实时荧光RT-PCR及RT-PCR快速检测肠道病毒71型和柯萨奇A16病毒

肠道病毒71型(Enterovirus 71,简称EV71)和柯萨奇病毒A组16型(Coxsackievirus A16,简称CA16)是引起手足口病的两种主要的病原体,常相伴造成手足口病的暴发流行,每次流行中两种病毒中所占的比例有所不同[1].EV71感染引起HFMD常有严重的神经系统并发症发生,CA16感染引起自限性的手足口病,极少发生严重并发症和死亡[2～6].2008年安徽、广东(包括深圳市)、浙江等28个省市出现手足口病暴发流行,因此,亟须一种特异性强,灵敏度高的方法对这两种病毒进行快速区分检测.     

肠道病毒71型和柯萨奇病毒A组16型实验室检测研究进展

肠道病毒71型（Enterovirus,EV71）和柯萨奇病毒A组16型（Coxsackievirus,CoxA 16）是引起手足口病的主要病原体,因此,快速准确地检测这2种病原体对于手足口病治疗和控制有着至关重要的作用。该文介绍了这2种病原体实验室检测技术的研究进展,主要包括病毒分离培养、血清学检测、分子生物学方法包括RT—PCR法、实时荧光RT—PCR法等。     

北京市儿童手足口病与肠道病毒71型和柯萨奇病毒A组16型感染有关

目的了解2007年5-6月北京市儿童中流行的手足口病的病原。方法于2007年5、6月间采集来首都儿科研究所附属儿童医院门诊就诊的手足口病患儿咽拭子标本6份和1份病情危重的手足口病并发神经系统症状而急诊收住院患儿（编号F4243，年龄9岁8个月，女性）的经气管插管呼吸道抽吸液、脑脊液及血清标本。将咽拭子和气管插管抽吸液标本接种Hep-2、MDCK和Vero细胞进行病毒分离。同时设计位于肠道病毒5^＋非编码区（UTR）的通用引物2对（巢式PCR）、肠道病毒71型（EV71）VPl基因不同位置的特异性引物2对半（PCR和半巢式PCR）和柯萨奇病毒A组16型（CAl6）的特异性引物1对检测标本中的病毒核酸。首先用随机引物将标本中的RNA逆转录成cDNA，然后分别用上述引物进行PCR扩增肠道病毒5^＋UTR和EV71／CAl6的VPl基因片段；对EV71阳性的标本还扩增了VPl全基因，扩增产物直接进行序列测定和分析。结果6例门诊就诊的普通手足口病患儿和F4243的呼吸道标本中均扩增到肠道病毒5^＋UTR基因片段，有2例门诊患儿和F4243的标本中还扩增到EV71的VPl基因片段，提示该组手足口病患儿均为肠道病毒感染，其中3例为EV71感染。序列测定和分析表明有1例普通手足口病患儿（F4211）和F4243标本确定为EV71。所有标本接种Hep-2和MDCK细胞均未出现病变，说明常见的呼吸道病毒为阴性。除1份咽拭子标本（F4283）外，其余6份接种Vero细胞的标本均出现细胞病变。用肠道病毒通用引物和EV71特异性引物对分离到的病毒株核酸的扩增结果显示这6株病毒均为肠道病毒，其中F4211和F4243为EV71，与直接从呼吸道标本中扩增的结果相符；其余4株为CAl6。结论北京市儿童中流行的手足口病与肠道病毒EV71和CAl6感染有关；EV71可以在5岁以上儿童中引起严重的神经系统并发症，应引起注意。     

实时荧光定量PCR检测手足口病肠道病毒71型与柯萨奇病毒16型临床分析

目的 探讨肠道病毒71型(EV71)与柯萨奇病毒A 16型(Cox A16)实时荧光定量PCR检测在早期诊断手足口病(HFMD)中的意义,为临床治疗提供参考依据。方法 对2012年3月-2013年6月157例疑似HFMD患儿的粪便、疱疹液、鼻咽拭子样本进行EV71、Cox A16、肠道病毒(EV)的实时荧光定量PCR检测,根据试剂说明书的标准判定;采用SPSS16.0进行数据分析。结果 粪便中EV、EV71、Cox A16的检出率分别为94.09%、42.04%、38.85%,高于鼻咽拭子样本,差异有统计学意义(P<0.05);而粪便与疱疹液中EV、EV71、Cox A16的阳性率相比,差异无统计学意义。结论 对HFMD患儿早期诊断、早期治疗,应该首选粪便标本采用实时荧光定量PCR法检测EV、EV71、Cox A16。     

肠道病毒71型和柯萨奇病毒A16型细胞受体的研究进展

近几年手足口病在亚太地区的流行大幅上升并伴随严重的中枢神经系统并发症,而针对其主要致病原肠道病毒71型和柯萨奇病毒A16型现在还缺乏有效的预防疫苗和抗病毒制剂.确定它们的特异性细胞受体对于进一步阐明病毒与宿主细胞早期互相作用的分子机制及其致病机制至关重要.过去一年中,P-选择素糖蛋白配体-1、B类清道夫受体Ⅱ和唾液酸化...     

肠道病毒71型和柯萨奇病毒A16型细胞受体的研究进展

近几年手足口病在亚太地区的流行大幅上升并伴随严重的中枢神经系统并发症,而针对其主要致病原肠道病毒71型和柯萨奇病毒A16型现在还缺乏有效的预防疫苗和抗病毒制剂.确定它们的特异性细胞受体对于进一步阐明病毒与宿主细胞早期互相作用的分子机制及其致病机制至关重要.过去一年中,P-选择素糖蛋白配体-1、B类清道夫受体Ⅱ和唾液酸化多聚糖类被分别发现和确定为肠道病毒71型和柯萨奇病毒A16型的功能性细胞受体.此文讨论了相关的实验结果并展望了未来值得探索和应用的方向.     

肠道病毒71型与柯萨奇病毒16型病原体检测在手足口病流行中的意义

目的探究肠道病毒71型(EV71)和柯萨奇病毒16型(Cox A16)病原体RT-PCR检测在预防手足口病传播中的意义。方法对70例疑似手足口病患儿和30例健康儿童的粪便进行EV71和Cox A16及肠道病毒(EV)RT-PCR检测。结果 70例有发热、口腔黏膜疹、手足皮疹的患儿粪便中EV病毒全阳性,EV71病毒阳性25例占35.71%,Cox A16病毒阳性22例占31.43%,二者均阳性的10例占14.29%,30例体检儿童粪便中EV病毒阳性5例,EV71和Cox A16全阴性。结论手足口病以EV71和Cox A16病原体感染为主,检测EV71和Cox A16病原体可以早期诊断手足口病,对预防和治疗手足口病具有重要临床意义。     

北京市入托体检健康儿童肠道病毒71型和柯萨奇病毒A组16型感染状况及就诊行为调查

目的 了解北京市入托儿童肠道病毒71型(EV71)和柯萨奇病毒A组16型(Cox A16)感染状况及其相关的就诊率,为估算手足口病疾病负担参考。方法 2013年8月20－31日对北京市东城区、朝阳区和怀柔区进行入托体检的健康儿童开展血清学调查,采用ELISA方法检测血清中EV71和Cox A16的IgG和IgM抗体。结果 共调查813名儿童,平均年龄为(3.5±1.0)岁。Cox A16 IgG阳性率为61.9%,IgM阳性率为4.4%;EV71 IgG阳性率为9.3%,IgM阳性率为1.1%;各种抗体阳性率在不同性别中的分布差异无统计学意义(P>0.05),在不同年龄组中的分布差异有统计学意义(P<0.05)。Cox A16抗体阳性者中7.8%有皮疹,EV71抗体阳性者中10.7%有皮疹。Cox A16或EV71抗体阳性者中,仅7.1%在父母陪伴下到医院就诊,而有皮疹的抗体阳性病例中,80.5%去医院就诊。结论 北京市健康入托儿童中,既往感染Cox A16的比例较大,EV71抗体阳性率明显低于Cox A16,提示托幼儿童对EV71普遍易感,应是手足口病防控重点人群。     

肠道病毒71型和柯萨奇病毒A16型抗体筛查在手足口病中的应用及相关性

目的 探讨肠道病毒71型(EV71)和柯萨奇病毒A16型(CA16)抗体筛查在手足口病中的应用及相关性.方法 采用酶联免疫吸附法对确诊的281例手足口病患儿进行血清EV71-IgM抗体和CA16-IgM抗体检测.结果 EV71-IgM抗体阳性144例(51.2％,144/281),CA16-IgM抗体阳性90例(32.0％,90/281),EV71-IgM和CA16-IgM双阳性17例(6.0％,17/281),即抗体阳性确切例数为217例,抗体阳性率为77.2％(217/281).EV71-IgM抗体阳性率与CA16-IgM抗体阳性率比较差异有统计学意义(x2=21.35,P＜0.01),总体男性与女性抗体阳性率[95.1％(135/142)比71.2％(99/139)]比较差异有统计学意义(x2=8.22,P＜0.01).结论 早期进行EV71-IgM和CA16-IgM抗体联合检测,对临床诊断手足口病及监控治疗具有重要意义.     

六个县母婴肠道病毒71型和柯萨奇病毒A组16型中和抗体水平调查

目的了解新生儿和新生儿母亲的肠道病毒71型(Enterovirus Type71,EV71)和柯萨奇病毒A组16型(Coxsackievirus Group AType16,CVAl6)中和抗体(Neutralizing Antibody,NA)水平。方法选取广西壮族自治区和江苏省的3个手足口病(Hand,Footand Mouth Disease;HFMD)高发县和3个低发县作为研究现场,在每个县选取一个HFMD高发乡和一个低发乡,每个乡选择10对新生儿及新生儿母亲,对所有调查对象采集静脉血,并对新生儿母亲进行问卷调查。结果母亲EV71和CVA16的NA阳性率和几何平均滴度(Geometric Mean Titer,GMT)分别为83.5%、33.1%和1∶26.61、1∶6.1l,新生儿EV7l和CVA16的NA阳性率和GMT分别为75.2%、35.5%和1∶22.05、1∶6.97,母婴EV71、CVA16的NA阳性率及GMT差异均无统计学意义(2EV71=2.52,P=0.1124;2CVA16=0.1650,P=0.6846;tEV7l=1.05,P=0.2953;tCVA16=1.30,P=0.1946)。EV71、CVA16的NA阳性率和GMT在HFMD高、低发县差异亦无统计学意义(2EV71=1.45,P=0.2288;2CVA16=1.28,P=0.2583;tEV7l=1.86,P=0.0643;tCVA16=0.2,P=0.8399)。母婴EV71和CVA16的NAGMT均存在相关性(rEV71=0.69,P<0.0001;rCVA16=0.4769,P<0.0001)。结论母亲EV71和CVA16的NA阳性率和GMT高,新生儿EV71和CVA16的NA相应也高,可为新生儿提供有效保护。     

Efficacy of an inactivated bivalent vaccine for enterovirus 71 and coxsackievirus A16 in mice immunized intradermally

Human hand, foot, and mouth disease (HFMD), an important infectious disease in children, is caused mainly by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, a bivalent inactivated EV71/CA16 vaccine is developed and evaluated in immunized BALB/c mice injected through the intradermal route. Q-RT-PCR detection of the mRNA of immune signal molecules in local epithelial tissues inoculated with the vaccine indicates activation of innate immunity, which includes upregulation of immune-related chemokines, interferons and CD molecules. Further, the finding that neutralizing antibodies and specific T cellular responses were elicited in adult mice after two immunizations with the vaccine at a 28-day interval, which endowed offspring mice to defend a viral challenge, suggests the successful induction of specific protective antiviral immunity. All these data suggest that immunization with this bivalent EV71/CA16 vaccine via the intradermal route elicits effective immunity against EV71 and CA16 infection.     

双重荧光定量RT-PCR快速检测A、B型流感病毒的研究

目的：建立双重荧光定量RT—PCR技术同时快速检测A、B型流感病毒，并应用于临床样本的检测。方法：在A型流感病毒M基因和B型流感病毒NP基因的保守区序列分别设计特异性引物和Taqman探针，建立优化单一和双重荧光RT—PCR反应体系，评价所建双重RT—PCR反应体系的特异性、敏感性和稳定性，并应用于疑似流感含漱液标本检测。结果：该方法对A、B型流感病毒检测具有高度特异性，检出限分别为0．1TCID50和0．01TCID50，可从疑似流感患者含漱液中直接检测到流感病毒核酸。构建的体系定量标准曲线相关系数分别为0．998和0．999，具有较好的稳定性。结论：本研究建立的双重荧光定量RT—PCR可以同时准确分型A、B型流感病毒，灵敏度高，稳定性好，是一种快速检测流感病毒的新方法。     

Chimeric enterovirus 71 virus-like particle displaying conserved coxsackievirus A16 epitopes elicits potent immune responses and protects mice against lethal EV71 and CA16 infection

Hand-foot-and-mouth disease (HFMD) is an infectious disease of infants and young children frequently caused by the enterovirus A species, mainly enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, we prepared the EV71 virus-like particle (EV71-VLP) and its chimeras using recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under CMV-IE promoter) proteins in Sf9 cells. EV71-VLP chimera ChiEV71(1E)-VLP or ChiEV71(4E)-VLP displayed single CA16 PEP71 epitope in VP1 or four conserved CA16 neutralizing epitopes (PEP71 in VP1, aa136-150 in VP2, aa176-190 in VP3 and aa48-62 in VP4) by substitution of the corresponding regions of EV71 structure proteins, respectively. In mice, EV71-VLP and its chimeras elicited similar EV71-specific IgG and neutralizing antibody (NAb) titers compared to inactivated EV71. Expectedly, vaccination of ChiEV71(1E)-VLP or ChiEV71(4E)-VLP resulted in significantly increased CA16-specific IgG and NAb production and improved cross-protection against CA16 infection compared to EV71-VLP. Interestingly, the VLPs induced potent cellular immune responses and significantly decreased Th2 type (IL-4 and IL-10) cytokines secretion in the splenocytes of immunized mice compared to inactivated EV71 or inactivated CA16. Neonatal mice born to dams immunized with the chimeric VLPs or neonatal mice passively transferred with sera of immunized mice were completely protected from lethal EV71 challenge and partially protected from lethal CA16 infection. Our study provides a novel bivalent or multivalent vaccine strategy to prevent EV71 and related-enterovirus infections.     

A novel inactivated enterovirus 71 vaccine can elicit cross-protective immunity against coxsackievirus A16 in mice

Hand, foot, and mouth disease (HFMD) is a highly contagious disease that mainly affects infants and children. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major pathogens of HFMD. Two EV71 vaccines were recently licensed in China and the administration of the EV71 vaccines is believed to significantly reduce the number of HFMD-related severe or fatal cases. However, a monovalent EV71 vaccine cannot cross-protect against CA16 infection, this may result in that it cannot effectively control the overall HFMD epidemic. In this study, a chimeric EV71, whose VP1/210–225 epitope was replaced by that of CA16, was constructed using a reverse genetics technique to produce a candidate EV71/CA16 bivalent vaccine strain. The chimeric EV71 was infectious and showed similar growth characteristics as its parental strain. The replacement of the VP1/210–225 epitope did not significantly affect the antigenicity and immunogenicity of EV71. More importantly, the chimeric EV71 could induce protective immunity against both EV71 and CA16, and protect neonatal mice against either EV71 or CA16 lethal infections, the chimeric EV71 constructed in this study was shown to be a feasible and promising candidate bivalent vaccine against both EV71 and CA16. The construction of a chimeric enterovirus also provides an alternative platform for broad-spectrum HFMD vaccines development.     

A virus-like particle based bivalent vaccine confers dual protection against enterovirus 71 and coxsackievirus A16 infections in mice

Enterovirus 71(EV71) and coxsackievirus A16 (CA16) are responsible for hand, foot and mouth disease which has been prevalent in Asia-Pacific regions, causing significant morbidity and mortality in young children. Co-circulation of and co-infection by both viruses underscores the importance and urgency of developing vaccines against both viruses simultaneously. Here we report the immunogenicity and protective efficacy of a bivalent combination vaccine comprised of EV71 and CA16 virus-like particles (VLPs). We show that monovalent EV71- or CA16-VLPs-elicited serum antibodies exhibited potent neutralization effect on the homotypic virus but little or no effect on the heterotypic one, whereas the antisera against the bivalent vaccine formulation were able to efficiently neutralize both EV71 and CA16, indicating there is no immunological interference between the two antigens with respect to their ability to induce virus-specific neutralizing antibodies. Passive immunization with monovalent VLP vaccines protected mice against a homotypic virus challenge but not heterotypic infection. Surprisingly, antibody-dependent enhancement (ADE) of disease was observed in mice passively transferred with mono-specific anti-CA16 VLP sera and subsequently challenged with EV71. In contrast, the bivalent VLP vaccine conferred full protection against lethal challenge by either EV71 or CA16, thus eliminating the potential of ADE. Taken together, our results demonstrate for the first time that the bivalent VLP approach represents a safe and efficacious vaccine strategy for both EV71 and CA16.     

Hepatitis B virus core particles containing multiple epitopes confer protection against enterovirus 71 and coxsackievirus A16 infection in mice

Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand-foot-and-mouth disease (HFMD). To investigate novel combined vaccines to prevent EV71 and CA16 infection, we constructed chimeric virus-like particles (tHBc/SPA or tHBc/SP VLPs) displaying conserved epitopes of EV71 (aa 208–222 of VP1 and aa 248–263 of VP2) and CA16 (aa271-285 of VP1) using a truncated hepatitis B virus core carrier (tHBc). Immunization with the chimeric VLPs induced epitope- or virus-specific IgG and neutralization antibodies against EV71 and CA16 in the mice. Compared with inactivated EV71, the chimeric VLPs induced significantly increased Th1 cytokine (IFN-γ, IL-2) production and decreased Th2 cytokine (IL-4, IL-10) responses. Neonatal mice born to dams immunized with the recombinant particles were completely protected from lethal EV71 and partially protected from CA16 infection. Co-expression of the conserved human MHC class I CD4+ T cell epitope (aa248-263 of VP2) did not improve the antiviral immunity of the chimeric VLP vaccine in mice. Our results demonstrate that experimental combination vaccines comprised of EV71 and CA16 epitopes induce both humoral and cellular immune responses and therefore support further preclinical and clinical development of a bivalent VLP vaccine targeting both CA16 and EV71.     

A combination vaccine comprising of inactivated enterovirus 71 and coxsackievirus A16 elicits balanced protective immunity against both viruses

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand, foot and mouth disease (HFMD), which is an infectious disease frequently occurring in children. A bivalent vaccine against both EV71 and CA16 is highly desirable. In the present study, we compare monovalent inactivated EV71, monovalent inactivated CA16, and a combination vaccine candidate comprising of both inactivated EV71 and CA16, for their immunogenicity and in vivo protective efficacy. The two monovalent vaccines were found to elicit serum antibodies that potently neutralized the homologous virus but had no or weak neutralization activity against the heterologous one; in contrast, the bivalent vaccine immunized sera efficiently neutralized both EV71 and CA16. More importantly, passive immunization with the bivalent vaccine protected mice against either EV71 or CA16 lethal infections, whereas the monovalent vaccines only prevented the homologous but not the heterologous challenges. Together, our results demonstrate that the experimental bivalent vaccine comprising of inactivated EV71 and CA16 induces a balanced protective immunity against both EV71 and CA16, and thus provide proof-of-concept for further development of multivalent vaccines for broad protection against HFMD.     

基于AllGlo探针的双重实时荧光定量PCR法检测高危型人乳头瘤病毒HPV16/18

目的建立基于AllGlo探针的双重实时荧光定量PCR检测高危型人乳头瘤病毒HPV16/18的方法。方法针对HPV16/18病毒基因组保守序列,设计引物和AllGlo探针,建立双重荧光定量PCR检测方法,评价其敏感性、特异性和稳定性,并与传统HC2方法比较。结果成功建立了双重实时荧光定量PCR检测HPV16/18的方法,检测限均为10copies/μL,敏感性和特异性均为100%。应用该方法检测160份宫颈肿瘤患者样本,与传统HC2法结果一致性为100%。结论该研究建立的快速、准确、可同时检测HPV16/18的双重实时荧光定量PCR法,具有较高的敏感性、特异性和稳定性,操作简单,可用于HPV16/18的临床筛查和诊断。     

2008年湖州市肠道病毒71型和COXA16型的监测分析

目的:了解湖州市肠道病毒的感染情况,为制定预防和控制肠道病毒感染提供依据。方法:采用荧光定量RT-PCR和RT-PCR方法,对湖州市101例疑似肠道病毒感染者的疱疹液、咽拭子及粪便标本进行肠道病毒通用核酸、肠道病毒71型和柯萨奇病毒A16的核酸检测。结果:肠道病毒通用核酸阳性87例,阳性率为86.14%(87/101)。其中EV71阳性20例,阳性率为19.80%(20/101);Cox A16阳性1例,阳性率为0.99%(1/101),二者均阴性14例。男女发病率分别为18.46%(12/65)和25.0%(9/36)。疱疹液中EV71检出率24.32%(9/37),Cox A l6检出率2.70%(1/37);咽拭子中EV71检出率18.96%(11/58),Cox A l6未检出;粪便标本中EV71和Cox A l6均未检出。结论:EV71是引起湖州市儿童手足口病的主要病原体,Ev71和Cox A16的发病率无性别差异,疱疹液、咽拭子标本中EV71及Cox A16的检出率明显高于粪便标本,要加强对Ev71和Cox A16的鉴别诊断,有利于提出更好的预防和控制措施。