Name: vector competence analyses on aedes aegypti mosquitoes using zika virus
Uploaded: 2020-05-31T14:00:00
Description: Watch this Scientific Journal Video about Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus at JoVE.com

Vi bekräftar personalen vid World Reference Center for Emerging Viruses and Arboviruses (WRCEVA): Dr. Robert Tesh, Hilda Guzman, Dr. Kenneth Plante, Dr. Jessica Plante, Dionna Scharton och Divya Mirchandani, för deras outtröttliga arbete med att kurera och tillhandahålla många av de virusstammar som används för våra och andra gruppers vektorkompetensexperiment. Det presenterade arbetet finansierades av McLaughlin Fellowship Fund (SRA) och NIH-bidrag ai120942 och AI121452.

Alla procedurer som utfördes i dessa protokoll utfördes i full överensstämmelse med protokoll som godkänts av Institutional Biosafety Committee och Institutional Animal Care and Use Committee vid University of Texas Medical Branch i Galveston.
1. Förstärk ZIKV i Vero-celler
<ol><li>Odla Vero-celler (CCL-81 eller VeroE6) i Dulbeccos modifiering av Eagles minimala essentiella medium (DMEM) kompletterat med 10% v/v värmeinaktiverat fetalt bovinserum (FBS), och 1% (v/v) penicillin-strept...

<ol>
	<li>Azar, S. R., Weaver, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vector+Competence:+What+Has+Zika+Virus+Taught+Us.">Vector Competence: What Has Zika Virus Taught Us.</a> Viruses. 11 (9), 867 (2019).</li><li>Souza-Neto, J. A., Powell, J. R., Bonizzoni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aedes+aegypti+vector+competence+studies:+A+review.">Aedes aegypti vector competence studies: A review.</a> Infection, Genetics and Evolution. 67, 191-209 (2019).</li><li>Smith, D. R., Adams, A. P., Kenney, J. L., Wang, E., Weaver, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venezuelan+Equine+Encephalitis+Virus+in+the+Mosquito+Vector+Aedes+taeniorhynchus:+Infection+Initiated+by+a+Small+Number+of+Susceptible+Epithelial+Cells+and+a+Population+Bottleneck.">Venezuelan Equine Encephalitis Virus in the Mosquito Vector Aedes taeniorhynchus: Infection Initiated by a Small Number of Susceptible Epithelial Cells and a Population Bottleneck.</a> Virology. 372 (1), 176-186 (2008).</li><li>Forrester, N. L., Coffey, L. L., Weaver, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arboviral+bottlenecks+and+challenges+to+maintaining+diversity+and+fitness+during+mosquito+transmission.">Arboviral bottlenecks and challenges to maintaining diversity and fitness during mosquito transmission.</a> Viruses. 6 (10), 3991-4004 (2014).</li><li>Kramer, L. D., Ciota, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+vectorial+capacity+for+mosquito-borne+viruses.">Dissecting vectorial capacity for mosquito-borne viruses.</a> Current Opinion in Virology. 15, 112-118 (2015).</li><li>Kramer, L. D., Hardy, J. L., Presser, S. B., Houk, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+Barriers+for+Western+Equine+Encephalomyelitis+Virus+in+Culex+tarsalis+infected+after+Ingestion+of+Low+Viral+Doses.">Dissemination Barriers for Western Equine Encephalomyelitis Virus in Culex tarsalis infected after Ingestion of Low Viral Doses.</a> American Journal of Tropical Medicine and Hygiene. 30 (1), 190-197 (1981).</li><li>Lounibos, L. P., Kramer, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasiveness+of+Aedes+aegypti+and+Aedes+albopictus+and+Vectorial+Capacity+for+Chikungunya+Virus.">Invasiveness of Aedes aegypti and Aedes albopictus and Vectorial Capacity for Chikungunya Virus.</a> The Journal of Infectious Diseases. 214, 453-458 (2016).</li><li>Heitmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+Salivation+as+a+Method+to+Analyze+Vector+Competence+of+Mosquitoes.">Forced Salivation as a Method to Analyze Vector Competence of Mosquitoes.</a> Journal of Visualized Experiments. (138), e57980 (2018).</li><li>Beerntsen, B. T., James, A. A., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+Mosquito+Vector+Competence.">Genetics of Mosquito Vector Competence.</a> Microbiology and Molecular Biology Reviews. 64 (1), 115-137 (2000).</li><li>Guo, X. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culex+pipiens+quinquefasciatus:+a+potential+vector+to+transmit+Zika+virus.">Culex pipiens quinquefasciatus: a potential vector to transmit Zika virus.</a> Emerging Microbes &amp; Infections. 5 (9), 102 (2016).</li><li>Secundino, N. F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+transmission+to+mouse+ear+by+mosquito+bite:+a+laboratory+model+that+replicates+the+natural+transmission+process.">Zika virus transmission to mouse ear by mosquito bite: a laboratory model that replicates the natural transmission process.</a> Parasites &amp; Vectors. 10 (1), 346 (2017).</li><li>Smith, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venezuelan+Equine+Encephalitis+Virus+Transmission+and+Effect+on+Pathogenesis.">Venezuelan Equine Encephalitis Virus Transmission and Effect on Pathogenesis.</a> Emerging Infectious Diseases. 12 (8), 1190-1196 (2006).</li><li>Lazear, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+of+Zika+Virus+Pathogenesis.">A Mouse Model of Zika Virus Pathogenesis.</a> Cell Host Microbe. 19 (5), 720-730 (2016).</li><li>Morrison, T. E., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Zika+Virus+Infection,+Pathogenesis,+and+Immunity.">Animal Models of Zika Virus Infection, Pathogenesis, and Immunity.</a> Journal of Virology. 91 (8), 9-17 (2017).</li><li>Reynolds, E. S., Hart, C. E., Hermance, M. E., Brining, D. L., Thangamani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Overview+of+Animal+Models+for+Arthropod-Borne+Viruses.">An Overview of Animal Models for Arthropod-Borne Viruses.</a> Comparative Medicine. 67 (3), 232-241 (2017).</li><li>Rossi, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Novel+Murine+Model+to+Study+Zika+Virus.">Characterization of a Novel Murine Model to Study Zika Virus.</a> American Journal of Tropical Medicine and Hygiene. 94 (6), 1362-1369 (2016).</li><li>Styer, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquitoes+inoculate+high+doses+of+West+Nile+virus+as+they+probe+and+feed+on+live+hosts.">Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts.</a> PLoS Pathogens. 3 (9), 1262-1270 (2007).</li><li>Azar, S. R., Diaz-Gonzalez, E. E., Danis-Lonzano, R., Fernandez-Salas, I., Weaver, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+infected+Aedes+aegypti+collected+during+a+Zika+virus+outbreak+have+viral+titres+consistent+with+transmission.">Naturally infected Aedes aegypti collected during a Zika virus outbreak have viral titres consistent with transmission.</a> Emerging Microbes &amp; Infections. 8 (1), 242-244 (2019).</li><li>Dzul-Manzanilla, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+vertical+transmission+and+co-circulation+of+chikungunya+and+dengue+viruses+in+field+populations+of+Aedes+aegypti+(L.)+from+Guerrero,+Mexico.">Evidence of vertical transmission and co-circulation of chikungunya and dengue viruses in field populations of Aedes aegypti (L.) from Guerrero, Mexico.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 110 (2), 141-144 (2016).</li><li>Grard, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+in+Gabon+(Central+Africa)+&#8211;+2007:+a+new+threat+from+Aedes+albopictus.">Zika virus in Gabon (Central Africa) &#8211; 2007: a new threat from Aedes albopictus.</a> PLoS Neglected Tropical Diseases. 8 (2), 2681 (2014).</li><li>Grubaugh, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+epidemiology+reveals+multiple+introductions+of+Zika+virus+into+the+United+States.">Genomic epidemiology reveals multiple introductions of Zika virus into the United States.</a> Nature. 546 (7658), 401-405 (2017).</li><li>Guerbois, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outbreak+of+Zika+Virus+Infection,+Chiapas+State,+Mexico,+2015,+and+First+Confirmed+Transmission+by+Aedes+aegyti+Mosquitoes+in+the+America.">Outbreak of Zika Virus Infection, Chiapas State, Mexico, 2015, and First Confirmed Transmission by Aedes aegyti Mosquitoes in the America.</a> The Journal of Infectious Diseases. 214 (9), 1349-1356 (2016).</li><li>Lundstrom, J. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sindbis+virus+polyarthritis+outbreak+signalled+by+virus+prevalence+in+the+mosquito+vectors.">Sindbis virus polyarthritis outbreak signalled by virus prevalence in the mosquito vectors.</a> PLoS Neglected Tropical Diseases. 13 (8), 0007702 (2019).</li><li>Miller, B. R., Monath, T. P., Tabachnik, W. J., Ezike, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemic+yellow+fever+caused+by+an+incompetent+mosquito+vector.">Epidemic yellow fever caused by an incompetent mosquito vector.</a> Tropical Medicine and Parasitology. 40 (4), 396-399 (1989).</li><li>Brown, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+Mosquito+Traps+in+Measuring+Species+Abundance+and+Composition.">Effectiveness of Mosquito Traps in Measuring Species Abundance and Composition.</a> Journal of Medical Entomology. 45 (3), 517-521 (2008).</li><li>Gorsich, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+assessment+of+adult+mosquito+trapping+methods+to+estimate+spatial+patterns+of+abundance+and+community+composition+in+southern+Africa.">A comparative assessment of adult mosquito trapping methods to estimate spatial patterns of abundance and community composition in southern Africa.</a> Parasites &amp; Vectors. 12 (1), 462 (2019).</li><li>Azar, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZIKV+Demonstrates+Minimal+Pathologic+Effects+and+Mosquito+Infectivity+in+Viremic+Cynomolgus+Macaques.">ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaques.</a> Viruses. 10 (11), 661 (2018).</li><li>Azar, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Vector+Competency+of+Aedes+albopictus+Populations+from+the+Americas+for+Zika+Virus.">Differential Vector Competency of Aedes albopictus Populations from the Americas for Zika Virus.</a> American Journal of Tropical Medicine and Hygiene. 97 (2), 330-339 (2017).</li><li>Hanley, K. A., Azar, S. R., Campos, R. K., Vasilakis, N., Rossi, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Support+for+the+Transmission-Clearance+Trade-Off+Hypothesis+from+a+Study+of+Zika+Virus+Delivered+by+Mosquito+Bite+to+Mice.">Support for the Transmission-Clearance Trade-Off Hypothesis from a Study of Zika Virus Delivered by Mosquito Bite to Mice.</a> Viruses. 11 (11), 1072 (2019).</li><li>Hart, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Vector+Competency+of+Mosquitoes,+Gulf+Coast,+United+States.">Zika Virus Vector Competency of Mosquitoes, Gulf Coast, United States.</a> Emerging Infectious Diseases. 23 (3), 559-560 (2017).</li><li>Karna, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonized+Sabethes+cyaneus,+a+Sylvatic+New+World+Mosquito+Species,+Shows+a+Low+Vector+Competence+for+Zika+Virus+Relative+to+Aedes+aegypti.">Colonized Sabethes cyaneus, a Sylvatic New World Mosquito Species, Shows a Low Vector Competence for Zika Virus Relative to Aedes aegypti.</a> Viruses. 10 (8), 434 (2018).</li><li>Roundy, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+Aedes+aegypti+Mosquito+Competence+for+Zika+Virus+Transmission.">Variation in Aedes aegypti Mosquito Competence for Zika Virus Transmission.</a> Emerging Infectious Diseases. 23 (4), 625-632 (2017).</li><li>Wilson, A. J., Harrup, L. 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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+Feeding+Patterns+of+Mosquitoes+in+a+Rural+Malaria-Endemic+Region+in+Hainan+Island,+China.">Host Feeding Patterns of Mosquitoes in a Rural Malaria-Endemic Region in Hainan Island, China.</a> Journal of the American Mosquito Control Association. 30 (4), 309-311 (2014).</li><li>Kuno, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+history+of+laboratory+breeding+of+Aedes+aegypti+(Diptera:+Culicidae)+focusing+on+the+origins+and+use+of+selected+strains.">Early history of laboratory breeding of Aedes aegypti (Diptera: Culicidae) focusing on the origins and use of selected strains.</a> Journal of Medical Entomology. 47 (6), 957-971 (2010).</li><li>Mayilsamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extremely+Long+Viability+of+Aedes+aegypti+(Diptera:+Culicidae)+Eggs+Stored+Under+Normal+Room+Condition.">Extremely Long Viability of Aedes aegypti (Diptera: Culicidae) Eggs Stored Under Normal Room Condition.</a> Journal of Medical Entomology. 56 (3), 878-880 (2019).</li><li>Althouse, B. 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Metoderna som beskrivs här ger ett generaliserat arbetsflöde för att genomföra vektorkompetensanalyser. Som en allmän ram bevaras många av dessa metoder i hela litteraturen. Det finns dock stort utrymme för ändringar (granskas i Azar och Weaver1). Virus (t.ex. virus härstamning, lagring av utmaningsvirus, viral passagehistoria), entomologi (t.ex. laboratoriekolonisering av myggpopulationer, medfödd immunitet, myggmikrobiom/virom) och experimentella variabler (t.ex. blodmjölssammansättn...

Vektorkompetens definieras som förmågan på art-, populations- och till och med en individs nivå hos en viss leddjur som mygga, fästing eller phlebotomine sandfluga, att förvärva och överföra ett medel biologiskt med replikering eller utveckling i leddjuret1,2. När det gäller myggor och leddjurburna virus (dvs. arbovirus) är medlet insuperat från en viremic värd av en kvinnlig mygga. Efter intag måste viruset produktivt infektera en av en liten population av midgut epitelceller3, övervinna olika fysiologiska hinder som proteolytisk nedbrytning av matsmältningsenzymer, närvaron av mikrobiota (midgut infektionsbarriären eller MIB) och den utsöndrade peritrofiska matrisen. Infektion av midgut epitel måste följas av replikering av viruset och eventuell flykt från midguten till myggans öppna cirkulationssystem, eller hemolymph, som representerar uppkomsten av en spridad infektion övervinna midgut escape barrier (MEB). Vid denna tidpunkt kan viruset fastställa infektioner i sekundära vävnader (t.ex. nerver, muskler och fettkroppar) och fortsätta att replikera, även om sådan sekundär replikering kanske inte är absolut nödvändig för att viruset ska infektera salivkörtlarnas acinära celler (övervinna salivkörtelinfektionsbarriären). Utgående från salivkörtelacincellerna i deras apikala håligheter och sedan rörelse i salivkanalen möjliggör inokulering av viruset till efterföljande värdar vid bitning och slutför överföringscykeln1,2,4,5,6,7.
Med tanke på denna väl karakteriserade och allmänt bevarade spridningsmekanism inom en myggvektor är laboratorievektorkompetensbedömningar ofta metodologiskt lika, även om skillnader i protokoll finns1,2. I allmänhet, efter oral virusexponering, dissekeras myggor så att enskilda vävnader som midgut, ben, äggstockar eller salivkörtlar kan analyseras för virusinfektion, spridad infektion, spridad infektion / potentiell transovariell överföring och spridad infektion / potentiell överföringskompetens, respektive8. Blotta förekomsten av ett virus i salivkörtlarna är dock inte definitiva bevis på överföringsförmåga, givet bevis på en salivary gland escape /egress barrier (SGEB) i vissa vektor/ viruskombinationer1,2,4,5,7,9. Standardmetoden för att bevisa överföringskompetens förblir myggöverföring till ett mottagligt djur10,11,12. Men med tanke på att för många arbovirus kräver detta användning av immunkomprometterade murinmodeller13,14,15,16, är denna metod ofta kostnadsöverkomlig. Ett vanligt alternativ är insamlingen av myggsaliv, som kan analyseras genom omvänd transkription-polymeraskedjereaktion (RT-PCR) eller en infektiös analys för att visa närvaron av virusgenomet respektive infektiösa partiklar. Det är värt att notera att sådana in vitro-metoder för insamling av saliv kan överskatta12 eller underskatta17 mängden virus som deponeras under in vivo-utfodring, vilket indikerar att sådana uppgifter måste tolkas med försiktighet. In vitro-metoden är dock mycket värdefull när den analyseras ur perspektivet av blotta närvaron av virus i saliven, vilket indikerar överföringspotential.
Två viktiga metoder finns för att bestämma myggvektorernas roll vid utbrott av arboviralsjukdomar. Den första metoden innebär fältövervakning, där myggor samlas in i samband med aktiv överföring18,19,20,21,22,23,24. Med tanke på att infektionsfrekvensen vanligtvis är ganska låg (t.ex. den uppskattade 0,061% infektionshastigheten för myggor i områden med aktiv Zika-virus (ZIKV) cirkulation i USA21), kan inskrivning av potentiella vektorarter vara kraftigt partisk genom fångstmetod25,26 och slumpmässig slump (t.ex. provtagning av en infekterad individ av 1 600 oinfekterade)21 . Med hänsyn till detta får en viss studie inte förvärva tillräckligt med myggor i både råantal eller artmångfald för att noggrant prova myggor som är involverade i överföring. Däremot görs vektorkompetensanalyser i laboratoriemiljö, vilket möjliggör strikt kontroll av parametrar som oral dos. Även om de inte är fullt kapabla att representera den verkliga komplexiteten hos mygginfektion och överföringskapacitet i en fältmiljö, förblir dessa laboratoriebedömningar kraftfulla verktyg inom arbovirologi.
Baserat på olika vektorkompetensanalyser med ZIKV i flera myggarter, populationer och metoder27,28,29,30,31,32, samt en nyligen genomgång av vektorkompetensbedömningar1, beskriver vi här flera av protokollen i samband med ett typiskt vektorkompetensarbetsflöde. I dessa experiment utsattes tre Ae. aegypti populationer från Amerika (staden Salvador, Brasilien; Dominikanska republiken; och den nedre Rio Grande Valley, TX, USA) för en enda stam av ZIKV (Mex 1-7, GenBank Accession: KX247632.1) vid 4, 5 eller 6 log10 fokusbildande enheter (FFU) / ml doser genom konstgjorda blodmjöl. Därefter analyserades de för bevis på infektion, spridas infektion och överföring kompetens efter olika tider av extrinsic inkubation (2, 4, 7, 10 och 14 dagar) med hjälp av dissekering och en cell kultur-baserade infektiös analys. Även om det nuvarande arbetsflödet/protokollen är optimerade för ZIKV, kan många element direkt översättas till andra myggburna arbovirus i leddjursinneslutning och biosäkerhetsnivåer 2 och 3 (ACL/BSL2 eller ACL/BSL3).

<table>
	<tbody>
		<tr>
			<td>3mL Standard Reservoir</td>
			<td>R37P30</td>
			<td>Hemotek Ltd</td>
			<td>Insectary Equipment</td>
		</tr>
		<tr>
			<td>7/32&#34; Stainless Steel 440 Grade C Balls</td>
			<td>4RJH9</td>
			<td>Grainger</td>
			<td>Grinding Media</td>
		</tr>
		<tr>
			<td>Acetone, Histological Grade, Fisher Chemicals, Poly Bottle, 4L, 4/Case</td>
			<td>A16-P4</td>
			<td>FisherScientific</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphospate disodium salt hydrat, microbial, BioReagent, suitable for cell culture</td>
			<td>A6419-1G</td>
			<td>MilliporeSigma</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Anti-Flavivirus Group Antigen Antibody, clone D1-4G2-4-15</td>
			<td>MAB10216</td>
			<td>MilliporeSigma</td>
			<td>Primary Antibody for focus forming assay</td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG (H+L) Antibody, Human Serum Adsorbed and Peroxidase-Labeled, 1.0mL/Bottle</td>
			<td>5450-0011</td>
			<td>KPL/Seracare</td>
			<td>Secondary Antibody for focus forming assay</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>NC0427256</td>
			<td>FisherScientific</td>
			<td>Decontamination</td>
		</tr>
		<tr>
			<td>Corning, Cell Culture Treated Flasks, 150cm2, Vented Cap, Case of 50</td>
			<td>10-126-34</td>
			<td>FisherScientific</td>
			<td>Cell culture consumable</td>
		</tr>
		<tr>
			<td>Costar Cell Culture Plates, 24-well, 5/bag, 100/case, Corning</td>
			<td>07-200-740</td>
			<td>FisherScientific</td>
			<td>Cell culture consumable</td>
		</tr>
		<tr>
			<td>Costar Cell Culture Plates, 96-well, 5/bag, 100/case, Corning</td>
			<td>07-200-91</td>
			<td>FisherScientific</td>
			<td>Cell culture consumable</td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>C0775-100G</td>
			<td>MilliporeSigma</td>
			<td>Stain</td>
		</tr>
		<tr>
			<td>Eppendorf Snap Cap Microcentrifuge Safe-Lock 2mL Tubes, 500/Case</td>
			<td>05-402-7</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Falcon 15mL Conical Centrigue Tubes</td>
			<td>14-959-70C</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Falcon 50mL Conical Centrigue Tubes</td>
			<td>14-959-49A</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Falcon Disposable Polystyrene Serological 10mL Pipets, 200/Case</td>
			<td>13-675-20</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Falcon Disposable Polystyrene Serological 1mL Pipets, 1000/Case</td>
			<td>13-675-15B</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Falcon Disposable Polystyrene Serological 25mL Pipets, 200/Case</td>
			<td>13-675-30</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Falcon Disposable Polystyrene Serological 5mL Pipets, 200/Case</td>
			<td>13-675-22</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Falcon Standard Tissue Culture Dishes</td>
			<td>08-772B</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum-Premium, 500mL</td>
			<td>S11150</td>
			<td>Atlanta Biologicals</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Fisherbrand Economy Plain Glass Microscope Slides</td>
			<td>12-550-A3</td>
			<td>FisherScientific</td>
			<td>Immobilization of Mosquitos</td>
		</tr>
		<tr>
			<td>FU1 Feeder</td>
			<td>FU1-0</td>
			<td>Hemotek Ltd</td>
			<td>Insectary Equipment; feeding units</td>
		</tr>
		<tr>
			<td>Gibco DPBS with Calcium and Magnesium, 10 x 500mL Bottles</td>
			<td>140-040-182</td>
			<td>FisherScientific</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Gibco Fungizone, Amphotericin B, 250&#956;g/mL, 50mL/Bottle</td>
			<td>15-290-026</td>
			<td>Fisher Scientific</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin (10,000 U/mL), 100mL/Bottle, 20 Bottles/Case</td>
			<td>15-140-163</td>
			<td>FisherScientific</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Gibco, Tryptsin-EDTA (.25%), Phenol red, 20 x 100mL Bottles</td>
			<td>25-200-114</td>
			<td>FisherScientific</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Gibcom DMEM, High Glucose, 10 x 500mL Bottles</td>
			<td>11-965-118</td>
			<td>FisherScientific</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Human Blood, Unspecified Gender, Na-Citrate, 1 Unit</td>
			<td>7203706</td>
			<td>Lampire</td>
			<td>Bloodmeal preparation</td>
		</tr>
		<tr>
			<td>InsectaVac Aspirator</td>
			<td>2809B</td>
			<td>Bioquip</td>
			<td>Insectary Equipment</td>
		</tr>
		<tr>
			<td>Methanol, Certified ACS, Fisher Chemicals, Amber Glass Bottle, 4L, 4/Case</td>
			<td>A412-4</td>
			<td>FisherScientific</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>Methyl cellulose, viscosity: 3,500-5,600 cP, 2 % in water(20 &#176;C), 250g/Bottle</td>
			<td>M0512-250G</td>
			<td>MilliporeSigma</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Micro-chem Plus Disinfectant Detergent</td>
			<td>C849T34</td>
			<td>Thomas Scientific</td>
			<td>Decontamination; working dilution of dual quaternary ammonium</td>
		</tr>
		<tr>
			<td>Mineral Oil, BioReagent, for molecular biology</td>
			<td>M5904-5X5ML</td>
			<td>MilliporeSigma</td>
			<td>Immobilization of Mosquitos</td>
		</tr>
		<tr>
			<td>O-rings</td>
			<td>OR37-25</td>
			<td>Hemotek Ltd</td>
			<td>Insectary Equipment</td>
		</tr>
		<tr>
			<td>Plastic Plugs</td>
			<td>PP5-250</td>
			<td>Hemotek Ltd</td>
			<td>Insectary Equipment</td>
		</tr>
		<tr>
			<td>PS6 Power Unit (110-120V)</td>
			<td>PS6120</td>
			<td>Hemotek Ltd</td>
			<td>Insectary Equipment; power source</td>
		</tr>
		<tr>
			<td>Rubis Forceps, Offset blades, superfine points</td>
			<td>4525</td>
			<td>Bioquip</td>
			<td>Insectary Equipment</td>
		</tr>
		<tr>
			<td>Sarstedt Inc, 2mL Screw Cap Microtube, Conical Bottom, O-ring Cap, Sterile, 1000/Case</td>
			<td>50-809-242</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>Sucrose, BioUltra, for molecular biology</td>
			<td>84097-250G</td>
			<td>MilliporeSigma</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>ThermoScientific, ART Barrier Low Retention 1000&#956;L Pipette Tips, 100 tips/Rack, 8 Racks/Pack, 4 Packs/Case</td>
			<td>21-402-487</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>ThermoScientific, ART Barrier Low Retention 200&#956;L Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case</td>
			<td>21-402-486</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>ThermoScientific, ART Barrier Low Retention 20&#956;L Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case</td>
			<td>21-402-484</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>ThermoScientific, ART Barrier Low Retention, Extended Reach 10&#956;L Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case</td>
			<td>21-402-482</td>
			<td>FisherScientific</td>
			<td>Plastic consumable</td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>85300</td>
			<td>QIAGEN</td>
			<td>Homogenization</td>
		</tr>
		<tr>
			<td>TrueBlue Peroxidase Substrate Kit, 200mL</td>
			<td>5510-0030</td>
			<td>Seracare</td>
			<td>Developing solution for focus forming assay</td>
		</tr>
		<tr>
			<td>Vero</td>
			<td>CCL-81</td>
			<td>American Type Culture Collection</td>
			<td>Mammalian cell line to amplify virus and conduct infectious assay</td>
		</tr>
		<tr>
			<td>Vero C1008 [Vero 76, clone E6, Vero E6]</td>
			<td>CRL-1586</td>
			<td>American Type Culture Collection</td>
			<td>Mammalian cell line to amplify virus and conduct infectious assay</td>
		</tr>
	</tbody>
</table>

vector competence analyses on aedes aegypti mosquitoes using zika virus

De förfaranden som presenteras beskriver en generaliserad metod för att infektera Aedes aegypti myggor med Zika virus under laboratorieförhållanden för att bestämma graden av infektion, spridad infektion och potentiell överföring av viruset i myggpopulationen i fråga. Dessa procedurer används i stor utsträckning med olika modifieringar i vektorkompetensutvärderingar globalt. De är viktiga för att bestämma den potentiella roll som en viss mygga (dvs. art, population, individ) kan spela vid överföring av ett visst medel.

Tre populationer av Ae. aegypti från Amerika (Salvador, Brasilien; Dominikanska republiken; och Rio Grande Valley, TX, USA) utsattes för en utbrottsstam av ZIKV från Amerika (ZIKV Mex 1-7, Chiapas State, Mexiko, 2015) över en rad blodmjölstitrar (4, 5 och 6 log10 FFU/mL) presenterade i en tvättad mänsklig erytocyt. Vid dag 2, 4, 7, 10 och 14 postinfection bearbetades undergrupper av myggor för att bestämma infektion, spridning och potentiella överföringshastigheter.
<p class="jove_conte...

Watch this Scientific Journal Video about Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus at JoVE.com

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Det presenterade protokollet kan bestämma vektorkompetensen hos Aedes aegypti myggpopulationer för ett visst virus, såsom Zika, i en inneslutningsmiljö.

Vektorkompetensanalyser på Aedes aegypti myggor med Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Research

JoVE Journal

Immunology and Infection

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

En Kalcium bioluminescence-analys för funktionsanalys av Mosquito ( Aedes aegypti) Och Tick ( Rhipicephalus MicroPlus) G proteinkopplade receptorer

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

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Ett protokoll för insamling och färgning hemocyter från gula febern Mosquito Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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Detektion av infektiösa virus från Field-in Myggor av vero-cellodling analys

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

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Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

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Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

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Utvärdering av Zika Virus-specifika T-cell svar i Immunoprivileged organ av infekterade Ifnar1- / - möss

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

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Räddning av rekombinant zikavirus från en bakterie konstgjord kromosom cDNA-klon

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines ...