Rift Valley Fever Phlebovirus Reassortment Study in Sheep.

Rift Valley fever (RVF) in ungulates and humans is caused by a mosquito-borne RVF phlebovirus (RVFV). Live attenuated vaccines are used in livestock (sheep and cattle) to control RVF in endemic regions during outbreaks. The ability of two or more different RVFV strains to reassort when co-infecting a host cell is a significant veterinary and public health concern due to the potential emergence of newly reassorted viruses, since reassortment of RVFVs has been documented in nature and in experimental infection studies. Due to the very limited information regarding the frequency and dynamics of RVFV reassortment, we evaluated the efficiency of RVFV reassortment in sheep, a natural host for this zoonotic pathogen. Co-infection experiments were performed, first in vitro in sheep-derived cells, and subsequently in vivo in sheep. Two RVFV co-infection groups were evaluated: group I consisted of co-infection with two wild-type (WT) RVFV strains, Kenya 128B-15 (Ken06) and Saudi Arabia SA01-1322 (SA01), while group II consisted of co-infection with the live attenuated virus (LAV) vaccine strain MP-12 and a WT strain, Ken06. In the in vitro experiments, the virus supernatants were collected 24 h post-infection. In the in vivo experiments, clinical signs were monitored, and blood and tissues were collected at various time points up to nine days post-challenge for analyses. Cell culture supernatants and samples from sheep were processed, and plaque-isolated viruses were genotyped to determine reassortment frequency. Our results show that RVFV reassortment is more efficient in co-infected sheep-derived cells compared to co-infected sheep. In vitro, the reassortment frequencies reached 37.9% for the group I co-infected cells and 25.4% for the group II co-infected cells. In contrast, we detected just 1.7% reassortant viruses from group I sheep co-infected with the two WT strains, while no reassortants were detected from group II sheep co-infected with the WT and LAV strains. The results indicate that RVFV reassortment occurs at a lower frequency in vivo in sheep when compared to in vitro conditions in sheep-derived cells. Further studies are needed to better understand the implications of RVFV reassortment in relation to virulence and transmission dynamics in the host and the vector. The knowledge learned from these studies on reassortment is important for understanding the dynamics of RVFV evolution.

Severe morbidity and hospital-based mortality from Rift Valley fever disease between November 2017 and March 2020 among humans in Uganda.

BACKGROUND: Rift Valley fever (RVF) is a zoonotic viral disease of increasing intensity among humans in Africa and the Arabian Peninsula. In Uganda, cases reported prior to 2016 were mild or not fully documented. We report in this paper on the severe morbidity and hospital-based mortality of human cases in Uganda. METHODS: Between November 2017 and March 2020 human cases reported to the Uganda Virus Research Institute (UVRI) were confirmed by polymerase chain reaction (PCR). Ethical and regulatory approvals were obtained to enrol survivors into a one-year follow-up study. Data were collected on socio-demographics, medical history, laboratory tests, potential risk factors, and analysed using Stata software. RESULTS: Overall, 40 cases were confirmed with acute RVF during this period. Cases were not geographically clustered and nearly all were male (39/40; 98%), median age 32 (range 11-63). The median definitive diagnosis time was 7 days and a delay of three days between presumptive and definitive diagnosis. Most patients (31/40; 78%) presented with fever and bleeding at case detection. Twenty-eight (70%) cases were hospitalised, out of whom 18 (64%) died. Mortality was highest among admissions in regional referral (11/16; 69%) and district (4/5; 80%) hospitals, hospitalized patients with bleeding at case detection (17/27; 63%), and patients older than 44 years (9/9; 100%). Survivors mostly manifested a mild gastro-intestinal syndrome with nausea (83%), anorexia (75%), vomiting (75%), abdominal pain (50%), and diarrhoea (42%), and prolonged symptoms of severe disease including jaundice (67%), visual difficulties (67%), epistaxis (50%), haemoptysis (42%), and dysentery (25%). Symptom duration varied between two to 120 days. CONCLUSION: RVF is associated with high hospital-based mortality, severe and prolonged morbidity among humans that present to the health care system and are confirmed by PCR. One-health composite interventions should be developed to improve environmental and livestock surveillance, prevent infections, promptly detect outbreaks, and improve patient outcomes.

HSP90 is part of a protein complex with the L polymerase of Rift Valley fever phlebovirus and prevents its degradation by the proteasome during the viral genome replication/transcription stage.

The mosquito-borne Rift Valley fever virus (RVFV) from the Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated the molecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication.

CK1 and PP1 regulate Rift Valley fever virus genome replication through L protein phosphorylation.

Rift Valley fever virus (RVFV) is an arbovirus in the Phenuiviridae family identified initially by the large 'abortion storms' observed among ruminants; RVFV can also infect humans. In humans, there is a wide variation of clinical symptoms ranging from subclinical to mild febrile illness to hepatitis, retinitis, delayed-onset encephalitis, or even hemorrhagic fever. The RVFV is a tri-segmented negative-sense RNA virus consisting of S, M, and L segments. The L segment encodes the RNA-dependent RNA polymerase (RdRp), termed the L protein, which is responsible for both viral mRNA synthesis and genome replication. Phosphorylation of viral RdRps is known to regulate viral replication. This study shows that RVFV L protein is serine phosphorylated and identified Casein Kinase 1 alpha (CK1α) and protein phosphatase 1 alpha (PP1α) as L protein binding partners. Inhibition of CK1 and PP1 through small molecule inhibitor treatment, D4476 and 1E7-03, respectively, caused a change in the phosphorylated status of the L protein. Inhibition of PP1α resulted in increased L protein phosphorylation whereas inhibition of CK1α decreased L protein phosphorylation. It was also found that in RVFV infected cells, PP1α localized to the cytoplasmic compartment. Treatment of RVFV infected cells with CK1 inhibitors reduced virus production in both mammalian and mosquito cells. Lastly, inhibition of either CK1 or PP1 reduced viral genomic RNA levels. These data indicate that L protein is phosphorylated and that CK1 and PP1 play a crucial role in regulating the L protein phosphorylation cycle, which is critical to viral RNA production and viral replication.

Exploring the transmission modalities of Bunyamwera virus.

Bunyamwera virus (BUNV) (Bunyamwera orthobunyavirus) has been found in Sub-Saharan Africa and demonstrated recently as cocirculating with Rift Valley Fever Virus (RVFV). Little is known regarding the breadth of transmission modalities of Bunyamwera. Given its co-occurence with RVFV, we hypothesized the transmission system of BUNV shared similarities to the RVFV system including transmission by Ae. aegypti mosquitoes and environmentally mediated transmission through fomites and environmental contamination. We exposed Ae. aegypti mosquitoes to BUNV and evaluated their ability to transmit both vertically and horizontally. Further, we investigated the potential for a novel transmission modality via environmental contamination. We found that the LSU colony of Ae. aegypti was not competent for the virus for either horizontal or vertical transmission; but, 20% of larva exposed to virus via contaminated aquatic habitat were positive. However, transstadial clearance of the virus was absolute. Finally, under simulated temperature conditions that matched peak transmission in Rwanda, we found that BUNV was stable in both whole blood and serum for up to 28 days at higher total volume in tubes at moderate quantities (103-5 genome copies/mL). In addition, infectiousness of these samples was demonstrated in 80% of the replicates. At lower volume samples (in plates), infectiousness was retained out to 6-8 days with a maximum infectious titer of 104 PFU/mL. Thus, the potential for contamination of the environment and/or transmission via contaminated fomites exists. Our findings have implications for biosafety and infection control, especially in the context of food animal production.

In silico identification of novel pre-microRNA genes in Rift valley fever virus suggest new pathomechanisms for embryo-fetal dysgenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the post-transcriptional expression of target genes. Virus-encoded miRNAs play an important role in the replication of viruses, modulate gene expression in both the virus and host, and affect their persistence and immune evasion in hosts. This renders viral miRNAs as potential targets for therapeutic applications, especially against pathogenic viruses that infect humans and animals. Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic RNA virus that causes severe disease in both humans and livestock. High mortality among newborn lambs and abortion storms are key characteristics of an RVF outbreak. To date, limited information is available on RVFV-derived miRNAs. In this study, computational methods were used to analyse the RVFV genome for putative pre-miRNA genes, which were then analysed for the presence of mature miRNAs. We detected 19 RVFV-encoded miRNAs and identified their potential mRNAs targets in sheep (Ovis aries), the most susceptible host. The identification of significantly enriched O. aries genes in association with RVFV miRNAs will help elucidate the molecular mechanisms underlying RVFV pathogenesis and potentially uncover novel drug targets for RVFV.

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.

Safety and Efficacy upon Infection in Sheep with Rift Valley Fever Virus ZH548-rA2, a Triple Mutant Rescued Virus.

The introduction of three single nucleotide mutations into the genome of the virulent RVFV ZH548 strain allows for the rescue of a fully attenuated virus in mice (ZH548-rA2). These mutations are located in the viral genes encoding the RdRp and the non-structural protein NSs. This paper shows the results obtained after the subcutaneous inoculation of ZH548-rA2 in adult sheep and the subsequent challenge with the parental virus (ZH548-rC1). Inoculation with the ZH548-rA2 virus caused no detectable clinical or pathological effect in sheep, whereas inoculation of the parental rC1 virus caused lesions compatible with viral infection characterised by the presence of scattered hepatic necrosis. Viral infection was confirmed via immunohistochemistry, with hepatocytes within the necrotic foci appearing as the main cells immunolabelled against viral antigen. Furthermore, the inoculation of sheep with the rA2 virus prevented the liver damage expected after rC1 virus inoculation, suggesting a protective efficacy in sheep which correlated with the induction of both humoral and cell-mediated immune responses.

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.

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.

Dishevelled Has Anti-Viral Activity in Rift Valley Fever Virus Infected Aedes aegypti.

Mosquitoes in the genera Aedes and Culex are vectors of Rift Valley fever virus (RVFV), which emerges in periodic epidemics in Africa and Saudi Arabia. Factors that influence the transmission dynamics of RVFV are not well characterized. To address this, we interrogated mosquito host-signaling responses through analysis of differentially expressed genes (DEGs) in two mosquito species with marked differences in RVFV vector competence: Aedes aegypti (Aae, low competence) and Culex tarsalis (Cxt, high competence). Mosquito-host transcripts related to three different signaling pathways were investigated. Selected genes from the Wingless (Wg, WNT-beta-catenin) pathway, which is a conserved regulator of cell proliferation and differentiation, were assessed. One of these, dishevelled (DSH), differentially regulates progression/inhibition of the WNT and JNK (c-Jun N-terminal Kinase) pathways. A negative regulator of the JNK-signaling pathway, puckered, was also assessed. Lastly, Janus kinase/signal transducers and activators of transcription (JAK-STAT) are important for innate immunity; in this context, we tested domeless levels. Here, individual Aae and Cxt were exposed to RVFV MP-12 via oral bloodmeals and held for 14 days. Robust decreases in DEGs in both Aae and Cxt were observed. In particular, Aae DSH expression, but not Cxt DSH, was correlated to the presence/absence of viral RNA at 14 days post-challenge (dpc). Moreover, there was an inverse relationship between the viral copy number and aaeDSH expression. DSH silencing resulted in increased viral copy numbers compared to controls at 3 dpc, consistent with a role for aaeDSH in antiviral immunity. Analysis of cis-regulatory regions for the genes of interest revealed clues to upstream regulation of these pathways.

A Novel Quadruple Gene-Deleted BoHV-1-Vectored RVFV Subunit Vaccine Induces Humoral and Cell-Mediated Immune Response against Rift Valley Fever in Calves.

Rift Valley fever virus (RVFV) is considered to be a high biodefense priority based on its threat to livestock and its ability to cause human hemorrhagic fever. RVFV-infected livestock are also a significant risk factor for human infection by direct contact with contaminated blood, tissues, and aborted fetal materials. Therefore, livestock vaccination in the affected regions has the direct dual benefit and one-health approach of protecting the lives of millions of animals and eliminating the risk of severe and sometimes lethal human Rift Valley fever (RVF) disease. Recently, we have developed a bovine herpesvirus type 1 (BoHV-1) quadruple gene mutant virus (BoHV-1qmv) vector that lacks virulence and immunosuppressive properties due to the deletion of envelope proteins UL49.5, glycoprotein G (gG), gE cytoplasmic tail, and US9 coding sequences. In the current study, we engineered the BoHV-1qmv further by incorporating a chimeric gene sequence to express a proteolytically cleavable polyprotein: RVFV envelope proteins Gn ectodomain sequence fused with bovine granulocyte-macrophage colony-stimulating factor (GMCSF) and Gc, resulting in a live BoHV-1qmv-vectored subunit vaccine against RVFV for livestock. In vitro, the resulting recombinant virus, BoHV-1qmv Sub-RVFV, was replicated in cell culture with high titers. The chimeric Gn-GMCSF and Gc proteins expressed by the vaccine virus formed the Gn-Gc complex. In calves, the BoHV-1qmv Sub-RVFV vaccination was safe and induced moderate levels of the RVFV vaccine strain, MP12-specific neutralizing antibody titers. Additionally, the peripheral blood mononuclear cells from the vaccinated calves had six-fold increased levels of interferon-gamma transcription compared with that of the BoHV-1qmv (vector)-vaccinated calves when stimulated with heat-inactivated MP12 antigen in vitro. Based on these findings, we believe that a single dose of BoHV-1qmv Sub-RVFV vaccine generated a protective RVFV-MP12-specific humoral and cellular immune response. Therefore, the BoHV-1qmv sub-RVFV can potentially be a protective subunit vaccine for cattle against RVFV.

Identification of Host Factors for Rift Valley Fever Phlebovirus.

Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication.

Host entry factors of Rift Valley Fever Virus infection.

Rift Valley Fever Virus (RVFV) is a negative sense segmented RNA virus that can cause severe hemorrhagic fever. The tri-segmented virus genome encodes for six (6) multifunctional proteins that engage host factors at a variety of different stages in the replication cycle. The S segment encodes nucleoprotein (N) and nonstructural protein S (NSs), the M segment encodes viral glycoproteins Gn and Gc as well as nonstructural protein M (NSm) and the L segment encodes the viral polymerase (L). Viral glycoproteins Gn and Gc are responsible for entry by binding to a number of host factors. Our recent studies identified a scavenger receptor, LDL receptor related protein 1 (Lrp1), as a potential pro-viral host factor for RVFV and related viruses, including Oropouche virus (OROV) infection. Coincidentally, several recent studies identified other LDL family proteins as viral entry factors and receptors for other viral families. Collectively, these observations suggest that highly conserved LDL family proteins may play a significant role in facilitating entry of viruses from several distinct families. Given the significant roles of viral and host factors during infection, characterization of these interactions is critical for therapeutic targeting with neutralizing antibodies and vaccines.

Interleukin-36γ is causative for liver damage upon infection with Rift Valley fever virus in type I interferon receptor-deficient mice.

Type I interferons (IFN) are pro-inflammatory cytokines which can also exert anti-inflammatory effects via the regulation of interleukin (IL)-1 family members. Several studies showed that interferon receptor (IFNAR)-deficient mice develop severe liver damage upon treatment with artificial agonists such as acetaminophen or polyinosinic:polycytidylic acid. In order to investigate if these mechanisms also play a role in an acute viral infection, experiments with the Bunyaviridae family member Rift Valley fever virus (RVFV) were performed. Upon RVFV clone (cl)13 infection, IFNAR-deficient mice develop a severe liver injury as indicated by high activity of serum alanine aminotransferase (ALT) and histological analyses. Infected IFNAR-/- mice expressed high amounts of IL-36γ within the liver, which was not observed in infected wildtype (WT) animals. In line with this, treatment of WT mice with recombinant IL-36γ induced ALT activity. Furthermore, administration of an IL-36 receptor antagonist prior to infection prevented the formation of liver injury in IFNAR-/- mice, indicating that IL-36γ is causative for the observed liver damage. Mice deficient for adaptor molecules of certain pattern recognition receptors indicated that IL-36γ induction was dependent on mitochondrial antiviral-signaling protein and the retinoic acid-inducible gene-I-like receptor. Consequently, cell type-specific IFNAR knockouts revealed that type I IFN signaling in myeloid cells is critical in order to prevent IL-36γ expression and liver injury upon viral infection. Our data demonstrate an anti-inflammatory role of type I IFN in a model for virus-induced hepatitis by preventing the expression of the novel IL-1 family member IL-36γ.

Lrp1 is essential for lethal Rift Valley fever hepatic disease in mice.

Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor-related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.

Alternative Splicing of RIOK3 Engages the Noncanonical NFκB Pathway during Rift Valley Fever Virus Infection.

Although the noncanonical NFκB pathway was originally identified as a cellular pathway contributing to lymphoid organogenesis, in the past 20 years, its involvement in innate immunity has become more appreciated. In particular, the noncanonical NFκB pathway has been found to be activated and even exploited by some RNA viruses during infection. Intriguingly, activation of this pathway has been shown to have a role in disrupting transcription of type 1 interferon (IFN), suggesting a rationale for why this response could be co-opted by some viruses. Rift Valley fever virus (RVFV) is a trisegmented ambisense RNA virus that poses a considerable threat to domestic livestock and human health. Previously, we showed the atypical kinase RIOK3 is important for mounting an IFN response to RVFV infection of human epithelial cells, and shortly following infection with RVFV (MP12 strain), RIOK3 mRNA is alternatively spliced to its X2 isoform that encodes a truncated RIOK3 protein. Alternative splicing of RIOK3 mRNA has an inhibitory effect on the IFN response but also stimulates an NFκB-mediated inflammatory response. Here, we demonstrate alternative splicing of RIOK3 mRNA is associated with activation of the noncanonical NFκB pathway and suggest this pathway is co-opted by RVFV (MP12) to enhance viral success during infection.

Rift Valley fever virus modulates apoptosis and immune response during infection of human astrocytes.

Rift Valley fever (RVF) is an arboviral disease of zoonotic origin that causes recurrent epidemics in Africa, the Arabic Peninsula, and islands of the South West of the Indian Ocean. RVF occurs mainly in livestock but also affects humans with severe clinical manifestations, including neurological disorders. However, human neuropathogenesis of Rift Valley fever virus (RVFV) is still poorly characterized. To study the interactions between RVFV and the central nervous system (CNS), we focused on RVFV infection of astrocytes, the major glial cells of the CNS that have several supporting roles including immune response regulation. We confirmed the permissiveness of astrocytes to RVFV infection and highlighted a strain-dependent infectivity. We showed that RVFV infection of astrocytes induced cell apoptosis and observed that the RVFV Non-Structural protein NSs, a known virulence factor, potentially delayed apoptosis by sequestrating activated-caspase 3 in the nucleus. Our study also showed that RVFV-infected astrocytes upregulated expression of genes associated with inflammatory and type I interferon responses at the mRNA level, but not at the protein level. This inhibition of immune response is potentially due to a NSs-dependent mechanism of mRNA nuclear export inhibition. Together, these results highlighted the direct impact of RVFV infection on the human CNS through the induction of apoptosis and a possible inhibition of early-onset immune responses that are crucial for the host survival.

Advances and perspectives in the development of vaccines against highly pathogenic bunyaviruses.

Increased human activities around the globe and the rapid development of once rural regions have increased the probability of contact between humans and wild animals. A majority of bunyaviruses are of zoonotic origin, and outbreaks may result in the substantial loss of lives, economy contraction, and social instability. Many bunyaviruses require manipulation in the highest levels of biocontainment, such as Biosafety Level 4 (BSL-4) laboratories, and the scarcity of this resource has limited the development speed of vaccines for these pathogens. Meanwhile, new technologies have been created, and used to innovate vaccines, like the mRNA vaccine platform and bioinformatics-based antigen design. Here, we summarize current vaccine developments for three different bunyaviruses requiring work in the highest levels of biocontainment: Crimean-Congo Hemorrhagic Fever Virus (CCHFV), Rift Valley Fever Virus (RVFV), and Hantaan virus (HTNV), and provide perspectives and potential future directions that can be further explored to advance specific vaccines for humans and livestock.

Antiviral and protective effect of small interfering RNAs against rift valley fever virus in vitro.

BACKGROUND: Rift Valley Fever Virus (RVFV) is an arbovirus, a zoonotic disease that resurfaces as a potential hazard beyond geographic boundaries. Fever that can proceed to encephalitis, retinitis, hemorrhagic fever, and death is the main manifestation observed in human infections. RVFV has no authorized medication. The RNA interference (RNAi) gene silencing pathway is extremely well conserved. By targeting specific genes, small interfering RNA (siRNA) can be used to suppress viral replication. The aim of this study was to design specific siRNAs against RVFV and evaluate their prophylactic and antiviral effects on the Vero cells. METHODS AND RESULTS: Various siRNAs were designed using different bioinformatics tools. Three unique candidates were tested against an Egyptian sheep cell culture-adapted strain BSL-2 that suppressed RVFV N mRNA expression. SiRNAs were transfected a day before RVFV infection (pre-transfection), and 1 h after the viral infection (post-transfection), and were evaluated to detect the silencing activity and gene expression decrease using real-time PCR and a TCID50 endpoint test. The degree of N protein expression was determined by western blot 48 h after viral infection. D2 which targets the (488-506 nucleotides), the middle region of RVFV N mRNA was the most effective siRNA at 30 nM concentration, it almost eliminates N mRNA expression when utilized as antiviral or preventive therapy. siRNAs had a stronger antiviral silencing impact when they were post-transfected into Vero cells. CONCLUSION: Pre and post-transfection of siRNAs significantly reduced RVFV titer in cell lines, offering novel and potentially effective anti-RVFV epidemics and epizootics therapy.