Name: propagation of homalodisca coagulata virus-01 via homalodisca vitripennis cell culture
Uploaded: 2014-09-25T15:00:00.000+00:00
Duration: 12 min 52 s
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
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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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><tbody><tr><td>Corning cell culture flasks</td><td>Sigma Aldrich</td><td>CLS430168</td><td>Surface area 25 cm&lt;sup&gt;2&lt;/sup&gt;, canted neck, cap (plug seal)</td></tr><tr><td>Olympus DP30BW, IX2-SP, IX71</td><td>Olympus</td><td></td><td>Inverted microscope and camera</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma Aldrich</td><td>T4049</td><td>0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA &amp;bull; 4Na per liter of Hanks&amp;prime; Balanced Salt Solution with phenol red</td></tr><tr><td>Greiner CELLSTAR multiwell culture plates</td><td>Sigma Aldrich</td><td>M8937</td><td>48 wells (TC treated with lid)</td></tr><tr><td>DETCA</td><td>Sigma Aldrich</td><td>228680</td><td>Sodium diethyldithiocarbamate trihydrate</td></tr><tr><td>Corning bottle-top vacuum filter system</td><td>Sigma Aldrich</td><td>CLS431206</td><td>Cellulose acetate membrane, pore size 0.45 &amp;mu;m, membrane area 54.5 cm&lt;sup&gt;2&lt;/sup&gt;, filter capacity 500 ml</td></tr><tr><td>Brij 52</td><td>Sigma Aldrich</td><td>388831</td><td>Polyethylene glycol hexadecyl ether</td></tr><tr><td>Phosphate buffer solution</td><td>Sigma Aldrich</td><td>P5244</td><td>Received as 100 mM diluted to 10 mM with sterile water</td></tr><tr><td>TRIzol LS</td><td>Life Technologies</td><td>10296-028</td><td></td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A5304</td><td>For electrophoresis</td></tr><tr><td>Ethidium bromide</td><td>Sigma Aldrich</td><td>E7637</td><td>BioReagent, for molecular biology, powder</td></tr><tr><td>QIAquick</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>QuantiTect qRT-PCR kit</td><td>Qiagen</td><td>204243</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td>Reagent grade, crystalline</td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P5368</td><td>Phosphate buffered saline</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A2153</td><td></td></tr><tr><td>Rhodamine red-conjugated phalloidin</td><td>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><td>DAPI</td><td>Sigma Aldrich</td><td>D9542</td><td></td></tr><tr><td>ProLong Gold Antifade Reagent</td><td>Life Technologies</td><td>P36934</td><td></td></tr><tr><td>LSM510 Meta Confocal System</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>LSM Zen 2007 Software</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Grace&amp;rsquo;s Insect medium (supplemented, 1x)</td><td>Sigma Aldrich</td><td>G8142</td><td>H2G+ leafhopper medium component</td></tr><tr><td>L-histidine monohydrate</td><td>Sigma Aldrich</td><td>H8125</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Medium 199 (10x)</td><td>Sigma Aldrich</td><td>M4530</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Medium 1066 (1x)</td><td>Sigma Aldrich</td><td>C0422</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salts (1x)</td><td>Sigma Aldrich</td><td>51322C</td><td>H2G+ leafhopper medium component</td></tr><tr><td>L-Glutamine (100x)</td><td>Sigma Aldrich</td><td>G3126</td><td>H2G+ leafhopper medium component</td></tr><tr><td>MEM, amino acid mix (50x)</td><td>Sigma Aldrich</td><td>56419C</td><td>H2G+ leafhopper medium component</td></tr><tr><td>1 M MgCl solution</td><td>Sigma Aldrich</td><td>M8266</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Pen-Strep (w/ glutamine)</td><td>Sigma Aldrich</td><td>G6784</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Nystatin</td><td>Sigma Aldrich</td><td>N6261</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Gentamycin</td><td>Sigma Aldrich</td><td>46305</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>D9434</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Fetal Bovine Serum</td><td>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 ...

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JoVE Journal

Immunology and Infection

11.7K 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

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Biology

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two main categories of these new mosquito-associated viruses are &#34;mosquito-borne viruses (MBVs)&#34; and &#34;mosquito-specific viruses (MSVs)&#34;. These novel viruses might be pathogenic to both vertebrates and mosquitoes, or they could just be symbiotic with mosquitoes. Entity viruses are essential to confirm the biological characters of these viruses. Thus, a detailed protocol has been described here for virus isolation and amplification from field-collected mosquitoes. First, the mosquito samples were prepared as supernatants of mosquito homogenates. After centrifugation twice, the supernatants were then inoculated into either mosquito cell line C6/36 or vertebrate cell line BHK-21 for virus amplification. After 7 days, the supernatants were collected as the P1 supernatants and stored at -80 &#176;C. Next, P1 supernatants were passaged twice more in C6/36 or BHK-21 cells while the cell status was being checked daily. When cytopathogenic effect (CPE) on the cells was discovered, these supernatants were collected and used to identify viruses. This protocol serves as the foundation for future research on mosquito-associated viruses, including MBVs and MSVs.

Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two ...

Cellular Impedance-Based Survival Kinetics Assay to Assess Virus Survival

Source: Labadie, T. et al., <a href="https://app.jove.com/v/61133">Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.</a> J. Vis. Exp. (2021).
This video describes a cellular impedance-based technique to study the survival kinetics of viruses that induce cytopathic effects. The method measures the electrical impedance of virus-infected adherent cells pre-treated with saline.

Source: Labadie, T. et al., Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis. J. Vis. Exp. (2021).
This video describes ...

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

Summary

Explore More Videos

Chapters in this video

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Influenza Virus Propagation in Embryonated Chicken Eggs

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

Cellular Impedance-Based Survival Kinetics Assay to Assess Virus Survival