白纹伊蚊抗溴氰菊酯品系的建立及其对登革病毒易感性的研究

发布时间：2018-03-30 16:49

本文选题：白纹伊蚊　切入点：抗药性　出处：《南方医科大学》2017年硕士论文

【摘要】：一、研究背景白纹伊蚊是登革热的重要传播媒介,在我国大部分地区广泛分布。登革热近年来在我国频繁暴发流行,由于目前没有成熟的疫苗或药物,对其预防控制主要是针对其传播媒介进行控制,特别是对白纹伊蚊成蚊化学杀虫剂的大量广泛应用成为避免疫情加重和扩散的主要措施。溴氰菊酯为经常使用的杀虫剂。然而在长期的溴氰菊酯筛选压力下,已造成白纹伊蚊对其耐受性的提高,且伴随着一系列生理和生化以及基因方面的变化。国外已有报道,媒介按蚊产生抗药性后,对疟原虫的传播能力受到影响;而白纹伊蚊对溴氰菊酯产生抗性后,对登革病毒的感染性和传播性有无改变,目前尚不清楚。本文在实验室建立了白纹伊蚊对溴氰菊酯的抗性品系,并对其对登革病毒的易感性进行了研究。二、研究目的1.应用溴氰菊酯对白纹伊蚊幼虫进行筛选,建立实验室抗性品系。2.对比白纹伊蚊敏感株和抗性株对登革病毒的易感性差异。三、实验方法1.抗性种群筛选应用世界卫生组织(WHO)推荐的蚊虫抗药性测定方法(幼虫浸溃法)检测白纹伊蚊抗性水平,测试种群的半数致死浓度(lethal concentration 50,LC50)。对1500只左右的四龄幼虫按LC50水平施加溴氰菊酯,存活个体继续饲养至羽化,喂血,产卵。按此方法,对每一代都进行溴氰菊酯加压筛选。成蚊抗性水平按WHO成蚊接触筒药膜滤纸接触法进行测试,采用WHO标准的0.05%溴氰菊酯药膜滤纸。2.C6/36细胞富集2型登革病毒,以106.83 TCID50/mL的病毒浓度经口饲感染抗性株和敏感株白纹伊蚊。3.抗原抗体间接免疫荧光实验检测2型登革病毒在中肠、卵巢、唾液腺的存在,初步检测感染效果。4.感染后0/4/7/10天,进行抗性株和敏感株白纹伊蚊中肠、卵巢、唾液腺的解剖、总RNA提取、2型登革病毒特异片段的逆转、聚合酶链式反应(RT-PCR)扩增、电泳以检测登革病毒是否存在。中肠感染率=阳性中肠样本数/所检测中肠的总样本数,作为病毒感染蚊虫的指标;卵巢感染率=阳性卵巢样本数/所检测卵巢的总样本数,作为病毒在蚊虫体内播散的指标;唾液腺感染率=阳性唾液腺样本数/所检测唾液腺的总样本数,作为蚊虫潜在传播病毒能力的指标。实时荧光定量聚合酶链式反应(RT-qPCR)测定阳性样本中病毒含量。四.实验结果1.经九代筛选之后,白纹伊蚊幼虫的对溴氰菊酯的抗性水平LC50由0.005mg/L 达到了 0.053mg/L,抗性系数(Resistance Ratio,RR)为 10.60,具有了中度抗性(WHO标准)。十三代幼虫LCso为0.052mg/L,RR为10.40,且九到十三代RR均维持在10-11之间。2.第九代成蚊的生测死亡率为80%。十三代成蚊的生测死亡率为67%。3.荧光显微镜下可观察到抗性株和敏感株感染后4/7/10天中肠、7/10天卵巢和唾液腺的绿色荧光。4.抗性株和敏感株中肠、卵巢、唾液腺感染后0/4/7/10天的RT-PCR检测结果抗性株和敏感株0天时,100%中肠可检测到登革病毒,说明成功使蚊虫摄入登革病毒。感染后4d、7d和10d,白纹伊蚊敏感株和抗性株的中肠阳性率在92.75%～97.18%之间,二者在各时间点均无统计学差异(P0.05)。感染后4d,可在卵巢检测到DENV-2,敏感株和抗性株的阳性率分别为36.11%和38.89%。感染后7d和10d,两种蚊虫的卵巢阳性率均较4天时有显著上升(P0.05),感染后7d达到84.7%和77.8%,感染后10d达到84.72%和86.11%,然而各时间点上二者卵巢阳性率之间无明显的统计学差异(P0.05)。蚊虫唾液腺呈阳性的检测时间点为感染后7d,此时敏感株和抗性株的唾液腺阳性率分别为80.56%和83.33%,无显著的统计学差异(P0.05)。感染后10d,两感染率均无明显升高。5.RT-qPCR检测显示,在0天时两蚊株中肠和各个时间点卵巢的病毒含量有统计学差异(中肠 0 天,P=0.005;卵巢 4dpi,P=0.012;7dpi,P=0.001;10dpi P=0.006),在随后的时间里病毒量的上升,卵巢中最为显著,抗性株的病毒含量高于敏感株。两蚊株唾液腺的病毒含量均随时间上升,但并无显著统计学差异。五.研究结论1.溴氰菊酯连续应用于白纹伊蚊幼虫会使其幼虫及成蚊对其抗性水平上升,已建立稳定实验室抗溴氰菊酯的中度抗性实验室品系。2.随感染时间的延长,白纹伊蚊不同组织中登革病毒感染率的增长不同。抗性株和敏感株之间并无统计学差异。3.第0天的抗性蚊株中肠有较高的病毒载量,提示其摄血能力较强。抗性株唾液腺较敏感株对2型登革病毒的易感性无显著改变。抗性株卵巢病毒量高于敏感株,说明抗性株卵巢更利于病毒的增殖。
[Abstract]:First, the research background of Aedes albopictus is an important medium for the spread of dengue fever, widely distributed in most areas of China. Dengue fever in China in recent years the frequent outbreaks, because there is no vaccine or medicine for the prevention and control of mature, mainly for its media control, especially the Aedes albopictus into a large number of widely used mosquito insecticide as main measures to avoid aggravation of epidemic and spread. Deltamethrin for frequently used pesticides. However, long-term selection pressure in deltamethrin, has caused the Aedes albopictus to improve its tolerance, and accompanied by a series of physiological and biochemical changes and genetic aspects. It has been reported that Anopheles resistance, impact on the spread of Plasmodium; and Aedes albopictus of deltamethrin resistance to infection after landing and spread of dengue virus has no change, It is not clear. This paper establishes Aedes albopictus to deltamethrin resistant strains in the laboratory, and the susceptibility to dengue virus on board were studied. Two objective: 1. application of deltamethrin to Aedes albopictus larvae were selected to establish different susceptibility to dengue virus strains of laboratory resistant strains of.2. sensitive and resistant strains of Aedes albopictus. Three experimental methods 1. resistant population screening application of WHO (WHO) method for the determination of resistance to the recommended mosquitoes (larvae dipping method) to detect Aedes albopictus population resistance level, half lethal concentration (lethal test concentration 50, LC50). Of the four instar larvae by only about 1500 the level of LC50 applied to deltamethrin, the survival of individuals continue to raise to eclosion, feeding blood, spawning. According to this method, for each generation of deltamethrin resistance level. Screened mosquito mosquito contact tube membrane filter press WHO The contact method was tested using WHO standard 0.05% deltamethrin membrane filter paper.2.C6/36 cell enrichment of dengue virus type 2 virus, with a concentration of 106.83 TCID50/mL by oral infection resistant and sensitive strains of Aedes albopictus.3. antigen antibody indirect immunofluorescence assay to detect dengue virus type 2 in the midgut, ovary, salivary gland, the initial detection of 0/4/7/10 infection days after.4. infection of resistant strains and sensitive strains of Aedes albopictus midgut, ovary, salivary gland anatomy, total RNA extraction, 2 dengue virus type specific fragment reverse polymerase chain reaction (RT-PCR) amplification, electrophoresis to detect dengue virus exists. The total sample mesenteronal infection rate = positive samples detected by midgut / midgut number as an indicator of mosquito virus infection; the total infection rate of ovarian samples = positive ovarian samples / ovarian detected number, as the virus in the mosquito body spread index; The total sample rate = the salivary gland of checking the number of positive samples of salivary gland / salivary gland infection, as potential mosquito spread the virus ability index. Real time fluorescence quantitative polymerase chain reaction (RT-qPCR) determination of content of virus positive samples. Four. Experimental results of 1. after nine generations of selection, resistance to deltamethrin in white LC50 Aedes albopictus larvae from 0.005mg/L to 0.053mg/L, the resistance coefficient (Resistance Ratio, RR) was 10.60, with moderate resistance (WHO). The thirteen generation larvae of LCso 0.052mg/L, RR is 10.40, and the nine to the thirteen generation of RR were maintained at 10-11.2. between ninth mosquito bioassay mortality was 80%. thirteen the mosquito bioassay for 67%.3. mortality was observed by fluorescent microscopy in sensitive and resistant strains were infected after 4/7/10 days 7/10 days of midgut, ovary and salivary gland green fluorescent.4. resistant strains and sensitive strains of midgut, ovary, salivary gland. The results of 0/4/7/10 days after dyeing of RT-PCR resistant strains and sensitive strains on day 0, 100% were detected by the dengue virus, indicating the success of the mosquito intake of dengue virus. After infection of 4D, 7d and 10d, Aedes albopictus in susceptible and resistant strains were positive rate in 92.75% to 97.18%, two in the at each time point were not statistically significant (P0.05). 4D after infection, can be detected in ovarian DENV-2 susceptible and resistant strains of the positive rates were 36.11% and 38.89%. after infection of 7D and 10d, the positive rate of ovarian two mosquito species were 4 days increased significantly (P0.05), 7d after infection reached 84.7% and 77.8% 10d after infection reached 84.72% and 86.11%, but no statistically significant difference between each time point of the two ovarian positive rate (P0.05). The mosquito salivary gland was positive for the detection time point after 7d infection, the susceptible and resistant strains of the salivary gland positive rate are 80.56% and 83.33%, No statistically significant difference (P0.05). 10d after infection, the infection rate was two and no significant increase of.5.RT-qPCR showed that on the 0 day there was significant difference in two strains of virus mosquito midgut and each time point of the ovary (midgut for 0 days, P=0.005; 7dpi, P=0.012; ovarian 4dpi, P=0.001; 10dpi, P=0.006) increased in the subsequent period of the amount of virus, the most significant in the ovary, the amount of virus resistant strains was higher than that of sensitive strains. Two strains of virus in mosquito salivary gland were increased, but there is no significant difference. Five. Conclusion 1. continuous application of deltamethrin on Aedes albopictus larvae of the larvae and adult mosquitoes the rise of the resistance level, has established a stable Laboratory of deltamethrin resistance in moderate resistant laboratory strain.2. with the infection time, Aedes albopictus in different tissues of dengue virus infection rate of growth. There is no unified different resistant and sensitive strains Meter resistant mosquito strains midgut difference.3. zeroth days have a high viral load, suggesting that the perturbation of blood ability. Resistant strains were sensitive to 2 strains of salivary gland dengue susceptibility to dengue virus had no significant change. The amount of virus resistant ovarian than susceptible strains, resistant strains of the ovary is more conducive to the proliferation of the virus.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R384.1

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