 minutes of breathing, 15 minutes of talking, and 15 minutes of singing., Results: Thirteen participants (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic and 1 presymptomatic patient. Viral loads ranged from 63-5821 N gene copies per expiratory activity per participant, with high person-to-person variation. Patients earlier in illness were more likely to emit detectable RNA. Two participants, sampled on day 3 of illness, accounted for 52% of total viral load. Overall, 94% of SARS-CoV-2 RNA copies were emitted by talking and singing. Interestingly, 7 participants emitted more virus from talking than singing. Overall, fine aerosols constituted 85% of the viral load detected in our study. Virus cultures were negative., Conclusions: Fine aerosols produced by talking and singing contain more SARS-CoV-2 copies than coarse aerosols and may play a significant role in SARS-CoV-2 transmission. Exposure to fine aerosols, especially indoors, should be mitigated. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging; whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an urgent enquiry necessitating larger-scale studies., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Early prognosis of abnormal vasculopathy is essential for effective clinical management of patients with severe dengue. An exaggerated interferon (IFN) response and release of vasoactive factors from endothelial cells cause vasculopathy. This study shows that dengue virus 2 (DENV2) infection of human umbilical vein endothelial cells (HUVEC) results in differentially regulated microRNAs (miRNAs) important for endothelial function. miR-573 was significantly downregulated in DENV2-infected HUVEC due to decreased peroxisome proliferator activator receptor gamma (PPARγ) activity. Restoring miR-573 expression decreased endothelial permeability by suppressing the expression of vasoactive angiopoietin 2 (ANGPT2). We also found that miR-573 suppressed the proinflammatory IFN response through direct downregulation of Toll-like receptor 2 (TLR2) expression. Our study provides a novel insight into miR-573-mediated regulation of endothelial function during DENV2 infection, which can be further translated into a potential therapeutic and prognostic agent for severe dengue patients. IMPORTANCE We need to identify molecular factors that can predict the onset of endothelial dysfunction in dengue patients. Increase in endothelial permeability during severe dengue infections is poorly understood. In this study, we focus on factors that regulate endothelial function and are dysregulated during DENV2 infection. We show that miR-573 rescues endothelial permeability and is downregulated during DENV2 infection in endothelial cells. This finding can have both diagnostic and therapeutic applications.

The ongoing SARS-CoV-2 pandemic has tested the capabilities of public health and scientific community. Since the dawn of the twenty-first century, viruses have caused several outbreaks, with coronaviruses being responsible for 2: SARS-CoV in 2007 and MERS-CoV in 2013. As the border between wildlife and the urban population continue to shrink, it is highly likely that zoonotic viruses may emerge more frequently. Furthermore, it has been shown repeatedly that these viruses are able to efficiently evade the innate immune system through various strategies. The strong and abundant antiviral innate immunity evasion strategies shown by SARS-CoV-2 has laid out shortcomings in our approach to quickly identify and modulate these mechanisms. It is thus imperative that there be a systematic framework for the study of the immune evasion strategies of these viruses, to guide development of therapeutics and curtail transmission. In this review, we first provide a brief overview of general viral evasion strategies against the innate immune system. Then, we utilize SARS-CoV-2 as a case study to highlight the methods used to identify the mechanisms of innate immune evasion, and pinpoint the shortcomings in the current paradigm with its focus on overexpression and protein-protein interactions. Finally, we provide a recommendation for future work to unravel viral innate immune evasion strategies and suitable methods to aid in the study of virus-host interactions. The insights provided from this review may then be applied to other viruses with outbreak potential to remain ahead in the arms race against viral diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tay, Lew, Chu and Tan.)

Enterovirus 71 (EV-A71) causes hand, foot, and mouth disease (HFMD) in children and has been associated with neurological complications. With no specific treatment and a monovalent vaccine limited to the Chinese market, HFMD remains a serious public health concern and an economic burden to affected societies. The molecular mechanisms underpinning EV-A71 neurovirulence have yet to be fully elucidated. In this work, we provide experimental evidence that a single amino acid substitution (I to K) at position 149 in structural protein VP2 of a non-mouse-adapted EV-A71 strain completely and specifically abrogated its infectivity in murine motor neuron-like NSC-34 cells. We showed that VP2 I149K mutant was impaired in murine SCARB2-mediated entry step but retained the ability to attach at the cell surface. In vivo , VP2 I149K mutant was fully attenuated in a symptomatic mouse model of progressive limb paralysis. While viral titers in limb muscles were comparable to mice infected with parental wild-type strain, significantly lower viral titers were measured in the spinal cord and brain, with minimal tissue damage, therefore indicating that VP2 I149K mutant is specifically impaired in its ability to invade the central nervous system (CNS). This study highlights the key role of amino acid at position 149 in VP2 in EV-A71 neurovirulence, and lends further support that the EF loop of VP2 represents a potential therapeutic target., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yeo, Chong, Chen, Lim, Ng, Yan, Chu, Chow and Alonso.)

Microbial resistance to current antibiotics therapies is a major cause of implant failure and adverse clinical outcomes in orthopaedic surgery. Recent developments in advanced antimicrobial nanotechnologies provide numerous opportunities to effective remove resistant bacteria and prevent resistance from occurring through unique mechanisms. With tunable physicochemical properties, nanomaterials can be designed to be bactericidal, antifouling, immunomodulating, and capable of delivering antibacterial compounds to the infection region with spatiotemporal accuracy. Despite its substantial advancement, an important, but under-explored area, is potential microbial resistance to nanomaterials and how this can impact the clinical use of antimicrobial nanotechnologies. This review aims to provide a better understanding of nanomaterial-associated microbial resistance to accelerate bench-to-bedside translations of emerging nanotechnologies for effective control of implant associated infections., Competing Interests: There is no conflict of interest., (© 2022 The Authors.)

Hand, foot and mouth disease (HFMD) caused by Human Enterovirus A71 (HEVA71) infection is typically a benign infection. However, in minority of cases, children can develop severe neuropathology that culminate in fatality. Approximately 36.9% of HEVA71-related hospitalizations develop neurological complications, of which 10.5% are fatal. Yet, the mechanism by which HEVA71 induces these neurological deficits remain unclear. Here, we show that HEVA71-infected astrocytes release CXCL1 which supports viral replication in neurons by activating the CXCR2 receptor-associated ERK1/2 signaling pathway. Elevated CXCL1 levels correlates with disease severity in a HEVA71-infected mice model. In humans infected with HEVA71, high CXCL1 levels are only present in patients presenting neurological complications. CXCL1 release is specifically triggered by VP4 synthesis in HEVA71-infected astrocytes, which then acts via its receptor CXCR2 to enhance viral replication in neurons. Perturbing CXCL1 signaling or VP4 myristylation strongly attenuates viral replication. Treatment with AZD5069, a CXCL1-specific competitor, improves survival and lessens disease severity in infected animals. Collectively, these results highlight the CXCL1-CXCR2 signaling pathway as a potential target against HFMD neuropathogenesis., (© 2022. The Author(s).)

Chikungunya virus (CHIKV) infection, a febrile illness caused by a mosquito-transmitted alphavirus, has afflicted millions of people worldwide. There is currently no approved effective antiviral treatment for CHIKV infection. In this study, we report a new class of small-molecule CHIKV inhibitors, the oxindole-labdanes, that potently block the replication of CHIKV with good selectivity. Andrographolide, a previously reported inhibitor of CHIKV infection, was used as the lead compound for our initial structure-activity relationship (SAR) study. From a focused library of 72 andrographolide analogues, we identified the lead compound (E)-2 with improved antiviral activities. Further optimization of (E)-2 led to the discovery of the normal-labdane 7-chloro-oxindole (E)-42 as potent inhibitor against two low-passage CHIKV isolates from human patients with an EC 50 of 1.55 μM against CHIKV-122508 and 0.14 μM against CHIKV-6708. Compound (E)-42 displayed minimal cytotoxic liability (CC 50  > 100 μM), thus furnishing good selectivity relative to the host cells. Mechanistically, (E)-42 does not inactivate the viral particles but rather acts on the host cells to interfere with the viral replication, demonstrating both prophylactic and therapeutic effects. Our findings open a new avenue for the development of oxindole-labdane compounds as promising antiviral drugs against CHIKV infection., 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.)

Research on the role of platelets in modulating innate and adaptive host immune responses has gaining importance in the last two decades. Since the virus can directly interact with platelet receptors and modulate the host immune response, understanding the role of platelets in viral pathogenesis would pave way for novel therapeutic means. The present review aims at presenting the important molecular aspects of platelet-flavivirus interactions and how it leads to platelet activation, thrombocytopenia, and vascular endothelial leakage. Besides, the role of some of the platelet-derived factors as biomarkers for the early prediction of disease outcome taking dengue infection as an example is reviewed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The constant mutation of SARS-CoV-2 has led to the emergence of new variants, which call for urgent effective therapeutic interventions. The trimeric spike (S) protein of SARS-CoV-2 is highly immunogenic with the receptor-binding domain (RBD) that binds first to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and is therefore the target of many neutralizing antibodies. In this study, we characterized a broadly neutralizing monoclonal antibody (mAb) 9G8, which shows potent neutralization against the authentic SARS-CoV-2 wild-type (WT), Alpha (B.1.1.7), and Delta (1.617.2) viruses. Furthermore, mAb 9G8 also displayed a prominent neutralizing efficacy in the SARS-CoV-2 surrogate virus neutralization test (sVNT) against the Epsilon (B.1.429/7), Kappa (B.1.617.1), Gamma (P.1), Beta (B.1.351), and Delta Plus (1.617.2.1) RBD variants in addition to the variants mentioned above. Based on our in vitro escape mutant studies, we proved that the mutations V483F and Y489H within the RBD were involved in ACE2 binding and caused the neutralizing evasion of the virus from mAb 9G8. The development of such a cross-reactive neutralizing antibody against majority of the SARS-CoV-2 variants provides an important insight into pursuing future therapeutic agents for the prevention and treatment of COVID-19.

Hand-foot-and-mouth disease (HFMD) caused by human enterovirus A71 (EV-A71) infection has been associated with severe neurological complications. With the lack of an internationally approved antiviral, coupled with a surge in outbreaks globally, EV-A71 has emerged as a neurotropic virus of high clinical importance. Andrographolide has many pharmacological effects including antiviral activity and its derivative, andrographolide sulfonate, has been used in China clinically to treat EV-A71 infections. This study sought to identify novel andrographolide derivatives as EV-A71 inhibitors and elucidate their antiviral mode of action. Using an immunofluorescence-based phenotypic screen, we identified novel EV-A71 inhibitors from a 344-compound library of andrographolide derivatives and validated them with viral plaque assays. Among these hits, ZAF-47 , a quinolinoxy-andrographolide, was selected for downstream mechanistic studies. It was found that ZAF-47 acts on EV-A71 post-entry stages and inhibits EV-A71 protein expression. Subsequent luciferase studies confirm that ZAF-47 targets EV-A71 genome RNA replication specifically. Unsuccessful attempts in generating resistant mutants led us to believe a host factor is likely to be involved which coincide with the finding that ZAF-47 exhibits broad-spectrum antiviral activity against other enteroviruses (CV-A16, CV-A6, Echo7, CV-B5, CV-A24 and EV-D68). Furthermore, ZAF-46 and ZAF-47 , hits from the screen, were derivatives of the same series containing quinolinoxy and olefin modifications, suggesting that an andrographolide scaffold mounted with these unique moieties could be a potential anti-EV-A71/HFMD strategy.

Identification of an effective antiviral for the treatment of COVID-19 is considered one of the holy grails in the bid to end the pandemic. However, the novelty of SARS-CoV-2, along with the little knowledge available about its infection characteristics at the beginning of this pandemic, challenges the scientific world on how one may be able to promptly identify promising drug candidates from a myriad of compound libraries. Here, we describe a cytopathic effect (CPE)-based drug screening assay for SARS-CoV-2 which allows for rapid assessment of drug compound libraries through pre- or posttreatment drug screening procedures and evaluation using a light microscope. By comparing the virus-induced CPE of the drug-treated cells against the vehicle and drug controls, potent drug candidates can be quickly identified for further downstream studies., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)