Name: protocols for investigating the host-tissue distribution, transmission-mode, and effect on the host fitness of a densovirus in the cotton bollworm
Uploaded: 2017-04-12T15:00:00
Description: Watch this Scientific Journal Video about Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm at JoVE.com

Dit werk werd ondersteund door de National Key Basic Research Program van China (Nr 2013CB127602) en de Wetenschap Fonds voor Creative Onderzoeksgroepen van de National Science Foundation of China (nr 31321004).

1. De bouw van de HaDV2-vrije katoenkever Colony <ol><li> Achterste katoenbolworm larven (H. armigera) op een kunstmatig dieet 32 gecontroleerde groeikamer of kunstmatige klimaatkamer bij 25 ± 1 ° C met 14 uur licht / 10 uur donker en 60% relatieve vochtigheid. </li><li> Steun enkelpaars paring nieuw Bijlage In motten door een plastic kooi per paar (10 cm hoog, diameter 5 cm) bedekt met een gaasje een goede ventilatie en dienen als een substraat ovipositie. Om de kans op het verkrijg...

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In de afgelopen decennia meeste studies over insecten virus interacties gericht op honingbij gezondheid 34, 35, 36, vectoren voor humane ziekten 37, plantenvirussen 38 Sommige insecten pathogene virussen die een groot potentieel als biologische controlemiddelen 39 zijn. Weinig aandacht is besteed aan geheime virussen bij insecten, vooral in lepidoptera. Hie...

In de afgelopen decennia is de ontwikkeling van sequentietechnologie zoals next-generation sequencing (NGS) vergemakkelijkt de ontdekking van vele nieuwe DNA- en RNA-virussen, met name niet-pathogene virussen, maar ook nieuwe isolaten van eerder bekende virussen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. In het modelorganisme Drosophila melanogaster, zijn meer dan 20 deelinfusie virusgenomen gedetecteerd middels metagenomic 13 technieken. Vele virale sequenties, inclusief nieuwe virussen zijn ook geïdentificeerd in andere insecten zoals bijen, mosquitoes, Aziatisch citrus bladvlooien, libellen, en meerdere soorten lepidoptera 14, 15, 16, 17, 18, 19, 20, 21. In de toekomst kan worden verwacht dat er meer nieuwe virussen zullen worden ontdekt bij insecten het gebruik van deze geavanceerde technologieën; derhalve kunnen ons begrip van de virus-gastheer interacties dienovereenkomstig veranderen 6, 9. Zo worden bijvoorbeeld virus-gastheer interacties beschouwd ingewikkelder dan eerder gedacht te zijn, omdat veel nieuwe virussen worden gedefinieerd als mutualistische partners in plaats van strikte ziekteverwekkers 22. Bijvoorbeeld, de mutualistische densovirus DplDNV in Dysaphis plantaginea induceert de gevleugelde morph en<html> vergroot de mobiliteit, vergemakkelijkt de verspreiding van de gastheer en het virus 23. Bovendien zijn mutualistic virussen beschreven met betrekking tot de gezondheid van zoogdieren, de droogte en koude tolerantie van planten, en de effecten van bacteriële infecties 24. Seneca valley virus-001 is aangetoond dat selectieve cytotoxiciteit mediëren richting tumorcellen met neuroendocriene kanker kenmerkt 25. Hepatitis A virusinfecties te onderdrukken hepatitis C virus replicatie en kunnen leiden tot terugwinning van hepatitis C 26. Herpesvirus latency verleent symbiotische bescherming tegen bacteriële infectie 27. De omhullende glycoproteïne van humaan endogeen retrovirus HERV-W induceert cellulaire resistentie tegen miltnecrosevirus 28. Curvularia thermische tolerantie virus (CThTV) vanaf een schimmel endofiet is betrokken bij de symbiotische interactie tussen deze schimmel en tropische paniek grasref &quot;&gt; 29. Vandaar dat de kennis van de interacties tussen de nieuw gevonden virussen en hun gastheren moeten nieuwe perspectieven op hun biologie en het management te genereren. Echter, de nieuwe virussen, in het bijzonder de geheime virussen tonen van geen duidelijke tekenen een typisch voorbeeld van een acute infectie, hebben zelden onderzocht, en we moeten een pijpleiding en protocollen om de gevolgen van de nieuw gevonden virussen te onderzoeken op hun gastheren. Eerder hebben we de prevalentie een nieuwe monosense densovirus Katoendaguil densovirus (HaDV2) in de katoenkever, Katoendaguil gemeld, en presenteerde het bewijs van een mutualistische relatie tussen HaDV2 en de katoenkever 30, 31. In dit artikel, zullen wij laboratoriumprotocol te bestuderen in detail de interactie tussen HaDV2 en katoenkever gastheer beschrijven. De hier gepresenteerde protocol kan ook zeer relevant voor onderzoekers onderzoeken r zijnole andere oraal overdraagbare virussen, in het bijzonder in lepidoptera.

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			<td>24-well plate</td>
			<td>Corning</td>
			<td>07-200-740</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr>
			<td>DNA extraction kit</td>
			<td>TIANGEN</td>
			<td>DP304-03</td>
			<td>Multiple suppliers available.</td>
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		<tr>
			<td>thermal cycler</td>
			<td>Veriti; Applied Biosystems</td>
			<td>4375786</td>
			<td></td>
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			<td>PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
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			<td>0.22 &#181;m membrane filter</td>
			<td>Millipore</td>
			<td>SLGS025NB</td>
			<td></td>
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			<td>pEASY-T Cloning Vector</td>
			<td>TransGen, Beijing, China</td>
			<td>CT301-02</td>
			<td></td>
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			<td>Tweezers</td>
			<td>IDEAL-TEK</td>
			<td>2.SA</td>
			<td></td>
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			<td>Premix Ex Taq (Probe qPCR)</td>
			<td>Takara</td>
			<td>RR390A</td>
			<td></td>
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		<tr>
			<td>Probes</td>
			<td>Invitrogen</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Invitrogen</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>microspectrophotometry NanoDrop 2000c&#160;</td>
			<td>Thermo scientific&#160;</td>
			<td>not available</td>
			<td></td>
		</tr>
		<tr>
			<td>7500 Real-Time PCR system</td>
			<td>Applied Biosystems</td>
			<td>not available</td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope SZX-16</td>
			<td>Olympus</td>
			<td>not available</td>
			<td></td>
		</tr>
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			<td>sucrose</td>
			<td></td>
			<td></td>
			<td>Multiple suppliers available.</td>
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			<td>vitamin complex</td>
			<td></td>
			<td></td>
			<td>Multiple suppliers available.</td>
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	</tbody>
</table>

protocols for investigating the host-tissue distribution, transmission-mode, and effect on the host fitness of a densovirus in the cotton bollworm

Veel nieuwe virussen zijn ontdekt in dierlijke gastheren met behulp van next-generation sequencing technologieën. Eerder berichtten we een mutualistische virus, Katoendaguil densovirus (HaDV2), in een lepidoptera soort, de katoenkever, Katoendaguil (Hubner). Hier beschrijven we de protocollen die momenteel worden gebruikt om het effect van HaDV2 op zijn gastheer te bestuderen. Ten eerste hebben we de oprichting van een HaDV2-vrije katoenkever kolonie van een enkel kweekkoppel. Dan hebben we oraal inoculeren enkele neonaat larvale nakomelingen met HaDV2 bevattende gefiltreerde vloeistof twee kolonies met dezelfde genetische achtergrond te produceren: een HaDV2 geïnfecteerde, geïnfecteerde andere. Een protocol om overlevingstafel parameters (bijvoorbeeld larven, popstadium en volwassen perioden en vruchtbaarheid) vergelijk tussen de HaDV2-geïnfecteerde individuen en -uninfected wordt ook getoond, evenals de protocollen voor het bepalen van de host-weefselverdeling en overdrachtsrendement van HaDV2. Deze protocollen would ook geschikt voor het onderzoeken van de effecten van andere mondeling overgedragen virussen op hun insect gastheren, lepidoptera gastheren in het bijzonder.

Nakomelingen van de ouders die HaDV2 free (figuur 3A) werden opgefokt als NONINF-stam. We met succes geamplificeerd het actine-gen met gelijke DNA templates, wat suggereert dat de DNA-matrijzen waren van goede kwaliteit (Figuur 3B). Bovendien, de willekeurig geselecteerde acht nakomelingen waren ook vrij van HaDV2 (figuur 3C), HaNPV (Figuur 3D) en Wolbachia (figuur 3E). Nogmaals, we gec...

Watch this Scientific Journal Video about Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm at JoVE.com

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm (Video) | JoVE

Hier presenteren we een protocol om de host-weefselverdeling, transmissiemodus en gevolgen voor de gastheer geschiktheid van een densovirus binnen Lepidoptera species, de katoenkever onderzoeken. Dit protocol kan ook worden gebruikt voor het bestuderen van de interactie tussen andere oraal overdraagbare virussen en insecten hun gastheer.

Protocollen voor het onderzoeken van de host-weefsel Distribution, transmissie-mode, en het effect op de Host Geschiktheid van een Densovirus in de katoenkever

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

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