Construction and in vivo detection of an enhanced green fluorescent protein-expressing strain of Saimiriine herpesvirus 1 (SaHV-1).
Saimiriine herpesvirus 1 (SaHV-1) is an alpha-herpesvirus of squirrel monkeys used in mice to study neural pathogenesis of herpesviruses. To trace dissemination of virus from a peripheral site of inoculation to the central nervous system tissues, a recombinant strain of SaHV-1 expressing enhanced green fluorescent protein (GFP) was constructed by site-specific insertion of a GFP expression cassette into a transcriptionally null point in the SaHV-1 genome. PCR and Southern blot confirmed insertion of a single GFP expression cassette into the target site of the SaHV-1 genome. The recombinant virus was shown to produce strong fluorescence in the cytoplasm of infected cells in vitro. Growth kinetic experiments demonstrated no differences between recombinant and wild type SaHV-1 in producing infectious progeny virions. The recombinant virus was comparable to wild type SaHV-1 in development of clinical disease, microscopic lesions and induction of an antibody response in mice following intramuscular inoculation. Using confocal microscopy, GFP expression was easily observed in formalin fixed, paraffin-embedded tissues of mice infected with the recombinant SaHV-1. This simple specimen processing technique preserves tissue morphology and allows detection of viral replication within various tissues of experimentally infected animals.
Breshears
M A
MA
Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, Oklahoma 74078, USA. melba98@okstate.edu
Black
D H
DH
Ritchey
J W
JW
Eberle
R
R
D000818:Animals; D015139:Blotting, Southern; D004274:DNA, Recombinant / Q000235:genetics; D005260:Female; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D016133:Polymerase Chain Reaction; D018139:Simplexvirus / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D012867:Skin / Q000473:pathology / Q000821:virology; D013116:Spinal Cord / Q000473:pathology / Q000821:virology; D013997:Time Factors; D014779:Virus Replication
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins