Name: propagation of homalodisca coagulata virus-01 via homalodisca vitripennis cell culture
Uploaded: 2014-09-25T15:00:00
Description: Watch this Scientific Journal Video about Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture at JoVE.com

We would like to thank the Texas Pierce&#8217;s Disease Research and Education Program and USDA-APHIS for their funding support for this project. We would also like to thank Hema Kothari at the University of Texas Health Science Center at Tyler for her assistance with confocal microscopy.

1. Cell Culture MERK: Homalodisca vitripennis cellelinjer fastsatt av Dr. Wayne Hunter laboratorium ved USDA Agricultural Research Service (. Ft Pierce, FL USA) ble brukt til å sette i gang en lab lager består av blandede celle stadier inkludert innledende fibroblaster og monolagene. <ol><li> Utfør de følgende fremgangsmåter i et sterilt laboratoriemiljøet opprettholdt ved en temperatur i området 20-24 ° C med 25 cm to kulturflasker. </li><li> Dyrke og vedlikeho...

<ol>
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Stigende bekymringer angående tilstrømningen av invasive landbruks arter har ført til en økt etterspørsel etter nye metoder for å forsvare seg mot nye skadedyr og patogener. Et fokus på sykdomsforebygging og ledelse innebærer ledelse av patogen vektorer og var den primære målet for denne studien. Økonomi spiller en viktig rolle i beslutningen om å produsere denne typen biopesticide å administrere patogen vektorer i landbruket fordi den praktiske anvendelsen må være store mengder over store områder, men t...

Den glassaktige vinger skarpskytter (Homalodisca vitripennis Germar 1821) har blitt identifisert som den dominerende vektor av Xylella fastidiosa (X. fastidiosa), den kausale agent Pierce sykdom i Grapevine (PD) i Nord-Amerika en. Insekt befolkningen ledelsen har raskt blitt fokus for forskning for å bekjempe denne ødeleggende problemet til vindyrking industrien i California og på tvers av det sørlige USA. En positiv-sense, single-strandet RNA-virus som tilhører familien Dicistroviridae, Homalodisca coagulata virus-01 (HoCV-01) har blitt identifisert i vill H. vitripennis populasjoner og vist seg å øke dødeligheten i disse populasjonene 2-4, mens du senker insekt motstand mot insektmidler. Utvikling av metoder for å effektivt bak smittet H. vitripennis til voksen i et laboratorium innstilling har vært vanskelig fordi <em&gt; H. vitripennis har ulike sceniske spesifikke ernæringsmessige behov som krever en rekke vertsplanter 5-8. Spesifikke anlegg er pålagt å bakre levende H. vitripennis i USA; Derfor, er cellekultur mer økonomisk og et levedyktig alternativ, så vel som i økende grad viktig for HoCV-01 deteksjon og replikasjon 2,9. Mens grunnleggende metoder for etablering av cellekulturer fra H. vitripennis er beskrevet, er disse metodene har enda ikke blitt anvendt for kommersiell produksjon av biologiske bekjempelsesmidler, for eksempel virus 2. Det overordnede målet med følgende prosedyrer er å produsere en høy konsentrasjon av HoCV-01 egnet for utnyttelse som en biologisk kontroll agent. Virusreplikasjon krever en levende celle, som er grunnen vellykket dyrking og optimalisere H. vitripennis kulturer er avgjørende for fremdriften av å produsere lønnsomme nivåer av virus.

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:93px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning&#160;cell culture flasks</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>CLS430168</td>
			<td>Surface area 25&#160;cm2, canted neck, cap (plug seal)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Olympus DP30BW, IX2-SP, IX71</td>
			<td style="width:93px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Inverted microscope and camera</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>T4049</td>
			<td>0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2&#160;g EDTA &#8226; 4Na per liter of Hanks&#8242; Balanced Salt Solution with phenol red</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Greiner CELLSTAR&#160;multiwell culture plates</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>M8937</td>
			<td>48 wells (TC treated with lid)</td>
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		<tr height="63">
			<td height="63" style="height:63px;">DETCA&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>228680</td>
			<td style="width:167px;">Sodium diethyldithiocarbamate trihydrate</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning&#160;bottle-top vacuum filter system</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>CLS431206</td>
			<td>Cellulose acetate membrane, pore size 0.45&#160;&#956;m, membrane area 54.5&#160;cm2, filter capacity 500&#160;mL</td>
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			<td height="42" style="height:42px;">Brij 52</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>388831</td>
			<td>Polyethylene glycol hexadecyl ether</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate buffer solution</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>P5244</td>
			<td>Recieved as 100mM diluted to 10mM with sterile water</td>
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		<tr height="63">
			<td height="63" style="height:63px;">TRIzol LS&#160;</td>
			<td style="width:93px;">Life Technologies</td>
			<td>10296-028</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agarose</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>A5304</td>
			<td>For electrophoresis</td>
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		<tr height="42">
			<td height="42" style="height:42px;">Ethidium bromide</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>E7637</td>
			<td>BioReagent, for molecular biology, powder</td>
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		<tr height="21">
			<td height="21" style="height:21px;">QIAquick</td>
			<td style="width:93px;">Qiagen</td>
			<td>28704</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">QuantiTect qRT-PCR kit&#160;</td>
			<td style="width:93px;">Qiagen</td>
			<td>204243</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">4% paraformaldehyde&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>P6148&#160;</td>
			<td>Reagent grade, crystalline</td>
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		<tr height="42">
			<td height="42" style="height:42px;">PBS&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>P5368</td>
			<td style="width:167px;">Phosphate buffered saline</td>
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		<tr height="42">
			<td height="42" style="height:42px;">Triton X-100&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bovine serum albumin (BSA)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Rhodamine red-conjugated phalloidin&#160;</td>
			<td style="width:93px;">Life Technologies</td>
			<td>R415</td>
			<td>Rhodamine phalloidin is a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine</td>
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			<td height="42" style="height:42px;">DAPI&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>D9542</td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;">ProLong Gold Antifade Reagent</td>
			<td style="width:93px;">Life Technologies</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM510 Meta Confocal System&#160;</td>
			<td style="width:93px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM Zen 2007 Software</td>
			<td style="width:93px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Grace&#8217;s Insect medium (supplemented, 1X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>G8142</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-histidine monohydrate</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>H8125</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium 199 (10X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>M4530</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium 1066 (1X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">C0422</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hank&#8217;s Balanced Salts (1X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>51322C</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-Glutamine (100X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>G3126</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM, amino acid mix (50X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>56419C</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1 M MgCl solution</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>M8266</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pen-Strep (w/ Glutamine)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>G6784</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nystatin</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>N6261</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gentamycin</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>46305</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextrose</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>D9434</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>F2442</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
	</tbody>
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propagation of homalodisca coagulata virus-01 via homalodisca vitripennis cell culture

Den glassaktige vinger skarpskytter (Homalodisca vitripennis) er en svært vagile og polyphagous insekt som finnes i hele det sørvestlige USA. Disse insektene er de dominerende vektorer av Xylella fastidiosa (X. fastidiosa), en xylem begrenset bakterie som er den kausale agent Pierce sykdom (PD) i Grapevine. Pierce sykdom er økonomisk ødeleggende; således, H. vitripennis har blitt et mål for patogen forvaltningsstrategier. En dicistrovirus identifisert som Homalodisca coagulata virus-01 (HoCV-01) har vært assosiert med økt dødelighet hos H. vitripennis populasjoner. Fordi en vertscelle er nødvendig for HoCV-01 replikasjon, gir en ensartet cellekultur miljø for målrettet replikasjon som er logistisk og økonomisk verdifulle for biopesticide produksjon. I denne studien, et system for storskala utbredelse av H. vitripennis celler via vev kultur ble utviklet, providing en virusreplikasjon mekanisme. HoCV-01 ble ekstrahert fra hele kroppen insekter og brukt til å inokulere kultivert H. vitripennis celler på forskjellige nivåer. Kulturmediet ble fjernet, hver 24 timer i 168 timer, RNA ble ekstrahert og analysert med QRT-PCR. Cellene ble farget med trypanblått og telles å kvantifisere celleoverlevelsesevne ved hjelp av lys mikroskopi. Hele viruspartikler ble ekstrahert opp til 96 timer etter infeksjon, som var tiden punktet bestemt til å være før total cellekultur sammenbrudd oppsto. Celler ble også utsatt for fluorescerende farging og vises ved hjelp av konfokal mikroskopi for å undersøke viral aktivitet på F-aktin feste og kjerner integritet. Konklusjonen av denne studien er at H. vitripennis celler er i stand til å bli dyrket, og som brukes for masseproduksjon av HoCV-01 ved et passende nivå for å tillate produksjon av en biopesticide.

Cellebinding og vekst ble sett innen 48 timer av passasje i både små og store kulturflasker, fra primærkulturer og fortsatt passasjer. Fibroblast vekst og utvikling ble også observert innenfor denne tidsrammen. Når nylig seeded kolber ble forstyrret før 48 timer, var det en synlig nedgang i cellebinding, som fører til langsommere voksende kulturer og noen ganger ikke noe feste eller vekst i det hele tatt. Celler som var omtrent 80% konfluent innen en uke etter passer og dannet et monosjikt i 10-14 dager (...

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Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Utbredelsen av<em&gt; Homalodisca coagulata virus-01</em&gt; Via<em&gt; Homalodisca vitripennis</em&gt; Cell Culture

Her presenterer vi en protokoll for å forplante Homalodisca vitripennis celler og HoCV-1 in vitro. Medium ble fjernet fra HoCV-1 positive kulturer og RNA ekstrahert hver 24. time i 168 timer. Cell overlevelsesevne ble kvantifisert ved trypanblått farging. Hele viruspartikler ble ekstrahert etter infeksjon. Ekstrahert RNA ble kvantifisert ved QRT-PCR.

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

Research

JoVE Journal

Immunology and Infection

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

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