We acknowledge the staff of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA): Dr. Robert Tesh, Hilda Guzman, Dr. Kenneth Plante, Dr. Jessica Plante, Dionna Scharton, and Divya Mirchandani, for their tireless work in curating and providing many of the viral strains used for our and other groups&#8217; vector competence experiments. The presented work was funded by the McLaughlin Fellowship Fund (SRA) and NIH grants AI120942 and AI121452.

All procedures performed in these protocols were performed in full compliance with protocols approved&#160;by the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee of the University of Texas Medical Branch at Galveston.
1. Amplify ZIKV in Vero cells
<ol>
	<li>Grow Vero cells (CCL-81 or VeroE6) in Dulbecco&#8217;s modification of Eagle&#8217;s minimal essential medium (DMEM) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS), and 1% (v/v) p...

<ol>
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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. 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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. 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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. 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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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The authors have nothing to disclose.

The methods described here provide a generalized workflow to conduct vector competence analyses. As a general framework, many of these methodologies are conserved throughout the literature. However, there is substantial room for modifications (reviewed in Azar and Weaver1). Virus (e.g., viral lineage, storage of challenge virus, viral passage history), entomology (e.g., laboratory colonization of mosquito populations, innate immunity, the mosquito microbiome/virome), and experimental variables (e....

Vector competence is defined as the ability at the level of species, population, and even an individual, of a given arthropod such as a mosquito, tick, or phlebotomine sand fly, to acquire and transmit an agent biologically with replication or development in the arthropod1,2. With respect to mosquitoes and arthropod-borne viruses (i.e., arboviruses), the agent is imbibed from a viremic host by a female mosquito. Following ingestion, the virus must productively infect one of a small population of midgut epithelial cells3, overcoming various physiological obstacles such as proteolytic degradation by digestive enzymes, the presence of the microbiota (midgut infection barrier, or MIB), and the secreted peritrophic matrix. Infection of the midgut epithelium must be followed by replication of the virus and eventual escape from the midgut into the mosquito&#8217;s open circulatory system, or hemolymph, which represents the onset of a disseminated infection overcoming the midgut escape barrier (MEB). At this point the virus can establish infections of secondary tissues (e.g., nerves, muscles, and fat bodies) and continue to replicate, although such secondary replication may not be strictly necessary for the virus to infect the acinar cells of the salivary glands (overcoming the salivary gland infection barrier). Egress from the salivary gland acinar cells into their apical cavities and then movement into the salivary duct enables inoculation of the virus into subsequent hosts on biting, and completes the transmission cycle1,2,4,5,6,7.
Given this well-characterized and generally conserved mechanism of spread within a mosquito vector, laboratory vector competence assessments are often methodologically similar, although differences in protocols do exist1,2. Generally, after oral virus exposure, mosquitoes are dissected so that individual tissues such as the midgut, legs, ovaries, or salivary glands can be assayed for viral infection, disseminated infection, disseminated infection/potential transovarial transmission, and disseminated infection/potential transmission competence, respectively8. The mere presence of a virus in the salivary glands, however, is not definitive evidence of transmission capability, given evidence of a salivary gland escape/egress barrier (SGEB) in some vector/virus combinations1,2,4,5,7,9. The standard method to prove transmission competence remains mosquito transmission to a susceptible animal10,11,12. However, given that for many arboviruses this necessitates the use of immunocompromised murine models13,14,15,16, this method is often cost-prohibitive. A commonly used alternative is the collection of the mosquito saliva, which can be analyzed by reverse transcription-polymerase chain reaction (RT-PCR) or an infectious assay to demonstrate the presence of the viral genome or infectious particles, respectively. It is worth noting that such in vitro saliva collection methods may overestimate12 or underestimate17 the amount of virus deposited during in vivo feeding, indicating that such data must be interpreted with caution. Nonetheless, the in vitro method is highly valuable when analyzed from the perspective of the mere presence of virus in the saliva, indicating transmission potential.
Two major approaches exist for determining the role of mosquito vectors in arboviral disease outbreaks. The first method involves field surveillance, in which mosquitoes are collected in the context of active transmission18,19,20,21,22,23,24. However, given that infection rates are typically quite low (e.g., the estimated 0.061% infection rate of mosquitoes in areas of active Zika virus (ZIKV) circulation in the United States21), incrimination of potential vector species can be heavily biased by trapping methodology25,26 and random chance (e.g., sampling one infected individual out of 1,600 uninfected)21. Taking this into account, a given study may not acquire sufficient mosquitoes in both raw numbers or species diversity to accurately sample mosquitoes involved in transmission. In contrast, vector competence analyses are undertaken in a laboratory setting, allowing for strict control of parameters such as oral dose. Although not fully capable of representing the true complexity of mosquito infection and transmission capability in a field setting, these laboratory assessments remain powerful tools in the field of arbovirology.
Based on various vector competence analyses with ZIKV in several mosquito species, populations, and methods27,28,29,30,31,32, as well as a recent review of vector competence assessments1, we describe here several of the protocols associated with a typical vector competence workflow. In these experiments, three Ae. aegypti populations from the Americas (the city of Salvador, Brazil; the Dominican Republic; and the lower Rio Grande Valley, TX, USA) were exposed to a single strain of ZIKV (Mex 1-7, GenBank Accession: KX247632.1) at 4, 5, or 6 log10 focus-forming units (FFU)/mL doses by way of artificial bloodmeals. Subsequently, they were analyzed for evidence of infection, disseminated infection, and transmission competence after various times of extrinsic incubation (2, 4, 7, 10, and 14 days) by means of dissection and a cell culture-based infectious assay. Although the present workflow/protocols are optimized for ZIKV, many elements are directly translatable to other mosquito-borne arboviruses in arthropod containment and biosafety levels 2 and 3 (ACL/BSL2 or 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

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Three populations of Ae. aegypti from the Americas (Salvador, Brazil; the Dominican Republic; and the Rio Grande Valley, TX, USA) were exposed to an outbreak strain of ZIKV from the Americas (ZIKV Mex 1-7, Chiapas State, Mexico, 2015) over a range of bloodmeal titers (4, 5, and 6 log10 FFU/mL) presented in a washed human erythrocyte-based artificial bloodmeal. At days 2, 4, 7, 10, and 14 postinfection, subsets of mosquitoes were processed to determine infection, dissemination, and potential transmissi...

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

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

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

An aprox mediated by arbovirus, as countermeasures are largely limited to abatement of mosquitoes. So vector competence analyses are critical prevention strategies because they determine what populations and species of mosquitoes can serve as vectors for a given arbovirus, and therefore should be targeted. So one of the most important components in our toolbox for assessing the role of mosquitoes, and other vectors in transmitting infectious diseases, is the vector competence study.These are experiments where we can very precisely control, not only the species of mosquito, or tick, for example, but also geographic strains, as well as very specific strains of a different virus or other infectious organism to get very precise and well-controlled data on the risk of different species for transmitting different agents in very specific parts of the world. Working with infected mosquitoes requires dedicated facilities, training, experience, and careful planning to minimize the risks to the experimenter, as well as the risks of an escaped, infected mosquito. It's therefore quite important for new individuals in these facilities to take the time to work with uninfected blood mosquitoes to familiarize themselves with the techniques and workflow.On the day of exposure, turn on the power source in an Arthropod containment facility to preheat the feeding units. Combine freshly harvested citrated or heparinized human blood with a viral stock at a one to one volume ratio, and overlay a standard three milliliter reservoir unit with the skin of a naive uninfected mouse. Place the covered reservoir on white paper towels, and add about two milliliters of infectious blood to the reservoir one milliliter at a time.Then, inspect the paper towels to confirm that there is no leakage. 24 hours prior to performing the infectious assay, prepare 24-well cell culture plates with Vero cells, and label each well with the identity of the mosquito and sample. On the morning that the mosquitoes will be exposed to the infectious blood meal, remove the water saturated cotton balls, and attach reservoirs with the artificial blood meals to the feeding unit leads with a clear plastic containment glove box.Place the cardboard carton with the starved mosquitoes into the glove box underneath the feeding unit. Upon completion of feeding, remove the reservoir, and immerse it in freshly prepared 10%bleach. Within the glove box, pour cold anesthetized mosquitoes into a Petri dish on ice, and sort the engorged from the un-engorged mosquitoes.Pour the engorged mosquitoes back into the cardboard carton, quickly covering it with a screen and lid. Trim the excess screen mesh off of the carton, and secure the lid with tape. Add a cotton ball saturated with sterile filtered 10%sucrose to the screen, and place the carton in a large plastic secondary container with a damp sponge to maintain humidity.Incubate the secondary container at 27 degrees celsius, allowing mosquitoes ad libitum access to the sucrose until completion of experiments. Use a mechanical aspirator to acquire a predetermined number of incubated mosquitoes from the carton, capping the collection tube with a cotton round when finished. Working within the glove box, pour cold anesthetized mosquitoes into a Petri dish on ice.Remove all six of each mosquitoes legs, and place them in a pre-labeled two milliliter micro centrifuge tube with a sterilized stainless steel ball bearing and 500 microliters of mosquito collection media, or MCM. Gently place the mosquito onto a drop of mineral oil, taking care that the oil does not contact the mosquito's head or proboscis. Insert the proboscis into a micro pipette tip filled with 10 microliters of heat inactivated FBS, and allow the mosquito to salivate for 30 minutes.Eject the pipette tip with the saliva into a micro centrifuge tube with 100 microliters of MCM, and place the carcass into a separate two millimeter tube with the sterilized steel ball bearing and 500 microliters of MCM. Transport the tubes to a bead milling tissue homogenization device within a bio safety cabinet, and triturate all body and leg samples at 26 hertz for five minutes to liberate viral particles into the supernatant. Clarify all samples by centrifugation at 200 times G for five minutes to pelite cellular debris.Immediately before inoculation with samples, remove the media from the previously prepare Vero cells. Carefully aliquot 100 microliters of clarified supernatants from the bodies or legs into each well, taking care not to disturb the mosquito cell debris from the pelite. Then, incubate the plates for one hour.After the incubation, add one milliliter of methylcellulose overlay to each well, and put the plates back in the incubator for three to seven days. Remove overlay plates from the incubator, and bring them to a bio safety cabinet. Discard the methylcellulose overlay into a tray pan containing 10%bleach, and wash each well twice with PBS, discarding each wash into the tray pan.Remove the PBS, and add one milliliter of methanol-acetone per well, and allow the cells to fix onto the plate for 30 minutes. Then, discard the fixative, and leave the plates to dry. Wash each well three times with non-sterile PBS on an orbital plate rocker for 15 minutes per wash, then add one milliliter of blocking solution to each well, and rock the plate for another 15 minutes.Add 100 microliters of anti Zika Virus, or anti flavivirus primary antibody to each well, and incubate the plate with rocking overnight. On the next day, remove the primary antibody, and repeat the washes with PBS. After the last wash, add 100 microliters of secondary antibodies, and incubate the plate while rocking for one hour.Repeat the washes with PBS, then add 100 microliters of substrate development reagent per well, and rock the plate for 15 minutes. When the foci or plaques develop, halt the reaction by rinsing the plate with tap water. Pour off the tap water, and allow the plates to air dry, then quantify the viral foci to determine the number of focus forming units present in the given sample.Three mosquito populations were exposed to an outbreak strain of Zika Virus over a range of blood meal titers. Over the following two weeks, subsets of mosquitoes were processed to determine infection, dissemination, and potential transmission rate. At the lower blood meal titers, the mosquitoes from Salvador, Brazil were infected after 4, 10, and 14 days of extrinsic incubation, with no evidence of disseminated infection observed in the assayed legs.At the highest titer, the virus was detected in the legs of the mosquitoes, but not in the saliva. The mosquitoes from the Dominican Republic proved to be the most susceptible to infection, and the most transmission competent at all tested blood meal titers. Mosquitoes fed all three doses demonstrated dissemination by seven days, and transmission competence at 14 days post infection.Mosquitoes from the Rio Grande Valley in Texas, exposed to lower blood meal titers, were infected as early as four days post infection, while disseminated infections were found after 14 days. In the cohort exposed to the highest titer, infection was observed beginning from the second day post infection, and reached peaks at 4, 10, and 14 days. Disseminated infections were observed at seven days, and only a single mosquito was transmission capable at 10 days.When attempting this protocol, it's important to work safely, as opposed to quickly. So always handle infected or potentially infected mosquitoes within the confines of a glove box or a bio safety cabinet, until they've had their legs or wings removed. The basic vector competence workflow can be adapted to produce infected mosquitoes for downstream analyses, such as transmission studies, as well as to examine the interactions between mosquitoes, the virus, and the microbiome.Vector competence has been a really important tool for understanding the role of mosquitoes in transmitting viruses during epidemics of viral disease, and this has been true for many decades. But it's also a method that's evolved over the years to include new information, as we learn it, about the interactions between these viruses and the mosquito vectors. Just by way of two examples, the genetic population of the virus, the complexity of the population within the mosquito turns out the be very important for its ability to transmit, and we can now include that in vector competence studies with new methods for sequencing the viral population.We also now know that the population of bacteria and other organisms that live in the gastrointestinal tract of the mosquito can also have a major impact on their ability to transmit, and we can also evaluate that population now with new methods in sequencing. So vector competence is a method that has evolved and continues to evolve as we learn more about these complex interactions.

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JoVE Journal

Immunology and Infection

2.8K Views.  University of Texas Medical Branch. An aprox mediated by arbovirus, as countermeasures are largely limited to abatement of mosquitoes. So vector competence analyses are critical prevention strategies because they determine what populations and species of mosquitoes can serve as vectors for a given arbovirus, and therefore should be targeted. So one of the most important components in our toolbox for assessing the role of mosquitoes, and other vectors in transmitting infectious diseases, is the vector competence study.The...