Herpes simplex virus type 2-mediated disease is reduced in mice lacking RNase L

Abstract

RNase L helps mediate the antiviral state induced by type I interferons (IFNalphabeta). Although herpes simplex virus (HSV) encodes inhibitors of the IFNalphabeta-induced antiviral response, the IFNalphabeta system serves the body as a first line of defense against HSV. We investigated whether RNase L limits HSV-2 replication and virulence. RNaseL(-/-) and wild-type C57BL/6 mice were infected intravaginally with HSV-2 strain 333. Although initial replication in the genital epithelium was similar, mice lacking RNase L developed less severe genital and neurologic disease than wild-type mice, survived longer, and contained lower viral titers in the nervous system. CD4(+) T cell infiltration into the genital tract and spinal cord of RNase L(-/-) mice was reduced, suggesting that a restricted inflammatory response may account for reduction in disease. Thus, RNase L does not play a significant role in control of HSV-2 infection in vivo; instead, RNase L may regulate aspects of the inflammatory response that contribute to disease.

Figure 1. Replication of HSV-2 in the genital mucosa of wild-type B6 and RNase L^−/− mice

Mice were infected i.vag. with 2×10⁶ pfu/mouse of HSV-2 strain 333. A) Titer of virus in vaginal swab samples taken 9 hr to 4 days post-infection was determined by standard plaque assay on Vero cells. Data points represent the geometric mean ± standard error of the mean for 6 mice per group in one of two experiments with similar results.

Figure 2. Severity of genital disease in wild-type B6 and RNase L^−/− mice infected with HSV-2

Mice were infected with 2×10⁶ pfu/mouse i.vag. with HSV-2 strain 333. A) Genital disease scores for 33 wild-type B6 and 43 RNase L^−/− mice, and B) change in weight for 22 wild-type B6 and 28 RNase L^−/− mice were recorded daily. Values represent the arithmetic mean ± standard error of the mean. *p value <0.001; **p value ≤0.0001.

Figure 3. Survival time of mice after infection with a lethal dose of HSV-2

Wild-type B6 and RNase L^−/− mice were infected i.vag. with 2×10⁶ pfu/mouse with HSV-2 strain 333 and survival was recorded daily. n=19 for wild-type and n=22 for RNase L^−/− mice. **p value=0.0003 on day 6; *p value= 0.0252 on day 7 post-infection.

Figure 4. Replication of HSV-2 in the nervous tissues of wild-type B6 and RNase L^−/− mice

A) Groups of mice surviving to day 6 post-infection with HSV-2 were sacrificed, and brain, brainstem, and spinal cord tissues were dissected. Viral titers in disrupted tissues were determined by plaque assay. The dashed line indicates the limit of detection. n=13 for wild-type B6 mice and n=22 for RNase L^−/− mice. *p value=0.0152; **p value=0.0116. B) Groups of mice were injected intracranially with 5×10³ PFU of HSV-2 and sacrificed 1, 2 or 3 days post-infection. Brain tissue was removed and viral titers in disrupted tissues were determined by plaque assay. Values represent the geometric mean ± SEM of 4 to 8 mice per group.

Figure 5. T lymphocyte infiltration into the genital tract and spinal cord

Groups of RNase L^−/− or wild-type mice were uninfected or infected with HSV-2. After 6 days, genital tracts and lumbosacral spinal cords were dissected. Mononuclear cells enzymatically released from tissues of individual mice were stained with anti-CD45-APC, anti-CD8-PE, anti-CD4-FITC, and anti-CD69-PerCP and analyzed by flow cytometry. Regions were defined by CD45⁺ cells of size and granularity typically associated with lymphocytes. Data from two individual experiments were pooled and represent A and B) the mean percentage ± SD of CD8⁺ and CD4⁺ T cells, respectively, recovered from 6 individual mice, and C and D) the mean number of CD8⁺ and CD4⁺ T cells, respectively, recovered. **p value=0.0301–0.0379; *p value=0.0438.