3D-QSAR, ADMET and Molecular Docking Study of a Series of 2-Substituted 1H-Benzimidazole-4-Carboxamide Derivatives Inhibitors Against Enteroviruses

A series of bioactive benzimidazoles were theoretically evaluated for their effect against enteroviruses, particularly coxsackievirus B and coxsackievirus A16. The structure-activity relationship allowed to deduce a very efficient model that can be used as a reference for the synthesis of compounds against this type of virus. The model used is essentially based on the RML and NN method, and its validation was carried out using cross-validation. The results obtained by molecular docking and ADMET of compound 9 as an inhibitor of enterovirus A16 give the proposal of the drug of this series.

Atypical hand foot mouth disease presenting as vesiculobullous lesion

Hand, food, and mouth disease (HFMD) is a highly contagious disease caused by enteroviruses affecting young children under 5 years. Among enteroviruses (EVs), the main pathogens of HFMD are coxsackievirus A16 (CV-A16) and EV-A71 (EV-71).1 The clinical features include a prodromal phase which has low-grade fever, malaise and sore throat. This initial phase is usually followed by enanthem and erythematous papular skin lesions, predominantly affecting palms and soles. The dorsal surface of hands, feet, and perioral skin are rarely affected. Atypical HFMD presents as a widely distributed rash with varying morphology that makes clinical diagnosis and treatment challenging.2 Our objective is to present atypical cutaneous manifestations of HFMD caused by CA6.

Molecular epidemiology of coxsackievirus A16 circulating in children in Beijing, China from 2010 to 2019

Background Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot and mouth disease (HFMD). This study aimed to investigate the molecular epidemiology and evolutionary characteristics of CVA16. Methods Throat swabs were collected from children with HFMD and suspected HFMD during 2010–2019. Enteroviruses (EVs) were detected and typed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR. The genotype, evolutionary rate, the most recent common ancestor, population dynamics and selection pressure of CVA16 were analyzed based on viral protein gene (VP1) by bioinformatics software. Results A total of 4709 throat swabs were screened. EVs were detected in 3180 samples and 814 were CVA16 positive. More than 81% of CVA16-positive children were under 5 years old. The prevalence of CVA16 showed obvious periodic fluctuations with a high level during 2010–2012 followed by an apparent decline during 2013–2017. However, the activities of CVA16 increased gradually during 2018–2019. All the Beijing CVA16 strains belonged to sub-genotype B1, and B1b was the dominant strain. One B1c strain was detected in Beijing for the first time in 2016. The estimated mean evolutionary rate of VP1 gene was 4.49 × 10–3 substitution/site/year. Methionine gradually fixed at site-23 of VP1 since 2012. Two sites were detected under episodic positive selection, one of which (site-223) located in neutralizing linear epitope PEP71. Conclusions The dominant strains of CVA16 belonged to clade B1b and evolved in a fast evolutionary rate during 2010–2019 in Beijing. To provide more favorable data for HFMD prevention and control, it is necessary to keep attention on molecular epidemiological and evolutionary characteristics of CVA16.

Coxsackievirus A16 in Southern Vietnam

Background: Hand, Foot and Mouth Disease (HFMD) is a major public health concern in the Asia-Pacific region. Most recent HFMD outbreaks have been caused by enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16), CVA10, and CVA6. There has been no report regarding the epidemiology and genetic diversity of CVA16 in Vietnam. Such knowledge is critical to inform the development of intervention strategies.Materials and Methods: From 2011 to 2017, clinical samples were collected from in- and outpatients enrolled in a HFMD research program conducted at three referral hospitals in Ho Chi Minh City (HCMC), Vietnam. Throat or rectal swabs positive for CVA16 with sufficient viral load were selected for whole genome sequencing and evolutionary analysis.Results: Throughout the study period, 320 CVA16 positive samples were collected from 2808 HFMD patients (11.4%). 59.4% of patients were male. The median age was 20.8 months (IQR, 14.96–31.41). Patients resided in HCMC (55.3%), Mekong Delta (22.2%), and South East Vietnam (22.5%). 10% of CVA16 infected patients had moderately severe or severe HFMD. CVA16 positive samples from 153 patients were selected for whole genome sequencing, and 66 complete genomes were obtained. Phylogenetic analysis demonstrated that Vietnamese CVA16 strains belong to a single genogroup B1a that clusters together with isolates from China, Japan, Thailand, Malaysia, France and Australia. The CVA16 strains of the present study were circulating in Vietnam some 4 years prior to its detection in HFMD cases.Conclusion: We report for the first time on the molecular epidemiology of CVA16 in Vietnam. Unlike EV-A71, which showed frequent replacement between subgenogroups B5 and C4 every 2–3 years in Vietnam, CVA16 displays a less pronounced genetic alternation with only subgenogroup B1a circulating in Vietnam since 2011. Our collective findings emphasize the importance of active surveillance for viral circulation in HFMD endemic countries, critical to informing outbreak response and vaccine development.

A hSCARB2-transgenic mouse model for Coxsackievirus A16 pathogenesis

Background Coxsackievirus A16 (CA16) is one of the neurotropic pathogen that has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD), but its pathogenesis is not yet clear. The limited host range of CA16 make the establishment of a suitable animal model that can recapitulate the neurological pathology observed in human HFMD more difficult. Because the human scavenger receptor class B, member 2 (hSCARB2) is a cellular receptor for CA16, we used transgenic mice bearing human SCARB2 and nasally infected them with CA16 to study the pathogenicity of the virus. Methods Coxsackievirus A16 was administered by intranasal instillation to groups of hSCARB2 transgenic mice and clinical signs were observed. Sampled at different time-points to document and characterize the mode of viral dissemination, pathological change and immune response of CA16 infection. Results Weight loss and virus replication in lung and brain were observed in hSCARB2 mice infected with CA16, indicating that these animals could model the neural infection process. Viral antigens were observed in the alveolar epithelia and brainstem cells. The typical histopathology was interstitial pneumonia with infiltration of significant lymphocytes into the alveolar interstitial in lung and diffuse punctate hemorrhages in the capillaries of the brainstem. In addition, we detected the expression levels of inflammatory cytokines and detected high levels of interleukin IL-1β, IL-6, IL-18, and IFN-γ in nasal mucosa, lungs and brain tissues. Conclusions The hSCARB2-transgenic mice can be productively infected with CA16 via respiratory route and exhibited a clear tropism to lung and brain tissues, which can serve as a model to investigate the pathogenesis of CA16 associated respiratory and neurological disease.

In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16

Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged <5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children.

Antiviral peptides targeting the helicase activity of enterovirus nonstructural protein 2C

Enteroviruses belong to the genus Enterovirus of the family Picornaviridae and include four human enterovirus groups (EV A-D), and the epidemic of enteroviruses such as human enterovirus A71 (EV-A71) and Coxsackievirus-A16 (CVA16) is a threat to global public health. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and possesses the RNA helicase activity that plays pivotal roles during enteroviral life cycles, which makes 2C an attractive target for developing the anti-enteroviral drugs. In this study, we designed a peptide, named 2CL, based on the structure of EV-A71 2C. This peptide effectively impaired the oligomerization of EV-A71 2C protein, and inhibited the RNA helicase activities of 2C proteins encoded by EV-A71 and CVA16, and both of which belong to EV-A, and showed potent antiviral efficacy against EV-A71 and CVA16 in cells. Moreover, the 2CL treatment elicited a strong in vivo protective efficacy against lethal EV-A71 challenge. Besides, the antiviral strategy of targeting the 2C helicase activity can be applied to inhibit the replication of EV-B. Either 2CL or B-2CL, the peptide redesigned based on the 2CL-corresponding sequence of EV-Bs, exerted effective antiviral activity against two important EV-Bs, Coxsackievirus B3 and Echovirus 11. Together, our findings demonstrated that targeting the helicase activity of 2C by rationally designed peptide is an efficient antiviral strategy against enteroviruses, and the 2CL and B-2CL showed promising clinical potentials to be further developed as broad-spectrum anti-enteroviral drugs. Importance Enteroviruses are a large group of positive-sense single-stranded RNA viruses, and include numerous human pathogens, such as enterovirus A71 (EV-A71), coxsackieviruses, and echoviruses. However, no approved antiviral drug is available. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and contains the RNA helicase activity critical for the viral life cycle. Herein, according to the structure of EV-A71 2C, we designed a peptide that effectively inhibited the RNA helicase activities of EV-A71-and coxsackievirus A16 (CVA16)-encoded 2C proteins. Moreover, this peptide exerted potent antiviral effects against EV-A71 and CVA16 in cells and elicited therapeutic efficacy against lethal EV-A71 challenge in vivo. Furthermore, we demonstrated that the strategy of targeting the 2C helicase activity can be used to other relevant enteroviruses, including coxsackievirus B3 and echovirus 11. In summary, our findings provide compelling evidence that the designed peptides targeting the helicase activity of 2C could be broad-spectrum antiviral for enteroviruses.