Rift Valley Fever Virus Strains Research Articles

Acute Rift Valley fever virus infection induces inflammatory cytokines and cell death in ex vivo rat brain slice culture.

Rift Valley fever virus (RVFV) is an emerging arboviral disease with pandemic potential. While infection is often self-limiting, a subset of individuals may develop late-onset encephalitis, accounting for up to 20 % of severe cases. Importantly, individuals displaying neurologic disease have up to a 53 % case fatality rate, yet the neuropathogenesis of RVFV infection remains understudied. In this study, we evaluated whether ex vivo postnatal rat brain slice cultures (BSCs) could be used to evaluate RVFV infection in the central nervous system. BSCs mounted an inflammatory response after slicing, which resolved over time, and they were viable in culture for at least 12 days. Infection of rat BSCs with pathogenic RVFV strain ZH501 induced tissue damage and apoptosis over 48 h. Viral replication in BSCs reached up to 1×107 p.f.u. equivalents/ml, depending on inoculation dose. Confocal immunofluorescent microscopy of cleared slices confirmed direct infection of neurons as well as activation of microglia and astrocytes. Further, RVFV-infected rat BSCs produced antiviral cytokines and chemokines, including MCP-1 and GRO/KC. This study demonstrates that rat BSCs support replication of RVFV for ex vivo studies of neuropathogenesis. This allows for continued and complementary investigation into RVFV infection in an ex vivo postnatal brain slice culture format.

Molecular detection and characterization of Rift Valley fever virus in humans and domestic ruminants in Nigeria

Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne disease of domestic livestock, wildlife and humans majorly in Africa and the Arabian Peninsula. The virus is a Phlebovirus belonging to the Family Phenuiviridae and constitutes a threat to both human and animal health. Although there has been serological evidence of RVFV in humans and animals in Nigeria, reports on its molecular detection and characterization are absent. Blood samples (n = 1101) were collected between January 2019 and March 2021 from humans, cattle and sheep in four States of Nigeria. The harvested plasma samples were tested using a commercial ELISA kit. All the ELISA-positive, ‘-doubtful’ and some ‘-negative’ samples (n = 436) were further analyzed using nested reverse transcriptase-polymerase chain reaction (nRT-PCR). Eight samples (humans, n = 1; cattle, n = 6; sheep, n = 1) that gave the expected band size of approximately 668 base pairs following gel electrophoresis were sequenced by Sanger method. The sequence data were edited using BioEdit software and RVFV was successfully identified in all of them. Nucleotide sequence and phylogenetic analyses revealed that these eight RVFV strains were 98.3–99.9% identical and clustered with the Ugandan Smithburn strain. Genotyping showed that they belonged to Lineage K. This report, which is the first molecular detection and characterization of RVFV in Nigeria, confirms the circulation of the virus in the country. It also highlights the need for large-scale surveillance for RVFV in Nigeria, including among mosquito vectors, to provide better understanding of its spatiotemporal epidemiology, and thus, aid in preventing potential RVF outbreaks in the country.

Universal primers for rift valley fever virus whole-genome sequencing

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease causing acute hemorrhagic fever. Accurate identification of mutations and phylogenetic characterization of RVF virus (RVFV) require whole-genome analysis. Universal primers to amplify the entire RVFV genome from clinical samples with low copy numbers are currently unavailable. Thus, we aimed to develop universal primers applicable for all known RVFV strains. Based on the genome sequences available from public databases, we designed eight pairs of universal PCR primers covering the entire RVFV genome. To evaluate primer universality, four RVFV strains (ZH548, Kenya 56 (IB8), BIME-01, and Lunyo), encompassing viral phylogenetic diversity, were chosen. The nucleic acids of the test strains were chemically synthesized or extracted via cell culture. These RNAs were evaluated using the PCR primers, resulting in successful amplification with expected sizes (0.8–1.7 kb). Sequencing confirmed that the products covered the entire genome of the RVFV strains tested. Primer specificity was confirmed via in silico comparison against all non-redundant nucleotide sequences using the BLASTn alignment tool in the NCBI database. To assess the clinical applicability of the primers, mock clinical specimens containing human and RVFV RNAs were prepared. The entire RVFV genome was successfully amplified and sequenced at a viral concentration of 108 copies/mL. Given the universality, specificity, and clinical applicability of the primers, we anticipate that the RVFV universal primer pairs and the developed method will aid in RVFV phylogenomics and mutation detection.

The attenuated Rift Valley fever virus strain clone 13 induces encephalitis in type 1 interferon competent mice

The attenuated Rift Valley fever virus strain clone 13 induces encephalitis in type 1 interferon competent mice

Comparison of RT-qPCR and RT-ddPCR with Rift valley fever virus (RVFV) RNA

Rift valley fever (RVF) is an important zoonotic disease caused by the Rift valley fever virus (RVFV) which can affect ruminants and humans. In this study, a comparison was done of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription-droplet digital PCR (RT-ddPCR) assays with synthesized RVFV RNA, cultured viral RNA, and mock clinical RVFV RNA samples. The genomic segments (L, M, and S) of three RVFV strains (BIME01, Kenya56, and ZH548) were synthesized and used as templates for in vitro transcription (IVT). Both the RT-qPCR and RT-ddPCR assays for RVFV did not react with any of the negative reference viral genomes. Thus, both the RT-qPCR and RT-ddPCR assays are specific to RVFV. The comparison of both the RT-qPCR and RT-ddPCR assays with serially diluted templates showed that the LoD of both assays are similar, and a concordant of the results was observed. The LoD of both assays reached the practical measurable minimum concentration. Taken altogether, the sensitivity of the RT-qPCR and RT-ddPCR assays is similar, and the material measured by RT-ddPCR can be used as a reference material for RT-qPCR.

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.

Intact Type I Interferon Receptor Signaling Prevents Hepatocellular Necrosis but Not Encephalitis in a Dose-Dependent Manner in Rift Valley Fever Virus Infected Mice.

Rift Valley fever (RVF) is a zoonotic and emerging disease, caused by the RVF virus (RVFV). In ruminants, it leads to “abortion storms” and enhanced mortality rates in young animals, whereas in humans it can cause symptoms like severe hemorrhagic fever or encephalitis. The role of the innate and adaptive immune response in disease initiation and progression is still poorly defined. The present study used the attenuated RVFV strain clone 13 to investigate viral spread, tissue tropism, and histopathological lesions after intranasal infection in C57BL/6 wild type (WT) and type I interferon (IFN-I) receptor I knockout (IFNAR−/−) mice. In WT mice, 104 PFU RVFV (high dose) resulted in a fatal encephalitis, but no hepatitis 7–11 days post infection (dpi), whereas 103 PFU RVFV (low dose) did not cause clinical disease or significant histopathological lesions in liver and the central nervous system (CNS). In contrast, IFNAR−/− mice infected with 103 PFU RVFV developed hepatocellular necrosis resulting in death at 2–5 dpi and lacked encephalitis. These results show that IFNAR signaling prevents systemic spread of the attenuated RVFV strain clone 13, but not the dissemination to the CNS and subsequent fatal disease. Consequently, neurotropic viruses may be able to evade antiviral IFN-I signaling pathways by using the transneuronal instead of the hematogenous route.

Epidemiology of Mosquito-Borne Viruses in Egypt: A Systematic Review.

There are at least five common mosquito-borne viruses (MBVs) recorded in Egypt, including dengue virus (DENV), Rift Valley fever virus (RVFV), West Nile virus (WNV), Chikungunya virus, and Sindbis virus. Unexpected outbreaks caused by MBVs reflect the deficiencies of the MBV surveillance system in Egypt. This systematic review characterized the epidemiology of MBV prevalence in Egypt. Human, animal, and vector prevalence studies on MBVs in Egypt were retrieved from Web of Science, PubMed, and Bing Scholar, and 33 eligible studies were included for further analyses. The monophyletic characterization of the RVFV and WNV strains found in Egypt, which spans about half a century, suggests that both RVFV and WNV are widely transmitted in this nation. Moreover, the seropositive rates of DENV and WNV in hosts were on the rise in recent years, and spillover events of DENV and WNV to other countries from Egypt have been recorded. The common drawback for surveillance of MBVs in Egypt is the lack of seroprevalence studies on MBVs, especially in this century. It is necessary to evaluate endemic transmission risk, establish an early warning system for MBVs, and develop a sound joint system for medical care and public health for managing MBVs in Egypt.

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.

Tra2beta-Dependent Regulation of RIO Kinase 3 Splicing During Rift Valley Fever Virus Infection Underscores the Links Between Alternative Splicing and Innate Antiviral Immunity.

Rift Valley fever virus (RVFV) is an emerging pathogen that has potential to cause severe disease in humans and domestic livestock. Propagation of RVFV strain MP-12 is negatively impacted by the actions of RIOK3, a protein involved in the cellular immune response to viral infection. During RVFV infection, RIOK3 mRNA is alternatively spliced to produce an isoform that correlates with the inhibition of interferon β signaling. Here, we identify splicing factor TRA2-β (also known as TRA2beta and hTRA2-β) as a key regulator governing the relative abundance of RIOK3 splicing isoforms. Using RT-PCR and minigenes, we determined that TRA2-β interaction with RIOK3 pre-mRNA was necessary for constitutive splicing of RIOK3 mRNA, and conversely, lack of TRA2-β engagement led to increased alternative splicing. Expression of TRA2-β was found to be necessary for RIOK3’s antiviral effect against RVFV. Intriguingly, TRA2-β mRNA is also alternatively spliced during RVFV infection, leading to a decrease in cellular TRA2-β protein levels. These results suggest that splicing modulation serves as an immune evasion strategy by RVFV and/or is a cellular mechanism to prevent excessive immune response. Furthermore, the results suggest that TRA2-β can act as a key regulator of additional steps of the innate immune response to viral infection.

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.

Tilorone-Dihydrochloride Protects against Rift Valley Fever Virus Infection and Disease in the Mouse Model

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East that can affect humans and ruminant livestock. Currently, there are no approved vaccines or therapeutics for the treatment of severe RVF disease in humans. Tilorone-dihydrochloride (Tilorone) is a broad-spectrum antiviral candidate that has previously shown efficacy against a wide range of DNA and RNA viruses, and which is clinically utilized for the treatment of respiratory infections in Russia and other Eastern European countries. Here, we evaluated the antiviral activity of Tilorone against Rift Valley fever virus (RVFV). In vitro, Tilorone inhibited both vaccine (MP-12) and virulent (ZH501) strains of RVFV at low micromolar concentrations. In the mouse model, treatment with Tilorone significantly improved survival outcomes in BALB/c mice challenged with a lethal dose of RVFV ZH501. Treatment with 30 mg/kg/day resulted in 80% survival when administered immediately after infection. In post-exposure prophylaxis, Tilorone resulted in 30% survival at one day after infection when administered at 45 mg/kg/day. These findings demonstrate that Tilorone has potent antiviral efficacy against RVFV infection in vitro and in vivo and supports further development of Tilorone as a potential antiviral therapeutic for treatment of RVFV infection.

Susceptibility and barriers to infection of Colorado mosquitoes with Rift Valley fever virus.

Rift Valley fever virus (RVFV) causes morbidity and mortality in humans and domestic ungulates in sub-Saharan Africa, Egypt, and the Arabian Peninsula. Mosquito vectors transmit RVFV between vertebrates by bite, and also vertically to produce infectious progeny. Arrival of RVFV into the United States by infected mosquitoes or humans could result in significant impacts on food security, human health, and wildlife health. Elucidation of the vectors involved in the post-introduction RVFV ecology is paramount to rapid implementation of vector control. We performed vector competence experiments in which field-collected mosquitoes were orally exposed to an epidemic strain of RVFV via infectious blood meals. We targeted floodwater Aedes species known to feed on cattle, and/or deer species (Aedes melanimon Dyar, Aedes increpitus Dyar, Aedes vexans [Meigen]). Two permanent-water-breeding species were targeted as well: Culiseta inornata (Williston) of unknown competence considering United States populations, and Culex tarsalis Coquillett as a control species for which transmission efficiency is known. We tested the potential for midgut infection, midgut escape (dissemination), ovarian infection (vertical transmission), and transmission by bite (infectious saliva). Tissues were assayed by plaque assay and RT-qPCR, to quantify infectious virus and confirm virus identity. Tissue infection data were analyzed using a within-host model under a Bayesian framework to determine the probabilities of infection outcomes (midgut-limited infection, disseminated infection, etc.) while estimating barriers to infection between tissues. Permanent-water-breeding mosquitoes (Cx. tarsalis and Cs. inornata) exhibited more efficient horizontal transmission, as well as potential for vertical transmission, which is contrary to the current assumptions of RVFV ecology. Barrier estimates trended higher for Aedes spp., suggesting systemic factors in the differences between these species and Cx. tarsalis and Cs. inornata. These data indicate higher potential for vertical transmission than previously appreciated, and support the consensus of RVFV transmission including a broad range of potential vectors.

Preliminary Evaluation of a Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Providing Full Protection against Heterologous Virulent Challenge in Cattle.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that causes periodic outbreaks of abortion in ruminant species and hemorrhagic disease in humans in sub-Saharan Africa. These outbreaks have a significant impact on veterinary and public health. Its introduction to the Arabian Peninsula in 2003 raised concerns of further spread of this transboundary pathogen to non-endemic areas. These concerns are supported by the presence of competent vectors in many non-endemic countries. There is no licensed RVF vaccine available for humans and only a conditionally licensed veterinary vaccine available in the United States. Currently employed modified live attenuated virus vaccines in endemic countries lack the ability for differentiating infected from vaccinated animals (DIVA). Previously, the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins, derived from the 1977 human RVFV isolate ZH548, was demonstrated in sheep. In the current study, cattle were vaccinated subcutaneously with the Gn only, or Gn and Gc combined, with either one or two doses of the vaccine and then subjected to heterologous virus challenge with the virulent Kenya-128B-15 RVFV strain, isolated from Aedes mosquitoes in 2006. The elicited immune responses by some vaccine formulations (one or two vaccinations) conferred complete protection from RVF within 35 days after the first vaccination. Vaccines given 35 days prior to RVFV challenge prevented viremia, fever and RVFV-associated histopathological lesions. This study indicates that a recombinant RVFV glycoprotein-based subunit vaccine platform is able to prevent and control RVFV infections in target animals.

Early Immune Response and Cytokine Induction of PBMC‐derived Bovine Macrophages to Infection with Rift Valley Fever Virus in the Presence and Absence of Culex tarsalis Mosquito Saliva

Rift Valley fever virus (RVFV) is a zoonotic arbovirus in the family Phenuiviridae. In Africa, outbreaks of Rift Valley fever (RVF) usually occur following periods of heavy rainfall that prompt population surges in mosquito vectors. RVF causes significant mortality in ruminants, especially in ovine fetuses and neonates (up to 100%), while human infections cause mild febrile illness to hemorrhagic fever and death. Macrophages play a critical role as phagocytes in the innate immune system and produce cytokines that stimulate the adaptive immune system. As in other arboviral diseases, macrophages and dendritic cells are thought to be early infection targets of RVFV, enabling the virus to suppress the immune response. To further investigate their role in RVF early immune response and cytokine induction, bovine peripheral blood mononuclear cell-derived macrophages were infected with RVFV MP-12 (an attenuated vaccine strain) in the presence and absence of Culex tarsalis saliva. We hypothesized that in macrophages treated with Culex saliva and RVFV will have increased transcription of Th2-associated cytokines compared to those infected with virus only due to immunomodulatory proteins present in mosquito saliva. The macrophage lineage of the cells and RVFV infection were confirmed by dual label immunofluorescence for IBA-1 (macrophage marker) and RVFV nucleoprotein. Total RNA was extracted from 3 biological replicates of macrophages treated respectively with saliva only, MP-12 only, MP-12 and saliva at 0, 8 and 24 hours post-infection (hpi) as well as untreated controls. RNA was reverse-transcribed and the resulting cDNA analyzed by qPCR of four housekeeping genes (ActinB, H3F3, PPIA, YWHAZ), 6 early response genes (TLR3, TLR7, TLR8, RIG1, MDA5, IFNa) and 9 cytokine genes (IL4, IL6, IL10, IL12b, IL18, IFNg, TNFa, IL1a, IL1b). Relative gene expression (RGE) was determined by the method described by Vandesompele which uses multiple reference genes for normalization and does not assume equal PCR efficiencies for the reference genes and genes of interest. RGEs for each target were log-transformed and analyzed with two-way ANOVAs and Tukey's post-hoc test with p-value correction for multiple tests to evaluate statistical differences between treatments and time points. There was no statistical increase in transcription of Th2-associated cytokines when bovine macrophages were infected with MP-12 in the presence of saliva at any of the time points when compared to virus only. In contrast, significant differences in gene expression were present when infected samples were compared to uninfected controls. At 8 hpi, transcription was significantly different than controls for all virus infected samples for TLR3, RIG1, MDA and IFNa as well as IL6, IL10, IL12b, IL18, TNFa, IL1a and IL1b. This trend continued at 24 hpi with the exception of IL12b. While Culex saliva may not enhance suppression of the early immune response in MP-12 infection, it may still have a role in virulent RVFV strain infections.

Genetic Diversity of Rift Valley Fever Strains Circulating in Namibia in 2010 and 2011.

Outbreaks of Rift Valley fever (RVF) occurred in Namibia in 2010 and 2011. Complete genome characterization was obtained from virus isolates collected during disease outbreaks in southern Namibia in 2010 and from wildlife in Etosha National Park in 2011, close to the area where RVF outbreaks occurred in domestic livestock. The virus strains were sequenced using Sanger sequencing (Namibia_2010) or next generation sequencing (Namibia_2011). A sequence-independent, single-primer amplification (SISPA) protocol was used in combination with the Illumina Next 500 sequencer. Phylogenetic analysis of the sequences of the small (S), medium (M), and large (L) genome segments of RVF virus (RVFV) provided evidence that two distinct RVFV strains circulated in the country. The strain collected in Namibia in 2010 is genetically similar to RVFV strains circulating in South Africa in 2009 and 2010, confirming that the outbreaks reported in the southern part of Namibia in 2010 were caused by possible dissemination of the infection from South Africa. Isolates collected in 2011 were close to RVFV isolates from 2010 collected in humans in Sudan and which belong to the large lineage containing RVFV strains that caused an outbreak in 2006–2008 in eastern Africa. This investigation showed that the RVFV strains circulating in Namibia in 2010 and 2011 were from two different introductions and that RVFV has the ability to move across regions. This supports the need for risk-based surveillance and monitoring.

Safety and efficacy of four-segmented Rift Valley fever virus in young sheep, goats and cattle

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes severe and recurrent outbreaks on the African continent and the Arabian Peninsula and continues to expand its habitat. RVFV induces severe disease in newborns and abortion in pregnant ruminants. The viral genome consists of a small (S), medium (M) and large (L) RNA segment of negative polarity. The M segment encodes a glycoprotein precursor protein that is co-translationally cleaved into the two structural glycoproteins Gn and Gc, which are involved in receptor attachment and cell entry. We previously constructed a four-segmented RVFV (RVFV-4s) by splitting the M genome segment into two M-type segments encoding either Gn or Gc. RVFV-4s replicates efficiently in cell culture but was shown to be completely avirulent in mice, lambs and pregnant ewes. Here, we show that a RVFV-4s candidate vaccine for veterinary use (vRVFV-4s) does not disseminate in vaccinated animals, is not shed or spread to the environment and does not revert to virulence. Furthermore, a single vaccination of lambs, goat kids and calves was shown to induce protective immunity against a homologous challenge. Finally, the vaccine was shown to provide full protection against a genetically distinct RVFV strain. Altogether, we demonstrate that vRVFV-4s optimally combines efficacy with safety, holding great promise as a next-generation RVF vaccine.

Rift Valley fever virus targets the maternal-foetal interface in ovine and human placentas.

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. 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. 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.

Rift Valley Fever Virus: Propagation, Quantification, and Storage.

Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic disease endemic to sub-Saharan Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever have had up to 100% mortality rates in fetal and neonatal sheep. Upon infection of ruminant and human hosts alike, RVFV infection causes an at times severe hepatitis and pathology in many other organs. The enveloped virion contains a tripartite, predominantly negative-sense, single-stranded RNA genome, which codes for the proteins the virus needs to replicate both in mammalian hosts and insect vectors. Endemic countries often use attenuated RVFV strains for vaccination of livestock but there are no commercially licensed vaccines for humans or livestock in non-endemic areas. In the laboratory, RVFV can be readily propagated and manipulated in vitro using cell culture systems. Presented in this article are techniques routinely used in RVFV research that have proven successful in our laboratories. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Propagation of Rift Valley fever virus in mammalian cells Basic Protocol 2: Quantification of Rift Valley fever virus by plaque assay Basic Protocol 3: Quantification of Rift Valley fever virus by 50% tissue culture infectious dose (TCID50 ) assay Basic Protocol 4: Quantification of Rift Valley fever virus by focus-forming assay Basic Protocol 5: Storage and disinfection Alternate Protocol 1: Propagation of Rift Valley fever virus in MRC-5 cells Alternate Protocol 2: Propagation of RVFV in mosquito-derived cells Alternate Protocol 3: TCID50 detection using fluorescence visualization Support Protocol 1: Calculation of the amount of virus needed to infect a flask at a chosen multiplicity of infection Support Protocol 2: Calculation of the virus titer by plaque assay or focus-forming assay Support Protocol 3: Calculation of the TCID50 titer by the method of Reed and Muench Support Protocol 4: Calculation of the antibody volume for the focus-forming assay.

Rift Valley Fever Viral RNA Detection by In Situ Hybridization in Formalin-Fixed, Paraffin-Embedded Tissues.

Sporadic outbreaks of Rift Valley fever virus (RVFV), a zoonotic, mosquito-borne Phlebovirus, cause abortion storms and death in sheep and cattle resulting in catastrophic economic impacts in endemic regions of Africa. More recently, with changes in competent vector distribution, growing international trade, and its potential use for bioterrorism, RVFV has become a transboundary animal disease of significant concern. New and sensitive techniques that determine RVFV presence, while lessening the potential for environmental contamination and human risk, through the use of inactivated, noninfectious samples such as formalin-fixed, paraffin-embedded (FFPE) tissues are needed. FFPE tissue in situ hybridization (ISH) enables the detection of nucleic acid sequences within the visual context of cellular and tissue morphology. Here, we present a chromogenic pan-RVFV ISH assay based on RNAscope® technology, which is able to detect multiple RVFV strains in FFPE tissues, enabling visual correlation of RVFV RNA presence with histopathologic lesions.