Diagnosis of hand,foot, and mouth disease caused by EV71 and other enteroviruses by a one‐step,single tube,duplex RT‐PCR

Hand, foot, and mouth disease (HFMD) is caused mainly by enterovirus 71 (EV71) and other enteroviruses (EVs) such as Coxsackie A16 in China. EV71 infection can lead to severe clinical manifestations and even death. Other EVs, however, generally cause mild symptoms. Thus, early and accurate distinction of EV71 from other EVs for HFMD will offer significant benefits. A one‐step, single tube, duplex RT‐PCR assay is described in the present study to detect simultaneously EV71 and other EVs. The primers used for the duplex RT‐PCR underwent screening and optimization. The detection threshold was 0.001 TCID₅₀/ml for EV71 and 0.01 TCID₅₀/ml for other EVs. The positive rate of enterovirus detection in 165 clinical samples reached 68.5%, including 46.1% for EV71 and 22.4% for other EVs. Of all the severe HFMD cases, EV71 was responsible for 85.3% cases. The positive rate of EV71 fell markedly by day 8 after onset. In addition, sequencing of EV71 specific amplicons from duplex RT‐PCR revealed that C4a was the predominant subgenotype of EV71 circulating in Nanjing, China. The accuracy and reliability of the assay suggest strongly that the one‐step, single tube, duplex RT‐PCR will be useful for early diagnosis and monitoring of EV71 and other EV infections. J. Med. Virol. 84:1803–1808, 2012. © 2012 Wiley Periodicals, Inc.     

EV71型手足口病乳鼠动物模型的建立及免疫、内分泌及病理特征研究

目的建立EV71型手足口病乳鼠动物模型并进行免疫、致病特性研究。方法将临床分离的EV71病毒株经蚀斑纯化,3T3细胞适应,乳鼠驯化,最终获得1株能致死7日龄乳鼠的EV71毒株,命名为BJ09/07(GenBank Accession NO.JQ319054,EV71-BJ)。EV71-BJ感染7日龄ICR乳鼠后,观测临床疾病得分、体质量变化、死亡率并测定病毒载量、免疫分子、内分泌水平、组织病理损伤等病毒、免疫、内分泌、病理指标。结果 EV71-BJ毒株感染7日龄ICR乳鼠,其病毒毒力为150 LD50/ml,感染后不同时期肌肉病毒载量均高于脑中,至第4天达到高峰,后不断下降。至感染后第6天发病达高峰时做病理检测,相对于脑组织,肌肉中有更严重的淋巴细胞浸润,引起更严重的炎性分子升高。肌肉研磨液和血清中MCP-1、IFN-γ、IL-6和TNF-α显著升高,而肾上腺素和皮质醇未见明显变化。结论初步建立了EV71型手足口病乳鼠动物模型,为药物筛选、疫苗研发及免疫机理研究奠定了基础。     

An outbreak of enterovirus 71 infection in Taiwan 1998: a comprehensive pathological, virological, and molecular study on a case of fulminant encephalitis.

BACKGROUND: In a recent enterovirus outbreak in Taiwan, serotype 71 was the culprit of encephalitis causing rapid clinical deterioration and death among young children. OBJECTIVES: Since knowledge of enterovirus 71 (EV71) infection in the central nervous system is still limited, the purpose of the present case study was attempted to uncover the pathogenesis of the virus. STUDY DESIGN: We performed a detailed pathological examination, virological and molecular studies on a case of EV71 infection with a rapidly fatal outcome. In addition, the whole genome of the virus was sequenced to determine the genetic relationships to other enteroviruses and two other EV71 strains (a prototype BrCr and a neurovirulent MS strain), and to provide the genetic basis of its neurovirulence of the new isolate, NCKU9822 strain. RESULTS: Characteristic features of acute encephalomyelitis were observed, with most prominent lesions in the spinal cord and brain stem. Mild myocarditis and pancreatitis were also noticed. EV71 antigen was localized to neurons on immunohistochemical staining. EV71 was recovered from all organs with inflammatory reaction. Sequence analysis showed that overall NCKU9822 and the two EV71 strains shared 80% nucleotide identity and 95% amino acid identity. It had only 45% amino acid and 52% nucleotide identities with polioviral P1 capsid region. CONCLUSION: The spinal cord and brain stem were the main targets of EV71 in the fatal cases in this outbreak, however, heart and pancreas might also be involved. Since the amino acid sequences in the P1 region are conserved (97% identity) among the three EV71 strains as compared to other enteroviruses and polioviruses, these EV71 neurovirulent strains might share the same mechanisms of neurovirulence, and the mechanisms might be different from those in polioviruses.     

我国广东、福建地区2000～2001年手足口病肠道病毒71型分离株的种系进化分析

目的 采用逆转录聚合酶链反应(RT-PCR)检测我国广东、福建地区2000-2001年手足口病(HFMD)散发病例中的肠道病毒71型(EV71)，进而通过扩增VP1节段的核苷酸序列，进行毒株的种系进化分析。方法以肠道病毒特异引物对EV-1、EV-2进行RT-PCR，经琼脂糖凝胶电泳鉴定为阳性的标本，进一步用EV71特异引物对159S、162A进行RT-PCR，扩增的VP1节段经纯化后与测序载体pGEM-T连接，转化大肠埃希菌DH5a，筛选后测序。所得序列与我国深圳、上海、武汉地区的EV71流行株，中国台湾1998年4例HFMD暴发分离的EV71毒株，及来自美国、日本、匈牙利等国家地区的EV71毒株的核苷酸序列，用ClustalX1．8和PHYuP3．5进行比对分析，构建种系进化树。结果 25份标本检测EV71的阳性率为20％，所得序列经种系进化分析，与肠道病毒71型的其他毒株同源，与深圳1998年HFMD散发分离的EV71毒株同源性为94％，与上海2000年HFMD暴发分离株同源性为94％～96％，与武汉1987年HFMD病例中分离的毒株同源性为91％，而与国外EV71毒株同源性仅为82％～84％。结论 EV71是我国南方地区HFMD的主要病原之一；我国大陆地区的EV71毒株在种系进化上有较高的同源性；与我国台湾地区大部分分离株亦有90％～91％的核苷酸同源性，但大陆EV71引起的HFMD发病症状较轻，有别于1998年台湾地区EV71的大暴发。     

Cost-effectiveness of bivalent versus monovalent vaccines against hand,foot and mouth disease

ObjectivesEnterovirus 71 (EV71) and coxsackievirus A16 (CA16) were responsible for 43.3% (235 123/543 243) and 24.8% (134 607/543 243) of all laboratory-confirmed hand, foot and mouth disease (HFMD) cases during 2010–2015 in China. Three monovalent EV71 vaccines have been licensed in China while bivalent EV71/CA16 vaccines are under development. A comparative cost-effectiveness analysis of bivalent EV71/CA16 versus monovalent EV71 vaccination would be useful for informing the additional value of bivalent HFMD vaccines in China.MethodsWe used a static model parameterized with the national HFMD surveillance data during 2010–2013, virological HFMD surveillance records from all 31 provinces in mainland China during 2010–2013 and caregiver survey data of costs and health quality of life during 2012–2013. We estimated the threshold vaccine cost (TVC), defined as the maximum additional cost that could be paid for a cost-effective bivalent EV71/CA16 vaccine over a monovalent EV71 vaccine, as the outcome. The base case analysis was performed from a societal perspective. Several sensitivity analyses were conducted by varying assumptions governing HFMD risk, costs, discounting and vaccine efficacy.ResultsIn the base case, choosing the bivalent EV71/CA16 over monovalent EV71 vaccination would be cost-effective only if the additional cost of the bivalent EV71/CA16 compared with the monovalent EV71 vaccine is less than €4.7 (95% CI 4.2–5.2). Compared with the TVC in the base case, TVC increased by up to €8.9 if all the test-negative cases were CA16-HFMD; decreased by €1.1 with an annual discount rate of 6% and exclusion of the productivity loss; and increased by €0.14 and €0.3 with every 1% increase in bivalent vaccine efficacy against CA16-HFMD and differential vaccine efficacy against EV71-HFMD, respectively.ConclusionsBivalent EV71/CA16 vaccines can be cost-effective compared with monovalent EV71 vaccines, if suitably priced. Our study provides further evidence for determining the optimal use of HFMD vaccines in routine paediatric vaccination programme in China.     

Molecular epidemiology analysis of enterovirus 71 strains isolated in Dak Lak,Vietnam, 2011-2016

Human enterovirus 71 (EV71) is the major etiologic agent of hand, foot, and mouth disease (HFMD). EV71 outbreaks have been reported in Dak Lak in recent years, however, the genotypes/subgenotypes information and phylogeny of circulating EV71 strains are limited. The objectives of this study were to determine the genotypes/subgenotypes and investigate the phylogeny of EV71 isolates in Dak Lak over a 6-year period. Viruses were isolated from clinical samples from patients with HFMD. In total, 43 EV71 isolates circulated in Dak Lak during 2011-2016 were used for the phylogenetic analysis using complete VP1 gene. The phylogenetic analysis of the VP1 gene revealed that two major genotypes, B and C, were found. Among the 43 EV71 strains, 29 belonged to subgenotype C4, 2 belonged to subgenotype C5, and 12 belonged to subgenotype B5. Of these, the subgenotype C4 was predominant in 2011-2013 and this was later replaced by the subgenotype B5 in 2014. The subgenotype B5 was dominant between 2014 and 2015, and then C4 recirculated in 2016. Our study also indicated that the subgenotypes C4 and B5 emerged into Dak Lak were closely related to variants causing epidemics of HFMD in the southern and central region of Vietnam and Thailand. Sequence analysis showed that nine amino acid mutations were detected in the VP1 region. Our results identified two significant amino acid substitutions (D31N and E145G/Q) associated with enhancing EV71 virulence.     

An outbreak of enterovirus 71 infection in Taiwan, 1998. II. Laboratory diagnosis and genetic analysis.

BACKGROUND: An epidemic of enterovirus 71 (EV71) occurred in Taiwan from April to December of 1998, with two peaks, one in June and the other in October. Many enteroviruses were isolated in our laboratory from 258 cases during this outbreak. Approximately half of the enteroviruses isolated were EV71 and one fifth were coxsackievirus A16. OBJECTIVES: To analyze laboratory findings in the EV71 epidemic of 1998 in Taiwan, various EV71 specimens in different cell lines were examined. In addition, genetic analysis of 5' non-coding region (NCR) was performed to analyze the strain variation in this outbreak. RESULTS: The cytopathic effect induced by EV71 was observed 2-13 (mean of 4.5) days post-inoculation in Vero cells and 4-15 (mean of 6.6) days in green monkey kidney (GMK) cells inoculated with throat swabs. Of the total positive EV71 cases, virus was most frequently obtained from throat swabs (91.7%), less from stools (64.8%), and none from cerebral spinal fluid (CSF). Molecular analyses of EV71 by sequencing the 5' NCR of 34 strains obtained from different clinical categories and various geographic areas showed that their sequences differed (0-13 bp in 681 bp sequenced) by approximately 0-2%. The sequences of these isolates differed from EV71 prototype BrCr or MS strain by 17.5-19%, with the exception of two samples which exhibited nucleotide variation by only 8.9 and 8.2%, when compared to the MS strain. CONCLUSION: EV71 was most frequently isolated from throat swab specimens in Vero cells. The molecular analyses of the 5' NCR of EV71 revealed that most isolates from this epidemic belonged to a group of closely related clones and only two were in a different group which was clustered with the EV71 MS strain.     

Association of Interleukin-17F Gene Polymorphism with Enterovirus 71 Encephalitis in Patients with Hand, Foot, and Mouth Disease

Enterovirus 71 (EV71) is one of the common pathogenic agents of hand, foot, and mouth disease (HFMD) and is associated with severe complications including encephalitis. Interleukin (IL)-17F plays an important role in tissue inflammation by inducing release of proinflammatory cytokines and chemokines. We investigated the association between EV71 encephalitis and of IL-17F 7488T/C (rs763780) gene polymorphism, which is known to cause a His-to-Arg substitution at amino acid 161. The study was performed in 58 Chinese patients with EV71 encephalitis and 127 Chinese patients with EV71-related HFMD without complications. Genotyping was determined by the polymerase chain reaction–restriction fragment length polymorphism technique. The patients with EV71 encephalitis had a significantly lower frequency of the IL-17F 7488TC+CC genotypes (10.3 %) as compared to the patients with EV71-related HFMD without complications (27.6 %, p?=?0.008). The frequency of IL-17F 7488C alleles was also significantly lower among the patients with EV71 encephalitis (5.2 %) as compared to that of the patients with EV71-related HFMD without complications (15 %, OR?=?0.310, 95 % CI?=?0.127–0.756, p?=?0.006). Furthermore, homozygotes with the T allele had significantly higher levels of C-reactive protein, white blood cell count, and neutrophil count as compared to the patients with CC+CT genotypes (p?=?0.004, 0.001, and 0.000, respectively). These findings suggested that the IL-17F 7488C allele could be significantly associated with protection against encephalitis in Chinese patients with EV71-related HFMD.     

肠道病毒71型手足口病的疫苗筛选和观察

目的 筛选安全有效新型EV71候选疫苗,为将来EV71疫苗开发应用奠定基础.方法 以重组蛋白VP1为疫苗设计靶点,不同候选疫苗包括灭活疫苗、DNA疫苗、VP1蛋白疫苗在0、2周、4周分别按照不同剂量和肌内注射途径免疫BALB/c雌性小鼠,在0、2周、4周、6周、8周、10周、16周分别采集小鼠尾静脉血,16周处死小鼠,收集小鼠脾脏细胞,检测小鼠体液免疫和细胞免疫功能,筛选评价候选疫苗的疗效和安全性.结果 灭活病毒疫苗、VP1 DNA质粒疫苗、VP1蛋白疫苗免疫小鼠2周后IgG抗体滴度即开始升高,4周后明显升高,8周后达高峰,至少维持16周以上;IgG亚类以IgG2a和IgG1为主.三种疫苗能诱导以γ-IFN和IL-4为主的细胞免疫反应.灭活疫苗疗效优于其他候选疫苗,未发现疫苗相关不良反应.结论 灭活病毒疫苗、VP1 DNA质粒疫苗、VP1蛋白疫苗均能诱导持久的特异性细胞免疫和中和抗体反应,以灭活病毒疫苗疗效较好,需要将来攻毒实验可进一步验证其免疫力.     

2008年深圳地区手足口病病原学调查

目的 研究2008年深圳地区手足口病病原体EV71和CoxA16感染情况,为手足口病的防治提供依据.方法 应用RT-PCR技术检测307例手足口病患儿标本病原体EV71、CoxA16基因,将PCR产物测序,并应用ChstaIW2在线分析软件进行序列分析、进化树分析.结果 不同标本中EV71的阳性率分别为:大便标本24.4%(75/307)、咽拭子为7.8%(24/307)、外周血12.5%(1/8);CoxA16的阳性率分别为,大便13.8%(28/203),咽拭子11.0%(20/181),其中EV71和CoxA16同时为阳性的有3份(0.98%);脑脊液标本未检测到EV71和CoxA16.与标准株BrCr序列比对分析,测序的14例标本中,8例的EV71新出现多个位点变异,其中2例标本的A2528G变异导致氨基酸改变,即N595D,1例标本的A2714G变异引起氨基酸改变,即1658V.进化树分析显示,有2例标本的病毒株与anhui毒株亲缘关系较近,与标准株BrCr及深圳株SHH02-6、SHZH02-40、SHZH03-58等亲缘关系较远;而其余12株与上述毒株亲缘关系较远.根据文献报道的EV71基因分型方法分析,测序的14株EV71基因型为C型.结论 在引起2008年深圳手足口病病原体中相当的比例是EV71,且可能部分是anhui病毒株的入侵.     

Antigenicity, animal protective effect and genetic characteristics of candidate vaccine strains of enterovirus 71

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease. Recently, several EV71 inactivated vaccine candidates have been development in mainland China. To compare the antigenicity and genetic characteristics of different candidate vaccine strains for EV71, the genomes of 11 EV71 strains were sequenced. The cross-protection capacities were determined by neutralizing antibody test and were further assessed in neonatal mice. The results showed 93.0%–99.8% genome homology of the VP1 regions, but their cross-neutralization capacities varied. These results showed that vaccine strains with good cross-neutralization capacities had the potential to be vaccine strains.     

Clinicopathologic features and molecular analysis of enterovirus 71 infection: report of an autopsy case from the epidemic of hand, foot and mouth disease in China

A 15-month boy with fatal hand, foot, and mouth disease (HFMD) exhibited atypical symptoms and progressed rapidly to death. An autopsy was performed the next day and tissue sections were stained for histopathological examination. His intestinal samples were tested for enterovirus 71 (EV71), and the whole-genome sequence of EV71 was analyzed. An autopsy revealed that the central nervous system, lungs, and gut displayed severe meningitis and brainstem encephalitis, remarkable pulmonary congestion, edema, moderate inflammatory infiltration, and hemorrhage as well as intestinal mucosal congestion, epithelial necrosis, thinning intestinal wall, and submucosal lymphoid follicular hyperplasia. The heart showed myocardial interstitial congestion, myocardial edema, and some inflammatory infiltrates. There were no significant alterations in the architecture of other organs. EV71 antigen and apoptotic cells were detected in brain, lung and intestine by immunohistochemical staining and TUNEL (TdT-mediated dUTP nick-end labeling) respectively. Intestinal contents and intestinal autopsy samples of this case were positive for EV71, and the EV71 strain was classified as subgenogroup C4. In China, the severe forms of HFMD were mostly caused by EV71 subgenogroup C4 infection. Severe intestinal damages may relate to EV71 subgenogroup C4 infection. Thus, children with severe EV71 HFMD may have serious pathological changes in their central nervous system, lungs, and gut. Physicians should pay special attention to infants with atypical symptoms, particularly in EV71 epidemic areas for early diagnosis and treatment.     

Transgenic expression of human P-selectin glycoprotein ligand-1 is not sufficient for enterovirus 71 infection in mice

Human PSGL-1 is a receptor for EV71 that facilitates EV71 entry and replication in mouse cells. We have evaluated the role of human PSGL-1 in EV71 infection in vivo using a transgenic mouse line. Expression of human PSGL-1 failed to enhance infectivity of clinical EV71 strains in mice; however, it promoted replication and symptom severity at an earlier stage in mice upon infection with a mouse-adapted EV71 strain. We therefore conclude that human PSGL-1 alone is not sufficient to modulate infection with the clinical EV71 strains of genotype C4 in mice.     

人源抗EV71病毒基因工程抗体的研究

目的 研制人源抗EV71病毒基因工程抗体.方法 采集多个患儿外周血淋巴细胞,构建人源抗EV71单链（scFv）噬菌体抗体基因文库.用纯化的EV71 VPI蛋白对抗体库进行筛选,将筛出抗体的轻链和重链通过序列测定确定抗体轻重链型别后分别克隆入全抗体表达载体pAC-LCH3R后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达.结果 成功地获得了1株抗EV71病毒VPI蛋白的人源单克隆抗体,利用ELISA、IFA对所获人源单克隆抗体的功能特性进行鉴定,表达成分泌型IgG全抗体在体外与A型及C4亚型EV71病毒的中和反应结果 显示为阴性.结论 从免疫库中获得了1株人源非中和单抗,为EV71病毒引起的手足口病的鉴别诊断奠定了基础.     

Genetic diversity of enterovirus 71 isolated from cases of hand,foot and mouth disease in Yokohama City between 1982 and 2000

Summary.  Enterovirus 71 (EV71) is known as one of the major causative agents of hand, foot and mouse disease (HFMD) and is also associated with neurological manifestations such as aseptic meningitis, polio-like paralysis and encephalitis. Recently, large HFMD outbreaks, involving severe neurological complications, have been experienced in Malaysia, Taiwan and some other countries in the Western-Pacific region. To investigate the genetic diversity of EV71 isolates in a single community in Japan, nucleotide sequences of the VP4 region of 52 EV71 isolates in Yokohama City from 1982 to 2000 were determined and the phylogenetic relationship was compared with other referential EV71 strains in Japan and in the world. There were two major genotypes of EV71 in Yokohama City through the 1980’s and 1990’s. Six EV71 isolates in the early 1980’s in Yokohama City were closely related to those from HFMD outbreaks in Japan and from outbreaks of polio-like paralysis in Europe in the 1970’s. During recent HFMD outbreaks in 1997 and 2000, two distinct genotypes of EV71 were co-circulating in Yokohama City as in HFMD outbreaks in Malaysia and Taiwan. However, the genetic diversity of EV71 in Yokohama City was not directly correlated with the severity of HFMD. The results confirmed the circulation of two distinct genotypes of EV71 over the past 20 years in Japan. Received June 25, 2002; accepted September 16, 2002     

IgM抗体检测对肠道病毒71型感染者诊断价值的探讨

目的 探讨IgM抗体检测对肠道病毒71型(EV71)感染者的诊断价值,并与核酸检测和中和试验方法进行比较分析.方法采用EV71 IgM抗体酶免检测试剂盒(EV71 -IgM试剂)、EV71核酸检测试剂盒(EV71-PCR试剂)和中和试验方法,检测115例EV71感染者的EV71 RNA、中和抗体和IgM抗体的水平,并进行比较性研究分析.结果 对115例EV71感染者,EV71 -IgM试剂对患者首份血清的检出率为80.9％ (93/115,95％ CI:72.5～87.6),对228份健康儿童血清,EV71 -IgM试剂的检出率为2.6％.同时,对病发1～2d采集的54份血清检出率为70.4％(38/54),显示了较好的早期诊断价值.EV71-PCR试剂对患者咽拭子标本的检出率为82.6％(95/115,95％ CI:74.4-89.0),与EV71-IgM试剂相比,差异无统计学意义.此外,EV71-IgM试剂与EV71-PCR试剂具有很好的互补性,两种方法联合使用可提高检测灵敏度约10个百分点达到92.2％.结论 EV71 -IgM试剂具有良好的早期诊断价值,与EV71-PCR试剂联用可提高诊断率.     

Association of polymorphisms in the vitamin D receptor gene with severity of hand,foot, and mouth disease caused by enterovirus 71

Severe hand, foot, and mouth disease (HFMD) is sometimes associated with critical complications that can cause substantial child mortality. Activity of the vitamin D receptor (VDR) may influence the outcomes of enterovirus 71 (EV71) infection. This case-control study aimed to assess the association of single-nucleotide polymorphisms (SNPs) in the gene encoding the VDR with the severity of EV71-associated HFMD. We selected four VDR SNPs based on linkage disequilibrium and functional prediction, and we tested them using the SNPscan multiple SNP typing method for potential association with severity of EV71-associated HFMD. We found a significant association in the case of rs11574129 (G vs A: odds ratio (OR), 0.3439; 95% confidence intervals (CI), 0.1778-0.6653) and rs739837 (T vs G: OR, 0.5580; 95%CI, 0.3352-0.9291). Our results suggest that these two SNPs may influence the severity of EV71-associated HFMD.     

肠道病毒71型IgM抗体检测在手足口病早期诊断中的价值

目的评估人肠道病毒7l型（EV71）IgM抗体在手足口病早期诊断中的价值。方法自发病后连续每日采集2010年我院收治的38例手足口病患儿血清及咽拭子标本,分别检测EV 71 IgM抗体、肠道病毒核酸、EV71特异核酸。结果38例手足口病患儿IgM抗体,按发病天数累加阳性率分别为：第1天60．5％、第2天71．1％、第3～4天81．5％、第5天92．1％、第6天92．1％;肠道病毒核酸阳性率为73．6％;EV71特异核酸阳性率为60．5％。结论EV 7l IgM抗体在手足口病发病的第1天即可出现,至第5天阳性率达到峰值,可作为手足口病早期诊断指标之一。