A Mouse Model of Zika Virus Sexual Transmission and Vaginal Viral Replication

Abstract

Case reports of Zika virus (ZIKV) sexual transmission and genital persistence are mounting. Venereal transmission and genital persistence threaten public health within and beyond the range of ZIKV's mosquito vectors. In this study, we administered ZIKV into the vaginas of AG129 mice and LysMCre⁺IFNAR^fl/fl C57BL/6 mice after hormonal treatments. Mice infected during estrus-like phase were resistant to vaginal infection. In contrast, when infected during diestrus-like phase, AG129 mice succumbed to infection, whereas LysMCre⁺IFNAR^fl/fl mice experienced transient illness. Patency of transgenital transmission (TGT) in diestrus-like mice was demonstrated by detection of viremia and ZIKV replication in spleen and brain, and viral RNA persisted in vaginal washes as late as 10 days post-infection. In these lethal and sublethal mouse models, this study indicates that intravaginal deposition of ZIKV can cause TGT, hormonal changes in the female reproductive tract (FRT) influence transmission, and ZIKV replication persists in the FRT for several days.

Keywords: Zika virus; diestrus; genital; mouse model; semen; sexual; sperm; vagina; venereal.

Figure 1. Mice in diestrus-like phase are susceptible to intravaginal ZIKV infection

Eight- to Twelve-week old AG129 (Figure 1A–C) and LysMCre⁺IFNAR^fl/fl C57BL/6 (Figure 1D–E) mice were intravaginally inoculated with 1 × 10⁵ FFU and 1 × 10⁶ FFU of ZIKV strain FSS13025, respectively. N = 4–5 mice per group. P-values were obtained using the Gehan-Breslow-Wilcoxon test (A–F), ** P≤0.01.

Figure 2. Viremia in diestrus-like mice with intravaginal ZIKV infection

Eight- to Twelve-week old AG129 (Figure 2A) and LysMCre⁺IFNAR^fl/fl C57BL/6 (Figure 2B) mice were intravaginally inoculated with 1 × 10⁵ FFU and 1 × 10⁶ FFU of ZIKV strain FSS13025, respectively. N = 4–5 mice per group. For figure 2A, two independent experiments were performed and data were pooled and expressed as mean ± SEM. Viral RNA levels were measured from serum collected on days 1, 3, 5, 7, and 10 for AG129 mice and on days 3, 5, 7, and 10 for LysMCre⁺IFNAR^fl/fl C57BL/6 mice. P-values were obtained using the parametric two-tailed unpaired t-test with Welch’s correction, * P≤0.05, ** P≤0.01, *** P≤0.001.

Figure 3. Estrus- and diestrus-like vaginal histopathology and immunohistochemical detection of ZIKV protein NS2B in diestrus-like AG129 mice with intravaginal ZIKV infection

At 18hpi, the vaginal mucosa was approximately 10 cell layers thick and covered by a thick layer of keratin in estrus-like mice (A), whereas the mucosa of diestrus-like mice (B) was approximately 5 cell layers thick and unkeratinized. At 18hpi NS2B immunoreactivity was detected in (C) rare cells in the vaginal epithelium and (D) round cells interpreted as macrophages in the subcapsular sinus of the iliac lymph nodes. Morphologically the subcapsular sinus is identified as a channel subjacent to the lymph node capsule that is lined by endothelium and usually does not contain erythrocytes. Little to no NS2B expression was seen in (E) spleen at 18hpi. At 72hpi, increased numbers of NS2B-positive cells were detected in (F) the uterine stroma, (G) iliac lymph node sinuses, and (J) cells within the splenic red pulp interpreted as macrophages. At 10dpi, there was strong NS2B expression in rare (I) neurons. No chromogenic reaction was present in control slides of each tissue incubated with non-specific IgG substituted for NS2B antibody [Lu, lumen; PF, perinodal fat; arrowheads, lymphatic endothelial cells; RP, red pulp; WP, white pulp; H&E, hematoxylin & eosin stain; arrows, NS2B-expressing cells; NovaRED chromogen; hematoxylin counterstain].

Figure 4. ZIKV vaginal RNA persists for up to 10 days post intravaginal infection

Eight- to Twelve-week old AG129 (Figure 4A) and LysMCre⁺IFNAR^fl/fl C57BL/6 (Figure 4B) mice were intravaginally inoculated with 1 × 10⁵ FFU and 1 × 10⁶ FFU of ZIKV strain FSS13025, respectively. N = 4–5 mice per group. For figure 4A, two independent experiments were performed and data were pooled and expressed as mean ± SEM. Viral RNA titers were measured from vaginal washes collected in PBS on days 1, 3, 5, 7, and 10 for AG129 mice and on days 3, 5, 7, and 10 for LysMCre⁺IFNAR^fl/fl C57BL/6 mice. P-values were obtained using the parametric two-tailed unpaired t-test with Welch’s correction, * P≤0.05, ** P≤0.01, *** P≤0.001.