Human Enterovirus 109: a Novel Interspecies Recombinant Enterovirus Isolated from a Case of Acute Pediatric Respiratory Illness in Nicaragua

Enteroviruses (Picornaviridae family) are a common cause of human illness worldwide and are associated with diverse clinical syndromes, including asymptomatic infection, respiratory illness, gastroenteritis, and meningitis. In this study, we report the identification and complete genome sequence of a novel enterovirus isolated from a case of acute respiratory illness in a Nicaraguan child. Unbiased deep sequencing of nucleic acids from a nose and throat swab sample enabled rapid recovery of the full-genome sequence. Phylogenetic analysis revealed that human enterovirus 109 (EV109) is most closely related to serotypes of human enterovirus species C (HEV-C) in all genomic regions except the 5′ untranslated region (5′ UTR). Bootstrap analysis indicates that the 5′ UTR of EV109 is likely the product of an interspecies recombination event between ancestral members of the HEV-A and HEV-C groups. Overall, the EV109 coding region shares 67 to 72% nucleotide sequence identity with its nearest relatives. EV109 isolates were detected in 5/310 (1.6%) of nose and throat swab samples collected from children in a pediatric cohort study of influenza-like illness in Managua, Nicaragua, between June 2007 and June 2008. Further experimentation is required to more fully characterize the pathogenic role, disease associations, and global distribution of EV109.The genus Enterovirus (EV) in the family Picornaviridae is a group of related viruses that are associated with a spectrum of disease, ranging from subclinical infections to acute respiratory and gastrointestinal illness to more severe manifestations, such as aseptic meningitis, encephalitis, and acute flaccid paralysis (16, 32). Enteroviruses are small, nonenveloped viruses that share a genomic organization. The RNA genome is a ∼7.5 kb single-stranded, positive-sense, polyadenylated molecule, with a single, long open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). The 5′ UTR is ∼700 nucleotides in length and contains highly structured secondary elements with internal ribosomal entry site (IRES) function. The ∼2,200-amino-acid (aa) polyprotein is cotranslationally processed by viral proteases to yield structural (VP4, VP2, VP3, and VP1) and nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins (32). Current enterovirus classification is based on the high sequence divergence within the VP1 capsid region, which has been shown to correspond with serotype neutralization (27, 28). Human enterovirus (HEV) types are currently classified into four species, human enterovirus A (HEV-A), HEV-B, HEV-C (including poliovirus), and HEV-D, based on the four phylogenetic clusters observed in comparisons of the coding region sequences. An enterovirus is considered a new type within a species if it possesses <75% nucleotide identity and <85% amino acid identity with known members across the VP1 sequence (27, 30). Molecular identification methods play a crucial role in rapid, sensitive enterovirus diagnostics and have led to the recent discovery of several novel enteroviruses (29, 31, 40, 42, 44). Most approaches target a limited number of conserved regions in the 5′ UTR and VP4-VP2 junction or seek to ascertain serotype information by probing antigenic regions, such as VP1 (5).Picornavirus RNA-dependent RNA polymerases are highly error prone and lack proofreading ability, resulting in a misincorporation frequency of 1 per 10³ to 10⁴ nucleotides (48). The relative infidelity of these polymerases is believed to enable rapid adaptability under selective pressure. Large-impact evolutionary events, such as recombination within and between enterovirus serotypes, also contribute to their evolution and genetic diversity (3, 8, 26, 39) and may lead to changes in disease associations with human enterovirus infections. Human enteroviruses are classified into four species based on coding region sequence phylogeny, and intraspecies recombination events between enteroviruses that are closely related in the coding region are well documented (26, 38, 39). All known enterovirus 5′ UTR sequences, however, cluster into two groups containing either HEV-A and -B sequences or HEV-C and -D sequences. Recent findings have described enterovirus genomes with a coding region that clusters with one species and a 5′ UTR that clusters with a different species, suggesting possible interspecies recombination events (41, 44). Understanding the recombination-driven evolution of HEV-C viruses is of particular public health concern due to the viruses'' ability to recombine with vaccine poliovirus, resulting in circulating, highly neurovirulent vaccine-derived polioviruses (17, 21, 34). It is unclear whether recombination events between poliovirus and HEV-C viruses allow for the rapid acquisition of traits that increase pathogenic and circulation potential.The enterovirus pathogenicity spectrum is related to tissue tropism and is largely determined by cellular receptor usage. Most picornaviruses use receptors from the immunoglobulin superfamily of proteins, such as intracellular adhesion molecule-1 (ICAM-1) or coxsackievirus-adenovirus receptor (CAR) (36). A distinct subgroup of HEV-C viruses, which includes coxsackievirus (CAV) A1, A19, and A22 and enterovirus 104, has not yet been grown successfully in cell culture, and the receptor molecule for this subgroup is unknown (6). HEV-C viruses are believed to be the ancestral source of poliovirus, which resulted from a capsid mutation that caused a cellular receptor switch from ICAM-1 to CD155 (poliovirus receptor [PVR]) (17).In this study, we report the discovery and characterization of a novel human enterovirus type within species HEV-C, for which we propose the designation human enterovirus 109 (EV109). Sequence analysis reveals considerable nucleotide divergence in the 5′ UTR between EV109 and other HEV-C types, and scanning bootstrap analysis supports the hypothesis that EV109 is the product of an interspecies recombination event with an ancestral member of the HEV-A group. Viral capsid amino acid alignments and homology modeling reveal the predicted three-dimensional arrangement of divergent and conserved residues of EV109 compared with other related enteroviruses. We also report highly similar EV109 isolates within multiple cases of acute pediatric respiratory illness in Managua, Nicaragua.     

Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice

Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni–NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.     

肠道病毒71型外壳蛋白VP1全长在大肠杆菌中的高效表达及初步活性测定

目的:重组表达肠道病毒71型(EV71)外壳蛋白VP1全长,用于研制血清学检测试剂和疫苗研发。方法:在获得EV71全长基因并测序正确的基础上,将外壳蛋白VP1全长基因克隆到表达载体pET28a(+)上,构建重组表达质粒pET28a(+)/VP1,转化大肠杆菌BL21,IPTG诱导表达,利用Ni2+亲和层析柱对重组蛋白进行纯化,采用双抗原夹心检测技术评价重组抗原与27份EV71抗体阳性血清和18份阴性血清的反应情况。结果:重组EV71-VP1蛋白在大肠杆菌中诱导6 h后可获得高效表达,能与27份EV71抗体阳性血清中的21份发生阳性反应,EV71双抗原夹心检测与中和血清测试结果具有很好的一致性(P0.05)。结论:实现了肠道病毒71型外壳蛋白VP1的高效表达,为肠道病毒71型诊断试剂和疫苗的研究奠定了基础。     

Population Dynamics and Genetic Diversity of C4 Strains of Human Enterovirus 71 in Mainland China, 1998–2010

Background

Since 1997, several countries within the Asian Pacific region have been affected by one or more massive outbreaks of Hand Foot and Mouth Disease (HFMD). Virus typing experiments revealed that these outbreaks were caused by strains of human enterovirus 71 (EV71) belonging to several different, recently emerged subgenogroups. In mainland China, a different situation was observed. The first outbreak, localized in Shangdong Province, was reported in 2007, and was followed by a wide-spread outbreak in mainland China in 2008. Since then, numbers of reported HFMD cases have been persistently high.

Methodology/Principal Findings

To gain insight in the epidemiological behavior of EV71 in China, we studied genetic diversity and EV71 population dynamics to address whether the increase in number of reported EV71 infections reflects a real increase in viral spread or is just the result of increased awareness and surveillance. We used systematically collected VP1 gene sequences of 257 EV71 strains collected in Guangdong province from 2008 to 2010 as part of HFMD surveillance activities, and supplemented them with 305 GenBank EV71 reference stains collected in China from 1998 to 2010. All isolates from Guangdong Province belonged to subgenogroup C4. Viral population dynamics indicated that the increased reporting of HFMD in China since 2007 reflects a real increase in viral spread and continued replacement of viral lineages through time. Amino acid sequence comparisons revealed substitution of amino acid in residues 22, 145 and 289 through time regularly with the VP1 gene of EV71 strains isolated in mainland China from 1998 to 2010.

Conclusions

EV71 strains isolated in mainland China mainly belonged to subgenogroup C4. There was exponential growth of the EV71 virus population in 2007 and 2008. There was amino acid substitution through time regularly with the VP1 gene which possibly increased viral spread and/or ability of the virus to circulate persistently among the Chinese population.     

Detection of human bocavirus mRNA in respiratory secretions correlates with high viral load and concurrent diarrhea

Human bocavirus (HBoV) is a parvovirus recently identified in association with acute respiratory infections (ARI). Despite its worldwide occurrence, little is known on the pathogenesis of HBoV infections. In addition, few systematic studies of HBoV in ARI have been conducted in Latin America. Therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of HBoV in outpatients and inpatients with symptoms of Acute Respiratory Infections (ARI) in Brazil. Nasopharyngeal aspirates (NPAs) from 1015 patients with respiratory symptoms were tested for HBoV DNA by PCR. All samples positive for HBoV were tested by PCR for all other respiratory viruses, had HBoV viral loads determined by quantitative real time PCR and, when possible, were tested by RT-PCR for HBoV VP1 mRNA, as evidence of active viral replication. HBoV was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. The range of respiratory symptoms was similar between HBoV-positive and HBoV-negative ARI patients. However, a higher rate of diarrhea was observed in HBoV-positive patients. High HBoV viral loads (>10⁸ copies/mL) and diarrhea were significantly more frequent in patients with exclusive infection by HBoV and in patients with detection of HBoV VP1 mRNA than in patients with viral co-infection, detected in 72.9% of patients with HBoV. In summary, our data demonstrated that active HBoV replication was detected in a small percentage of patients with ARI and was correlated with concurrent diarrhea and lack of other viral co-infections.     

Hand foot and mouth disease due to enterovirus 71 in Malaysia

Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth.The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents.Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae.EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD),aseptic meningitis,encephalitis and poliomyelitis-like acute flaccid paralysis.A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997,and caused 41deaths amongst young children.In late 2000,a recurrence of an outbreak of HFMD occurred in Malaysia with S fatalities in peninsular Malaysia.Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities.A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006.The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010.As with other HFMD outbreaks in Malaysia,both EV71 and CA16 were the main aetiological viruses isolated.In similarity with the HFMD outbreak in 2005,the isolation of CA16 preceded the appearance of EV71.Based on the VP 1 gene nucleotide sequences,4 sub-genogroups of EV71 (C1,C2,B3 and B4) co-circulated and caused the outbreak of hand,foot and mouth disease in peninsular Malaysia in 1997.Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak.EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003.In the 2005 outbreak,besides the EV71 strains of sub-genogroup C1,EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003.The four EV71 strains isolated from clinical specimens of patients with hand,foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5.Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years.In each of the past outbreaks,more than one sub-genogroup of the virus co-circulate.     

中国浙江地区儿童下呼吸道感染KI多瘤病毒的鉴定

呼吸道病毒感染可以导致大面积人群致病[1],近些年,临床上由于不明原因导致的小儿下呼吸道感染病例屡见不鲜,对于该类患儿一般采取抗生素混合治疗的方法,但是治疗效果不佳.尽管过去几年开展的多项研究已经鉴定到了很多的病原体,如腺病毒,鼻病毒属病毒,冠状病毒属病毒,呼吸道合胞体病毒,流感病毒,副流感病毒等.     

Detection and genetic characterization of enteroviruses circulating among wild populations of chimpanzees in Cameroon: relationship with human and simian enteroviruses

Enteroviruses (EVs), members of the family Picornaviridae, are a genetically and antigenically diverse range of viruses causing acute infections in humans and several Old World monkey (OWM) species. Despite their known wide distribution in primates, nothing is currently known about the occurrence, frequency, and genetic diversity of enteroviruses infecting apes. To investigate this, 27 chimpanzee and 27 gorilla fecal samples collected from undisturbed jungle areas with minimal human contact in Cameroon were screened for EVs. Four chimpanzee samples were positive, but none of the gorilla samples were positive. Genetic characterization of the VP1, VP4, and partial VP2 genes, the 5' untranslated region, and partial 3Dpol sequences enabled chimpanzee-derived EVs to be identified as (i) the species A type, EV76, (ii) a new species D type assigned as EV111, along with a human isolate from the Democratic Republic of Congo previously described by the International Committee on the Taxonomy of Viruses, and (iii) a new species B type (assigned as EV110) most closely related to, although a distinct type from, the SA5 isolate recovered from a vervet monkey. The identification of EVs infecting chimpanzees related to those circulating in human and OWM populations provides evidence for cross-species transmission of EVs between primates. However, the direction of transfer and the existence of primate sources of zoonotic enterovirus infections in humans require further investigation of population exposure and more extensive characterization of EVs circulating in wild ape populations.     

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。     

GeXP多重基因表达遗传分析系统在手足口病病原分型检测中的应用

利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应(RT-PCR)方法,同时检测引起手足口病的9种常见的人肠道病毒—人肠道病毒71型(HEV71)、柯萨奇病毒A组(CVA)16、4、5、9、10型和柯萨奇病毒B组(CVB)1、3、5型。优化多重反应体系中针对5’UTR区的肠道病毒通用引物和11对针对9种血清型人肠道病毒VP1区的特异性引物的浓度比例,分别以病毒细胞培养物和阳性粪便标本来验证多重反应体系的特异性,以TCID50定量的细胞培养物和克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度。结果表明,优化后的多重检测体系,可扩增出人肠道病毒共有的保守片段的和型特异性片段,HEV71和CVA16细胞培养物的检测下限为100.5TCID50/μL,并可在103copies/μL水平同时、特异地检测出9种病毒RNA。该方法灵敏度高、特异性强,可快速对大量临床样本进行高通量检测,用于手足口病的分子流行病学调查。     

miR-142-3p is essential for hematopoiesis and affects cardiac cell fate in zebrafish

Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.     

2007年北京地区儿童手足口病病原的初步筛查

2007年4～6月儿童手足口病流行期间,对北京地区51例皮损症状典型、伴/不伴发热、无重症合并症的手足口病患儿采样,建立RT-PCR方法,以5'非编码区(5'UTR)肠道病毒通用引物、CA16和EV71 VP1区特异性引物直接对82份临床标本进行了初步筛查,肠道病毒阳性率达70.6%。检测病例中CA16阳性25例(25/51)、EV71阳性4例(4/51)、非CA16和EV71的肠道病毒阳性病例7例(7/51),三者比例约为6:1:2。2007年北京地区儿童轻症手足口病主要病原包括CA16和EV71,同时还存在一定比例其它肠道病毒。部分EV71毒株经测序验证及系统进化分析显示为C4基因亚型。     

Clinical characteristics and molecular epidemiology of human metapneumovirus in children with acute lower respiratory tract infections in China, 2017 to 2019: A multicentre prospective observational study

Human metapneumovirus (HMPV) infection is one of the leading causes of hospitalization in young children with acute respiratory illness. In this study, we prospectively collected respiratory tract samples from children who were hospitalized with acute lower respiratory tract infection in six hospitals in China from 2017 to 2019. HMPV was detected in 145 out of 2733 samples (5.3%) from the hospitalized children. The majority of HMPV-positive children were under the age of two (67.6%), with a median age of one year. HMPV can independently cause acute lower respiratory tract infection in young children, while all patients showed mild clinical symptoms. Of all the co-infected patients, HMPV was most commonly detected with enterovirus (EV) or rhinovirus (RhV) (38.0%, followed by respiratory syncytial virus (RSV) (32.0%). The highest detection rate occurred from March to May in both northern and southern China. Out of 145 HMPV positive samples, 48 were successfully typed, of which 36 strains were subgrouped into subtypes A2c (75%), eight strains were included in subtype B1 (16.7%), and four strains were included in subtype B2 (8.3%). Moreover, 16 A2c strains contained 111-nucleotide duplications in the G gene. Twenty-seven complete HMPV genomes were successfully obtained, and 25 (92.6%) strains belonged to subtype A2c, whereas one strain was included in subgroup B1 and another was included in subgroup B2. A total of 277 mutations were observed in the complete genomes of 25 A2c strains. All results presented here improve our understanding of clinical characteristics and molecular epidemiology of HMPV infection in children.     

儿童急性呼吸道博卡病毒感染

了解博卡病毒(Human Bocavirus,HBoV)在我国儿童急性呼吸道疾病中的感染情况。采用PCR扩增的方法对2005年10月~2006年1月收集的72例急性呼吸道感染的住院儿童鼻咽抽吸物(nasopharyngeal aspirates,NPA)进行了HBoV基因检测。将PCR阳性产物进行TA克隆,测序,并将所测序列与GenBank中HBoV序列进行比较分析。72份标本中共检测到6份HBoV阳性扩增产物,阳性率为8.3%(6/72),该6例HBoV阳性患儿临床均有肺炎或支气管肺炎症状。由此可以初步看出HBoV可能也是儿童急性呼吸道感染中较为重要的一个病原,且可能与儿童急性下呼吸道感染存在相关性。     

Complete Genome Sequence of an Enterovirus 80 Strain Isolated in China

Enterovirus 80 (EV80) is a newly identified serotype of the species Human enterovirus B. An EV80 strain designated HZ01/SD/CHN/2004 was isolated from an acute flaccid paralysis case in Shandong, China, in 2004. Complete genome comparison revealed 79.5% similarity with the prototype strain and an insertion of 36 nucleotides in the 3′ end of the VP1 coding region. Intertypic recombination with other serotypes was observed. This is the first report of the complete genome of EV80 in China.     

北京地区住院患儿WU多瘤病毒感染的流行病学研究

自2007年Gaynor A M等人通过高通量测序在肺炎患儿呼吸道样本中发现了WU多瘤病毒(Washington University polyomavirus,WUPyV)以来,WUPyV在世界各地被广泛检出。为了解北京地区急性呼吸道感染住院儿童中WUPyV感染情况及其临床特征,收集北京地区2017年4月至2018年3月共1 276份呼吸道感染住院患儿的鼻咽抽吸物样本,使用real time PCR方法对样本进行WUPyV检测,同时对WUPyV阳性样本进行17种常见呼吸道病毒筛查。共检出WUPyV阳性样本76份(5.96%,76/1 276),4岁以下儿童居多(92.11%,70/76);WUPyV感染全年可见,无显著季节性;多伴随其他呼吸道病毒混合感染(60.53%,46/76),最常见混合感染为鼻病毒和A型流感病毒;WUPyV单一感染者与混合感染者病毒载量无显著性差异,临床诊断和表现基本一致;WUPyV感染患儿的常见诊断为支气管炎(68.42%,52/76)和肺炎(30.26%,23/76);临床症状主要表现为高热、咳嗽、咳痰。研究结果提示WUPyV是北京地区急性呼吸道感染住院患儿呼吸道样本中常见病毒之一,多见于4岁以下儿童。     

肠道病毒71型北京分离株VP4基因序列及蛋白抗原性分析

为探讨肠道病毒71型(EV71)VP4基因序列与手足口病(HFMD)不同临床类型之间的关系,分析重组蛋白EV71 VP4的抗原性,并初步探讨其与柯萨奇病毒A16(CA16)重组蛋白VP4是否存在交叉反应性,对2007~2009年从北京患HFMD儿童标本分离到的10株EV71的VP4基因进行克隆测序,运用生物学软件对测序结果进行比较分析,并选择其中1株与1株同期分离的CA16的VP4分别进行原核表达;用表达产物对189份正常体检的成人及来首都儿科研究所就医的非HFMD患儿血清中的IgG进行Western Blot检测,并分析14份确诊为EV71感染和12份CA16感染患者急性期血清中的IgM抗体。分析表明这10株EV71 VP4基因核苷酸同源性为94.20%~100.00%,所推导的氨基酸序列则完全相同,从重症与轻症患儿分离的毒株之间VP4的核苷酸序列未见一致性的差异,基于EV71 VP4基因核苷酸序列的进化树分析表明2007~2009年北京地区所流行的毒株均属于C4亚型;本研究中EV71和CA16的VP4核苷酸序列的同源性为69.60%,所推导的氨基酸序列的同源性为78.60%,在运用Western Blot检测189份血清中的VP4特异性IgG时,EV71 VP4的血清阳性率为38.10%,说明其具有良好的抗原性,CA16 VP4的血清阳性率为58.20%,两者差别具有显著统计学意义(2χ=15.30,P<0.01),提示EV71 VP4与CA16 VP4没有交叉反应性;在用表达的VP4检测已确诊为相应病毒的特异性IgM时,两者皆为阴性,提示感染后机体对VP1和VP4产生不同的反应。     

Incidence rates of enterovirus 71 infections in young children during a nationwide epidemic in Taiwan, 2008-09

Objective

Enterovirus 71 (EV71) is causing life-threatening outbreaks in tropical Asia. In Taiwan and other tropical Asian countries, although nationwide EV71 epidemics occur cyclically, age-specific incidence rates of EV71 infections that are critical to estimate disease burden and design vaccine trials are not clear. A nationwide EV71 epidemic occurred in 2008–09 in Taiwan, which provided a unique opportunity to estimate age-specific incidence rates of EV71 infections.

Study Design

We prospectively recruited 749 healthy neonates and conducted follow-ups from June 2006 to December 2009. Sera were obtained from participants at 0, 6, 12, 24, and 36 months of age for measuring EV71 neutralizing antibody titers. If the participants developed suspected enterovirus illnesses, throat swabs were collected for virus isolation.

Results

We detected 28 EV71 infections including 20 symptomatic and 8 asymptomatic infections. Age-specific incidence rates of EV71 infection increased from 1.71 per 100 person-years at 0–6 months of age to 4.09, 5.74, and 4.97 per 100 person-years at 7–12, 13–24, and 25–36 months of age, respectively. Cumulative incidence rate was 15.15 per 100 persons by 36 months of age, respectively.

Conclusions

Risk of EV71 infections in Taiwan increased after 6 months of age during EV71 epidemics. The cumulative incidence rate was 15% by 36 months of age, and 29% of EV71 infections were asymptomatic in young children.