Progressive saphenous vein graft (SVG) narrowing and occlusion remains a major limitation of coronary artery bypass grafting and is an important target for gene therapy. Ex vivo adenoviral gene transfer of tissue inhibitor of metalloproteinase 3 (TIMP-3) reduces adverse SVG remodelling postarterialization, but concerns remain over the use of viral vectors in patients. Ultrasound exposure (USE) in the presence of echocontrast microbubbles (ECM) substantially enhances nonviral gene delivery. We investigated the effects of ultrasound-enhanced gene delivery (UEGD) of TIMP-3 plasmid on vascular remodelling in porcine SVG. Maximal luciferase activity (3000-fold versus naked plasmid alone) and TIMP-3 transgene expression in porcine vascular smooth muscle cells in vitro was achieved using USE at 1 MHz, 1.8 mechanical index (MI), 6% duty cycle (DC) in the presence of 50% (v/v) BR14 ECM (Bracco). These conditions were therefore utilized for subsequent studies in vivo. Yorkshire White pigs received carotid interposition SVG that were untransfected or had undergone ex vivo UEGD of lacZ (control) or TIMP-3 plasmids. At 28 d postgrafting, lumen and total vessel area were significantly greater in the TIMP-3 group (10.171.2 and 25.572.2 mm 2 , respectively) compared to untransfected (6.3470.5 and 20.871.9 mm 2 ) or lacZ-transfected (6.170.7 and 19.771.2 mm 2 ) controls (Po0.01). These data indicate that nonviral TIMP-3 plasmid delivery by USE achieves significant biological effects in a clinically relevant model of SV grafting, and is the first study to demonstrate the potential for therapeutic UEGD to prevent SVG failure.

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that prior DENV-2 exposure enhanced ZIKV infection of maternal-fetal interface tissues in macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate.

For more than three decades it has been recognized that small amounts of vector saliva can significantly alter the infectivity of vector-borne pathogens and subsequent in vivo dynamics. Mouse and nonhuman primate models now serve as useful platforms to study Zika virus (ZIKV) pathogenesis, candidate therapies, and vaccines, but they rely on needle inoculation of virus: the effects of mosquito-borne infection on disease outcome have not been explored in these models. To model vector-borne transmission of ZIKV in nonhuman primates, we infected Aedes aegypti mosquitoes with ZIKV and allowed them to feed on four ZIKV-naive rhesus macaques. We compared ZIKV replication kinetics and tissue distribution between animals that were subcutaneously inoculated with 104 plaque-forming units of ZIKV and those that were exposed via mosquito bite. Here, we show that infection via mosquito bite delays ZIKV replication to peak viral loads in rhesus macaques. Importantly, in mosquito-infected animals ZIKV tissue distribution was limited to hemolymphatic tissues, female reproductive tract tissues, kidney, and liver, potentially emulating key features of human ZIKV infections, most of which are characterized by mild or asymptomatic disease. This newly developed system will be valuable for studying ZIKV disease because it more closely mimics human infection by mosquito bite than needle-based inoculations.

31Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We 32 infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The 33 pregnancy was complicated by preterm premature rupture of membranes (PPROM) and fetal 34 demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal 35 interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the 36 myometrial radial arteries. ZIKV RNA was disseminated throughout the fetus tissues and 37 maternal immune system at necropsy, as assessed by quantitative RT-PCR for viral RNA. 38Replicating ZIKV was identified in fetal tissues, maternal lymph node, and maternal spleen by 39 fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included 40 a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is 41 the first report of ocular pathology and prolonged viral replication in both maternal and fetal 42 tissues following congenital ZIKV infection in rhesus macaques. PPROM followed by fetal 43 demise and severe pathology of the visual system have not been described in macaque 44 congenital infection previously; further nonhuman primate studies are needed to determine if an 45 increased risk for PPROM is associated with congenital Zika virus infection. 46 47 Author summary 48 A ZIKV infection during pregnancy is associated with malformations in fetal development 49including, but not limited to, ocular and brain anomalies, such as microcephaly, and stillbirth. The 50 3 development of an accurate pregnancy model to study the effects of ZIKV will provide insight into 51 vertical transmission, ZIKV tissue distribution, and fetal injury and malformations. Non-human 52 primates closely resemble human in terms of the reproductive system, immunity, placentation 53 and pregnancy. Our study demonstrates that the rhesus macaque is a compelling model in which 54 to study ZIKV during pregnancy due to similar outcomes between the human and rhesus 55 macaque. These similarities include prolonged viremia, vertical transmission, adverse pregnancy 56 outcomes and fetal pathology, including defects in the visual system. 57 58