No abstract

Zika virus (ZIKV) infection during pregnancy in humans is associated with an increased incidence of congenital anomalies including microcephaly as well as fetal death and miscarriage and collectively has been referred to as Congenital Zika Syndrome (CZS). Animal models for ZIKV infection in pregnancy have been developed including mice and non-human primates (NHPs). In macaques, fetal CZS outcomes from maternal ZIKV infection range from none to significant. In the present study we develop the olive baboon (Papio anubis), as a model for vertical transfer of ZIKV during pregnancy. Four mid-gestation, timed-pregnant baboons were inoculated with the French Polynesian ZIKV isolate (104 ffu). This study specifically focused on the acute phase of vertical transfer. Dams were terminated at 7 days post infection (dpi; n = 1), 14 dpi (n = 2) and 21 dpi (n = 1). All dams exhibited mild to moderate rash and conjunctivitis. Viremia peaked at 5–7 dpi with only one of three dams remaining mildly viremic at 14 dpi. An anti-ZIKV IgM response was observed by 14 dpi in all three dams studied to this stage, and two dams developed a neutralizing IgG response by either 14 dpi or 21 dpi, the latter included transfer of the IgG to the fetus (cord blood). A systemic inflammatory response (increased IL2, IL6, IL7, IL15, IL16) was observed in three of four dams. Vertical transfer of ZIKV to the placenta was observed in three pregnancies (n = 2 at 14 dpi and n = 1 at 21 dpi) and ZIKV was detected in fetal tissues in two pregnancies: one associated with fetal death at ~14 dpi, and the other in a viable fetus at 21 dpi. ZIKV RNA was detected in the fetal cerebral cortex and other tissues of both of these fetuses. In the fetus studied at 21 dpi with vertical transfer of virus to the CNS, the frontal cerebral cortex exhibited notable defects in radial glia, radial glial fibers, disorganized migration of immature neurons to the cortical layers, and signs of pathology in immature oligodendrocytes. In addition, indices of pronounced neuroinflammation were observed including astrogliosis, increased microglia and IL6 expression. Of interest, in one fetus examined at 14 dpi without detection of ZIKV RNA in brain and other fetal tissues, increased neuroinflammation (IL6 and microglia) was observed in the cortex. Although the placenta of the 14 dpi dam with fetal death showed considerable pathology, only minor pathology was noted in the other three placentas. ZIKV was detected immunohistochemically in two placentas (14 dpi) and one placenta at 21 dpi but not at 7 dpi. This is the first study to examine the early events of vertical transfer of ZIKV in a NHP infected at mid-gestation. The baboon thus represents an additional NHP as a model for ZIKV induced brain pathologies to contrast and compare to humans as well as other NHPs.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus with devastating outcomes seen recently in the Americas due to the association of maternal ZIKV infection with fetal microcephaly and other fetal malformations not previously associated with flavivirus infections. Here, we have developed the olive baboon () as a non-human primate (NHP) translational model for the study of ZIKV pathogenesis and associated disease outcomes to contrast and compare with humans and other major NHPs such as macaques. Following subcutaneous inoculation of adult male and non-pregnant female baboons, viremia was detected at 3 and 4 days post infection (dpi) with the concordant presentation of a visible rash and conjunctivitis similar to human ZIKV infection. Furthermore, virus was detected in the mucosa and cerebrospinal fluid. A robust ZIKV-specific IgM and IgG antibody response was also observed in all animals. These data show striking similarity between humans and the olive baboon following infection with ZIKV suggesting our model as a suitable translational NHP model to study ZIKV pathogenesis and potential therapeutics. ZIKV was first identified in 1947 in a sentinel rhesus monkey in Uganda and subsequently spread to Southeast Asia. Until 2007, only a small number of cases were reported, and ZIKV infection was relatively minor until the South Pacific and Brazilian outbreaks where more severe outcomes were reported. Here we present the baboon as a non-human primate model for contrast and comparison to other published animal models of ZIKV such as the mouse and macaque species. Baboons breed year around and are not currently a primary non-human primate species used in biomedical research marking them more readily available for studies other than Human Immunodeficiency virus studies, which many macaque species are designated for. This taken together with the similarities baboons have with humans, such as immunology, reproduction, genetics and size, makes the baboon an attractive NHP model for ZIKV studies in comparison to other non-human primates.

Zika virus (ZIKV) infection in pregnant women is a serious threat to the development and viability of the fetus. The primary mode of ZIKV transmission to humans is through mosquito bites, but sexual transmission has also been well documented in humans. However, little is known of the short- and long-term effects of ZIKV infection on the human male reproductive system. This study examines the effects of ZIKV infection on the male reproductive organs and semen and the immune response of the olive baboon (Papio anubis). Nine mature male baboons were infected with ZIKV (French Polynesian strain) subcutaneously. Six animals were euthanized at 41 days, while three animals were euthanized at 10 or 11 days postinfection (dpi). Viremia and clinical evidence of infection were present in all nine baboons. ZIKV RNA was present in the semen of five of nine baboons. ZIKV was present in the testes of two of three males euthanized at 10 or 11 dpi, but in none of six males at 41 dpi. Immunofluorescence of testes suggested the presence of ZIKV in sperm progenitor cells, macrophage penetration of seminiferous tubules, and increased tumor necrosis factor alpha (TNF-α), particularly in vascular walls. These data demonstrate that male olive baboons approximate the male human ZIKV response, including viremia, the adaptive immune response, and persistent ZIKV in semen. Although gross testicular pathology was not seen, the demonstrated breach of the testes-blood barrier and targeting of spermatogenic precursors suggest possible long-term implications in ZIKV-infected primates. IMPORTANCE Zika virus (ZIKV) is an emerging flavivirus spread through mosquitoes and sexual contact. ZIKV infection during pregnancy can lead to severe fetal outcomes, including miscarriage, fetal death, preterm birth, intrauterine growth restriction, and fetal microcephaly, collectively known as congenital Zika syndrome. Therefore, it is important to understand how this virus spreads, as well as the resulting pathogenesis in translational animal models that faithfully mimic ZIKV infection in humans. Such models will contribute to the future development of efficient therapeutics and prevention mechanisms. Through our previous work in olive baboons, we developed a nonhuman primate model that is permissive to ZIKV infection and transfers the virus vertically from mother to fetus, modeling human observations. The present study contributes to understanding of ZIKV infection in male baboon reproductive tissues and begins to elucidate how this may affect fertility, reproductive capacity, and sexual transmission of the virus.

Zika virus (ZIKV) infection is now firmly linked to congenital Zika syndrome (CZS), including fetal microcephaly. While Aedes species of mosquito are the primary vector for ZIKV, sexual transmission of ZIKV is a significant route of infection. ZIKV has been documented in human, mouse, and nonhuman primate (NHP) semen. It is critical to establish NHP models of the vertical transfer of ZIKV that recapitulate human pathogenesis. We hypothesized that vaginal deposition of ZIKV-infected baboon semen would lead to maternal infection and vertical transfer in the olive baboon (Papio anubis). Epidemiological studies suggest an increased rate of CZS in the Americas compared to the original link to CZS in French Polynesia; therefore, we also compared the French Polynesian (FP) ZIKV isolate to the Puerto Rican (PR) isolate. Timed-pregnant baboons (n = 6) were inoculated via vaginal deposition of baboon semen containing 106 focus-forming units (FFU) of ZIKV (n = 3 for FP isolate H/PF/2013; n = 3 for PR isolate PRVABC59) at midgestation (86 to 95 days of gestation [dG]; term, 183 dG) on day 0 (all dams) and then at 7-day intervals through 3 weeks. Maternal blood, saliva, and cervicovaginal wash (CVW) samples were obtained. Animals were euthanized at 28 days (n = 5) or 39 days (n = 1) after the initial inoculation, and maternal/fetal tissues were collected. Viremia was achieved in 3/3 FP ZIKV-infected dams and 2/3 PR ZIKV-infected dams. ZIKV RNA was detected in CVW samples of 5/6 dams. ZIKV RNA was detected in lymph nodes but not the ovaries, uterus, cervix, or vagina in FP isolate-infected dams. ZIKV RNA was detected in lymph nodes (3/3), uterus (2/3), and vagina (2/3) in PR isolate-infected dams. Placenta, amniotic fluid, and fetal tissues were ZIKV RNA negative in the FP isolate-infected dams, whereas 2/3 PR isolate-infected dam placentas were ZIKV RNA positive. We conclude that ZIKV-infected semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate appeared more capable of widespread dissemination to tissues, including reproductive tissues and placenta, than the FP isolate. IMPORTANCE Zika virus remains a worldwide health threat, with outbreaks still occurring in the Americas. While mosquitos are the primary vector for the spread of the virus, sexual transmission of Zika virus is also a significant means of infection, especially in terms of passage from an infected to an uninfected partner. While sexual transmission has been documented in humans, and male-to-female transmission has been reported in mice, ours is the first study in nonhuman primates to demonstrate infection via vaginal deposition of Zika virus-infected semen. The latter is important since a recent publication indicated that human semen inhibited, in a laboratory setting, Zika virus infection of reproductive tissues. We also found that compared to the French Polynesian isolate, the Puerto Rican Zika virus isolate led to greater spread throughout the body, particularly in reproductive tissues. The American isolates of Zika virus appear to have acquired mutations that increase their efficacy.