title: Single cell detection of latent cytomegalovirus reactivation in host tissue.
abstract: The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor-GFP reporter mice, we never observed reactivation originating from CX3CR1(+) monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b(+) and CX3CR1(+) subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.
authors.affiliation: Institute of Virology, Hannover Medical School, Hannover, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Marquardt
authors.affiliation: Institute of Immunology, Hannover Medical School, Hannover, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Halle
authors.affiliation: Institute for Virology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.
authors.forename: Christof K
authors.initials: CK
authors.lastname: Seckert
authors.affiliation: Institute for Virology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.
authors.forename: Niels A W
authors.initials: NAW
authors.lastname: Lemmermann
authors.affiliation: Department of Immunology, Allergology and Immunotoxicology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany.
authors.forename: Tibor Z
authors.initials: TZ
authors.lastname: Veres
authors.affiliation: Department of Immunology, Allergology and Immunotoxicology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany.
authors.forename: Armin
authors.initials: A
authors.lastname: Braun
authors.affiliation: Department of Experimental Pneumology, Hannover Medical School, Hannover, Germany.
authors.forename: Ulrich A
authors.initials: UA
authors.lastname: Maus
authors.affiliation: Institute of Immunology, Hannover Medical School, Hannover, Germany.
authors.forename: Reinhold
authors.initials: R
authors.lastname: Förster
authors.affiliation: Institute for Virology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.
authors.forename: Matthias J
authors.initials: MJ
authors.lastname: Reddehase
authors.affiliation: Institute of Virology, Hannover Medical School, Hannover, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Messerle
authors.affiliation: Institute of Virology, Hannover Medical School, Hannover, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Busche
mesh_terms: D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics / Q000502:physiology; D003586:Cytomegalovirus Infections / Q000821:virology*; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D006566:Herpesviridae Infections / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000821:virology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018146:Muromegalovirus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D059010:Single-Cell Analysis / Q000379:methods*; D014775:Virus Activation; D017735:Virus Latency; D000097573:Red Fluorescent Protein
chemical_list: D008156:Luciferases; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein