Organotypic epithelial raft cultures as a model for evaluating compounds against alphaherpesviruses

Abstract

The course of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) infections in squamous epithelial cells cultured in a three-dimensional organotypic raft culture was tested. In these raft cultures, normal human keratinocytes isolated from neonatal foreskins grown at the air-liquid interface stratified and differentiated, reproducing a fully differentiated epithelium. Typical cytopathic changes identical to those found in the squamous epithelium in vivo, including ballooning and reticular degeneration with the formation of multinucleate cells, were observed throughout the raft following infection with HSV and VZV at different times after lifting the cultures to the air-liquid interface. For VZV, the aspects of the lesions depended on the stage of differentiation of the organotypic cultures. The activity of reference antiviral agents, acyclovir (ACV), penciclovir (PCV), brivudin (BVDU), foscarnet (PFA), and cidofovir (CDV), was evaluated against wild-type and thymidine kinase (TK) mutants of HSV and VZV in the raft cultures. ACV, PCV, and BVDU protected the epithelium against cytopathic effect induced by wild-type viruses in a concentration-dependent manner, while treatment with CDV and PFA proved protective against the cytodestructive effects induced by both TK+ and TK- strains. The quantification of the antiviral effects in the rafts were accomplished by measuring viral titers by plaque assay for HSV and by measuring viral DNA load by real-time PCR for VZV. A correlation between the degree of protection as determined by histological examination and viral quantification could be demonstrated The three-dimensional epithelial raft culture represents a novel model for the study of antiviral agents active against HSV and VZV. Since no animal model is available for the evaluation of antiviral agents against VZV, the organotypic cultures may be considered a model to evaluate the efficacy of new anti-VZV antivirals before clinical trials.

FIG. 1.

Pattern of HSV-1 (KOS strain) infection in cultures infected at day 0 (B), or 2 days (C), 4 days (D), 6 days (E), or 8 days (F) after lifting compared to noninfected cultures (A), where the stratum corneum (a), the well-differentiated epithelium (b) and the collagen matrix with the feeder cells (c) can be seen. Magnification 40.

FIG. 2.

Pattern of VZV (OKA and 07-1 strains) infection in cultures infected at day 0 (A), or 2 days (B) 4 days (C), 6 days (D), or 8 days (E) after lifting. Magnification ×40.

FIG. 3.

Effects of ACV (A) and PCV (B) on organotypic epithelial rafts cultures infected with HSV-1 (KOS strain) at 8 days after lifting. Compounds were added to the culture medium on the day of infection and remained in contact with the cells until the rafts were fixed (i.e., at 12 days after lifting).

FIG. 3.

Effects of ACV (A) and PCV (B) on organotypic epithelial rafts cultures infected with HSV-1 (KOS strain) at 8 days after lifting. Compounds were added to the culture medium on the day of infection and remained in contact with the cells until the rafts were fixed (i.e., at 12 days after lifting).

FIG. 4.

Quantification of virus yield in organotypic epithelial raft cultures infected with HSV-1 and HSV-2 TK+ and TK⁻ strains at 8 days postlifting. Compounds were added to the culture medium the day of infection (i.e., 8 days postdifferentiation) and remained in contact with the cells till the rafts were frozen (i.e., at 12 days after lifting) for determination of virus production by plaque assay.

FIG. 5.

Effects of different antiviral compounds on organotypic epithelial raft cultures infected with 07-1 TK⁻ strain at 5 days after lifting. Compounds were added to the culture medium the day of infection and remained in contact with the cells until the rafts were fixed (i.e., at 12 days after lifting).

FIG. 6.

Quantification of VZV-DNA by real-time PCR in organotypic epithelial raft cultures infected with Oka (TK⁺) or 07-1 (TK⁻). Cidofovir was added to the culture medium the day of infection (5 days postdifferentiation) and remained in contact with the cells till the epithelium was harvested and DNA isolated (i.e., at 12 days after lifting).