基孔肯雅病毒检测方法及进展

基孔肯雅病毒是引起基孔肯雅热的病原体,主要经伊蚊传播,感染者的症状主要以发热、皮疹和关节疼痛为主。该病毒主要分布在非洲、东南亚等地区,近年来在印度洋地区造成大规模流行。我国主要以输入性病例为主,未发生大规模流行。对基孔肯雅病毒的实验室检测方法及最新研究进展进行了综述。     

白纹伊蚊和埃及伊蚊对基孔肯雅病毒的易感性和传播性的研究

用４株基孔肯雅病毒经口感染白纹伊蚊和埃及伊蚊,进行了易感性和传播性的研究。结果表明,这两种蚊虫对基孔肯雅病毒易感。无论白纹伊蚊或埃及伊蚊,感染后第５－６天即可通过吸血将病毒传播给乳鼠,至第８－１３天,传播率可高达５５．５５％－１００％。感染蚊亦可经叮咬将病毒传播给小鸡。埃及伊蚊的易感性和传播率高于白纹伊蚊。实验还发现,不同来源毒株之间存在一定差异,如分离自云南白纹伊蚊的Ｍ８１株的感染率和传播率均高于其它毒株。这些结果表明,白纹伊蚊和埃及伊蚊在基孔肯雅病毒的保存和传播中起重要作用。     

基孔肯雅热

基孔肯雅热(Chikungunya fever,CHIK)是由基孔肯雅病毒(Chikungunya virus,CHIKV)病毒引起的,伊蚊叮咬传播的,以发热、关节疼痛等为主要特征的自限性传染性疾病。1952年在坦桑尼亚发生的暴发流行中首次分离CHIKV[1-2],亚洲地区于     

基孔肯雅病毒疫苗的研究进展

基孔肯雅热是由基孔肯雅病毒(Chikungunya virus,CHIKV)引起的一种急性传染病。基孔肯雅病毒属披膜病毒科甲病毒属。近年来,该病毒死灰复燃,在全球多处引发大规模疫情暴发,是重要的全球性公共卫生问题之一。目前尚无针对CHIKV的疫苗和有效抗病毒药物。疫苗一直是CHIKV研究的热点领域,目前已经取得了很大的进展,就基孔肯雅病毒疫苗的研究进展进行了综述。     

广州首起输入性基孔肯雅热的调查分析

广州网络报告的疑似"登革热"患者经广州市疾病预防控制中心流行病学调查、血清学检测、病原学分离培养等做出的诊断为首例输入性基孔肯雅热。又经中国疾病预防控制中心和广东省疾病预防控制中心对疑似基孔肯雅热患者作进一步基孔肯雅病毒抗体和核酸等检测,并对扩增出的基孔肯雅病毒特异性片段进行了序列测定和分析。核苷酸同源性比较显示,E2区(336nt)和NS1区(314nt)的核苷酸序列与意大利基孔肯雅病毒分离株ITA07-RA1及多株印度的基孔肯雅病毒分离株的序列高度同源(98%以上)。根据现场流行病学和实验流行病学的调查结果,可以确认这例疑似境外感染的输入性登革热实为基孔肯雅热病例,也是中国首例输入性基孔肯雅热病例。     

基孔肯雅病毒浓缩提纯及多肽抗原研究

采用聚乙二醇(PEG 6000)沉淀法(粗提)和蔗糖密度梯度速率区带离心(精提)浓缩纯化基孔肯雅病毒获得满意效果。以SDS-PAGE和免疫印迹法分析基孔肯雅病毒结构蛋白的组成及抗原性。结果证实:基孔肯雅病毒含有三种结构蛋白(E_1、E_2和C),另一种病毒特异性蛋白E_3与完整毒粒无结构上的联系;病毒粗提样品与精提样品的免疫印迹结果基本一致,尽管在SDS-PAGE图谱上二者相差甚大,因而就某些研究或应用目的而言,病毒的纯化程序可简化。文中初步探讨了以基孔肯雅病毒包膜糖蛋白为特异性抗原用于流行病学调查和临床     

基孔肯雅病毒囊膜蛋白假病毒模型的构建

目的:以HIV为骨架构建单次复制性的含基孔肯雅病毒囊膜蛋白的假病毒模型,观察其对哺乳动物细胞的侵染性。方法:PCR合成基孔肯雅病毒囊膜蛋白基因,克隆到真核表达载体上,与HIV慢病毒包装系统质粒共转染293FT细胞,48 h后收培养上清,在8μg/mL Polybrene存在下感染293FT细胞,感染48 h后在荧光显微镜下观察结果。结果:PCR合成了基孔肯雅病毒囊膜蛋白基因并克隆到真核表达载体上,测序结果正确;共转染293FT细胞后,检测到基孔肯雅病毒囊膜蛋白的表达并包装成假病毒,感染新鲜293FT细胞后能够检测到绿色荧光蛋白。结论:合成的基孔肯雅病毒囊膜蛋白基因能正确表达并包装成假病毒,含基孔肯雅病毒囊膜蛋白的假病毒能感染293FT细胞并表达绿色荧光蛋白,可用该假病毒模型进一步研究基孔肯雅病毒的感染性,筛选评价抗基孔肯雅病毒药物。     

树鼩实验感染基孔肯雅病毒的研究

选用3株基孔肯雅病毒人工感染成年树鼩,进行了病毒血症、抗体动态变化、内脏组织病理改变和病毒在宿主体内定位的研究。结果表明,感染树鼩能产生2～6天的病毒血症。血凝抑制(Hi)抗体第6天产生,第30～50天达高峰:中和(NT)抗体在第10天产生,第30～40天达高峰,二者相关性非常显著(P<0.01)。补体结合(CF)抗体第14天产生,第40～50天为高峰,以后逐渐下降。第8～12天能在其脑、肺、肝、脾和肾等组织查到病毒,经病理检查这些内脏组织呈炎性改变和出血倾向,表明该病毒能侵袭树鼩各主要脏器。试验认为树鼩对基孔肯雅病毒敏感。     

基孔肯雅病毒浓缩提纯及多肽抗原研究

采用聚乙二醇(PEG 6000)沉淀法(粗提)和蔗糖密度梯度速率区带离心(精提)浓缩纯化基孔肯雅病毒获得满意效果。以SDS-PAGE和免疫印迹法分析基孔肯雅病毒结构蛋白的组成及抗原性。结果证实:基孔肯雅病毒含有三种结构蛋白(E₁、E₂和C),另一种病毒特异性蛋白E₃与完整毒粒无结构上的联系;病毒粗提样品与精提样品的免疫印迹结果基本一致,尽管在SDS-PAGE图谱上二者相差甚大,因而就某些研究或应用目的而言,病毒的纯化程序可简化。文中初步探讨     

云南基孔肯雅病毒生物学性状的研究

本文对分离自云南的基孔肯雅病毒进行了生物学特性研究。实验证明基孔肯雅病毒对乳鼠有强而稳定的致病性,对C6/36,BHK21及Vero细胞有致病变作用,能与鹅和鸽红细胞凝集,最适pH5.75。电镜观察病毒呈圆形,有膜,外径66.8nm,内径47.7nm。交互血凝抑制及中和试验表明,云南毒株之间或与国外原株在抗原性上无明显差别,为同一血清型,并与同亚组病毒反应较弱,与其它虫媒病毒无交叉。本文还讨论了云南株与国外原株在某些生物学特性方面的异同。     

Multiplication of chikungunya virus in salivary glands of Aedes albopictus (Oahu strain) mosquitoes: an electron microscopic study

Aedes albopictus as well as Aedes aegypti is an important vector of chikungunya and dengue viruses. Electron microscopic observations on the salivary glands of Ae. albopictus infected with chikungunya virus were performed in comparing with those of Ae. aegypti infected with dengue virus. No virus budding from the cell surface of the chikungunya-infected mosquito's salivary glands was found as shown in dengue-infected ones, in contrast to the findings of the mammalian cells such as Vero, KB, IMR, J-111 and BHK-21 cells infected with chikungunya and/or dengue virus(es).     

Larval development of Aedes aegypti and Aedes albopictus in peri-urban brackish water and its implications for transmission of arboviral diseases

Aedes aegypti (Linnaeus) and Aedes albopictus Skuse mosquitoes transmit serious human arboviral diseases including yellow fever, dengue and chikungunya in many tropical and sub-tropical countries. Females of the two species have adapted to undergo preimaginal development in natural or artificial collections of freshwater near human habitations and feed on human blood. While there is an effective vaccine against yellow fever, the control of dengue and chikungunya is mainly dependent on reducing freshwater preimaginal development habitats of the two vectors. We show here that Ae. aegypti and Ae. albopictus lay eggs and their larvae survive to emerge as adults in brackish water (water with <0.5 ppt or parts per thousand, 0.5-30 ppt and >30 ppt salt are termed fresh, brackish and saline respectively). Brackish water with salinity of 2 to 15 ppt in discarded plastic and glass containers, abandoned fishing boats and unused wells in coastal peri-urban environment were found to contain Ae. aegypti and Ae. albopictus larvae. Relatively high incidence of dengue in Jaffna city, Sri Lanka was observed in the vicinity of brackish water habitats containing Ae. aegypti larvae. These observations raise the possibility that brackish water-adapted Ae. aegypti and Ae. albopictus may play a hitherto unrecognized role in transmitting dengue, chikungunya and yellow fever in coastal urban areas. National and international health authorities therefore need to take the findings into consideration and extend their vector control efforts, which are presently focused on urban freshwater habitats, to include brackish water larval development habitats.     

基孔肯亚病毒垂直传播的研究进展

基孔肯亚病毒(chikungunya virus,CHIKV)是一种由埃及伊蚊和白纹伊蚊传播的蚊媒病毒,属于披膜病毒科甲病毒属.从2005年起,CHIKV在非洲、亚洲和美洲多次发生大规模疫情,其感染率较高,能引起发热性疾病,常伴有关节炎,关节炎症状可维持数月甚至数年,对患者生活和社会生产造成极大影响.除了蚊媒传播,妊娠...     

Are Aedes albopictus or other mosquito species from northern Italy competent to sustain new arboviral outbreaks?

The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae), native to Southeast Asia, has extended its geographical distribution to invade new temperate and tropical regions. This species was introduced in 1990 to Italy and has since become the main pest in urban settings. It was incriminated as a principal vector in the first European outbreak of chikungunya virus (CHIKV) in the province of Ravenna (Italy) in 2007. This outbreak was associated with CHIKV E1-226V, efficiently transmitted by Ae. albopictus . The occurrence of this outbreak in a temperate country led us to estimate the potential of Ae. albopictus to transmit CHIKV and dengue virus (DENV), and to determine the susceptibility to CHIKV of other mosquito species collected in northern Italy. Experimental infections showed that Ae. albopictus exhibited high disseminated infection rates for CHIKV (75.0% in Alessandria; 90.3% in San Lazzaro) and low disseminated infection rates for DENV-2 (14.3% in San Lazzaro; 38.5% in Alessandria). Moreover, Ae. albopictus was able to attain a high level of viral replication, with CHIKV detectable in the salivary glands at day 2 after infection. In addition, the other three mosquito species, Anopheles maculipennis Meigen, Aedes vexans vexans (Meigen) and Culex pipiens L., showed variable susceptibilities to infection with CHIKV, of 0%, 7.7% and 0–33%, respectively. This information on vector competence is crucial in assessing the risk for an outbreak of CHIKV or DENV in Italy.     

Adaptation of the BG-Sentinel trap to capture male and female Aedes albopictus mosquitoes

In recent years, the remarkable spread of Aedes albopictus (Skuse) (Diptera: Culicidae) throughout the world has drawn attention to this hitherto poorly studied species, particularly after its role in outbreaks of chikungunya fever in the western Indian Ocean and in Italy. Variants of sterile insect technique (SIT), including the release of transgenic males with a dominant lethal gene (RIDL), have been proposed in the search for new and innovative methods of control. Knowledge of male dispersal, mating behaviour and longevity will be critical to the success of this approach. We present an effective and practical method for trapping both male and female Ae. albopictus using a mouse-baited BG-Sentinel trap.     

Aedes albopictus, vector of chikungunya and dengue viruses in Reunion Island: biology and control

Chikungunya virus (CHIKV) and dengue virus (DENV) are mosquito-borne viruses transmitted by the Aedes genus. Dengue is considered as the most important arbovirus disease throughout the World. Chikungunya, known from epidemics in continental Africa and Asia, has up to now been poorly studied. It has been recently responsible for the severe 2004-2007 epidemic reported in the Indian Ocean (IO), which has caused several serious health and economic problems. This unprecedented epidemic of the IO has shown severe health troubles with morbidity and death associated, which had never been observed before. The two major vectors of those arboviruses in the IO area are Aedes aegypti and Aedes albopictus. The latest is considered as the main vector in most of the islands of the area, especially in Reunion Island. Ae. albopictus showed strong ecological plasticity. Small disposable containers were the principal urban breeding sites, and preferred natural developmental sites were bamboo stumps and rock holes in peri-urban and gully areas. The virus has been isolated from field collected Ae. albopictus females, and in two out of 500 pools of larvae, demonstrating vertical transmission. Experimental works showed that both Ae. albopictus and Ae. aegypti from west IO islands are efficient vectors of dengue and chikungunya viruses. Since 2006 and all along the epidemic of CHIKV, measures for the control of larvae (temephos then Bacillus thuringiensis) and adults (fenitrothion, then deltamethrine) of Ae. albopictus where applied along with individual and collective actions (by the use of repellents, and removal of breeding sites around houses) in Reunion Island. In order to prevent such epidemics, a preventive plan for arboviruses upsurge is ongoing processed. This plan would allow a quicker response to the threat and adapt it according to the virus and its specific vector.     

Colorado Tick Fever Virus: Growth in a Mosquito Cell Line

Two strains of Colorado tick fever virus grew in Singh's Aedes albopictus cells. In one of three experiments, virus growth continued for 7 weeks.     

福建省白纹伊蚊中登革病毒的分离与鉴定

近年来福建省登革热(Dengue fever,DF)输入性病例持续存在,且登革热的主要传播媒介白纹伊蚊在全省内广泛分布,为了解福建省福州市登革热的媒介白纹伊蚊携带登革病毒(Dengue virus,DENV)状况,2017年10月7日在福州市台江区元一花园小区内开展伊蚊监测,采用双层叠帐法捕获255只白纹伊蚊蚊体研磨液上清提取核酸后用实时荧光RT-PCR法检测DENV特异性核酸,将检测阳性的蚊体研磨液上清接种C6/36细胞进行病毒分离,成功分离到1株DENV病毒株mosquito13/Fujian/2017;经实时荧光RT-PCR法鉴定所分离病毒株的血清型为I型;利用型特异性引物通过RT-PCR扩增病毒E基因并测序进行分子遗传特性分析;E基因核苷酸和氨基酸同源性分析显示,该毒株与2017年10月17日同小区本地登革热病例血清中分离得到的登革毒株E基因序列完全一致,与越南2014年分离株KT825033/Vietnam/2014核苷酸(99.7%)和氨基酸(99.8%)同源性最高;系统进化树分析表明所分离登革病毒毒株的基因型为I型,与东南亚地区的越南,泰国,柬埔寨等国家进化关系相近,可能输入来源于东南亚国家。本研究证实了登革热外潜伏期的存在以及白纹伊蚊在登革热疫情传播过程中的媒介作用,提示在登革热的防控工作中媒介登革病毒监测、检测的重要性,也提示福建省需要加强输入来源监测,特别是东南亚入境人员的监测。     

Chikungunya virus infection of cell lines: analysis of the East, Central and South African lineage

Chikungunya virus (CHIKV) is a re-emerging mosquito borne alphavirus that has caused large scale epidemics in the countries around the Indian Ocean, as well as leading to autochthonous transmission in some European countries. The transmission of the disease has been driven by the emergence of an African lineage of CHIKV with enhanced transmission and dissemination in Aedes mosquito hosts. Two main genotypes of this lineage have been circulating, characterized by the presence of a substitution of a valine for an alanine at position 226 of the E1 protein. The outbreak, numbering in millions of cases in the infected areas, has been associated with increasing numbers of cases with non-classical presentation including encephalitis and meningitis. This study sought to compare the original Ross strain with two isolates from the recent outbreak of chikungunya fever in respect of infectivity and the induction of apoptosis in eight mammalian cell lines and two insect cell lines, in addition to generating a comprehensive virus production profile for one of the newer isolates. Results showed that in mammalian cells there were few differences in either tropism or pathogenicity as assessed by induction of apoptosis with the exception of Hela cells were the recent valine isolate showed less infectivity. The Aedes albopictus C6/36 cell line was however significantly more permissive for both of the more recent isolates than the Ross strain. The results suggest that the increased infectivity seen in insect cells derives from an evolution of the CHIKV genome not solely associated with the E1:226 substitution.