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Rift Valley fever virus (RVFV) is a mosquito-transmitted bunyavirus that causes severe outbreaks among wild and domesticated ruminants, of which sheep are the most susceptible. Outbreaks are characterised by high mortality rates among new-born lambs and abortion storms, in which all pregnant ewes in a flock may abort their foetuses. In endemic areas, Rift Valley fever (RVF) can be controlled by vaccination with either inactivated or live-attenuated vaccines. Inactivated vaccines are safe for animals during all physiological stages, including pregnancy. However, optimal efficacy of these vaccines depends on multiple vaccinations and yearly re-vaccination. Live-attenuated vaccines are generally highly efficacious after a single vaccination, but currently available live-attenuated vaccines may transmit to the ovine foetus, resulting in stillbirths, congenital malformations or abortion. We have previously reported the development of a novel live-attenuated RVFV vaccine, named RVFV-4s. This vaccine virus was created by splitting the M genome segment and deleting the major virulence determinant NSs, and was shown to be safe even for the most susceptible species, including pregnant ewes. The demonstrated efficacy and safety profile suggests that RVFV-4s holds promise for veterinary and human application. The RVFV-4s vaccine for veterinary application, here referred to as vRVFV-4s, was shown to provide complete protection after a single vaccination of lambs, goats and cattle. In this work, we evaluated the efficacy of the vRVFV-4s vaccine in pregnant ewes. Anticipating on the extremely high susceptibility of pregnant ewes for RVFV, both a single vaccination and double vaccination were evaluated in two independent experiments. The combined results suggest that a single vaccination with vRVFV-4s is sufficient to protect pregnant ewes and to prevent transmission to the ovine foetus.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that is pathogenic to ruminants and humans. The virus is endemic to Africa and the Arabian Peninsula where outbreaks are characterized by abortion storms and mortality of newborns, particularly in sheep herds. Vector competence experiments in laboratory settings have suggested that over 50 mosquito species are capable of transmitting RVFV. Transmission of mosquito-borne viruses in the field is however influenced by numerous factors, including population densities, blood feeding behavior, extrinsic incubation period, longevity of vectors, and viremia levels in vertebrate hosts. Animal models to study these important aspects of RVFV transmission are currently lacking. In the present work, RVFV was transmitted to European (Texel-swifter cross-breed) lambs by laboratory-reared Aedes aegypti mosquitoes that were infected either by membrane feeding on a virus-spiked blood meal or by feeding on lambs that developed viremia after intravenous inoculation of RVFV. Feeding of mosquitoes on viremic lambs resulted in strikingly higher infection rates as compared to membrane feeding. Subsequent transmission of RVFV from lamb to lamb by infected mosquitoes was highly efficient in both models. The animal models described here can be used to study mosquito-mediated transmission of RVFV among the major natural target species and to evaluate the efficacy of vaccines against mosquito-mediated RVFV infection.

Compared to free antigens, antigens immobilized on scaffolds, such as nanoparticles, generally show improved immunogenicity. Conventionally, antigens are conjugated to scaffolds through genetic fusion or chemical conjugation, which may result in impaired assembly or heterogeneous binding and orientation of the antigens. By combining two emerging technologies—i.e., self-assembling multimeric protein scaffold particles (MPSPs) and bacterial superglue—these shortcomings can be overcome and antigens can be bound on particles in their native conformation. In the present work, we assessed whether this technology could improve the immunogenicity of a candidate subunit vaccine against the zoonotic Rift Valley fever virus (RVFV). For this, the head domain of glycoprotein Gn, a known target of neutralizing antibodies, was coupled on various MPSPs to further assess immunogenicity and efficacy in vivo. The results showed that the Gn head domain, when bound to the lumazine synthase-based MPSP, reduced mortality in a lethal mouse model and protected lambs, the most susceptible RVFV target animals, from viremia and clinical signs after immunization. Furthermore, the same subunit coupled to two other MPSPs (Geobacillus stearothermophilus E2 or a modified KDPG Aldolase) provided full protection in lambs as well.

Background Rift Valley fever virus (RVFV) is an arbovirus of the order Bunyavirales that causes severe disease in ruminants and humans. Outbreaks in sheep herds are characterised by newborn fatalities and abortion storms. The association of RVFV infections with abortions of ovines and other ruminants is well recognized, whereas the pathology resulting in abortion has remained undescribed. Accumulating evidence suggests that RVFV is abortogenic in humans as well, warranting more research on the interaction of RVFV with the ruminant and human placenta. Methodology/Principal findings Pregnant ewes were inoculated with a highly virulent strain of RVFV and necropsied at different days post infection. Tissues were collected and analysed by PCR, virus isolation, and immunohistochemistry. The results show that RVFV replicates efficiently in maternal placental epithelial cells before the virus infects foetal trophoblasts. Moreover, the virus was shown to bypass the maternal epithelial cell layer by directly targeting foetal trophoblasts in the haemophagous zone, a region of the ovine placenta where maternal blood is in direct contact with foetal cells. Abortion was associated with widespread necrosis of placental tissues accompanied with severe haemorrhages. Experiments with human placental explants revealed that the same virus strain replicates efficiently in both cyto- and syncytiotrophoblasts. Conclusions/Significance This study demonstrates that RVFV targets the foetal-maternal interface in both ovine and human placentas. The virus was shown to cross the ovine placental barrier via two distinct routes, ultimately resulting in placental and foetal demise followed by abortion. Our finding that RVFV replicates efficiently in human trophoblasts underscores the risk of RVFV infection for human pregnancy., Author summary Rift Valley fever virus (RVFV) is a mosquito-borne RNA virus that causes severe disease in ruminants, wildlife and humans in Africa and the Arabian Peninsula. Outbreaks are characterised by high mortality rates among newborn lambs and abortion storms in sheep herds. The severe outcome of RVFV infection during pregnancy in livestock is well documented, whereas the pathological changes that result in abortion have not yet been described. To investigate how RVFV crosses the placenta and how infection results in abortion, pregnant ewes were infected with RVFV and target cells in maternal and foetal tissues were identified at different time points after inoculation. We show that epithelial cells of the ovine placenta and foetal trophoblasts are primary target cells of RVFV and that placental demise is the primary cause of abortion. The same RVFV strain was shown to replicate efficiently in human placental explants, targeting both cyto- and syncytiotrophoblasts.

Zika virus (ZIKV) is a mosquito-borne flavivirus that became associated with microcephaly in newborns and Guillain-Barré syndrome in adults after its emergence in the Pacific and the Americas in 2015. Newly developed rodent and nonhuman primate models have already revealed important insights into ZIKV-induced neuropathology. Nonhuman primates are phylogenetically closely related to humans and are therefore preferred human surrogates in ZIKV research. However, the use of nonhuman primates, particularly during gestation, raises ethical issues. Considering that pigs also share many anatomical and physiological features with humans, this species may be an attractive alternative human surrogate for ZIKV research. Here, we inoculated 20 porcine fetuses in utero and assessed the effect of ZIKV on brain development 4 weeks later. All inoculated fetuses presented mild to severe neuropathology, characterized by a depletion of neurons in the cerebral cortex. In most cases, neuronal depletion was confined to specific cerebral lobes without affecting brain size, whereas in severe cases a more generalized depletion resulted in microencephaly. Although the virus was widespread in the sows' placenta at the time of necropsy only low levels of viral RNA were detected in fetal brain samples, thereby preventing the identification of primary target cells. Our findings suggest that pigs can be used to study ZIKV-induced neurodevelopmental defects as currently observed in human neonates, varying from stunted brain growth to localized cortical neuronal depletion in the absence of major macroscopic abnormalities.

Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family, causes recurrent outbreaks with severe disease in ruminants and occasionally humans. The virus comprises a segmented genome consisting of a small (S), medium (M) and large (L) RNA segment of negative polarity. The M-segment encodes a glycoprotein precursor (GPC) protein that is co-translationally cleaved into Gn and Gc, which are required for virus entry and fusion. Recently we developed a four-segmented RVFV (RVFV-4s) by splitting the M-genome segment, and used this virus to study RVFV genome packaging. Here we evaluated the potential of a RVFV-4s variant lacking the NSs gene (4s-ΔNSs) to induce protective immunity in sheep. Groups of seven lambs were either mock-vaccinated or vaccinated with 10(5) or 10(6) tissue culture infective dose (TCID50) of 4s-ΔNSs via the intramuscular (IM) or subcutaneous (SC) route. Three weeks post-vaccination all lambs were challenged with wild-type RVFV. Mock-vaccinated lambs developed high fever and high viremia within 2 days post-challenge and three animals eventually succumbed to the infection. In contrast, none of the 4s-ΔNSs vaccinated animals developed clinical signs during the course of the experiment. Vaccination with 10(5) TCID50 via the IM route provided sterile immunity, whereas a 10(6) dose was required to induce sterile immunity via SC vaccination. Protection was strongly correlated with the presence of RVFV neutralizing antibodies. This study shows that 4s-ΔNSs is able to induce sterile immunity in the natural target species after a single vaccination, preferably administrated via the IM route.

Rift Valley fever virus (RVFV) is transmitted among susceptible animals by mosquito vectors. Although the virus can be isolated from nasal and oral swabs of infected animals and is known to be highly infectious when administered experimentally via oral or respiratory route, horizontal transmission of the virus is only sporadically reported in literature. We considered that immunosuppression resulting from stressful conditions in the field may increase the susceptibility to horizontally transmitted RVFV. Additionally, we reasoned that horizontal transmission may induce immune responses that could affect the susceptibility of contact-exposed animals to subsequent infection via mosquito vectors. To address these two hypotheses, viremic lambs were brought into contact with sentinel lambs. One group of sentinel lambs was treated with the immunosuppressive synthetic glucocorticosteroid dexamethasone and monitored for signs of disease and presence of virus in the blood and target organs. Another group of contact-exposed sentinel lambs remained untreated for three weeks and was subsequently challenged with RVFV. We found that none of the dexamethasone-treated contact-exposed lambs developed detectable viremia, antibody responses or significant increases in cytokine mRNA levels. Susceptibility of immunocompetent lambs to RVFV infection was not influenced by previous contact-exposure. Our results are discussed in light of previous findings.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.

Rift Valley fever virus (RVFV) is an important pathogen that affects ruminants and humans. Recently we developed a vaccine based on nonspreading RVFV (NSR) and showed that a single vaccination with this vaccine protects lambs from viremia and clinical signs. However, low levels of viral RNA were detected in the blood of vaccinated lambs shortly after challenge infection. These low levels of virus, when present in a pregnant ewe, could potentially infect the highly susceptible fetus. We therefore aimed to further improve the efficacy of the NSR vaccine. Here we report the expression of Gn, the major immunogenic protein of the virus, from the NSR genome. The resulting NSR-Gn vaccine was shown to elicit superior CD8 and CD4-restricted memory responses and improved virus neutralization titers in mice. A dose titration study in lambs revealed that the highest vaccination dose of 10(6.3) TCID50/ml protected all lambs from clinical signs and viremia. The lambs developed neutralizing antibodies within three weeks after vaccination and no anamnestic responses were observed following challenge. The combined results suggest that sterile immunity was achieved by a single vaccination with the NSR-Gn vaccine.

A ring trial was organized to evaluate Rift Valley fever virus (RVFV) ELISAs by European laboratories. A total of five ELISAs, two of which specific for IgM antibodies, were evaluated by six participants. Sera were derived from cattle or sheep and originated from either a RVFV endemic area, a RVFV-free area or from experimental infection studies. Cohen's kappa analysis showed higher than 90% agreement of two commercially available ELISAs with the virus neutralization test, suggesting that primary screening as well as serological confirmation using these ELISAs is feasible. More extensive validations with sera of known IgM status are, however, required to determine agreement between IgM ELISAs. © 2012 Elsevier B.V.

Rift Valley fever virus (RVFV) is a zoonotic bunyavirus that causes abortions in domesticated ruminants. Sheep breeds exotic to endemic areas are reportedly the most susceptible to RVFV infection. Within the scope of a risk assessment program of The Netherlands, we investigated the susceptibility of a native breed of gestating sheep to RVFV infection. Ewes were infected experimentally during the first, second, or third trimester of gestation. Mortality was high among ewes that developed viremia. Four of 11 inoculated ewes, however, did not develop detectable viremia nor clinical signs and did not seroconvert for immunoglobulin G (IgG) or IgM antibodies. Surprisingly, these ewes were found to contain viral RNA in maternal and fetal organs, and the presence of live virus in fetal organs was demonstrated by virus isolation. We demonstrate that RVFV can be transmitted vertically in the absence of detectable maternal viremia.

Rift Valley fever virus (RVFV) causes recurrent large outbreaks among humans and livestock. Although the virus is currently confined to the African continent and the Arabian Peninsula, there is a growing concern for RVFV incursions into countries with immunologically naive populations. The RVFV structural glycoproteins Gn and Cc are preferred targets in the development of subunit vaccines that can be used to control future outbreaks. We here report the production of Gn and Cc by a recombinant vaccine strain of the avian paramyxovirus Newcastle disease virus (NDV) and demonstrate that intramuscular vaccination with this experimental NDV-based vector vaccine provides complete protection in mice. We also demonstrate that a single intramuscular vaccination of lambs, the main target species of RVFV, is sufficient to elicit a neutralizing antibody response. (C) 2010 Elsevier Ltd. All rights reserved.