Pharmacokinetic Analyses of a Lipid Nanoparticle-Encapsulated mRNA-Encoded Antibody against Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) could cause an emergency illness characterized by fever, muscle pain, and even death in humans or ruminants. However, there are no approved antiviral drugs that prevent or treat RVFV infection. While therapeutic antibodies have shown promising potential for prevention or treatment in several studies, many studies are ongoing, especially in the field of infectious diseases. Among these studies, the mRNA-LNP platform shows great potential for application, following the COVID-19 pandemic. Previously, we have obtained a neutralizing antibody against RVFV, which was named A38 protein and verified to have a high binding and neutralization ability. In this study, we aimed to identify an effectively optimized sequence and expressed the prioritized mRNA-encoded antibody in vitro. Notably, we effectively expressed mRNA-encoded protein and used the mRNA-LNP platform to generate A38-mRNA-LNP. Pharmacokinetic experiments were conducted in vivo and set up in two groups of mRNA-A38 group and A38 protein group, which were derived from mRNA-LNP and plasmid DNA-expressed proteins, respectively. A38-mRNA-LNPs were administrated by intramuscular injection, A38 proteins were administrated by intravenous administration, and their unique ability to maintain long-lasting protein concentrations by mRNA-encoded protein was demonstrated with the mRNA-encoded protein providing a longer circulating half-life compared to injection of the free A38 protein. These preclinical data on the mRNA-encoded antibody highlighted its potential to prevent infectious diseases in the future.

Increasing evolution, prevalence, and outbreaks for rift valley fever virus in the process of breaking geographical barriers.

BACKGROUND: Rift valley fever (RVF) is listed as one of prioritized diseases by WHO. This study aims to describe RVF virus' landscape distribution globally, and to insight dynamics change of its evolution, prevalence, and outbreaks in the process of breaking geographical barriers. METHODS: A systematic literature review and meta-analyses was conducted to estimate RVF prevalence by hosts using a random-effect model. Molecular clock-based phylogenetic analyses were performed to estimate RVF virus nucleotide substitution rates using nucleotide sequences in NCBI database. RVF virus prevalence, nucleotide substitution rates, and outbreaks were compared before and after breaking geographical barriers twice, respectively. RESULTS: RVF virus was reported from 26 kinds of hosts covering 48 countries from 1930 to 2022. Since RVF broke geographical barriers, (1) nucleotide substitution rates significantly increased after firstly spreading out of Africa in 2000, (2) prevalence in humans significantly increased from 1.92 % (95 % CI: 0.86-3.25 %) to 3.03 % (95 % CI: 2.09-4.12 %) after it broke Sahara Desert geographical barriers in 1977, and to 5.24 % (95 % CI: 3.81-6.82 %) after 2000, (3) RVF outbreaks in humans and the number of wildlife hosts presented increasing trends. RVF virus spillover may exist between bats and humans, and accelerate viral substitution rates in humans. During outbreaks, the RVF virus substitution rates accelerated in humans. 60.00 % RVF outbreaks occurred 0-2 months after floods and (or) heavy rainfall. CONCLUSION: RVF has the increasing risk to cause pandemics, and global collaboration on "One Health" is needed to prevent potential pandemics.

Reverse Genetics System for Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is an important mosquito-borne virus that can cause severe disease manifestations in humans including ocular damage, vision loss, late-onset encephalitis, and hemorrhagic fever. In ruminants, RVFV can cause high mortality rates in young animals and high rates of abortion in pregnant animals resulting in an enormous negative impact on the economy of affected regions. To date, no licensed vaccines in humans or anti-RVFV therapeutics for animal or human use are available. The development of reverse genetics has facilitated the generation of recombinant infectious viruses that serve as powerful tools for investigating the molecular biology and pathogenesis of RVFV. Infectious recombinant RVFV can be rescued entirely from cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis and generate live-attenuated vaccines. In this chapter, we will describe the experimental procedures for the implementation of RVFV reverse genetics.

Safety and immunogenicity of inactivated Rift Valley Fever Smithburn viral vaccine in sheep.

BACKGROUND: The live-attenuated Rift Valley Fever Smithburn (SB) vaccine is one of the oldest products widely used in ruminants for control of RVF infections. Vaccinations with RVF Smithburn result in residual pathogenic effect and is limited for use in non-pregnant animals. Commercially available RVFV inactivated vaccines are considered safer options to control the disease. These products are prepared from virulent RVFV isolates and present occupational safety concerns. This research study evaluates the ability of an inactivated SB vaccine strain to elicit neutralising antibody response in sheep. METHODS: The RVF Smithburn vaccine was inactivated with binary ethylenimine at 37 °C. Inactivated RVFV cultures were adjuvanted with Montande™ Gel-01 and aluminium hydroxide (Al (OH)3) gel for immunogenicity and safety determination in sheep. The commercial RVF inactivated vaccine and a placebo were included as positive and negative control groups, respectively. RESULTS: Inactivated RVFV vaccine formulations were safe with all animals showing no clinical signs of RVFV infection and temperature reactions following prime-boost injections. The aluminium hydroxide formulated vaccine induced an immune response as early as 14 days post primary vaccination with neutralising antibody titre of 1:20 and a peak antibody titre of 1:83 was reached on day 56. A similar trend was observed in the animal group vaccinated with the commercial inactivated RVF vaccine obtaining the highest antibody titre of 1:128 on day 56. The neutralizing antibody levels remained within a threshold for the duration of the study. Merino sheep vaccinated with Montanide™ Gel-01-Smithburn were characterised with overall lower immune response when compared to aluminium hydroxide vaccine emulsions. CONCLUSIONS: These finding suggests that the inactivated RVF Smithburn vaccine strain adjuvanted with aluminium-hydroxide can be used an alternative to the products prepared from virulent RVFV isolates for protection of ruminants against the disease. The vaccine can further be evaluated for safety in pregnant ewes.

The increasing threat of Rift Valley fever virus globalization: strategic guidance for protection and preparation.

Rift Valley fever virus (RVFV) (Bunyavirales: Phlebovirus) is a prominent vector-borne zoonotic disease threat to global agriculture and public health. Risks of introduction into nonendemic regions are tied to changing climate regimes and other dynamic environmental factors that are becoming more prevalent, as well as virus evolutionary factors and human/animal movement. Endemic to the African continent, RVFV has caused large epizootics at the decadal scale since the early 20th century but has spread to the Arabian Peninsula and shows increasing patterns of interepizootic transmission on the annual scale. This virus can be transmitted by mosquitoes as well as through direct contact with infected tissues and can cause sporadic to widespread morbidity and mortality in domestic ungulate livestock as well as humans. High viremias in infected livestock moved for legal and illegal trade as well as in infected mosquitoes or human travelers can spread this virus worldwide. With increasing global commerce, it is likely RVFV will be introduced to new areas with suitable hosts, mosquito vector species, and environments. However, the strong mosquito component of RVFV epidemiology combined with advancements in vaccines, diagnostics, and virus evolutionary factors create opportunities for strategies to leverage models of connectivity among potential source and emerging regions to target surveillance and mitigation activities to reduce the risk of RVFV introduction, or contain the virus should it be introduced, into new regions.

Knowledge, attitudes and practices on rift valley fever among pastoral and agropastoral communities of Ngorongoro in the rift valley ecosystem, Tanzania, conducted in 2021/2022.

Epidemics of Rift Valley fever (RVF), a mosquito-borne zoonotic disease caused by RVF virus, have been linked to exceptionally heavy rainfall and widespread flooding. The disease is endemic in most African countries and pose a major global health risk. Given that the disease was reported in various districts of Tanzania, we hypothesized a lack of knowledge about RVF epidemiology among agropastoral and pastoral communities. The research took place in a period of 7 months, from July, 2021 to January, 2022. The aim of this study was to assess the knowledge, attitudes, and practices (KAP) among the agropastoral and pastoral communities of Ngorongoro district towards RVF. The survey employed a mixed method system, which included 3 focus groups (each comprised 12 individuals), 20 key informant interviews and administration of questionnaire (N = 352) in agropastoral and pastoral community members of Ngorongoro district. The relationship between demographic characteristics and communities' knowledge, attitudes, and practices regarding RVF was observed using a multiple logistic regression model. A total of 352 participants were interviewed, with the majority (67.61%) being male and 32.39% being female, majority (39.5%) attending primary school, and majority (58.2%) being pastoralists. The findings showed that only 36.1%, 38.64% and 16.19% of participants had good knowledge, positive attitude and good practices regarding RVF respectively. Significant demographic factors related with knowledge included: gender (OR = 1.9, CI = 1.03-3.56, P = 0.041), education levels (primary: OR = 3.97, CI = 2-8.16, P = 0.000; secondary: OR = 15.27, CI = 5.5-46.23, P = 0.000 and college: OR = 34. 23, CI = 5.4-67.22, P = 0.000), and locality (Pinyinyi: OR = 0.14, CI = 0.05-0.38, P = 0.000 and Sale: OR = 0.14, CI = 0.04-0.44, P = 0.001). Male participants showed significant positive attitude towards RVF compared to female (OR = 2.37, CI = 1.35-4.17, P = 0.003). Individuals with formal education showed a significant positive attitude toward RVF compared to informal (OR>1, P<0.05). Agropastoral members showed a significant negative attitude toward RVF compared to pastoralists (OR = 0.51, CI = 0.26-0.99, P = 0.048). The calculated RVF prevention practices values were insignificantly (P = 0.853) correlated with knowledge values. The significant correlation between knowledge and attitude, as well as attitude and practice were found (P<0.05). In general, the study revealed poor knowledge, negative attitude and poor practices of communities towards RVF. The lack of regular education programs to make the communities aware of the disease was implicated for these findings. This recommends that provision of health education should be a long-term practice among agropastoral and pastoral communities in order to prevent further RVF outbreaks in Tanzania.

Safety and immunogenicity of a ChAdOx1 vaccine against Rift Valley fever in UK adults: an open-label, non-randomised, first-in-human phase 1 clinical trial.

BACKGROUND: Rift Valley fever is a viral epidemic illness prevalent in Africa that can be fatal or result in debilitating sequelae in humans. No vaccines are available for human use. We aimed to evaluate the safety and immunogenicity of a non-replicating simian adenovirus-vectored Rift Valley fever (ChAdOx1 RVF) vaccine in humans. METHODS: We conducted a phase 1, first-in-human, open-label, dose-escalation trial in healthy adults aged 18-50 years at the Centre for Clinical Vaccinology and Tropical Medicine, Oxford, UK. Participants were required to have no serious comorbidities or previous history of receiving an adenovirus-based vaccine before enrolment. Participants were non-randomly allocated to receive a single ChAdOx1 RVF dose of either 5 × 109 virus particles (vp), 2·5 × 1010 vp, or 5 × 1010 vp administered intramuscularly into the deltoid of their non-dominant arm; enrolment was sequential and administration was staggered to allow for safety to be assessed before progression to the next dose. Primary outcome measures were assessment of adverse events and secondary outcome measures were Rift Valley fever neutralising antibody titres, Rift Valley fever GnGc-binding antibody titres (ELISA), and cellular response (ELISpot), analysed in all participants who received a vaccine. This trial is registered with ClinicalTrials.gov (NCT04754776). FINDINGS: Between June 11, 2021, and Jan 13, 2022, 15 volunteers received a single dose of either 5 × 109 vp (n=3), 2·5 × 1010 vp (n=6), or 5 × 1010 vp (n=6) ChAdOx1 RVF. Nine participants were female and six were male. 14 (93%) of 15 participants reported solicited local adverse reactions; injection-site pain was the most frequent (13 [87%] of 15). Ten (67%) of 15 participants (from the 2·5 × 1010 vp and 5 × 1010 vp groups only) reported systemic symptoms, which were mostly mild in intensity, the most common being headache (nine [60%] of 15) and fatigue (seven [47%]). All unsolicited adverse events reported within 28 days were either mild or moderate in severity; gastrointestinal symptoms were the most common reaction (at least possibly related to vaccination), occurring in four (27%) of 15 participants. Transient decreases in total white cell, lymphocyte, or neutrophil counts occurred at day 2 in some participants in the intermediate-dose and high-dose groups. Lymphopenia graded as severe occurred in two participants in the 5 × 1010 vp group at a single timepoint, but resolved at the subsequent follow-up visit. No serious adverse events occurred. Rift Valley fever neutralising antibodies were detectable across all dose groups, with all participants in the 5 × 1010 vp dose group having high neutralising antibody titres that peaked at day 28 after vaccination and persisted through the 3-month follow-up. High titres of binding IgG targeting Gc glycoprotein were detected whereas those targeting Gn were comparatively low. IFNγ cellular responses against Rift Valley fever Gn and Gc glycoproteins were observed in all participants except one in the 5 × 1010 vp dose group. These IFNγ responses peaked at 2 weeks after vaccination, were highest in the 5 × 1010 vp dose group, and tended to be more frequent against the Gn glycoprotein. INTERPRETATION: ChAdOx1 RVF was safe, well tolerated, and immunogenic when administered as a single dose in this study population. The data support further clinical development of ChAdOx1 RVF for human use. FUNDING: UK Department of Health and Social Care through the UK Vaccines Network, Oak Foundation, and the Wellcome Trust. TRANSLATION: For the Swahili translation of the abstract see Supplementary Materials section.

Pseudotyped Viruses for Phlebovirus.

Rift Valley fever virus (RVFV) is a member of the Phlebovirus genus, one of the 20 genera in the Phenuiviridae family. RVFV causes disease in animals and humans and is transmitted by sandflies or ticks. However, research into RVFV is limited by the requirement for biosafety level 3 (BSL-3) containment. Pseudotyped virus overcomes this limitation as it can be handled in a BSL-2 environment. Pseudotyped RVFV possesses an identical envelope protein structure to that of the authentic virus, simulating the same process of receptor binding and membrane fusion to host cells. Pseudotyped phleboviruses are therefore useful tools to study the infection mechanism of these viruses and for the screening of inhibitory drugs and the development of therapeutic monoclonal antibodies.

Exploring potential risk pathways with high risk groups for urban Rift Valley fever virus introduction, transmission, and persistence in two urban centers of Kenya.

Rift Valley fever virus (RVFV) is a zoonotic arbovirus that has profound impact on domestic ruminants and can also be transmitted to humans via infected animal secretions. Urban areas in endemic regions across Africa have susceptible animal and human hosts, dense vector distributions, and source livestock (often from high risk locations to meet the demand for animal protein). Yet, there has never been a documented urban outbreak of RVF. To understand the likely risk of RVFV introduction to urban communities from their perspective and guide future initiatives, we conducted focus group discussions with slaughterhouse workers, slaughterhouse animal product traders, and livestock owners in Kisumu City and Ukunda Town in Kenya. For added perspective and data triangulation, in-depth interviews were conducted one-on-one with meat inspector veterinarians from selected slaughterhouses. A theoretical framework relevant to introduction, transmission, and potential persistence of RVF in urban areas is presented here. Urban livestock were primarily mentioned as business opportunities, but also had personal sentiment. In addition to slaughtering risks, perceived risk factors included consumption of fresh milk. High risk groups' knowledge and experience with RVFV and other zoonotic diseases impacted their consideration of personal risk, with consensus towards lower risk in the urban setting compared to rural areas as determination of health risk was said to primarily rely on hygiene practices rather than the slaughtering process. Groups relied heavily on veterinarians to confirm animal health and meat safety, yet veterinarians reported difficulty in accessing RVFV diagnostics. We also identified vulnerable public health regulations including corruption in meat certification outside of the slaughterhouse system, and blood collected during slaughter being used for food and medicine, which could provide a means for direct RVFV community transmission. These factors, when compounded by diverse urban vector breeding habitats and dense human and animal populations, could create suitable conditions for RVFV to arrive an urban center via a viremic imported animal, transmit to locally owned animals and humans, and potentially adapt to secondary vectors and persist in the urban setting. This explorative qualitative study proposes risk pathways and provides initial insight towards determining how urban areas could adapt control measures and plan future initiatives to better understand urban RVF potential.

Immunogenicity of a Candidate Live Attenuated Vaccine for Rift Valley Fever Virus with a Two-Segmented Genome.

Rift Valley fever virus (RVFV) is an emerging arbovirus that affects both ruminants and humans. RVFV causes severe and recurrent outbreaks in Africa and the Arabian Peninsula with a significant risk for emergence into new locations. Although there are a variety of RVFV veterinary vaccines for use in endemic areas, there is currently no licensed vaccine for human use; therefore, there is a need to develop and assess new vaccines. Herein, we report a live-attenuated recombinant vaccine candidate for RVFV, based on the previously described genomic reconfiguration of the conditionally licensed MP12 vaccine. There are two general strategies used to develop live-attenuated RVFV vaccines, one being serial passage of wild-type RVFV strains to select attenuated mutants such as Smithburn, Clone 13, and MP12 vaccine strains. The second strategy has utilized reverse genetics to attenuate RVFV strains by introducing deletions or insertions within the viral genome. The novel candidate vaccine characterized in this report contains a two-segmented genome that lacks the medium viral segment (M) and two virulence genes (nonstructural small and nonstructural medium). The vaccine candidate, named r2segMP12, was evaluated for the production of neutralizing antibodies to RVFV in outbred CD-1 mice. The immune response induced by the r2segMP12 vaccine candidate was directly compared to the immune response induced by the rMP12 parental strain vaccine. Our study demonstrated that a single immunization with the r2segMP12 vaccine candidate at 105 plaque-forming units elicited a higher neutralizing antibody response than the rMP12 vaccine at the same vaccination titer without the need for a booster.

Rift Valley fever MP-12 vaccine elicits an early protective immune response in mice.

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen that causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The development of an effective veterinary and human vaccine to protect against Rift Valley fever (RVF) disease remains a high priority. The live attenuated RVFV MP-12 is a promising vaccine candidate for the prevention of RVF in both human and domestic ruminants. The aim of this study was to determine the onset of protective immunity elicted in mice by a single dose of this vaccine. Groups of CD-1 mice were vaccinated intraperitoneally with RVFV MP-12 vaccine and challenged on days 2, 5, 6 and 7 post-vaccination (PV) with a lethal dose of virulent RVFV. The mice were observed once daily for terminal morbidity and blood samples were obtained from the retro-orbital sinus complex on days 23 and 28 PV of surviving mice to determine RVFV neutralizing antibody titers. In one test, 2 of 3 mice challenged on day 2 PV survived and all 3 mice challenged at days 5 and 7 PV also survived. A second test of 10 mice per group was performed, and half (5) of those challenged at day 2 PV survived while all (10) survived challenge at day 4 and 6 PV. All surviving animals develop antibody that ranged from 1:80 to 1:1,280 PV. In a separate experiment, RVFV MP-12 vaccinated CD-1 mice, but not challenged developed a low viremia for the first 3 days PV and neutralzing antibody was detected on days 5 through day 28 PV. These findings demonstrated that the RVFV MP-12 vaccine elicited a rapid protective immune response in mice as early as 2 days PV, thus further supporting the effectiveness of this vaccine candidate for preventing RVF among humans and domestic ruminants.

Single-dose of a replication-competent adenovirus-vectored vaccine provides sterilizing protection against Rift Valley fever virus challenge.

Rift Valley fever virus (RVFV) is one of the most important virulent pathogens causing severe disease in animals and humans. However, there is currently no approved vaccine to prevent RVFV infection in humans. The use of human adenovirus serotype 4 (Ad4) as a vector for an RVFV vaccine has not been reported. Here, we report the generation of a replication-competent recombinant Ad4 vector expressing codon-optimized forms of the RVFV glycoproteins Gn and Gc (named Ad4-GnGc). Intramuscular immunization with Ad4-GnGc elicited robust neutralizing antibodies against RVFV and cellular immune responses in mice. A single low-dose vaccination with Ad4-GnGc completely protected interferon-α/ß receptor-deficient A129 mice from lethal RVFV infection. More importantly, Ad4-GnGc efficacy was not affected by pre-existing immunity to adenovirus serotype 5, which currently exists widely in populations. These results suggest that Ad4-GnGc is a promising vaccine candidate against RVFV.

Discovery of Rift Valley fever virus natural pan-inhibitors by targeting its multiple key proteins through computational approaches.

The Rift Valley fever virus (RVFV) is a zoonotic arbovirus and pathogenic to both humans and animals. Currently, no proven effective RVFV drugs or licensed vaccine are available for human or animal use. Hence, there is an urgent need to develop effective treatment options to control this viral infection. RVFV glycoprotein N (GN), glycoprotein C (GC), and nucleocapsid (N) proteins are attractive antiviral drug targets due to their critical roles in RVFV replication. In present study, an integrated docking-based virtual screening of more than 6000 phytochemicals with known antiviral activities against these conserved RVFV proteins was conducted. The top five hit compounds, calyxin C, calyxin D, calyxin J, gericudranins A, and blepharocalyxin C displayed optimal binding against all three target proteins. Moreover, multiple parameters from the molecular dynamics (MD) simulations and MM/GBSA analysis confirmed the stability of protein-ligand complexes and revealed that these compounds may act as potential pan-inhibitors of RVFV replication. Our computational analyses may contribute toward the development of promising effective drugs against RVFV infection.

Using RVFV as a Vector Platform for the Expression of Ruminant Disease Antigens.

Live attenuated viruses remain as vaccine agents with unparalleled performance in terms of duration, magnitude, and breadth of induced immune responses. As the yellow fever-attenuated vaccine strain Y17D, attenuated Rift Valley fever virus shares features suitable to be used as a viral vector for heterologous antigen expression and bivalent vaccine development. Current reverse genetics technology showed the successful rescue of RVFV carrying foreign antigens with little immunogenicity loss in experimental animal models. We show here the basic experimental protocol to achieve the expression of candidate vaccine antigens from other important diseases of ruminants using RVFV as a vector platform as well as preliminary steps for the characterization of immunogenicity in vivo.

Paving the way for human vaccination against Rift Valley fever virus: A systematic literature review of RVFV epidemiology from 1999 to 2021.

BACKGROUND: Rift Valley fever virus (RVFV) is a lethal threat to humans and livestock in many parts of Africa, the Arabian Peninsula, and the Indian Ocean. This systematic review's objective was to consolidate understanding of RVFV epidemiology during 1999-2021 and highlight knowledge gaps relevant to plans for human vaccine trials. METHODOLOGY/PRINCIPAL FINDINGS: The review is registered with PROSPERO (CRD42020221622). Reports of RVFV infection or exposure among humans, animals, and/or vectors in Africa, the Arabian Peninsula, and the Indian Ocean during the period January 1999 to June 2021 were eligible for inclusion. Online databases were searched for publications, and supplemental materials were recovered from official reports and research colleagues. Exposures were classified into five groups: 1) acute human RVF cases, 2) acute animal cases, 3) human RVFV sero-surveys, 4) animal sero-surveys, and 5) arthropod infections. Human risk factors, circulating RVFV lineages, and surveillance methods were also tabulated. In meta-analysis of risks, summary odds ratios were computed using random-effects modeling. 1104 unique human or animal RVFV transmission events were reported in 39 countries during 1999-2021. Outbreaks among humans or animals occurred at rates of 5.8/year and 12.4/year, respectively, with Mauritania, Madagascar, Kenya, South Africa, and Sudan having the most human outbreak years. Men had greater odds of RVFV infection than women, and animal contact, butchering, milking, and handling aborted material were significantly associated with greater odds of exposure. Animal infection risk was linked to location, proximity to water, and exposure to other herds or wildlife. RVFV was detected in a variety of mosquito vectors during interepidemic periods, confirming ongoing transmission. CONCLUSIONS/SIGNIFICANCE: With broad variability in surveillance, case finding, survey design, and RVFV case confirmation, combined with uncertainty about populations-at-risk, there were inconsistent results from location to location. However, it was evident that RVFV transmission is expanding its range and frequency. Gaps assessment indicated the need to harmonize human and animal surveillance and improve diagnostics and genotyping. Given the frequency of RVFV outbreaks, human vaccination has strong potential to mitigate the impact of this now widely endemic disease.

Safety study of Rift Valley Fever human vaccine candidate (DDVax) in mosquitoes.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen with significant human and veterinary health consequences that periodically emerges in epizootics. RVFV causes fetal loss and death in ruminants and in humans can lead to liver and renal disease, delayed-onset encephalitis, retinitis, and in some cases severe haemorrhagic fever. A live attenuated vaccine candidate (DDVax), was developed by the deletion of the virulence factors NSs and NSm from a clinical isolate, ZH501, and has proven safe and immunogenic in rodents, pregnant sheep and non-human primates. Deletion of NSm also severely restricted mosquito midgut infection and inhibited vector-borne transmission. To demonstrate environmental safety, this study investigated the replication, dissemination and transmission efficiency of DDVax in mosquitoes following oral exposure compared to RVFV strains MP-12 and ZH501. Infection and dissemination profiles were also measured in mosquitoes 7 days after they fed on goats inoculated with DDvax or MP-12. We hypothesized that DDVax would infect mosquitoes at significantly lower rates than other RVFV strains and, due to lack of NSm, be transmission incompetent. Exposure of Ae. aegypti and Cx. tarsalis to 8 log10 plaque forming units (PFU)/ml DDVax by artificial bloodmeal resulted in significantly reduced DDVax infection rates in mosquito bodies compared to controls. Plaque assays indicated negligible transmission of infectious DDVax in Cx. tarsalis saliva (1/140 sampled) and none in Ae. aegypti saliva (0/120). Serum from goats inoculated with DDVax or MP-12 did not harbour detectable infectious virus by plaque assay at 1, 2 or 3 days post-inoculation. Infectious virus was, however, recovered from Aedes and Culex bodies that fed on goats vaccinated with MP-12 (13.8% and 4.6%, respectively), but strikingly, DDvax-positive mosquito bodies were greatly reduced (4%, and 0%, respectively). Furthermore, DDVax did not disseminate to legs/wings in any of the goat-fed mosquitoes. Collectively, these results are consistent with a beneficial environmental safety profile.