1 Introduction

Rift Valley fever virus (RVFV) (family Phenuiviridae , genusPhlebovirus ) is a mosquito-borne virus that causes periodic epizootic outbreaks across Africa and the Arabian peninsula(Al-Afaleq & Hussein, 2011; Nguku et al., 2010). In ruminants, primarily sheep, goats, camels and camelids, RVF is often characterized by sudden epizootics marked by near universal fetal death at all stages of gestation, congenital malformations(Coetzer, 1982) and significant adult animal deaths often due to acute virus induced hepatic and renal pathology(Wichgers Schreur et al., 2021). Though most human cases are typically self-limiting with mild to moderate symptoms (McElroy, Harmon, Flietstra, Nichol, & Spiropoulou, 2018), cases of delayed onset encephalitis, kidney and/or eye damage, severe anemia, hemorrhagic fever and miscarriage can occur(Baudin et al., 2016; Coetzer, 1982; Madani et al., 2003; Oymans, Wichgers Schreur, van Keulen, Kant, & Kortekaas, 2020).
Over 40 species of mosquitoes have been demonstrated as competent vectors for RVFV(reviewed in(Lumley et al., 2017)), some of which range on multiple continents. Following periods of heavy rainfall, which stimulate rapid increases in vector mosquito populations, RVFV re-emerges periodically in explosive epizootics(Al-Afaleq & Hussein, 2011; Nguku et al., 2010). In the absence of humans and livestock, RVFV cycles between mosquitoes and wild ruminants(Britch et al., 2013; Clark, Warimwe, Di Nardo, Lyons, & Gubbins, 2018). Between epizootics, there is also support for low level maintenance of RVFV in livestock in inter-epidemic periods(Lichoti et al., 2014).
Due to these health implications and the potential to cause a public health emergency, in 2018 the World Health Organization listed RVFV as a research and development blueprint priority pathogen(Mehand, Al-Shorbaji, Millett, & Murgue, 2018). Availability of a safe and effective human vaccine against RVFV is essential to protect the health of people in endemic regions and a preparatory measure for the anticipated cross-border spread and establishment of RVFV into new geographic areas. A number of vaccine candidates have been developed for RVFV, including formalin inactivated (Pittman et al., 1999; Randall, Gibbs, Aulisio, Binn, & Harrison, 1962) and live attenuated strains(Ikegami et al., 2015; Smithburn, 1949). However residual teratogenic effects in animals or the need for boosters to maintain protective immunity(Bird, Ksiazek, Nichol, & Maclachlan, 2009; Botros et al., 2006) present challenges for further development of these earlier candidates. To date, there is currently no commercially available and fully FDA-approved RVFV human vaccine.
To meet this critical health need, a human vaccine candidate (DDVax), a double deletion construct of the parental wild-type strain ZH501 was generated using a reverse genetics approach wherein both the NSs (non-structural, S segment) and NSm (non-structural, M segment) virulence genes were removed(Bird et al., 2008). NSs is expressed in an ambisense fashion from the viral S segment(Ikegami et al., 2009) and is a multi-functional protein that antagonizes host cell interferon responses(Le May et al., 2008). The viral M segment encodes 2 major glycoproteins and multiple open reading frames in the NSm coding regions, which is required for efficient dissemination in mosquitoes(Crabtree et al., 2012). Neither NSs nor NSm are required for viral replication in interferon-deficient cell culture, and the attenuated DDVax vaccine candidate has proven to be safe and immunogenic in a variety of animal species with the added benefit of inhibited replication and transmission in mosquitoes(Bird et al., 2008; Bird et al., 2011; Crabtree et al., 2012; Kading et al., 2014).
The objective of this study was to confirm that the newly rescued version of DDVax produced for development under Good Manufacturing Practices (GMP) behaved as previously described and exhibited a highly favorable environmental safety profile by not being transmitted by potential mosquito vectors. Here, we describe characterization of RVFV DDVax in mosquitoes using both in vitro and in vivoapproaches. These vector assessments were divided into two experimental phases: 1) mosquito oral challenges via artificial feeding and 2) mosquito feeding on DDVax inoculated goats. Features of vector competence were measured in two competent mosquito species, Culex tarsalis Coquillett and Aedes aegypti L., to determine infection, dissemination and transmission potential, using reverse transcriptase- quantitative PCR (RT-qPCR) and infectious virus plaque assay. Vertebrate-to-vector transmission from DDVax-inoculated goats to mosquitoes was also measured. Collectively, these experiments provided important comparison of vector competence of mosquitoes exposed to DDVax(Bird et al., 2008), ZH501, the parental wild-type virus and MP-12, an existing vaccine virus strain(Turell & Rossi, 1991).