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.

Molecular Epidemiological Analysis of Echovirus 19 Isolated From an Outbreak Associated With Hand, Foot, and Mouth Disease (HFMD) in Shandong Province of China

To elucidate the genetic characterization and molecular epidemiological features of Echovirus 19 （El9） isolates collected from an outbreak associated with hand, foot and mouth disease （HFMD） in Tai＇an city of Shandong Province of China from July to September, 2003. Methods Thirty seven Echovirus 19 isolates were isolated from stool specimens and throat swabs collected during the outbreak, then major capsid （VP1） genomic sequence was determined, and phylogenetic tree was done based on the VP1 sequences among these 37 and other El9 viruses deposited in the Genbank. Also a representative strain named CHN-SD03-TN12 was selected for sequencing of 5′-untranslated regions （5′-UTR）. Results The identity rate was about 98.9%-100% among all these 37 El9 viruses. The genetic relationships between these 37 El9 isolates and other strains reported were also depicted. The identity rate was about 78.4%-78.9% compared with El9 reference strain Burke. The substitutions in the sequence of 5′-UTR resulted in changes in the conjectural properties of 5′-UTR of El9 viruses. Condusion The genetic features of El9 viruses isolated during the outbreak in Shandong Province in 2003 may be associated with a genetic and antigenic drift that changes the virulence of the Shandong isolates, but the molecular changes in Shandong El9 viruses contributing to their phenotype remain to be further illuminated. However, the sequences described in this paper substantiate the changes taken place in capsid VPI and 5′UTR regions. These substitutions may contribute to their tropism and virulence, and play a significant role in pathogenesis and clinical manifestations of the disease.

Construction of Plant Expression Vector for Hand,Foot and Mouth Virus EV71-VP1 Gene and Its Expression in Tomato

EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.

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,

The Comparative Study on Two Models of Syndrome Differentiation of the Hand, Foot and Mouth Disease: An Investigation Analysis of the Signs and Symptoms on 2325 Cases

Objective To realize the characteristics of "zheng" differentiation-treatment for hand, foot and mouth disease(HFMD), a new methodology of syndrome differentiation for different stages of HFMD has been explored. Methods Total of 2 325 cases with HFMD were recorded by distributing them into exterior syndrome stage, interior syndrome stage, severe syndrome stage and recovered syndrome stage, respectively, and the main symptoms and subsidiary symptoms of different stages of HFMD have been observed. The major and minor pathogenesis of HFMD in different stages were obtained, and compared with the "2010 Guideline for the Diagnosis and Treatment of HFMD". Results It was found that the major pathogenesis of exterior stage was defined as "the invation of the wenevil to the defender of the body with the collaterals got involved ", and the minor as "qi deficiency"; in interior stage, "the fury of Gan-Yang" was the main pathogenesis, and "qi in chaos and qi deficiency" was the minor; in severe syndrome stage, "the damage of heart, liver and lung" was the main pathogenesis, and "qi in chaos" was the minor; and the pathogenesis of recovered stage was "qi-yin deficiency". Compared with the "2010 Guideline for the Diagnosis and Treatment of HFMD", it showed that "the obstruction of the fei-pi qi by the mixture of shi-re evil" and "the mixture of shi-re" in vivo was quite difficult to be explained in completely different context in the general situation; in the severe stage, the TCM clinical characteristics of syndrome differentiation might lose; in the early acute severe cases, the phenomenon that xin-yang and fei-qi almost ran out was difficult to be observed, then, the line between the severe and the acute severe became vague.Conclusions The theory of syndrome differentiation by stages of HFMD was reasonable in the actual situation of clinical description on HFMD which was expected to be further tested and widely applied in the "zheng" differentiation-treatment of HFMD in the future.

Regional characteristics and spatiotemporal differentiation of the prevalence of hand, foot, and mouth disease in Xinjiang, China

In recent years,the number of hand,foot,and mouth disease(HFMD)cases in China has continued to grow,and the disease has become a serious public health issue.Xinjiang Uygur Autonomous Region is located in the arid areas of Northwest China,where the epidemiological trend of HFMD is gradually increasing and characterized by geographical heterogeneity.In this study,based on the HFMD case data in all counties and cities in Xinjiang,we employed statistical and GIS spatial analyses,and geographic probe models to characterize the spatiotemporal differentiation of HFMD epidemics in Xinjiang during the period of 2009–2018,and quantitatively analyzed the factors influencing the spatial differentiation of HFMD epidemics.The results showed that HFMD incidence rate in Xinjiang had non-stationary temporal characteristics on the interannual and monthly scales,and the monthly variation characteristics of HFMD epidemic were quite different in southern and northern Xinjiang.The spatial distribution characteristics of HFMD epidemics showed a north–south spatial differentiation pattern with the Tianshan Mountains as the boundary;cold spot and hot spot of HFMD epidemics in Xinjiang have shifted from scattered to concentrated,and the spatial differentiation pattern had gradually stabilized.Moreover,the dominant factors influencing the spatial differentiation of HFMD epidemics in Xinjiang were socioeconomic factors,such as per capita GDP and urbanization rate,while the basic factors affecting its spatial differentiation were natural environmental factors.The spatial differentiation and evolution patterns of HFMD epidemics differed between northern and southern Xinjiang.Specifically,the leading role of socioeconomic factors is more obvious in southern Xinjiang than in northern Xinjiang,while natural environmental factors(e.g.,dryness and relative humidity)contribute to the prevalence of HFMD epidemics in northern Xinjiang,and the perturbing effect of these factors was more prominent than other factors.The results of this study can provide a scientific basis for the prevention of HFMD epidemics and early warning of HFMD epidemics in Xinjiang.

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.

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.

From Monovalent to Multivalent Vaccines, the Exploration for Potential Preventive Strategies Against Hand, Foot, and Mouth Disease(HFMD)

Hand,foot,and mouth disease(HFMD)recently emerged as a global public threat.The licensure of inactivated enterovirus A71(EV-A71)vaccine was the first step in using a vaccine to control HFMD.New challenges arise from changes in the pathogen spectrum while vaccines directed against other common serotypes are in the preclinical stage.The mission of a broad-spectrum prevention strategy clearly favors multivalent vaccines.The development of multivalent vaccines was attempted via the simple combination of potent monovalent vaccines or the construction of chimeric vaccines comprised of epitopes derived from different virus serotypes.The present review summarizes recent advances in HFMD vaccine development and discusses the next steps toward a safe and effective HFMD vaccine that is capable of establishing a crossprotective antibody response.

National Epidemiology and Evolutionary History of Four Hand, Foot and Mouth Disease-Related Enteroviruses in China from 2008 to 2016

Hand, foot and mouth disease(HFMD) is a major public health concern in China. The most predominant enteroviruses that cause HFMD have traditionally been attributed to enterovirus A71(EVA71) and coxsackievirus A16(CVA16). Since its first large outbreak in 2008, the dominant HFMD pathogens are constantly changing. In 2013 and 2015, CVA6 exceeded both EVA71 and CVA16 to become the leading cause of HFMD in some provinces. However, there still lacks a comprehensive overview on the molecular epidemiology and evolution of HFMD-related enteroviruses at the national level. In this study, we performed systematic epidemiological analyses of HFMD-related enteroviruses using the data of 64 published papers that met the inclusion criteria, and conducted phylogenetic analyses based on 12,080 partial VP1 sequences identified in China before 31 st June 2018. We found that EVA71 prevalence has decreased sharply but other enteroviruses have increased rapidly from 2008 to 2016 and that one subtype of each enterovirus is represented during the epidemic. In addition, four genotypes EVA71_C4, CVA16_B1, CVA6_D and CVA10_C are the most predominant enterovirus strains and collectively they cause over 90% of all HFMD cases in China according to the phylogenetic trees using representative partial VP1 sequences. These four major enterovirus genotypes have different geographical distributions, and they may cocirculate with other genotypes and serotypes. These results suggest that more molecular epidemiological studies should be performed on several enteroviruses simultaneously, and such information should have implications for virological surveillance, disease management, vaccine development and policy-making on the prevention and control of HFMD.

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.

Achievements, challenges and prospects for the development of broadly protective multivalent vaccines and therapeutic antibodies against hand, foot and mouth disease

Hand, foot and mouth disease(HFMD) is a significant health concern in the Asia–Pacific regions for infants and young children in recent years. However, no vaccines or therapeutics are available at present. The causative agents for HFMD include human enterovirus 71(EV71), coxsackievirus A16(CVA16) and some other viruses. Recently, tremendous progress has been made in the development of monovalent and bivalent vaccines against HFMD. A few neutralizing monoclonal antibodies against EV71 or CVA16 have been identified and characterized. Here, we reviewed some achievements for the development of broadly protective vaccines and neutralizing antibodies against HFMD, and discussed challenges and prospects toward broadly protective multivalent vaccines and therapeutic antibodies against HFMD.

天津市手足口病防控知识科普宣讲活动及效果评价

2023年6—10月,对天津市16个区进行分层抽样,抽取4个宣传地区及其24所托幼机构,对849位幼儿家长、705名校(园)医、129名社区医生开展手足口病防控知识科普宣讲活动,宣传形式包括家长课堂宣讲,校医、社区医生现场活动和答疑解惑,以科普文章制作转发、视频播放等形式传播,并针对现场宣讲活动人群开展问卷调查。科普宣讲活动后,手足口病日常预防知识知晓率及防控行为率明显升高。提出对于需要多次强化或不易记忆的日常防护知识,可采用科普宣讲和短视频循环播放相结合的方式。

深圳市宝安区手足口病经济负担及影响因素

目的:了解深圳市宝安区2021年手足口病经济负担并分析其影响因素。方法:收集某三级甲等医院2021年9月1日—12月1日就诊的1 225例手足口病病人,设计调查问卷,采用电话询问及医院信息系统查询收集资料,评估手足口病造成的经济负担并分析其影响因素。结果:1 225例病人中,门诊病人1 195例(97.55%),住院病人30例(2.45%),无重症病例。1 195例手足口病门诊病人的总体经济负担为422.57(336.70,604.54)元/例,30例手足口病住院病人的总体经济负担为(4 386.88±1 624.61)元/例。年龄、报销类型以及诊疗次数是手足口病门诊病人总体经济负担的影响因素(P<0.05)。结论:手足口病住院病人经济负担高于门诊病人,年龄、报销类型以及诊疗次数是手足口病门诊病人经济负担的影响因素。早发现、早诊断、早治疗能有效降低疾病经济负担。

上海市长宁区儿童家长肠道病毒71型灭活疫苗接种意愿及影响因素分析

目的 了解上海市长宁区托幼机构儿童家长为孩子接种肠道病毒71型灭活疫苗(EV71疫苗)的意愿并分析影响因素,为推广EV71疫苗接种提出建议。方法 采用随机抽样的方式抽取长宁区托幼机构就读的儿童,向儿童家长发放问卷进行调查。结果 有75.40%的家长愿意为孩子接种EV71疫苗。Logistic回归分析,不认为EV71会产生强烈副作用、疫苗犹豫量表得分越高、主动获取知识得分越低的家长更愿意为孩子接种EV71疫苗。结论 家长为儿童接种EV71疫苗的意愿受到对EV71疫苗副作用的认知、疫苗犹豫和主动获取知识情况的影响,应对家长开展相关健康教育,以提升接种率。

The Application Research of WBC Morphology Combined with NAP in the HFMD Co-Infection of Children

In this manuscript the authors have studied the application value of White blood cell morphology combined with neutrophils alkaline phosphatase staining integral value in treatment of pediatric HFMD combined with infection. They showed that comparing the NAP positive rate of HFMD co-infection with the NAP positive rate of other three groups respectively—reference group, HFMD group and anti-infection HFMD group—the t value is 25.7964, 28.2326, 28.3696 and the values of P are 0.0000;Comparing the NAP integral value of HFMD co-infection with the NAP integral value of other three groups respectively, which are reference group, HFMD group and anti-infection HFMD, the t value is 35.8687, 36.2664, 36.1374 and the values of P are 0.0000. They concluded that it is useful to apply WBC morphological examination combined with the positive rate and integral value of NAP staining to diagnose and treatment HFMD.

2016至2022年杭州地区14259例儿童肠道病毒流行特征分析

目的了解杭州地区手足口病(hand-foot-mouth disease,HFMD)病原体肠道病毒(entero virus,EV)的阳性检出情况及流行特征,为今后制定杭州地区HFMD防控策略提供科学依据。方法收集整理2016年1月至2022年12月期间杭州市儿童医院检出的EV通用型核酸阳性的14259例的病例信息,对年龄、性别、检出时间等因素进行回顾性分析。结果EV历年平均阳性检出率为45.48%(14259/31354),其中男童阳性检出率为27.20%,女童为18.28%,差异有统计学意义(P<0.01)。不同年龄段患儿EV阳性检出率随年龄增大呈下降趋势,5岁以下儿童的EV阳性检出率占所有阳性检出患儿的90.38%。在季节方面,EV夏季高发,历年夏季5月至7月的总阳性检出数占总检出数的49.16%,自2016年以来EV阳性检出率呈逐年下降趋势。结论杭州地区EV感染具有性别差异,男童易感染,尤其需要重视5岁以下儿童的EV防护。另外HFMD存在一定的季节性,夏季高发,历年检出率自2016年后逐年下降,可能与疫苗推广和健康意识提高存在一定关系。

辽宁省首株柯萨奇病毒B组5型全基因组序列分析

研究引起辽宁地区手足口病的柯萨奇病毒B组5型(coxsackievirus B5,CV-B5)基因组特征。对2018年从辽宁省688份肠道病毒核酸阳性的标本中分离到的1株CV-B5进行高通量测序,并对其全基因组进行遗传进化分析。结果表明,CV-B5辽宁分离株与国内流行株的全基因组核苷酸序列同源性为78.5%~97%,氨基酸序列同源性为75.3%~96.7%。基于全基因组的进化分析将CV-B5流行株分为A~D四个基因型,辽宁分离株属于D基因型。通过重组分析发现其在P3区的3D区段发生重组。首次在辽宁地区手足口病患儿中分离出CV-B5,辽宁省分离株(LN2018-23-21/CHN/2018)可能为重组株。

2018—2020年黔东南少数民族自治州肠道病毒A71型基因特征分析

目的对2018—2020年黔东南州手足口病(hand,foot and mouth disease,HFMD)进行鉴定分析,并对肠道病毒A组71型(enterovirus A71,EV-A71)VP1编码区进行基因特征分析。方法2018—2020年黔东南州共采集HFMD病例标本2789份,首先,采用荧光定量RT-PCR初筛肠道病毒核酸,阳性病例标本采用人类横纹肌肉瘤(human rhabdomyosarcoma,RD)细胞进行病毒分离,对分离到的病毒株提取病毒核酸,经RT-PCR方法扩增VP1编码区核苷酸序列并进行序列测定。使用MEGA 11.0软件构建遗传进化树进行基因特征分析。结果2789份HFMD标本中检出EV-A71核酸阳性标本为328份,阳性率为11.8%。从部分EV-71阳性患者的咽拭子及肛拭子中,分离培养获得7株病毒株,其中6株来自于轻症病例,另1株为重症病例,均属于EV-A71基因型C4a基因亚型,分成2个不同的簇。7株EV-A71 VP1序列之间核苷酸和氨基酸同源性分别为89.8%~99.9%和98.0%~99.7%;与BrCr原型参考株间的核苷酸和氨基酸同源性分别为77.1%~78.8%和94.8%~96.6%;并在VP1有18处氨基酸位点发生变异。结论2018—2020年黔东南州EV-A71分离株属于C4a基因亚型,多处氨基酸位点存在变异,可导致多条传播链循环。