Molecular Epidemiology of Enteroviruses Associated with Hand, Foot, and Mouth Disease in the Mainland of China

Hand, foot, and mouth disease （HFMD） is a common contagious illness which occurs worldwide both sporadically and in epidemics. The disease mainly affects, children and the typical symptoms, which may resolve spontaneously, include mucocutaneous papulovesicular lesions on the hands, feet, mouth, and buttocks. In rare cases, however, the patients may also develop neurological complications such as neurogenic pulmonary edema,

Detection and Identification of Enteroviruses RNA by Using Polymerase Chain Reaction

For rapid diagnosis of enteroviral infection in clinic practice, we developed a reverse transcription and polymerase chain reaction (RT-PCR) assay.Primers homologous to the conserved 5' non-coding region were designed by analyzing enteroviral genomes, and then they were used to enzymatically amplify RNA from 31 prototype enteroviral strains and enteroviruses (EV) in cerebrospinal fluid (CSF) of 34 cases of aseptic meningitis and 11 cases of aseptic encephalitis. The RT-PCR products generated with these enteroviral primers were analyzed by agar gel electrophoresis and dot blot hybridization analysis. 31 EV strains showed an obvious monoclonal amplification band, and all dot blot hybridization results were positive. Four other viruses and cells cultured were all negative. The study of sensitivity of the RT-PCR showed that amplification production were positive to 10(-2)- 10(-3) 50% tissue culture infective doses. With this assay, 21(61.8%) of 34 aseptic meningitis and 8 (72. 7%) of 11 aseptic encephalitis contained EV RNA in CSF samples. Two cases of meningitis and one of encephalitis with EV infection were still positive during convalescence. Our results suggest that this RT-PCR method was a fast, sensitive and specific technique for detection of common EV infection.

Can Non-Polio Enteroviruses Be Tamed with a Vaccine to Minimize Paralysis Caused by Them?

Background: While we are inching towards global eradication of polio, the paralysis due to non-polio viruses (NPEV) poses greater challenge. Factors responsible for causing Acute Flaccid Paralysis (AFP) were studied in 3596 AFP patients in 64 districts of Uttar-Pradesh, India, to observe indirect relationship of AFP with wild polio as well as NPEV. A recent study suggests the need to investigate polio virus negative but NPEV positive AFP cases. Methods: The lab results of the stool samples of these children were line listed and analysed to observe the association of various factors with respect to presence of paralysis on 60 follow-up days. Taking zero OPV dose AFP cases as a biological base, we studied the relationship of presence of paralysis at 60 follow-up days to that of presence of NPEV in stool samples while polio virus was present or absent. Results: 70 of the 86 AFP cases (81%) with zero OPV dose and having only NPEV isolated in stool samples were having paralysis at 60 follow-up days. There were 4.54% (162) AFP cases, which did not carry any polio virus but were having NPEV isolated in the stool samples and paralysis at 60 follow-up days. 79% (75/95) of zero OPV dose children, who were having residual weakness at 60 follow-up days, were carrying both polio virus as well as NPEV in their stool samples. Total AFP cases, having residual weakness at 60 follow-up days and having NPEV in stool samples, decreased with increase in OPV doses;a behavior similar to what wild polio viruses (WPV) have to OPV. Conclusions: Maybe polio like NPEV is active for causing severe paralysis in children and is responding to the OPV. As is evident in the studies by M. Margalith, B. Fattal et al. [1] that there is an antibody response to the enteroviruses, we can think of coming out with a vaccine against the enteroviruses. Therefore, enterovirus vaccine can be produced on similar lines to that of OPV, as now we have enough isolates of NPEV. Effective NPEV surveillance system also needs to be in place.

In vitro screening of traditionally used medicinal plants in China against Enteroviruses

AIM： To search for new antiviral agents from traditional Chinese medicine, specifically anti-enterovirosuses agents. METHODS： The aqueous extracts （AE） of more than 100 traditionally used medicinal plants in China were evaluated for their in vitro anti-Coxsackie virus B3 activities with a MTT-based colorimetric assay. RESULTS： The test for AE of 16 plants exhibited anti- Coxsackie virus B3 activities at different magnitudes of potency. They can inhibit three steps （inactivation, adsorption and replication） during the infection. Among the 16 plants, Sargentodoxa cuneata （Oliv.） Rehd. et Wils., Sophora tonkinensis Gapnep., Paeonia veitchii Lynch, Spatholobus suberectus Dunn. and Cyrtorniurn fortunei J, sm. also have activity against other enterovirus, including Coxsackie virus 135, Polio virus I, Echo virus 9 and Echo virus 29. Cell cytotoxic assay demonstrated that all tested AE had CC50 values higher than their EC50 values. CONCLUSION： The sixteen traditionally used medicinal plants in China possessed antMral activity, and some of them merit further investigations.

Detection and quantification of enteroviruses in coastal seawaters from Bohai Bay,Tianjin,China

An 8-month survey was conducted to detect and quantify enteroviruses in Tianjin coastal seawaters of Bohai Bay to assess coastal water quality. Ten water samples were collected from Bohai Bay for the detection and quantification of enteroviruses by conventional reverse transcription polymerase chain reaction （RT-PCR） and SYBR Green real-time quantitative RT-PCR （qRT-PCR）. Total viral nucleic acid was extracted from 500 mL of seawater samples concentrated by Centricon plus-70 centrifugal filter devices. The viral recovery rate was 29.1% based on viral seeding study. The centrifugal ultrafiltration method applied is effective for viral recovery from small volume of polluted water, which may have broader applications to monitoring human virus in aquatic environment. Our results indicated that there was a severe viral contamination in seawater of Bohai Bay. Enteroviruses were detected at concentrations ranging from 1.7 × 10^6 to 6.3 × 10^7 copies/L by qRT-PCR. Sequencing analyses identified that all of the twenty clones as poliovirus type 2. This is the first quantitative report of human viruses in coastal waters of a metropolitan city in China. This study emphasized the importance for the local and central governmertts to monitor and assess the water quality.

Functional Dyspepsia and Chronic Gastritis Associated with Enteroviruses

After decades of research, functional dyspepsia (FD) remains one of the most elusive gastrointestinal disorders. Endoscopic appearance of mild inflammation of the gastric mucosa without ulceration and microscopic evidence of mild chronic inflammation are often considered as normal findings since no etiology could be found other than H. Pylori. Enteroviruses infect the gastrointestinal tract and have been shown to persist in the stomach of symptomatic patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). In this study, we evaluated FD patients with and without the diagnosis of ME/CFS, and were able to support the viral protein staining with finding of double-stranded RNA in 63% of the same stomach biopsies by immunoperoxidase staining. Furthermore, we clarified the possible cross-reaction with creatine kinase brain subtype (CKB), present in parietal cells, using antibody competition experiments and western blot analysis of stomach proteins. Viral protein+ and dsRNA+ biopsies were infectious in SCID mice. More research is needed to elucidate the mechanism of enterovirus infection of the stomach associated with FD and chronic gastritis.

Evolutionary pattern of 5'-UTR of enteroviruses and primer update for the detection of enteroviral RNA in environmental samples

Objective:To study the recombination events among enterovirus strains and the development of specific primers for the detection of enteroviruses in environmental samples.Methods: Nucleotide sequence analysis of enteroviruses deposited in the international database GenBank （www.ncbi.nlm.nih.gov/Genbank） was conducted to develop specific primers for the detection of these viruses.The specificity and sensitivity of the method were tested using coxackicvirus B3 strain Nancy,environmental isolate of human hepatitis A virus and human rotavirus strain WA.Seventy sewage samples were analyzed.Results:Enterovirus genome was detected in all positive samples.The genome of enterovirus was not detected in negative samples.The level of detection of these viruses was 102 TCID50mI..Conclusions:The development of new primers is an important issue for the detection of enteroviruses in the environment and the assessment of risk factors to human health.

Integration of HiBiT into enteroviruses:A universal tool for advancing enterovirus virology research

The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications,enabling the development of effective therapeutic and preventive strategies.In this study,our initial attempts to introduce a NanoLuc luciferase(NLuc)reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies.We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus.Therefore,we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71(EV-A71),coxsackievirus A10(CVA10),coxsackievirus A7(CVA7),coxsackievirus A16(CVA16),namely EV-A71-HiBiT,CVA16-HiBiT,CVA10-HiBiT,CVA7-HiBiT,and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency.Notably,the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses.A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics.Moreover,these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages.In addition,the HiBiT-tagged viruses were successfully tested in antiviral drug assays,and the sensitivity of the viruses to drugs was not affected by the HiBiT tag.Ultimately,our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal,convenient,and invaluable method for advancing research in the realm of enterovirus virology.Furthermore,HiBiT-tagged enteroviruses exhibit great potential for diverse applications,including the development of antivirals and the elucidation of viral infection mechanisms.

Seroprevalence of neutralizing antibodies against HFMD associated enteroviruses among healthy individuals in Shanghai,China,2022

Dear Editor,Hand,foot and mouth disease(HFMD)is one of the most common infectious diseases,particularly in the Asia-Pacific region.In the past two decades,HFMD rises to prominence for its heavy burden,with over one million cases reported annually.Before 2013,enterovirus A71(EV-A71)and Coxsackievirus A16(CVA16)were the main pathogens leading to HFMD in the mainland of China(Yang et al.,2017).In recent years,non-EV-A71-non-CVA16 other enteroviruses,such as Coxsackievirus A6(CVA6),Coxsackievirus A10(CVA10)and Coxsackievirus A4(CVA4),were frequently reported and replaced EV-A71 and CVA16 becoming the major causative agents of HFMD(Zhou et al.,2021;Wang et al.,2022).

Stability and infectivity of enteroviruses on dry surfaces:Potential for indirect transmission control

Hand,foot,and mouth disease(HFMD)is a contagious disease mainly occurring in young children,and outbreaks commonly occur among young children in the Asia–Pacific region including Thailand.Moreover,the World Health Organization(WHO)monitors HFMD in the Western Pacific region to detect outbreaks and other significant events by the Regional Event Based Surveillance System.HFMD is mainly caused by a group of enteroviruses(EVs)transmitted through direct contact(person to person)and indirect contact with contaminated objects(surface-to-hand).However,few studies have examined the surface stability of EVs.In this study,we investigated the stability of enterovirus A71(EV-A71)and coxsackievirus A16(CVA16)on three different dry surfaces(wood,plastic,and stainless steel)using the endpoint titration using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)staining of viable cells and real-time polymerase chain reaction(viral genome detection).The results revealed that virus infectivity dramatically decreased within a few hours on dry surfaces.However,viral RNA could be detected on dry surfaces for up to 28 days.Concerning heat inactivation,both EV-A71 and CVA16 were inactivated after exposure to 60°C for 15 min.Information on virus stability on different dry surfaces will provide useful information for HFMD transmission control.

Laboratory diagnosis of nonpolio enteroviruses:A review of the current literature

Infections by nonpolio enteroviruses(EVs)are highly prevalent,particularly among children and neonates,where they may cause substantial morbidity and mortality.Laboratory diagnosis of these viral infections is important in patient prognosis and guidance of clinical management.Although the laboratory diagnosis of non-polio EVs is mainly based on molecular techniques,classical virus-isolation techniques are still used in refer-ence laboratories.Other techniques,such as antigen detection and serology,are becoming obsolete and rarely used in diagnosis.An important part of diagnosis and surveillance of EV infections is viral typing by VP1 gene sequencing using conventional Sanger technique and more recently,full-genome next-generation sequencing.The latter allows the typing of all EVs,better investigation of EV outbreaks,detection of coinfec-tion,and identification of severity markers in the EV genome.

A one-step reverse-transcription recombinase aided PCR assay for the rapid and sensitive detection of human enteroviruses

Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR),the gold-standard method,still has shortfalls in diagnostic sensitivity and timeliness.Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay(RT-RAP)to detect HEV fragment within an hour.The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains.Among 15 types of HEV(species A-C),the sensitivity of RT-RAP was approximately 2-8 folds lower than that of the qRT-PCR in 9 types,and no-cross reaction with other viruses was observed.RT-RAP was further applied to analyze CSF and fecal specimens;the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results.These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.

Antiviral Drugs(Synthetic Small Molecule Inhibitors and Nature Drugs)Against EV71 in Enteroviruses:Advances and Perspectives

Background:Enterovirus 71(EV71)is a major virus that causes hand-foot-mouth disease.In cases of infants and young children,EV71 infection has been associated with severe neurological disease and potentially fatal systemic complications.The sporadic outbreak worldwide is increasingly prevalent in the Asia-Pacific region,where it has become a major public health concern.Objective:No specific antiviral drugs are currently approved for the treatment of EV71 infection.The purpose of this study is to comprehensively review the research progress of anti-EV71 drugs(synthetic small molecule inhibitors and nature drugs)in the past twenty years,and further to promote the research and development of antiviral drugs against enterovirus infection.Methods:This study reviewed the drugs on anti EV71 in the past decades.The literature search in PubMed database was conducted for original studies and review articles on drugs against enterovirus 71.Related articles published in English were selected for study and discussion.Results:As reviewed in this paper,bioactive molecules include receptor analogues,protease inhibitors,natural drugs derived from traditional chinese medicine or natural medicine.These bioactive molecules have shown significant effectiveness in inhibiting the entry and replication of EV71 in vitro and in vivo experiments.Conclusion:This review demonstrated that the entry receptor of EV71 into host cells has been studied,and receptor drugs against enterovirus have been made some progress,but most receptor analogues have not been reported.Further research is needed in this area in the future.On the other hand,the protease inhibitors have always been a major aspect of anti-enterovirus research and can be developed as antiviral agents for clinical application.In terms of natural drugs,many monomers derived from traditional chinese medicine or natural medicine have good antiviral activity and little toxic and side effects on host cells,but in view of their multi-target properties,the mechanism of drug action needs to be further studied.

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.

Simultaneous detection of enteroviruses from surface waters by real-time RT-PCR with universal primers

In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green I was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy （GEC）/μL, the intraand inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship （r 2 = 0.997） between the logarithm of viral density and cycle threshold in a wide range of 2.31 × 10 0 to 2.31 × 10 9 GEC/μL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.

Ectopic Expression of TRIM25 Restores RIG-I Expression and IFN Production Reduced by Multiple Enteroviruses 3Cpro

Enteroviruses(EVs) 3C proteins suppress type I interferon(IFN) responses mediated by retinoid acid-inducible gene I(RIG-I), while an E3 ubiquitin ligase, tripartite motif protein 25(TRIM25)-mediated RIG-I ubiquitination is essential for RIG-I antiviral activity. Therefore, whether the effect of EVs 3C on RIG-I is associated with TRIM25 expression is worth to be further investigated. Here, we demonstrate that 3C proteins of EV71 and coxsackievirus B3(CVB3) reduced not only RIG-I expression but also TRIM25 expression through protease cleavage activity, while overexpression of TRIM25 restored RIG-I expression and IFN-b production reduced by 3C proteins. Further investigation confirmed that the two amino acids and functional domains in TRIM25 required for RIG-I ubiquitination and TRIM25 structural conformation were essential for the recovery of RIG-I expression. Moreover, we also observed that TRIM25 could rescue RIG-I expression reduced by 3C proteins of CVA6 and EV-D68 but not CVA16. Our findings provide an insightful interpretation of 3C-mediated host innate immune suppression and support TRIM25 as an attractive target against multiple EVs infection.

Gp78 regulates PMP22 and causes ER stress and autophagy in EV71-VP1-overexpressing mouse Schwann cells

Background:During Enterovirus type 71(EV71)infection,the structural viral protein 1(VP1)activates endoplasmic reticulum(ER)stress associated with peripheral myelin protein 22(PMP22)accumulation and induces autophagy.However,the specific mechanism behind this process remains elusive.Methods:In this research,we used the VP1-overexpressing mouse Schwann cells(SCs)models co-transfected with a PMP22 silencing or Autocrine motility factor receptor(AMFR/gp78)overexpressing vector to explore the regulation of gp78 on PMP22 and its relationship with autophagy and apoptosis.Results:The activity of gp78 could be influenced by EV71-VP1,leading to a decrease in the ubiquitination and degradation of PMP22,resulting in PMP22 accumulation in ER.In VP1-overexpressing mouse SCs,all three ER stress sensors,including pancreatic endoplasmic reticulum kinase(PERK),activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1)and the related downstream signals(C/EBP-homologous protein(CHOP)and Caspase 12)were activated,as well as the ER-resident chaperone Glucose-regulated protein 78(GRP78).In addition,VP1 upregulated the autophagy marker Microtubule-associated protein 1 light chain 3 beta(LC3B),while PMP22 silencing or gp78 overexpression reversed the phenomenon.Meanwhile,PMP22 silencing or gp78 overexpression increased proliferation of EV71-VP1-transfected mouse SCs.Conclusion:Gp78 could regulate PMP22 accumulation through ubiquitination degradation and cause ER stress and autophagy in EV71-VP1-overexpressing mouse SCs.Therefore,the gp78/PMP22/ER stress axis might emerge as a promising therapeutic target for myelin and neuronal damage induced by EV71 infection.

Epidemiology and etiology of hand,foot,and mouth disease in Zhengzhou,China,from 2009 to 2021

Background:Hand,foot,and mouth disease(HFMD)is a common childhood infectious disease caused by a variety of enteroviruses(EVs).To explore the epidemiological characteristics and etiology of HFMD in Zhengzhou,China,we conducted a systematic analysis of HFMD surveillance data from Zhengzhou Center for Disease Control and Prevention from January 2009 to December 2021(https://wjw.zhengzhou.gov.cn/).Methods:Surveillance data were collected from Zhengzhou Center for Disease Control and Prevention from January 2009 to December 2021(https://wjw.zhengzhou.gov.cn/).Cases were analyzed according to the time of onset,type of diagnosis,characteristics,viral serotype,and epidemiological trends.Results:We found that the primary causative agent responsible for the HFMD outbreaks in Zhengzhou was Enterovirus A71(EVA-71)(48.56%)before 2014.After 2015,other EVs gradually became the dominant strains(57.68%).The data revealed that the HFMD epidemics in Zhengzhou displayed marked seasonality,with major peaks occurring from April to June,followed by secondary peaks from October to November,except in 2020.Both the severity and case-fatality ratio of HFMD decreased following the COVID-19 pandemic(severity‰:13.46 vs.0.17;case-fatality‰:0.21 vs.0,respectively).Most severe cases were observed in patients aged 1 year and below,accounting for 45.81%.Conclusions:Overall,the incidence rate of HFMD decreased in Zhengzhou following the introduction of the EVA-71 vaccine in 2016.However,it is crucial to acknowledge that HFMD prevalence continues to exhibit a distinct seasonal pattern and periodicity,and the occurrence of other EV infections poses a new challenge for children’s health.

A review of the recombination events,mechanisms and consequences of Coxsackievirus A6

Hand,foot,and mouth disease(HFMD)is one of the most common class C infectious diseases,posing a seri-ous threat to public health worldwide.Enterovirus A71(EV-A71)and coxsackievirus A16(CV-A16)have been regarded as the major pathogenic agents of HFMD;however,since an outbreak caused by coxsackievirus A6(CV-A6)in France in 2008,CV-A6 has gradually become the predominant pathogen in many regions.CV-A6 infects not only children but also adults,and causes atypical clinical symptoms such as a more generalized rash,eczema herpeticum,high fever,and onychomadesis,which are different from the symptoms associated with EV-A71 and CV-A16.Importantly,the rate of genetic recombination of CV-A6 is high,which can lead to changes in virulence and the rapid evolution of other characteristics,thus posing a serious threat to public health.To date,no specific vaccines or therapeutics have been approved for CV-A6 prevention or treatment,hence it is essential to fully understand the relationship between recombination and evolution of this virus.Here,we systematically review the genetic recombination events of CV-A6 that have occurred worldwide and explore how these events have promoted virus evolution,thus providing important information regarding future HFMD surveillance and prevention.

Expression and clinical significance of pattern recognition receptor-associated genes in hand, foot and mouth disease

Objective:To explore which pattern recognition receptors(PRRs)play a key role in the development of hand,foot,and mouth disease(HFMD)by analyzing PRR-associated genes.Methods:We conducted a comparative analysis of PRR-associated gene expression in human peripheral blood mononuclear cells(PBMCs)infected with enterovirus 71(EV-A71)which were derived from patients with HFMD of different severities and at different stages.A total of 30 PRR-associated genes were identified as significantly upregulated both over time and across different EV-A71 isolates.Subsequently,ELISA was employed to quantify the expression of the six most prominent genes among these 30 identified genes,specifically,BST2,IRF7,IFI16,TRIM21,MX1,and DDX58.Results:Compared with those at the recovery stage,the expression levels of BST2(P=0.027),IFI16(P=0.016),MX1(P=0.046)and DDX58(P=0.008)in the acute stage of infection were significantly upregulated,while no significant difference in the expression levels of IRF7(P=0.495)and TRIM21(P=0.071)was found between different stages of the disease.The expression levels of BST2,IRF7,IFI16 and MX1 were significantly higher in children infected with single pathogen than those infected with mixed pathogens,and BST2,IRF7,IFI16 and MX1 expression levels were significantly lower in coxsackie B virus(COXB)positive patients than the negative patients.Expression levels of one or more of BST2,IRF7,IFI16,TRIM21,MX1 and DDX58 genes were correlated with PCT levels,various white blood cell counts,and serum antibody levels that reflect disease course of HFMD.Aspartate aminotransferase was correlated with BST2,MX1 and DDX58 expression levels.Conclusions:PRR-associated genes likely initiate the immune response in patients at the acute stage of HFMD.