A cell culture model of facial palsy resulting from reactivation of latent herpes simplex type 1

Abstract

Hypothesis: Reactivation of herpes simplex virus type 1 (HSV-1) in geniculate ganglion neurons (GGNs) is an etiologic mechanism of Bell's palsy (BP) and delayed facial palsy (DFP) after otologic surgery.

Background: Several clinical studies, including temporal bone studies, antibody, titers, and intraoperative studies, suggest that reactivation of HSV-1 from latently infected GGNs may lead to both BP and DFP. However, it is difficult to study these processes in humans or live animals.

Methods: Primary cultures of GGNs were latently infected with Patton strain HSV-1 expressing a green fluorescent protein-late lytic gene chimera. Four days later, these cultures were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1 in other neurons. Cultures were monitored daily by fluorescent microscopy. Titers of media from lytic, latent, and latent/TSA treated GGN cultures were obtained using plaque assays on Vero cells. RNA was harvested from latently infected GGN cultures and examined for the presence of viral transcripts using reverse transcription-polymerase chain reaction.

Results: Latently infected GGN cultures displayed latency-associated transcripts only, whereas lytically infected and reactivated latent cultures produced other viral transcripts, as well. The GGN cultures displayed a reactivation rate of 65% after treatment with TSA. Media from latently infected cultures contained no detectable infectious HSV-1, whereas infectious virus was observed in both lytically and latently infected/TSA-treated culture media.

Conclusion: We have shown that cultured GGNs can be latently infected with HSV-1, and HSV-1 in these latently infected neurons can be reactivated using TSA, yielding infectious virus. These results have implications for the cause of both BP and DFP.

FIG. 1

HSV1 lifecycle. Following harvest of GGNs from young rats, cultures are pretreated with ACV and then infected with HSV1. Treatment with ACV is continued for 4 additional days. Following withdrawal of ACV, cultures are treated with TSA to induce viral reactivation. Cultures are monitored with fluorescent microscopy for the expression of GFP.

FIG. 2

Time course of latent HSV1 infection and reactivation experiments.

FIG 3

Light microscopy (A) and fluorescent microscopy (B) of lytically infected GGN cultures. Light microscopy (C) and fluorescent microscopy (D) of latently-infected GGN cultures (D). Light microscopy (E) and fluorescent microscopy (F) of latently infected GGN cultures treated with TSA. Size bar = 30 microns.

FIG. 4

(A) HSV1 reactivation in latently-infected GGN cultures measured as cumulative percentage of GFP-positive wells. Open circles = baseline reactivation, black squares = TSA treated. (B) HSV1 titer measured by plaque assay on media from lytically infected (lytic), latently infected (latent), and latently-infected, TSA treated cultures (+TSA). pfu = plaque-forming units.

FIG. 5

End-point reverse transcription polymerase chain reaction of total RNA extracted from GGN cultures lytically (lytic) and latently (latent) infected with HSV1.