Coitus-Free Sexual Transmission of Zika Virus in a Mouse Model

Abstract

Zika virus (ZIKV) is an arboviral infection that may be sexually transmitted. The present study aims to determine if accessory sex glands are a potential source of infectious virus and important in sexual transmission. Male interferon type I receptor knockout (Ifnar^-/-) mice were challenged subcutaneously with a Puerto Rican ZIKV isolate. Reproductive tissues were harvested seven days after viral challenge and artificial insemination fluid derived from epididymis or homogenized accessory sex glands (seminal plasma) was obtained. Naïve interferon type I and II receptor knockout (AG129) females were pre-treated with progesterone, and inoculated intravaginally with either epididymal flush or seminal plasma from ZIKV-infected males. ZIKV RNA was demonstrated in the artificial insemination fluid and ZIKV antigen was detected in epididymal epithelial cells but not within seminiferous tubules at the time of artificial insemination fluid collection. Peripheral viremia, demonstrated by ZIKV RNA in whole blood samples of females from each challenge group was observed. Infectious virus was present in both epididymal fluid and seminal plasma. These studies provide evidence of passage of virus from epididymal flush and seminal plasma to naïve females via artificial insemination and provides a model for the study of sexual transmission of ZIKV.

Figure 1

Normal histology and histopathology of the testicle and epididymis of control and Zika vius infected Ifnar^−/− males. (A) Normal testicle from a sham-infected male. The interstitium contain Leydig (interstitial) cells and blood vessels. (B) Testis from a Zika virus infected Ifnar^−/− male with mild orchitis. Mild neutrophilic infiltration (arrow) is observed in the interstitium. (C) Normal epididymis from a sham-infected male. (D) Epididymis from a Zika virus infected male exhibiting mild epididymitis. There is neutrophilic infiltration (arrow) of interstitium and individual epithelial cell necrosis (arrowhead). All sections 400x magnification; hematoxylin and eosin stain; bar = 50 µm.

Figure 2

Immunohistochemical staining for Zika virus antigen in the epididymis. (A) Epididymis from a sham-infected male (400×). (B) Epididymis from a ZIKV-infected male. Focally, moderate numbers of epididymal epithelial cells exhibit cytoplasmic ZIKV antigen immunoreactivity (400×).

Figure 3

Relative Genome Equivalents of Zika Virus RNA (A) Relative genome equivalents of Zika virus (ZIKV) in the artificial insemination of treatment groups. Group 3 (b), epididymal flush from a sham-infected male and seminal plasma from a ZIKV-infected male, had statistically significant lower ZIKV RNA than groups 1 and 2 (a). (B) Relative genome equivalents of Zika virus in peripheral whole blood of intra-vaginally inoculated females. High viral RNA loads were observed in treatment groups one, two, three and four.+P = progesterone pre-treated; −P = no progesterone treatment.

Figure 4

Female Morbidity and Mortality (A) Survival of intravaginally inoculated females in each treatment group. One hundred percent survival was observed in the negative control group, six. In addition, the non-progesterone treated replicates of treatments four and five had 100% survival. (B) Percent weight change of intravaginally inoculated females. Weight change was compared between the day of intravaginal inoculation and the weight on the day of death. An average weight loss was observed in treatments 1, 2, and 4. (C) Survival of subcutaneously inoculated females. Complete cohort survival was only observed in treatment 6, artificial insemination fluid derived from sham-infected males. One hundred percent mortality was observed in treatments 2, 4 and 5. (D) Percent weight change of subcutaneously inoculated females. Significant weight loss was observed in treatments 2, 4 and 5. Initial weight loss was observed in treatment 1 with surviving females recovering weight following days 11 post infection.