Potential relevance of salivary legumain for the clinical diagnostic of hand, foot, and mouth disease.

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.

Enterovirus A71 Infection Activates Human Immune Responses and Induces Pathological Changes in Humanized Mice.

Since the discovery of enterovirus A71 (EV-A71) half a century ago, it has been recognized as the cause of large-scale outbreaks of hand-foot-and-mouth disease worldwide, particularly in the Asia-Pacific region, causing great concern for public health and economic burdens. Detailed mechanisms on the modulation of immune responses after EV-A71 infection have not been fully known, and the lack of appropriate models hinders the development of promising vaccines and drugs. In the present study, NOD-scid IL2Rγ-/- (NSG) mice with a human immune system (humanized mice) at the age of 4 weeks were found to be susceptible to a human isolate of EV-A71 infection. After infection, humanized mice displayed limb weakness, which is similar to the clinical features found in some of the EV-A71-infected patients. Histopathological examination indicated the presence of vacuolation, gliosis, or meningomyelitis in brain stem and spinal cord, which were accompanied by high viral loads detected in these organs. The numbers of activated human CD4+ and CD8+ T cells were upregulated after EV-A71 infection, and EV-A71-specific human T cell responses were found. Furthermore, the secretion of several proinflammatory cytokines, such as human gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-17A, was elevated in the EV-A71-infected humanized mice. Taken together, our results suggested that the humanized mouse model permits insights into the human immune responses and the pathogenesis of EV-A71 infection, which may provide a platform for the evaluation of anti-EV-A71 drug candidates in the future.IMPORTANCE Despite causing self-limited hand-food-and-mouth disease in younger children, EV-A71 is consistently associated with severe forms of neurological complications and pulmonary edema. Nevertheless, only limited vaccines and drugs have been developed over the years, which is possibly due to a lack of models that can more accurately recapitulate human specificity, since human is the only natural host for wild-type EV-A71 infection. Our humanized mouse model not only mimics histological symptoms in patients but also allows us to investigate the function of the human immune system during infection. It was found that human T cell responses were activated, accompanied by an increase in the production of proinflammatory cytokines in EV-A71-infected humanized mice, which might contribute to the exacerbation of disease pathogenesis. Collectively, this model allows us to delineate the modulation of human immune responses during EV-A71 infection and may provide a platform to evaluate anti-EV-A71 drug candidates in the future.

Peptidomimetic ethyl propenoate covalent inhibitors of the enterovirus 71 3C protease: a P2-P4 study.

Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC50 value 3.4 ± 0.4 versus 7.3 ± 0.8 µM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities.

Binding of the 5'-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription.

Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5'-untranslated region (5'-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5'-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem-loop I of IBV 5'-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.

The host-targeting compound peruvoside has a broad-spectrum antiviral activity against positive-sense RNA viruses.

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.

Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants.

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.

Novel capsid binder and PI4KIIIbeta inhibitors for EV-A71 replication inhibition.

The Hand, Foot and Mouth Disease (HFMD) is a highly contagious viral illness generally manifests as a mild disease in young children and immunocompromised adults. It has however emerged as a significant public health threat in recent years as outbreaks have been occurring regularly, especially in the Asia-Pacific. The disease can result from infections by a wide variety of human enteroviruses, particularly, Enterovirus A71 (EV-A71) has garnered more attention due to its association with severe disease in infected patients. Despite the potential to result severe neurological complications or even fatality, there is currently no effective antiviral for treatment of EV-A71 infections and the only vaccines available are restricted to distribution in China. In this study, we report the in vitro and in vivo evaluation of two candidate antiviral compounds active against EV-A71, a viral capsid inhibitor (G197) and a novel host-targeting phosphatidylinositol 4-kinase III beta inhibitor (N373) which, especially when used in combination, can significantly improve the survival and pathology of infected mice.

Drug repurposing of pyrimidine analogs as potent antiviral compounds against human enterovirus A71 infection with potential clinical applications.

Enterovirus A71 (EV-A71) is one of the aetiological agents for the hand, foot and mouth disease (HFMD) in young children and a potential cause of neurological complications in afflicted patients. Since its discovery in 1969, there remains no approved antiviral for EV-A71 and other HFMD-causing enteroviruses. We set out to address the lack of therapeutics against EV-A71 by screening an FDA-approved drug library and found an enrichment of hits including pyrimidine antimetabolite, gemcitabine which showed 90.2% of inhibition on EV-A71 infection. Gemcitabine and other nucleoside analogs, LY2334737 and sofosbuvir inhibition of EV-A71 infection were disclosed using molecular and proteomic quantification, and in vitro and in vivo efficacy evaluation. Gemcitabine displayed a significant reduction of infectious EV-A71 titres by 2.5 logs PFU/mL and was shown to target the early stage of EV-A71 viral RNA and viral protein synthesis process especially via inhibition of the RNA dependent RNA polymerase. In addition, the drug combination study of gemcitabine's synergistic effects with interferon-ß at 1:1 and 1:2 ratio enhanced inhibition against EV-A71 replication. Since gemcitabine is known to metabolize rapidly in vivo, other nucleoside analogs, LY2334737 and sofosbuvir conferred protection in mice against lethal EV-A71 challenge by potentially reducing the death rate, viral titers as well on virus-induced pathology in the limb muscle tissue of mice. Additionally, we found that gemcitabine is competent to inhibit other positive-sense RNA viruses of the Flaviviridae and Togaviridae family. Overall, these drugs provide new insights into targeting viral factors as a broad-spectrum antiviral strategy with potential therapeutic value for future development and are worthy of potential clinical application.

In utero infection of Zika virus leads to abnormal central nervous system development in mice.

The World Health Organization has declared ZIKA virus (ZIKV) a global public health emergency, prompted by the association of ZIKV infections with severe brain abnormalities in the human fetus. ZIKV preferentially targets human neuronal precursor cells (NPCs) in both monolayer and cortical brain organoid culture systems and stunts their growth. Although ZIKV is well recognized to cause microcephaly, there is no systematic analysis to demonstrate the effect of ZIKV on central nervous system (CNS) development, including brain malformations and spinal cord dysfunction. Here, we conducted a longitudinal analysis to show that a novel mouse model (infected in utero and monitored after birth until adulthood) recapitulates the effects of ZIKV infection affecting neural stem cells fate and leads to a thinner cortex and a smaller brain. Furthermore, we demonstrate the effect of ZIKV on spinal cord function. Specifically, we found significant reductions in neuron numbers in the anterior horn of grey matter of the spinal cord and muscle dystrophy with a significant decrease in forepaw grip strength in the ZIKV group. Thus, the established mouse model of ZIKV infection leading to abnormal CNS development will help to further advance our understanding of the disease pathogenesis.

Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells.

The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged beta-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection.

Coronavirus infectious bronchitis virus non-structural proteins 8 and 12 form stable complex independent of the non-translated regions of viral RNA and other viral proteins.

The cleavage products from coronavirus polyproteins, known as the non-structural proteins (nsps), are believed to make up the major components of the viral replication/transcription complex. In this study, several nsps encoded by avian gammacoronavirus infectious bronchitis virus (IBV) were screened for RNA-binding activity and interaction with its RNA-dependent RNA polymerase, nsp12. Nsp2, nsp5, nsp8, nsp9 and nsp10 were found to bind to untranslated regions (UTRs), while nsp8 was confirmed to interact with nsp12. Nsp8 has been reported to interact with nsp7 and functions as a primase synthesizing RNA primers for nsp12. Further characterization revealed that nsp8-nsp12 interaction is independent of the UTRs of viral RNA, and nsp8 interacts with both the N- and C-terminal regions of nsp12. These results have prompted a proposal of how the nsp7-nsp8 complex could possibly function in tandem with nsp12, forming a highly efficient complex that could synthesize both the RNA primer and viral RNA during coronavirus infection.

An evaluation of Chloroquine as a broad-acting antiviral against Hand, Foot and Mouth Disease.

A common childhood affliction of viral origin in young children and immunocompromised adults, the Hand, Foot and Mouth Disease (HFMD) has become a significant public health concern in the Asia-Pacific Region. Characterized by the appearance of vesiculopapular rashes on the hands, feet and mouth, the disease is generally mild and self-limiting. In a minority of cases, patients can develop neurological complications that could result in permanent morbidity or even fatality. In the absence of a specific antiviral for treatment, medical care is limited to supportive and symptomatic relief, presenting a need for more research into an effective antiviral to be used in the management of the disease. In this study, we evaluated the efficacy of chloroquine, a FDA-approved lysosomotropic agent, against several serotypes of HFMD-associated enteroviruses, including EV-A71, in reducing infectious virus production. We have also evaluated chloroquine in a murine model of EV-A71 infection to ascertain its antiviral efficacy in vivo. The results suggest that chloroquine could be a broad-acting antiviral effective against HFMD-associated enteroviruses.

The nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its N-terminal domain and multimerization properties.

The coronavirus nucleocapsid (N) protein packages viral genomic RNA into a ribonucleoprotein complex. Interactions between N proteins and RNA are thus crucial for the assembly of infectious virus particles. The 45 kDa recombinant nucleocapsid N protein of coronavirus infectious bronchitis virus (IBV) is highly sensitive to proteolysis. We obtained a stable fragment of 14.7 kDa spanning its N-terminal residues 29-160 (IBV-N29-160). Like the N-terminal RNA binding domain (SARS-N45-181) of the severe acute respiratory syndrome virus (SARS-CoV) N protein, the crystal structure of the IBV-N29-160 fragment at 1.85 A resolution reveals a protein core composed of a five-stranded antiparallel beta sheet with a positively charged beta hairpin extension and a hydrophobic platform that are probably involved in RNA binding. Crosslinking studies demonstrate the formation of dimers, tetramers, and higher multimers of IBV-N. A model for coronavirus shell formation is proposed in which dimerization of the C-terminal domain of IBV-N leads to oligomerization of the IBV-nucleocapsid protein and viral RNA condensation.

The Important Role of Lipid Raft-Mediated Attachment in the Infection of Cultured Cells by Coronavirus Infectious Bronchitis Virus Beaudette Strain.

Lipid raft is an important element for the cellular entry of some viruses, including coronavirus infectious bronchitis virus (IBV). However, the exact role of lipid rafts in the cellular membrane during the entry of IBV into host cells is still unknown. In this study, we biochemically fractionated IBV-infected cells via sucrose density gradient centrifugation after depleting plasma membrane cholesterol with methyl-ß-cyclodextrin or Mevastatin. Our results demonstrated that unlike IBV non-structural proteins, IBV structural proteins co-localized with lipid raft marker caveolin-1. Infectivity assay results of Vero cells illustrated that the drug-induced disruption of lipid rafts significantly suppressed IBV infection. Further studies revealed that lipid rafts were not required for IBV genome replication or virion release at later stages. However, the drug-mediated depletion of lipid rafts in Vero cells before IBV attachment significantly reduced the expression of viral structural proteins, suggesting that drug treatment impaired the attachment of IBV to the cell surface. Our results indicated that lipid rafts serve as attachment factors during the early stages of IBV infection, especially during the attachment stage.

Inhibition of enterovirus VP4 myristoylation is a potential antiviral strategy for hand, foot and mouth disease.

The Hand, Foot and Mouth Disease (HFMD) can result from infections by a plethora of human enteroviruses of the species Enterovirus A and B. These infections are highly contagious, resulting in regular outbreaks especially in the Asia-Pacific Region in the recent decade. Although this disease is generally a childhood affliction which manifests as a mild, febrile illness accompanied by the vesicles on the hands, feet and mouth, permanent morbidity or even fatality can result from severe forms of the disease in a subset of the infected patients. The N-terminal myristoylation signal (MGXXXS) of viral capsid protein VP4, one of the four viral structural proteins, is an extremely well conserved feature of enteroviruses, a potential antiviral target that may yield broad-spectrum inhibitors of HFMD. In this study, we have confirmed through the use of small interfering RNAs, human N-myristoyltransferase 1 plays an integral role in human Enterovirus 71 replication. Subsequent studies by inhibition of myristoylation using different myristic acid analogues elicited differential effects on the virus replication in human rhabdomyosarcoma cells. In particular, 2-hydroxymyristic acid specifically inhibited the cleavage between VP4 and VP2, part of the virion maturation process required to ensure infectivity of progeny virions while 4-oxatetradecanoic acid reduced the synthesis of viral RNA. These findings suggest that the requirement of a myristate moiety in viral structural protein precursor cleavage can serve as a viable antiviral target for further research.

Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease.

Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery.

Recent progress in studies of arterivirus- and coronavirus-host interactions.

Animal coronaviruses, such as infectious bronchitis virus (IBV), and arteriviruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), are able to manifest highly contagious infections in their specific native hosts, thereby arising in critical economic damage to animal industries. This review discusses recent progress in studies of virus-host interactions during animal and human coronavirus and arterivirus infections, with emphasis on IBV-host cell interactions. These interactions may be directly involved in viral replication or lead to the alteration of certain signaling pathways, such as cell stress response and innate immunity, to facilitate viral replication and pathogenesis.