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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Immunology and Infection

Vektor kompetence analyser på Aedes aegypti Myg ved hjælp af Zika Virus

Published: May 31, 2020
doi:
10.3791/61112
,
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

Den præsenterede protokol kan bestemme vektorkompetenceN for Aedes aegypti mygpopulationer for en given virus, såsom zika, i en indeslutningsindstilling.

Abstract

De fremlagte procedurer beskriver en generel metode til at inficere Aedes aegypti myg med zikavirus under laboratorieforhold for at bestemme infektionsraten, formidlet infektion og potentiel overførsel af virus i den pågældende myggepopulation. Disse procedurer er bredt udnyttet med forskellige ændringer i vektor kompetence evalueringer globalt. De er vigtige for at bestemme den potentielle rolle, som en given myg (dvs. arter, population, individ) kan spille i transmissionen af et givet middel.

Introduction

Vektor kompetence er defineret som evnen på niveau med arter, befolkning, og endda en individuel, af en given leddyr såsom en myg, kryds, eller phlebotomine sand flyve, at erhverve og overføre et middel biologisk med replikation eller udvikling i leddyr1,2. Med hensyn til myg og leddyrbårne vira (dvs. arbovirus) er midlet imbibed fra en viremisk vært af en kvindelig myg. Efter indtagelse skal virussen produktivt inficere en af en lille population af midgut epitelceller3, overvinde forskellige fysiologiske forhindringer som proteolytisk nedbrydning af fordøjelsesenzymer, tilstedeværelsen af mikrobiota (midgut infektionsbarriere eller MIB) og den uds sekitære pertrofisk matrix. Infektion af midgut epitel skal efterfølges af replikation af virus og eventuel flugt fra midgut ind i myggens åbne kredsløbssystem, eller hæmolymph, som repræsenterer starten af en formidlet infektion overvinde midgut flugt barriere (MEB). På dette tidspunkt virus kan etablere infektioner i sekundære væv (f.eks nerver, muskler, og fedt organer) og fortsætte med at replikere, selv om en sådan sekundær replikation kan ikke være strengt nødvendigt for virus til at inficere acinare celler i spytkirtlerne (overvinde spytkirtel infektion barriere). Udgang fra spytkirtlen acinar celler i deres apikale hulrum og derefter bevægelse ind i spytkanalen muliggør podning af virus i efterfølgende værter på bidende, og fuldfører transmission cyklus1,2,4,5,6,7.

I betragtning af denne velkarakteriseret og generelt bevarede mekanisme for spredning inden for en myg vektor, laboratorie vektor kompetence vurderinger er ofte metodisk ens, selv om forskelle i protokoller findes1,2. Generelt, efter oral virus eksponering, myg dissekeres, således at individuelle væv såsom midgut, ben, æggestokke, eller spytkirtler kan analyseres for virusinfektion, formidlet infektion, formidlet infektion / potentielle transovarial transmission, og formidles infektion / potentiel transmission kompetence, henholdsvis8. Den blotte tilstedeværelse af en virus i spytkirtlerne er imidlertid ikke endelige beviser for overførselsevne, da der er tegn på en spytkirteludgang / udgangsbarriere (SGEB) i nogle vektor / viruskombinationer1,2,4,5,7,9. Standardmetoden til at bevise transmissionskompetence forbliver myggeoverførsel til et modtageligt dyr10,11,12. Men i betragtning af, at for mange arbovirus dette nødvendiggør brugen af immunkompromitterede murin modeller13,14,15,16, denne metode er ofte omkostnings-uoverkommelige. Et almindeligt anvendt alternativ er indsamlingen af myg spyt, som kan analyseres ved omvendt transskription-polymerase kædereaktion (RT-PCR) eller en smitsom analyse for at påvise tilstedeværelsen af den virale genom eller infektiøse partikler, henholdsvis. Det er værd at bemærke, at sådanne in vitro spyt indsamling metoder kan overvurdere12 eller undervurdere17 mængden af virus deponeret under in vivo fodring, hvilket indikerer, at sådanne data skal fortolkes med forsigtighed. Ikke desto mindre er in vitro-metoden meget værdifuld, når den analyseres ud fra den blotte tilstedeværelse af virus i spyt, hvilket indikerer transmissionspotentiale.

To vigtige tilgange findes til bestemmelse af myg vektorer i arboviral sygdom udbrud. Den første metode involverer feltovervågning, hvor myg indsamles i forbindelse med aktiv transmission18,19,20,21,22,23,24. Men i betragtning af at infektionsraterne typisk er ret lave (f.eks. kan den anslåede infektionsrate på 0,061 % af myg i områder med aktiv zikavirus (ZIKV) i USA21), inkriminering af potentielle vektorarter være stærkt forudindtaget ved at fange metode25,26 og tilfældig tilfældighed (f.eks. prøveudtagning af en inficeret person ud af 1.600 ikke-inficerede)21 . Under hensyntagen til dette kan en given undersøgelse ikke erhverve tilstrækkelige myg i både rå tal eller artsdiversitet til nøjagtigt at prøve myg, der er involveret i transmission. I modsætning hertil foretages vektorkompetenceanalyser i laboratorieindstilling, hvilket giver mulighed for streng kontrol af parametre som oral dosis. Selv om disse laboratorievurderinger ikke fuldt ud er i stand til at repræsentere den sande kompleksitet af myggeinfektion og transmissionskapacitet i en feltindstilling, forbliver de stadig kraftfulde værktøjer inden for arbovirologi.

Baseret på forskellige vektorkompetenceanalyser med ZIKV i flere myggearter, populationer og metoder27,28,29,30,31,32samt en nylig gennemgang af vektorkompetencevurderinger1beskriver vi her flere af protokollerne forbundet med en typisk vektorkompetencearbejdsgang. I disse forsøg blev tre Ae. aegypti-populationer fra Amerika (byen Salvador, Brasilien, Den Dominikanske Republik og den nedre Rio Grande Valley, TX, USA) udsat for en enkelt stamme af ZIKV (Mex 1-7, GenBank Accession: KX247632.1) ved 4, 5 eller 6 log10 fokusdannende enheder (FFU)/mL doser i form af kunstige blodmel. Efterfølgende blev de analyseret for tegn på infektion, formidlet infektion og overførselskompetence efter forskellige tidspunkter med ekstrinsisk inkubation (2, 4, 7, 10 og 14 dage) ved hjælp af dissektion og en cellekulturbaseret infektiøs analyse. Selvom den nuværende arbejdsgang/protokoller er optimeret til ZIKV, kan mange elementer oversættes direkte til andre myggebårne arbovirus i leddyrs indeslutning og biosikkerhedsniveau 2 og 3 (ACL/BSL2 eller ACL/BSL3).

Protocol

Alle procedurer, der udføres i disse protokoller, blev udført i fuld overensstemmelse med protokoller, der blev godkendt af den institutionelle biosikkerhedskomité og den institutionelle dyrepleje- og brugskomité ved University of Texas Medical Branch i Galveston. 1. Forstærk ZIKV i Vero-celler Grow Vero-celler (CCL-81 eller VeroE6) i Dulbeccos modifikation af Eagles minimale essentielle medium (DMEM) suppleret med henholdsvis 10 % v/v varmeinaktiveret fosterkvægsserum (FBS) og…

Representative Results

Tre populationer af Ae. aegypti fra Amerika (Salvador, Brasilien, Den Dominikanske Republik, og Rio Grande Valley, TX, USA) blev udsat for et udbrud stamme af ZIKV fra Amerika (ZIKV Mex 1-7, Chiapas Stat, Mexico, 2015) over en række blodmeal titers (4, 5, og 6 log10 FFU / mL) præsenteret i en vasket menneskelig erythrocyt-baserede kunstige blodmel. På dag 2, 4, 7, 10 og 14 postinfection blev delmængder af myg behandlet for at bestemme infektion, spredning og potentielle transmissionsrater. <p …

Discussion

De metoder, der er beskrevet her, giver en generaliseret arbejdsgang til at udføre vektorkompetenceanalyser. Som en generel ramme bevares mange af disse metoder i hele litteraturen. Der er dog plads til ændringer (gennemgået i Azar og Weaver1). Virus (f.eks. viral afstamning, opbevaring af udfordringsvirus, viral passagehistorie), entomologi (f.eks. laboratoriekolonisering af mygpopulationer, medfødt immunitet, myggemikrobiomet/viromet) og eksperimentelle variabler (f.eks. blodmelssammensætni…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi anerkender personalet i World Reference Center for Emerging Virus and Arboviruses (WRCEVA): Dr. Robert Tesh, Hilda Guzman, Dr. Kenneth Plante, Dr. Jessica Plante, Dionna Scharton og Divya Mirchandani, for deres utrættelige arbejde med kuratering og levering af mange af de virale stammer, der anvendes til vores og andre gruppers vektorkompetence eksperimenter. Det præsenterede arbejde blev finansieret af McLaughlin Fellowship Fund (SRA) og NIH giver AI120942 og 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
DOWNLOAD MATERIALS LIST

References

  1. Azar, S. R., Weaver, S. C. Vector Competence: What Has Zika Virus Taught Us. Viruses. 11 (9), 867 (2019).
  2. Souza-Neto, J. A., Powell, J. R., Bonizzoni, M. Aedes aegypti vector competence studies: A review. Infection, Genetics and Evolution. 67, 191-209 (2019).
  3. 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).
  4. 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).
  5. Kramer, L. D., Ciota, A. T. Dissecting vectorial capacity for mosquito-borne viruses. Current Opinion in Virology. 15, 112-118 (2015).
  6. 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).
  7. 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).
  8. Heitmann, A., et al. Forced Salivation as a Method to Analyze Vector Competence of Mosquitoes. Journal of Visualized Experiments. (138), e57980 (2018).
  9. Beerntsen, B. T., James, A. A., Christensen, B. M. Genetics of Mosquito Vector Competence. Microbiology and Molecular Biology Reviews. 64 (1), 115-137 (2000).
  10. Guo, X. X., et al. Culex pipiens quinquefasciatus: a potential vector to transmit Zika virus. Emerging Microbes & Infections. 5 (9), 102 (2016).
  11. 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).
  12. Smith, D. R., et al. Venezuelan Equine Encephalitis Virus Transmission and Effect on Pathogenesis. Emerging Infectious Diseases. 12 (8), 1190-1196 (2006).
  13. Lazear, H. M., et al. A Mouse Model of Zika Virus Pathogenesis. Cell Host Microbe. 19 (5), 720-730 (2016).
  14. Morrison, T. E., Diamond, M. S. Animal Models of Zika Virus Infection, Pathogenesis, and Immunity. Journal of Virology. 91 (8), 9-17 (2017).
  15. 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).
  16. 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).
  17. 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).
  18. 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).
  19. 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).
  20. 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).
  21. Grubaugh, N. D., et al. Genomic epidemiology reveals multiple introductions of Zika virus into the United States. Nature. 546 (7658), 401-405 (2017).
  22. 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).
  23. 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).
  24. 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).
  25. Brown, H. E., et al. Effectiveness of Mosquito Traps in Measuring Species Abundance and Composition. Journal of Medical Entomology. 45 (3), 517-521 (2008).
  26. 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).
  27. Azar, S. R., et al. ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaques. Viruses. 10 (11), 661 (2018).
  28. 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).
  29. 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).
  30. Hart, C. E., et al. Zika Virus Vector Competency of Mosquitoes, Gulf Coast, United States. Emerging Infectious Diseases. 23 (3), 559-560 (2017).
  31. 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).
  32. Roundy, C. M., et al. Variation in Aedes aegypti Mosquito Competence for Zika Virus Transmission. Emerging Infectious Diseases. 23 (4), 625-632 (2017).
  33. Wilson, A. J., Harrup, L. E. Reproducibility and relevance in insect-arbovirus infection studies. Current Opinion in Insect Science. 28, 105-112 (2018).
  34. Hagan, R. W., et al. Dehydration prompts increased activity and blood feeding by mosquitoes. Scientific Reports. 8 (1), 6804 (2018).
  35. 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).
  36. 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).
  37. 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).
  38. 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).
  39. 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).
  40. Vasilakis, N., et al. Potential of ancestral sylvatic dengue-2 viruses to re-emerge. Virology. 358 (2), 402-412 (2007).

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Here Are The Key Keywords From The Given Text:Vector CompetenceAedes AegyptiZika VirusArbovirusMosquitoesVector Competence AnalysesInfectious DiseasesVectorExperimentsInfected MosquitoesArthropod Containment FacilityBloodViral StockVero CellsInfectious Blood Meal

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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).
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