登革Ⅱ型病毒在白纹伊蚊体内分布的研究

利用蚊虫连续石蜡切片免疫组织化学技术,对登革Ⅱ型病毒(DEN-2)感染白纹伊蚊Aedes albopictus后的散播时间、程度及组织器官的感染顺序进行监测,以了解DEN-2在媒介白纹伊蚊体内的分布规律。结果表明：大剂量感染登革Ⅱ型病毒后,在蚊虫消化道的主要部位以及大多数组织器官包括神经及内分泌系统在内,如涎腺、脑、神经节等亦检测到病毒抗原。登革Ⅱ型病毒一旦感染并逸出中肠会迅速侵染其它组织。从各组织感染率的高低推断,病毒逸出中肠后通过血淋巴传播到其它组织的顺序通常为：前肠、涎腺、咽部神经节、脑及食管下神经节、后肠及复眼的小眼等。     

埃及伊蚊浓核病毒(AaeDV)对致倦库蚊的致病性研究

致倦库蚊(Culex quinquefasciatus)是西尼罗病毒(West Nile virus,WNV)、圣路易斯脑炎病毒等重要的媒介。蚊浓核病毒(Mosquito densovirus,MDV)是特异性感染蚊类的病毒,对蚊虫具有明显的致病性。为研究MDV对致倦库蚊的致病效应,我们使用不同浓度埃及伊蚊浓核病毒(Aedes aegypti densovirus,AaeDV)感染致倦库蚊不同发育阶段的幼虫、蛹与成蚊,测定其感染率与死亡率,结果证实AaeDV对致倦库蚊1~2龄幼虫、3~4龄幼虫、蛹和雌雄成蚊的感染率分别为93.33%±2.49%、66%±3.74%、2.67%±0.94%、23.33%±3.40%和24.00%±1.63%,感染率与其发育阶段有关而与性别无关。AaeDV对幼虫的杀伤效果具有剂量依赖性,且与发育阶段有关,在1×1011拷贝/mL浓度下,新孵化龄幼虫和3~4龄幼虫的死亡率高达95.33%±0.74%和61.67%±2.07%。因此AaeDV对致倦库蚊具有生物防制的潜在价值。     

登革Ⅱ型病毒经白纹伊蚊滞育卵的传递

采用C6/36细胞培养分离病毒的方法检测感染登革Ⅱ型病毒的白纹伊蚊Aedes albopictus滞育卵孵化的F₁代蚊虫感染率,从第一个生殖营养周期子代蚊虫中未分离到病毒,第二与第三生殖营养周期子代蚊虫最低感染率没有显著性差异（χ²=0.01,P＞0.0 5）,感染子代的批阳性率为9.1%,最低感染率为1∶330;间接免疫荧光检测结果表明感染登革Ⅱ型病毒的白纹伊蚊滞育卵孵化的子代成蚊能通过叮咬将登革病毒传播给敏感乳鼠。这些研究结果表明登革病毒能在媒介滞育卵内存活并传至子代,子代蚊虫能通过叮咬敏感宿主水平传播病毒。     

我国D2-43病毒株PrM-E基因的复制型载体质粒DNA的免疫原性(英文)

观察含我国登革 2型病毒株 (D2 4 3)的PrM E基因的复制型SFV(semlikiforestvirus)重组质粒DNA的免疫原性 ,为登革新型疫苗的研制提供依据 .将PrM E基因自T载体上切下 ,插入复制型SFV病毒载体质粒DNA中 .将此重组质粒DNA以电穿孔法导入BHK2 1细胞 ,用间接免疫荧光法在感染细胞内可检测到登革 2型病毒特异蛋白的表达 .采用去除内毒素的质粒提取试剂盒制备重组质粒DNA ,然后以不同剂量通过肌肉多点注射途径免疫Balb c鼠 ,获得的鼠血清可与登革D2 4 3感染的C6 36抗原片起特异的抗原抗体反应 .结果表明 ,含登革 2型病毒PrM E基因的复制型SFV病毒载体质粒DNA在Balb c鼠中可诱导登革 2型病毒特异抗体的产生 ,但抗体水平较低 .     

中国尖音库蚊复合组杂交的研究

本文对分布在我国的尖音库蚊复合组的尖音库蚊,致倦库蚊和淡色库蚊进行杂交研究,结果表明致倦库蚊与尖音库蚊及淡色库蚊以及两者尖交可以产生能育的Ｆ１代,它们之间没有传统意义上的生殖隔离,致倦库蚊应为尖音库蚊是的一个亚种,而不是如国外有些学者认为的独立种,另外,杂种Ｆ２代成活率很低,这有可能是尖音库蚊（广义）种下分化在遗传学上的反映。     

致倦库蚊几丁质酶家族基因的鉴定与分析

利用生物信息学方法鉴定致倦库蚊23条几丁质酶基因,并对它们进行命名、分组,以及基因结构、蛋白结构域、蛋白理化特性和系统发育分析,随后使用q PCR方法检测Cq Cht10在致倦库蚊生长各时期的相对表达量。结果表明,致倦库蚊几丁质酶基因长度为1 360~19 218 bp,蛋白长度为353~2 681 aa,分子量为39.557~279.503 k D,等电点为4.76~8.69。它们属于GH18家族,每条几丁质酶包含信号肽区域、催化结构域、几丁质结合域和连接区域,但包含的数量不同。此外,每条几丁质酶的催化结构域均包含保守序列FDGXDLDWEYP。本研究将致倦库蚊几丁质酶划分为12组,组Ⅰ~组Ⅶ沿用早期研究的分组标准,此外将组Ⅷ更名为组Ⅸ,再新增4个分组。Cq Cht10的q PCR实验结果显示,该基因在Ⅱ龄~Ⅳ龄幼虫期呈高表达,在蛹期和成虫期呈低表达,而在卵期和Ⅰ龄幼虫期呈微量表达,此外在雌蚊和雄蚊中无显著差异表达,提示该基因与致倦库蚊的蜕皮相关,且与性别关联不大。本研究的分析结果能够为致倦库蚊几丁质酶基因的功能研究提供有价值的信息。     

2006年广州登革热病原体的分离鉴定及其生物学性质的观察

目的：对2006年广州流行登革热病原进行分离鉴定及生物学性质研究。方法：采用传代蚊细胞微量培养方法对2006年广州登革热病原进行分离,并通过脑内途径观察其对乳鼠的致病性;经间接免疫荧光和RT-PCR技术,对患者血清标本中的病毒特异抗体及新分离的病原体进行检测和鉴定;将此次分离的病原体与1980年分离的同型毒株进行生物学性质比较。结果：从57份患者血清标本中分离出10株病毒,在传代蚊细胞中可产生稳定的细胞病变并对乳鼠致病;其基因组为登革1型病毒特异的RNA分子,经鉴定为登革1型病毒;此次分离的登革1型病毒与1980年分离的同型毒株在致细胞产生病变的时间和严重程度,蚀斑的大小、形态以及致乳鼠发病的时间等生物学性质上有所不同。结论：2006年广州流行登革热病原为登革1型病毒,且与1980年分离的同型毒株在生物学性质方面存在明显差异。     

一株新的致倦库蚊细胞系的建立与鉴定

【目的】以致倦库蚊Culex pipiens quinquefasciatus初孵幼虫组织为材料建立细胞系并对其进行分子鉴定。【方法】将实验室筛选的对硫磷抗性的致倦库蚊深桂品系的初孵幼虫剪碎后放入添加20%胎牛血清的改良M199培养基原代培养,并对原代细胞用胰蛋白酶传代培养成细胞系;通过显微镜观察其生物学特性以及DNA扩增指纹图谱和内转录间隔区序列鉴定其组织来源。【结果】该细胞系Cxq-1在M199完全培养基中生长良好,已成功传代培养12个月,并于液氮中冻存,并多次成功复苏。通过DNA扩增指纹图谱鉴定,Cxq-1与实验室培养的其他昆虫细胞DNA指纹图谱的条带各异,表明确为同源昆虫致倦库蚊细胞系。通过内转录间隔区序列测定技术鉴定,Cxq-1的r DNA-ITS1和r DNA-ITS2序列与Gen Bank中致倦库蚊相应核酸序列的一致性大于99%,表明其来源组织确为致倦库蚊。【结论】新建立的蚊初孵幼虫细胞系Cxq-1确为致倦库蚊细胞系。该细胞系为实验室今后开展杀虫剂抗性分子机制、蚊媒病毒、寄生虫等研究提供了重要平台。     

中国尖音库蚊复合组生物分类学的研究(英文)

本文对我国尖音库蚊复合组(Culex pipiens complex)进行了研究,其中包括从新疆采集尖音库蚊。建立实验室株,进行杂交、不同地理株形态学数值分析、杂种F_1代形态学的数值分析,表皮碳氢化合物、体内脂肪酸和单糖气相色谱分析以及卵蛋白质的电泳分析等。在我国首次发现了骚扰库蚊。根据研究结果,我国异域分布的尖音库蚊复合组实际上包含着同一个种的4个亚种,即指名亚种(Culex pipiens pipiens),淡色库蚊亚种(Culex pipiens pallens)、致倦库蚊亚种(Culex pipiens quinquenfasciatus)和骚扰库蚊亚种(Culex pipiens molestus)。致倦库蚊不应作为独立的蚊种,淡色库坟也不应作为所谓“中间型”。在亲缘关系上根据形态学特征聚类分析等结果表明,尖音库蚊与致倦库蚊的距离较远。     

有机磷抗性致倦库蚊种群中酯酶基因扩增的定量分析

致倦库蚊Culex qinquefasciatus是丝虫病的主要传染媒介。通过生物测定、单个蚊虫酯酶α2和β2基因拷贝数分析和酯酶β基因序列比较, 分析了抗性水平、抗性相关基因在种群中的分布及其基因拷贝数等的抗性分子特征。应用快速PCR仪（realtime quantitatIve PCRs）直接检测库蚊中酯酶基因和mRNA拷贝数。结果显示：上海致倦库蚊对对硫磷的抗性LC₅₀为8.12, 酯酶活性升高是上海致倦库蚊种群对有机磷杀虫药剂产生抗性的主要机理。编码致倦库蚊酯酶β的氨基酸序列同编码尖音库蚊酯酶B1的氨基酸序列相比同源性为98%;同致倦库蚊酯酶B2氨基酸序列相比同源性为100%,同环蹶库蚊酯酶B3氨基酸序列相比同源性为90%, 上海致倦库蚊中酯酶α和β基因均扩增。有机磷抗性的上海和PellRR蚊虫种群中单个蚊虫酯酶α2 和β2定量基因拷贝数均不同,其同一蚊虫个体的酯酶α2 比酯酶β2基因的拷贝数高,但没有明显的规律性,酯酶结构基因的扩增是上海致倦库蚊种群对有机磷杀虫药剂抗性的主要机理,估计在野外种群的杂合个体中存在多种调控机制。     

白纹伊蚊感染登革Ⅱ型病毒后的屏障

以高、低两种浓度病毒液分别经胸接种和经口感染海南株白纹伊蚊Aedes albopictus后,经不同时间内对蚊虫中肠、脑、神经节和涎腺进行IFA染色检查.比较各器官感染率后可知该蚊存在中肠感染屏障(mesenteronal infection barrier MIB)、中肠释放屏障(mesenteronal escape barrier,MEB)和涎腺感染屏障(salivary gland infection barrier,SGIB)并且各屏障率分别与时间及剂量呈负相关.     

竹叶花椒果实精油对两种蚊虫的毒杀活性研究

采用浸液法测试了竹叶花椒果实的水蒸汽蒸馏精油对白纹伊蚊和致倦库蚊幼虫的毒杀效果,并用三角瓶密闭熏蒸法研究了精油对这两种成蚊的熏杀活性;此外,采用GC-MS分析了该精油的化学成分。研究结果:(1)精油对白纹伊蚊和致倦库蚊的Ⅰ、Ⅱ、Ⅲ、Ⅳ龄期幼虫及蛹的LC50值分别为25.634/61.472、31.763/76.431、52.356/110.172、258.497/121.884和198.263/162.048mg.L-1;(2)精油对白纹伊蚊和致倦库蚊成蚊的LC50值分别为24.957和29.517μg.cm-3;(3)在147.52μg.cm-3熏杀剂量下,精油对白纹伊蚊和致倦库蚊成蚊的KT50值分别为3.493和2.993min,24h致死率均为100%;(4)共鉴定出18种化合物,其中萜烯类10种,占精油总量的67.122%,为竹叶花椒果实精油的主要成分。竹叶花椒果实精油对白纹伊蚊和致倦库蚊均有明显的致死作用,且作用速度快,具有开发为植物源杀蚊剂的潜力。     

几种蚊虫线粒体DNA-16SrRNA序列及其相互关系的研究

测定我国尖音库蚊复合组4亚种（尖音库蚊、淡色库蚊、致倦库蚊和骚扰库蚊）、三带喙库蚊、白纹伊蚊和中华按蚊的线粒体DNA 16S rRNA基因（mtDNA-16S rRNA)序列,发现我国尖音库蚊复合组4亚种mtDNA-16S rRNA序列基本一致,长度为555bp(554bp),GC含量为25．41％。该序列与其他3种蚊虫在种间存在差异,与三带喙库蚊的种间差异为0．54％;与白纹伊蚊的种间差异为5．77％;与中华按蚊的种间差异为9．62％。分子系统关系分析表明该序列与传统分类系统的同属或同亚科种类近似性相一致。     

Susceptibility of mosquitoes in central Taiwan to natural infections of Dirofilaria immitis

From October 1997 to September 1998, 3085 Culex quinquefasciatus (Say) (Diptera: Culicidae), 584 Cx. tritaeniorhynchus (Giles) (Diptera: Culicidae), 392 Cx. annulus (Theobald) (Diptera: Culicidae), 374 Aedes albopictus (Skuse) (Diptera: Culicidae) and 102 Armigeres subalbatus (Coquillet) (Diptera: Culicidae) were collected and examined for Dirofilaria immitis (Leidy) (Spirurida: Filariidae) infection. However, only Cx. quinquefasciatus and Ae. albopictus were infected, with a prevalence of 4.28% and 3.74%, respectively. The intensity of D. immitis found in Ae. albopictus (3.43 larvae/mosquito) was higher than that found in Cx. quinquefasciatus (2.89 larvae/mosquito). After being fed with canine blood containing 7500 microfilariae (mf) per mL, Cx. quinquefasciatus ingested approximately two times as many mf as Ae. albopictus (mean of 31.73 in comparison to 16.47). However, almost three times as many third-stage infective larvae developed in Ae. albopictus as in Cx. quinquefasciatus (mean of 3.25 as compared with 1.10), with a vector efficiency index (VEI) of 19.73 and 3.47, respectively. The results showed that Cx. quinquefasciatus and Ae. albopictus served as natural vectors of D. immitis in central Taiwan. Although Ae. albopictus was more efficient for heartworm transmission, Cx. quinquefasciatus may play a more prominent role on the transmission of dirofilariasis in central Taiwan.     

小蓬草精油对两种蚊虫的毒杀活性和成分分析

【目的】明确小蓬草Conyza canadensis ( L.) Cronq.精油的杀虫潜力及其活性成分。【方法】通过浸虫法和密闭熏蒸法测试了小蓬草精油对白纹伊蚊Aedes albopictus和致倦库蚊Culex pipiens quinquefasciatus幼虫及成蚊的毒杀活性, 并利用气相色谱 质谱联用仪（GC-MS）对精油的挥发性成分进行了定性分析。【结果】 小蓬草精油对白纹伊蚊1-4龄期幼虫及蛹的24 h LC50值分别为25.01, 45.88, 56.94, 64.60和346.23 μg/mL; 对致倦库蚊幼虫1-4龄期幼虫及蛹的24 h LC₅₀值分别为9.16, 8.65, 32.12, 43.68和197.83 μg/mL。在剂量分别为48, 64, 80, 96, 112和128 μg/cm3时, 小蓬草精油对白纹伊蚊成蚊的KT₅₀值分别为28.81, 22.31, 20.38, 17.05, 13.92和9.74 min; 对致倦库蚊的KT50值分别为34.90, 32.97, 23.97, 19.60, 15.20和10.34 min。 24 h熏蒸对白纹伊蚊和致倦库蚊成蚊的LC50值分别是75.46和99.19 μg/cm³。小蓬草精油的GC MS定性分析共分离鉴定出31种化合物, 其中单萜类物质6种, 倍半萜烯类物质17种, 含氧萜烯类6种。【结论】结果表明小蓬草精油对这两种蚊虫的毒杀活性较高, 具有深开发潜力。     

First report of the oriental mosquito Aedes albopictus on the West African island of Bioko,Equatorial Guinea

The invasive oriental mosquito Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) was detected on Bioko Island for the first time in November 2001. It was found to be well established breeding in artificial containers at Planta, near Malabo, the capital of Equatorial Guinea. Associated species of mosquito larvae were Aedes aegypti (L.), Ae. africanus (Theobald), Culex near decens Theobald, Cx. duttoni Theobald, Cx. quinquefasciatus Say, Cx. tigripes De Grandpré & De Charmoy, Eretmapodites quinquevittatus Theobald and Mansonia africana (Theobald). This is the third tropical African country to be invaded by Ae. albopictus, which has recently spread to many parts of the Americas and Europe--with vector competence for dengue, yellow fever and other arboviruses. In the Afrotropical environment, it will be interesting to monitor the ecological balance and/or displacement between introduced Ae. albopictus and indigenous Ae. aegpyti (domestic, peri-domestic and sylvatic populations).     

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections.     

Growth characteristics of the veterinary vaccine candidate ChimeriVax-West Nile (WN) virus in Aedes and Culex mosquitoes

In 1999 West Nile (WN) virus was introduced to North America where this flavivirus has spread rapidly among wildlife (especially birds) transmitted by various species of mosquitoes (Diptera: Culicidae). Increasing numbers of cases and deaths among humans, horses and other domestic animals require development of effective vaccines. 'ChimeriVax-West Nile(vet)' is being developed for use as a veterinary vaccine to protect against WN infection. This chimeric virus contains the pre-membrane (prM) and envelope (E) genes from the wild-type WN NY99 virus (isolated from a flamingo in New York zoo during the 1999 WN epidemic) in the backbone of yellow fever (YF) 17D vaccine virus. Replication kinetics of ChimeriVax-WN(vet) virus were evaluated in mosquito cell culture (Aedes albopictus C6/36), in WN vector mosquitoes [Culex tritaeniorhynchus Giles, Cx. nigripalpus Theobald and Cx. quinquefasciatus Say (Diptera: Culicidae)] and in YF vectors [Aedes aegypti (L) and Ae. albopictus (Skuse)], to determine whether these mosquitoes become infected through feeding on a viraemic vaccine, and their potential infectivity to transmit the virus. Growth of ChimeriVax-WN(vet) virus was found to be restricted in mosquitoes, compared to WN virus in Ae. albopictus C6/36 cells. When inoculated intrathoracically, ChimeriVax-WN(vet) and YF 17D viruses did not replicate in Cx. tritaeniorhynchus or Cx. nigripalpus; replication was very restricted compared to the wild-type WN virus in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. When fed on hanging drops with ChimeriVax-WN(vet) virus (7.7 log10 PFU/mL), none of the Culex mosquitoes became infected; one Ae. albopictus and 10% of the Ae. aegypti became infected, but the titre was very low and virus did not disseminate to head tissue. ChimeriVax-WN(vet) virus had a replication profile similar to that of the attenuated vaccine virus YF 17D, which is not transmitted by mosquitoes. These results suggest that the natural mosquito vectors of WN and YF viruses, which may incidentally take a bloodmeal from a vaccinated host, will not become infected with ChimeriVax-WN(vet) virus.     

椿叶花椒果实精油对两种蚊虫的生物活性及成分分析

为筛选环保型的植物源灭蚊活性物质, 研究了椿叶花椒 Zanthoxylum ailanthoides 果实精油对白纹伊蚊Aedes albopictus和致倦库蚊Culex pipiens quinquefasciatus成蚊的熏杀活性, 并测试了精油对这两种蚊幼虫的毒杀效果。此外, 采用GC-MS分析了该精油的化学成分。 结果表明：在102.145 μg·cm^-3熏杀剂量下, 精油对白纹伊蚊和致倦库蚊成蚊的KT50值分别为2.788和3.505 min, 24 h致死率分别为100%和97.53%。该精油对白纹伊蚊和致倦库蚊4龄幼虫的LC50值分别为45.237和20.759 mg·L^-1。从椿叶花椒果实精油中共鉴定出14种化合物, 其中2-十一酮为主要成分, 相对含量为89.86%。结果说明椿叶花椒果实精油对两种蚊虫均有明显的杀虫活性, 具有开发成植物源灭蚊剂的潜力。