Transcriptomic Profiling Reveals Intense Host-Pathogen Dispute Compromising Homeostasis during Acute Rift Valley Fever Virus Infection.

Rift Valley fever virus (RVFV) (family Phenuiviridae) can cause severe disease, and outbreaks of this mosquito-borne pathogen pose a significant threat to public and animal health. Yet many molecular aspects of RVFV pathogenesis remain incompletely understood. Natural RVFV infections are acute, characterized by a rapid onset of peak viremia during the first days post-infection, followed by a rapid decline. Although in vitro studies identified a major role of interferon (IFN) responses in counteracting the infection, a comprehensive overview of the specific host factors that play a role in RVFV pathogenesis in vivo is still lacking. Here, the host in vivo transcriptional profiles in the liver and spleen tissues of lambs exposed to RVFV are studied using RNA sequencing (RNA-seq) technology. We validate that IFN-mediated pathways are robustly activated in response to infection. We also link the observed hepatocellular necrosis with severely compromised organ function, which is reflected as a marked downregulation of multiple metabolic enzymes essential for homeostasis. Furthermore, we associate the elevated basal expression of LRP1 in the liver with RVFV tissue tropism. Collectively, the results of this study deepen the knowledge of the in vivo host response during RVFV infection and reveal new insights into the gene regulation networks underlying pathogenesis in a natural host. IMPORTANCE Rift Valley fever virus (RVFV) is a mosquito-transmitted pathogen capable of causing severe disease in animals and humans. Outbreaks of RVFV pose a significant threat to public health and can result in substantial economic losses. Little is known about the molecular basis of RVFV pathogenesis in vivo, particularly in its natural hosts. We employed RNA-seq technology to investigate genome-wide host responses in the liver and spleen of lambs during acute RVFV infection. We show that RVFV infection drastically decreases the expression of metabolic enzymes, which impairs normal liver function. Moreover, we highlight that basal expression levels of the host factor LRP1 may be a determinant of RVFV tissue tropism. This study links the typical pathological phenotype induced by RVFV infection with tissue-specific gene expression profiles, thereby improving our understanding of RVFV pathogenesis.

Rift Valley Fever Virus Propagates in Human Villous Trophoblast Cell Lines and Induces Cytokine mRNA Responses Known to Provoke Miscarriage.

The mosquito-borne Rift Valley fever (RVF) is a prioritised disease that has been listed by the World Health Organization for urgent research and development of counteraction. Rift Valley fever virus (RVFV) can cause a cytopathogenic effect in the infected cell and induce hyperimmune responses that contribute to pathogenesis. In livestock, the consequences of RVFV infection vary from mild symptoms to abortion. In humans, 1-3% of patients with RVFV infection develop severe disease, manifested as, for example, haemorrhagic fever, encephalitis or blindness. RVFV infection has also been associated with miscarriage in humans. During pregnancy, there should be a balance between pro-inflammatory and anti-inflammatory mediators to create a protective environment for the placenta and foetus. Many viruses are capable of penetrating that protective environment and infecting the foetal-maternal unit, possibly via the trophoblasts in the placenta, with potentially severe consequences. Whether it is the viral infection per se, the immune response, or both that contribute to the pathogenesis of miscarriage remains unknown. To investigate how RVFV could contribute to pathogenesis during pregnancy, we infected two human trophoblast cell lines, A3 and Jar, representing normal and transformed human villous trophoblasts, respectively. They were infected with two RVFV variants (wild-type RVFV and RVFV with a deleted NSs protein), and the infection kinetics and 15 different cytokines were analysed. The trophoblast cell lines were infected by both RVFV variants and infection caused upregulation of messenger RNA (mRNA) expression for interferon (IFN) types I-III and inflammatory cytokines, combined with cell line-specific mRNA expression of transforming growth factor (TGF)-ß1 and interleukin (IL)-10. When comparing the two RVFV variants, we found that infection with RVFV lacking NSs function caused a hyper-IFN response and inflammatory response, while the wild-type RVFV suppressed the IFN I and inflammatory response. The induction of certain cytokines by RVFV infection could potentially lead to teratogenic effects that disrupt foetal and placental developmental pathways, leading to birth defects and other pregnancy complications, such as miscarriage.

Experimental Infection of Domestic Piglets (Sus scrofa) with Rift Valley Fever Virus.

Rift Valley fever phlebovirus (RVFV) is a mosquito-transmitted phlebovirus (Family: Phenuiviridae, Order: Bunyavirales) causing severe neonatal mortality and abortion primarily in domestic ruminants. The susceptibility of young domestic swine to RVFV and this species' role in geographic expansion and establishment of viral endemicity is unclear. Six commercially bred Landrace-cross piglets were inoculated subcutaneously with 105 plaque-forming units of RVFV ZH501 strain and two piglets received a sham inoculum. All animals were monitored for clinical signs, viremia, viral shedding, and antibody response for 14 days. Piglets did not develop evidence of clinical disease, become febrile, or experience decreased weight gain during the study period. A brief lymphopenia followed by progressive lymphocytosis was observed following inoculation in all piglets. Four piglets developed a brief viremia for 2 days post-inoculation and three of these had detectable virus in oronasal secretions three days post-inoculation. Primary inoculated piglets seroconverted and those that developed detectable viremias had the highest titers assessed by serum neutralization (1:64-1:256). Two viremic piglets had a lymphoplasmacytic encephalitis with glial nodules; RVFV was not detected by immunohistochemistry in these sections. While young piglets do not appear to readily develop clinical disease following RVFV infection, results suggest swine could be subclinically infected with RVFV.

Rift Valley fever virus 78kDa envelope protein attenuates virus replication in macrophage-derived cell lines and viral virulence in mice.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines.

Further Characterization of Rio Grande Virus and Potential for Cross Reactivity with Rift Valley Fever Virus Assays.

Phleboviruses (genus Phlebovirus, family Phenuiviridae) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep (Ovis aries) and horses (Equus caballus) in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus (South and Central America) and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.

Insights into the Pathogenesis of Viral Haemorrhagic Fever Based on Virus Tropism and Tissue Lesions of Natural Rift Valley Fever.

Rift Valley fever phlebovirus (RVFV) infects humans and a wide range of ungulates and historically has caused devastating epidemics in Africa and the Arabian Peninsula. Lesions of naturally infected cases of Rift Valley fever (RVF) have only been described in detail in sheep with a few reports concerning cattle and humans. The most frequently observed lesion in both ruminants and humans is randomly distributed necrosis, particularly in the liver. Lesions supportive of vascular endothelial injury are also present and include mild hydropericardium, hydrothorax and ascites; marked pulmonary congestion and oedema; lymph node congestion and oedema; and haemorrhages in many tissues. Although a complete understanding of RVF pathogenesis is still lacking, antigen-presenting cells in the skin are likely the early targets of the virus. Following suppression of type I IFN production and necrosis of dermal cells, RVFV spreads systemically, resulting in infection and necrosis of other cells in a variety of organs. Failure of both the innate and adaptive immune responses to control infection is exacerbated by apoptosis of lymphocytes. An excessive pro-inflammatory cytokine and chemokine response leads to microcirculatory dysfunction. Additionally, impairment of the coagulation system results in widespread haemorrhages. Fatal outcomes result from multiorgan failure, oedema in many organs (including the lungs and brain), hypotension, and circulatory shock. Here, we summarize current understanding of RVF cellular tropism as informed by lesions caused by natural infections. We specifically examine how extant knowledge informs current understanding regarding pathogenesis of the haemorrhagic fever form of RVF, identifying opportunities for future research.

Production and Characterization of Monoclonal Antibodies Against N Protein of Rift Valley Fever Virus.

The DNA fragment encoding predicted main antigenic region, aa 14-245 on N protein of Rift Valley virus (RVFV) was cloned into the vector pET-28a (+) and p3xFLAG-CMV-10. The recombinant pET-28a-N1 protein was expressed in Escherichia coli BL21 (DE3) with 1 mM isopropyl-b-thio-galactopyranoside at 37°C for 5 hours, and purified by protein purifier. Three monoclonal antibodies (mAbs) named 3A5, 3A6, and 3A7 against N protein were obtained by fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from pET-28a-N1 protein-immunized mice. Finally, the mAbs were characterized by enzyme-linked immunosorbent assays, indirect immunofluorescent assays, and Western blot. The results show that all the mAbs possess high specificity and react with both prokaryotic and eukaryotic N protein, which could provide important materials for the research on the function of N protein and the diagnostic methods of RVFV.

Visualizing the ribonucleoprotein content of single bunyavirus virions reveals more efficient genome packaging in the arthropod host.

Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.

Rift Valley Fever: a Threat to Pregnant Women Hiding in Plain Sight?

The potential for emerging mosquito-borne viruses to cause fetal infection in pregnant women was overlooked until the Zika fever outbreak several years ago. Rift Valley fever virus (RVFV) is an emerging arbovirus with a long history of fetal infection and death in pregnant livestock. The effect of RVFV infection on pregnant women is not well understood. This Gem examines the effects that this important emerging pathogen has during pregnancy, its potential impact on pregnant women, and the current research efforts designed to understand and mitigate adverse effects of RVFV infection during pregnancy.

Reproducing the Rift Valley fever virus mosquito-lamb-mosquito transmission cycle.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that is pathogenic to ruminants and humans. The virus is endemic to Africa and the Arabian Peninsula where outbreaks are characterized by abortion storms and mortality of newborns, particularly in sheep herds. Vector competence experiments in laboratory settings have suggested that over 50 mosquito species are capable of transmitting RVFV. Transmission of mosquito-borne viruses in the field is however influenced by numerous factors, including population densities, blood feeding behavior, extrinsic incubation period, longevity of vectors, and viremia levels in vertebrate hosts. Animal models to study these important aspects of RVFV transmission are currently lacking. In the present work, RVFV was transmitted to European (Texel-swifter cross-breed) lambs by laboratory-reared Aedes aegypti mosquitoes that were infected either by membrane feeding on a virus-spiked blood meal or by feeding on lambs that developed viremia after intravenous inoculation of RVFV. Feeding of mosquitoes on viremic lambs resulted in strikingly higher infection rates as compared to membrane feeding. Subsequent transmission of RVFV from lamb to lamb by infected mosquitoes was highly efficient in both models. The animal models described here can be used to study mosquito-mediated transmission of RVFV among the major natural target species and to evaluate the efficacy of vaccines against mosquito-mediated RVFV infection.

Reanalysis of the 2000 Rift Valley fever outbreak in Southwestern Arabia.

The first documented Rift Valley hemorrhagic fever outbreak in the Arabian Peninsula occurred in northwestern Yemen and southwestern Saudi Arabia from August 2000 to September 2001. This Rift Valley fever outbreak is unique because the virus was introduced into Arabia during or after the 1997-1998 East African outbreak and before August 2000, either by wind-blown infected mosquitos or by infected animals, both from East Africa. A wet period from August 2000 into 2001 resulted in a large number of amplification vector mosquitoes, these mosquitos fed on infected animals, and the outbreak occurred. More than 1,500 people were diagnosed with the disease, at least 215 died, and widespread losses of domestic animals were reported. Using a combination of satellite data products, including 2 x 2 m digital elevation images derived from commercial satellite data, we show rainfall and potential areas of inundation or water impoundment were favorable for the 2000 outbreak. However, favorable conditions for subsequent outbreaks were present in 2007 and 2013, and very favorable conditions were also present in 2016-2018. The lack of subsequent Rift Valley fever outbreaks in this area suggests that Rift Valley fever has not been established in mosquito species in Southwest Arabia, or that strict animal import inspection and quarantine procedures, medical and veterinary surveillance, and mosquito control efforts put in place in Saudi Arabia following the 2000 outbreak have been successful. Any area with Rift Valley fever amplification vector mosquitos present is a potential outbreak area unless strict animal import inspection and quarantine proceedures are in place.

The NSs Protein Encoded by the Virulent Strain of Rift Valley Fever Virus Targets the Expression of Abl2 and the Actin Cytoskeleton of the Host, Affecting Cell Mobility, Cell Shape, and Cell-Cell Adhesion.

Rift Valley fever virus (RVFV) is a highly pathogenic zoonotic arbovirus endemic in many African countries and the Arabian Peninsula. Animal infections cause high rates of mortality and abortion among sheep, goats, and cattle. In humans, an estimated 1 to 2% of RVFV infections result in severe disease (encephalitis, hepatitis, or retinitis) with a high rate of lethality when associated with hemorrhagic fever. The RVFV NSs protein, which is the main virulence factor, counteracts the host innate antiviral response to favor viral replication and spread. However, the mechanisms underlying RVFV-induced cytopathic effects and the role of NSs in these alterations remain for the most part unknown. In this work, we have analyzed the effects of NSs expression on the actin cytoskeleton while conducting infections with the NSs-expressing virulent (ZH548) and attenuated (MP12) strains of RVFV and the non-NSs-expressing avirulent (ZH548ΔNSs) strain, as well as after the ectopic expression of NSs. In macrophages, fibroblasts, and hepatocytes, NSs expression prevented the upregulation of Abl2 (a major regulator of the actin cytoskeleton) expression otherwise induced by avirulent infections and identified here as part of the antiviral response. The presence of NSs was also linked to an increased mobility of ZH548-infected cells compared to ZH548ΔNSs-infected fibroblasts and to strong changes in cell morphology in nonmigrating hepatocytes, with reduction of lamellipodia, cell spreading, and dissolution of adherens junctions reminiscent of the ZH548-induced cytopathic effects observed in vivo Finally, we show evidence of the presence of NSs within long actin-rich structures associated with NSs dissemination from NSs-expressing toward non-NSs-expressing cells.IMPORTANCE Rift Valley fever virus (RVFV) is a dangerous human and animal pathogen that was ranked by the World Health Organization in 2018 as among the eight pathogens of most concern for being likely to cause wide epidemics in the near future and for which there are no, or insufficient, countermeasures. The focus of this work is to address the question of the mechanisms underlying RVFV-induced cytopathic effects that participate in RVFV pathogenicity. We demonstrate here that RVFV targets cell adhesion and the actin cytoskeleton at the transcriptional and cellular level, affecting cell mobility and inducing cell shape collapse, along with distortion of cell-cell adhesion. All these effects may participate in RVFV-induced pathogenicity, facilitate virulent RVFV dissemination, and thus constitute interesting potential targets for future development of antiviral therapeutic strategies that, in the case of RVFV, as with several other emerging arboviruses, are presently lacking.

Vector-borne viruses in Turkey: A systematic review and bibliography.

Turkey serves as a natural hub for the dissemination of vector-borne viruses and provides many suitable habitats with diverse ecologies for introduction and establishment of new pathogens. This manuscript provides an updated systematic review and meta-analysis of the vector-borne viruses documented in Turkey. Following web-based identification, screening and eligibility evaluation, 291 published reports were reviewed. The publications were categorized and listed as a supplementary bibliography accompanying the manuscript. In brief, Crimean-Congo hemorrhagic fever virus (CCHFV) and West Nile virus (WNV) are currently documented as prominent tick and mosquito-borne viral pathogens in Turkey. CCHFV produces a significant number of infections annually, with severe outcome or death in a portion of cases. WNV gained attention following the clustering of cases in 2010. Exposure and infections with sandfly-borne phleboviruses, such as Toscana virus, are indigenous and widespread. Epidemiology, risk factors, symptomatic infections in susceptible hosts, vectors and reservoirs for these pathogens have been explored in detail. Detection of novel viruses in mosquitoes, sandflies and ticks from several regions is of particular interest, despite scarce information on their epidemiology and pathogenicity in vertebrates. Introduction and emergence of viruses transmitted by invasive Aedes mosquitoes constitute a threat, albeit only imported infections have so far been documented. Detection of Rift valley fever virus exposure is also of concern, due to its detrimental effects on livestock and spillover infections in humans. Vigilance to identify and diagnose probable cases as well as vector surveillance for established and potential pathogens is therefore, imperative.

Ovine Fetal and Placental Lesions and Cellular Tropism in Natural Rift Valley Fever Virus Infections.

Infection with Rift Valley fever phlebovirus (RVFV) causes abortion storms and a wide variety of outcomes for both ewes and fetuses. Sheep fetuses and placenta specimens were examined during the 2010-2011 River Valley fever (RVF) outbreak in South Africa. A total of 72 fetuses were studied of which 58 were confirmed positive for RVF. Placenta specimens were available for 35 cases. Macroscopic lesions in fetuses were nonspecific and included marked edema and occasional hemorrhages in visceral organs. Microscopically, multifocal hepatic necrosis was present in 48 of 58 cases, and apoptotic bodies, foci of liquefactive hepatic necrosis (primary foci), and eosinophilic intranuclear inclusions in hepatocytes were useful diagnostic features. Lymphocytolysis was present in all lymphoid organs examined with the exception of thymus and Peyer's patches, and pyknosis or karyorrhexis was often present in renal glomeruli. The most significant histologic lesion in the placenta was necrosis of trophoblasts and endothelial cells in the cotyledonary and intercotyledonary chorioallantois. Immunolabeling for RVFV was most consistent in trophoblasts of the cotyledon or caruncle. Other antigen-positive cells included hepatocytes, renal tubular epithelial, juxtaglomerular and extraglomerular mesangial cells, vascular smooth muscle, endothelial and adrenocortical cells, cardiomyocytes, Purkinje fibers, and macrophages. Fetal organ samples for diagnosis must minimally include liver, kidney, and spleen. From the placenta, the minimum recommended specimens for histopathology include the cotyledonary units and caruncles from the endometrium, if available. The diagnostic investigation of abortion in endemic areas should always include routine testing for RVFV, and a diagnosis during interepidemic periods might be missed if only limited specimens are available for examination.

NSs amyloid formation is associated with the virulence of Rift Valley fever virus in mice.

Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer's disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.

Selected wetland soil properties correlate to Rift Valley fever livestock mortalities reported in 2009-10 in central South Africa.

Outbreaks of Rift Valley fever have devastating impacts on ruminants, humans, as well as on regional and national economies. Although numerous studies on the impact and outbreak of Rift Valley fever exist, relatively little is known about the role of environmental factors, especially soil, on the aestivation of the virus. This study thus selected 22 sites for study in central South Africa, known to be the recurrent epicenter of widespread Rift Valley fever outbreaks in Southern Africa. Soils were described, sampled and analyzed in detail at each site. Of all the soil variables analyzed for, only eight (cation exchange capacity, exchangeable Ca2+, exchangeable K+, exchangeable Mg2+, soluble Ca2+, medium sand, As, and Br) were statistically identified to be potential indicators of sites with reported Rift Valley fever mortalities, as reported for the 2009-2010 Rift Valley fever outbreak. Four soil characteristics (exchangeable K+, exchangeable Mg2+, medium sand, and Br) were subsequently included in a discriminant function that could potentially be used to predict sites that had reported Rift Valley fever-associated mortalities in livestock. This study therefore constitutes an initial attempt to predict sites prone to Rift Valley fever livestock mortality from soil properties and thus serves as a basis for broader research on the interaction between soil, mosquitoes and Rift Valley fever virus. Future research should include other environmental components such as vegetation, climate, and water properties as well as correlating soil properties with floodwater Aedes spp. abundance and Rift Valley fever virus prevalence.

Genetic dissection of Rift Valley fever pathogenesis: Rvfs2 locus on mouse chromosome 11 enables survival to early-onset hepatitis.

Infection of mice with Rift Valley fever virus (RVFV) reproduces major pathological features of severe human disease, notably the early-onset hepatitis and delayed-onset encephalitis. We previously reported that the Rvfs2 locus from the susceptible MBT/Pas strain reduces survival time after RVFV infection. Here, we used BALB/cByJ (BALB) mice congenic for Rvfs2 (C.MBT-Rvfs2) to investigate the pathophysiological mechanisms impacted by Rvfs2. Clinical, biochemical and histopathological features indicated similar liver damage in BALB and C.MBT-Rvfs2 mice until day 5 after infection. However, while C.MBT-Rvfs2 mice succumbed from acute liver injury, most BALB mice recovered and died later of encephalitis. Hepatocytes of BALB infected liver proliferated actively on day 6, promoting organ regeneration and recovery from liver damage. By comparison with C.MBT-Rvfs2, BALB mice had up to 100-fold lower production of infectious virions in the peripheral blood and liver, strongly decreased RVFV protein in liver and reduced viral replication in primary cultured hepatocytes, suggesting that the BALB Rvfs2 haplotype limits RVFV pathogenicity through decreased virus replication. Moreover, bone marrow chimera experiments showed that both hematopoietic and non-hematopoietic cells are required for the protective effect of the BALB Rvfs2 haplotype. Altogether, these results indicate that Rvfs2 controls critical events which allow survival to RVFV-induced hepatitis.

NSs Filament Formation Is Important but Not Sufficient for RVFV Virulence In Vivo.

Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that represents as a serious health threat to both domestic animals and humans. The viral protein NSs is the key virulence factor of RVFV, and has been proposed that NSs nuclear filament formation is critical for its virulence. However, the detailed mechanisms are currently unclear. Here, we generated a T7 RNA polymerase-driven RVFV reverse genetics system based on a strain imported into China (BJ01). Several NSs mutations (T1, T3 and T4) were introduced into the system for investigating the correlation between NSs filament formation and virulence in vivo. The NSs T1 mutant showed distinct NSs filament in the nuclei of infected cells, the T3 mutant diffusively localized in the cytoplasm and the T4 mutant showed fragmented nuclear filament formation. Infection of BALB/c mice with these NSs mutant viruses revealed that the in vivo virulence was severely compromised for all three NSs mutants, including the T1 mutant. This suggests that NSs filament formation is not directly correlated with RVFV virulence in vivo. Results from this study not only shed new light on the virulence mechanism of RVFV NSs but also provided tools for future in-depth investigations of RVFV pathogenesis and anti-RVFV drug screening.

The Role of Reference Materials in the Research and Development of Diagnostic Tools and Treatments for Haemorrhagic Fever Viruses.

Following the Ebola outbreak in Western Africa in 2013-16, a global effort has taken place for preparedness for future outbreaks. As part of this response, the development of vaccines, treatments and diagnostic tools has been accelerated, especially towards pathogens listed as likely to cause an epidemic and for which there are no current treatments. Several of the priority pathogens identified by the World Health Organisation are haemorrhagic fever viruses. This review provides information on the role of reference materials as an enabling tool for the development and evaluation of assays, and ultimately vaccines and treatments. The types of standards available are described, along with how they can be applied for assay harmonisation through calibration as a relative potency to a common arbitrary unitage system (WHO International Unit). This assures that assay metrology is accurate and robust. We describe reference materials that have been or are being developed for haemorrhagic fever viruses and consider the issues surrounding their production, particularly that of biosafety where the viruses require specialised containment facilities. Finally, we advocate the use of reference materials at early stages, including research and development, as this helps produce reliable assays and can smooth the path to regulatory approval.

Chimaeric Rift Valley Fever Virus-Like Particle Vaccine Candidate Production in Nicotiana benthamiana.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live-attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus-like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail-encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg-1 fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne-HA particles of 49-60 nm which are immunogenic, eliciting Gn-specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne-HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants.