Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10–12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.

Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PLOS ONE | https://doi.org/10.1371/journal.pone

. Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-B signaling pathways in murine RAW 264.7 macrophages. Am J Physiol Cell Physiol 286: C923-C930, 2004. First published December 18, 2003; 10.1152/ajpcell.00417.2003.-Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X 7. In this regard, after exposure to bacterial LPS, P2X 7 activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X 7 has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-B, we tested the hypothesis that LPS and nucleotides regulate NF-B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X 7-selective ligand 2Ј-3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate (BzATP) results in the cooperative activation of NF-B DNA-binding activity and a sustained attenuation of levels of the NF-B inhibitory protein IB␣. Interestingly, a persistent reduction in IB␣ levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating IB␣ protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IB␣ levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-B/ IB-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-B and Ras/MEK/ MAPK-associated pathways. nucleotide receptors; mitogen-activated protein kinases; nuclear factor-B; monocytes/macrophages; cytokines ALTHOUGH ATP IS KNOWN to be an important source of intracellular energy, numerous studies have revealed that high concentrations of ATP (5 mM) in the extracellular milieu following tissue damage or platelet degranulation can play a key role in the regulation of biological responses such as inflammation, platelet aggregation, and smooth muscle contraction (13,20,25,38). Nucleotides have been shown to greatly enhance the effects of bacterial LPS on macrophage and monocyte activation by augmenting the production of mediators such as nitric oxide (NO) and other free radicals, in addition to numerous cytokines, such as interleukin (IL)-1␤ and tumor necrosis factor (TNF)-␣ (26, 27, 39, 51, 55). The overproduction of these mediators, although important for the activation of the immune system and for bactericidal effects, can als...

To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell rounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lines. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors. Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF. The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-␣ by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or Cterminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration. EGF1 triggers many biological responses, including cell proliferation and differentiation (1). In addition, EGF has been shown to induce the reorganization of the actin cytoskeleton, and the EGF receptor has been found to be associated with actin filaments (2-7). In this regard, EGF has been reported to stimulate rapid cell rounding, extensive membrane ruffling, extension of filopodia, retraction of cells from the substratum (8, 9), extensive cortical actin polymerization, and depolymerization of actin stress fibers (10, 11). Moreover, numerous studies have shown that activation of the EGF receptor leads to increased cell motility (12-19) and production of ECM degrading proteases (20 -23), thereby supporting a role for the EGF receptor in normal development and pathophysiological events such...

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10-12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated. Author summaryMaternal ZIKV infection in pregnancy is associated with severe fetal anomalies, including microcephaly. It has been shown that infection manifests differently in pregnancy than in the non-pregnant state, with prolonged maternal viremia. ZIKV is spread by mosquitos and through sexual contact and since its first detection in early 2015, has become endemic to the Americas. While much has been learned from studying infected human pregnancies, there are still many questions concerning transmission of ZIKV from mother to fetus. Investigating ZIKV infection in non-human primates could help answer these questions due to similarities in the immune system, and the tissues separating the fetus from the mother during pregnancy. Our study serves to model ZIKV transmission in early and late pregnancy, as well as study the effects of this infection on the fetus and mother at these different times in pregnancy. The data collected provides an important insight on ZIKV in pregnancy where the pregnancies have been monitored throughout the entire infection period until term, and suggests that vertical transmission may be very efficient, although severe fetal outcomes are uncommon.