title: Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain.
abstract: Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.
authors.affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109-0618.
authors.forename: R
authors.initials: R
authors.lastname: Ramakrishnan
authors.forename: D J
authors.initials: DJ
authors.lastname: Fink
authors.forename: G
authors.initials: G
authors.lastname: Jiang
authors.forename: P
authors.initials: P
authors.lastname: Desai
authors.forename: J C
authors.initials: JC
authors.lastname: Glorioso
authors.forename: M
authors.initials: M
authors.lastname: Levine
mesh_terms: D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D016679:Genome, Viral; D006561:Herpes Simplex / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000472:pathogenicity*; D006624:Hippocampus / Q000382:microbiology; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012264:Ribonucleotide Reductases / Q000235:genetics; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics; D014774:Virulence; D017735:Virus Latency / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D012264:Ribonucleotide Reductases