title: Development and pathogenic evaluation of recombinant herpes simplex virus type 1 expressing two fluorescent reporter genes from different lytic promoters.
abstract: To develop means to explore viral gene expression in ganglia without laborious histological sectioning and staining, we created a two color fluorescent recombinant HSV-1, in which enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) are expressed from the glycoprotein B (gB) and glycoprotein C (gC) promoters respectively. We show that this virus retained growth and pathogenic capacity both in vitro and in vivo compared to wild type HSV-1; established latent infections with similar genome copy number in trigeminal ganglia (TG); induced a similar HSV-specific CD8(+) T cell infiltrate; did not induce CD8(+) T cells reactive to EGFP or RFP; and reactivated from latency with normal kinetics in ex vivo TG cultures. Fluorescent EGFP expression in plaques surrounding neurons preceded RFP expression and provided highly sensitive detection of reactivation and different stages of infection in ex vivo TG cultures. Expression of both EGFP and RFP in neurons was readily detectable in whole mounts of TG excised during acute infection and following in vivo sodium butyrate-induced reactivation from latency. This virus constitutes a useful reagent for monitoring lytic viral promoter activity in sensory neurons in vivo and in vitro.
authors.affiliation: Graduate Program in Molecular Microbiology and Virology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Srividya
authors.initials: S
authors.lastname: Ramachandran
authors.forename: Jared E
authors.initials: JE
authors.lastname: Knickelbein
authors.forename: Christina
authors.initials: C
authors.lastname: Ferko
authors.forename: Robert L
authors.initials: RL
authors.lastname: Hendricks
authors.forename: Paul R
authors.initials: PR
authors.lastname: Kinchington
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002522:Chlorocebus aethiops; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008967:Molecular Biology / Q000379:methods*; D009474:Neurons / Q000821:virology; D009924:Organ Culture Techniques; D011401:Promoter Regions, Genetic; D012668:Trigeminal Ganglion / Q000821:virology; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics; D010948:Viral Plaque Assay; D014775:Virus Activation; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D014759:Viral Envelope Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein; C040495:glycoprotein B, Simplexvirus; C038535:glycoprotein gC, herpes simplex virus type 1