Institute for Experimental Pathology, University of Iceland
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Jónsson, S. R., Andrésdóttir, V. Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein. J. Vis. Exp. (56), e3483, doi:10.3791/3483 (2011).
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.
The molecular clone is contained in two plasmids, p8XSp5-egfp and p67r, of 12 kb and 4.5 kb respectively.9,10
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.
2. Titration of virus
3. FACS analysis of infected cells
Note: For rapid monitoring of replication of the virus, infected cells can be examined by flow cytometry.
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).
Figure 1. FACScan analysis of SCP cells infected with KV1772-egfp virus after 7 days of infection.
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).
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 construct is only 5-15%, and using a pEGFP-N3 vector the transfection efficiency is 15-20% compared to over 90% using 293-T cells. 293-T cells have been tested as packaging cells for MVV vectors, but the resulting virus particles proved non-infectious both in sheep cells and in 293-T cells12 probably due to unidentified restriction factors. The infectious clone is contained in two plasmids, which is another shortcoming. This is due to the fact that the full-length clone is unstable in E. coli. However, it should be possible to clone the full-length clone in a low copy plasmid.
Plasmids and cells can be obtained from the authors.
No conflicts of interest declared.
This work was funded by the Icelandic Research Fund and the University of Iceland Research Fund.
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