免疫荧光技术检测室内感染蚊虫体内登革病毒的研究

目的 探讨检测媒介蚊虫体内登革病毒的方法。方法 以埃及伊蚊和白纹伊蚊两种有效媒介为研究对象，在人工经口感染大剂量登革2型病毒（DEN-2）的基础上，通过蚊细胞培养病毒分离和蚊头部压片免疫荧光技术进行病毒抗原检测。结果 埃及伊蚊感染DEN-2病毒1d后，接种C6/36细胞，盲传一代后第5d出现空斑现象，确证细胞发病，白纹伊蚊经口感染登革病毒后，取吸血雌蚊每日进行头部压片免疫荧光检测，结果显示，从蚊虫感染后第1d-第20d内均能检测到病毒抗原。结论 用免疫荧光技术，可直接检测出感染蚊体内是否携带登革病毒，是较为简便，省时，敏感的方法。     

登革热

<正>登革热病因1.传染源患者和隐性感染者为主要传染源。患者在发病1日至发病后3日内传染性最强。少数患者在热退后第3 d还可从血液中分离到病毒。此外蝙蝠、猴、鸟类和狗等动物体内可检测到登革病毒抗体,可能为登革病毒的自然宿主,从而有可能成为本病的传染源。2.传播媒介蚊虫是本病的主要传播媒介,其中伊蚊是传播登革病毒的主要蚊种,包括埃及伊蚊和白纹伊蚊。3.易感性在新流行区各年龄组均易感但以青壮年发病者居多。     

白纹伊蚊DENV 2载量随饲养时长的变化趋势研究北大核心CSCD

目的探讨白纹伊蚊感染DENV 2后,体内病毒载量随饲养时长变化趋势,为相关研究提供参考依据。方法305只白纹伊蚊100%人工感染后,分别于第2、4、6、8、10d随机取蚊,共3个重复,每个重复至少20只蚊虫,应用实时荧光定量PCR检测蚊体内DENV 2载量,记录CT值。计算并比较各组蚊虫病毒载量、阳性率等。结果白纹伊蚊感染DENV 2后第2、4、6、8、10d病毒载量中位数为2.24×103-1.99×106 copies/ml,差异具有统计学意义(χ2=51.08,P<0.01);阳性率为81.67%-100%,差异具有统计学意义(Fisher确切概率P<0.01)。其中,白纹伊蚊体内病毒载量变化特点为:以第2d时病毒载量为基线值,第4d显著降低,第6d回升至第2d水平,第8、10d逐步升高;白纹伊蚊DENV 2阳性率变化特点:感染后第2d阳性率100%,第4d时降至81.67%,且随后保持稳定。结论白纹伊蚊感染DENV 2载量随饲养时长变化先降低后升高,阳性率先升高后降低,因此中应根据A科研需要合理选择饲养时长。     

白纹伊蚊DENV 2载量随饲养时长的变化趋势研究

目的探讨白纹伊蚊感染DENV 2后,体内病毒载量随饲养时长变化趋势,为相关研究提供参考依据。方法305只白纹伊蚊100%人工感染后,分别于第2、4、6、8、10d随机取蚊,共3个重复,每个重复至少20只蚊虫,应用实时荧光定量PCR检测蚊体内DENV 2载量,记录CT值。计算并比较各组蚊虫病毒载量、阳性率等。结果白纹伊蚊感染DENV 2后第2、4、6、8、10d病毒载量中位数为2.24×103-1.99×106 copies/ml,差异具有统计学意义(χ2=51.08,P0.01);阳性率为81.67%-100%,差异具有统计学意义(Fisher确切概率P0.01)。其中,白纹伊蚊体内病毒载量变化特点为:以第2d时病毒载量为基线值,第4d显著降低,第6d回升至第2d水平,第8、10d逐步升高;白纹伊蚊DENV 2阳性率变化特点:感染后第2d阳性率100%,第4d时降至81.67%,且随后保持稳定。结论白纹伊蚊感染DENV 2载量随饲养时长变化先降低后升高,阳性率先升高后降低,因此中应根据A科研需要合理选择饲养时长。     

白纹伊蚊感染登革病毒体内屏障的实验观察

以登革病毒Ⅱ型经口和胸内接种感染白纹伊蚊,观察蚊体内屏障对感染登革病毒的影响。白纹伊蚊不同途径感染同剂量登革病毒Ⅱ型后,胸内接种感染的蚊脑感染率明显高于经口感染,提示中肠屏障的作用。以不同剂量登革病毒Ⅱ型胸内接种感染白纹伊蚊,蚊唾液腺的感染率均低于蚊脑感染率,提示白纹伊蚊唾液腺对登革病毒感染屏障的存在,且接种病毒剂量与唾液腺感染率呈密切的直线相关,表明唾液腺感染屏障作用决定于剂量。     

登革病毒在白纹伊蚊体内的扩散和分布

研究登革病毒在蚊体内的扩散途径及分布对分析蚊体内的屏障、决定政体对病毒的敏感器官及评价经卵传递病毒在流行病学上的作用具有一定的意义。国内作者对蚊虫感染及传播登革病毒、病毒在蚊体内的繁殖动态进行过报道。本文对登革病毒在白纹伊蚊体内的扩散和分布进行观察。     

不同温度对DEN-2型病毒在白纹伊蚊体内外潜伏期的影响

摘要：目的 研究温度对外潜伏期的影响。方法 登革2型病毒人工经口感染白纹伊蚊,冻麻挑出吸饱血的雌蚊放入新的蚊笼,分别置于18℃、２1℃、26℃、31℃和36℃中饲养,第3、4、5、7、10、14、20、25天分别收集并解剖白纹伊蚊,间接免疫荧光法分别检测蚊虫头部、唾液腺和胸腹部的登革2型病毒抗原。结果 置于18、21、26、31和36℃饲养的白纹伊蚊分别在第25、7、5、4、3天时胸腹部中开始检出阳性。21、26、31和36℃饲养的蚊虫唾液腺阳性检出的时间分别为10、7、4、4天,18℃在感染后的25天内,唾液腺均未检出阳性。结论 随着温度的升高,白纹伊蚊的外潜伏期逐渐缩短。     

中老边境地区江城县登革热媒介及病毒感染调查

目的了解中老边境江城县登革热媒介的种群密度、孳生习性以及登革病毒自然感染情况,为边境登革热的防控提供参考。方法 2015年6-10月在中老边境江城县选择3个自然村作为固定监测点,采用人诱捕法诱捕白天成蚊,同时在监测点居民区进行幼虫密度和孳生习性调查。捕获的成蚊用液氮低温保存带回实验室用RT-PCR进行登革病毒检测。结果白天人诱法共捕获7属20种2 247只成蚊,其中白纹伊蚊1 405只,占捕获总数的62.53%,其次为骚扰阿蚊438只,占捕获总数的19.49%,未捕到埃及伊蚊,白纹伊蚊成蚊平均密度为16.7只/人工小时;白纹伊蚊幼虫的平均房屋指数、布雷图指数、容器指数和千人指数分别为12.0、18.3、2.2和25.4。8、9月是白纹伊蚊孳生和活动的高峰;白蚊伊纹幼虫以轮胎、罐子和竹筒为主要孳生容器,3种容器占总阳性容器数比例分别为26.3%、23.4%和20.4%;人诱法捕获成蚊RT-PCR检测37组蚊虫,黄病毒属阳性17组,批阳性率为46.0%,未检出登革病毒。结论中老边境江城县登革热传播媒介白纹伊蚊密度较高且分布广,蚊虫携带黄病毒属病毒比例高,应加强当地登革热监测工作。     

核酸杂交和McAb—IFA快速检出鉴定白纹伊蚊体内登革热病毒…

本文报道用实验感染和自然叮咬吸血感染带毒的雌性白纹伊蚊，用克隆的Ｄｅｎ．Ｖ－ｃＤＮＡ探针作斑点杂交检测蚊体内病毒ＲＮＡ和Ｄｅｎ．ｖ－ＭｃＡｂ－ＩＦＡ检查蚊头压片的病毒抗原。探针检出病毒ＲＮＡ的时间分别为实验感染后第２ｄ，自然叮咬血后第１ｄ。ＭｃＡｂ－ＩＦＡ检出病毒抗原的时间分别为实验感染后第３ｄ，自然叮咬后第２天。从登革热监测点中，检出Ｂ组虫媒病毒的存在，提示早期检出蚊虫携带病毒水平，可预测Ｂ组虫     

逆转录聚合酶链反应检测蚊体内黄病毒的研究

本文报告了应用自行设计的黄病毒通用引物和逆转录聚合酶键反应成功地扩增了实验室脑内接种感染白纹伊蚊体内登革病毒RNA和海南省非流行区野外捕获的白纹伊蚊和骚扰阿蚊体内黄病毒RNA。该法灵敏度高,特异性强,获得结果迅速。能检出5只蚊虫中含有1只阳性蚊的混合标本中的登革病毒RNA。整个过程只要数小时就可完成。这对蚊体内黄病毒的快速检测及流行病学调查、防治监测等研究,具有重要意义。     

Growth characteristics of ChimeriVax-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes

The chimeric yellow fever (YF) 17D-dengue type 2 (ChimeriVax-DEN2) vaccine virus developed by Acambis, Inc. (Cambridge, MA) contains the prM and E genes of wild-type (wt) dengue 2 (DEN-2) (strain PUO-218) virus in the YF vaccine virus (strain 17D) backbone. The potential of ChimeriVax-DEN2 virus to infect and be transmitted by Aedes aegypti, the principal DEN and YF virus mosquito vector, and Aedes albopictus, a species that occurs in areas of active transmission of YF and DEN viruses, was evaluated. Mosquitoes were intrathoracically (IT) inoculated with virus or were fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN2 were compared with the wt DEN-2 and YF 17D vaccine viruses. Replication of YF 17D virus is attenuated in cultured Ae. albopictus C6/36 mosquito cells and in Ae. aegypti and Ae. albopictus mosquitoes. Growth of ChimeriVax-DEN2 virus similarly was restricted in C6/36 cells and in mosquitoes. ChimeriVax-DEN2 replicated in 56% of IT inoculated Ae. aegypti, and virus disseminated to head tissue in 36%, with a mean viral titer of 1.8 log10 PFU/mosquito. Of mosquitoes, 16% of Ae. aegypti and 24% of Ae. albopictus were infected 14 days after a blood meal containing ChimeriVax-DEN2, but virus did not disseminate to head tissue. In contrast, DEN-2 replicated in all IT inoculated and orally infected Ae. aegypti (mean titer 5.5 log10 PFU/mosquito), and virus disseminated to head tissue in 95%. Of Ae. albopictus, 84% were infected after a blood meal containing DEN-2 virus; dissemination occurred in 36%. Replication of ChimeriVax-DEN2 virus in mosquitoes corresponded to that of YF 17D vaccine virus, which is restricted in its ability to infect and replicate in mosquitoes. Therefore, transmission of ChimeriVax-DEN2 virus by vector mosquitoes is unlikely.     

Introduction of Aedes albopictus in Gabon: what consequences for dengue and chikungunya transmission?

The 2007 outbreak of chikungunya in Gabon has indicated the potential of this disease to spread beyond its usual range ensuing from the expansion of the mosquito Aedes albopictus . A few cases of dengue (DEN) infection were also detected. Because little is known about the potential for Gabonese mosquito species to transmit both chikungunya and DEN viruses (DENV), we conducted studies to determine the susceptibility of Ae. albopictus and Aedes aegypti collected in Libreville to both viruses by experimental infections. Disseminated infection rates were high for Ae. albopictus infected with chikungunya virus (CHIKV) (66.7–86%) and low with DENV (13–21.4%). Moreover, Ae. aegypti sp. formosus was a less efficient vector of CHIKV than Ae. albopictus . The recent introduction and dissemination of chikungunya associated with the invasion of Ae. albopictus in Africa illustrates the potential for CHIKV to spread to other parts of the world.     

Sexual transmission of dengue viruses by Aedes albopictus

Male Aedes albopictus experimentally infected with dengue virus types 1, 2, 3, or 4 transmitted their infection sexually to female Ae. albopictus. Such transmission was enhanced if the females had taken a bloodmeal 2 to 7 days prior to mating. Male Ae. albopictus also transmitted dengue virus vertically to their F1 progeny. Infected progeny were found among those derived from eggs laid greater than or equal to 73 hr after mating but not among those derived from eggs laid prior to that time. This suggests that virus probably was not transmitted directly to ova but, rather, underwent prior replication in the female genital tract. Female Ae. albopictus experimentally infected with dengue type 1 virus did not transmit their infection sexually to males. This finding supports the hypothesis that male mosquitoes naturally infected with dengue virus acquired their infection vertically.     

Double infection of heteroserotypes of dengue viruses in field populations of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) and serological features of dengue viruses found in patients in southern Thailand

In order to understand more about the epidemiology of DHF, a study of the type of dengue viruses and vectors under natural conditions was carried out. Mosquito vectors in the field and the serum of DHF patients in southern Thailand were examined. The two mosquito species are abundant and DHF incidence remains high in this region. Dengue viruses were examined in field-caught mosquitoes by RT-PCR technique. The mosquitoes were caught in 4 provinces: Krabi, Phuket, Phang-Nga and Surat Thani during the late dry season until the early rainy season in 2005. Three dengue serotypes (DEN-2, DEN-3, DEN-4) were detected in Ae. aegypti males and females, and 2 (DEN-2, DEN-3) were detected in Ae. albopictus females. Double infection with 2 serotypes of dengue viruses (DEN-2 and DEN-3) were detected in Ae. aegypti males and females and Ae. albopictus females. DEN-2 and DEN-1 were the most prevalent serotypes found in the serum of the patients in this area, followed by DEN-4 and DEN-3. The prevalence of the predominant dengue serotype varied from province to province. Detection of viruses in adult male mosquitoes reveals the role of transovarial transmission of dengue viruses in field populations of DHF vectors and elucidates circulation of dengue viruses in vectors in the natural environment of endemic areas. The incidence of multiple serotypes of dengue virus in Ae. aegypti and Ae. albopictus in the same area points toward a high risk for an epidemic of DHF. These findings provide greater understanding of the relationship among mosquito vectors, virus transmission and DHF epidemiology in endemic areas.     

Distribution and seasonality of vertically transmitted dengue viruses in Aedes mosquitoes in arid and semi-arid areas of Rajasthan, India

BACKGROUND & OBJECTIVES: Transovarial transmission of dengue virus is a crucial etiological phenomenon responsible for persistence of virus during inter-epidemic period of the disease. Distribution and seasonality of this phenomenon in disease endemic areas may contribute to explain emergence of dengue and its subsequent prevention. The study on seasonal and area distribution of transovarial transmission of dengue virus in Aedes aegypti, Ae. albopictus and Ae. vittatus has been made in desert and non-desert districts of Rajasthan, India from 2006 to 2007. The observations revealed role of different Aedes species in transmission and retention of dengue virus. METHODS: The larvae of Ae. aegypti, Ae. albopictus and Ae. vittatus were collected during each of the study seasons from rural and urban areas of three districts-Jodhpur, Jaipur and Kota. The larvae were collected from domestic and peri-domestic containers and from tree holes of peri-urban foci such as gardens and parks and were reared into adults in the laboratory at room temperature. The laboratory reared adults were subjected to Indirect fluorescence antibody test (IFAT). The laboratory-reared adult mosquitoes showing positive IFA were treated as the sample showing vertically transmitted dengue virus. RESULTS: Pooled data for all the four seasons revealed maximum (15.7%) mosquito infectivity in Ae. albopictus followed by Ae. aegypti (12.6%) in Jodhpur district. In Jaipur district, Ae. vittatus showed highest infection (20%) of vertically transmitted virus followed by Ae. albopictus (18.7%) and least in Ae. aegypti (13.3%). In Kota district, pooled data for all the four seasons showed maximum vertical infection of mosquitoes in Ae. albopictus (14.2%). INTERPRETATION & CONCLUSION: Transovarial transmission of dengue virus by available vector species in a dengue endemic setting could be the key etiological phenomenon responsible for re-emergence of the disease from inter-epidemic to epidemic phase of disease onset. The observations in the present study suggest that during winter season which is not the active transmission season of dengue in Rajasthan, Ae. albopictus has shown maximum percentage of vertically transmitted virus. Our observation substantiates with the earlier studies that how Ae. albopictus is horbouring virus during inter-epidemic period of dengue. Another important lead emerging through present study is the high mosquito infectivity of Ae. aegypti during summer and rainy seasons especially from desert districts, Jodhpur and semi district. This observation suggests that in Rajasthan, owing to tendency of overstorage of domestic water by the inhabitants, mosquito and vertically transmitted virus get pronounced during summer season which could precedes the active transmission season of dengue during following rainy season.     

Replication of dengue type 2 virus in Culex quinquefasciatus (Diptera: Culicidae)

We were able to infect Culex quinquefasciatus by the parenteral route with dengue virus type 2. The percentage of mosquitoes infected was dose dependent and we obtained a rate of 45.6% infected Cx. quinquefasciatus when a 10(5.9) MID50 (mosquito infectious dose for 50% of the individuals as measured in Aedes aegypti) of dengue virus type 2 per mosquito was used. Infection was detected by an immunofluorescent assay performed on mosquito head squashes 14 days after infection. The replication of dengue virus in Cx. quinquefasciatus was either at a very low level of magnitude or generated a large number of noninfectious particles since the triturated bodies of infected Cx. quinquefasciatus did not infect Ae. aegypti mosquitoes when inoculated parenterally. We were unable to infect Cx. quinquefasciatus females orally with an artificial meal that infected 100% of Ae. aegypti females. These findings lead us to agree with the consensus that Cx. quinquefasciatus should not be considered a biological vector of dengue viruses.     

Infectivity of West Nile/dengue chimeric viruses for West Nile and dengue mosquito vectors

West Nile virus (WN), an agent of significant human and veterinary disease, is endemic in the Old World and rapidly spreading throughout the Americas. Vaccines are needed to halt the geographic expansion of this virus and prevent disease where it is established. However, to preclude introduction of a vaccine virus into the environment, a live attenuated WN vaccine should have low potential for transmission by mosquitoes. A chimeric WN vaccine candidate was previously generated by replacing the membrane and envelope structural protein genes of recombinant dengue type 4 virus (rDEN4) with those of WN; a derivative of this virus, WN/DEN4-3'delta30, contains a 30-nucleotide deletion in the 3' untranslated region. To assess the potential for transmission by mosquitoes of these vaccine candidates, the ability of each chimeric virus to infect the mosquito midgut, disseminate to the head, and pass into the saliva was compared to that of their wild-type parental WN and DEN4 viruses in three vector species. The WN/DEN4 chimeric viruses were significantly attenuated in both Culex tarsalis, a vector able to transmit WN but not dengue, and in Ae. aegypti, a vector able to transmit dengue but not WN. However, the chimeric viruses were as infectious as either wild-type virus for Ae. albopictus, a vector able to transmit both dengue and WN. These results indicate that chimerization caused a contraction in vector host range rather than universal attenuation for mosquitoes per se. This restriction in potential vectors renders it less likely that WN/DEN4 and WN/DEN4-3'delta30 would be transmitted from vaccinees to mosquitoes.     

Natural vertical transmission of dengue virus in Aedes albopictus (Diptera: Culicidae) in Kerala, a southern Indian state

The natural occurrence of vertical transmission of dengue viruses in Aedes albopictus (Skuse) mosquitoes was examined in the state of Kerala in southern India. Adults and larvae of Ae. albopictus collected from Kerala were screened for dengue viruses by using enzyme-linked immunosorbent assay with dengue-specific monoclonal antibodies. The possibility of the vertical transmission of dengue virus in Ae. albopictus was further evidenced by the detection of the virus in field-collected adult males as well as females emerged from field-collected larvae. Two pools, one pool from the adult males and one pool from the emerged females derived from field-collected larvae, were collected in the relatively hot months of June and March, respectively, and found to be positive for dengue virus antigen. Dengue serotype 2 virus was isolated from field-collected male adults in Kerala. These findings suggest that dengue virus is maintained in Ae. albopictus mosquitoes during the dry season by vertical transmission in nature.