Name: Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein (Video) | JoVE Journal
Uploaded: 2011-10-09T14:00:00.000+00:00
Duration: 8 min 49 s
Description: Learn to propagate an infectious molecular clone of maedi-visna virus expressing green fluorescent protein in sheep choroid plexus cells. This method enables visualization of viral replication via fluorescence microscopy and flow cytometry.

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5 minutes

This work was funded by the Icelandic Research Fund and the University of Iceland Research Fund.

1. Transfection
The molecular clone is contained in two plasmids, p8XSp5-egfp and p67r, of 12 kb and 4.5 kb respectively.9,10
<ol>
 <li>Cut equimolar quantities of the two plasmids, i.e. 4.4 μg of p8XSp5-egfp and 1.6 μg of p67r with XbaI and ligate before transfection.</li>
 <li>For ligation, use 1 Weiss unit of ligase in a 50 μl reaction and incubate at 16°C overnight.</li>
 <li>When ligation is completed, incubate the ligation mix at 65°C for 15 min for inactivation of the ligase. This step may be omitted.</li>
 <li>Two days before transfection, seed 106 SCP cells in a T25 tissue culture flask, and grow cells for two days in 5 ml DMEM + 10% lamb serum and 2mM glutamine without antibiotics to a monolayer of 90% confluency. If the cells have not reached 90% confluency after two days, leave the cells to grow one additional day.</li>
</ol>
Note: The lamb serum can be substituted by fetal bovine serum (FBS). We routinely use lamb serum, since some strains of MVV are inhibited by FBS; this MVV strain is not inhibited by FBS, however. 
<ol start="5">
 <li>When the cells are ready for transfection, change medium to DMEM with 1% serum (lamb serum or FBS) without antibiotics. 
 We use Lipofectamin 2000 (Invitrogen) for transfection. </li>
 <li>Dilute the DNA in 500μl Opti-MEM medium. </li>
 <li>Dilute 20μl Lipofectamin 2000 in 500μl Opti-MEM medium and leave at room temperature (RT) for 5 min.</li>
 <li>After the 5 min incubation, combine the diluted DNA with diluted Lipofectamine 2000 (total volume = 1000 μl). Mix gently and incubate for 20-30 min at RT.</li>
 <li>Add the transfection mix (1 ml) to the T25 flask containing SCP cells and medium (5 ml). Mix by rocking gently.</li>
 <li>Incubate at 37°C and 5% CO2 overnight (18 – 24 h). </li>
 <li>Change medium the following day to DMEM with 1% serum, 2mM glutamine, 100IU penicillin and 100IU streptomycin.</li>
 <li>Monitor the production of GFP in the SCP cells by inverted fluorescent microscopy. Low transfection efficiency can be expected with these primary cells, usually 5 – 15%.</li>
 <li>Incubate further at 37°C, 5% CO2 for several days to allow spreading of the virus in the flask. It usually takes 7-14 days for full infection.</li>
 <li>Harvest the virus by aliquoting the supernatant into Eppendorf tubes. Spin in a microfuge at 3000 rpm for 5 min to remove cell debris. Transfer the supernatant containing the virus to new tubes. Store at -80°C.</li>
</ol>
2. Titration of virus
<ol>
 <li>Seed 3x104 SCP cells in 100 μl DMEM medium supplemented with 10% serum, 2mM glutamine, 100IU/ml penicillin and 100IU/ml streptomycin, per well in a 96 well plate. Incubate at 37°C, 5% CO2.</li>
 <li>If the cells are confluent the following day, change medium to DMEM with 1% serum, 2mM glutamine, 100IU/ml penicillin and 100IU/ml streptomycin, 100 μl in each well.</li>
 <li>Dilute the virus in the following way: 
 <ol>
 <li>Add 180 μl DMEM with 1% serum, 2mM glutamine, 100IU/ml penicillin and 100IU/ml streptomycin per well to 4 x 10 wells in a 96 well round bottom or V-bottom plate.</li>
 <li>For serial tenfold dilutions in quadruplicate, add 20 μl of virus to each of 4wells in the first column of the plate, and using a four-channel pipette, pipette up and down to ensure that the samples are thoroughly mixed. Change pipette tips and transfer 20 μl to the next 4 wells and so on. Leave the last wells without virus for a negative control. </li>
 </ol>
 </li>
 <li>Add 100 μl of the virus dilutions to the SCP cells and medium.</li>
 <li>Incubate at 37°C and 5% CO2. Observe for GFP fluorescence and cytopathic effects for two weeks. 
 Cytopathic effects consist of cells becoming rounded and less transparent than normal cells with processes stretching out, ending with multinuclear cells appearing and cell death.</li>
 <li>Calculate virus titer (TCID50) using the Reed Muench method11.</li>
</ol>
3. FACS analysis of infected cells 
Note: For rapid monitoring of replication of the virus, infected cells can be examined by flow cytometry. 
<ol>
 <li>Trypsinize infected cells (ca 105), wash 1x with PBS and resuspend in 500μl PBS.</li>
 <li>Add 500 μl 4% paraformaldehyde solution (2% final concentration), incubate at RT for 30 min.</li>
 <li>Pellet cells in a microfuge at 3000 rpm for 5 min.</li>
 <li>Wash with PBS 2x5 min and analyze on a FACScan analyzer. Count 10,000 events.</li>
</ol>
4. Representative results:
This virus should be fully infectious and replicate to a titer of 106 – 107 TCID50/ml. Infected cells are fluorescent and can be detected by flow cytometry and fluorescence microscopy. Typically, GFP can be detected in more than 60% of the cells in the infected cell cultures using flow cytometry (Fig. 1). Likewise, when visualized by fluorescence microscopy, a majority of the cells are fluorescent (Fig. 2A and B).
<img src="/files/ftp_upload/3483/3483fig1.jpg" alt="Flow cytometry scatter plot; GFP expression analysis; experimental data visualization." data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. FACScan analysis of SCP cells infected with KV1772-egfp virus after 7 days of infection.
<img src="/files/ftp_upload/3483/3483fig2.jpg" alt="Cell culture comparison; microscopy images A) unstained cells, B) fluorescence-stained cells." data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. Phase contrast microscopy of SCP cells infected with KV1772-egfp after 7 days of incubation (A). Same cells visualized by fluorescence microscopy (B).

<ol><li>Sigurdsson, B., Palsson, P. A., Grimsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visna%2C+a+demyelinating+transmissable+disease+of+sheep%5BTitle%5D)+AND+%22J.+Neuropathol.+Exp.+Neurol%22%5BJournal%5D)">Visna, a demyelinating transmissable disease of sheep.</a> J. Neuropathol. Exp. Neurol. 14, 389-403 (1957).</li>
<li>Gorrell, M. D., Brandon, M. R., Sheffer, D., Adams, R. J., Narayan, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovine+lentivirus+is+macrophagetropic+and+does+not+replicate+productively+in+T+lymphocytes%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.</a> J. Virol. 66, 2679-2688 (1992).</li>
<li>Andresdottir, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+and+genetic+differences+between+lung-+and+brain-derived+isolates+of+maedi-visna+virus%5BTitle%5D)+AND+%22Virus+Genes%22%5BJournal%5D)">Biological and genetic differences between lung- and brain-derived isolates of maedi-visna virus.</a> Virus Genes. 16, 281-293 (1998).</li>
<li>Georgsson, G., Houwers, D. J., Palsson, P. A., Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+viral+antigens+in+the+central+nervous+system+of+visna-infected+sheep%3A+an+immunohistochemical+study+on+experimental+visna+induced+by+virus+strains+of+increased+neurovirulence%5BTitle%5D)+AND+%22Acta+Neuropathol%22%5BJournal%5D)">Expression of viral antigens in the central nervous system of visna-infected sheep: an immunohistochemical study on experimental visna induced by virus strains of increased neurovirulence.</a> Acta Neuropathol. 77, 299-306 (1989).</li>
<li>Turelli, P., Guiguen, F., Mornex, J. F., Vigne, R., Querat, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((dUTPase-minus+caprine+arthritis-encephalitis+virus+is+attenuated+for+pathogenesis+and+accumulates+G-to-A+substitutions%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">dUTPase-minus caprine arthritis-encephalitis virus is attenuated for pathogenesis and accumulates G-to-A substitutions.</a> J. Virol. 71, 4522-4530 (1997).</li>
<li>Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visna+virus+dUTPase+is+dispensable+for+neuropathogenicity%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Visna virus dUTPase is dispensable for neuropathogenicity.</a> J. Virol. 72, 1657-1661 (1998).</li>
<li>Sigurdsson, B., Thormar, H., Palsson, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cultivation+of+visna+virus+in+tissue+culture%5BTitle%5D)+AND+%22Arch+Gesamte+Virusforsch%22%5BJournal%5D)">Cultivation of visna virus in tissue culture.</a> Arch Gesamte Virusforsch. 10, 368-380 (1960).</li>
<li>Gudmundsdottir, H. S., Olafsdottir, K., Franzdottir, S. R., Andresdottir, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+and+characterization+of+an+infectious+molecular+clone+of+maedi-visna+virus+that+expresses+green+fluorescent+protein%5BTitle%5D)+AND+%22J.+Virol.+Methods%22%5BJournal%5D)">Construction and characterization of an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein.</a> J. Virol. Methods. 168, 98-102 (2010).</li>
<li>Andresson, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nucleotide+sequence+and+biological+properties+of+a+pathogenic+proviral+molecular+clone+of+neurovirulent+visna+virus%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus.</a> Virology. 193, 89-105 (1993).</li>
<li>Skraban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naturally+occurring+mutations+within+39+amino+acids+in+the+envelope+glycoprotein+of+maedi-visna+virus+alter+the+neutralization+phenotype%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype.</a> J. Virol. 73, 8064-8072 (1999).</li>
<li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+method+of+estimating+fifty+percent+endpoints%5BTitle%5D)+AND+%22American+Journal+of+Hygiene%22%5BJournal%5D)">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27, 493-497 (1938).</li>
<li>Berkowitz, R. D., Ilves, H., Plavec, I., Veres, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+transfer+systems+derived+from+Visna+virus%3A+analysis+of+virus+production+and+infectivity%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Gene transfer systems derived from Visna virus: analysis of virus production and infectivity.</a> Virology. 279, 116-129 (2001).</li>
</ol>

No conflicts of interest declared.

The GFP expressing molecular clone of MVV presented here should be useful for analyzing the host-cell interactions and pathogenicity of MVV both in vitro and in vivo. The virus obtained after 7-14 days of replication has undoubtedly acquired some mutations due to the inaccuracy of reverse transcriptase. However, there are limitations to the use of this clone as a single cycle vector. One is that use of the clone is restricted to cells of sheep and goat origin. The transfection efficiency of these cells using our construc...

<table><tbody><tr><td>DMEM</td><td>GIBCO, by Life Technologies</td><td>10938025</td><td></td></tr><tr><td>Opti-MEM</td><td>GIBCO, by Life Technologies</td><td>51985018</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668019</td><td></td></tr></tbody></table>

propagating and detecting an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

The overall goal of this procedure is to propagate and detect an infectious clone of Mildy vs. No virus that expresses green fluorescent protein. This green fluorescent protein has been cloned into the gene coating for D utp Ace in the Myy Ner virus genome.The infectious clone is contained in two plasmids. First, the two plasmids carrying the myy Ner sequences are cut with xpa one restriction enzyme and ligated. Second sheep choroid plexus cells are transfected with the construct.Finally, myy vino virus infected cells are detected by fluorescent microscopy and flow cytometry. Hi, I'm Stefano Neron at the TER Institute for Experimental Pathology at the University of Iceland. Today we will show you a procedure for propagating and detecting an infectious molecular clone of my vascular virus that expresses green fluorescent protein.The molecular clone is contained into plasmids P eight XSP five EGFP, and P six seven or of 12 kilobases and 4.5 kilobases respectively. Cut equimolar quantities of the two plasmids, namely 4.4 micrograms of P eight XSP five EGFP, and 1.6 micrograms of P six seven or with XPA one and ligate before transfection. When ligation is completed, incubate the ligation mix at 65 degrees Celsius for 15 minutes two days before transfection seed 10 to the six SCP cells in a T 25 tissue culture flask and grow cells for two days in five milliliter DMEM plus 10%lamb serum and two millimolar glutamine without antibiotics to a monolayer of 90%co fluency.If the cells have not reached 90%CO after two days, leave the cells to grow one additional day. The lamb serum can be substituted by fetal bovine serum FBS. We routinely use lamb serum since some strains of MVV are inhibited by FBS.This MVV strain, however, is not inhibited by FBS. When the cells are ready for transfection change medium to DMEM with 1%serum lamb serum or FBS without antibiotics. We use Lipofectamine 2000 for transfection, dilute the DNA in 500 microliters of opti MEM medium dilute 20 microliters of lipofectamine 2000 in 500 microliters of opti MEM medium, and leave at room temperature for five minutes.After the five minute incubation, combine the diluted DNA with diluted lipofectamine 2000 total volume 1000 microliters. Mix gently and incubate for 2230 minutes. At room temperature, add the transfection mix to the T 25 flask containing SCP cells and medium mix by rocking gently.Incubator 37 degrees Celsius and 5%carbon dioxide overnight. Change medium the following day to DMEM with 1%serum two millimolar glutamine, 100 IU per milliliter, penicillin, and 100 IU per milliliter.Streptomycin. Monitor the production of GFP and the SCP cells by inverted fluorescent microscopy.Low transfection efficiency can be expected with these primary cells, usually five to 15%Incubate further at 37 degrees Celsius, 5%carbon dioxide for several days to allow spreading of the virus in the flask. It usually takes seven to 14 days for full infection. Harvest the virus by Ali, watting the supernatant into einor tubes.Spin in a micro fige at 3000 or PM for five minutes. To remove cell debris. Transfer the supernatant containing the virus to new tubes.Store at minus 80 degrees Celsius seed three times 10 to the four SCP cells. In 100 microliters of DMEM medium supplemented with 10%serum two millimolar glutamine, 100 IU per milliliter penicillin, and 100 IU per milliliter. Streptomycin per well.In a 96 well plate incubate at 37 degrees Celsius, 5%carbon dioxide. If the cells are confluent the following day, change the medium to DMEM with 1%serum. Having 100 microliters in each, well dilute the virus in the following way.Add 180 microliters DMEM with 1%serum per well to four times 10 wells in a 96 well round bottom or V bottom plate for serial tenfold dilution in quad duplicate. Add 20 microliters of virus to each of the four wells in the first column of the plate. And using a four channel pipee pipet up and down to ensure the samples are thoroughly mixed.Change pipette tips and transfer 20 microliters to the next four wells and so on. Leave the last wells without virus for a negative control. Add 100 microliters of the virus dilution to the SEP cells and medium incubator.37 degrees Celsius and 5%carbon dioxide. Observe for GFP fluorescence and cytopathic effects for two weeks. Cytopathic effects consist of cells becoming rounded and less transparent than normal cells with processes stretching out, ending with multi nuclear cells appearing and cell death.Calculate virus titer using the Reid Munch method for rapid monitoring of replication of the virus. Infected cells can be examined by flow cytometry trypsin. Eyes around 10 to the five infected cells.Wash once with PBS and RESUSPEND in 500 microliters. PBS add 500 microliters of 4%Para formaldehyde solution 2%final concentration incubated room temperature for 30 minutes. Pellet cells at 3000 or PMM for five minutes.Wash with PBS two times for five minutes and analyze on a fact scan analyzer. Count 10, 000 events. The GFP expressing molecular clone of my virus presented here can be useful for analyzing virus host interactions and pathogenicity of my neurovirus, both in vivo and in vitro.Although it has not been shown that my neurovirus can in affect human cells, it is important to observe all biosafety level precautions both cells and plasmid can be obtained from our laboratory. Good luck with your experiments.

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Learn to propagate an infectious molecular clone of maedi-visna virus expressing green fluorescent protein in sheep choroid plexus cells. This method enables visualization of viral replication via fluorescence microscopy and flow cytometry.

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

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