![]() ![]() Including agRNA2 with the RRV replicon produced observable virions. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. ![]() The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. The results of the studies support the unprecedented safety profile of both vaccines for animals and humans. In contrast, both vaccines were well tolerated with no evidence of dissemination and shedding while inducing potent neutralizing antibody responses. Both wild-type strains induced high viremia and disseminated to primary target organs, associated with mild-to-severe morbidity. To accurately assess the attenuation of both vaccine viruses, separate groups of marmosets were inoculated with the parent wild-type RVFV strains. 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. 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). ![]() 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. 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. No human vaccine is available and commercialized veterinary vaccines do not optimally combine efficacy with safety. While animals are exclusively infected via mosquito bites, humans can also be infected via contact with contaminated tissues or blood. Rift Valley fever virus (RVFV) is an emerging mosquito-borne bunyavirus that is highly pathogenic to wild and domesticated ruminants, camelids, and humans. ![]()
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