BACKGROUND:Chikungunya virus (CHIKV) reemerged and caused millions of human infections since 2004. The disease could be established, when the virus has been introduced to areas where the appropriate vectors are endemic. The differential diagnosis of CHIKV infection varies based on place of residence, travel history, and exposures. Serological tests are commonly used to diagnose CHIKV infection, but their availability and assessments of the performance of the diagnostics have been limited.OBJECTIVES:To develop and evaluate antibodies detection methods for chikungunya diagnosis and serological investigation.METHODS:Recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (Mac-ELISA) and double antigen sandwich ELISA (Das-ELISA) for detection of antibodies to Chikungunya virus were developed and evaluated. The repeatability was evaluated by testing of three reference sera at single dilutions in triplicated for 5 times. The sensitivity, specificity, accuracy, and agreement of the MAC-ELISA and Das-ELISA were obtained by comparing the detection results of 225 serum samples (45 positive; 180 negative) with a real-time RT-PCR assay and an IFA commercial tests manufactured by Euroimmun.RESULTS:The established ELISA assays were standardized by determining the optimal concentrations of the key reagents. The coefficient values of repeat testing were within 10% and 20% for intraassay and interassay precision, respectively. A sensitivity of 60.0% and 52.5%, a specificity of 96.2% and 96.8%, and an accuracy of 89.8% and 88.9% were obtained for the Mac-ELISA and Das-ELISA, respectively, when compared to a CHIKV qRT-PCR method. And a sensitivity of 100%, a specificity of 97.5% and 99.5%, and an accuracy of 97.8% and 99.6% were yielded respectively when using the IIFT as a reference method, which showed a highly consistence to the commercial IIFT assay with a Kappa value greater than 0.90.CONCLUSIONS:The Mac-ELISA and Das-ELISA based on recombinant E2 protein of CHIKV were developed and standardized, which could detect IgM or total antibodies against CHIKV in 2-3 hours with acceptable sensitivities and specificities. These assays can be used for laboratory diagnosis and serological investigation of CHIKV infections to evaluate the risk of CHIKV transmission.

建立可检测琉球病毒(Ryukyu virus,RYKV),索尔韦齐病毒(Solwezi virus,SOLV)以及苏里斯病毒(Souris virus,SOUV)等三种沙粒病毒的实时荧光定量RT-PCR检测方法.分析三种病毒流行病学分布特征,从国际公共数据库搜索下载基因组序列,进行比对分析,确定检测靶标,借助primer premier 6.0生物信息学软件,设计引物和探针,建立实时荧光定量RT-PCR检测方法,利用化学合成和体外转录方法制备模拟样本,比较评价三种方法的检测限、特异性、重复性特征.所建实时荧光定量RT-PCR检测方法均可有效扩增检测病毒RNA靶标,检测限分别为40拷贝/μL、7拷贝/μL和15拷贝/μL,检测汉城病毒、汉滩病毒、登革病毒Ⅰ～Ⅳ型分离株、发热伴血小板减少综合病毒及30份健康人血清样本无非特异性扩增,三种病毒相互间以及其他8种出血热相关沙粒病毒RNA间无交叉反应,重复性比较分析显示变异系数均在2％以内.本研究建立的检测RYKV、SOLV和SOUV三种沙粒病毒的实时荧光RT-PCR方法具备用于相关疑似感染者临床样本、宿主动物标本以及进出口物品的筛查检测的潜力,但因未经基于实际病毒感染样本的比较评价,检测结果的解释仍具有一定的局限性.

Flexal病毒（Flexal virus,FLEV）和Whitewater Arroyo病毒（Whitewater Arroyo virus,WWAV）属沙粒病毒,经鼠传播,可引起人类严重传染病,主要分布于美洲,有因野外工程而感染或国际物流和人员往来而输入的风险。本研究通过检索、分析FLEV和WWAV基因组序列,参照同科属病毒遗传进化特征,确定病毒基因组保守区,筛选分子检测靶基因,设计PCR扩增引物和检测探针,建立两种沙粒病毒的实时荧光RT-PCR检测方法,并进行灵敏性、特异性和重复性初步评价。结果表明,所建立的FLEV和WWAV检测方法与其他病毒无交叉反应,扩增效率大于95%,检出限分别为100copies/μL和20copies/μL,变异系数在2%以内。上述结果显示,本研究所建立的FLEV和WWAV检测方法的特异性强,灵敏性与重复性均处于常规病毒性传染病分子诊断可接受范围,可应用于疑似病例的快速检测和相关病原的筛查和流行病学调查。

Objective:To establish real-time fluorescent RT-PCR methods for detection of the three viruses in nairovirus genus, including dugbe virus (DUGV), qalyub virus (QALV) and thiafro virus (TFAV).Methods:The S-segment sequences of the three virus genomes published in the international public database were collected, collated, compared and analyzed to define the detection targets, and the viral specific primers and probes were designed accordingly using bioinformatic software. Real-time fluorescent quantitative RT-PCR detection methods were established with protocol optimization. The simulated samples prepared by in vitro transcription technique, specimens of infections with other viruses, virus strains and blood samples from healthy human were used to evaluate the detection limit, specificity and reproducibility of the methods. Results:The established real-time fluorescent quantitative RT-PCR detection methods could effectively amplify and detect the viral target RNA, with detection limits of 160 copies/μL, 20 copies /μL and 10 copies/μL, respectively. No nonspecific amplification in the samples of Kyasanur Forest disease virus, influenza BV and BY viruses and influenza A(H3N2) virus, yellow fever virus, encephalitis B virus, Crimean-Congo hemorrhagic fever virus, Tahyna virus. No cross reaction was detected among the three nairoviruses. The coefficient of variation was within 2% in the reproducibility analysis.Conclusions:The real-time RT-PCR methods for the detection of DUGV, QALV and TFAV established in this study could be used for screening and detection of related clinical samples, vectors, host animal samples and import and export articles.

Flexal病毒(Flexal virus,FLEV)和Whitewater Arroyo病毒(Whitewater Arroyo virus,WWAV)属沙粒病毒,经鼠传播,可引起人类严重传染病,主要分布于美洲,有因野外工程而感染或国际物流和人员往来而输入的风险.本研究通过检索、分析FLEV和WWAV基因组序列,参照同科属病毒遗传进化特征,确定病毒基因组保守区,筛选分子检测靶基因,设计PCR扩增引物和检测探针,建立两种沙粒病毒的实时荧光RT-PCR检测方法,并进行灵敏性、特异性和重复性初步评价.结果 表明,所建立的FLEV和WWAV检测方法与其他病毒无交叉反应,扩增效率大于95％,检出限分别为100copies/μL和20copies/μL,变异系数在2％以内.上述结果显示,本研究所建立的FLEV和WWAV检测方法的特异性强,灵敏性与重复性均处于常规病毒性传染病分子诊断可接受范围,可应用于疑似病例的快速检测和相关病原的筛查和流行病学调查.

拉沙热主要流行于西非,经鼠传播,人群普遍易感,病死率高,暴发疫情频发,跨境传播时有发生.拉沙病毒易于传播,病毒分离、培养需在生物安全四级实验室(BSL-4).2018年,世界卫生组织将其列为年度重点关注传染病,需加快研制防治关键技术手段.随着中非关系的日益紧密,贸易往来频繁,中国面临拉沙热的威胁显著增加.本文检索20世纪50年代首例拉沙热病例报道以来公开发表的主要文献,归纳拉沙热临床表现、病原学特征、流行病学特征、实验室检测以及跨境传播风险等,并对GenBank发布的全部287条拉沙病毒S基因编码区全长序列进行复核分析,以加强人们对拉沙热的了解,提高防控意识.

目的 分析裂谷热病毒自发现以来,疫情发生、疫源地分布以及病毒遗传进化和流行特征,为科学防控提供依据.方法 应用描述性流行病学方法收集、分析裂谷热疫情的流行情况,筛选全球有明确分离时间和地区的病毒的全基因组序列,利用BEAST 2等软件进行系统进化分析.结果 截止2018年,全球共22个国家报告了裂谷热本地疫情,人间报告病例39 404例,死亡病例2 323例.共筛选出183条S片段、138条M片段和135条L片段基因序列.系统进化树显示,裂谷热病毒的S、M和L片段可分为7个基因亚型(A～G),A亚型和B亚型是引起3次大流行的主要亚型.2016年中国发现首例输入性病例,国际跨境传播趋势有所增强.结论 裂谷热病毒在非洲分布范围广,实际流行形势被明显低估,应加强对该病毒的监测,注重信息收集与发布,提高有关部门和群众的防控意识,保护公民健康.

Objective To establish a method for the simultaneous identification of Zika, Chikungunya and Mayaro viruses. Methods The complete genome sequences of Zika, Chikungunya and Mayaro virus were retrieved from Global Shared Database for comparative analysis, estimate its conservative region and determine the target gene location, specific primers and probes were designed, then a triplex real-time RT-PCR assay was developed. The specificity, sensitivity and repeatability of the assay were assessed by viral nucleic acid of Zika virus, Chikungunya virus a, in vitro transcriptional RNA of Mayaro virus, normal human serum and related virus simulation sample. Results The result showed that the established method could detect Zika virus, Chikungunya virus, as well as simulated Mayaro virus samples, the limit of detection (LOD) of Zika and Chikungunya virus was 16.22 Copy/PCR and 12.02 Copy/PCR, respectively, the LOD for simulated Mayaro virus RNA was 2.82 Copy/PCR, no significant difference was detected between the triplex and monoplex assays. No cross reaction was found in the detection of dengue virus, Hantavirus, severe fever with thrombocytopenia syndrome (SFTS) virus, yellow fever virus and influenza virus, and 100 healthy adults blood samples, the specificity of the method was 100%. The repeatability result showed that the standard deviation of all three detections were blow 0.5 and the coefficient of variation was less than 2% by selecting viral nucleic acids or transcribed RNA with high, medium and low concentration gradients. Conclusions A triplex real-time RT-PCR assay for detection of Zika, Chikungunya and Mayaro virus has been established with an acceptable specificity, sensitivity and repeatability. Key words: Zika virus; Chikungunya virus; Mayaro virus; Triplex Real-time RT-PCR