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
NOTE: Homalodisca vitripennis cell lines established by the Dr. Wayne Hunter laboratory at the USDA Agricultural Research Service (Ft. Pierce, FL USA) were used to initiate a lab stock composed of mixed cell stages including initial fibroblasts and monolayers.
<ol>
	<li>Perform the following procedures in a sterile laboratory environment maintained at a temperature range of 20-24 &#176;C with 25 cm2 culture flasks.</li>
	<li>Cultivate and maintain cultures in 25 cm2 tissue culture flasks using H2G+ Leafhopper medium, a modified WH2 honeybee media10 (Table 1).</li>
	<li>Incubate culture flasks at 24 &#176;C with ~53% humidity.</li>
	<li>Use an inverted microscope at a total magnification (TM) of 100X to monitor culture growth, e.g., check for any abnormal cellular growth, monitor for any potential contaminants, and check growth progress across the culture surface.</li>
	<li>Perform a complete medium change (~4 ml culture medium per flask) every 7 - 10 days without disturbing the culture surface.</li>
	<li>Pass cultures when the culture surface is approximately 80% covered by cell growth (confluent) using 0.25% trypsin containing ethylenediaminetetraacetic acid (EDTA) to dissociate cells. Add ~2 - 3 ml of trypsin to each culture flask and expose culture(s) to the enzyme for short periods of time (5 - 10 min) to achieve complete cell dissociation.</li>
	<li>Add an equal amount of fresh medium to the culture flask(s) (~2 - 3 ml) to stop enzyme activity and transfer the whole solution to a conical tube for centrifugation.</li>
	<li>Pellet cells by centrifugation at 4 &#176;C for 6 min at 350 x g.</li>
	<li>Remove the supernatant without disturbing the cell pellet and add ~8 ml of fresh medium to each tube. Gently homogenize cells using a pipette.</li>
	<li>Split cultures at a 1:2 ratio for 25 cm2 flasks (~4 ml of cell solution per flask) and leave freshly passed cultures undisturbed for 48 hr to allow cells in suspension to attach securely to the surface of the flask. 
	NOTE: Cultivation of cells in 48-well sterile tissue culture plates with a growth surface of 1 cm2 is also possible and they can be maintained in the same manner as culture flasks with a reduction in volume of culture medium to ~250 &#956;l.</li>
</ol>
2. Whole Virus Extraction
<ol>
	<li>Homogenize whole bodies of virus positive H. vitripennis in phosphate buffer, pH ~7.2, with 0.02% sodium diethyldithiocarbamate trihydrate (DETCA) by vortexing for ~10 sec intervals until there are no more large clumps of tissue present. Extract virus via superspeed centrifugation at 124,500 x g for 4 hr at 4 &#176;C or 22,000 x g for 16 hr at 4 &#176;C. If a precipitate forms at the top, remove it with a sterile cotton swab11.</li>
	<li>Discard the supernatant.</li>
	<li>Collect the pellet and dissolve with 5 ml of 10 mM phosphate buffer (no DETCA), pH ~7.2, containing 0.4% Na-deoxycholic acid and 4% polyethylene glycol hexadecyl ether (Brij 52).
	<ol>
		<li>To mix the pellet well, remove the pellet from the side of the tube and crush up until in solution if necessary.</li>
		<li>Add more 10 mM phosphate buffer in ~5 ml increments to help the pellet go into solution if needed and combine into 2 tubes11.</li>
	</ol>
	</li>
	<li>Centrifuge the solution at 300 x g for 15 min11.</li>
	<li>Remove the supernatant and pass the solution through a 0.45 &#956;m filter, and collect the filtrate in large collection tube11.</li>
	<li>Transfer filtrate to a dialysis membrane with a molecular weight cut off (MWCO) of 3.5 kD, using small amounts of 10 mM phosphate buffer (pH 7) containing no DETCA if needed11.</li>
	<li>Place the dialysis membrane in a large beaker filled with ddH2O at 4 &#176;C. Change the ddH2O every hour for 5 - 6 hr, until a white precipitate forms in the membrane11.</li>
	<li>Collect the purified virus from within the membrane and subject the 100% virus solution to a 10-fold dilution series by adding 10 &#956;l of solution to 90 &#956;l of ddH2O. Subsequently add 10 &#956;l of the dilution to another 90 &#956;l of ddH2O until reaching a dilution of 1:100,000.</li>
	<li>Store virus solution at -80 &#176;C.</li>
</ol>
3. Viral Replication
<ol>
	<li>Seed cells in 48-well culture plates and grow all rows until cell growth is 80% confluent (approximately 72 hr post-pass if cells are growing at an average rate).</li>
	<li>Inoculate each row of cells with the serial diluted virus once cell growth reaches confluency, i.e., one row of a 1:10 dilution, one row of a 1:100 dilution, etc., except for the top row. Use the top row as a control and add 10 &#956;l of ddH2O to each well as a volume control.
	<ol>
		<li>In order to establish a starting baseline cell concentration for comparison with experimental cell counts, dissociate and count the first well of each row prior to the initial inoculation of any wells in the cultures with virus.</li>
	</ol>
	</li>
	<li>Monitor culture plates every 24 hr after viral inoculation for any color change in the medium indicating a pH change and for any changes in cell morphology.</li>
	<li>Image one column of the test plate at each time point, using an inverted microscope at 100X TM.</li>
	<li>Remove all medium from the column that was imaged at each 24 hr time point and store it short-term at -20 &#176;C for RNA extraction and viral quantification or long term at -80 &#176;C.</li>
	<li>Dissociate cells from the wells after removing the medium as previously described in steps 1.6 &#8211; 1.9, using only ~250 &#956;l of 0.25% trypsin EDTA and fresh medium to stop enzyme activity and ~250 &#956;l of fresh medium to re-suspend the cell pellet.</li>
	<li>Add 10 &#956;l of 0.4% trypan blue stain to each tube containing cells to perform cell counts after cell dissociation for each 24 hr period over one week. Allow stain to sit for 10 min before performing cell counts.
	<ol>
		<li>Perform cell counts within 1 hr of exposure to stain or viable cells will begin to uptake stain as well as non-viable cells.</li>
	</ol>
	</li>
	<li>Slowly add 10 &#956;l of the stained cell solution to each side of a standard hemocytometer using a micropipette. Allow the solution to be taken up by capillary action to avoid air bubbles.</li>
	<li>Count the number of viable (non-stained) cells on both sides of the hemocytometer (the equivalent of 4 counts of the 16 squares in the hemocytometer). Perform cell counts for each well of the column that was removed.</li>
	<li>Average the cell counts from each column and use the following formula to obtain the total cell count number: (average number of cells / 4) x dilution factor = number of cells x 104 ml. This is the cell density or number of viable cells in the culture.</li>
</ol>
4. Virus Extraction from Cell Culture
<ol>
	<li>Remove treated H. vitripennis cells from culture flasks as previously described in steps 1.6 &#8211; 1.8, using only ~250 &#956;l of 0.25% trypsin EDTA and fresh medium to stop enzyme activity.</li>
	<li>Discard the supernatant.</li>
	<li>Extract virus from culture cells following the protocol previously described in steps 2.3 &#8211; 2.8.</li>
</ol>
5. RNA Extraction
<ol>
	<li>Extract RNA from medium samples collected during each one-week virus trial using a guanidinium thiocyanate-phenol-chloroform extraction designed for liquid samples per the manufacturer&#8217;s protocol.</li>
	<li>Store extracted samples at -80 &#176;C.</li>
</ol>
6. RT-PCR
<ol>
	<li>Establish viral standards for RT-PCR by running traditional PCR using the primer pair HoCV RT-PCR primer 1 (forward 5&#8242;-GCTCCCCGGCTTTGCTGGTT-3&#8242;, reverse 5&#8242;-ACGACGGATCTGCGTGCCAA-3&#8242;) with virus isolate from whole body H. vitripennis.</li>
	<li>Subject PCR product to gel electrophoresis for 60 min at 120 V in a 2% agarose gel containing 0.1% ethidium bromide.</li>
	<li>Excise the bands from the gel and purify using a gel extraction kit as per the manufacturer&#8217;s protocol.</li>
	<li>Pool all excised and gel purified product and further purify by basic ethanol precipitation. Elute the precipitation product in approximately 30 &#956;l of Tris EDTA (TE) to increase the overall sample concentration.</li>
	<li>Quantify the level of cDNA in pooled samples via spectrophotometry using 260/230 wavelength settings for nucleic acid detection.</li>
	<li>Perform a ten-fold serial dilution series of the purified sample ranging from 57 ng/&#956;l to 57 ag/&#956;l (10-18). Perform the serial dilution similar to the one described in 2.8 with adjustments made for the difference in concentration requirements.</li>
	<li>Determine detection limits of the dilution series by performing qRT-PCR using a qRT-PCR kit with reliable quantification of low-abundance transcripts. 
	NOTE: Viral concentrations lower than 5 x 10-3 copies are not detectable.</li>
	<li>Extract RNA from experimental samples as described previously in step 5.1 and quantify using spectrophotometry.</li>
	<li>Normalize all the extracted samples to 5 ng/&#956;l using nuclease free water.</li>
	<li>Perform qRT-PCR on all samples, in duplicates, as 25 &#956;l reactions using a one-step qRT-PCR kit with the ability to sense low copy numbers as follows: 50 &#176;C hold for 10 min; 95 &#176;C hold for 5 min; 30 cycles of 95 &#176;C for 10 sec, 60 &#176;C for 30 sec; melt from 50 - 99 &#176;C for 5 sec on each step.
	<ol>
		<li>Make the master mix so that each reaction mixture contains 12.5 &#956;l of 1x master mix, 1.0 &#956;l (0.3 &#956;M) of forward primer, 1.0 &#956;l (0.3 &#956;M) of reverse primer, 0.25 &#956;l of reverse transcriptase and variable amounts of template based on standardization values.</li>
		<li>Bring the total reaction volume to 25 &#956;l with RNase free water.</li>
		<li>Include five standard concentrations in each PCR run with the following copy numbers: 5 x 10-10, 5 x 10-8, 5 x 10-6, 5 x 10-4, and 5 x 10-2 copies. Set the threshold for each run to just below a fluorescence of 10x-2.5 to reduce noise during early acquisition at the beginning of each run.</li>
	</ol>
	</li>
</ol>
7. Confocal Microscopy
<ol>
	<li>Grow Homalodisca vitripennis cells in a twelve well plate containing glass coverslips measuring 18 mm in diameter in each well.</li>
	<li>Inoculate one column on the plate every 24 hr for a period of four days, once a monolayer is achieved. Ensure that each column contains a control well, a low viral dilution (1:10) well and a high viral dilution (1:100,000) well. Use virus solutions obtained from step 2.8.</li>
	<li>Remove all media on the fifth day and wash cells twice with 1x PBS (pH 7.4).</li>
	<li>Fix the cells with cold 4% paraformaldehyde at 4 &#176;C for 30 min.</li>
	<li>Add 500 &#956;l of 1x Phosphate buffered saline (PBS) to the cells and wash them for 10 min at RT on a rocker at low speed. Wash the cells three times.</li>
	<li>Add 500 &#956;l of 0.1% Triton X-100 to the cells to permeabilize them. Let sit for 10 min at RT.</li>
	<li>Wash cells again as previously described in step 7.5.</li>
	<li>Add 500 &#956;l of a 5% bovine serum albumin (BSA) solution to block cells at RT. Let sit for 2 hr and then remove.</li>
	<li>Dilute stock Rhodamine red-conjugated phalloidin (RCP) 1:250 in 1x PBS containing 5% BSA and add 250 &#956;l of the dilution to each well to stain for F-actin.</li>
	<li>Cover plate in aluminum foil to prevent the dye from bleaching and incubate cells at 4 &#176;C O/N.</li>
	<li>Remove the RCP after 12 hr of incubation and replace it with 250 &#956;l of 4&#8242;,6-diamidino-2-phenylindole (DAPI) (250 &#956;g/ml) diluted in 1x PBS containing 5% BSA, to stain the nuclei of the cells.</li>
	<li>Incubate the cells containing DAPI at RT for 1 hr.</li>
	<li>Wash cells three times with 1x PBS as previously described in step 7.5.</li>
	<li>Gently remove the coverslips from the wells and mount to microscope slides using a mounting media with an anti-fade reagent.</li>
	<li>Allow the slides to dry in lightproof boxes until viewed under the confocal microscope. View prepared slides as soon as possible as dyes can fade rapidly.</li>
	<li>Image stained cells using a confocal system equipped with a microscope containing a 63X (oil) plan-apochromate lens.
	<ol>
		<li>Set the laser wavelengths to 543 &#177; 10 nm excitation and 575 &#177; 10 nm emission for Rhoadmine red-conjucated phalloidin, and 369 &#177; 10 nm excitation and 450 &#177; 30 nm emission for DAPI.</li>
		<li>Use identical gain and off-set settings for the detector to obtain all images.</li>
		<li>Process images using appropriate software for image processing and sorting and import desired images into an image managing software.</li>
	</ol>
	</li>
</ol>

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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+capsid+protein+of+Homalodisca+coagulata+Virus-1,+a+new+leafhopper-infecting+virus+from+the+glassy-winged+sharpshooter,+Homalodisca+coagulata.">Molecular analysis of capsid protein of Homalodisca coagulata Virus-1, a new leafhopper-infecting virus from the glassy-winged sharpshooter, Homalodisca coagulata.</a> J. Insect Sci. 6 (28), 1-10 (2006).</li><li>Hunnicutt, L. E., Hunter, W. B., Cave, R. D., Powell, C. A., Mozoruk, J. 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The authors have nothing to disclose.

Rising concerns regarding the influx of invasive agricultural species have lead to an increased demand for new methodologies to defend against emerging pests and pathogens. A focus of disease prevention and management involves the management of pathogen vectors and was the primary target of this study. Economics play a vital role in the decision to produce this type of biopesticide to manage pathogen vectors in agriculture because the practical application needs to be large quantities over large areas but at a low cost<s...

The glassy-winged sharpshooter (Homalodisca vitripennis Germar 1821) has been identified as the predominant vector of Xylella fastidiosa (X. fastidiosa), the causal agent of Pierce&#8217;s disease of grapevine (PD) in North America1. Insect population management has quickly become the focus of research to combat this devastating problem to the viticulture industry in California and across the southern United States. A positive-sense, single-stranded RNA virus belonging to the family Dicistroviridae, Homalodisca coagulata virus-01 (HoCV-01) has been identified in wild H. vitripennis populations and shown to increase mortality in those populations2-4, while lowering the insect&#8217;s resistance to insecticides.
Development of methods to effectively rear infected H. vitripennis to adulthood in a laboratory setting have been difficult because H. vitripennis have different stage-specific nutritional needs that require a variety of host plants5-8. Specific facilities are required to rear live H. vitripennis in the United States; therefore, cell culture is more economical and a viable alternative, as well as increasingly vital for HoCV-01 detection and replication2,9. While basic methods for establishing cell cultures of H. vitripennis are described, these methods have not yet been utilized for commercial production of biological control agents, such as viruses2.
The overall goal of the following procedures is to produce a high concentration of HoCV-01 suitable for utilization as a biological control agent. Viral replication requires a living cell, which is why successfully cultivating and optimizing H. vitripennis cultures is vital to the progress of producing profitable levels of virus.

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<tbody>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
</table>

propagation of homalodisca coagulata virus-01 via homalodisca vitripennis cell culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Cell attachment and growth was seen within 48 hr of passage in both small and large culture flasks, from primary cultures and continued passages. Fibroblast growth and development was also observed within this time frame. When newly seeded flasks were disturbed before 48 hr, there was a visible decline in cell attachment, leading to slower growing cultures and sometimes no attachment or growth at all. Cells were approximately 80% confluent within one week of passing and formed a monolayer in 10-14 days (Figure 1<...

Watch this Scientific Journal Video about Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture at JoVE.com

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

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

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

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

propagation-homalodisca-coagulata-virus-01-via-homalodisca

Research

JoVE Journal

Immunology and Infection

11.6K Views.  University of Texas at Tyler. Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

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

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). 
The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).
The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...