Human trigeminal ganglionic explants as a model to study alphaherpesvirus reactivation

Abstract

Varicella zoster virus (VZV) latency is characterized by limited virus gene expression and the absence of virus DNA replication. Investigations of VZV latency and reactivation have been hindered by the lack of an in vitro model of virus latency. Since VZV is an exclusively human pathogen, we used naturally infected human trigeminal ganglia (TG) obtained at autopsy to study virus latency. Herein, we report optimization of medium to maintain TG integrity as determined by histology and immunohisto-chemistry. Using the optimized culture medium, we also found that both herpes simplex virus-1 (HSV-1) and VZV DNA replicated in TG explants after 5 days in culture. The increase in HSV-1 DNA was fourfold greater than the increase in VZV DNA. Overall, we present a model for alphaherpesvirus latency in human neurons in which the key molecular events leading to virus reactivation can be studied.

A comparison of herpes simplex virus type 1 and varicella-zoster virus latency and reactivation

“…There is one report of VZV reactivation from human ganglia that had been removed 1-5 days post-mortem (Saitoh et al, 2013), but this observation has not been confirmed. Furthermore, only minimal amounts of VZV DNA replication, presumably an event preceding virus reactivation, have been demonstrated within this time frame (Azarkh et al, 2012).…”

“…Following animation, HSV-1 gene transcription undergoes a shift from generalized to organized transcription that orchestrates protein synthesis with DNA replication resulting in release of progeny infectious virus by 24 h for HSV-1. To date, VZV reactivation with release of infectious virus from human ganglia explants has not been demonstrated, but VZV DNA replication has been detected by quantitative PCR analysis of human ganglia explants (Azarkh et al, 2012). Lower-right graph: y-axis, HSV-1 DNA copy number (black line) and VZV DNA copy number (red line); x-axis, time the human ganglia explants were incubated in optimized culture medium; data represent mean¡SEM for virus DNA quantification by real-time, TaqMan-based PCR.…”

“…Whilst this stage occurs within 24 h for HSV-1, VZV DNA replication can be seen as late as 5 days in human trigeminal ganglionic explants (Azarkh et al, 2012). Ultimately, virus components are assembled, transported through the nerve to the skin and complete virions are released with subsequent infection of neighbouring cells.…”

Alphaherpesvirus DNA replication in dissociated human trigeminal ganglia

“…Thus, we focused our present analyses on adult human trigeminal ganglia (TG), where both HSV-1 and VZV are latent (Cohrs et al 2005). Previously, both HSV-1 and VZV DNA replication was observed in human TGs cut into equal pieces and cultured for 0, 1 and 5 days (Azarkh et al 2012). However, latent HSV-1 and VZV DNA are not uniformly distributed in human TG (Cohrs et al 2005; Hüfner et al 2009) and a more accurate analysis of virus DNA replication in human TG requires confirmation that dissociated ganglia are randomly distributed among multiple samples.…”

“…TG were washed and mechanically dissociated. Cells were collected (1000 g , 10 min, 4°C), resuspended in 3 ml of complete culture medium #2 lacking anti-mitotic drugs (Azarkh et al 2012) and distributed into 7 wells in each of three 96-well poly-L-ornithine/laminin-coated plates (BD-Biocoat, Fisher Biosciences, Vacaville, CA). Cells were not sorted before use and contained neurons, satellite cells, which enhance neuronal survival (Hanai et al 2011), and other non-neuronal cells.…”