Measures directed at reducing the incidence of necrotizing enterocolitis may reduce morbidity in neonates with hypoplastic left heart syndrome and reduce cost by decreasing hospital length of stay. A standardized feeding protocol instituted to address these problems likely contributed to reducing the incidence of necrotizing enterocolitis in this high-risk population.

No abstract

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.

44 45 ZIKV infection is associated with pregnancy loss, fetal microcephaly and other 46 malformations. While Aedes sp. of mosquito are the primary vector for ZIKV, sexual 47 transmission of ZIKV is a significant route of infection. ZIKV has been documented in 48 human, mouse and non-human primate (NHP) semen. It is critical to establish NHP 49 models of vertical transfer of ZIKV that recapitulate human ZIKV pathogenesis. We 50 hypothesized that vaginal deposition of ZIKV infected baboon semen would lead to 51 maternal infection and vertical transfer in the olive baboon (Papio anubis). Timed 52 pregnant baboons (n=6) were inoculated via vaginal deposition of baboon semen 53 containing 10 6 ffu ZIKV (n=3, French Polynesian isolate:H/PF/2013, n=3 Puerto Rican 54 isolate:PRVABC59) at mid-gestation (86-95 days gestation [dG]; term 183dG) on day (d) 550 (all dams), and then at 7 day intervals through three weeks. Maternal blood, saliva and 56 cervico-vaginal washes were obtained at select days post-inoculation. Animals were 57 euthanized at 28 days post initial inoculation (dpi; n=5) or 39 dpi (n=1) and maternal/fetal 58 tissues collected. vRNA was quantified by qPCR. Viremia was achieved in 3/3 FP ZIKV 59 infected dams and 2/3 PR ZIKV. ZIKV RNA was detected in cvw (5/6 dams;). ZIKV RNA 60 was detected in lymph nodes, but not ovary, uterus, cervix or vagina in the FP ZIKV 61 dams but was detected in uterus, vagina and lymph nodes. Placenta, amniotic fluid and 62 all fetal tissues were ZIKV RNA negative in the FP infected dams whereas 2/3 PR 63 infected dam placentas were ZIKV RNA positive. We conclude that ZIKV infected 64 semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate 65 appeared more capable of wide spread dissemination to tissues, including placenta 66 compared to the FP strain. 67 68 69 IMPORTANCE 70 Due to its established link to pregnancy loss, microcephaly and other major congenital 71 anomalies, Zika virus (ZIKV) remains a worldwide health threat. Although mosquitoes 72 are the primary means of ZIVK transmission, sexual transmission in human populations 73 is well documented and provides a means for widespread dissemination of the virus. 74 Differences in viremia, tissue distribution, immune responses and pregnancy outcome 75 from sexually transmitted ZIKV compared to the subcutaneous route of infection are 76 needed to better clinically manage ZIKV in pregnancy. Through our previous work, we 77 have developed the olive baboon as a non-human primate model of ZIKV infection that 78 is permissible to ZIKV infection via the subcutaneous route of inoculation and transfer of 79 ZIKV to the fetus in pregnancy. The current study evaluated the course of ZIKV infection 80 after vaginal inoculation of ZIKV in pregnant baboons at mid-gestation using baboon 81 semen as the carrier and comparing two isolates of ZIKV, the French Polynesian isolate 82 first associated with microcephaly and the Puerto Rican isolate, associated with an 83 increased risk of microcephaly observed in the America...