Efficient reactivation of latent herpes simplex virus from mouse central nervous system tissues

Abstract

For decades, numerous ex vivo studies have documented that latent herpes simplex virus (HSV) reactivates efficiently from ganglia, but rarely from the central nervous systems (CNS), of mice when assayed by mincing tissues before explant culture, despite the presence of viral genomes in both sites. Here we show that 88% of mouse brain stems reactivated latent virus when they were dissociated into cell suspensions before ex vivo explant culture. The efficient reactivation of HSV from the mouse CNS was demonstrated with more than one viral strain, viral serotype, and mouse strain, further indicating that the CNS can be an authentic latency site for HSV with the potential to cause recurrent disease.

FIG. 1.

Correlation between reactivation frequency and acute replication or number of latent viral genomes in brain regions. The reactivation frequencies were plotted against the average viral titers at day 5 p.i. (A) or against the average number of latent viral genomes (B) in brain stems (open diamond), cerebellums (solid triangle), olfactory bulbs (open circle), frontal cortices (solid circle), and hippocampi (open triangle). The best-fit lines were generated by linear regression of values, and the correlation coefficients (r²) are displayed in each panel.

FIG. 2.

Kinetics of reactivation of HSV from mouse neural tissues. Brain stems (BS) (filled symbols) and trigeminal ganglia (TG) (open symbols) of mice inoculated with (A) HSV-1 KOS, (B) HSV-1 McKrae, or (C) HSV-2 333 were harvested at day 30 p.i. to assay for viral reactivation by the dissociation method.

FIG. 3.

The dissociation method sustains the cell viability and viral infectivity of brain stem cultures. (A) Brain stems (BS) and trigeminal ganglia (TG) harvested from uninfected BALB/c mice were minced or dissociated and then cultured before the cell viabilities of minced and dissociated cultures were evaluated by trypan blue exclusion. One hundred nucleated cells were counted, and the unstained (viable) cells were recorded. (B) BS and TG harvested from uninfected BALB/c mice were minced or dissociated and cultured. HSV-1 KOS (2,000 PFU) was added to cultures, and cultures were harvested for titration of infectious virus at 24 h p.i. Data shown in panels A and B are the means ± standard errors of the means from three independent experiments, each done in duplicate. *, P < 0.05 by Student's t test. (C) Dissociated BS, but not TG, preparations damaged less confluent Vero and human neuronal (SK-N-SH) cell monolayers. Dissociated cells from one-third of a BS or one TG were added to monolayers of Vero or SK-N-SH cells and cultured for 48 h before being stained with crystal violet. The data shown represent two experiments.