Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening.

Enterovirus A71 (EV-A71)-related hand, foot, and mouth disease (HFMD) imposes a substantial clinical burden in the Asia Pacific region. To inform policy on the introduction of the EV-A71 vaccine into the National Immunization Programme, we investigated the seroepidemiological characteristics of EV-A71 in two prospective cohorts of children in southern China conducted between 2013 and 2018. Our results show that maternal antibody titres declined rapidly in neonates, with over half becoming susceptible to EV-A71 at 1 month of age. Between 6 months and 2 years of age, over 80% of study participants were susceptible, while one third remained susceptible at 5 years old. The highest incidence of EV-A71 infections was observed in children aged 5-6 months. Our findings support EV-A71 vaccination before 6 months for birth cohorts in southern China, potentially with a one-time catch-up vaccination for children 6 months-5 years old. More regionally representative longitudinal seroepidemiological studies are needed to further validate these findings.

Respiratory syncytial virus (RSV) is a major pathogen that causes severe lower respiratory tract infection in infants, the elderly and the immunocompromised worldwide. At present no approved specific drugs or vaccines are available to treat this pathogen. Recently, several promising candidates targeting RSV entry and multiplication steps are under investigation. However, it is possible to lead to drug resistance under the long-term treatment. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we tested in vitro two-drug combinations of fusion inhibitors (GS5806, Ziresovir and BMS433771) and RNA-dependent RNA polymerase complex (RdRp) inhibitors (ALS8176, RSV604, and Cyclopamine). The statistical program MacSynergy II was employed to determine synergism, additivity or antagonism between drugs. From the result, we found that combinations of ALS8176 and Ziresovir or GS5806 exhibit additive effects against RSV in vitro, with interaction volume of 50 µM2% and 31 µM2% at 95% confidence interval, respectively. On the other hand, all combinations between fusion inhibitors showed antagonistic effects against RSV in vitro, with volume of antagonism ranging from −50 µM2 % to −176 µM2 % at 95% confidence interval. Over all, our results suggest the potentially therapeutic combinations in combating RSV in vitro could be considered for further animal and clinical evaluations.

Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6.

Background:Hand, foot, and mouth disease (HFMD) represents a substantial disease burden in the Western Pacific region. We investigated the spectrum of causative enteroviruses of HFMD, and evaluated different clinical samples' diagnostic yield for enteroviruses. Methods:We enrolled pediatric patients hospitalized for HFMD among 6 hospitals in Anhua County, Hunan Province, China between October 2013 and September 2016. Throat swabs and stool samples (or rectal swabs) were collected to detect the enterovirus serotypes by real-time reverse-transcription polymerase chain reaction (PCR) or nested PCR. Results:Among the 2836 patients, only 1 developed severe illness. Seventeen serotypes were identified in 2401 patients (85%), with the most frequently detected being CV-A16 (29% [814]), CV-A6 (28% [784]), EV-A71 (17% [491]), CV-A10 (4% [114]), and CV-A4 (2% [53]). Children were younger in CV-A6, CV-A10, and CV-A4 infections (median, 12 months; interquartile range [IQR], 12-24 months) than EV-A71 and CV-A16 infections (median, 24 months; IQR, 12-36 months; P < .05). The predominant enterovirus serotype shifted between CV-A16 and CV-A6 during the 3 years. Stool had a higher diagnostic yield (89%) than rectal (77%) and throat swabs (74%). Detection rates reached 93% when testing stools followed by throat swabs if stools were negative, and 89% when testing rectal swabs followed by throat swabs if rectal swabs were negative. Conclusions:Our results provide a virological benchmark for future surveillance and diagnostics. Continuous comprehensive virological surveillance is essential, especially after implementation of the EV-A71 vaccine in China, to monitor serotype replacement and the vaccine's impact.

Enterovirus 71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD). Infection with EV-A71 is more often associated with neurological complications in children and is responsible for the majority of fatalities, but currently there is no approved antiviral therapy for treatment. Here, we identified auraptene, formononetin, and yangonin as effective inhibitors of EV-A71 infection in the low-micromolar range from screening of a natural product library. Among them, formononetin and yangonin selectively inhibited EV-A71 while auraptene could inhibit viruses within the enterovirus species A. Time of addition studies showed that all the three inhibitors inhibit both attachment and postattachment step of entry. We found mutations conferring the resistance to these inhibitors in the VP1 and VP4 capsid proteins and confirmed the target residues using a reverse genetic approach. Interestingly, auraptene- and formononetin-resistant viruses exhibit cross-resistance to other inhibitors while yangonin-resistant virus still remains susceptible to auraptene and formononetin. Moreover, auraptene and formononetin, but not yangonin protected EV-A71 against thermal inactivation, indicating a direct stabilizing effect of both compounds on virion capsid conformation. Finally, neither biochanin A (an analog of formononetin) nor DL-Kavain (an analog of yangonin) exhibited anti-EV-A71 activity, suggesting the structural elements required for anti-EV-A71 activity. Taken together, these compounds could become potential lead compounds for anti-EV-A71 drug development and also serve as tool compounds for studying virus entry.

Suramin was previously shown to bind to the EV-A71 capsid through its naphthalenetrisulfonic acid groups, thereby reducing virus-cell binding and inhibiting viral replication. Here, we identify VP1-145 as the critical amino acid that accounts for the differential sensitivity of EVA-71 viruses to suramin. A single Q or G to E substitution at VP1-145 results in an approximately 30-fold shift of IC 50 or IC 90 values reproducing the inhibition profile observed with field isolates expressing either the 145Q or E mutation. Our data support the conclusion that suramin binds to the positively charged region surrounding the 5-fold axis of the capsid and consequently blocks the virus attachment and entry into host cells. In order to assess the antiviral-spectrum of suramin, we analyzed 18 representative enteroviruses: A (n = 7), B (n = 5), C (n = 5) and D (n = 1). We show that suramin potency is restricted to enterovirus A species. Clinical development of suramin is further supported by pharmacokinetic data demonstrating bioactive plasma levels after a single dose intramuscular administration in macaques. Altogether, our findings support the clinical development of suramin as a novel entry inhibitor for the treatment of enterovirus A infections.

Enterovirus 71 (EV-A71) is a major causative pathogen of hand, foot, and mouth disease (HFMD) epidemics. No antiviral therapies are currently available for treating EV-A71 infections. Here, we selected five reported enterovirus inhibitors (suramin, itraconazole [ITZ], GW5074, rupintrivir, and favipiravir) with different mechanisms of action to test their abilities to inhibit EV-A71 replication alone and in combination. All selected compounds have anti-EV-A71 activities in cell culture. The combination of rupintrivir and ITZ or favipiravir was synergistic, while the combination of rupintrivir and suramin was additive. The combination of suramin and favipiravir exerted a strong synergistic antiviral effect. The observed synergy was not due to cytotoxicity, as there was no significant increase in cytotoxicity when compounds were used in combinations at the tested doses. To investigate the potential inhibitory mechanism of favipiravir against enterovirus, two favipiravir-resistant EV-A71 variants were independently selected, and both of them carried an S121N mutation in the finger subdomain of the 3D polymerase. Reverse engineering of this 3D S121N mutation into an infectious clone of EV-A71 confirmed the resistant phenotype. Moreover, viruses resistant to ITZ or favipiravir remained susceptible to other inhibitors. Most notably, combined with ITZ, rupintrivir prevented the development of ITZ-resistant variants. Taken together, these results provide a rational basis for the design of combination regimens for use in the treatment of EV-A71 infections.

Human enterovirus 71 (EV-A71) causes hand, foot and mouth disease (HFMD). EV-A71 circulates in many countries and has caused large epidemics, especially in the Asia-Pacific region, since 1997. In April 2012, an undiagnosed fatal disease with neurological involvement and respiratory distress occurred in young children admitted to the Kantha Bopha Children's Hospital in Phnom Penh, Cambodia. Most died within a day of hospital admission, causing public panic and international concern. In this study, we describe the enterovirus (EV) genotypes that were isolated during the outbreak in 2012 and the following year. From June 2012 to November 2013, 312 specimens were collected from hospitalized and ambulatory patients and tested by generic EV and specific EV-A71 reverse transcription PCR. EV-A71 was detected in 208 clinical specimens while other EVs were found in 32 patients. The VP1 gene and/or the complete genome were generated. Our phylogenetic sequencing analysis demonstrated that 80 EV-A71 strains belonged to the C4a subgenotype and 3 EV-A71 strains belonged to the B5 genotype. Furthermore, some lineages of EV-A71 were found to have appeared in Cambodia following separate introductions from neighboring countries. Nineteen EV A (CV-A6 and CV-A16), 9 EV B (EV-B83, CV-B3, CV-B2, CV-A9, E-31, E-2 and EV-B80) and 4 EV C (EV-C116, EV-C96, CV-A20 and Vaccine-related PV-3) strains were also detected. We found no molecular markers of disease severity. We report here that EV-A71 genotype C4 was the main etiological agent of a large outbreak of HFMD and particularly of severe forms associated with central nervous system infections. The role played by other EVs in the epidemic could not be clearly established.

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in infants and young children worldwide, yet no vaccine or effective antiviral treatment is available. To search for new anti-RSV agents, we developed a cell-based assay that measures inhibition of RSV-induced cytopathic effect (CPE) and identified cyclopiazonic acid (CPA), an intracellular calcium ATPase inhibitor as a RSV inhibitor (EC50 values 4.13 μM) by screening of natural product library. CPA inhibited the replication of RSV strains belonging to both A and B subgroups and human parainfluenza virus type 3, but not Enterovirus 71. Mechanism of action study by time-of-addition assay and minigenome assay revealed that CPA acts at the step of virus genome replication and/or transcription. Moreover, two other calcium ATPase inhibitors (Thapsigargin and BHQ) and calcium ionophores (A23187 and ionomycin), but not calcium channel blockers (nifedipine, nimodipine, and tetrandrine), also had similar effect. These results indicate that an increase in intracellular calcium concentration is detrimental to RSV replication. Thus, our findings provide a new strategy for anti-RSV therapy via increasing intracellular calcium concentration.

Human parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive in vitro inhibitor of the hPIV-3 haemagglutinin-neuraminidase (HN). Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 μM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs.

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection.

ABSTRACT There is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections, which remain a substantial threat to public health. To discover inhibitors that can be immediately repurposed for treatment of enterovirus infections, we developed a high-throughput screening assay that measures the cytopathic effect induced by enterovirus 71 (EV71) to screen an FDA-approved drug library. Itraconazole (ITZ), a triazole antifungal agent, was identified as an effective inhibitor of EV71 replication in the low-micromolar range (50% effective concentrations [EC 50 s], 1.15 μM). Besides EV71, the compound also inhibited other enteroviruses, including coxsackievirus A16, coxsackievirus B3, poliovirus 1, and enterovirus 68. Study of the mechanism of action by time-of-addition assay and transient-replicon assay revealed that ITZ targeted a step involved in RNA replication or polyprotein processing. We found that the mutations (G5213U and U5286C) conferring the resistance to the compound were in nonstructural protein 3A, and we confirmed the target amino acid substitutions (3A V51L and 3A V75A) using a reverse genetic approach. Interestingly, posaconazole, a new oral azole with a molecular structure similar to that of ITZ, also exhibited anti-EV71 activity. Moreover, ITZ-resistant viruses do not exhibit cross-resistance to posaconazole or the enviroxime-like compound GW5074, which also targets the 3A region, indicating that they may target a specific site(s) in viral genome. Although the protective activity of ITZ or posaconazole (alone or in combination with other antivirals) remains to be assessed in animal models, our findings may represent an opportunity to develop therapeutic interventions for enterovirus infection.

Dandruff is a scalp disorder characterized by the formation of flaky white-yellowish scales due to an altered proliferation and differentiation status; a disrupted barrier function; a decrease in the level of hydration and of natural moisturizing factors (NMF) in the scalp, with a persistent and relapsing inflammatory condition. It was recently reported that an imbalance between bacterial and fungal species colonizing the scalp of French volunteers was associated with dandruff condition. The purpose of the present study was to analyze the major bacterial and fungal species present on the scalp surface of Chinese volunteers and to investigate possible region-related variation in the microbiota linked to dandruff condition. The data obtained from the Chinese populations were highly similar to those obtained in France, confirming that dandruff scalps are associated with a higher incidence of Malassezia restricta and Staphylococcal sp. The ratios of Malassezia to Propionibacterium and Propionibacterium to Staphylococcus were also significantly higher in the dandruff volunteers as compared to normal volunteers, suggesting that equilibrium between the major bacterial and fungal taxa found on the normal scalps is perturbed in the dandruff scalps. The main difference between the French and Shanghai subjects was in their Staphylococcal biota. The results obtained in China and in France suggest that targeting one particular Malassezia sp. by antifungals instead of using large spectrum antifungals and rebalancing the dandruff scalp microbiota could be common approach to improve dandruff condition in the two countries.

ABSTRACT Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children, which can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues in VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine changes were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra- and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to the EV71 entry inhibitor suramin, which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles. IMPORTANCE Infection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are currently available for the prevention or treatment of EV71 infection. Understanding the determinants of virion assembly and entry will facilitate vaccine development and drug discovery. Here, we identified 23 out of 27 positively charged residues in VP1 which impaired or blocked the production of infectious particles. The defect could be rescued by second-site mutations within the VP1 protein. Our findings highlight the importance of positively charged residues in VP1 during infectious particle production and reveal a potential strategy for blocking EV71 infections by inhibiting intra- or intermolecular interactions of the VP1 protein.

Le virus respiratoire syncytial (VRS) est le principal agent responsable de maladies respiratoires graves (bronchiolites, pneumonies) chez l’homme, principalement chez les nouveau-nes, personnes âgees ou immunodeprimees. En l’absence de vaccin efficace contre ce virus, le developpement rationnel de traitements antiviraux constitue un enjeu de taille. Appartenant a la famille des Paramyxoviridae, le VRS est un virus enveloppe dont le genome est constitue d’un ARN simple brin de polarite negative, codant pour 11 proteines. Le genome est replique et transcrit par le complexe ARN polymerase dependant de l’ARN viral. Ce complexe est compose de la nucleoproteine N, de la polymerase L, de la phosphoproteine P et du facteur de transcription M2-1 qui assure la « processivite » de la polymerase au cours de la transcription virale. Le complexe polymerase, ne presentant pas d’equivalent dans la cellule, constitue une cible privilegiee pour le developpement d’inhibiteurs specifiques. Une meilleure comprehension des mecanismes d’interaction entre les differentes proteines du complexe polymerase est donc indispensable afin d’identifier des cibles potentielles. En collaboration avec le groupe de John N. Barr (Leeds University, UK), nous avons resolu la structure cristalline tridimensionnelle de la proteine M2-1 sous forme tetramerique et avons etudie le role de certains residus par mutagenese dirigee. Nous avons notamment montre l’importance de certains residus pour les interactions avec la phosphoproteine et les ARN viraux.Le groupe de Ralf Altmeyer - Institut Pasteur de Shanghai, a selectionne, apres criblage, un compose inhibant la replication du VRS in vitro. La culture du virus en presence de concentrations croissantes de ce compose, a permis de selectionner des virus mutants resistants. Trois mutations ponctuelles ont ete identifiees uniquement sur la proteine M2-1. Par genetique inverse, nous avons valide le role de ces residus dans la resistance a ce compose et ainsi demontre que la proteine M2-1 est la cible de ce compose antiviral.

In 2012 a large outbreak of hand, foot, and mouth disease (HFMD) widely spread over China, causing more than 2 million cases and 567 deaths. Our purpose was to characterize the major pathogens responsible for the 2012 HFMD outbreak and analyze the genetic characterization of the enterovirus 71 (EV71) strains in Shanghai; also, to analyze the dynamic patterns of neutralizing antibody (NAb) against EV71 and evaluate the diagnostic value of several methods for clinical detection of EV71.

There is currently no approved antiviral therapy for treatment of Ebola virus disease. To discover readily available approved drugs that can be rapidly repurposed for treatment of Ebola virus infections, we screened 1280 FDA-approved drugs and identified glycopeptide antibiotic teicoplanin inhibiting Ebola pseudovirus infection by blocking virus entry in the low micromolar range. Teicoplanin could be evaluated further and incorporated into ongoing clinical studies.

BACKGROUND:An effective enterovirus 71 (EV71) vaccine is needed to control the annual outbreaks of hand, foot and mouth disease (HFMD) in China. Adequate epidemiologic data relating to HFMD are needed to make decisions about appropriate public health interventions and implementation of the new EV71 vaccine.METHODS:We analyzed the population-based epidemiologic characteristics, clinical outcome and laboratory investigation of the 2011 HFMD outbreak in children based on the citywide surveillance system in Shanghai.RESULTS:The incidence rate of HFMD was 25.8 per 1000 in children <10 years of age in Shanghai in 2011, ranging from 2.5 per 1000 in the age group 7 to 9.9 years to 48.4 per 1000 in the age group 3 to 3.9 years. Children 1 to 1.9 years were at the highest risk of developing severe complications and most susceptible to HFMD. Boys and migrant children had significantly increased risks of contracting HFMD and developing severe disease. More institutional clusters/outbreaks occurred in the winter peak months than in the summer peak months. Migrant young children played a central role in the spread of HFMD in the community. EV71 was identified in 39.7% of mild HFMD outpatients, 47.4% of hospitalized patients, 92.1% of severe inpatients with complications, 50% of outbreaks and 38.8% of clusters in institutions.CONCLUSION:HFMD and EV71 infections have a significant health effect on Shanghai children.