Molecular epidemiology of enterovirus 71 in peninsular Malaysia, 1997–2000

Summary. Human enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae) has been responsible for sporadic cases and outbreaks of hand-foot-and-mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like disease in Europe, the U.S.A., Australia and Asia. Recently, there has been an increase in EV71 activity in the Asia-Pacific region, with many outbreaks of HFMD associated with brainstem encephalitis manifesting as neurogenic pulmonary oedema with a high case fatality rate. In 1997, and again in 2000, EV71 outbreaks occurred in peninsular Malaysia. Variations in VP1 gene sequences have been shown to divide all known EV71 field isolates into three distinct genogroups (A, B and C). Consequently we examined the VP1 gene sequences of 43 EV71 strains isolated in peninsular Malaysia between 1997 and 2000 in order to determine the genogroup prevalence over the period. In this study we show that four subgenogroups (B3, B4, C1 and C2) of EV71 circulated in peninsular Malaysia between 1997 and 2000. Subgenogroups B3, B4 and C1 have been identified as the primary cause of the outbreaks of EV71 in peninsular Malaysia. Subgenogroup C1 also displayed endemic circulation from 1997 to 2000 and subgenogroup C2 was present at a low level during the 1997 outbreak.Received September 6, 2002; accepted January 20, 2003 Published online June 2, 2003     

Molecular characterization and complete genome analysis of human enterovirus 71 and coxsackievirus A16 from children with hand, foot and mouth disease in Thailand during 2008-2011

Hand, foot and mouth disease (HFMD) has mostly been caused by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). CA 16 was the most common cause of HFMD in 2010. EV71 had a high prevalence in 2008-2009 and has been identified with a higher frequency since 2011. Nearly complete genome sequences of three EV71 strains (2008-2009 strains) and two CA16 strains (2010 strains) obtained from outbreaks in Thailand in 2008 to 2010 were characterized. Based on a phylogenetic tree of the complete VP1 region, three EV71 strains grouped into the B5, C1 and C4 genotypes, and two CA16 strains grouped into the C genotype. Based on sequence analysis, nucleotide changes were found to cluster in the internal ribosome entry site (IRES) element of the 5′-untranslated region (5′-UTR). Amino acid differences identified in all strains were located in the non-structural protein. These data also provide the molecular epidemiology of EV71 and CA16 outbreaks in Thailand.     

Molecular epidemiology of enterovirus 71 in Taiwan

Summary.  Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5–24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 × 10⁻³vs. 1.4 × 1010⁻³) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance. Received June 13, 2000 Accepted July 29, 2000     

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.     

Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia

Thirteen enterovirus 71 (EV71) isolates were obtained from both fatal and non-fatal infections of patients seen in Peninsula Malaysia and in Sarawak during an outbreak of hand, foot and mouth disease (HFMD) in Malaysia in 1997, with incidences of fatal brainstem encephalomyelitis. The isolates were identified using immunofluorescence staining, neutralization assays, and partial sequencing of the 5' untranslated regions (UTR). Assessment of the potential genetic relationships of the isolates using the partial 5'UTR sequences suggested clustering of the isolates into at least two main clusters. Isolates from Peninsula Malaysia were found in both clusters whereas Sarawak-derived isolates clustered only in cluster II. Isolates derived from fatal infections, however, occurred in both clusters and no distinctive nucleotide sequences could be attributed to the fatal isolates. Examination of the nucleotide sequences revealed at least 13 nucleotide positions in all the isolates which differ completely from the previously reported EV71 5'UTR sequences. In addition, at least 11 nucleotide position differences within the 5'UTR were noted which differentiated cluster I from cluster II. Predicted secondary RNA structures drawn using the nucleotide sequences also suggested differences between isolates from the two clusters. These findings suggest the presence of at least two potentially virulent EV71 co-circulating in Malaysia during the 1997 HFMD outbreak.     

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。     

An outbreak of enterovirus 71 infection in Taiwan, 1998: epidemiologic and clinical manifestations.

BACKGROUND: An outbreak of enterovirus infections occurred throughout Taiwan in 1998. The diseases were manifectated with hand, foot, and mouth disease (HFMD), some associated with meningitis, encephalitis, or acute flaccid paralysis (AFP). OBJECTIVES: This study is aimed to characterize and analyze the epidermologic and clinical features during the outbreak. STUDY DESIGN: The epidemiologic information was collected from the Ministry of Health on passive surveillance; clinical and virological investigations were carried out at National Cheng Kung University Medical Center. RESULTS: Between April and December 1998, 405 children were hospitalized, and 78 patients died during this outbreak in Taiwan. There were 119 cases identified to be EV71 infection in Tainan and Chiayi areas; 105 cases by virus isolation and 14 by serological assay. The outbreak had a biphasic curve with peak in June and October, especially in the southern Taiwan. Seventy-two percent of patients were below 3 years of age. The spectrum of disease included HFMD in 54, HFMD with central nerve system (CNS) involvement in 37, herpangina in 12, aseptic meningitis in three, encephalitis/ meningoencephalitis in ten, acute flaccid paralysis in three. There was nine fatal cases complicated with neurogenic pulmonary edema. Myoclonus with sleep disturbance was the most important early sign of EV71 infection with CNS involvement. CONCLUSION: Our experience demonstrated that the EV71 isolated in Taiwan had strong dermatotropic as well as neurotropic tendencies. Early detecting CNS involvement and commencing aggressive therapy may reduce the mortality.     

Hand, foot and mouth disease: seroprevalence of Coxsackie A16 and Enterovirus 71 in Germany

Coxsackie A16 (CA16) and Enterovirus 71 (EV71) are members of the picornaviridae family and are associated with hand, foot and mouth disease (HFMD), in rare cases also to acute neurological diseases. HFMD outbreaks have been reported from many parts of the world, especially Southeast Asia. The objective of the study was to analyze CA16 and EV71 seroepidemiologically in the population of Frankfurt/M., Germany. A total of 696 individuals (349 males and 347 females, divided into seven different age groups, 1–4, 5–9, 10–14, 15–19, 20–39, 40–59 and >60 years) were tested for serum antibodies against CA16 and EV71 by the use of a microneutralization test. Sera were collected at the Frankfurt university hospital from patients suffering from other diseases between March and September 2006. CA16 and EV71 infections were observed to be widely present in the population. The age-adjusted seroprevalence for individuals ≥1 year was found to be 62.9% for CA16 and 42.8% for EV71 without a gender-specific significant difference. Only 12.0 and 27.0% of the children aged 1–4 had antibodies to EV71 and CA16, respectively – indicating that 88 and 73% of the children in this age group were susceptible to the infection. A total of 213 individuals (30.6%) was seropositive for both viruses, 303 (43.5%) showed neutralizing antibodies (NtAb) to at least one of the two viruses. A total of 180 individuals (25.9%) revealed no antibodies. High CA16 and EV71 antibody titers were found especially in the age group of the 10- to 14-year-olds, without gender-specific difference. The seroprevalence study demonstrates a common spread of CA16 and EV71 in Germany, but a relatively high susceptibility of the younger population to CA16 and EV71. Obviously, the manifestation rate, i.e., distinct disease of these infections is low.     

Emergence of enterovirus 71 “double-recombinant” strains belonging to a novel genotype D originating from southern China: first evidence for combination of intratypic and intertypic recombination events in EV71

Hand–foot–mouth disease due to enterovirus 71 (EV71) and coxsackievirus A16 (CA16) has recently caused large outbreaks in mainland China in 2008. We performed complete genome sequencing on two EV71 (SZ/HK08-5 and SZ/HK08-6) and two CA16 (SZ/HK08-3 and SZ/HK08-7) strains from patients in Shenzhen, China. Phylogenetic, similarity plot and bootscan analyses revealed recombination between EV71 genotypes B and C at the 2A–2B junction, and between EV71 genotype B and CA16 strain G-10 in the 3C region for EV71 strains. A similar phenomenon was also found upon further gene sequencing with other EV71 strains. Recombination between CA16 strain G-10 and EV71 genotype A at the 2A–2B junction was also observed for CA16 strains. The present “double-recombinant” EV71 strains circulating in China and other EV71 subgenotype “C4” strains represent an additional genotype, D. CA16 strains should also be classified into two genotypes. This represents the first evidence for a combination of intratypic and intertypic recombination in EV71 strains.     

Clinical and Etiological Characteristics of Enterovirus 71-Related Diseases during a Recent 2-Year Period in Korea

Human enterovirus 71 (EV 71) has caused large-scale outbreaks of hand-foot-and-mouth disease (HFMD), particularly in the Asian-Pacific region. In this study, we report a major outbreak of EV 71 infection in Korea and describe the clinical differences between EV 71 and non-EV 71 enterovirus infections. We prospectively enrolled patients with suspected viral infections during a recent 2-year period through a nationwide surveillance system. We identified 719 patients with suspected HFMD or herpangina using real-time PCR and genotyping based on VP1 sequence analysis. The major pathogen causing HFMD changed substantially from 2008 to 2009, with EV 71 becoming the most common cause of HFMD in Korea in 2009. We successfully identified the enteroviral genotypes for 218 of the 719 patients. Patients with EV 71 infections tended to be younger than those with non-EV 71 enteroviral infections and presented with HFMD and meningoencephalitis. In addition, the occurrence of fever, headache, and neck stiffness was significantly higher in patients with EV 71 infections. Multivariable analysis showed that for patients presenting with HFMD, fever, or a sore throat, each covariate was independently associated with EV 71 infection; the adjusted odds ratios (with 95% confidence intervals in parentheses) for these variables were 31.86 (10.04 to 101.09), 4.76 (1.71 to 13.25), and 0.18 (0.04 to 0.77), respectively. Our results indicate that EV 71 was a major cause of HFMD in Korea during the study period. In addition, we found that clinical symptoms may be helpful in the early identification of patients with EV 71 infections.Human enterovirus 71 (EV 71) is an important emerging pathogen of hand-foot-and-mouth disease (HFMD) (1, 6, 30). In particular, several major outbreaks of EV 71 have been documented in the Asia-Pacific region since 1997 (3, 4, 7, 9, 25). EV 71 infection causes HFMD, a common exanthema of young children that is characterized by a fever, rashes on the palms and the bottoms of the feet, and ulcers in the oral cavity. In general, patients with HFMD experience a mild course of disease; however, some patients develop severe neurological complications, especially as a result of EV 71 (18).Given that EV 71 infection could cause severe neurological complications, its early detection in patients with HFMD is an important part of intensive care efforts and efforts to prevent mortality. Unfortunately, current methods for the detection of EV 71 do not enable early detection (23). Here we report an outbreak of EV 71 infection in Korea. As part of this nationwide surveillance study, we examined the clinical differences between EV 71 and non-EV 71 enteroviral infections, and we investigated whether these differences are clinically applicable to the early detection of EV 71 in patients with complications of HFMD.     

Phylogenetic and serological characterization of echovirus 11 and echovirus 19 strains causing uveitis

Summary.  The strains of echovirus 19 (EV19) and echovirus 11 (EV11), isolated from infants with similar clinical symptoms of acute enterovirus uveitis (EU) in Russia (Siberia) in 1980–1989, were investigated phylogenetically (nucleotide sequence of a 300 nt fragment in 5′ NTR and VP4 junction) and serologically. The result confirmed that viruses belong to the Enterovirus genus, with 58–80% nt sequence homology with previously sequenced enteroviruses, and showed the genetical identity between the strains isolated during each of five outbreaks of the EU. The results also demonstrated that isolates from the last three outbreaks of EU belong to the same phylogenetic group despite the remarkable spatial and temporal distance between the outbreaks. The results confirm the role of these echoviruses in the etiology of the EU. Based on phylogenetic and serological comparisons the studied strains were divided into three distinct groups: group I, EV19/K (Krasnoyarsk, 1980–1981), group II, EV11/A (Krasnoyarsk, 1982), group III, EV11/B (Krasnoyarsk, 1986; Omsk, 1987–1988; Irkutsk, 1989). Minor details of the epidemiology of the outbreaks were also revealed. Received February 16, 2001 Accepted July 13, 2001     

Reemerging of enterovirus 71 in Taiwan: the age impact on disease severity

Enterovirus 71 (EV71) infection commonly strike children under the age of 3 years, with an occasionally unfavorable outcome in children. This study was designed to explore the relationship between age and the severity of complications, which may associate with antibody-dependent enhancement (ADE) in EV71. All EV71-infected patients during the outbreak of 2008 were recruited. In total, 134 patients were enrolled and categorized into two age groups, 0–12 months (n = 18) and >12 months (n = 116). Pulmonary edema/hemorrhage more commonly occur in patients younger than 12 months. No difference in the occurrence of herpangina/hand-foot-and-mouth disease (HFMD), uncomplicated brainstem encephalitis (BE), or autonomic nervous system (ANS) dysregulation was noted between the two age groups. Patients with pulmonary edema/hemorrhage (11.9 ± 14.7 months) were younger than patients with herpangina/HFMD (35.8 ± 26.4 months) or ANS dysregulation (33.9 ± 20.9 months). Our findings are in agreement with the data regarding the outbreak in Taiwan, in which a decrease in age corresponded to an increase in disease severity with regard to central nervous system complications. A reduction of maternal antibodies to the subneutralizing level within 1 year of age may be associated with the ADE of the infection. This study could provide possible clinical significance with regard to ADE phenomena in young infants infected by EV71.     

2008至2009年北京地区分离的肠道病毒71型全基因组序列分析

目的了解2008至2009年从北京地区手足口病（HFMD）患儿分离到的肠道病毒71型（EV71）全基因组序列特点（未包括多聚腺苷尾）,以探讨基因序列的改变是否与病毒的致病性有关。方法选取首都儿科研究所病毒研究室2008年分离到的5株EV71毒株和2009年分离到的4株EV71毒株,其中4株来源于重症HFMD患儿（伴高热、持续抽搐及意识丧失等中枢神经系统症状）,5株来源于轻症HFMD患儿。设计覆盖病毒全基因组的10对特异性引物,对9株EV71毒株进行RT-PCR扩增、全基因组序列测定和分析。结果 9株EV71毒株的全基因组长度为7406bp或7405bp,部分毒株在5′UTR存在1处1个碱基的缺失。9株EV71毒株的全基因组核苷酸和氨基酸同源性分别为96.3%～99.4%和98.2%～99.6%,在VP1区核苷酸和氨基酸同源性分别为96.9%～99.9%和98.3%～100.0%。重症HFMD来源的4株毒株中有3株在VP2蛋白第144位及3D聚合酶（3Dpol）第140和263位同时出现相同的氨基酸变异（T144S、R140K和I263V）,并且在5′UTR区第208和254位同时出现相同的碱基变异（G208A和A254G）。9株EV71毒株的全基因组与C4亚型毒株具有最高的核苷酸同源性,在VP1区为94.3%～95.5%;在3D及3′UTR区与CV-A16/G10的同源性（84.3%～85.0%和89.0%～91.5%）高于与EV71-B型、A型及C型（C1～3、C5）的同源性。VP1和3D基因的遗传进化分析显示,9株EV71毒株与C4亚型毒株属同一分支。结论 VP2蛋白第144位氨基酸突变（T→S）、3Dpol第140和263位氨基酸突变（R→K和I→V）及5′UTR区第254位碱基突变（A→G）可能与EV71感染后引起的不同临床症状有关。根据VP1核苷酸序列,2008至2009年北京地区流行的EV71属于C4亚型;非结构蛋白基因在EV71进化中可能有一定的作用。     

Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase

In 1998, an enterovirus 71 (EV71) epidemic in Taiwan resulted in 78 deaths; however, the molecular basis of EV71 pathogenicity remains poorly understood. Comparison of the deduced amino acid sequences in 3D polymerases of EV71clinical isolates showed the T251V or T251I substitution from 1986 and 1998 outbreaks. An EV71 replicon system showed that introducing an I251T mutation did not affect luciferase activities at 35 °C when compared with wild type; however, lower luciferase activities were observed when they were incubated at 39.5 °C. In addition, the I251T mutation in the EV71 infectious clone not only reduced viral replication at 39.5 °C in vitro but also decreased the virulence of the mouse adaptive strain MP4 in neonatal mice in an i.p. infection model. Therefore, these results suggested that the threonine at position 251 results in a temperature sensitivity phenotype of EV71 which may contribute to the attenuation of circulating strains.     

Rapid detection of enterovirus 71 by real-time TaqMan RT-PCR.

BACKGROUND: Enterovirus 71 (EV71) is the main etiological agent of Hand, Foot and Mouth Disease (HFMD) and has been associated with neurological complications which resulted in fatalities during recent outbreaks in Asia Pacific region. OBJECTIVE: Develop a real-time TaqMan RT-PCR for rapid detection of EV71. STUDY DESIGN: Specific primers and probe were designed based on highly conserved VP1 region of EV71. The sensitivity of the real-time RT-PCR was evaluated with 67 clinical specimens collected from pediatric patients with suspected HFMD. RESULTS: Our real-time TaqMan RT-PCR showed 100% specificity in detecting EV71 and showed an analytical sensitivity of 5 viral copies. High sensitivity was also achieved in detecting EV71 directly from clinical specimens. CONCLUSIONS: Real-time TaqMan RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.     

Seroprevalence and molecular epidemiology of enterovirus 71 in Germany

Enterovirus 71 (EV71) has emerged as a significant pathogen with potential to cause large outbreaks. Because little is known about its seroprevalence and molecular epidemiology in Germany, data for 1997–2007 are presented. Four hundred thirty-six sera from persons aging 10 months to 75 years were tested in a neutralisation test; 63.4% of pre-school children were seronegative, whereas about 75% of adults had antibodies to EV71. Phylogenetic analysis of 28 isolates associated with neurological or cutaneous manifestations showed that isolates belonging to genogroup C1 predominated in 2000–2005, followed by a change to genogroup C2 in 2006 and 2007. This shows the importance of monitoring the diversity of one of the most relevant neurotropic enteroviruses.     

Encephalomyelitis caused by enterovirus type 71 in children

Enterovirus type 71 (EV71) is a causative agent of large outbreaks of hand, foot, and mouth disease (HFMD) in Europe (Bulgaria, 1975; Hungary, 1978) and South-East Asia (Malaysia, 1977; Taiwan, 1998; Singapore, 2000-2007; People's Republic of China, 2007-2009). HFMD afflicted children less than 10 years of age and resulted in recovery within 3-7 days. In a small percentage of infants (aged 6 months to 3 years), HFMD was accompanied by acute neurological complications, such as serous meningitis, poliomyelitis-like syndrome (extremity pareses and muscle paralyses); brain stem encephalitis (myoclonic jerks, tremor, lethargy, swallowing and speech disorders, cardiopulmonary failure, pulmonary edema, shock, coma, death). X-ray study revealed pulmonary hemorrhages and edema. Mortality rates were as high as 82-94% in severe cases. Incapacitating motor, respiratory, and psychoemotional disorders persisted in some surviving children. Pathomorphologically, patients with central nervous system disease and cardiopulmonary failure were found to have acute inflammation of the grey matter of the brain stem (medulla oblongata, pons) and spinal cord. Inflammatory changes in the lung and myocardial tissues were negligible or absent. Fatal pulmonary edema was neurogenic in origin and resulted from damage to the respiratory and vasomotor centers of the brain stem.     

惠州市重症手足口病病原谱及EV71型VP1区基因特征分析

目的分析惠州市手足口病重症病例的病原谱构成,了解惠州市肠道病毒71型分离株的VPl区基因特征．为科学防治手足口病提供科学依据。方法采集300例重症手足口病（HFMD）患者标本,采用实时荧光RT-PCR检测肠道病毒核酸,并对肠道病毒7l型（EV71）和柯萨奇病毒A16型（CoxA16）进行分型检测;选择8株EV71分离株进行VPl区基因全长序列测定,测序结果利用DNASTAR软件进行核苷酸、氨基酸序列分析和同源性比较。并用Mega5．0软件构建亲缘性进化树。结果通过实时荧光RT-PCR特异性检测,EV71阳性结果154份,阳性率为51．33％;CoxAl6阳性结果38份,阳性率为12．67％。测序结果表明,8株EV71之间的VPl基因核苷酸同源性为96．9％～99．2％。氨基酸同源性为99．3％～100％。VPI区基因遗传进化分析表明。8株EV71分离株与c4基因亚型的代表株处于同一分支,均属于C4基因亚型的C4a进化分支。结论惠州市手足口病疫情主要病原EV71病毒均属于C4a基因亚型,与2004年以来的中国大陆EV71病毒流行的基因型一致,未产生明显的抗原漂移及变异。