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

我々は、新興ウイルスおよびアルボウイルスのための世界参照センター(WRCEVA):ロバート・テッシュ博士、ヒルダ・グスマン、ケネス・プランテ博士、ジェシカ・プランテ博士、ディオンナ・シャルトン、ディヴィヤ・ミルチャンダニ博士が、私たちの他のグループのベクター能力実験に使用されるウイルス株の多くをキュレーションし、提供するたゆまぬ努力をしていることを認めます。提示された作品は、マクラフリン・フェローシップ・ファンド(SRA)とNIH助成金AI120942とAI121452によって資金提供されました。

これらのプロトコルで行われたすべての手順は、ガルベストンのテキサス大学医学部の機関バイオセーフティ委員会および制度的動物ケアおよび使用委員会によって承認されたプロトコルに完全に準拠して実行されました。
1. VERO細胞でZIKVを増幅
<ol><li>10%v/v熱不活性化ウシ胎児血清(FBS)と1%(v/v)ペニシリンストレプトマイシン(100 U/mLおよび100 μg/mL)を補ったイーグルの最?...

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ここで説明する方法は、ベクトル能力分析を行う一般化されたワークフローを提供します。一般的な枠組みとして、これらの方法論の多くは文献全体に保存されています。しかし、変更の余地は大きくあります(AzarとWeaver1でレビュー)。ウイルス(例えば、ウイルス系統、チャレンジウイルスの保存、ウイルス通過歴)、昆虫学(例えば、蚊集団の実験室コロニー形成、先天性免...

ベクター能力は、種、集団、さらには個体のレベルでの能力として定義され、蚊、ダニ、またはフレボトミン砂飛のような与えられた節足動物の、節足動物1,2における複製または発生を伴う生物学的に薬剤を取得および伝達する。蚊および節足動物媒介ウイルス(すなわち、アルボウイルス)に関して、薬剤は雌の蚊によってビレミック宿主から浸透する。摂取後、ウイルスは、消化酵素によるタンパク質分解分解、微生物叢(ミドグ感染障壁、またはMIB)の存在、および分泌された腹膜マトリックスなどの様々な生理学的障害を克服し、中腸上皮細胞3の小集団の1つに生産的に感染しなければならない。中腸上皮の感染は、ウイルスの複製と最終的に蚊の開放的な循環系への中腸からの脱出、または中腸脱出障壁(MEB)を克服する播種感染の発症を表す血中リンパに続かなければならない。この時点で、ウイルスは二次組織(例えば、神経、筋肉、脂肪体)の感染を確立し、複製を続けることができますが、そのような二次複製は、ウイルスが唾液腺の体内細胞に感染する(唾液腺感染障壁を克服する)ために厳密に必要ではないかもしれません。唾液腺から補助的な空洞への出射口から唾液管への移動は、噛み付きの後続の宿主へのウイルスの接種を可能にし、伝達周期1、2、4、5、6、7を完了する。
このよく特徴づけられ、一般的に蚊ベクター内に広がるメカニズムが一般的に保存されていることを考えると、実験室のベクター能力評価はしばしば方法論的に類似しているが、プロトコルの違いは1,2である。一般的に、経口ウイルス暴露後、蚊は、ウイルス感染、播種感染、感染/潜在的なトランスリアル感染、および感染/潜在的な感染能力をそれぞれ8回受精させるため、中腸、脚、卵巣、唾液腺などの個々の組織をアッセイできるように解剖される。しかし、唾液腺にウイルスが存在することは、唾液腺脱出/出口バリア(SGEB)の証拠を与えられた1、2、4、5、7、9の伝達能力の決定的な証拠ではない。伝染能力を証明する標準的な方法は、感染の影響を受けやすい動物10、11、12への蚊の伝染のままである。しかし、多くのアルボウイルスでは、免疫不全マウスモデル13、14、15、16の使用が必要であることを考えると、この方法はしばしばコストが非常に高い。一般的に使用される代替手段は、逆転写ポリメラーゼ連鎖反応(RT-PCR)または感染性アッセイによって分析することができる蚊の唾液の採取であり、それぞれウイルスゲノムまたは感染性粒子の存在を実証する。このようなインビトロ唾液採取方法は、生体内給餌中に堆積したウイルスの量を12個過大評価または過小評価する可能性があり、そのようなデータは慎重に解釈しなければならないことを示している。しかし、インビトロ法は唾液中のウイルスの存在の観点から分析した場合に非常に価値が高く、感染の可能性を示す。
アルボウイルス性疾患の発生における蚊ベクターの役割を決定するための2つの主要なアプローチが存在する。最初の方法は、活動的な伝達18、19、20、21、22、23、24のコンテキストで蚊が収集されるフィールド監視を含みます。しかし、感染率が典型的には非常に低い(例えば、米国21の活動性ジカウイルス(ZIKV)循環地域における蚊の推定0.061%感染率)を考えると、潜在的な種の犯罪は、方法論25、26およびランダムな確率(例えば、1,600人の感染した個人をサンプリングする)によって大きく偏る可能性がある。.これを考慮して、特定の研究は、伝染に関与する蚊を正確にサンプリングするのに十分な生の数または種の多様性の両方で十分な蚊を獲得しない可能性があります。対照的に、ベクター能力分析は実験室で行われ、経口投与などのパラメータを厳密に制御することができます。蚊の感染と伝染能力の真の複雑さをフィールド環境で完全に表す能力はありませんが、これらの実験室評価はアーボビロ学の分野で強力なツールのままです。
いくつかの蚊種、集団、および方法27、28、29、30、31、32の様々なベクトル能力分析に基づいて、ベクター能力評価の最近のレビュー1、典型的なベクトル能力ワークフローに関連するプロトコルのいくつかをここで説明する。これらの実験では、南北アメリカ(サルバドール市、ブラジル、ドミニカ共和国、および米国の低リオグランデバレー、TX、米国)からの3つのAe.aegypti集団が、4、5、または6ログ10の焦点形成単位(FFU/m線量)でZIKV(Mex 1-7、GenBank加盟:KX247632.1)の単一株にさらされました。その後、外挿術の様々な時間(2、4、7、10、および14日間)の後に、解剖および細胞培養ベースの感染アッセイによって感染、播種感染、および伝染能力の証拠について分析した。現在のワークフロー/プロトコルはZIKV用に最適化されていますが、多くの要素は節足動物の封じ込めおよびバイオセーフティレベル2および3(ACL/BSL2またはACL/ BSL3)の他の蚊媒介性アルボウイルスに直接翻訳可能です。

<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>
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		<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>
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		<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>
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		<tr>
			<td>Bleach</td>
			<td>NC0427256</td>
			<td>FisherScientific</td>
			<td>Decontamination</td>
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		<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>
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		<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>
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		<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

提示された手順は、問題の蚊集団における感染率、播種感染、およびウイルスの潜在的な感染率を決定するために、実験室の条件下で Aedes aegypti 蚊をジカウイルスに感染させる一般的な方法論を記述する。これらの手順は、ベクター能力評価における様々な変更をグローバルに広く利用されています。彼らは、与えられた蚊(すなわち、種、集団、個体)が与えられた薬剤の伝達において果たす可能性のある役割を決定する上で重要である。

Aeの3つの集団。 アメリカ大陸(サルバドール、ブラジル、ドミニカ共和国、およびリオグランデバレー、TX、米国)からの aegypti は、血液をベースにした血液中のチター(4、5、および6ログ10 FFU/mL)の範囲にわたって、アメリカ大陸(ZIKV Mex 1-7、チアパス州、メキシコ、2015)からのZIKVの発生株にさらされました。2日目、4日目、7日目、10日目、14日目に、蚊のサブセットを処理して感?...

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

提示されたプロトコルは、封じ込めの設定で、ジカのような特定のウイルスに対する Aedes aegypti 蚊集団のベクター能力を決定することができる。

ジカウイルスを用いた Aedes aegypti蚊の ベクターコンピテンス分析

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

Research

JoVE Journal

Immunology and Infection

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

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

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

蚊の機能解析のためのカルシウムの生物発光アッセイ（ネッタイシマカ）とダニ（ Rhipicephalus microplus）Gタンパク質共役受容体

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

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

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

黄熱病の蚊から血球を収集し、染色のためのプロトコルネッタイシマカ</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

Vero細胞培養アッセイによるフィールドコレクト蚊から感染性ウイルスの検出

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

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

ハイブリダイゼーション<em&gt;その場で</em&gt;唾液腺、卵巣、および媒介蚊の胚の

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

ジカウイルス感染細胞培養系と<em&gt;インビトロ</emインターフェロンの&gt;予防効果

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

新世界Zikaウイルス感染クローンからの組換えウイルスの救出とキャラクタリゼーション

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

ジカ特定診断エピトープの探索

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

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

単純なフローサイトメトリーによるジカ回復期血清を用いたデング ウイルスのIn Vitro抗体依存強化測定法

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

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

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

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

細菌人工染色体cDNAクローンからの組換えジカウイルスの救出

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

ジカウイルスを用いた Aedes aegypti蚊の ベクターコンピテンス分析

ジカウイルスを用いた Aedes aegypti蚊のベクターコンピテンス分析