Seroepidemiology and Molecular Epidemiology of Enterovirus 71 in Russia

Enterovirus 71 (EV71) is an emerging human pathogen causing massive epidemics of hand, foot and mouth disease with severe neurological complications in Asia. EV71 also circulates in Europe, however it does not cause large outbreaks. The reason for distinct epidemiological patterns of EV71 infection in Europe and Asia and the risk of EV71 epidemic in Europe and Russia remain unknown. Seroepidemiology of EV71 and molecular epidemiology of occasional EV71 isolates were studied to explore circulation of EV71 in Russia. In six regions of Russian Federation, seroprevalence of EV71 in sera collected in 2008 ranged from 5% to 20% in children aged 1–2 years and from 19% to 83% in children aged 3–5 years. The seroprevalence among elder children was significantly higher (41–83% vs. 19–27%) in Asian regions of Russia. EV71 strains identified in Russia in 2001–2011 belonged to subtypes C1 and C2, while genotype C4 that was causing epidemics in Asia since 1998 emerged in 2009 and became dominant in 2013.     

Antigenic and Genetic Diversity of Human Enterovirus 71 from 2009 to 2012, Taiwan

Different subgenogroups of enterovirus 71 (EV-71) have caused numerous outbreaks of hand, foot, and mouth disease worldwide, especially in the Asia-Pacific region. During the development of a vaccine against EV-71, the genetic and antigenic diversities of EV-71 isolates from Taiwan were analyzed by phylogenetic analyses and neutralization tests. The results showed that the dominant genogroups had changed twice, from B to C and from C to B, between 2009 and 2012. The subgenogroup B5 (B5b cluster) was dominant in 2008-2009 but was replaced by subgenogroup C4 in 2010-2011. From the end of 2011 to 2012, the re-emerging subgenogroup B5 (B5c cluster) was identified as the dominant subgenogroup of EV-71 outbreaks, and subgenogroups C2 and C4 were detected in sporadic cases. Interestingly, the amino acid substitution at position 145 in the VP1 gene was observed in some strains isolated from patients with acute flaccid paralysis. Furthermore, thirty-five strains and their corresponding serum samples were used to analyze the cross-protections and antigenic diversities among different subgenogroups (C4a, C5, B4, B5b, B5c, and C2-like) of EV-71. Evident antigenic diversity existed only for the C2-like subgenogroup, which was not effectively neutralized by other serum samples. In contrast, the anti-C2-like serum sample showed broad cross-reactivity against all other subgenogroups. Therefore, these results may provide valuable information for the selection of EV-71 vaccine candidates and the evolution of EV-71 subgenogroups in Taiwan from 2009 to 2012.     

肠道病毒71型分子流行病学研究进展

肠道病毒71型(Enterovirus type71,EV71),自1974年首次报道以来,在世界范围内引起多次爆发与流行[1].EV71感染主要引起患者手足口病(hand,foot and mouth disease,HFMD),在临床上与柯萨奇病毒A16(Coxsakie A16,CA16)感染所引起的手足口病难以区别,但EV71还能够引起多种与神经系统相关的疾病[2].近年来,EV71病毒的流行在亚太地区呈上升趋势[3～5],其中最令人关注的是在该地区的EV71感染引起越来越严重的中枢神经系统症状.     

人肠道病毒71型动物模型研究进展

人肠道病毒71型是婴幼儿手足口病的致病原之一,其严重的并发症可导致神经系统疾病,甚至死亡,是近期威胁中国儿童健康的因素之一。目前尚无临床疫苗可以预防该病毒感染,而EV71的动物模型是进行致病机理、疫苗评价和药物等研究的基础。本文对EV71的两种常用动物模型：小鼠和猕猴（Macaca mulatta）模型进行了描述,并对其在研究中的应用给与概括,为研究者选择合适的动物模型提供了依据。     

Molecular Epidemiology of HIV-1 Infection among Men who Have Sex with Men in Taiwan in 2012

The number of men who have sex with men (MSM) infected with HIV-1 in Taiwan has increased rapidly in the past few years. The goal of this study was to conduct a molecular epidemiological study of HIV-1 infection among MSM in Taiwan to identify risk factors for intervention. Voluntary counseling program and anonymous testing were provided to patrons at 1 gay bar, 7 night clubs and 3 gay saunas in Taipei and New Taipei Cities in 2012. HIV-1 subtypes were determined using gag subtype-specific PCR and phylogenetic analysis by env sequences. Recent HIV-1 infection was determined using LAg-Avidity EIA. In-depth interviews and questionnaires were used to identify risk factors. The prevalence and incidence of HIV-1 among MSM in Taiwan were 4.38% (53/1,208) and 3.29 per 100 person-years, respectively. Of 49 cases genotyped, 48 (97.9%) were infected with subtype B and 1 with CRF01_AE (2%). Phylogenetic analysis of 46 HIV-1 strains showed that 25 (54.4%) subtype B strains formed 9 clusters with each other or with other local strains. The CRF01_AE case clustered with a reference strain from a Thai blood donor with bootstrap value of 99. Multivariate logistic regression analysis showed that risk factors associated with HIV-1 infection included use of oil-based solution as lubricant (vs. saliva or water-based lubricants, OR= 4.23; p <0.001); exclusively receptive role (vs. insertive role, OR= 9.69; p <0.001); versatile role (vs. insertive role, OR= 6.45; p= 0.003); oral sex (vs. insertive role, OR= 11.93; p= 0.044); times of sexual contact per week (2-3 vs. zero per week, OR= 3.41; p= 0.021); illegal drug use (OR= 4.12; p <0.001); and history of sexually transmitted diseases (OR= 3.65; p= 0.002). In conclusion, there was no new HIV-1 subtype or circulating recombinant form responsible for the increase of HIV-1 among MSM in Taiwan in 2012. Misuse of oil-based solution as lubricant is a new risk factor identified among MSM in Taiwan. The Taiwan’s Centers for Disease Control has created a video (www.youtube.com/watch?v=BinExvvOTMM&feature=iv&src_vid=BW81-PfmY3E&annotation_id=annotation_2436493705) to correct such misconception in its AIDS prevention campaign.     

Molecular epidemiology and recombination of Enterovirus A71 in mainland China from 1987 to 2017

International Microbiology - Enterovirus A71 (EV-A71) is an important pathogen of severe hand, foot, and mouth disease (HFMD) in young children. This study aimed to retrospectively analyze the...     

The Association between Enterovirus 71 Infections and Meteorological Parameters in Taiwan

Background

Enterovirus 71 (EV71) infections are a significant cause of neurological disorder and death in children worldwide. Seasonal variations in EV71 infections have been recognized, but the mechanisms responsible for this phenomenon remain unknown. The purpose of this study was to examine the relationship between meteorological parameters and EV71 infection.

Methods and Findings

We analyzed the number of EV71 infections and daily climate data collected in Taiwan between 1998 and 2008 and used Poisson regression analysis and case-crossover methodology to evaluate the association between weather variability and the incidence of EV71 infection. A total of 1,914 EV71-infected patients were reported between 1998 and 2008. The incidence of EV71 infections reflected significant summertime seasonality (for oscillation, p<0.001). The incidence of EV71 infections began to rise at temperatures above 13°C (r² = 0.76, p<0.001); at temperatures higher than approximately 26°C (r² = 0.94, p<0.05), the incidence began to decline, producing an inverted V-shaped relationship. The increase in the incidence with increasing relative humidity was positive and linear (r² = 0.68, p<0.05). EV71 infection was most highly correlated with temperature and relative humidity in the period that likely preceded the infection.

Conclusion

Our study provides quantitative evidence that the rate of EV71 infection increased significantly with increasing mean temperature and relative humidity in Taiwan.     

肠道病毒71型安徽、河南株的分离与VP1序列进化分析

旨在研究手足口病患者中肠道病毒71型分离株的病毒基因型特征。采集手足口病患者的粪便标本,进行病毒分离和逆转录-聚合酶链式反应(RT-PCR)特异性扩增进行鉴定,同时选取其中9株EV71分离株,对其抗原决定簇部位VP1区进行核酸序列测定,并参考EV71 A、B、C各基因型的参考株和以往中国EV71的分离株进行同源分析和构建系统发生树。结果显示,所分析的9株病毒株均为C4亚型,3株安徽株H7、H8和H9的VP1序列相似度很高(≥98.8%,其中H7、H9的相似度为100.0%),4株河南株H3、H4、H5和H6相似度较高(≥98.4%,H3、H4和H5≥99.6%,其中H3、H4的相似度为100.0%),它们同河南株H1、H5的相似度也较高(≥97.2%),河南株H2虽然与其他河南株具有较高的序列相似度,但进化分析表明,其与安徽株同源性较高。结果表明,安徽株H7、H8和H9株变异速率明显加快,这可能导致了手足口病在安徽省的率先爆发与大流行,河南株H2最初可能由安徽传入河南。     

白介素18启动子区基因多态性与儿童EV71感染相关性研究

目的:研究白介素18基因启动子多态性与儿童EV71感染遗传易感性的关系.方法:收集EV71感染患儿177例,单纯HFMD组127例,HFMD并脑炎组50例,提取外周血DNA,用序列特异性引物-聚合酶链反应(SSP-PCR)技术及基因测序法检测IL-18启动子区-137G/C、-607A/C位点的基因多态性.结果:EV71感染患儿与健康儿童IL-18-607基因型以CA为主,AA、CC次之,EV71感染患儿AA基因型、A等位基因分布频率显著高于健康儿童.EV71感染HFMD并脑炎组患儿AA基因型分布显著高于单纯HFMD组患儿,差异有统计学意义.EV71感染患儿与健康儿童IL-18-137基因型以CC为主,CG次之,GG占少数.该位点基因型及等位基因在EV71感染组与健康儿童、单纯HFMD与HFMD并脑炎组的分布无显著差异.结论:IL-18基因多态性与EV71感染相关,IL-18-607AA基因型、A等位基因携带儿童更易感染EV71病毒,且AA基因型患儿易并发脑炎,-607AA基因型可能为EV71感染的易感基因型.-137C/G位点基因多态性与EV71感染无相关性.     

Enterovirus71 (EV71) Utilise Host microRNAs to Mediate Host Immune System Enhancing Survival during Infection

Hand, Foot and Mouth Disease (HFMD) is a self-limiting viral disease that mainly affects infants and children. In contrast with other HFMD causing enteroviruses, Enterovirus71 (EV71) has commonly been associated with severe clinical manifestation leading to death. Currently, due to a lack in understanding of EV71 pathogenesis, there is no antiviral therapeutics for the treatment of HFMD patients. Therefore the need to better understand the mechanism of EV71 pathogenesis is warranted. We have previously reported a human colorectal adenocarcinoma cell line (HT29) based model to study the pathogenesis of EV71. Using this system, we showed that knockdown of DGCR8, an essential cofactor for microRNAs biogenesis resulted in a reduction of EV71 replication. We also demonstrated that there are miRNAs changes during EV71 pathogenesis and EV71 utilise host miRNAs to attenuate antiviral pathways during infection. Together, data from this study provide critical information on the role of miRNAs during EV71 infection.     

EV71病毒核酸快速检测方法的比较

PCR方法检测肠道病毒71型(EV71)的分子诊断技术十分快速敏感。目前已有多篇文献报道了不同的检测EV71的PCR方法,但对不同方法检测结果的比较还少有报道。本研究将检测肠道病毒通用5’UTR基因的3种定量PCR(rRT-PCR)方法和1种普通PCR(cRT-PCR)方法和特异性检测EV71的2种rRT-PCR和1种cRT-PCR方法进行特异性和敏感性比较评估。所有试验方法都有较好的特异性,无交叉反应。核酸最低检测限值为从8.19×101～8.19×105个拷贝,rRT-PCR比cRT-PCR更敏感。所有的rRT-PCR方法检测50份临床标本时都较敏感。     

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2A^pro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID₅₀) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2A^pro region of the EV71 genome exerted antiviral effects in vitro.     

Conformational Plasticity of the 2A Proteinase from Enterovirus 71

The 2A proteinase (2A^pro) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A^pro from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A^pro was comprised of an N-terminal domain of a four-stranded antiparallel β sheet and a C-terminal domain of a six-stranded antiparallel β barrel with a tightly bound zinc atom. Unlike in other 2A^pro structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A^pro of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII β strands. This was supported by molecular dynamic simulation. The structural variation among different 2A^pro structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target.     

新乡地区2011年肠道病毒71型VP1基因特征分析及手足口病流行特点

为了解新乡地区2011年肠道病毒71型(EV71)VP1基因特征及手足口病流行特点,采用荧光RT-PCR对临床诊断的粪便标本进行总肠道病毒(EV)、柯萨奇病毒A16(CA16)和EV71检测;选取10例EV71阳性标本进行VPl序列扩增并测序,所得序列进行同源性分析和构建系统发生树;对2011年新乡市手足口病疫情监测数据进行分析。结果显示,重症标本的EV71阳性率(73%)显著高于CA16阳性率(19%)(P<0.01);10株新乡EV71分离株的核苷酸及氨基酸差异分别为2.8%和0.9%,属于C4亚型的C4a簇;9株VP1区第170位氨基酸为A,1株为V;与近缘的C4a型代表株相比,新乡优势株的氨基酸变异一般发生在VP1第292位氨基酸(T→A);2011年新乡市共上报手足口临床诊断病例1118例,92%的发病年龄在3岁以下,发病高峰分别出现在4和12月份,提示一定要加强手足口病预防控制,寒冷天气尤其不能忽视。     

EV71小鼠适应株制备及体内外感染特点

目的用1日龄ICR小鼠传代制备EV71小鼠适应株,研究EV71亲代株与小鼠适应株的体内外感染特点,建立EV71感染ICR小鼠动物模型,为病毒疫苗和抗病毒药物的研究提供实用的动物评价工具。方法用1日龄ICR小鼠进行EV71病毒(Fuyang-0805)的传代,得到小鼠传代株。以一定浓度亲代株和传代株病毒分别接种RD、Vero、SY5Y、Caco-2四种细胞,定量方法检测各时间点不同毒株在四种细胞上的复制数量,CCK8方法测定各时间点细胞的存活率;同时,两毒株分别腹腔注射感染1日龄小鼠,定期安乐死动物,采集肺、小肠、骨骼肌、大脑四种器官组织,进行动物体内病毒半定量和定量分析,同时进行各器官组织病理学观察、免疫组织化学鉴定。结果与亲代毒株相比较,小鼠传代株(EV71-MMP4)表现出更强的肌肉来源细胞嗜性与毒性;同时,两毒株腹腔注射感染1日龄小鼠后,EV71-MMP4感染的小鼠体重增长较正常小鼠体重增长缓慢;半定量和定量RT-PCR显示,在小鼠肌肉中的病毒载量于感染后1d和5d达到高峰。EV71-MMP4感染组感染率较高、病毒组织分布较广、感染持续性较好、病毒载量较高,高剂量病毒感染后小鼠小肠、心肌和骨骼肌可观察到细胞空泡变性、淋巴细胞浸润等病理变化。免疫组织化学显示感染后小鼠骨骼肌有EV71病毒特异分布。结论阜阳EV71小鼠适应株表现出较亲代毒株更好的小鼠易感性、细胞毒性,所建立的动物模型可用于EV71病毒致病机制、感染特点的研究和病毒疫苗及药物的评价。     

Histopathological Features and Viral Antigen Distribution in the Lung of Fatal Patients with Enterovirus 71 Infection

正Dear Editor,Enterovirus 71(EV71)infection causes hand-foot-andmouth disease(HFMD)in infants and children.Patients with HFMD usually have good prognosis;however,in some extreme cases the infection can be accompanied by central nervous system diseases,eventually leading to cardiorespiratory failure,and even death.Currently,EV71     

肠道病毒71型的研究进展

肠道病毒71型(enterovirus71,EV71)是小RNA病毒科(Picornaviridae)肠道病毒属(Enterovirus)成员,其感染主要引起患者手足口病(hand-foot-and-mouth disease,HFMD).通常情况下,EV71感染引起的HFMD在临床症状等方面与柯萨奇病毒A16(Coxsackie A16,CA16)引起的手足口病难以区别,但EV71感染除了引起HFMD以外,还能够引起无菌性脑膜炎(aseptic meningitis)、脑干脑炎(brainstem encephalitis)和脊髓灰质炎样的麻痹(poliomyelitis-like paralysis)等多种与神经系统相关的疾病[1].自1974年首次报道[2]以来,EV71已在世界范围内引起十多次爆发与流行[3-6].近年来,EV71病毒的流行在亚太地区呈上升趋势[7-9].根据病毒衣壳蛋白VP1核苷酸序列的差异,可将EV71分为A、B、C 3个基因型,其中,B型和C型又进一步分为B1、B2、B3、B4以及C1和C2亚型[10-12].     

The Clinical Correlation of Regulatory T Cells and Cyclic Adenosine Monophosphate in Enterovirus 71 Infection

Background

Brainstem encephalitis (BE) and pulmonary edema (PE) are notable complications of enterovirus 71 (EV71) infection.

Objective

This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment.

Study Design

Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD) or BE group, and the autonomic nervous system (ANS) dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP) levels, and the regulatory T cell (Tregs) profiles of the patients were determined.

Results

Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4⁺CD25⁺Foxp3⁺ and CD4⁺Foxp3⁺ T cells compared with patients with HFMD or BE. The expression frequency of CD4⁻CD8⁻ was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4⁺Foxp3⁺ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment.

Conclusions

These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels.