使用寨卡病毒对 埃及伊 蚊的载体能力分析
1Institute for Human Infections and Immunity,University of Texas Medical Branch, 2Department of Pathology,University of Texas Medical Branch, 3Department of Microbiology and Immunology,University of Texas Medical Branch, 4World Reference Center for Emerging Viruses and Arboviruses,University of Texas Medical Branch
Summary
所提出的方案可以确定 埃及伊蚊 种群在收容环境中对特定病毒（如寨卡病毒）的载体能力。
Abstract
所介绍的程序描述了在实验室条件下用寨卡病毒感染 埃及伊蚊 的通用方法，以确定病毒在相关蚊子种群中的感染率、播散性感染率和潜在传播率。这些程序在全球矢量能力评估中经过各种修改后被广泛使用。它们对于确定特定蚊子（即物种，种群，个体）在给定病原体的传播中可能发挥的潜在作用非常重要。
Introduction
载体能力被定义为在物种，种群甚至个体水平上，给定节肢动物（如蚊子，蜱或放血沙蝇）在生物上获取和传播病原体的能力，并在节肢动物1，2中复制或发育。对于蚊子和节肢动物传播的病毒（即虫媒病毒），该病原体由雌性蚊子从病毒血症宿主体内吸收。摄入后，病毒必须有效地感染一小群中肠上皮细胞3，克服各种生理障碍，如消化酶的蛋白水解降解，微生物群的存在（中肠感染屏障，或MIB）和分泌的营养周基质。中肠上皮感染后必须复制病毒并最终从中肠逃逸到蚊子的开放循环系统或血淋巴中，这代表了克服中肠逃逸屏障（MEB）的播散性感染的发作。此时，病毒可以建立次级组织（例如，神经，肌肉和脂肪体）的感染并继续复制，尽管这种二次复制对于病毒感染唾液腺的腺泡细胞（克服唾液腺感染屏障）可能不是严格必要的。从唾液腺腺泡细胞流出到它们的顶端腔，然后移动到唾液管中，使病毒在叮咬时接种到随后的宿主中，并完成传播周期1，2，4，5，6，7。
鉴于这种表征良好且通常保守的蚊子媒介传播机制，实验室媒介能力评估通常在方法上相似，尽管方案确实存在差异1，2。通常，在口腔病毒暴露后，对蚊子进行解剖，以便可以分别测定中肠，腿部，卵巢或唾液腺等单个组织是否存在病毒感染，播散性感染，播散性感染/潜在经卵巢传播和播散性感染/潜在传播能力8。然而，仅仅唾液腺中存在病毒并不是传播能力的明确证据，因为在某些载体/病毒组合1，2，4，5，7，9中存在唾液腺逃逸/出口屏障（SGEB）。证明传播能力的标准方法仍然是蚊子传播给易感动物10，11，12。然而，鉴于对于许多虫媒病毒，这需要使用免疫功能低下的小鼠模型13，14，15，16，这种方法通常成本过高。一种常用的替代方案是收集蚊子唾液，可以通过逆转录聚合酶链反应（RT-PCR）或感染性测定进行分析，以分别证明病毒基因组或感染性颗粒的存在。值得注意的是，这种体外唾液收集方法可能高估了体内进食过程中沉积的12种或17种病毒的量，表明必须谨慎解释此类数据。尽管如此，当从唾液中仅存在病毒的角度进行分析时，体外方法非常有价值，这表明了传播潜力。
确定蚊媒在虫媒病毒病暴发中的作用有两种主要方法。第一种方法涉及现场监测，其中在主动传播的背景下收集蚊子18，19，20，21，22，23，24。然而，鉴于感染率通常相当低（例如，在美国寨卡病毒（ZIKV）传播活跃地区，蚊子的感染率估计为0.061%21），诱捕方法25、26和随机机会（例如，从1，600名未感染者中抽样一名受感染者中抽样一名）可能严重偏向于潜在病媒物种的犯罪21.考虑到这一点，一项特定的研究可能无法获得足够的蚊子的原始数量或物种多样性，以准确检测参与传播的蚊子。相比之下，载体能力分析在实验室环境中进行，允许严格控制口服剂量等参数。虽然不能完全代表现场环境中蚊子感染和传播能力的真正复杂性，但这些实验室评估仍然是arbovirology领域的有力工具。
基于ZIKV在几种蚊子物种，种群和方法27，28，29，30，31，32中的各种载体能力分析，以及最近对载体能力评估1的回顾，我们在这里描述了与典型载体能力工作流程相关的几种方案。在这些实验中，来自美洲（巴西萨尔瓦多市;多米尼加共和国;和美国德克萨斯州里奥格兰德河谷下游）的三个埃及伊蚊种群通过人工血粉以4，5或6对数10聚焦单位（FFU）/ mL剂量暴露于单一的ZIKV菌株（Mex 1-7，GenBank加入：KX247632.1）。随后，通过解剖和基于细胞培养的感染测定，在不同时间外在孵育（2，4，7，10和14天）后，对他们进行感染，播散性感染和传播能力的证据。尽管目前的工作流程/方案针对ZIKV进行了优化，但许多元素可直接转化为节肢动物收容和生物安全等级2和3（ACL / BSL2或ACL / BSL3）中的其他蚊子传播的虫媒病毒。
Protocol
这些协议中执行的所有程序均完全符合德克萨斯大学加尔维斯顿医学分校机构生物安全委员会和机构动物护理和使用委员会批准的协议。 1. 扩增Vero细胞中的ZIKV 在Dulbecco的Eagle最小必需培养基（DMEM）的修饰中培养Vero细胞（CCL-81或VeroE6），在150cm 2组织培养瓶中补充10%V / v热灭活胎儿牛血清（FBS）和1%（v / v）青霉素- 链霉素（分别为100 U / mL和100μg/ mL）的5%CO2至80-9…
Representative Results
来自美洲（巴西萨尔瓦多;多米尼加共和国;和美国德克萨斯州里奥格兰德河谷）的三个 埃及伊 蚊种群暴露于来自美洲的ZIKV爆发菌株（ZIKV Mex 1-7，恰帕斯州，墨西哥，2015年），超过一系列血粉滴度（4，5和6 log10 FFU / mL），这些滴度在洗涤后基于红细胞的人造血粉中呈递。在感染后第2天、第4天、第7天、第10天和第14天，对蚊子亚群进行处理，以确定感染、传播和潜在传播率。 <p cl…
Discussion
此处描述的方法提供了执行矢量能力分析的通用工作流。作为一个一般框架，其中许多方法在整个文献中都是保守的。但是，有很大的修改空间（在Azar和Weaver1中进行了审查）。已知病毒（例如，病毒谱系、激发病毒的储存、病毒传代史）、昆虫学（例如，蚊子种群的实验室定植、先天免疫、蚊子微生物组/病毒组）和实验变量（例如，血粉组成、顺序喂养和孵育温度）都会影响?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢世界新兴病毒和虫媒病毒参考中心（WRCEVA）的工作人员：Robert Tesh博士，Hilda Guzman博士，Kenneth Plante博士，Jessica Plante博士，Dionna Scharton和Divya Mirchandani，他们在策划和提供许多用于我们和其他团体的载体能力实验的病毒株方面所做的不懈努力。所介绍的工作由麦克劳克林奖学金基金（SRA）资助，NIH资助AI120942和AI121452。
Materials
| 3mL Standard Reservoir | R37P30 | Hemotek Ltd | Insectary Equipment |
| 7/32" Stainless Steel 440 Grade C Balls | 4RJH9 | Grainger | Grinding Media |
| Acetone, Histological Grade, Fisher Chemicals, Poly Bottle, 4L, 4/Case | A16-P4 | FisherScientific | Fixative |
| Adenosine 5'-triphospate disodium salt hydrat, microbial, BioReagent, suitable for cell culture | A6419-1G | MilliporeSigma | Reagent |
| Anti-Flavivirus Group Antigen Antibody, clone D1-4G2-4-15 | MAB10216 | MilliporeSigma | Primary Antibody for focus forming assay |
| Anti-Mouse IgG (H+L) Antibody, Human Serum Adsorbed and Peroxidase-Labeled, 1.0mL/Bottle | 5450-0011 | KPL/Seracare | Secondary Antibody for focus forming assay |
| Bleach | NC0427256 | FisherScientific | Decontamination |
| Corning, Cell Culture Treated Flasks, 150cm2, Vented Cap, Case of 50 | 10-126-34 | FisherScientific | Cell culture consumable |
| Costar Cell Culture Plates, 24-well, 5/bag, 100/case, Corning | 07-200-740 | FisherScientific | Cell culture consumable |
| Costar Cell Culture Plates, 96-well, 5/bag, 100/case, Corning | 07-200-91 | FisherScientific | Cell culture consumable |
| Crystal Violet | C0775-100G | MilliporeSigma | Stain |
| Eppendorf Snap Cap Microcentrifuge Safe-Lock 2mL Tubes, 500/Case | 05-402-7 | FisherScientific | Plastic consumable |
| Falcon 15mL Conical Centrigue Tubes | 14-959-70C | FisherScientific | Plastic consumable |
| Falcon 50mL Conical Centrigue Tubes | 14-959-49A | FisherScientific | Plastic consumable |
| Falcon Disposable Polystyrene Serological 10mL Pipets, 200/Case | 13-675-20 | FisherScientific | Plastic consumable |
| Falcon Disposable Polystyrene Serological 1mL Pipets, 1000/Case | 13-675-15B | FisherScientific | Plastic consumable |
| Falcon Disposable Polystyrene Serological 25mL Pipets, 200/Case | 13-675-30 | FisherScientific | Plastic consumable |
| Falcon Disposable Polystyrene Serological 5mL Pipets, 200/Case | 13-675-22 | FisherScientific | Plastic consumable |
| Falcon Standard Tissue Culture Dishes | 08-772B | FisherScientific | Plastic consumable |
| Fetal Bovine Serum-Premium, 500mL | S11150 | Atlanta Biologicals | Cell culture reagent |
| Fisherbrand Economy Plain Glass Microscope Slides | 12-550-A3 | FisherScientific | Immobilization of Mosquitos |
| FU1 Feeder | FU1-0 | Hemotek Ltd | Insectary Equipment; feeding units |
| Gibco DPBS with Calcium and Magnesium, 10 x 500mL Bottles | 140-040-182 | FisherScientific | Cell culture reagent |
| Gibco Fungizone, Amphotericin B, 250μg/mL, 50mL/Bottle | 15-290-026 | Fisher Scientific | Cell culture reagent |
| Gibco Penicillin-Streptomycin (10,000 U/mL), 100mL/Bottle, 20 Bottles/Case | 15-140-163 | FisherScientific | Cell culture reagent |
| Gibco, Tryptsin-EDTA (.25%), Phenol red, 20 x 100mL Bottles | 25-200-114 | FisherScientific | Cell culture reagent |
| Gibcom DMEM, High Glucose, 10 x 500mL Bottles | 11-965-118 | FisherScientific | Cell culture reagent |
| Human Blood, Unspecified Gender, Na-Citrate, 1 Unit | 7203706 | Lampire | Bloodmeal preparation |
| InsectaVac Aspirator | 2809B | Bioquip | Insectary Equipment |
| Methanol, Certified ACS, Fisher Chemicals, Amber Glass Bottle, 4L, 4/Case | A412-4 | FisherScientific | Fixative |
| Methyl cellulose, viscosity: 3,500-5,600 cP, 2 % in water(20 °C), 250g/Bottle | M0512-250G | MilliporeSigma | Cell culture reagent |
| Micro-chem Plus Disinfectant Detergent | C849T34 | Thomas Scientific | Decontamination; working dilution of dual quaternary ammonium |
| Mineral Oil, BioReagent, for molecular biology | M5904-5X5ML | MilliporeSigma | Immobilization of Mosquitos |
| O-rings | OR37-25 | Hemotek Ltd | Insectary Equipment |
| Plastic Plugs | PP5-250 | Hemotek Ltd | Insectary Equipment |
| PS6 Power Unit (110-120V) | PS6120 | Hemotek Ltd | Insectary Equipment; power source |
| Rubis Forceps, Offset blades, superfine points | 4525 | Bioquip | Insectary Equipment |
| Sarstedt Inc, 2mL Screw Cap Microtube, Conical Bottom, O-ring Cap, Sterile, 1000/Case | 50-809-242 | FisherScientific | Plastic consumable |
| Sucrose, BioUltra, for molecular biology | 84097-250G | MilliporeSigma | Reagent |
| ThermoScientific, ART Barrier Low Retention 1000μL Pipette Tips, 100 tips/Rack, 8 Racks/Pack, 4 Packs/Case | 21-402-487 | FisherScientific | Plastic consumable |
| ThermoScientific, ART Barrier Low Retention 200μL Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case | 21-402-486 | FisherScientific | Plastic consumable |
| ThermoScientific, ART Barrier Low Retention 20μL Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case | 21-402-484 | FisherScientific | Plastic consumable |
| ThermoScientific, ART Barrier Low Retention, Extended Reach 10μL Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case | 21-402-482 | FisherScientific | Plastic consumable |
| TissueLyser II | 85300 | QIAGEN | Homogenization |
| TrueBlue Peroxidase Substrate Kit, 200mL | 5510-0030 | Seracare | Developing solution for focus forming assay |
| Vero | CCL-81 | American Type Culture Collection | Mammalian cell line to amplify virus and conduct infectious assay |
| Vero C1008 [Vero 76, clone E6, Vero E6] | CRL-1586 | American Type Culture Collection | Mammalian cell line to amplify virus and conduct infectious assay |
Riferimenti
- Azar, S. R., Weaver, S. C. Vector Competence: What Has Zika Virus Taught Us. Viruses. 11 (9), 867 (2019).
- Souza-Neto, J. A., Powell, J. R., Bonizzoni, M. Aedes aegypti vector competence studies: A review. Infection, Genetics and Evolution. 67, 191-209 (2019).
- Smith, D. R., Adams, A. P., Kenney, J. L., Wang, E., Weaver, S. C. Venezuelan Equine Encephalitis Virus in the Mosquito Vector Aedes taeniorhynchus: Infection Initiated by a Small Number of Susceptible Epithelial Cells and a Population Bottleneck. Virology. 372 (1), 176-186 (2008).
- Forrester, N. L., Coffey, L. L., Weaver, S. C. Arboviral bottlenecks and challenges to maintaining diversity and fitness during mosquito transmission. Viruses. 6 (10), 3991-4004 (2014).
- Kramer, L. D., Ciota, A. T. Dissecting vectorial capacity for mosquito-borne viruses. Current Opinion in Virology. 15, 112-118 (2015).
- Kramer, L. D., Hardy, J. L., Presser, S. B., Houk, E. J. Dissemination Barriers for Western Equine Encephalomyelitis Virus in Culex tarsalis infected after Ingestion of Low Viral Doses. American Journal of Tropical Medicine and Hygiene. 30 (1), 190-197 (1981).
- Lounibos, L. P., Kramer, L. D. Invasiveness of Aedes aegypti and Aedes albopictus and Vectorial Capacity for Chikungunya Virus. The Journal of Infectious Diseases. 214, 453-458 (2016).
- Heitmann, A., et al. Forced Salivation as a Method to Analyze Vector Competence of Mosquitoes. Journal of Visualized Experiments. (138), e57980 (2018).
- Beerntsen, B. T., James, A. A., Christensen, B. M. Genetics of Mosquito Vector Competence. Microbiology and Molecular Biology Reviews. 64 (1), 115-137 (2000).
- Guo, X. X., et al. Culex pipiens quinquefasciatus: a potential vector to transmit Zika virus. Emerging Microbes & Infections. 5 (9), 102 (2016).
- Secundino, N. F. C., et al. Zika virus transmission to mouse ear by mosquito bite: a laboratory model that replicates the natural transmission process. Parasites & Vectors. 10 (1), 346 (2017).
- Smith, D. R., et al. Venezuelan Equine Encephalitis Virus Transmission and Effect on Pathogenesis. Emerging Infectious Diseases. 12 (8), 1190-1196 (2006).
- Lazear, H. M., et al. A Mouse Model of Zika Virus Pathogenesis. Cell Host Microbe. 19 (5), 720-730 (2016).
- Morrison, T. E., Diamond, M. S. Animal Models of Zika Virus Infection, Pathogenesis, and Immunity. Journal of Virology. 91 (8), 9-17 (2017).
- Reynolds, E. S., Hart, C. E., Hermance, M. E., Brining, D. L., Thangamani, S. An Overview of Animal Models for Arthropod-Borne Viruses. Comparative Medicine. 67 (3), 232-241 (2017).
- Rossi, S. L., et al. Characterization of a Novel Murine Model to Study Zika Virus. American Journal of Tropical Medicine and Hygiene. 94 (6), 1362-1369 (2016).
- Styer, L. M., et al. Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts. PLoS Pathogens. 3 (9), 1262-1270 (2007).
- Azar, S. R., Diaz-Gonzalez, E. E., Danis-Lonzano, R., Fernandez-Salas, I., Weaver, S. C. Naturally infected Aedes aegypti collected during a Zika virus outbreak have viral titres consistent with transmission. Emerging Microbes & Infections. 8 (1), 242-244 (2019).
- Dzul-Manzanilla, F., et al. Evidence of vertical transmission and co-circulation of chikungunya and dengue viruses in field populations of Aedes aegypti (L.) from Guerrero, Mexico. Transactions of the Royal Society of Tropical Medicine and Hygiene. 110 (2), 141-144 (2016).
- Grard, G., et al. Zika virus in Gabon (Central Africa) – 2007: a new threat from Aedes albopictus. PLoS Neglected Tropical Diseases. 8 (2), 2681 (2014).
- Grubaugh, N. D., et al. Genomic epidemiology reveals multiple introductions of Zika virus into the United States. Nature. 546 (7658), 401-405 (2017).
- Guerbois, M., et al. Outbreak of Zika Virus Infection, Chiapas State, Mexico, 2015, and First Confirmed Transmission by Aedes aegyti Mosquitoes in the America. The Journal of Infectious Diseases. 214 (9), 1349-1356 (2016).
- Lundstrom, J. O., et al. Sindbis virus polyarthritis outbreak signalled by virus prevalence in the mosquito vectors. PLoS Neglected Tropical Diseases. 13 (8), 0007702 (2019).
- Miller, B. R., Monath, T. P., Tabachnik, W. J., Ezike, V. I. Epidemic yellow fever caused by an incompetent mosquito vector. Tropical Medicine and Parasitology. 40 (4), 396-399 (1989).
- Brown, H. E., et al. Effectiveness of Mosquito Traps in Measuring Species Abundance and Composition. Journal of Medical Entomology. 45 (3), 517-521 (2008).
- Gorsich, E. E., et al. A comparative assessment of adult mosquito trapping methods to estimate spatial patterns of abundance and community composition in southern Africa. Parasites & Vectors. 12 (1), 462 (2019).
- Azar, S. R., et al. ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaques. Viruses. 10 (11), 661 (2018).
- Azar, S. R., et al. Differential Vector Competency of Aedes albopictus Populations from the Americas for Zika Virus. American Journal of Tropical Medicine and Hygiene. 97 (2), 330-339 (2017).
- Hanley, K. A., Azar, S. R., Campos, R. K., Vasilakis, N., Rossi, S. L. Support for the Transmission-Clearance Trade-Off Hypothesis from a Study of Zika Virus Delivered by Mosquito Bite to Mice. Viruses. 11 (11), 1072 (2019).
- Hart, C. E., et al. Zika Virus Vector Competency of Mosquitoes, Gulf Coast, United States. Emerging Infectious Diseases. 23 (3), 559-560 (2017).
- Karna, A. K., et al. Colonized Sabethes cyaneus, a Sylvatic New World Mosquito Species, Shows a Low Vector Competence for Zika Virus Relative to Aedes aegypti. Viruses. 10 (8), 434 (2018).
- Roundy, C. M., et al. Variation in Aedes aegypti Mosquito Competence for Zika Virus Transmission. Emerging Infectious Diseases. 23 (4), 625-632 (2017).
- Wilson, A. J., Harrup, L. E. Reproducibility and relevance in insect-arbovirus infection studies. Current Opinion in Insect Science. 28, 105-112 (2018).
- Hagan, R. W., et al. Dehydration prompts increased activity and blood feeding by mosquitoes. Scientific Reports. 8 (1), 6804 (2018).
- Guo, X. X., et al. Host Feeding Patterns of Mosquitoes in a Rural Malaria-Endemic Region in Hainan Island, China. Journal of the American Mosquito Control Association. 30 (4), 309-311 (2014).
- Kuno, G. Early history of laboratory breeding of Aedes aegypti (Diptera: Culicidae) focusing on the origins and use of selected strains. Journal of Medical Entomology. 47 (6), 957-971 (2010).
- Mayilsamy, M. Extremely Long Viability of Aedes aegypti (Diptera: Culicidae) Eggs Stored Under Normal Room Condition. Journal of Medical Entomology. 56 (3), 878-880 (2019).
- Althouse, B. M., et al. Potential for Zika Virus to Establish a Sylvatic Transmission Cycle in the Americas. PLoS Neglected Tropical Diseases. 10 (12), 0002055 (2016).
- Vasilakis, N., Cardosa, J., Hanley, K. A., Holmes, E. C., Weaver, S. C. Fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health. Nature Reviews Microbiology. 9 (7), 532-541 (2011).
- Vasilakis, N., et al. Potential of ancestral sylvatic dengue-2 viruses to re-emerge. Virology. 358 (2), 402-412 (2007).
Citazione di questo articolo
Azar, S. R., Weaver, S. C. Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus. J. Vis. Exp. (159), e61112, doi:10.3791/61112 (2020).