Immune correlates of protection of the four-segmented Rift Valley fever virus candidate vaccine in mice.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

Single-Molecule Visualization of Bunyavirus Genome Segments Using Fluorescence In Situ Hybridization.

The genome of most bunyaviruses is divided over three (S, M, and L) single-stranded RNA segments of negative polarity. The three viral RNA segments are essential to establish a productive infection. RNA fluorescence in situ hybridization (FISH) enables the detection, localization, and quantification of RNA molecules at single-molecule resolution. This chapter describes an RNA FISH method to directly visualize individual segment-specific bunyavirus RNAs in fixed infected cells and in mature virus particles, using Rift Valley fever virus as an example. Imaging of bunyavirus RNA segments is a valuable experimental tool to investigate fundamental aspects of the bunyavirus life cycle, such as virus replication, genome packaging, and virion assembly, among others.

Safety and immunogenicity of the live-attenuated hRVFV-4s vaccine against Rift Valley fever in healthy adults: a dose-escalation, placebo-controlled, first-in-human, phase 1 randomised clinical trial.

BACKGROUND: Rift Valley fever virus, a pathogen to ruminants, camelids, and humans, is an emerging mosquito-borne bunyavirus currently endemic to Africa and the Arabian Peninsula. Although animals are primarily infected via mosquito bites, humans mainly become infected following contact with infected tissues or fluids of infected animals. There is an urgent need for adequate countermeasures, especially for humans, because effective therapeutics or vaccines are not yet available. Here we assessed the safety, tolerability, and immunogenicity of a next-generation, four-segmented, live-attenuated vaccine candidate, referred to as hRVFV-4s, in humans. METHODS: A first-in-human, single-centre, randomised, double-blind, placebo-controlled trial was done in Belgium in which a single dose of hRVFV-4s was administered to healthy volunteers aged 18-45 years. Participants were randomly assigned using an interactive web response system. The study population encompassed 75 participants naive to Rift Valley fever virus infection, divided over three dosage groups (cohorts) of 25 participants each. All participants were followed up until 6 months. Using a staggered dose escalating approach, 20 individuals of each cohort were injected in the deltoid muscle of the non-dominant arm with either 104 (low dose), 105 (medium dose), or 106 (high dose) of 50% tissue culture infectious dose of hRVFV-4s as based on animal data, and five individuals per cohort received formulation buffer as a placebo. Primary outcome measures in the intention-to-treat population were adverse events and tolerability. Secondary outcome measures were vaccine-induced viraemia, vaccine virus shedding, Rift Valley fever virus nucleocapsid antibody responses (with ELISA), and neutralising antibody titres. Furthermore, exploratory objectives included the assessment of cellular immune responses by ELISpot. The trial was registered with the EU Clinical Trials Register, 2022-501460-17-00. FINDINGS: Between August and December, 2022, all 75 participants were vaccinated. No serious adverse events or vaccine-related severe adverse events were reported. Pain at the injection site (51 [85%] of 60 participants) was most frequently reported as solicited local adverse event, and headache (28 [47%] of 60) and fatigue (28 [47%] of 60) as solicited systemic adverse events in the active group. No vaccine virus RNA was detected in any of the blood, saliva, urine, or semen samples. Rift Valley fever virus nucleocapsid antibody responses were detected in most participants who were vaccinated with hRVFV-4s (43 [72%] of 60 on day 14) irrespective of the administered dose. In contrast, a clear dose-response relationship was observed for neutralising antibodies on day 28 with four (20%) of 20 participants responding in the low-dose group, 13 (65%) of 20 responding in the medium-dose group, and all participants (20 [100%] of 20) responding in the high-dose group. Consistent with the antibody responses, cellular immune responses against the nucleocapsid protein were detected in all dose groups, whereas a more dose-dependent response was observed for the Gn and Gc surface glycoproteins. Neutralising antibody titres declined over time, whereas nucleocapsid antibody responses remained relatively stable for at least 6 months. INTERPRETATION: The hRVFV-4s vaccine showed a high safety profile and excellent tolerability across all tested dose regimens, eliciting robust immune responses, particularly with the high-dose administration. The findings strongly support further clinical development of this candidate vaccine for human use. FUNDING: The Coalition for Epidemic Preparedness Innovations with support from the EU Horizon 2020 programme.

Infection of wild-caught wood mice (Apodemus sylvaticus) and yellow-necked mice (A. flavicollis) with tick-borne encephalitis virus.

The distribution of tick-borne encephalitis virus (TBEV) is expanding to Western European countries, including the Netherlands, but the contribution of different rodent species to the transmission of TBEV is poorly understood. We investigated whether two species of wild rodents native to the Netherlands, the wood mouse Apodemus sylvaticus and the yellow-necked mouse Apodemus flavicollis, differ in their relative susceptibility to experimental infection with TBEV. Wild-caught individuals were inoculated subcutaneously with the classical European subtype of TBEV (Neudoerfl) or with TBEV-NL, a genetically divergent TBEV strain from the Netherlands. Mice were euthanised and necropsied between 3 and 21 days post-inoculation. None of the mice showed clinical signs or died during the experimental period. Nevertheless, TBEV RNA was detected up to 21 days in the blood of both mouse species and TBEV was also isolated from the brain of some mice. Moreover, no differences in infection rates between virus strains and mouse species were found in blood, spleen, or liver samples. Our results suggest that the wood mouse and the yellow-necked mouse may equally contribute to the transmission cycle of TBEV in the Netherlands. Future experimental infection studies that include feeding ticks will help elucidate the relative importance of viraemic transmission in the epidemiology of TBEV.

Transcriptomic Profiling Reveals Intense Host-Pathogen Dispute Compromising Homeostasis during Acute Rift Valley Fever Virus Infection.

Rift Valley fever virus (RVFV) (family Phenuiviridae) can cause severe disease, and outbreaks of this mosquito-borne pathogen pose a significant threat to public and animal health. Yet many molecular aspects of RVFV pathogenesis remain incompletely understood. Natural RVFV infections are acute, characterized by a rapid onset of peak viremia during the first days post-infection, followed by a rapid decline. Although in vitro studies identified a major role of interferon (IFN) responses in counteracting the infection, a comprehensive overview of the specific host factors that play a role in RVFV pathogenesis in vivo is still lacking. Here, the host in vivo transcriptional profiles in the liver and spleen tissues of lambs exposed to RVFV are studied using RNA sequencing (RNA-seq) technology. We validate that IFN-mediated pathways are robustly activated in response to infection. We also link the observed hepatocellular necrosis with severely compromised organ function, which is reflected as a marked downregulation of multiple metabolic enzymes essential for homeostasis. Furthermore, we associate the elevated basal expression of LRP1 in the liver with RVFV tissue tropism. Collectively, the results of this study deepen the knowledge of the in vivo host response during RVFV infection and reveal new insights into the gene regulation networks underlying pathogenesis in a natural host. IMPORTANCE Rift Valley fever virus (RVFV) is a mosquito-transmitted pathogen capable of causing severe disease in animals and humans. Outbreaks of RVFV pose a significant threat to public health and can result in substantial economic losses. Little is known about the molecular basis of RVFV pathogenesis in vivo, particularly in its natural hosts. We employed RNA-seq technology to investigate genome-wide host responses in the liver and spleen of lambs during acute RVFV infection. We show that RVFV infection drastically decreases the expression of metabolic enzymes, which impairs normal liver function. Moreover, we highlight that basal expression levels of the host factor LRP1 may be a determinant of RVFV tissue tropism. This study links the typical pathological phenotype induced by RVFV infection with tissue-specific gene expression profiles, thereby improving our understanding of RVFV pathogenesis.

Perspectives of Next-Generation Live-Attenuated Rift Valley Fever Vaccines for Animal and Human Use.

Live-attenuated Rift Valley fever (RVF) vaccines transiently replicate in the vaccinated host, thereby effectively initiating an innate and adaptive immune response. Rift Valley fever virus (RVFV)-specific neutralizing antibodies are considered the main correlate of protection. Vaccination with classical live-attenuated RVF vaccines during gestation in livestock has been associated with fetal malformations, stillbirths, and fetal demise. Facilitated by an increased understanding of the RVFV infection and replication cycle and availability of reverse genetics systems, novel rationally-designed live-attenuated candidate RVF vaccines with improved safety profiles have been developed. Several of these experimental vaccines are currently advancing beyond the proof-of-concept phase and are being evaluated for application in both animals and humans. We here provide perspectives on some of these next-generation live-attenuated RVF vaccines and highlight the opportunities and challenges of these approaches to improve global health.

Quantifying Rift Valley fever virus transmission efficiency in a lamb-mosquito-lamb model.

Rift Valley fever virus (RVFV) is a (re)emerging mosquito-borne pathogen impacting human and animal health. How RVFV spreads through a population depends on population-level and individual-level interactions between vector, host and pathogen. Here, we estimated the probability for RVFV to transmit to naive animals by experimentally exposing lambs to a bite of an infectious mosquito, and assessed if and how RVFV infection subsequently developed in the exposed animal. Aedes aegypti mosquitoes, previously infected via feeding on a viremic lamb, were used to expose naive lambs to the virus. Aedes aegypti colony mosquitoes were used as they are easy to maintain and readily feed in captivity. Other mosquito spp. could be examined with similar methodology. Lambs were exposed to either 1-3 (low exposure) or 7-9 (high exposure) infectious mosquitoes. All lambs in the high exposure group became viremic and showed characteristic signs of Rift Valley fever within 2-4 days post exposure. In contrast, 3 out of 12 lambs in the low exposure group developed viremia and disease, with similar peak-levels of viremia as the high exposure group but with some heterogeneity in the onset of viremia. These results suggest that the likelihood for successful infection of a ruminant host is affected by the number of infectious mosquitoes biting, but also highlights that a single bite of an infectious mosquito can result in disease. The per bite mosquito-to-host transmission efficiency was estimated at 28% (95% confidence interval: 15 - 47%). We subsequently combined this transmission efficiency with estimates for life traits of Aedes aegypti or related mosquitoes into a Ross-McDonald mathematical model to illustrate scenarios under which major RVFV outbreaks could occur in naïve populations (i.e., R0 >1). The model revealed that relatively high vector-to-host ratios as well as mosquitoes feeding preferably on competent hosts are required for R0 to exceed 1. Altogether, this study highlights the importance of experiments that mimic natural exposure to RVFV. The experiments facilitate a better understanding of the natural progression of disease and a direct way to obtain epidemiological parameters for mathematical models.

Incomplete bunyavirus particles can cooperatively support virus infection and spread.

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented arthropod-borne Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations unable to spread autonomously are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that complementing incomplete virus particles can co-infect mosquitoes, resulting in the reconstitution of infectious virus that is able to disseminate to the mosquito salivary glands. Computational models of infection dynamics predict that incomplete virus particles can positively impact virus spread over a wide range of conditions, with the strongest effect at intermediate multiplicities of infection. Our findings suggest that incomplete particles may play a significant role in within-host spread and between-host transmission, reminiscent of the infection cycle of multipartite viruses.

Heterogeneity of Rift Valley fever virus transmission potential across livestock hosts, quantified through a model-based analysis of host viral load and vector infection.

Quantifying the variation of pathogens' life history traits in multiple host systems is crucial to understand their transmission dynamics. It is particularly important for arthropod-borne viruses (arboviruses), which are prone to infecting several species of vertebrate hosts. Here, we focus on how host-pathogen interactions determine the ability of host species to transmit a virus to susceptible vectors upon a potentially infectious contact. Rift Valley fever (RVF) is a viral, vector-borne, zoonotic disease, chosen as a case study. The relative contributions of livestock species to RVFV transmission has not been previously quantified. To estimate their potential to transmit the virus over the course of their infection, we 1) fitted a within-host model to viral RNA and infectious virus measures, obtained daily from infected lambs, calves, and young goats, 2) estimated the relationship between vertebrate host infectious titers and probability to infect mosquitoes, and 3) estimated the net infectiousness of each host species over the duration of their infectious periods, taking into account different survival outcomes for lambs. Our results indicate that the efficiency of viral replication, along with the lifespan of infectious particles, could be sources of heterogeneity between hosts. Given available data on RVFV competent vectors, we found that, for similar infectious titers, infection rates in the Aedes genus were on average higher than in the Culex genus. Consequently, for Aedes-mediated infections, we estimated the net infectiousness of lambs to be 2.93 (median) and 3.65 times higher than that of calves and goats, respectively. In lambs, we estimated the overall infectiousness to be 1.93 times higher in individuals which eventually died from the infection than in those recovering. Beyond infectiousness, the relative contributions of host species to transmission depend on local ecological factors, including relative abundances and vector host-feeding preferences. Quantifying these contributions will ultimately help design efficient, targeted, surveillance and vaccination strategies.

Safety and immunogenicity of four-segmented Rift Valley fever virus in the common marmoset.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne bunyavirus that is highly pathogenic to wild and domesticated ruminants, camelids, and humans. While animals are exclusively infected via mosquito bites, humans can also be infected via contact with contaminated tissues or blood. No human vaccine is available and commercialized veterinary vaccines do not optimally combine efficacy with safety. We previously reported the development of two novel live-attenuated RVF vaccines, created by splitting the M genome segment and deleting the major virulence determinant NSs. The vaccine candidates, referred to as the veterinary vaccine vRVFV-4s and the human vaccine hRVFV-4s, were shown to induce protective immunity in multiple species after a single vaccination. Anticipating accidental exposure of humans to the veterinary vaccine and the application of hRVFV-4s to humans, the safety of each vaccine was evaluated in the most susceptible nonhuman primate model, the common marmoset (Callithrix jacchus). Marmosets were inoculated with high doses of each vaccine and were monitored for clinical signs as well as for vaccine virus dissemination, shedding, and spreading to the environment. To accurately assess the attenuation of both vaccine viruses, separate groups of marmosets were inoculated with the parent wild-type RVFV strains. Both wild-type strains induced high viremia and disseminated to primary target organs, associated with mild-to-severe morbidity. In contrast, both vaccines were well tolerated with no evidence of dissemination and shedding while inducing potent neutralizing antibody responses. The results of the studies support the unprecedented safety profile of both vaccines for animals and humans.

Suitability of transiently expressed antibodies for clinical studies: product quality consistency at different production scales.

Transgenic human monoclonal antibodies derived from humanized mice against different epitopes of the Middle East respiratory syndrome coronavirus (MERS-CoV), and chimeric llama-human bispecific heavy chain-only antibodies targeting the Rift Valley fever virus (RVFV), were produced using a CHO-based transient expression system. Two lead candidates were assessed for each model virus before selecting and progressing one lead molecule. MERS-7.7G6 was used as the model antibody to demonstrate batch-to-batch process consistency and, together with RVFV-107-104, were scaled up to 200 L. Consistent expression titers were obtained in different batches at a 5 L scale for MERS-7.7G6. Although lower expression levels were observed for MERS-7.7G6 and RVFV-107-104 during scale up to 200 L, product quality attributes were consistent at different scales and in different batches. In addition to this, peptide mapping data suggested no detectable sequence variants for any of these candidates. Functional assays demonstrated comparable neutralizing activity for MERS-7.7G6 and RVFV-107-104 generated at different production scales. Similarly, MERS-7.7G6 batches generated at different scales were shown to provide comparable protection in mouse models. Our study demonstrates that a CHO-based transient expression process is capable of generating consistent product quality at different production scales and thereby supports the potential of using transient gene expression to accelerate the manufacturing of early clinical material.

Creation of Multimeric Single-Domain Antibodies Using Bacterial Superglues.

Multimerization of single-domain antibodies (sdAbs) is instrumental for construction of antibody molecules with high avidity, extended in vivo half-life, and tailor-made biological activity. Two-component superglues, based on bacterium-derived peptides (Tags) and small protein domains (Catchers) that form isopeptide bonds when in close proximity, enable the creation of multimers by simply mixing of the individual components. Here, we provide detailed methods for the construction of sdAbs and scaffolds bearing genetically fused superglue components and their assembly into multimeric complexes.

Zoonoses Anticipation and Preparedness Initiative, stakeholders conference, February 4 & 5, 2021.

The Zoonoses Anticipation and Preparedness Initiative (ZAPI) was set up to prepare for future outbreaks and to develop and implement new technologies to accelerate development and manufacturing of vaccines and monoclonal antibodies. To be able to achieve surge capacity, an easy deployment and production at multiple sites is needed. This requires a straightforward manufacturing system with a limited number of steps in upstream and downstream processes, a minimum number of in vitro Quality Control assays, and robust and consistent platforms. Three viruses were selected as prototypes: Middle East Respiratory Syndrome (MERS) coronavirus, Rift Valley fever virus, and Schmallenberg virus. Selected antibodies against the viral surface antigens were manufactured by transient gene expression in Chinese Hamster Ovary (CHO) cells, scaling up to 200 L. For vaccine production, viral antigens were fused to multimeric protein scaffold particles using the SpyCatcher/SpyTag system. In vivo models demonstrated the efficacy of both antibodies and vaccines. The final step in speeding up vaccine (and antibody) development is the regulatory appraisal of new platform technologies. Towards this end, within ZAPI, a Platform Master File (PfMF) was developed, as part of a licensing dossier, to facilitate and accelerate the scientific assessment by avoiding repeated discussion of already accepted platforms. The veterinary PfMF was accepted, whereas the human PfMF is currently under review by the European Medicines Agency, aiming for publication of the guideline by January 2022.

Predictive Value of Precision-Cut Lung Slices for the Susceptibility of Three Animal Species for SARS-CoV-2 and Validation in a Refined Hamster Model.

In assessing species susceptibility for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and in the search for an appropriate animal model, multiple research groups around the world inoculated a broad range of animal species using various SARS-CoV-2 strains, doses and administration routes. Although in silico analyses based on receptor binding and diverse in vitro cell cultures were valuable, exact prediction of species susceptibility based on these tools proved challenging. Here, we assessed whether precision-cut lung slices (PCLS) could facilitate the selection of animal models, thereby reducing animal experimentation. Pig, hamster and cat PCLS were incubated with SARS-CoV-2 and virus replication was followed over time. Virus replicated efficiently in PCLS from hamsters and cats, while no evidence of replication was obtained for pig PCLS. These data corroborate the findings of many research groups that have investigated the susceptibility of hamsters, pigs and cats towards infection with SARS-CoV-2. Our findings suggest that PCLS can be used as convenient tool for the screening of different animal species for sensitivity to newly emerged viruses. To validate our results obtained in PCLS, we employed the hamster model. Hamsters were inoculated with SARS-CoV-2 via the intranasal route. Susceptibility to infection was evaluated by body weight loss, viral loads in oropharyngeal swabs and respiratory tissues and lung pathology. The broadly used hamster model was further refined by including activity tracking of the hamsters by an activity wheel as a very robust and sensitive parameter for clinical health. In addition, to facilitate the quantification of pathology in the lungs, we devised a semi-quantitative scoring system for evaluating the degree of histological changes in the lungs. The inclusion of these additional parameters refined and enriched the hamster model, allowing for the generation of more data from a single experiment.

Development of a Modular Vaccine Platform for Multimeric Antigen Display Using an Orthobunyavirus Model.

Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.

Animal board invited review: Risks of zoonotic disease emergence at the interface of wildlife and livestock systems.

The ongoing coronavirus disease 19s pandemic has yet again demonstrated the importance of the human-animal interface in the emergence of zoonotic diseases, and in particular the role of wildlife and livestock species as potential hosts and virus reservoirs. As most diseases emerge out of the human-animal interface, a better understanding of the specific drivers and mechanisms involved is crucial to prepare for future disease outbreaks. Interactions between wildlife and livestock systems contribute to the emergence of zoonotic diseases, especially in the face of globalization, habitat fragmentation and destruction and climate change. As several groups of viruses and bacteria are more likely to emerge, we focus on pathogenic viruses of the Bunyavirales, Coronaviridae, Flaviviridae, Orthomyxoviridae, and Paramyxoviridae, as well as bacterial species including Mycobacterium sp., Brucella sp., Bacillus anthracis and Coxiella burnetii. Noteworthy, it was difficult to predict the drivers of disease emergence in the past, even for well-known pathogens. Thus, an improved surveillance in hotspot areas and the availability of fast, effective, and adaptable control measures would definitely contribute to preparedness. We here propose strategies to mitigate the risk of emergence and/or re-emergence of prioritized pathogens to prevent future epidemics.

Vaccine Efficacy of Self-Assembled Multimeric Protein Scaffold Particles Displaying the Glycoprotein Gn Head Domain of Rift Valley Fever Virus.

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.

Visualizing the ribonucleoprotein content of single bunyavirus virions reveals more efficient genome packaging in the arthropod host.

Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.

Reproducing the Rift Valley fever virus mosquito-lamb-mosquito transmission cycle.

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.

A single vaccination with four-segmented rift valley fever virus prevents vertical transmission of the wild-type virus in pregnant ewes.

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.