Alphaherpesvirus DNA replication in dissociated human trigeminal ganglia

Abstract

Analysis of the frequency and PCR-quantifiable abundance of herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) DNA in multiple biological replicates of cells from dissociated, randomly distributed human trigeminal ganglia (TG) of 4 subjects revealed an increase in both parameters and in both viruses during 5 days of culture, with no further change by 10 days. Dissociated TG provides a platform to analyze initiation of latent virus DNA replication within 5 days of culture.

Herpes Simplex Virus Establishment, Maintenance, and Reactivation: In Vitro Modeling of Latency

“…Defining the specific site of HSV latency, one aspect necessary in building a relevant model, has been historically challenging. The current consensus is that HSV is most frequently found in sensory neurons that innervate the primary site of infection [ 47 ]. For example, latent HSV-1 can be found in the trigeminal ganglia (TG) that innervate the lips, gingiva, and eyes, and latent HSV-2 can be found in the dorsal root ganglia (DRG) that innervate the skin and muscle of the back as well as the mucosa of the genitalia [ 48 ].…”

Induction of varicella zoster virus DNA replication in dissociated human trigeminal ganglia

“…The mean 14.4-fold increase in VZV DNA was significantly greater than the amount of VZV DNA present at day 0 (Wilconon signed-rank test; p = 0.0005) and was greater than the <3.5-fold increase at day 5 when dissociated individual human TG were cultured at higher density in 96-well tissue culture plates, although the abundance of VZV DNA in either culture condition was not increased at day 10 (Fig. 2; Cohrs et al 2016). …”

“…Latent HSV-1 can be recovered from about half of human TG explants 10–24 days in culture (Plummer 1973; Warren et al 1977). Although VZV has never been isolated from latently infected human TG (Plotkin et al 1977), virus DNA does replicate in human TG explants (Azarkh et al 2012) and dissociated ganglia (Cohrs et al 2016). …”

“…The two TG from each subject were combined, washed with PBS, and mechanically dissociated into sizes sufficiently small to pass through the opening of a 1000-μl pipette tip. This critical step ensured that neurons were randomly distributed among samples to reduce sampling error upon assessment of VZV DNA content (Cohrs et al 2016). While we did not count neurons or non-neuronal cells in the current experiments, human TG contain on average 27,400 ± 4800 neurons with approximately 100:1 non-neuronal cell/neuron ratio (Laguardia et al 2000) and our dissociation protocol did not specifically eliminate non-neuronal cells or add antimitotic drugs to kill actively dividing cells.…”

“…1). VZV DNA copy number was determined by TaqMan-based quantitative PCR of total DNA extracted from each sample (Cohrs et al 2016). …”