Diagnostic performance of different specimens in detecting enterovirus A71 in children with hand,foot and mouth disease

Hand,foot and mouth disease(HFMD)is a major public health problem among children in the Asia-Pacific region.The optimal specimen for HFMD virological diagnosis remains unclear.Enterovirus A71(EV-A71)neutralizing antibody titres detected in paired sera were considered the reference standard for calculating the sensitivity,specificity,positive and negative predictive value of throat swabs,rectal swabs,stool,blood samples and cerebrospinal fluid(CSF)by RT-PCR or ELISA assay.In this study,clinical samples from 276 HFMD patients were collected for analysing the sensitivity of different kind of specimens.Our results showed that stool had the highest sensitivity(88%,95%CI:74%–96%)and agreement with the reference standard(91%).The order of diagnostic yield for EV-A71 infection was stool samplerectal swab>throat swab>blood sample>CSF sample,and using a combination of clinical samples improved sensitivity for enterovirus detection.The sensitivity of ELISA for IgM antibody detection in sterile-site specimens was significantly higher than that of RT-PCR(serum/plasma:62%vs.2%,CSF:47%vs.0%)(P<0.002).In conclusion,our results suggest that stool has the highest diagnostic yield for EV-A71-infected HFMD.If stool is unavailable,rectal swabs can be collected to achieve a similar diagnostic yield.Otherwise,throat swabs may be useful in detecting positive samples.Although IgM in blood or CSF is diagnostically accurate,it lacks sensitivity,missing 40%–50%of cases.The higher proportion of severe cases and shorter interval between onset and sampling contributed to the increase in congruency between clinical testing and the serological reference standard.

Seroprevalence of human enterovirus A71 in Guangzhou,China,2019-2021

Enterovirus A71 (EV-A71) is a significant hand-foot-mouth disease (HFMD) etiology. The inactivated EV-A71 vaccines were approved in China in 2016. However, the seroprevalence of EV-A71 after the vaccine application and its potential association with the EV-A71 epidemic in the population are rarely studied. In this study, we analyzed the incidence of EV-A71 infection and seroepidemiology in Guangzhou City, China. From 2019 to 2021, 167,920 clinically confirmed HFMD cases were reported in Guangzhou. In 6,868 enterovirus-positive samples, Coxsackievirus A6 and Coxsackievirus A16 were dominant genotypes, and only 3 EV-A71-positive samples were detected, highlighting the deficient epidemic activity of EV-A71. Microneutralization assay was performed on 1,000 representative serum samples. Notably, the seroprevalence and geometric mean titer (GMT) decreased significantly in 2020, and that in the < 3-year age group were increased and even higher than that in 3–5-year age group in 2019 and 2021, which was contrary to our previous surveillance result and other studies in Guangzhou. Furthermore, a moderate decline of GMT level was observed following the vaccination, but the seropositive serums were still detected for 49 months after second immunization, suggesting the long-term persistence of the immunity. Our seroepidemiology study revealed relatively higher neutralizing antibody activity in the susceptible population after the EV-A71 vaccine was adopted in 2016 in Guangzhou. It may be one of the reasons for the lower epidemic activity of EV-A71 in Guangzhou from 2019 to 2021.

The impact of EV71 vaccination program on hand,foot and mouth disease in Zhejiang Province,China:A negative control study

Objective:To estimate the potential causal impact of Enterovirus A71(EV71)vaccination program on the reduction of EV71-infected hand,foot,and mouth disease(HFMD)in Zhejiang Province.Methods:We utilized the longitudinal surveillance dataset of HFMD and EV71 vaccination in Zhejiang Province during 2010-2019.We estimated vaccine efficacy using a Bayesian structured time series(BSTS)model,and employed a negative control outcome(NCO)model to detect unmeasured confounding and reveal potential causal association.Results:We estimated that 20,132 EV71 cases(95%CI:16,733,23,532)were prevented by vaccination program during 2017-2019,corresponding to a reduction of 29%(95%CI:24%,34%).The effectiveness of vaccination increased annually,with reductions of 11%(95%CI:6%,16%)in 2017 and 66%(95%CI:61%,71%)in 2019.Children under 5 years old obtained greater benefits compared to those over 5 years.Cities with higher vaccination coverage experienced a sharper EV71 reduction compared to those with lower coverage.The NCO model detected no confounding factors in the association between vaccination and EV71 cases reduction.

Enterovirus A71 antivirals:Past,present,and future

Enterovirus A71(EV-A71)is a significant human pathogen,especially in children.EV-A71 infection is one of the leading causes of hand,foot,and mouth diseases(HFMD),and can lead to neurological complications such as acute flaccid myelitis(AFM)in severe cases.Although three EV-A71 vaccines are available in China,they are not broadly protective and have reduced efficacy against emerging strains.There is currently no approved antiviral for EV-A71.Significant progress has been made in developing antivirals against EV-A71 by targeting both viral proteins and host factors.However,viral capsid inhibitors and protease inhibitors failed in clinical trials of human rhinovirus infection due to limited efficacy or side effects.This review discusses major discoveries in EV-A71 antiviral development,analyzes the advantages and limitations of each drug target,and highlights the knowledge gaps that need to be addressed to advance the field forward.

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71

Enterovirus A71(EV-A71)is one of the etiological pathogens leading to hand,foot,and mouth disease(HFMD),which can cause severe neurological complications.The neuropathogenesis of EV-A71 infection is not well understood.The mislocalization and aggregation of TAR DNA-binding protein 43(TDP-43)is the pathological hallmark of amyotrophic lateral sclerosis(ALS).However,whether TDP-43 was impacted by EV-A71 infection is unknown.This study demonstrated that TDP-43 was cleaved during EV-A71 infection.The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection.TDP-43 is cleaved by viral protease 3 C between the residues 331 Q and332 S,while mutated TDP-43(Q331 A)was not cleaved.In addition,mutated 3 C which lacks the protease activity failed to induce TDP-43 cleavage.We also found that TDP-43 was translocated from the nucleus to the cytoplasm,and the mislocalization of TDP-43 was induced by viral protease 2 A rather than 3 C.Taken together,we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection,implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71 infection.

Structural and functional analysis of a potent human neutralizing antibody against enterovirus A71

Enterovirus A71(EV-A71) causes major outbreaks of hand,foot,and mouth disease(HFMD) in many countries,most frequently affecting children,and a small proportion of cases may lead to death.Currently,no vaccine is available in most endemic regions,and no licenced treatments for EV-A71 infection are available.Here,we characterize a human monoclonal antibody(Hu MAb),E1,by screening a Fab antibody phage library derived from patients who recovered from EV-A71 infection.E1 exhibits strong neutralizing activity against EV-A71 virus in cells.The cryo-electron microscopy(cryo-EM) structures of the EV-A71 virion in complex with E1 Fab fragments demonstrated that E1 recognized an epitope formed by residues in the BC and HI loops of VP1.In a mouse model,E1 effectively protected against lethal EV-A71 challenge in both prophylactic and therapeutic treatment.In particular,E1 significantly reduces virus titers and muscle damage.E1 might represent a potential adjunct to EV-A71 treatment.

Genetic Variation of Multiple Serotypes of Enteroviruses Associated with Hand, Foot and Mouth Disease in Southern China

Enteroviruses(EVs)species A are a major public health issue in the Asia–Pacific region and cause frequent epidemics of hand,foot and mouth disease(HFMD)in China.Mild infections are common in children;however,HFMD can also cause severe illness that affects the central nervous system.To molecularly characterize EVs,a prospective HFMD virological surveillance program was performed in China between 2013 and 2016.Throat swabs,rectal swabs and stool samples were collected from suspected HFMD patients at participating hospitals.EVs were detected using generic real-time and nested reverse transcription-polymerase chain reactions(RT-PCRs).Then,the complete VP1 regions of enterovirus A71(EV-A71),coxsackievirus A16(CVA16)and CVA6 were sequenced to analyze amino acid changes and construct a viral molecular phylogeny.Of the 2836 enrolled HFMD patients,2,517(89%)were EV positive.The most frequently detected EVs were CVA16(32.5%,819),CVA6(31.2%,785),and EV-A71(20.4%,514).The subgenogroups CVA16B1 b,CVA6D3 a and EV-A71C4 a were predominant in China and recombination was not observed in the VP1 region.Sequence analysis revealed amino acid variations at the 30,29 and 44 positions in the VP1 region of EV-A71,CVA16 and CVA6(compared to the respective prototype strains Br Cr,G10 and Gdula),respectively.Furthermore,in 21 of 24(87.5%)identified EV-A71 samples,a known amino acid substitution(D31 N)that may enhance neurovirulence was detected.Our study provides insights about the genetic characteristics of common HFMD-associated EVs.However,the emergence and virulence of the described mutations require further investigation.