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

Abstract

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis 1 . The primary target cells of MVV in vivo are considered to be of the monocyte lineage 2 . Certain strains of MVV can replicate in other cell types, however 3,4 . 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 CAEV 5 . 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 vivo 6 . 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 virus 7 . The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages 8 . 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 vivo 8 . Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=3483 Protocol TransfectionThe 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.