Herpes simplex virus type 1 corneal infection results in periocular disease by zosteriform spread

Abstract

In humans and animal models of herpes simplex virus infection, zosteriform skin lesions have been described which result from anterograde spread of the virus following invasion of the nervous system. Such routes of viral spread have not been fully examined following corneal infection, and the possible pathologic consequences of such spread are unknown. To investigate this, recombinant viruses expressing reporter genes were generated to quantify and correlate gene expression with replication in eyes, trigeminal ganglia, and periocular tissue. Reporter activity peaked in eyes 24 h postinfection and rapidly fell to background levels by 48 h despite the continued presence of viral titers. Reporter activity rose in the trigeminal ganglia at 60 h and peaked at 72 h, concomitant with the appearance and persistence of infectious virus. Virus was present in the periocular skin from 24 h despite the lack of significant reporter activity until 84 h postinfection. This detection of reporter activity was followed by the onset of periocular disease on day 4. Corneal infection with a thymidine kinase-deleted reporter virus displayed a similar profile of reporter activity and viral titer in the eyes, but little or no detectable activity was observed in trigeminal ganglia or periocular tissue. In addition, no periocular disease symptoms were observed. These findings demonstrate that viral infection of periocular tissue and subsequent disease development occurs by zosteriform spread from the cornea to the periocular tissue via the trigeminal ganglion rather than by direct spread from cornea to the periocular skin. Furthermore, clinical evidence is discussed suggesting that a similar mode of spreading and disease occurs in humans following primary ocular infection.

FIG. 1

Maps of reporter gene viruses used in this study. (A) Prototypical arrangement of the HSV-1 genome, showing the unique long (U_L) and unique short (U_S) segments flanked by internal (a′, b′, c′) and terminal (a, b, c) repeats. The locations of reporter cassette insertions and the thymidine kinase deletion are shown. (B) KOS6β was constructed by insertion of a cassette containing the early ICP6 promoter regulating the expression of β-galactosidase (10) into the BglII site at map position 106750. (C) KOS6βΔtk was constructed by a 360-bp deletion of the thymidine kinase optical reading frame within the context of KOS6β (16). (D) KOS/Dlux/oriS was constructed by insertion of a cassette carrying the ICP4/22/47 and oriS regulatory region driving firefly and Renilla luciferase expression into the BglII site.

FIG. 2

Single-step growth kinetics and reporter gene activity for KOS (A), KOS6β and KOS6βΔtk (B), and KOS/Dlux/oriS (C). Vero cells were infected at an MOI of 5. Data represent standard errors of the means of three independent experiments. The limit of detection is 10 PFU/ml. RLU, relative light units.

FIG. 3

Reporter gene activity under cycloheximide reversal or control conditions. Vero cells were pretreated for 1 h and then were infected at an MOI of 5 in the presence or absence of cycloheximide (100 μg/ml) for 8 h, after which time cells were washed and plated in the presence of actinomycin D (10 μg/ml) for 4 h. Monolayers were harvested and assayed for reporter activity. RLU, relative light units.

FIG. 4

Periocular disease scores in mice following corneal scarification and infection with 2 × 10⁶ PFU per eye of KOS/Dlux/oriS, KOS6β, and KOS6βΔtk. Animals were scored as follows: 0, no lesions; 1, minimal eyelid swelling; 2, moderate swelling and crusty ocular discharge; 3, severe swelling, moderate periocular hair loss, and skin lesions; 4, severe swelling with eyes crusted shut, severe periocular hair loss, and skin lesions. Data represent combined averages of at least 10 mice per time point.

FIG. 5

In vivo growth and reporter gene expression of KOS/Dlux/oriS after ocular infection. Mice were infected via corneal scarification and inoculation of 2 × 10⁶ PFU per eye. At various times postinfection tissues were harvested and assayed for infectious virus and luciferase activity. Data points represent 12 tissues from two independent experiments with three mice. In periocular skin, both luciferase activity and titer were significantly increased (P < .05) at 84 h relative to earlier time points. RLU, relative light units.

FIG. 6

In vivo growth and reporter gene expression of KOS6β and KOS6βΔtk after ocular infection. Mice were infected via corneal scarification and inoculation of 2 × 10⁶ PFU of KOS6β or 8.5 × 10⁷ PFU of KOS6βΔtk per eye. At various times postinfection tissues were harvested and assayed for infectious virus and β-galactosidase activity. Data points represent 12 tissues from two independent experiments with three mice. In KOS6β infections, β-galactosidase activity and titer were significantly increased (P < 0.05) at 84 h relative to earlier time points and significantly higher than those of KOS6βΔtk (P < 0.05) from 72 h onward.

FIG. 7

Growth of KOS6β and KOS6βΔtk in periocular tissue explants after ocular infection. Mice were infected via corneal scarification and inoculation of 2 × 10⁶ PFU of KOS6β or 8.5 × 10⁷ PFU of KOS6βΔtk per eye. Twelve hours postinfection tissue was harvested and explanted in medium. Supernatants were titers at various times postinfection. Data represent the mean of data from four mice from two independent experiments.

FIG. 8

Human clinical correlate to periocular disease in mice. Five days following initial presentation with primary corneal disease, the subject shown had periorbital swelling and redness. Discrete vesicles and lesions, characteristic of HSV, are visible on the lid margin and brow.