温度对白纹伊蚊传播登革2型病毒易感性影响的研究

目的 研究不同温度下白纹伊蚊对登革病毒的易感性差异.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℃、33℃和36℃的人工气候箱中饲养,经外潜伏期后,解剖蚊虫,间接免疫荧光法分别检测蚊虫的头部、唾液腺和胸腹部内的登革2型病毒抗原,并计算蚊虫的播散感染率.结果 18℃白纹伊蚊感染率最低,胸腹部感染率为8％,头部和唾液腺未检出.31℃胸腹部感染率最高为82％,头部和唾液腺感染率33℃时最高.36℃各部位感染率均较33℃下降.18℃的播散感染率最低为0,36℃播散感染率最高,达到了100％.结论 白纹伊蚊感染率随温度的升高,呈现先升后降趋势.18℃可能是白纹伊蚊传播登革病毒的最低温度.播散感染率随温度的升高逐渐升高.     

围食膜在蚊虫对登革Ⅱ型病毒中肠感染屏障中的作用

通过透射电镜观察了致倦库蚊和白纹伊蚊围食膜的形成特点及登革Ⅱ型病毒感染蚊虫中肠上皮细胞的规律 ,针对围食膜在蚊虫对登革Ⅱ型病毒中肠感染屏障中的作用进行了探讨。结果表明 :DEN 2能在吸血 2h内侵染白纹伊蚊上皮细胞并大量复制 ,而经过一定的外潜伏期仍不能侵染致倦库蚊的上皮细胞 ,提示致倦库蚊对DEN 2存在中肠感染屏障。而从围食膜形成的时间及病毒感染的规律推测 ,围食膜在中肠感染屏障中不是决定性的因子     

白纹伊蚊感染登革2型病毒后基因表达变化的研究

白纹伊蚊感染登革 2型病毒 2 4h后 ,和对照蚊虫一起提取RNA ,用差异显示PCR (DDRT PCR)技术对蚊虫感染病毒后基因表达的变化进行研究。白纹伊蚊感染登革 2型病毒后 ,经过 8条随机引物和 3条 3′端锚定引物配对扩增 ,发现了 6条表达有差异的片段 ,其中 5条为感染后表达量增加的片段 ,1条为感染后表达量减少的片段 ,并对这些片段进行了克隆和测序 ,通过GenBank查询 ,其中 5条为未知序列 ,1条表达量增高的片段和果蝇核糖体蛋白S5基因有较高的同源性。这些差异片段的发现可能对探讨媒介蚊虫对登革病毒的易感性机理有意义     

实验感染登革Ⅱ型病毒的2种蚊媒RT-PCR检测defensin A结果分析

白纹伊蚊和埃及伊蚊在经口大剂量感染登革Ⅱ型病毒1天、2天、3天和11天后,用RT-PCR方法都未能在其体内检测到defensin A的mRNA的表达,提示登革Ⅱ型病毒感染其有效媒介宿主白纹伊蚊和埃及伊蚊后并不诱导defensin A的表达,或者表达量极低用RT-PCR方法不能检测到.     

白纹伊蚊干燥卵保存登革2型病毒的实验研究

感染登革2型病毒的白纹伊蚊卵在(25±1)℃、光照14h/天,75%±5%RH的相对干燥环境中保存42天,分别采用C6/36细胞培养分离病毒和RT-PCR方法,检测卵孵化的F1代蚊虫感染率。第1个生殖营养周环F1代蚊虫未检测和分离到病毒,PCR检测第2生殖营养周环蚊虫批阳性率为26.7%,最低感染率为1∶112.5;第3生殖营养周环蚊虫批阳性率为27.8%,最低感染率为1∶108。统计学检验结果表明第2和第3生殖营养周环卵孵化的F1代蚊虫感染率没有达到显著性水平(P>0.05)。结果表明感染白纹伊蚊卵在75%±5%RH的相对干燥环境中保存42天后,能在孵化的子代蚊虫中检测和分离到病毒,证实伊蚊卵在干燥环境中能够保存卵内病毒。     

埃及伊蚊唾液腺激肽对Balb/C小鼠感染登革病毒的增强作用

登革热 登革出血热是我国重要的蚊媒病之一 ,主要通过埃及伊蚊和白纹伊蚊传播。有关登革病毒感染传播机制还不够清楚。本研究以Balb C小鼠为实验动物 ,探讨人工合成埃及伊蚊唾液腺激肽对登革病毒感染宿主的作用。结果表明唾液腺激肽对登革病毒感染Balb C实验小鼠具有一定的增强作用 ,病毒血症时间延长 ,抗体滴度降低。与埃及伊蚊唾液的增强作用近似 ,但具体机制还有待进一步探讨     

白纹伊蚊实验种群感染沃尔巴体Wolbachia的研究

为了了解白纹伊蚊实验种群沃尔巴体Wolbachia感染率、感染品系、组织分布及系统发育,运用引物PCR方法检测实验种群白纹伊蚊Wolbachia 的感染情况.解剖白纹伊蚊,提取头部、卵巢/睾丸、脂肪体、唾液腺/胸部、马氏管、中肠组织的基因组DNA,PCR法扩增Wolbachia表面膜蛋白（wsp）,扩增产物进行克隆及测序,并用BLAST软件对其进行系统发育分析.将随机抽取的白纹伊蚊雌雄各50只分别检测,均为阳性.卵巢/睾丸、脂肪体和唾液腺/胸部均存在着Wolbachia A、B组超感染;雌蚊头部、雄蚊马氏管和中肠为B组单感染;雄蚊头部则未检测到Wolbachia.将wsp基因克隆测序后进行系统发育分析显示白纹伊蚊体内Wolbachia与尖音库蚊、菜蛾、宽边黄粉蝶四者同源性较高达99%,这表明四者体内所感染的Wolbachia可能来源于同一个分支.     

登革2型病毒在C6/36细胞上受体蛋白的鉴定

应用VOPBA (病毒辅覆蛋白结合分析 )技术 ,结合免疫印迹化学发光的方法在C6 36细胞上鉴定出了登革 2型病毒唯一的大小约为 35kDa的受体蛋白成分。为研究病毒对白纹伊蚊的感染机理打下了良好的基础。     

利用Aag2细胞过表达体系筛选埃及伊蚊对DENV2易感相关免疫基因

埃及伊蚊Aedes aegypti是登革病毒的主要传播媒介,埃及伊蚊感染登革Ⅱ型病毒(dengue virus typeⅡ,DENV2)后产生先天免疫反应的相关研究,有助于进一步理解埃及伊蚊感染DENV2的过程和机制.本研究通过埃及伊蚊和Aag2细胞感染DENV2进行转录组分析,与未染毒的蚊虫和细胞进行对比,筛选出12...     

垂直感染登革病毒对白纹伊蚊滞育卵孵化及F1代成蚊产卵量的影响

本文用低氧水刺激感染登革Ⅱ型病毒的白纹伊蚊滞育卵孵化,按时间顺序共收集孵化的7批幼虫,仅在最先孵化的两批幼虫中检测到病毒,而在此之后孵化的幼虫都没有检测到病毒,推测感染病毒的滞育卵在低氧水的刺激作用下比没有感染病毒的滞育卵更易解除滞育,使得先孵化的蚊虫检测的阳性率较高。感染滞育卵孵化的幼虫最低感染率为1∶131.25(0.76%),明显低于非滞育卵最低感染率1∶82.5(1.21%),即垂直感染登革病毒影响卵的孵化和低龄幼虫的成活。单管饲养法获得经感染滞育卵孵化的F1代单只雌蚊产卵量,被感染的白纹伊蚊平均单只雌蚊产卵量与未感染雌蚊没有显著性差异(P>0.05),即垂直感染病毒对F1代雌蚊产卵量无显著影响。     

Experimental studies on the susceptibility of Aedes vittatus to dengue viruses.

Ae. vittatus mosquitoes were infected by oral route and by intrathoracic inoculation with dengue (DEN) viruses and tested for the presence of dengue virus antigen in their head squashes and salivary glands by indirect immunofluorescence. The results indicate that this species was susceptible to all four types of DEN viruses and supported the growth of DEN-2 virus.     

Vector competence of Aedes hendersoni (Diptera: Culicidae) for La Crosse virus: lack of impaired function in virus-infected salivary glands and enhanced virus transmission by sporozoite-infected mosquitoes.

Previous research has shown Aedes hendersoni Cockerell to be an incompetent vector of La Crosse (LAC) virus because of a salivary gland escape (SGE) barrier; that is, the salivary glands are infected but the mosquito fails to transmit the virus orally. Intradermal probing behavior and ability to locate blood were studied in infected mosquitoes as indicators of salivary gland impairment to determine if the SGE barrier was due to virus-induced pathology of the salivary glands. No evidence of salivary gland impairment as a result of virus infection was detected in infected Ae. hendersoni. This was also true for Aedes triseriatus (Say), a competent vector of LAC virus, which was used as a control. However, coinfection of Ae. hendersoni with Plasmodium gallinaceum and LAC virus dramatically increased virus transmission (72 versus 8%), whereas transmission by coinfected Ae. triseriatus was not significantly affected. Possible causes for the SGE barrier in Ae. hendersoni are discussed.     

Replication and dissemination of La Crosse virus in the competent vector Aedes triseriatus and the incompetent vector Aedes hendersoni and evidence for transovarial transmission by Aedes hendersoni (Diptera: Culicidae)

The time course and pattern of the replication and dissemination of La Crosse virus was studied in orally infected Aedes triseriatus (Say) and Ae. hendersoni Cockerell. Development of La Crosse virus was approximately the same in both species when plaque assay titers of intact mosquitos or dissected tissues were compared. The mosquitoes were equally susceptible to infection; all Ae. hendersoni and 99% of the Ae. triseriatus tested showed detectable midgut infections. Virus was first detected in hemolymph, salivary glands, and ovaries 10-13 d after infection in both species. The pattern of infection suggests virus dissemination beyond the midgut to be via the hemolymph. By 21 d after infection, 100% (10 of 10) of Ae. triseriatus and 70% (7 of 10) of Ae. hendersoni had infected salivary glands, and the geometric mean titer of Ae. hendersoni salivary glands was 10 times higher than the geometric mean titer of those of Ae. triseriatus, However, when tested for transmission 22 d after infection by refeeding on suckling mice, only 9% (2 of 22) of the Ae. hendersoni with disseminated infections transmitted virus versus 71% (12 of 17) of the Ae. triseriatus. A salivary gland escape barrier was shown to be primarily responsible for the failure of Ae. hendersoni to orally transmit La Crosse virus. However, eight parenterally infected Ae. hendersoni females transovarially transmitted the virus to 25% (5 of 20) of their progeny.     

蚊虫抗登革病毒天然免疫效应

埃及伊蚊和白纹伊蚊是传播登革病毒（Dengue viruses）的主要媒介,通过一次感染血餐,登革病毒被蚊摄取,病毒首先感染蚊中肠组织,病毒越过蚊中肠到达蚊其它组织,包括涎腺。当感染病毒的蚊再次叮吸另一健康人血时,将涎腺中病毒传播给下一个人。蚊利用它的天然免疫系统抗登革病毒而使其“不发病”,并能生存传播这种病毒。蚊抗登革病毒天然免疫主要涉及血细胞、Toll、JAK—STAT、RNAi通道等。本文就蚊抗登革病毒天然免疫效应做了综述。     

Detection of dengue virus type 2 in Aedes albopictus by nucleic acid hybridization with strand-specific RNA probes.

A molecular hybridization technique with radiolabeled, strand-specific RNA probes was developed to detect dengue virus type 2 RNA in pools of infected Aedes albopictus mosquitoes. One infected mosquito in a pool of 25 could be detected, corresponding to a dengue virus type 2 titer of 2.75 log10 50% tissue culture infectious doses.     

The effect of temperature on the extrinsic incubation period and infection rate of dengue virus serotype 2 infection in Aedes albopictus

Dengue fever is an acute mosquito-borne viral disease caused by dengue virus (DENV). Temperature may affect the efficiency of the mosquito vectors in spreading DENV. Aedes albopictus mosquitoes were infected orally with a DENV2 suspension and incubated at different temperatures. Subsequently, DENV2 antigen was collected from salivary gland and thorax-abdomen samples on different days postinfection and tested using an immunofluorescence assay to determine the extrinsic incubation period and infection rate. As the temperature increased, the extrinsic DENV2 incubation period in Ae. albopictus gradually shortened, and infection rates showed a tendency to initially increase, followed by a subsequent decrease.     

Bioecology and vectorial capacity of Aedes albopictus (Diptera: Culicidae) in Macao, China, in relation to dengue virus transmission

Until 2001, the Chinese Territory of Macao had not registered any autochthonous dengue cases, despite the abundance of Aedes albopictus (Skuse), a known vector. This work describes a bioecological characterization of the local Ae. albopictus adult population, with the purpose of estimating the receptivity of Macao to dengue introduction. In the wet seasons of 1997 and 1998 and the dry season of 1998, Ae. albopictus was the most abundant human-biting mosquito. Daily biting rates of 314 mosquitoes per person were recorded in the wet season with a reduction to 94 in the dry season. Ae. albopictus was mainly exophagic and exophilic and had a human blood index of 44%. The parity rate of field-collected mosquitoes was 57%. Daily survival rate ranged from 91 to 97%. Estimates of vectorial capacity ranged from 144 to 880, depending on what parameter values were used. These estimates indicated a great receptivity for the introduction of dengue viruses, as the 2001 outbreak came to prove.     

Multiplication and distribution of type 2 dengue and Japanese encephalitis viruses in Toxorhynchites splendens after intrathoracic inoculation

Summary The nonhematophagous mosquitoToxorhynchites (Tx.) splendens was found to be the most susceptible to type 2 dengue (D-2) and Japanese encephalitis (JEV) viruses among three hosts examined by virus titration and replication assays. After inoculation with D-2, the number of viral antigen positive cells in the head, thorax and abdomen increased up to day 15 and D-2 reached the maximum titer of 8.4 log₁₀ PFU/g in the head on day 15. Hemocytes were the earliest cell type that could be detected as D-2 antigen positive on day 2. Multiplication of JEV was faster than that of D-2 in the mosquito. The number of JEV antigen positive cells in each part of the mosquito increased up to day 3, JEV reaching the maximum titer of 8.0 log₁₀ PFU/g in the abdomen on day 3. Hemocytes and fat body cells (FBC) could be detected as JEV antigen positive cells on day 1. The time course of D-2 and JEV infection suggested that intrathoracically inoculated viruses were probably initially phagocytosed by hemocytes and/or FBC, and multiplied primarily in their cytoplasm. The infected hemocytes were then transported by the flow of body fluid and viruses were disseminated to other susceptible organs, such as ganglia, salivary glands, etc. The results obtained indicate that the course of infection of D-2 and JEV inTx. splendens is similar to that in vector mosquitoes.Tx. splendens is therefore very useful for the study of these viruses.     

贵州白纹伊蚊对登革病毒易感性的研究

目的 用细胞、分子生物学技术进行贵州省白纹伊蚊不同地理株对登革病毒(DEN)易感性的研究。方法 采集贵州省9个地(州)市共计15个县(区)白纹伊蚊幼虫标本，饲养为成蚊；取羽化后3～5日龄期的贵州不同地理株白纹伊蚊，用不同型别的DEN分别经口连续感染3d，于首次感染后的4、7、10、14d收集感染成蚊标本；制备蚊悬液，碘化钠法提取RNA，用DENNS1基因区通用引物经逆转录．聚合酶链反应(RT-PCR)检测DEN核酸；蚊悬液接种C6／36细胞进行病毒分离，制作细胞抗原片，经间接免疫荧光法检测DEN抗原；同时感染白纹伊蚊海南株作为对照。结果 DEN1-4型国际参考株感染白纹伊蚊贵州省不同地方株，其感染比率分别为12／15、12／15、8／15和13／15。结论 白纹伊蚊贵州省不同地方株对DEN1-4型国际参考株普遍易感，表明贵州省具备引起登革热流行的条件。