Reconhecemos a equipe do Centro Mundial de Referência para Vírus Emergentes e Arboviroses (WRCEVA): Dr. Robert Tesh, Hilda Guzman, Dr. Kenneth Plante, Dr. Jessica Plante, Dionna Scharton e Divya Mirchandani, por seu trabalho incansável na curadoria e fornecimento de muitas das cepas virais usadas para nossos e outros grupos experimentos de competência vetorial. O trabalho apresentado foi financiado pelas bolsas McLaughlin Fellowship Fund (SRA) e nih grants AI120942 e AI121452.

Todos os procedimentos realizados nesses protocolos foram realizados em pleno cumprimento dos protocolos aprovados pelo Comitê de Biossegurança Institucional e pelo Comitê Institucional de Cuidados e Uso de Animais da Filial Médica da Universidade do Texas em Galveston.
1. Amplie o ZIKV em células Vero
<ol><li>Cultivar células Vero (CCL-81 ou VeroE6) na modificação de Dulbecco do meio essencial mínimo (DMEM) da Eagle suplementado com 10% v/v de soro bovino fetal inativado (FBS) e 1% (v/v) penicilina-estreptomicina (100 U/mL e 100 μg/mL, respectivamente) em uma incubadora umidificada de 37 °C com 5% de CO2 a entre 80-90% de confluência em um frasco de cultura tecidual de 150 cm2.</li>
	<li>Em um armário de biossegurança (BSC), remova o meio e descarte em alvejante de 10% ou uma diluição de trabalho de amônio quaternário duplo(Tabela de Materiais). Inocular imediatamente a monocamada com 1 mL de estoque viral, visando 0,1-1 célula de partícula viral infecciosa. Agitar o frasco imediatamente de tal forma que o inóculo contate a monocamadas em sua totalidade.</li>
	<li>Cubra o médio a um volume de 5 mL usando DMEM suplementado com FBS inativados por v/v de 2% v/v e penicilina de 1% (v/v) penicilina-estreptomicina. Em seguida, mova o frasco para a incubadora umidificada de 37 °C com 5% de CO2 por 60 min.</li>
	<li>Remova o frasco da incubadora e leve-o para um BSC. Adicione meio adicional a um volume total de 15 mL usando DMEM suplementado com FBS inativados por v/v de 2% v/v e penicilina-estreptomicina de 1% (v/v). Mova o frasco para uma incubadora umidificada de 37 °C com 5% de CO2.</li>
	<li>Examine o frasco diariamente sob microscopia de contraste de fase para obter evidências de efeitos citopáticos (CPE). Prossiga para a colheita viral (etapa 1.7) quando apenas aproximadamente 40-50% das células permanecem na monocamada, geralmente 3-5 dias após a infecção, dependendo da cepa de ZIKV sendo utilizada.</li>
	<li>Aspire o supernante e coloque em um frasco cônico de 50 mL. Esclareça o sobrenatante de detritos celulares por centrifugação (3.500 x g por 20 min). NOTA: Se vários frascos foram infectados de forma idêntica, supernacantes de vários frascos podem ser combinados em tubos cônicos de 50 mL.</li>
	<li>Remova o supernatante do tubo cônico de 50 mL para um novo, tomando cuidado para não interromper a pelota. Suplemente o supernatante com FBS inativado a calor a uma concentração final de 30% (v/v). Aliquotar esta mistura em tubos individuais de tampa de parafuso e congelar a -80 °C até usar.</li>
</ol>2. Preparação de farinhas de sangue artificiais
<ol><li>No dia em que os mosquitos devem ser expostos a farinhas infecciosas, ligue a fonte de energia (Tabela de Materiais) em uma instalação de contenção de artrópodes, de modo que as unidades de alimentação(Tabela de Materiais) estejam pré-aquecidos quando os mosquitos estiverem preparados para exposição.</li>
	<li>Prepare farinhas de sangue artificiais usando um dos métodos descritos abaixo.<ol>
<li>Método 1: Combinar sangue humano recém-colhido (dentro de uma semana) citado ou heparinizado comprado comercialmente 1:1 v/v com estoque viral (preparado conforme descrito na seção 1). NOTA: Este método depende da ausência de quaisquer anticorpos para a família vírus/vírus estar presente no sangue.</li>
		<li>Se a ausência de anticorpos não puder ser confirmada ou a fonte de sangue for conhecida por ter exposição prévia ao flavivírus, lave e embale manualmente os eritrócitos.<ol>
<li>Em um BSC, adicione 30 mL de sangue humano inteiro a um tubo cônico de 50 mL e cubra até 50 mL com soro fisiológico tamponado de fosfato (PBS).</li>
			<li>Centrifugar a 3.500 x g por 20 min.</li>
			<li>Aspire o supernatante, derramando suavemente em uma panela de bandeja contendo 10% de alvejante ou diluição de trabalho de amônio quaternário duplo, tomando cuidado para não descartar a pelota de eritrócito.</li>
			<li>Adicione 10 mL de PBS e bata suavemente a parte inferior do tubo cônico contra a parte inferior do BSC de tal forma que a pelota eritrócito tenha sido reconstituída. Leve o volume da suspensão até 50 mL com PBS. Misture por inversão suave.</li>
			<li>Repetição de passos 2.2.2.1−2.2.2.4 um total de 4-6x. Confirme se o supernatante é claro ou apenas ligeiramente rosa e não mais opaco. Remova todo o supernatante usando uma pipeta sorológica. Resuspend a pelota eritrócito em 1-2 mL de PBS.</li>
			<li>Monte a farinha de sangue: 350 μL de eritrócitos embalados, 100 μL de 10% de sacarose, 200 μL de FBS inativados pelo calor, ATP recombinante de 900 μM e 2 mL de estoque de vírus apropriadamente diluído usando DMEM complementado com FBS inativados por calor de 2% v/v e 1% (v/v) penicilina-estreptomicina.</li>
		</ol>
</li>
	</ol>
</li>
	<li>Sobreponha uma unidade de reservatório padrão de 3 mL(Tabela de Materiais) com a pele de um rato não infectado (outras opções incluem filme de parafina, membranas colágenas ou invólucro de salsicha).</li>
	<li>Coloque o reservatório coberto em toalhas de papel branco. Adicione ~2 mL de farinha de sangue infecciosa ao reservatório 1 mL de cada vez. Inspecione a toalha debaixo do alimentador para obter qualquer evidência de vazamento. Se houver vazamentos, recupere a farinha de sangue dos alimentadores e descarte as tampas. Sele os alimentadores com plugues. Mais uma vez confirme que não há vazamentos.</li>
</ol>3. Backtitration of bloodmeals/plaque assay
<ol><li>Utilizando o volume restante da farinha de sangue preparada, realize uma série de diluição serial de 10x (ou seja, 6 diluições, variando de diluído 10x a 1.000.000x) utilizando DMEM complementado com FBS inativados por v/v de 2% v/v e penicilina-estreptomicina de 1% (v/v).</li>
	<li>Alíquota 100 μL das diluições nos poços de 24 ou 12 placas de poços das mais diluídas para as mais concentradas.</li>
	<li>Incubar por 1h em uma incubadora de 37 °C, 5% de CO2.</li>
	<li>Ao final do período de incubação de 1 h, leve as placas de volta ao BSC e adicione 1 mL ou 2 mL de sobreposição de metilcelulose aos 24 poços ou 12 placas de poço, correspondentemente.</li>
	<li>Coloque as placas sobrepostas de volta em uma incubadora de 37 °C, 5% de CO2 e incubar por 3-7 dias (dependente de cepa do vírus).</li>
	<li>Após a incubação, remova as placas da incubadora e leve para o BSC. Descarte a sobreposição de metilcelulose em uma panela de bandeja contendo 10% de alvejante ou desinfetante de amônio quaternário duplo.</li>
	<li>Lave cada poço 2x com PBS, descartando a lavagem em uma panela de bandeja contendo 10% de alvejante ou amônio quaternário duplo.</li>
	<li>Adicione ~1 mL de metanol:acetona (1:1 v:v) e deixe as células fixarem na placa por pelo menos 30 minutos no BSC à temperatura ambiente (RT). Descarte metanol:acetona de acordo com a política institucional sobre resíduos orgânicos.</li>
	<li>Visualize o ZIKV usando um dos dois métodos descritos abaixo.<ol>
<li>Após a remoção do metanol:acetona, colora imediatamente com uma solução violeta cristalina (0,25% w/v em 30% de metanol) por 5 min. Enxágüe 2x na água da torneira e deixe secar, depois visualize diretamente de olho para evidências de placas ou destruição de monocamadas.</li>
		<li>Alternativamente, realize o ensaio de formação de foco.<ol>
<li>Deixe as placas secarem até que não haja restos orgânicos. NOTA: Este deve levar ~2−3 h fora do BSC, mas pode ser acelerado por secagem de ar em um BSC ou capô de fumaça química.</li>
			<li>Lave cada poço 3x por 15 min cada em PBS não ester (Mg2+ e Ca2+ livre) em um roqueiro de placa orbital. Remova o PBS e adicione 1 mL de solução de bloqueio (PBS + 3% FBS) a cada poço e arrase por 15 min no RT.</li>
			<li>Adicione 100 μL por poço de α-ZIKV ou anticorpo primário α-flavivirus (por exemplo, híbridoma do grupo flavivírus D1-4G2-4-15 [4G2]) a uma solução de diluição de 1:2.000 na solução de bloqueio. Incubar com balanço por um mínimo de 4h (preferencialmente durante a noite, não excedendo 18 h) na RT.</li>
			<li>Remova o anticorpo primário e lave 3x por 15 min cada com PBS (Mg2+ e Ca2+ livre) em um roqueiro de placa orbital.</li>
			<li>Adicione 100 μL por poço do anticorpo secundário (cabra α mouse hrp-rotulado) diluído 1:2.000 no buffer de bloqueio. Incubar com balanço por 1h na RT.</li>
			<li>Lave 3x por 15 min cada com PBS (Mg2+ e Ca2+ livre) em um roqueiro de placa orbital.</li>
			<li>Alíquota 100 μL de reagente de desenvolvimento de substrato(Tabela de Materiais) por poço. Placas de rocha na RT por 15 minutos.</li>
			<li>Interrompa a reação após o desenvolvimento de focos/placas removendo o substrato e enxaguando as placas 2x com água da torneira. Despeje a água da torneira e deixe as placas secarem antes de quantificar.</li>
			<li>Conte focos virais para determinar o número de FFU presente na amostra dada.</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. Administração de farinhas de sangue
<ol><li>Use mosquitos Ae. aegypti 2-4 dias após a eclosão. Classifique mosquitos fêmeas em caixas de papelão 0,5 L com tampas de tela e prive-os de açúcar (geralmente 36-48 h antes da farinha de sangue infecciosa). Forneça água ad libitum através de bolas de algodão saturadas de água.</li>
	<li>Remova as bolas de algodão saturadas de água na manhã em que os mosquitos serão expostos à farinha de sangue infecciosa.</li>
	<li>Anexar reservatórios contendo farinhas de sangue artificiais à unidade de alimentação leva dentro de uma caixa de luvas de plástico transparente.</li>
	<li>Dentro do porta-luvas, coloque uma caixa de papelão 0,5 L com tampa de tela, contendo 50-100 mosquitos Ae. aegypti famintos, sob a unidade de alimentação ligada ao reservatório. NOTA: Mosquitos apropriadamente famintos geralmente se alimentam dentro de 20 minutos. A alimentação pode ser prolongada conforme necessário para aumentar o tamanho da amostra em populações mais lentas, embora isso não deva se estender além de 60 minutos, porque o título viral (ZIKV) pode diminuir dentro do alimentador após ~60 min.</li>
	<li>Após a conclusão da alimentação, remova o reservatório e mergulhe em alvejante recém-feito 10%.</li>
	<li>Mosquitos anestesizem a frio por incubação para 30 s a -20 °C ou por 5 min em uma geladeira.</li>
	<li>Dentro do porta-luvas, despeje os mosquitos em uma placa de Petri no gelo. Conte e classifique as fêmeas engorgadas de mosquitos não engorgados. Descarte os mosquitos não engorgados por imersão em um tubo cônico de tubo de 50 mL cheio de 70% de etanol. Enquanto os mosquitos ainda estão anestesiados, despeje-os de volta na caixa de papelão 0,5 L e cubra rapidamente com a tela e a tampa. Corte a malha de tela em excesso da caixa e fixe a malha com fita.</li>
	<li>Adicione uma bola de algodão saturada com sacarose estéril filtrada 10% aquosa na tela de cada caixa. Coloque todas as caixas de mosquitos em um grande recipiente secundário de plástico com uma esponja úmida para manter a umidade.</li>
	<li>Coloque recipiente secundário contendo caixas de mosquitos em uma incubadora com temperatura de 27 ± 1 °C (ou conforme apropriado para simular condições na região de interesse) com 80% ± 10% de umidade relativa e um ciclo claro de 16:8:escuro). Mantenha os mosquitos com acesso a ad libitum a 10% de sacarose até a conclusão dos experimentos.</li>
</ol>5. Aquisição e processamento de amostras
<ol><li>Nos dias de pós-alimentação especificados, aspire um número predeterminado de mosquitos das caixas apropriadas usando um aspirador mecânico dentro de um porta-luvas. Tampe o tubo de coleta com uma rodada de algodão após a aquisição do número necessário de mosquitos.</li>
	<li>Mosquitos anesthetize frio por incubação por 30 segundos a -20 °C ou por 5 minutos a 4 °C.</li>
	<li>Dentro do porta-luvas, despeje os mosquitos em uma placa de Petri no gelo. Usando dois pares de fórceps, remova todas as seis pernas de cada mosquito e coloque em um tubo de microcentrifusidade de fundo redondo de 2 mL pré-rotulado contendo um rolamento de esfera de aço inoxidável esterilizado (7/32") e 500 μL de mídia de coleta de mosquitos (MCM) composta de DMEM, 2% FBS, 1% penicilina-estreptomicina e 2,5 μg/mL de anfotericina.</li>
	<li>Coloque suavemente o mosquito em uma gota de óleo mineral para contê-lo, tomando cuidado para não permitir qualquer contato entre o óleo e a cabeça do mosquito e proboscis.</li>
	<li>Insira o proboscis do mosquito em uma ponta de pipeta de 10 μL preenchida com 10 μL de FBS inativados pelo calor. NOTA: Alternativamente, a pipeta pode ser preenchida com sacarose, sangue ou óleo mineral. O óleo permite visualização direta de bolhas de saliva através de microscopia leve.</li>
	<li>Deixe o mosquito salivar por 30 minutos. Ejete a ponta de micropipette contendo FBS + saliva em um tubo de microcentrifuuge contendo 100 μL de MCM, em seguida, coloque a carcaça em um tubo de microcentrifuuge fundo redondo separado de 2 mL contendo um rolamento de esfera de aço esterilizado e 500 μL de MCM. Certifique-se de que os tubos utilizados para os corpos, pernas e saliva sejam rotulados para que fique claro que todas as três amostras se originaram do mesmo mosquito.</li>
	<li>Enquanto os mosquitos estão salivando, realize etapas 5.3-5.5 nos mosquitos restantes.</li>
	<li>Transporte os tubos contendo os corpos e pernas para um dispositivo de homogeneização de tecido de moagem de contas contido dentro de um BSC. Triturar todas as amostras de corpo e perna a 26 Hz por 5 minutos para liberar partículas virais para o supernante. Esclareça todas as amostras por centrifugação a 200 x g por 5 min para pelotas de detritos celulares. NOTA: Neste ponto, as amostras podem ser congeladas a -80 °C, ou avaliadas imediatamente.</li>
</ol>6. Detecção de ZIKV por ensaio infeccioso
<ol><li>Em um BSC, prepare 24 placas de cultura de tecido bem com células Vero (105 células por poço) 24 h antes do início do ensaio infeccioso. Rotule cada poço com a identidade de um único mosquito/amostra.</li>
	<li>Se as amostras foram congeladas a -80 °C, deixe descongelar.</li>
	<li>Remova a mídia das placas celulares Vero antes da inoculação com amostras de uma placa de cada vez.</li>
	<li>Para amostras contendo corpos ou pernas, alíquota cuidadosamente de 100 μL de supernanato clarificado em cada poço, tomando cuidado para não perturbar os detritos celulares do mosquito da pelota. NOTA: As amostras de saliva podem ser diluídas 1:1 (v/v) com MCM antes da inoculação nas células para conservar amostras para titulação posterior, se necessário.</li>
	<li>Mova as placas para uma incubadora de 37 °C, 5% de CO2 e incubar por 1h.</li>
	<li>Devolva as placas ao BSC e adicione ~1 mL de sobreposição de metilcelulose a cada poço. Coloque as placas sobrepostas de volta em uma incubadora de 37 °C, 5% de CO2 e incubar por 3-7 dias (vírus/dependente de cepas).</li>
	<li>Realize fixação e visualização conforme descrito nas etapas 3.6-3.9.</li>
	<li>Quanto à pontuação através de um ensaio de formação de foco, quantifique os poços positivos por exame sob um microscópio leve. A detecção de coloração intracytoplasmática das células em um poço indica a presença de vírus. As amostras são pontuadas apenas como focus-positive ou -negativo.</li>
</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=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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+and+relevance+in+insect-arbovirus+infection+studies.">Reproducibility and relevance in insect-arbovirus infection studies.</a> Current Opinion in Insect Science. 28, 105-112 (2018).</li><li>Hagan, R. W., 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=Dehydration+prompts+increased+activity+and+blood+feeding+by+mosquitoes.">Dehydration prompts increased activity and blood feeding by mosquitoes.</a> Scientific Reports. 8 (1), 6804 (2018).</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=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. 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=Potential+for+Zika+Virus+to+Establish+a+Sylvatic+Transmission+Cycle+in+the+Americas.">Potential for Zika Virus to Establish a Sylvatic Transmission Cycle in the Americas.</a> PLoS Neglected Tropical Diseases. 10 (12), 0002055 (2016).</li><li>Vasilakis, N., Cardosa, J., Hanley, K. A., Holmes, E. C., 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=Fever+from+the+forest:+prospects+for+the+continued+emergence+of+sylvatic+dengue+virus+and+its+impact+on+public+health.">Fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health.</a> Nature Reviews Microbiology. 9 (7), 532-541 (2011).</li><li>Vasilakis, N., 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=Potential+of+ancestral+sylvatic+dengue-2+viruses+to+re-emerge.">Potential of ancestral sylvatic dengue-2 viruses to re-emerge.</a> Virology. 358 (2), 402-412 (2007).</li></ol>

Os autores não têm nada a revelar.

Os métodos aqui descritos fornecem um fluxo de trabalho generalizado para a realização de análises de competência vetorial. Como estrutura geral, muitas dessas metodologias são conservadas ao longo da literatura. No entanto, há espaço substancial para modificações (revisadas em Azar e Weaver1). Vírus (por exemplo, linhagem viral, armazenamento de vírus de desafio, histórico de passagem viral), entomologia (por exemplo, colonização laboratorial de populações de mosquitos, imunidade...

A competência vetorial é definida como a capacidade no nível de espécies, população e até mesmo um indivíduo, de um determinado artrópode, como um mosquito, carrapato ou mosca de areia de flebotomina, para adquirir e transmitir um agente biologicamente com replicação ou desenvolvimento no artrópode1,2. Com relação a mosquitos e vírus transmitidos por artrópodes (ou seja, arboviroses), o agente é absorvido de um hospedeiro virêmico por uma fêmea de mosquito. Após a ingestão, o vírus deve infectar produtivamente uma pequena população de células epiteliais midgut3, superando vários obstáculos fisiológicos, como a degradação proteolítica por enzimas digestivas, a presença da microbiota (barreira de infecção midgut, ou MIB), e a matriz peritrofídica secretada. A infecção do epitélio midgut deve ser seguida pela replicação do vírus e eventual fuga do midgut para o sistema circulatório aberto do mosquito, ou hemoglifo, o que representa o aparecimento de uma infecção disseminada superando a barreira de escape midgut (MEB). Neste ponto, o vírus pode estabelecer infecções de tecidos secundários (por exemplo, nervos, músculos e corpos de gordura) e continuar a se replicar, embora essa replicação secundária possa não ser estritamente necessária para que o vírus infecte as células acinares das glândulas salivares (superando a barreira da infecção da glândula salivar). O lagross das células acinares da glândula salivar em suas cavidades apical e, em seguida, o movimento no ducto salivar permite a inoculação do vírus em hospedeiros subsequentes na mordida, e completa o ciclo de transmissão1,2,4,5,6,7.
Dado esse mecanismo bem caracterizado e geralmente conservado de disseminação dentro de um vetor de mosquitos, as avaliações de competência vetorial laboratorial são muitas vezes metodologicamente semelhantes, embora existam diferenças nos protocolos1,2. Geralmente, após a exposição ao vírus oral, os mosquitos são dissecados para que tecidos individuais como o midgut, pernas, ovários ou glândulas salivares possam ser testados para infecção viral, infecção disseminada, infecção disseminada/transmissão transovarial potencial e competência de transmissão disseminada/potencial,respectivamente 8. A mera presença de um vírus nas glândulas salivares, no entanto, não é evidência definitiva da capacidade de transmissão, dada a evidência de uma barreira de fuga/saída da glândula salivar (SGEB) em algumas combinações vetoriais/vírus1,2,4,5,7,9. O método padrão para comprovar a competência de transmissão continua sendo a transmissão do mosquito para um animal suscetível10,11,12. No entanto, dado que para muitas arboviroses isso requer o uso de modelos murinas imunocomprometidos13,14,15,16, este método muitas vezes é proibitivo. Uma alternativa comumente utilizada é a coleta da saliva do mosquito, que pode ser analisada por reação de cadeia transcrição reversa-polimerase (RT-PCR) ou um ensaio infeccioso para demonstrar a presença do genoma viral ou partículas infecciosas, respectivamente. Vale ressaltar que tais métodos de coleta de saliva in vitro podem superestimar12 ou subestimar17 a quantidade de vírus depositado durante a alimentação in vivo, indicando que tais dados devem ser interpretados com cautela. No entanto, o método in vitro é altamente valioso quando analisado na perspectiva da mera presença de vírus na saliva, indicando potencial de transmissão.
Existem duas abordagens importantes para determinar o papel dos vetores de mosquitos em surtos de doenças arbovirais. O primeiro método envolve a vigilância em campo, na qual os mosquitos são coletados no contexto de transmissão ativa18,19,20,21,22,23,24. No entanto, dado que as taxas de infecção são tipicamente bastante baixas (por exemplo, a taxa estimada de infecção de 0,061% de mosquitos em áreas de circulação ativa do vírus Zika (ZIKV) nos Estados Unidos21), a incriminação de espécies vetoriais potenciais pode ser fortemente tendenciosa pela metodologia de captura25,26 e chance aleatória (por exemplo, amostragem de um indivíduo infectado de 1.600 não infectados)2 . Levando isso em conta, um determinado estudo pode não adquirir mosquitos suficientes tanto em números brutos quanto na diversidade de espécies para amostrar com precisão os mosquitos envolvidos na transmissão. Em contrapartida, as análises de competência vetorial são realizadas em ambiente laboratorial, permitindo um controle rigoroso de parâmetros como a dose oral. Embora não sejam totalmente capazes de representar a verdadeira complexidade da infecção por mosquitos e da capacidade de transmissão em um ambiente de campo, essas avaliações laboratoriais permanecem ferramentas poderosas no campo da arbovirologia.
Com base em diversas análises de competência vetorial com ZIKV em diversas espécies de mosquitos, populações e métodos27,28,29,30,31,32, bem como uma revisão recente das avaliações de competência vetorial1,descrevemos aqui vários dos protocolos associados a um típico fluxo de trabalho de competência vetorial. Nestes experimentos, três Ae. populações de aegypti das Américas (cidade de Salvador, Brasil; República Dominicana; e do baixo Vale do Rio Grande, TX, EUA) foram expostas a uma única cepa de ZIKV (Mex 1-7, GenBank Accession: KX247632.1) a 4, 5 ou 6 log10 unidades de formação focal (FFU)/mL por meio de doses de sangue artificial. Posteriormente, foram analisadas evidências de infecção, infecção disseminada e competência de transmissão após vários momentos de incubação extrínseca (2, 4, 7, 10 e 14 dias) por meio de dissecção e ensaio infeccioso baseado em cultura celular. Embora o fluxo de trabalho/protocolos atual seja otimizado para ZIKV, muitos elementos são diretamente traduzíveis para outras arboviroses transmitidas por mosquitos nos níveis de contenção de artrópodes e biossegurança 2 e 3 (ACL/BSL2 ou 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

Os procedimentos apresentados descrevem uma metodologia generalizada para infectar mosquitos Aedes aegypti com zika vírus em condições laboratoriais para determinar a taxa de infecção, infecção disseminada e potencial transmissão do vírus na população do mosquito em questão. Esses procedimentos são amplamente utilizados com várias modificações nas avaliações de competência vetorial globalmente. São importantes na determinação do papel potencial que um determinado mosquito (ou seja, espécie, população, indivíduo) pode desempenhar na transmissão de um determinado agente.

Três populações de Ae. aegypti das Américas (Salvador, Brasil; República Dominicana; e o Vale do Rio Grande, TX, EUA) foram expostos a um surto de ZIKV das Américas (ZIKV Mex 1-7, Estado de Chiapas, México, 2015) sobre uma gama de de farinha de sangue (4, 5 e 6 log10 FFU/mL) apresentados em um eritrócito humano lavado. Nos dias 2, 4, 7, 10 e 14 pós-infecção, subconjuntos de mosquitos foram processados para determinar as taxas de infecção, disseminação e potencial de transmissão.
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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

O protocolo apresentado pode determinar a competência vetorial das populações do mosquito Aedes aegypti para um determinado vírus, como o Zika, em um ambiente de contenção.

Análises de competência vetorial sobre mosquitos Aedes aegypti que usam zika vírus

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

vector-competence-analyses-on-aedes-aegypti-mosquitoes-using-zika

Research

JoVE Journal

Immunology and Infection

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

2.9K visualizações.  University of Texas Medical Branch. O protocolo apresentado pode determinar a competência vetorial das populações do mosquito Aedes aegypti para um determinado vírus, como o Zika, em um ambiente de contenção.

Vídeo: 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

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

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

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Um Ensaio Bioluminescência Cálcio de análise funcional do Mosquito ( Aedes aegypti) E Tick ( Rhipicephalus microplus) G receptores acoplados à proteína

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

Imunologia e Infecção

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

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

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

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

Um Protocolo de Coleta e coloração Hemócitos do mosquito da febre amarela 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 ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Detecção de vírus infecciosos de Campo mosquitos coletados por Vero Ensaio Cultura de Células

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

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Hibridização<em&gt; In situ</em&gt; Das glândulas salivares, ovários e embriões de mosquitos vetores

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

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Virus Zika sistema de cultura celular Infecciosas e do<em&gt; In Vitro</em&gt; Efeito profilático de interferões

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Salvamento e Caracterização do Vírus Recombinante de um Clone Infeccioso do Vírus do Novo Mundo Zika

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

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika vírus específico diagnóstico Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

A citometria de fluxo simples com base em ensaio para determinar In Vitro anticorpo dependente do realce do vírus da Dengue, usando soro convalescente vírus Zika

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

Quantificação de realce dependente de anticorpo do vírus nas células humanas primárias Zika

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial&#160;propagation.

The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

Resgate do vírus Zika recombinante de um clone de cDNA de cromossomo artificial bacteriano

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