Recurrent vaginal shedding of herpes simplex type 2 virus in the mouse and effects of antiviral therapy

Abstract

A mouse model of recurrent herpes simplex type 2 (HSV-2) would improve our understanding of the immunobiology of recurrent disease and provide a useful model for evaluating antiviral treatments. We developed a model to evaluate recurrent vaginal HSV-2 shedding using high-dose acyclovir (ACV) therapy beginning at 3 days post infection (dpi). Treatment with 150mg/kg of ACV for 10 days increased survival to 80% following vaginal challenge with HSV-2 strain 186 and to 100% after challenge with strain MS. We then evaluated recurrent vaginal HSV-2 shedding in surviving mice. Although infectious virus was not detected in vaginal samples after 21dpi, viral DNA was detectable by PCR in 80% of mice (47/59) on at least 1 day, while no animal was positive for virus on every day. ACV therapy administered from day 21 to 31 significantly reduced recurrent virus shedding during this period from 7.3% (8/109 swabs) to 0.8% (1/126 swabs) (p=0.013). Lastly, ACV-rescued HSV-2-infected mice treated with cyclophosphamide at 35 and 38dpi rapidly succumbed, indicating that this model can be used to study immune control of the persistent infection. Thus, this model provides an inexpensive model for evaluating therapeutic strategies and immune control of persistent HSV.

Figure 1. Effect of ACV treatment on levels of replicating HSV-2 in the dorsal root ganglia

Dorsal root ganglia (DRG) were harvested from HSV-2-infected mice at 0, 3, 5, 8, 12, and 15 dpi and the viral titer in tissue homogenates was quantified by plaque assay. For each day, the average viral titer ***(N=3, except the untreated group on day 15 (N=1) is indicated. ACV-treated animals received 100 mg/kg IP BID for 10 days beginning 3 dpi.

Figure 2. Survival and vaginal HSV-2 shedding in ACV rescued mice

HSV-2-infected mice were administered ACV at 100 mg/kg IP BID for 10 days beginning 3 dpi. Animals were monitored daily for mortality and vaginal swabs were collected from surviving animals at 20, 23, 26, 29, 32, and 41 dpi. (A) Survival of ACV-treated and untreated mice. (B) Nested PCR was used to determine the percentage of swabs containing HSV-2 DNA on each day. The fractions above each bar indicate the number of positive swabs over the total from each day. Results shown are combined data from two independent experiments (ACV treated, N=72; untreated, N=7).

Figure 3. Symptom and Vaginal shedding patterns in ACV rescued mice

(A) Symptom and shedding patterns from 0–41 dpi observed in 6 selected mice, infected vaginally with HSV-2 strain 186 are shown in panels a–f. The symbols as shown in the figure depict a PCR+ vaginal sample, erythema, ruffled fur, hair loss and erythema, hair loss, time of sacrifice, and virus+ DRG (replicating virus). All mice were positive for replicating virus at 2 dpi and positive for viral DNA in DRG at time of sacrifice (not shown in figure). (B) Results from a typical PCR analysis is depicted for 20 and 26 dpi. The 124 bp gB product is shown by an arrow.

Figure 4. Comparison of symptoms and survival in mice infected with either HSV-2 strain 186 or HSV-2 strain MS

Mice were intravaginally infected with either 1×10⁴ pfu (strain 186, N=14) or 5×10³ pfu (strain MS, N=12) HSV-2. All mice received ACV at 150 mg/kg IP BID for 10 days beginning 3 dpi. (A) The percentage of animals in each group experiencing the combined symptoms of hair loss and erythema, and (B) the percentage of surviving animals on each day are indicated.

Figure 5. Comparison of vaginal HSV shedding in mice infected with either HSV-2 strain 186 or HSV-2 strain MS

Vaginal swabs were collected from surviving animals at 20, 23, 26, 29, 32, and 35 dpi, and the presence of HSV-2 DNA was determined by nested PCR. The fractions above each bar indicate the number of positive swabs over the total from each day.

Figure 6. Effect of a second ACV treatment on survival and vaginal HSV-2 shedding

All mice were intravaginally infected with 5×10³ pfu HSV-2 strain MS and given ACV at 150 mg/kg IP BID for 10 days beginning 3 dpi. From 21 to 31 days post infection, Group B (ACV, ACV) (N=21) received an additional course of ACV at 150 mg/kg BID while Group A (ACV, −) (N=21) remained untreated on these days. (A). Animals were monitored daily for mortality. (B) Vaginal swabs were collected from surviving animals at 22, 24, 26, 28, 30, and 32 dpi, and the percentage of swabs containing HSV-2 DNA was determined by nested PCR. The P=0.0133 is depicted for the difference in shedding between groups A and B. The ACV treatment times are indicated by boxes.

Figure 7. Effect of immunosuppresion on survival of HSV-2 infected mice and the levels of replicating virus in neural tissues

HSV-infected mice were immunosuppressed with 150 mg/kg of cyclophosphamide at 35 and 38 dpi (CY), or remained untreated (No CY). (A) The combined survival of untreated mice and those receiving immunosuppression are shown. (B) Dorsal root ganglia (DRG) and spinal cords (SC) were harvested from sickly mice requiring euthanization and healthy mice at experiment termination (105 dpi). Replicating virus was detected in tissue homogenates by plaque assay, and the percentages of animals with detectable viral replication in each tissue are shown.