DUSP4 modulates RIG-I- and STING-mediated IRF3-type I IFN response.

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.

Chelerythrine chloride inhibits Zika virus infection by targeting the viral NS4B protein.

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.

Characterization of oral virome and microbiome revealed distinctive microbiome disruptions in paediatric patients with hand, foot and mouth disease.

While the underlying determinants are unclear, hand, foot and mouth disease (HFMD) presents a wide spectrum of clinical manifestations with varying severity in different individuals. Recently, many studies identified the human microbiome as a critical factor in the pathogenesis of various diseases. Therefore, we here investigated the ecological dynamics of the oral microbiome changes during the HFMD infection. After targeted enrichment of all known vertebrate viruses, the virome profiles of symptomatic and asymptomatic HFMD patients were examined and revealed to be significantly altered from those of healthy individuals, with nine discriminative viruses detected. Further characterization of the prokaryotic microbiome revealed an elevated level of Streptococcus sp. as the most important signature of the symptomatic HFMD cohort, positively correlating to the level of enterovirus A RNA. In addition, we found that while coxsackievirus A5 is detected in saliva RNA of all asymptomatic cases, coxsackievirus A6 dominates the majority of the symptomatic cohort.

An epidemiological surveillance of hand foot and mouth disease in paediatric patients and in community: A Singapore retrospective cohort study, 2013-2018.

BACKGROUND: While hand, foot and mouth disease (HFMD) is primarily self-resolving-soaring incidence rate of symptomatic HFMD effectuates economic burden in the Asia-Pacific region. Singapore has seen a conspicuous rise in the number of HFMD cases from 2010s. Here, we aims to identify the serology and genotypes responsible for such outbreaks in hospitals and childcare facilities. METHODS: We studied symptomatic paediatric HFMD cases from 2013 to 2018 in Singapore. Surveillance for subclinical enterovirus infections was also performed in childcares at the same time period. RESULTS: Genotyping 101 symptomatic HFMD samples revealed CV-A6 as the major etiological agent for recent outbreaks. We detected infections with CV-A6 (41.0%), EV-A71 (7%), CV-A16 (3.0%), coxsackievirus A2, CV-A2 (1.0%) and coxsackievirus A10, CV-A10 (1.0%). Phylogenetic analysis of local CV-A6 strains revealed a high level of heterogeneity compared against others worldwide, dissimilar to other HFMD causative enteroviruses for which the dominant strains and genotypes are highly region specific. We detected sub-clinical enterovirus infections in childcare centres; 17.1% (n = 245) tested positive for enterovirus in saliva, without HFMD indicative symptoms at the point of sample collection. CONCLUSIONS: CV-A6 remained as the dominant HFMD causative strain in Singapore. Silent subclinical enteroviral infections were detected and warrant further investigations.

Adenovirus vectored IFN-α protects mice from lethal challenge of Chikungunya virus infection.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that is responsible for numerous large and geographical epidemics, causing millions of cases. However, there is no vaccine or therapeutics against CHIKV infection available. Interferon-alpha (IFN-α) has been shown to produce potent antiviral responses during viral infection. Herein we demonstrated the use of an adenovirus-vectored expressed mouse IFN-α (mDEF201) as a prophylactic and therapeutic treatment against CHIKV in vivo. 6-day-old BALB/c mice were pre- or post-treated intranasally with single dose of mDEF201 at 5 x 106 PFU per mouse and challenged with lethal dose of CHIKV. Complete survival protection was observed in mice upon a single dose of mDEF201 administration 1 days prior to virus challenge. Viral load in the serum and multiple organs were significantly reduced upon mDEF201 administration in a dose dependent manner as compare with adenovirus 5 vector placebo set. Histological analysis of the mice tissue revealed that mDEF201 could significantly reduce the tissue morphological abnormities, mainly infiltration of immune cells and muscle fibre necrosis caused by CHIKV infection. In addition, administration of mDEF201 at 6 hours post CHIKV challenge also showed promising inhibitory effect against viral replication and dissemination. In conclusion, single-dose of intranasal administration with mDEF201 as a prophylactic or therapeutic agent within 6 hours post CHIKV infection is highly protective against a lethal challenge of CHIKV in the murine model.

Prunin suppresses viral IRES activity and is a potential candidate for treating enterovirus A71 infection.

Human enterovirus A71 (HEVA71) causes hand, foot, and mouth disease (HFMD) in young children and is considered a major neurotropic pathogen but lacks effective antivirals. To identify potential therapeutic agents against HFMD, we screened a 502-compound flavonoid library for compounds targeting the HEVA71 internal ribosome entry site (IRES) that facilitates translation of the HEVA71 genome and is vital for the production of HEVA71 viral particles. We validated hits using cell viability and viral plaque assays and found that prunin was the most potent inhibitor of HEVA71. Downstream assays affirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex 1, or chikungunya virus. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants with five mutations that mapped to the viral IRES. Knockdown studies showed that the mutations allowed HEVA71 to overcome treatment-induced suppression by differentially regulating recruitment of the IRES trans-acting factors Sam68 and hnRNPK without affecting the hnRNPA1-IRES interaction required for IRES translation. Furthermore, prunin effectively reduced HEVA71-associated clinical symptoms and mortality in HEVA71-infected BALB/c mice and suppressed hepatitis C virus at higher concentrations, suggesting a similar mechanism of prunin-mediated IRES inhibition for both viruses. These studies establish prunin as a candidate for further development as a HEVA71 therapeutic agent.

Pomegranate activates TFEB to promote autophagy-lysosomal fitness and mitophagy.

Mitochondrial dysfunction underscores aging and diseases. Mitophagy (mitochondria + autophagy) is a quality control pathway that preserves mitochondrial health by targeting damaged mitochondria for autophagic degradation. Hence, molecules or compounds that can augment mitophagy are therapeutic candidates to mitigate mitochondrial-related diseases. However, mitochondrial stress remains the most effective inducer of mitophagy. Thus, identification of mitophagy-inducing regimes that are clinically relevant is favorable. In this study, pomegranate extract (PE) supplementation is shown to stimulate mitophagy. PE activates transcription factor EB (TFEB) to upregulate the expression of autophagy and lysosomal genes for mitochondrial quality control under basal and stress conditions. Basally, PE alters mitochondrial morphology and promotes recruitment of autophagosomes to the mitochondria (mitophagosome formation). Upon onset of mitochondrial stress, PE further augments mitophagosome formation, and engages PINK1 and Parkin to the mitochondria to potentiate mitophagy. This cellular phenomenon of PE-induced mitophagy helps to negate superfluous mitochondrial reactive oxygen species (ROS) production and mitochondrial impairment. Overall, our study highlights the potential of PE supplementation as a physiological therapy to modulate TFEB activity to alleviate mitochondrial dysfunction in aging and mitochondrial-related diseases.

Circulating Salivary miRNA hsa-miR-221 as Clinically Validated Diagnostic Marker for Hand, Foot, and Mouth Disease in Pediatric Patients.

Enhancements in the diagnostic capabilities using host biomarkers are currently much needed where sensitivity and specificity issues plague the diagnosis of Hand, Foot and Mouth Disease (HFMD) in pediatrics clinical samples. We investigated miRNome profiles of HFMD saliva samples against healthy children and developed miRNA-based diagnosis models. Our 6-miRNA scoring model predicted HFMD with an overall accuracy of 85.11% in the training set and 92.86% in the blinded test set of Singapore cohort. Blinded evaluation of the model in Taiwan HFMD cases resulted in 77.08% accuracy with the 6-miRNA model and 68.75% with the 4-miRNA model. The strongest predictor of HFMD in all of the panels, hsa-miR-221 was found to be consistently and significantly downregulated in all of our HFMD cohorts. This is the first study to prove that HFMD infection could be diagnosed by circulating miRNAs in patient's saliva. Moreover, this study also serves as a stepping stone towards the future development of other infectious disease diagnosis workflows using novel biomarkers.

A flavonoid compound library screen revealed potent antiviral activity of plant-derived flavonoids on human enterovirus A71 replication.

Hand Foot Mouth Disease (HFMD), resulting from human enterovirus A71 (HEVA71) infection can cause severe neurological complications leading to fatality in young children. Currently, there is no approved antiviral for therapeutic treatment against HEVA71 infection. In this study, a 500-compound flavonoid library was screened to identify potential inhibitors of HEVA71 using high-throughput immunofluorescence-based phenotypic screening method. Two lead flavonoid compounds, ST077124 and ST024734 at the non-cytotoxic concentration of 50 µM were found to be effective antivirals that inhibited replication of HEVA71, reducing infectious viral titers by 3.5 log10 PFU/ml and 2.5 log10 PFU/ml respectively. Our study revealed that ST077124 is a specific antiviral compound that inhibits human enteroviruses while ST024734 exhibited antiviral activity against human enteroviruses as well as dengue virus type-2. We also identified that both compounds affected the viral RNA transcription and translation machinery of HEVA71 but did not interfere with the viral internal ribosomal entry site (IRES) activity. Hence, our findings strongly suggest that ST077124 and ST024734 are effective antiviral compounds of minimal cytotoxicity and could serve as promising therapeutic agents against HEVA71 infection.

Antiviral Phosphorodiamidate Morpholino Oligomers are Protective against Chikungunya Virus Infection on Cell-based and Murine Models.

Chikungunya virus (CHIKV) infection in human is associated with debilitating and persistent arthralgia and arthritis. Currently, there is no specific vaccine or effective antiviral available. Anti-CHIKV Phosphorodiamidate Morpholino Oligomer (CPMO) was evaluated for its antiviral efficacy and cytotoxcity in human cells and neonate murine model. Two CPMOs were designed to block translation initiation of a highly conserved sequence in CHIKV non-structural and structural polyprotein, respectively. Pre-treatment of HeLa cells with CPMO1 significantly suppressed CHIKV titre, CHIKV E2 protein expression and prevented CHIKV-induced CPE. CPMO1 activity was also CHIKV-specific as shown by the lack of cross-reactivity against SINV or DENV replication. When administered prophylactically in neonate mice, 15 µg/g CPMO1v conferred 100% survival against CHIKV disease. In parallel, these mice demonstrated significant reduction in viremia and viral load in various tissues. Immunohistological examination of skeletal muscles and liver of CPMO1v-treated mice also showed healthy tissue morphology, in contrast to evident manifestation of CHIKV pathogenesis in PBS- or scrambled sCPMO1v-treated groups. Taken together, our findings highlight for the first time that CPMO1v has strong protective effect against CHIKV infection. This warrants future development of morpholino as an alternative antiviral agent to address CHIKV infection in clinical applications.