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 immunohistochemistry. 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.

Fig. 1

Integrity of human trigeminal ganglia. Human trigeminal ganglia were cultured for 0 days (a–c) or 5 days (d–k) in culture medium 1 (d, e), 2 (f, g), 3 (h, i), and 4 (j, k). Sections were stained with hematoxylin/eosin (a, d, f, h, and j) or immunostained with antibodies directed against the heavy subunit of human neurofilament (b, e, g, i, and k). To detect background autofluorescence, normal IgG was substituted for neurofilament antibody (c)

Fig. 2

Efficiency of PCR amplification. Dilutions of VZV or HSV-1 DNA were PCR-amplified with primers specific for each virus

Fig. 3

Quantification of VZV and HSV-1 DNA in human trigeminal ganglion explants. Human trigeminal ganglia from subject 2 were divided into three portions and incubated for various times as indicated in “Methods” section. Total DNA was extracted and the abundance of VZV and HSV-1 DNA quantified by real-time PCR. TG-L left trigeminal ganglion, TG-R right trigeminal ganglion