V. Hukkanen et al., Herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture, J GEN VIROL, 80, 1999, pp. 2149-2155
This study describes the outcome of herpes simplex virus type 1 (HSV-1) inf
ection in an organotypic raft culture of spontaneously immortalized HaCat k
eratinocytes and human fibroblasts, as related to the virus load and epithe
lial stratification and differentiation. In this model, a confluent monolay
er of HaCat keratinocytes was formed 60 h after seeding. Inoculation of HSV
-1 before induction of differentiation by lifting of the culture to the air
-liquid interface always resulted in a productive infection, but the virus
yield was highest when the inoculation took place 72 h after seeding. Even
at 0.1 p.f.u. per culture, the HaCat cultures became HSV positive. Infectio
n of the full-thickness epithelium at 5 p.f.u. per culture resulted in a pr
oductive infection of the whole epithelium. The HaCat cells were about 10 t
imes more sensitive to HSV-1 infection than the Vero cells in which the vir
us stocks were titrated. The raft cultures infected 30 min after lifting we
re negative by HSV-1 culture, and no HSV-1 antigen was detected by immunocy
tochemistry, PCR showed the presence of HSV-I DNA and in situ hybridization
showed reactivity with a latency-associated RNA probe, indicating the pres
ence of a non-productive infection. Two different patterns of virus spread
in epithelia were found: (i) lateral spread through the superficial layers
of the epithelium and (ii) a demarcated infection throughout the whole thic
kness of the epithelium at the margins of the culture.