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 arbeidet ble støttet av National Key Basic Research Program of China (nr 2013CB127602) og Science Fund for kreativ forskning Grupper av Science Foundation National of China (nr 31321004).

1. Bygging av HaDV2 frie Cotton Bollworm Colony <ol><li> Bakre bomulls bollworm med larver (H. armigera) på en kunstig diett 32 i et kontrollert vekst rom eller en kunstig klimakammer ved 25 ± 1 ° C, med 14 timers lys / 10 timers mørke og 60% relativ fuktighet. </li><li> Hjelpe til enkeltparet parring av nylig eclosed møll ved hjelp av en plast bur pr par (10 cm høyde, diameter 5 cm) som er dekket med bomullsgas for å sikre god ventilasjon og for å tjene som et substrat egglegg...

<ol>
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I de siste tiårene har de fleste studier på insekt-virus interaksjoner fokusert på honningbie helse 34, 35, 36, vektorer av menneskelig sykdom 37, virus plante 38, og noen insekt patogene virus som har stort potensial som biologiske bekjempelsesmidler 39. Lite oppmerksomhet har blitt betalt til hemmelige virus i insekter, spesielt i sommerfugl-skadedyr. ...

I de siste tiårene, har utviklingen av sekvenseringsteknologi slik som neste generasjons sekvensering (NGS) lettes oppdagelsen av mange nye DNA og RNA-virus, spesielt ikke-patogene virus, men også nye isolater av tidligere kjente virus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. I modellorganisme Drosophila melanogaster, har mer enn 20 nye partielle virusgenomene blitt detektert ved bruk av teknikker metagenomic 13. Mange virussekvenser, inkludert nye virus, har også blitt identifisert i andre insekter, så som honningbier, mosquitoes, asiatisk sitrus psyllids, øyenstikkere, og flere sommerfugl-arter 14, 15, 16, 17, 18, 19, 20, 21. I fremtiden kan det forventes at flere nye virus blir oppdaget i insekter som bruker disse avanserte teknologier; dermed kan vår forståelse av virus og vert interaksjon endres tilsvarende 6, 9. For eksempel er virus-vert interaksjoner anses å være mer komplisert enn tidligere antatt, fordi mange nye virus blir definert som mutualistic partnere fremfor strenge patogener 22. For eksempel, den mutualistic densovirus DplDNV i Dysaphis plantaginea induserer den bevingede morf og<html> øker mobiliteten, som letter spredning av verten, så vel som viruset 23. Dessuten har mutualistic virus blitt beskrevet med hensyn til pattedyr helse, tørke og kulde toleranse av planter, og virkningen av bakterielle infeksjoner 24. Seneca dal virus-001 er vist å mediere selektiv cytotoksisitet mot tumorceller med nevroendokrine kreft funksjoner 25. Hepatitt A virusinfeksjoner undertrykke hepatitt C-virusreplikasjon, og kan føre til utvinning av hepatitt C-26. Herpesvirus latens confers symbiotisk beskyttelse mot bakteriell infeksjon 27. Konvolutten glykoprotein of human endogenous retrovirus Herv-W induserer cellulær resistens overfor milten nekrosevirus 28. Curvularia termisk toleranse virus (CThTV) fra en sopp endophyte er involvert i den mutualistic interaksjonen mellom denne soppen og en tropisk panikk gressref &quot;&gt; 29. Derfor er kunnskap om samspillet mellom de nylig funnet virus og deres verter skal generere nye perspektiver på deres biologi og forvaltning. Men nye virus, spesielt de skjulte virus viser ingen åpenbare tegn typisk for akutt infeksjon, har sjelden vært etterforsket, og vi trenger en rørledning og protokoller for å undersøke konsekvensene av de nylig funnet virus på sine verter. Tidligere har vi rapportert prevalens en ny monosense densovirus pestfagerfly densovirus (HaDV2) i bomull bollworm, pestfagerfly, og presenterte bevis for en mutualistic forholdet mellom HaDV2 og bomull bollworm 30, 31. I denne artikkelen vil vi beskrive laboratoriet protokollen til å studere i detalj samspillet mellom HaDV2 og dens bomull bollworm vert. Protokollen som presenteres her kan også være svært relevant for forskere undersøker role andre oralt overfør virus, spesielt i sommerfugl-skadedyr.

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

Mange nye virus har blitt oppdaget i dyre verter som bruker neste generasjons sekvensering teknologi. Tidligere rapporterte vi en mutualistic virus, pestfagerfly densovirus (HaDV2), i en sommerfugl-arter, bomull bollworm, pestfagerfly (Hubner). Her beskriver vi protokollene som er brukt for å studere effekten av HaDV2 på sin vert. Først etablerer vi en HaDV2 fritt bomull bollworm koloni fra en enkelt avl par. Da vi oral inokulering av noen nyfødte larver avkom med HaDV2 holdig filtrerte væske for å fremstille to kolonier med det samme genetiske bakgrunn: en HaDV2-infiserte, ikke-infiserte den andre. En protokoll for å sammenligne liv tabell parametere (f.eks larve, puppe, og voksen perioder og fecundity) mellom de HaDV2-infiserte individer og -uninfected blir også presentert, som er protokollene for å bestemme vertsvevet fordeling og overføring effektivitet av HaDV2. Disse protokollene would også være egnet for undersøkelse av virkningene av andre oralt overfør virus på sine insekt verter, sommerfugl-verter i særdeleshet.

Progenies fra foreldrene som ble HaDV2-fri (figur 3A) er blitt oppdrettet som NONINF-stammen. Vi lykkes forsterket aktingenet med de samme DNA-maler, noe som tyder på at DNA-maler var av god kvalitet (figur 3B). I tillegg er de tilfeldig utvalgte åtte progenies var også fri for HaDV2 (figur 3C), HaNPV (figur 3D), og Wolbachia (figur 3E). Igjen, konkluderte vi at DNA-prøvene av avkom...

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

Her presenterer vi en protokoll for å undersøke vertsvevet distribusjon, overføring modus, og effekt på verts egnethet av en densovirus løpet av sommerfugl-arter, bomull bollworm. Denne protokollen kan også bli anvendt for å studere interaksjonen mellom andre oralt overfør virus og deres insekt verter.

Protokoller for Gransker Host-vev distribusjon, overføring-modus, og innvirkning på vertsmaskinen Fitness av en Densovirus i Cotton Bollworm

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

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