Spectrum of Enterovirus Serotypes Causing Uncomplicated Hand, Foot, and Mouth Disease and Enteroviral Diagnostic Yield of Different Clinical Samples.

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.

Identification of Positively Charged Residues in Enterovirus 71 Capsid Protein VP1 Essential for Production of Infectious Particles.

UNLABELLED: 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.

In Vitro Assessment of Combinations of Enterovirus Inhibitors against Enterovirus 71.

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.

Enterovirus 71 infection in children with hand, foot, and mouth disease in Shanghai, China: epidemiology, clinical feature and diagnosis.

BACKGROUND: 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. METHODS: Clinical samples including stool, serum and CSF were collected from 396 enrolled HFMD inpatients during the peak seasons in 2012. We analyzed the molecular epidemiology, clinical feature, and diagnostic tests of EV71 infection. RESULTS: EV71 was responsible for 60.35 % of HFMD inpatients and 88.46 % of severe cases. The circulating EV71 strains belonged to subgenogroup C4a. The nucleotide sequences of VP1 between severe cases and uncomplicated cases shared 99.2 ~ 100 % of homology. Among 218 cases with EV71 infection, 211 (96.79 %) serum samples showed NAb positive against EV71 and NAb titer reached higher level 3 days after disease onset. Of 92 cases with EV71-associated meningitis or encephalitis, 5 (5.43 %) of 92 had EV71 RNA detected in CSF samples. The blood anti-EV71 IgM assay showed a sensitivity of 93.30 % and a specificity of 50 %. CONCLUSIONS: EV71 C4a remained the predominant subgenotype circulating in Shanghai. The severity of the EV71 infection is not associated with the virulence determinants in VP1. RT-PCR together with IgM detection can enhance the early diagnosis of severe EV71-associated HFMD.

Seroepidemiology of enterovirus A71 infection in prospective cohort studies of children in southern China, 2013-2018.

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.

Characterization of three small molecule inhibitors of enterovirus 71 identified from screening of a library of natural products.

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.

Molecular epidemiology of human enterovirus 71 at the origin of an epidemic of fatal hand, foot and mouth disease cases in Cambodia.

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.

The population-based health effect of hand, foot and mouth disease in children in Shanghai.

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.

The cytokine and chemokine profiles in patients with hand, foot and mouth disease of different severities in Shanghai, China, 2010.

BACKGROUND AND PURPOSE: Systemic upregulation of inflammatory cytokines is characteristic of critical severe hand, foot, and mouth disease (HFMD) with pulmonary edema. Thus, immunomodulatory medicines such as steroids, including methylprednisolone, have been proposed to treat patients with severe HFMD in China, because it is postulated that inflammatory cytokines play a role in the development of severe complications. This study is to further investigate the inflammatory response in the relatively mild HFMD patients, and whether steroid treatment has a beneficial effect on the suppression of inflammation in HFMD patients. METHOD: We measured the levels of 50 kinds of chemokines, cytokines, growth factors and soluble receptors in serum samples from control patients without HFMD and the HFMD patients with or without prior treatment of intravenous methylprednisolone. RESULTS: Our present study found that even relatively mild HFMD patients without central nervous system (CNS) complications had elevated serum levels of inflammatory cytokines, including interleukin (IL)-3, IL-6, IL-12p40, and tumor necrosis factor (TNF)-α, which suggested systemic inflammation. In contrast, these patients also have decreased levels of other serum biomarkers, including IL-1Ra, IL-8, IL-16, soluble ICAM-1, CXCL-1, and CCL27. The dysregulation of cytokine and chemokine expression may be involved in CNS complications and unbalanced circulating leukocytes in HFMD patients. Surprisingly, patients treated with methylprednisolone had no difference in the expression levels of HFMD-associated biomarkers instead had slightly increased levels of IL-17A, which was not associated with the occurrence of HFMD. CONCLUSION: Whether steroid treatment has any beneficial effect on the prognosis of HFMD patients requires to be further investigated.

Elevated antigen-specific Th2 type response is associated with the poor prognosis of hand, foot and mouth disease.

The immunopathogenesis of severe hand, foot and mouth disease (HFMD) remains elusive. This study revealed that enterovirus 71 (EV71) epitope-specific CD4+ T cell responses of HFMD patients were skewed toward a Th2 cytokine profile. Patients that demonstrated higher levels of IL-4 expression in their CD4 T cells following antigen stimulation in vitro tended to have a more prolonged period of high fevers and a longer duration of illness. Thus, an increase of EV71 epitope-specific Th2 type response may portend the poor prognosis for some HFMD patients.

Children of rural-to-urban migrant workers in China are at a higher risk of contracting severe hand, foot and mouth disease and EV71 infection: a hospital-based study.

The incidence and severity of hand, foot and mouth disease have increased in mainland China since 2008. Therapies and vaccines are currently at different stages of development. This study aimed to determine the social factors associated with the outbreaks and severity of the disease in Chinese children. A multicentre, prospective, case-controlled study was conducted in Shanghai, Chongqing, Guangzhou and Shantou to identify the sociodemographic and behavioural risk factors for hand, foot and mouth disease. Children hospitalized for hand, foot and mouth disease were randomly enrolled from April to November 2011. Stool samples were collected to test for the presence of enterovirus 71 (EV71). A total of 443 children between 1.6 and 68 months of age were enrolled; 304 were uncomplicated cases and 139 were severe cases with central nervous system involvement. The overall detection rate of EV71 was 54.2%, and the positivity rate of EV71 was significantly higher in the severe group than in the uncomplicated group (82.0% versus 40.9%, odds ratio (OR): 8.35, P=0.000). The children of migrant workers (OR: 3.014, P=0.000) and children attending kindergarten (OR: 2.133, P=0.002) were significantly associated with a severe outcome of the disease (OR: 1.765, P=0.026). Our findings indicate that kindergarten attendance and migrant worker parents are the major risk factors associated with severe hand, foot and mouth disease in children <5 years of age. Future public health intervention vaccination campaigns should consider the particular difficulties of achieving high compliance with multiple-dose vaccination regimens in the children of migrant workers.

Seroepidemiology of Enterovirus 71 infection prior to the 2011 season in children in Shanghai.

BACKGROUND: In 2010, China experienced the largest outbreak on record of Enterovirus 71 (EV71)-associated Hand Foot and Mouth Disease (HFMD) with more than 1.7 million cases, 27,000 patients with severe neurological complications and 905 deaths. Understanding of the seroprevalence of neutralizing antibodies (NAb) against EV71 and their protective role against HFMD in children is crucial for the implementation of future therapeutic and prophylactic intervention. OBJECTIVES: To correlate the prevalence of NAb against EV71 genotype C4a in children prior to the 2011 epidemic season with severe EV71-associated HFMD disease during the subsequent 2011 epidemic season. STUDY DESIGN: 614 sera samples were collected from children without HFMD. EV71 NAb were tested by a quantitative PCR assay. Samples with NAb ≥1:8 were scored as positive. RESULTS: 122 (19.9%) of 614 sera were EV71-seropositive. The NAb seroprevalence was highest in infants 0-5 months of age (28.6%) and lowest in children 1-1.9 years of age (13.4%). 64.1% of severe EV71-associated HFMD occurred in children 1-2.9 years. CONCLUSIONS: Despite the large 2010 outbreak, the overall seroprevalence of EV71 in children is relatively low. The seropositive rate of EV71 NAb prior to the 2011 season was inversely correlated with the number of EV71-infected severe cases in 2011. Loss of maternal antibodies in infants and lack of acquired anti-EV71 immunity are responsible for increased proportion of severe HFMD in the 1-2 years age group. Our data suggest that future vaccination campaigns should be initiated as early as 6 months.

A dominant EV71-specific CD4+ T cell epitope is highly conserved among human enteroviruses.

CD4+ T cell-mediated immunity plays a central role in determining the immunopathogenesis of viral infections. However, the role of CD4+ T cells in EV71 infection, which causes hand, foot and mouth disease (HFMD), has yet to be elucidated. We applied a sophisticated method to identify promiscuous CD4+ T cell epitopes contained within the sequence of the EV71 polyprotein. Fifteen epitopes were identified, and three of them are dominant ones. The most dominant epitope is highly conserved among enterovirus species, including HFMD-related coxsackieviruses, HFMD-unrelated echoviruses and polioviruses. Furthermore, the CD4+ T cells specific to the epitope indeed cross-reacted with the homolog of poliovirus 3 Sabin. Our findings imply that CD4+ T cell responses to poliovirus following vaccination, or to other enteroviruses to which individuals may be exposed in early childhood, may have a modulating effect on subsequent CD4+ T cell response to EV71 infection or vaccine.

Open-label phase 1b pilot study to assess the antiviral efficacy of simvastatin combined with sertraline in chronic hepatitis C patients.

BACKGROUND: 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors inhibit HCV replication in vitro. The combination of sertraline and simvastatin has synergistic antiviral activity in vitro, but there are no prior in vivo studies. Our aims were to prospectively assess the antiviral efficacy and safety of this drug combination in chronic hepatitis C (CHC) patients. METHODS: A total of 15 CHC adults (including 1 control subject) that were treatment-naive or prior partial responders/relapsers to standard-of-care therapy were enrolled at four centres (2 in Singapore and 2 in the US). Patients received simvastatin 40 mg once daily and sertraline 50 mg once daily for 7 days, and then 80 mg once daily and 100 mg once daily, respectively, for another 21 days with a 14-day follow-up. RESULTS: Of the 15 CHC patients, 13 completed the study. Subjects were mostly Caucasian (8/15), mean age 49.1 ±9 years and the genotype distribution was 1=10, 2=2 and 3=3. No subject discontinued dosing due to adverse events. Mean HCV RNA change from baseline was from -0.005 to -0.236 log(10) IU/ml across study intervals. Three subjects had transient >1 log(10) HCV RNA declines. No subject achieved >2 log(10) HCV RNA decline. CONCLUSIONS: The combination of sertraline and simvastatin is well-tolerated over the short-term, but has no significant antiviral or anti-inflammatory response in CHC patients. This may reflect in vivo differences in synergy between statin and/or selective serotonin reuptake inhibitors and incomplete inhibition of membrane protein prenylation with statin therapy.