title: Rapid determination of Epstein-Barr virus latent or lytic infection in single human cells using in situ hybridization.
abstract: Epstein-Barr (EBV) virus is associated with malignancies such as lymphoma and carcinoma. Infection of cells with EBV may result in either lytic infection with production of viral particles, characterized by the presence of linear DNA forms, or latent infection, characterized by either episomal or integrated DNA forms. To examine whether the different lytic and latent EBV DNA forms can reliably be distinguished in single human cells, in situ hybridization was performed in EBV-positive cell lines. Immunocytochemistry and Southern blot analysis were performed supplementary to in situ hybridization. In latent infection, three in situ hybridization patterns were observed: large-disperse (episomal), small-punctate (integrated) and combined (both), signal types 1, 2 and 3 respectively. These were associated with expression of latent membrane protein 1, but not with Z fragment of Epstein-Barr replication activator or viral capsid antigen. In lytic infection, three additional in situ hybridization patterns were observed: nuclear membrane associated, bubble (filling up the nucleus) and spillover (covering the lysed cells) signals types 4, 5 and 6 respectively. Signal types 4 and 5 were associated with expression of latent membrane protein 1 and Z fragment of Epstein-Barr replication activator but not viral capsid antigen, whereas type 6 was associated with expression of viral capsid antigen only. Southern blot analysis confirmed these results; however, low copy numbers of integrated virus were often missed by Southern blot, confirming that in situ hybridization is more sensitive in determining the presence of all types of EBV DNA. In situ hybridization may prove useful in rapidly screening large series of tissue microarrays and other clinical specimens for the presence of lytic or latent EBV.
authors.affiliation: Research Institute of Radiology and Roentgenology, St Petersburg, Russia.
authors.forename: Elena E
authors.initials: EE
authors.lastname: Leenman
authors.forename: Renate E
authors.initials: RE
authors.lastname: Panzer-Grümayer
authors.forename: Susanna
authors.initials: S
authors.lastname: Fischer
authors.forename: Heather A
authors.initials: HA
authors.lastname: Leitch
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Horsman
authors.forename: Thomas
authors.initials: T
authors.lastname: Lion
authors.forename: Helmut
authors.initials: H
authors.lastname: Gadner
authors.forename: Peter F
authors.initials: PF
authors.lastname: Ambros
authors.forename: Valia S
authors.initials: VS
authors.lastname: Lestou
mesh_terms: D015139:Blotting, Southern; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D009195:Peroxidase; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics; D017735:Virus Latency / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins; D009195:Peroxidase