直肠滴入中药对CoxA16、EV71肠道病毒感染手足口病的疗效

目的观察直肠滴入中药对CoxA16、EV71肠道病毒感染手足口病的疗效。方法选取2019年8月—2022年8月贵州水矿控股集团有限公司总医院CoxA16、EV71肠道病毒感染手足口病患儿85例,按照随机数表法分为观察组(42例)和对照组(43例),其中对照组患儿给予利巴韦林治疗,观察组患儿给予直肠滴入中药治疗。对比两组患儿治疗前后口腔疱疹、发热、皮疹等症状的评分,治疗前后患儿炎症因子、免疫指标水平及两组患儿治疗后不良反应发生率。结果治疗前,两组患儿的口腔疱疹、发热、皮疹评分差异无统计学意义(P>0.05),治疗后,两组患儿的口腔疱疹、发热、皮疹评分均降低,并且治疗后观察组患儿的以上症状评分低于对照组(P<0.05);治疗后,两组患儿白细胞介素(IL)-6、IL-8、肿瘤坏死因子(TNF)-α均降低,并且治疗后观察组患儿的以上降低幅度大于对照组(P<0.05);治疗后,两组患儿CD4^(+)、CD3^(+)、CD4^(+)/CD8^(+)水平均升高,CD8^(+)水平降低,并且治疗后观察组患儿的变化幅度大于对照组(P<0.05);观察组患儿不良反应发生率为4.76%,对照组患儿不良反应发生率为18.60%,观察组不良反应发生率低于对照组(P<0.05)。结论给予CoxA16、EV71肠道病毒感染手足口病患儿直肠滴入中药治疗,能减轻患儿临床症状及炎性反应,改善免疫功能,且不良反应少,值得借鉴。

Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.

手足口患儿EV71、CoxA16和其他肠道病毒感染情况及基因型分析

目的探究2018—2020年平顶山市新华区手足口患儿EV71、CoxA16和其他肠道病毒感染情况及基因型状况。方法选择平顶山市新华区疾病预防控制中心2018年7月至2020年12月收治的104例手足口病患儿作为研究对象,通过患儿粪便及肛拭子标本对患儿病毒核酸进行检测,观察患儿肠道病毒病原学构成情况及分析患儿EV71病毒及CoxA16病毒VP1基因中各基因型同源性,并记录手足口病患儿临床症状表现及外周血白细胞水平。结果104例手足口病阳性样本中,EV71病毒感染54例(51.92%),CoxA16病毒感染7例(6.73%),其他肠道病毒感染43例(41.35%),EV71病毒感染比例高于CoxA16病毒感染比例,差异有统计学意义(P<0.05);CoxA16 VP1基因序列与A、B、C基因型代表株核苷酸同源性为82.6%~82.9%,与D基因型代表株核苷酸同源性为92.7%~99.2%;EV71 VP1基因序列与C4基因型代表株核酸同源性为92.4%~96.8%,与C4a基因型代表株核苷酸同源性为94.3%~96.8%,与C4b基因型代表株核苷酸同源性为92.4%~93.7%;EV71病毒、CoxA16病毒及其他肠道病毒感染组之间发热、皮疹等临床症状所占比例及外周血白细胞水平比较,差异均无统计学意义(P>0.05)。结论手足口病患儿中EV71病毒感染比例相比于CoxA16病毒感染比例更高;且CoxA16及EV71 VP1基因序列均存在高同源性的病毒株;但各病毒株感染后临床症状表现几乎一致。

2022—2023年新冠疫情期间及疫情后手足口病流行病学及分型分析

目的对比了解2022—2023年新冠疫情期间及疫情后广州市和佛山市手足口病流行病学及非肠道病毒71型(enterovirus 71,EV71)非柯萨奇病毒A组16型(Coxsaekievirus group A 16,CA16)病毒的分子流行病学特征,为治疗和防控提供依据。方法运用荧光逆转录聚合酶链反应(RT-PCR)对2022—2023年疑似手足口病患者标本同时进行肠道病毒(enterovirus,EV)通用型、EV71、CA16检测,并选取EV71和CA16是阴性而EV通用型是阳性的标本进行型别鉴定,设计5′非编码区(UTR)引物,RT-PCR扩增后进行序列测定,序列用BLAST程序进行序列的EV型别确定。结果2022年,同时进行EV通用型、EV71、CA163种病毒检测的疑似手足口病例标本共362份,总阳性47份,阳性率为12.98%;其中EV71阳性0份;CA16阳性5份,占1.38%;非EV71非CA16的EV阳性标本42份,占11.60%。2023年,同时进行EV通用型、EV71、CA163种病毒检测的疑似手足口病标本有297份,总阳性100份,阳性率为33.67%,全年没有检出EV71,CA16全年检出率为2.36%(7/297),非EV71非CA16的EV阳性率为31.31%(93/297)。51份非EV71非CA16阳性标本的序列分析表明,2022—2023检出的非EV71非CA16的EV有9种型别,分别为:CA6、CA10、CA4、CA2、CA8、柯萨奇病毒B组5型(CB5)、CB2、CB3、埃可病毒30型(E30),其中CA6占主要,为27.45%(14/51),其次是CA10,占15.69%(8/51)。结论新冠疫情结束后的2023年比疫情期间的2022年EV阳性率高。2022—2023年手足口病主要以非EV71非CA16为主,序列分析表明非EV71非CA16病毒中以CA6和CA10为主,应加强对CA6、CA10为主要非EV71非CA16病毒进行研究及监测。

2011-2013年手足口病病原谱及临床特征分析

目的：探讨2011-2013年手足口病（HFMD）病原体变化趋势。方法采集4064例在我院就诊的HFMD患者的咽拭子标本并提取核酸，采用实时荧光定量PCR方法检测肠道病毒通用型（EV），肠道病毒71型（EV71）及柯萨奇病毒A组16型（CA16）。结果3年间6岁以上患儿占96.0%；发病以5~7月份为主，占73.5%。2011-2013年HFMD 患者 EV71、CA16和未分型肠道病毒的检出率依次为42.6%、35.3%、18.5%，16.1%、22.6%、11.5%，41.3%、42.1%、70.0%，差异有统计学意义（x2=343.3，P＜0.05）。2013年EV71、CA16检出率明显低于2011、2012年，未分型病毒检出率明显增高（P均＜0.017）。613例重症HFMD患儿中，EV71感染占59.2%、CA16感染占11.1%，未分型病毒感染占29.7%，且均以肺炎合并脑干脑炎为主。结论2011-2013年HFMD病原谱发生了变化，未分型肠道病毒增多，需引起重视。

Ubiquitin-specific protease 24 promotes EV71 infection by restricting K63-linked polyubiquitination of TBK1

TANK-binding kinase 1(TBK1)is an essential protein kinase for activation of interferon regulatory factor 3(IRF3)and induction of the type I interferons(IFN-I).Although the biochemical regulation of TBK1 activation has been studied,little is known about how enterovirus 71(EV71)employs the deubiquitinases(DUBs)to regulate TBK1 activation for viral immune evasion.Here,we found that EV71 infection upregulated the expression of ubiquitinspecific protease 24(USP24).Further studies revealed that USP24 physically interacted with TBK1,and can reduce K63-linked polyubiquitination of TBK1.Knockdown of USP24 upregulated TBK1 K63-linked polyubiquitination,promoted the phosphorylation and nuclear translocation of IRF3,and in turn improved IFN-I production during EV71 infection.As a consequence,USP24 knockdown dramatically inhibited EV71 infection.This study revealed USP24 as a novel regulator of TBK1 activation,which promotes the understanding of immune evasion mechanisms of EV71 and could provide a potential strategy for treatment of EV71 infection.

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.

PLX8394,a RAF inhibitor,inhibits enterovirus 71 replication by blocking RAF/MEK/ERK signaling

Enterovirus 71(EV71)poses a serious threat to human health,with scattered outbreaks worldwide.There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection.Therefore,it is urgent and of significance to develop anti-EV71 drugs.Here,we found that PLX8394,a RAF inhibitor,possesses high antiviral activity against EV71 in vitro,being superior to the traditional clinical drug ribavirin.Moreover,PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses.Notably,in a suckling mouse model,PLX8394 provided a 70%protection rate for EV71-infected mice,reduced the viral load in liver and heart tissues,and relieved the inflammatory response.A mechanistic study showed that PLX8394 inhibited EV71 by suppressing the RAF/MEK/ERK signaling pathway.Thus,PLX8394 lays a foundation for the development of new drugs against EV71.

小儿重症手足口病的护理体会

总结近2a来小儿重症手足口病的护理措施，预防并发症及后遗症，降低病死率。201O年6月～2012年6月入住我科的手足口病患儿共512例，有2／3以上为重症患儿，其中包括9例极重症手足口病患儿。近2a来手足口病重症病例有所增多，临床工作者应高度重视，加强诊断．根据病情制定周密的护理计划，预防并发症，大大提高治愈率，降低病死率。

EV71疫苗研究进展

肠道病毒71型(EV71),可引起手足口病(HFMD)和严重神经系统疾病。近年来,在世界多个国家和地区报道了EV71的爆发。由于目前缺乏有效的抗病毒药物,因此研制有效的疫苗是控制EV71流行最为有效的策略。近年来很多报道显示在EV71疫苗研究上取得了重要进展,本文以此为重点概括了EV71灭活疫苗、重组疫苗、DNA疫苗、多肽疫苗、减毒活疫苗等以及动物模型方面的研究进展。

Regulation of RDN on Th1/ILC1 cell imbalance in HFMD patients caused by EV71 infection

Enterovirus 71(EV71)infection is more likely to cause hand,foot and mouth disease(HFMD)in children,which can lead to neurogenic complications and higher mortality.As a commonly used clinical medicine,Reduning injection(RDN)helps to shorten the symptoms of patients with HFMD and facilitate the early recovery of children.However,the regulatory mechanism of RDN on the HFMD immune system disorder caused by EV71 remains to be discussed.This study collected detailed treatment data of56 children with HFMD who entered the affiliated Children’s Hospital of Nanjing Medical University during 2019.Retrospective analysis of clinical data showed that the symptoms of the RDN treatment group were improved compared with the untreated group.To explore its mechanism,the relevant detection indicators were detected by flow cytometry,enzyme-linked immunosorbent assay and realtime quantitative PCR.It was found that the number and function of innate immune(ILCs)and adaptive immunity(Th1,Th2 and secreted cytokines)were reduced,suggesting that RDN plays a role by regulating cellular immunity.The in vitro differentiation inhibition test further confirmed that RDN affected Th1 differentiation by inhibiting the expression of transcription factors on the basis of Th1 cell differentiation in vitro.

IFN-β1b induces OAS3 to inhibit EV71 via IFN-β1b/JAK/STAT1 pathway

Enterovirus 71(EV71) caused hand, foot and mouth disease(HFMD) is a serious threat to the health of young children. Although type I interferon(IFN-I) has been proven to control EV71 replication, the key downstream IFNstimulated gene(ISG) remains to be clarified and investigated. Recently, we found that 2’-5’-oligoadenylate synthetases 3(OAS3), as one of ISG of IFN-β1b, was antagonized by EV71 3C protein. Here, we confirm that OAS3is the major determinant of IFN-β1b-mediated EV71 inhibition, which depends on the downstream constitutive RNase L activation. 2’-5’-oligoadenylate(2-5A) synthesis activity deficient mutations of OAS3 D816A, D818A,D888A, and K950A lost resistance to EV71 because they could not activate downstream RNase L. Further investigation proved that EV71 infection induced OAS3 but not RNase L expression by IFN pathway. Mechanically, EV71 or IFN-β1b-induced phosphorylation of STAT1, but not STAT3, initiated the transcription of OAS3 by directly binding to the OAS3 promoter. Our works elucidate the immune regulatory mechanism of the host OAS3/RNase L system against EV71 replication.

ANXA2 Facilitates Enterovirus 71 Infection by Interacting with 3D Polymerase and PI4KB to Assist the Assembly of Replication Organelles

Similar to that of other enteroviruses, the replication of enterovirus 71(EV71) occurs on rearranged membranous structures called replication organelles(ROs). Phosphatidylinositol 4-kinase Ⅲ(PI4KB), which is required by enteroviruses for RO formation, yields phosphatidylinositol-4-phosphate(PI4P) on ROs. PI4P then binds and induces conformational changes in the RNA-dependent RNA polymerase(Rd Rp) to modulate Rd Rp activity. Here, we targeted 3D polymerase, the core enzyme of EV71 ROs, and found that the host factor Annexin A2(ANXA2) can interact with 3 D polymerase and promote the replication of EV71. Then, an experiment showed that the annexin domain of ANXA2, which possesses membranebinding capacity, mediates the interaction of ANXA2 with EV71 3 D polymerase. Further research showed that ANXA2 is localized on ROs and interacts with PI4KB. Overexpression of ANXA2 stimulated the formation of PI4P, and the level of PI4P was decreased in ANXA2-knockout cells. Furthermore, ANXA2, PI4KB, and 3D were shown to be localized to the viral RNA replication site, where they form a higher-order protein complex, and the presence of ANXA2 promoted the PI4 KB-3D interaction. Altogether, our data provide new insight into the role of ANXA2 in facilitating formation of the EV71 RNA replication complex.

Myristoylation of EV71 VP4 is Essential for Infectivity and Interaction with Membrane Structure

The Enterovirus 71(EV71)VP4 is co-translationally linked to myristic acid at its amino-terminal glycine residue.However,the role of this myristoylation in the EV71 life cycle remains largely unknown.To investigate this issue,we developed a myristoylation-deficient virus and reporter(luciferase)pseudovirus with a Gly-to-Ala mutation(G2A)on EV71 VP4.When transfecting the EV71-G2 A genome encoding plasmid in cells,the loss of myristoylation on VP4 did not affect the expression of viral proteins and the virus morphology,however,it did significantly influence viral infectivity.Further,in myristoylation-deficient reporter pseudovirus-infected cells,the luciferase activity and viral genome RNA decreased significantly as compared to that of wild type virus;however,cytopathic effect and viral capsid proteins were not detected in myristoylation-deficient virus-infected cells.Also,although myristoylation-deficient viral RNA and proteins were detected in the second blind passage of infection,they were much fewer in number compared to that of the wild type virus.The replication of genomic RNA and negative-strand viral RNA were both blocked in myristoylation-deficient viruses,suggesting that myristoylation affects viral genome RNA release from capsid to cytoplasm.Besides,loss of myristoylation on VP4 altered the distribution of VP4-green fluorescent protein protein,which disappeared from the membrane structure fraction.Finally,a liposome leakage assay showed that EV71 myristoylation mediates the permeability of the model membrane.Hence,the amino-terminal myristoylation of VP4 is pivotal to EV71 infection and capsidmembrane structure interaction.This study provides novel molecular mechanisms regarding EV71 infection and potential molecular targets for antiviral drug design.

2012年武汉地区手足口病肠道病毒分型与临床特征分析

目的:了解武汉地区手足口病肠道病毒分型和临床特征,为手足口病预防及治疗提供依据。方法:采集手足口病患者咽拭子标本,采用实时荧光定量法检测CA16、EV71和通用型肠道病毒,同时搜集手足口病患儿的临床资料,分析不同病毒型手足口病患儿临床特征。结果:2012年1-12月共收集手足口病咽拭子标本3 083例,其中EV71阳性765例,阳性率为24.81%;CA16病毒阳性559份,阳性率为18.13%;EV71和CA16双阳性61例,阳性率为1.99%;通用型肠道病毒阳性1 698例,阳性率55.07%。1-3岁是手足口病高发年龄段,各病毒型均表现为男性多于女性。3 083例手足口病患者,临床上均有手、足、口或臀部皮疹,大部分患者有发热,部分患者伴有神经系统症状。EV71感染者出现发热、神经系统症状及白细胞增多的比例高于CA16和通用型肠道病毒阳性者。通用型肠道病毒阳性患者出现肺部并发症比例多于EV71感染者。结论:武汉市2012年手足口病主要由EV71、CA16及通用型肠道病毒引起,且以通用型肠道病毒为主。了解不同病毒型手足口病临床特征,对于手足口病预防和治疗具有重要意义。

2010～2016年济宁市手足口病流行病学特征分析

目的了解济宁市2010~2016年手足口病的流行病学特征,手足口病防控提供科学依据。方法利用中国疾病预防控制信息系统,采用描述流行病学方法对济宁市2010~2016年手足口病发病情况进行统计分析。结果济宁市2010~2016年累计报告手足口病25 851例,重症26例,死亡3例,年均发病率为44.99/10万。发病高峰在5~6月(12 063例),年均发病率最高的是任城区(107.23/10万),市区的发病率(75.85/10万)高于县城(38.34/10万)(P<0.01)。男性发病数为16 863例,女性为8 988例,1~3岁儿童占所有病例的73.06%,散居儿童占所有病例的78.64%。实验室诊断病例2 632例,EV71阳性所占比例为32.40%,其他肠道病毒为40.46%。结论济宁市手足口病有明显的区域性、季节性、人群差异,主要病原是EV71型和其他肠道病毒。

2012年度211例手足口病住院患儿病原学检测及分析

目的：掌握2012年度北京大学北京地坛医院教学医院手足口病住院患儿的病原体分布情况与变化趋势，为手足口病的防治提供科学依据。方法本研究收集北京大学北京地坛医院教学医院儿科2012年度211例手足口病住院患儿的咽拭子标本，提取病毒RNA，采用实时荧光聚合酶链反应（RT-PCR）法，进行肠道病毒（EV）通用型、肠道病毒71（EV71）型和柯萨奇A16（CoxA16）型肠道病毒核酸检测。EV（＋）标本判为EV阳性，EV（＋）EV71（＋）标本判为EV71阳性，EV （＋）CoxA16（＋）标本判为CoxA16阳性，EV（＋）且EV71（-）CoxA16（-）标本判为非EV71非CoxA16型肠道病毒阳性。结果2012年度211例本院手足口病住院患儿中EV阳性标本共118例，占55.92%。病毒分型结果显示，非EV71非CoxA16型肠道病毒阳性标本共46例，占22.81%；EV71阳性标本共45例，占21.32%；CoxA16阳性标本共27例，占12.80%。病原学分布分析结果显示，5～7月份为发病高峰期；不同年龄、性别组患儿之间病原体构成无显著差异；患儿入院前3d肠道病毒检出率较3 d后高；不同型别肠道病毒感染患儿之间平均住院天数差异无统计学意义。结论2012年度本院手足口病住院患儿不同型别EV感染率由高到低依次为：非EV71非CoxA16型肠道病毒、EV71、CoxA16，非EV71非CoxA16型肠道病毒感染率较往年具有升高趋势，尚待进一步研究。

2010年-2013年枣庄市手足口病核酸检测结果分析

目的 了解本市手足口病肠道病毒的型别、分布特征和流行趋势,为进一步预防控制该病的流行提供实验室技术支持。方法 采集手足口病疑似患者的咽拭子标本,提取RNA,应用逆转录-聚合酶链式反应（RT-PCR）法进行肠道病毒通用型、EV71型及CoxA16型的核酸检测,统计检测结果并分析。结果 共检测手足口病患者标本947份,总阳性率为58.61%,其中EV71型阳性率为37.17%、CoxA16型阳性率为8.45%,其他EV病毒阳性率为12.99%。男性阳性率为58.39%,女性阳性率为59.05%,差异无统计学意义（P〉0.05）。农村患者阳性率为61.12%,城市患者阳性率为52.24%,二者的检出率差异有统计学意义（χ^2=6.247,P〈0.05）。1岁-3岁组低龄儿童占确诊患者的78.38%。结论 枣庄市手足口病病原以肠道病毒EV71型为主。有明显的时间和人群分布特征。每年的5月-7月为发病高峰期。农村1岁-3岁组低龄儿童是手足口病的主要发病群体。

A microfluidic cell chip for virus isolation via rapid screening for permissive cells

Virus identification is a prerequisite not only for the early diagnosis of viral infectious diseases but also for the effective prevention of epidemics.Successful cultivation is the gold standard for identifying a virus,according to the Koch postulates.However,this requires screening for a permissive cell line,which is traditionally time-,reagent-and labor-intensive.Here,a simple and easy-to-operate microfluidic chip,formed by seeding a variety of cell lines and culturing them in parallel,is reported for use in virus cultivation and virus-permissive host-cell screening.The chip was tested by infection with two known viruses,enterovirus 71(EV71)and influenza virus H1N1.Infection with EV71 and H1N1 caused significant cytopathic effects(CPE)in RD and MDCK cells,respectively,demonstrating that virus cultivation based on this microfluidic cell chip can be used as a substitute for the traditional plate-based culture method and reproduce the typical CPE caused by virus infection.Using this microfluidic cell chip method for virus cultivation could make it possible to identify an emerging virus in a highthroughput,automatic,and unprecedentedly fast way.