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

Dette arbejde blev støttet af National Key Basic Research Program for Kina (nr 2013CB127602) og Science Fund for Creative Research Grupper af National Science Foundation of China (nr 31.321.004).

1. Konstruktion af HaDV2-fri bomuldsfrøkapselorm Colony <ol><li> Bageste bomuldsfrøkapselorm larver (H. armigera) på en kunstig diæt 32 på kontrolleret vækst rum eller et kunstigt klimakammer ved 25 ± 1 ° C, med 14 h lys / 10 timer mørke og 60% relativ fugtighed. </li><li> Sag single-pair parring af nyligt eclosed møl ved anvendelse af et plastbur per par (10 cm højde, 5 cm i diameter), dækket med bomuldsgaze at sikre god ventilation og at tjene som en oviposition substrat....

<ol>
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I de seneste årtier har de fleste undersøgelser af insekt-virus interaktioner med fokus på honningbien sundhed 34, 35, 36, vektorer for sygdomme hos mennesker 37, plante virus 38, og nogle insekt sygdomsfremkaldende vira, der har et stort potentiale som biologiske bekæmpelsesmidler 39. Lidt opmærksomhed er blevet betalt til hemmelige virus i insekter, ...

I de seneste årtier har udviklingen af sekventering teknologi såsom næste generation sekventering (NGS) lettet opdagelsen af mange hidtil ukendte DNA og RNA-vira, især ikke-patogene vira, men også hidtil ukendte isolater af tidligere kendte virus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. I modelorganismen Drosophila melanogaster er der påvist mere end 20 nye partielle virusgenomer anvendelse metagenomisk teknikker 13. Mange virale sekvenser, herunder hidtil ukendte vira, er også blevet identificeret i andre insekter, såsom honningbier mosquitoes, asiatiske citrus psyllids, guldsmede, og flere lepidoptera arter 14, 15, 16, 17, 18, 19, 20, 21. I fremtiden kan det forventes, at flere nye virus vil blive opdaget i insekter ved hjælp af disse avancerede teknologier; derfor kan vores forståelse af virus-vært interaktion ændres tilsvarende 6, 9. For eksempel er virus-værtssammenspil anses for at være mere kompliceret end hidtil antaget, fordi mange hidtil ukendte vira defineres som mutualistic partnere snarere end strenge patogener 22. For eksempel mutualistisk densovirus DplDNV i Dysaphis plantaginea inducerer vingede morph og<html> øger mobiliteten, lette spredning af værten samt virus 23. Desuden har mutualistic vira blevet beskrevet med hensyn til pattedyr sundhed, tørke og kulde tolerance af planter, og virkningen af bakterieinfektioner 24. Seneca dal virus-001 er vist at mediere selektiv cytotoksicitet i retning tumorceller med neuroendokrin kræft features 25. Hepatitis A virus infektioner undertrykke hepatitis C-virus-replikation og kan føre til bedring fra hepatitis C 26. Herpesvirus latens bibringer symbiotisk beskyttelse mod bakterieinfektion 27. Kappeglycoproteinet af human endogen retrovirus HERV-W inducerer cellulær resistens over for miltnekrosevirus 28. Curvularia termisk tolerance virus (CThTV) fra en fungal endofyt er involveret i mutualistisk interaktionen mellem denne svamp og et tropisk panik græsref &quot;&gt; 29. Derfor kendskab til samspillet mellem de nyligt fundne vira og deres værter skal generere nye perspektiver på deres biologi og ledelse. Men den nye virus, især de hemmelige virus viser ingen tydelige tegn typiske for akut infektion, har sjældent været undersøgt, og vi har brug for en rørledning og protokoller til at undersøge virkningerne af de nyligt fundne virus på deres værter. Vi har tidligere rapporteret forekomsten af en ny monosense densovirus Helicoverpa armigera densovirus (HaDV2) i bomuldsfrøkapselorm, Helicoverpa armigera, og præsenterede beviser på en mutualistisk forhold mellem HaDV2 og bomuldsfrøkapselorm 30, 31. I dette papir vil vi beskrive laboratorieprotokol at studere i detaljer interaktionen mellem HaDV2 og dens bomuldsfrøkapselorm vært. Protokollen præsenteres her, kan også være yderst relevante for forskere undersøger role andre oralt overførte vira, især i skadelige sommerfugle.

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	<tbody>
		<tr>
			<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>
		</tr>
		<tr>
			<td>thermal cycler</td>
			<td>Veriti; Applied Biosystems</td>
			<td>4375786</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m membrane filter</td>
			<td>Millipore</td>
			<td>SLGS025NB</td>
			<td></td>
		</tr>
		<tr>
			<td>pEASY-T Cloning Vector</td>
			<td>TransGen, Beijing, China</td>
			<td>CT301-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>IDEAL-TEK</td>
			<td>2.SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Premix Ex Taq (Probe qPCR)</td>
			<td>Takara</td>
			<td>RR390A</td>
			<td></td>
		</tr>
		<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>
		<tr>
			<td>sucrose</td>
			<td></td>
			<td></td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr>
			<td>vitamin complex</td>
			<td></td>
			<td></td>
			<td>Multiple suppliers available.</td>
		</tr>
	</tbody>
</table>

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

Mange nye vira er blevet opdaget i animalske værter ved hjælp af næste generation sekventering teknologier. Tidligere har vi rapporteret en mutualistisk virus, Helicoverpa armigera densovirus (HaDV2), i en sommerfugle arter, den bomuldsfrøkapselorm, Helicoverpa armigera (Hubner). Her beskriver vi de protokoller, der i øjeblikket anvendes til at undersøge effekten af ​​HaDV2 på sin vært. Først, vi etablere en HaDV2-fri bomuldsfrøkapselorm koloni fra et enkelt ynglende par. Derefter, vi oralt pode nogle nyfødte larve afkom med HaDV2-holdig filtrerede væske at frembringe to kolonier med samme genetiske baggrund: en HaDV2-smittet, anden ikke-inficerede. En protokol til at sammenligne livet tabelparametrene (fx larvestadiet, puppe og voksne perioder og frugtbarhed) mellem HaDV2-inficerede og -uninfected individer ligeledes vist, som er protokollerne for bestemmelse af værten-vævsfordeling og sendeeffektivitet HaDV2. Disse protokoller WOuld også være egnet til at undersøge virkningerne af andre oralt overførte virus på deres insekt værter, lepidoptera værter i særdeleshed.

Afkom fra forældre, der befandt HaDV2-fri (figur 3A) blev opdrættet som NONINF-stamme. Vi med succes amplificeret actin-genet ved anvendelse af de samme DNA-templates, hvilket antyder, at DNA-templates var af god kvalitet (figur 3B). Derudover tilfældigt udvalgte otte afkom var også fri for HaDV2 (figur 3C), HaNPV (figur 3D), og Wolbachia (figur 3E). Igen, konkluderede vi, at DNA-pr...

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

Her præsenterer vi en protokol til at undersøge vært-væv distribution, transmission tilstand og effekt på værten egnethed af en densovirus inden for sommerfugle arter, den bomuldsfrøkapselorm. Denne protokol kan også anvendes til undersøgelse af interaktionen mellem andre oralt overførte vira og deres insekt værter.

Protokoller til Undersøgelse af Host-væv Distribution, Transmission-tilstand, og Effekt på Host Fitness af en densovirus i bomuldsfrøkapselorm

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

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