Abstract
BackgroundDetection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells.Methodology/Principal findingsMurine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow.Conclusions/SignificanceThe use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections.
Highlights
The study of host-virus interactions in individual cell populations in vivo is important for the development of disease control strategies
Correct insertion of the Cre expression cassette within the Murine gammaherpesvirus 68 (MHV-68) bacterial artificial chromosome (BAC) genome was confirmed by restriction digestion of BAC DNA (Figure 1B) followed by Southern analysis, as well as by direct DNA sequence analysis of a PCR product spanning the insertion site
BAC sequences were removed from Cre expressing clones by selfexcision in BHK cells, indicating that the RSV LTR promoter is active during lytic infection or by passage through a Cre-expressing cell line
Summary
The study of host-virus interactions in individual cell populations in vivo is important for the development of disease control strategies This is the case for viruses which establish life long latent or persistent infections, frequently within low numbers of cells with minimal virus gene expression, often in a number of different cell types. One approach, which has been investigated for several latent herpesvirus infections including herpes simplex virus, pseudorabies virus and Marek’s disease virus involves insertion of a reporter gene such as b-galactosidase or green fluorescent protein within the viral genome [1,2,3,4,5] This technique requires the use of a latency associated promoter to overcome the general repression of viral promoters during latency, including foreign promoters inserted into the viral genome [6,7,8,9]. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells
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