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. Cellekultur BEMÆRK: Homalodisca vitripennis cellelinjer oprettet af Dr. Wayne Hunter laboratorium på USDA Agricultural Research Service (. Ft Pierce USA) blev anvendt til at indlede en lab lager bestående af blandede celle faser, herunder indledende fibroblaster og monolag. <ol><li> Udfør følgende procedurer i et sterilt laboratorium miljø holdes ved en temperatur i området 20-24 ° C med 25 cm 2 dyrkningskolber. </li><li> Dyrk og vedligeholde kulturer i 25 <s...

<ol>
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Stigende bekymringer vedrørende tilstrømningen af ​​invasive arter af afgrøder, har ført til en øget efterspørgsel efter nye metoder til at forsvare sig mod nye skadedyr og patogener. Fokus forebyggelse og håndtering sygdommen indebærer håndtering af patogene vektorer og var det primære mål for denne undersøgelse. Økonomi spiller en afgørende rolle i beslutningen om at producere denne type biopesticid at styre patogene vektorer i landbruget, fordi den praktiske anvendelse skal være store mængder over...

Den glasagtige-vingede skarpskytte (Homalodisca vitripennis Germar 1821) er blevet identificeret som den fremherskende vektor af Xylella fastidiosa (X. fastidiosa), det agens af Pierces sygdom i Grapevine (PD) i Nordamerika 1. Insekt befolkning ledelse er hurtigt blevet fokus for forskning til bekæmpelse af denne ødelæggende problem for vinavl industrien i Californien og på tværs af sydlige USA. En positiv-sense, enkeltstrenget RNA-virus, der tilhører familien Dicistroviridae, Homalodisca coagulata virus-01 (HoCV-01) er blevet identificeret i vild H. vitripennis befolkninger og vist sig at øge dødeligheden i disse populationer 2-4, og samtidig sænke insektets resistens over for insekticider. Udvikling af metoder til effektivt bag inficeret H. vitripennis til fuldvoksen i et laboratorium indstilling har været vanskeligt, fordi <em&gt; H. vitripennis har forskellige trinspecifik ernæringsmæssige behov, som kræver en bred vifte af værtsplanter 5-8. Særlige faciliteter er forpligtet til bageste levende H. vitripennis i USA; Derfor cellekultur er mere økonomisk og et levedygtigt alternativ, samt i stigende grad afgørende for HoCV-01 detektion og replikation 2,9. Mens grundlæggende metoder til oprettelse af cellekulturer af H. vitripennis er beskrevet, er endnu ikke blevet udnyttet disse metoder til kommerciel produktion af biologiske bekæmpelsesmidler, som virus 2. Det overordnede mål med følgende procedurer er at producere en høj koncentration af HoCV-01 er egnet til anvendelse som et biologisk bekæmpelsesmiddel. Viral replikation kræver en levende celle, hvilket er grunden til succes dyrke og optimere H. vitripennis kulturer er afgørende for fremskridt producere rentable niveauer af 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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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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		<tr height="42">
			<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>
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		<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>
		</tr>
		<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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		<tr height="42">
			<td height="42" style="height:42px;">DAPI&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<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 glasagtige-vingede skarpskytte (Homalodisca vitripennis) er en yderst vagile og polyphagous insekt fundet i hele det sydvestlige USA. Disse insekter er de fremherskende vektorer for Xylella fastidiosa (X. fastidiosa), en veddet begrænset bakterie, der er det agens af Pierce sygdom (PD) for Grapevine. Pierce sygdom er økonomisk skadelig; således, H. vitripennis er blevet et mål for patogen ledelsesstrategier. En dicistrovirus identificeret som Homalodisca coagulata virus-01 (HoCV-01) er blevet forbundet med en øget dødelighed i H. vitripennis befolkninger. Fordi en værtscelle er nødvendig for HoCV-01 replikation, cellekultur giver et ensartet miljø for målrettet replikation, som er logistisk og økonomisk værdifulde for biopesticid produktion. I denne undersøgelse, et system for storstilet udbredelse af H. vitripennis celler via vævskultur blev udviklet, sroviding en viral replikation mekanisme. HoCV-01 blev ekstraheret fra hele kroppen insekter og anvendt til at inokulere dyrkede H. vitripennis celler med forskellige niveauer. Dyrkningsmediet blev fjernet hver 24 timer til 168 timer, RNA ekstraheret og analyseret med QRT-PCR. Celler blev farvet med trypanblåt og talt for at kvantificere celle overlevelsesevne ved hjælp af lysmikroskopi. Hele viruspartikler blev ekstraheret op til 96 timer efter infektion, hvilket var den tid, der bestemmes til at være, før den samlede cellekultur sammenbrud forekom. Cellerne blev også udsat for fluorescensfarvede og vises ved hjælp af konfokal mikroskopi til at undersøge viral aktivitet på F-actin tilknytning og kerner integritet. Konklusionen på denne undersøgelse er, at H. vitripennis celler er i stand til at blive dyrket og anvendt til masseproduktion af HoCV-01 på et passende niveau for at muliggøre produktionen af en biopesticid.

Cellebinding og vækst sås inden for 48 timer af passage i både små og store dyrkningskolber, fra primære kulturer og fortsatte passager. Fibroblast vækst og udvikling blev også observeret inden for denne tidsramme. Når nyligt seedede kolber blev forstyrret før 48 timer, var der en synlig nedgang i celle vedhæftet fil, der fører til langsommere voksende kulturer og undertiden ingen tilknytning eller vækst overhovedet. Cellerne var omtrent 80% sammenflydende inden for en uge passerer og dannede et monolag i 10...

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

Formering af<em&gt; Homalodisca coagulata virus-01</em&gt; Via<em&gt; Homalodisca vitripennis</em&gt; Cell Culture

Her præsenterer vi en protokol til at udbrede Homalodisca vitripennis celler og HoCV-1 in vitro. Medium blev fjernet fra HoCV-1-positive kulturer og RNA ekstraheres hver 24 timer til 168 timer. Cell overlevelsesevne blev kvantificeret ved trypanblåt-farvning. Hele viruspartikler blev ekstraheret efter infektion. Ekstraheret RNA blev kvantificeret 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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