Studies On The Neurons Of Inner Ear Infected By Herpes Simplex Virus Type 1 In Vitro

Part I Study on the cultured spiral ganglion neurons infected by herpes simplex virus-1 in vitroObjectives:To establish cell culture models of spiral ganglion neurons (SGNs) in vitro both lytically and latently infected by herpes simplex virus type 1 (HSV-1), as well as a reactivation model in latently infected neurons.Methods:The spiral ganglion of postnatal day 4-6 Sprague-Dawley rats were dissected out. SGNs were cultured by dissociated culture method and were planted in the 96-well plates. Parts of the wells were directly added with HSV-1 which was incorporated with Ureen Fluorescent Protein (GFP) in the tegument protein of the virus. Other wells were added with HSV-1 in the presence of acyclovir, at which case SGNs were latently infected with HSV-1. Four days later, these cells were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1. Cultures were monitored daily by fluorescent microscopy. TCID50 was used to measure the titers of virus in cultures on Vero cells. RNA from cultures was detected for the presence of transcripts of ICP27 and latency associated transcript (LAT) using reverse-transcription polymerase chain reaction.Results:There was no detectable infectious HSV-1 in latently infected cultures, whereas they could be observed in both lytically infected and latently infected/TSA treated cultures. The SGN cultures showed a reactivation rate of 68% after application of TSA. LAT was the only detectable transcript during latent infection, whereas lytic ICP27 transcript was detected in lytically infected and latently infected/TSA treated cultures.Conclusion:Cultured SGNs can be both latently and lytically infected with HSV-1. Furthermore, latently infected SGNs can be reactivated using TSA, yielding infectious virus.Part Ⅱ The role of NSAIDs in multiplication and reactivation of HSV-1 in cultured vestibular ganglion neurons in vitroObjectives:To establish cell culture models of vestibular ganglion neurons (VGNs) in vitro both lytically and latently infected by HSV-1. And to measure the effects of non-steroidal anti-inflammatory drugs (NSAIDs) including indomethacin and celecoxib on multiplication and reactivation of HSV-1 in VGN cultures.Methods:The vestibular ganglions of postnatal day 4-6 Sprague-Dawley rats were dissected out. VGNs were cultured by dissociated culture method and were planted in the 96-well plates. Cultured VGNs were infected with HSV-1 and expression level of cyclooxygenase was measured by real-time PCR and Western blot. Lytically infected cultures were maintained in cycloxygenase (COX) inhibitors of different concentrations for two days. The cells and supernants were collected to measure the viral titers using TCID50 and were compared. The latently infected neurons were treated with indomethacin after TSA application. Cultures were checked daily for the appearance of GFP-positive wells. RNA from cultures was detected for the presence of transcripts of ICP27 and LAT using reverse-transcription polymerase chain reaction.Results:Cycloxygenase-2, which is the target of NSAIDs, was induced by HSV-1 in the lytically infected cultures, with an increase of 14 folds. However, it appeared that indomethacin and celecoxib showed limited but concentration-dependent inhibition effects on viral production under our condition. Indomethacin (100 μmol/L) decreased yields, in this case by 10-fold at 48 hours post infection, and celecoxib (15 μmol/L) caused a reduction of 5.6-fold during this period. This inhibitory effect could be partly reversed by treatment with PGE2. Indomethacin decreased reactivation rate of HSV-1 by about 20%.Conclusion:HSV-1 can induce the expression of COX-2 in VGN cultures; COX inhibitors, indomethacin and celecoxib showed concentration-dependent inhibitory effects on the multiplication of HSV-1. Indomethacin decreased the reactivation rate of HSV-1 in latently infected VGNs.