Rift Valley Fever Virus Pathogenesis Research Articles

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

ABSTRACT 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.

Limiting viral replication in hepatocytes alters Rift Valley fever virus disease manifestations.

Rift Valley fever virus (RVFV) causes mild to severe disease in humans and livestock. Outbreaks of RVFV have been reported throughout Africa and have spread outside Africa since 2000, calling for urgent worldwide attention to this emerging virus. RVFV directly infects the liver, and elevated transaminases are a hallmark of severe RVFV infection. However, the specific contribution of viral replication in hepatocytes to pathogenesis of RVFV remains undefined. To address this, we generated a recombinant miRNA-targeted virus, RVFVmiR-122, to limit hepatocellular replication. MicroRNAs are evolutionarily conserved non-coding RNAs that regulate mRNA expression by targeting them for degradation. RVFVmiR-122 includes an insertion of four target sequences of the liver-specific miR-122. In contrast to control RVFVmiR-184, which contains four target sequences of mosquito-specific miR-184, RVFVmiR-122 has restricted replication in vitro in primary mouse hepatocytes. RVFVmiR-122-infected C57BL/6 mice survived acute hepatitis and instead developed late-onset encephalitis. This difference in clinical outcome was eliminated in Mir-122 KO mice, confirming the specificity of the finding. Interestingly, C57BL/6 mice infected with higher doses of RVFVmiR-122 had a higher survival rate which was correlated with faster clearance of virus from the liver, suggesting a role for activation of host immunity in the phenotype. Together, our data demonstrate that miR-122 can specifically restrict the replication of RVFVmiR-122 in liver tissue both in vitro and in vivo, and this restriction alters the clinical course of disease following RVFVmiR-122 infection. IMPORTANCE Rift Valley fever virus (RVFV) is a hemorrhagic fever virus that causes outbreaks in humans and livestock throughout Africa and has spread to continents outside Africa since 2000. However, no commercial vaccine or treatment is currently available for human use against RVFV. Although the liver has been demonstrated as a key target of RVFV, the contribution of viral replication in hepatocytes to overall RVFV pathogenesis is less well defined. In this study we addressed this question by using a recombinant miRNA-targeted virus with restricted replication in hepatocytes. We gained a better understanding of how this individual cell type contributes to the development of disease caused by RVFV. Techniques used in this study provide an innovative tool to the RVFV field that could be applied to study the consequences of limited RVFV replication in other target cells.

Discovery of potential immune epitopes and peptide vaccine design - a prophylactic strategy against Rift Valley fever virus

Background: Rift Valley fever virus (RVFV) is an emerging arbovirus infecting both animals and humans. Any form of direct contact with body fluids, blood or tissue of infected animals is the mode of transmission of this pathogen. Despite being an emerging virus, no proper vaccinations are yet available for the public. Our objective is to compose a multiepitope vaccine utilizing immuno-bioinformatics as a strategy against RVFV. Methods: To identify immunodominant epitopes and design a potent vaccine candidate, we applied a series of immunoinformatic approaches with molecular dynamics and immune response simulation frameworks. Results: A glycoprotein with the highest antigenicity was selected and employed for determining promising epitopes. We selected T cell epitopes based on their immunological potencies and cytokine inducing properties, while B cell epitopes were selected based on their antigenic features. Finally, we selected four cytotoxic T-lymphocyte, two helper T-lymphocyte, and three linear B-lymphocyte epitopes that were arranged into a vaccine construct with appropriate adjuvants and linkers. The chimera protein was modeled, refined, and validated prior to docking against toll-like receptor 4. Docking studies suggest strong binding interactions while dynamics simulation revealed the stable nature of the docked complex. Furthermore, the immune simulation showed robust and prolonged immune responses with rapid antigen clearance. Finally, codon optimization and cloning conducted with Escherichia coli K12 suggests high translation efficiency within the host system. Conclusion: We believe that our designed multiepitope vaccine is a promising prophylactic candidate against RVFV pathogenesis.

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.

Transcriptome profiling in Rift Valley fever virus infected cells reveals modified transcriptional and alternative splicing programs.

Rift Valley fever virus (RVFV) is a negative-sense RNA virus belonging to the Phenuiviridae family that infects both domestic livestock and humans. The NIAID has designated RVFV as a Category A priority emerging pathogen due to the devastating public health outcomes associated with epidemic outbreaks. However, there is no licensed treatment or vaccine approved for human use. Therefore it is of great interest to understand RVFV pathogenesis in infected hosts in order to facilitate creation of targeted therapies and treatment options. Here we provide insight into the host-pathogen interface in human HEK293 cells during RVFV MP-12 strain infection using high-throughput mRNA sequencing technology. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes showed robust innate immune and cytokine-mediated inflammatory pathway activation as well as alterations in pathways associated with fatty acid metabolism and extracellular matrix receptor signaling. We also analyzed the promoter regions of DEGs for patterns in transcription factor binding sites, and found several that are known to act synergistically to impact apoptosis, immunity, metabolism, and cell growth and differentiation. Lastly, we noted dramatic changes in host alternative splicing patterns in genes associated with mRNA decay and surveillance, RNA transport, and DNA repair. This study has improved our understanding of RVFV pathogenesis and has provided novel insight into pathways and signaling modules important for RVFV diagnostics and therapeutic development.

-Microglia mount a robust immune response to Rift Valley Fever virus that is dependent on MAVS and independent of TLR7

Abstract The mosquito-borne Rift Valley Fever virus (RVFV) is a category A biodefense pathogen due to the potential for intentional spread by aerosol and the lack of any licensed vaccine or antiviral therapeutics. Animal studies and limited data from human infection have demonstrated that RVFV pathogenesis following aerosol exposure differs from subcutaneous exposure, and is characterized by severe encephalitis, high viral titers in the brains, and long lasting neural pathology in human survivors of infection. Despite the brain being a major target of viral replication and tissue damage, little is known about innate immune responses to RVFV in the brain following aerosol infection. Thus, we are investigating the responses of the resident innate immune cells of the brain microglia and astrocytes to RVFV infection. Microglia mount a robust response to RVFV infection evidenced by upregulation of immunoregulatory genes, secretion of cytokines and upregulation of surface markers of activation. The response of microglia to RVFV is dependent upon MAVS and independent of TLR7, with cells derived from MAVS knockout animals unresponsive to infection while cells derived from TLR7 knock out animals responded similarly to infection as wild type cells. Experiments to determine the in vivo innate immune response to RVFV in the brain are ongoing. Understanding the in vivo innate immune response in the brain will be will be critical to the development of vaccines and antiviral therapeutics that are successful in preventing viral encephalitis caused by RVFV.

Combination Kinase Inhibitor Treatment Suppresses Rift Valley Fever Virus Replication.

Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection.

Rapamycin modulation of p70 S6 kinase signaling inhibits Rift Valley fever virus pathogenesis

Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.

Reverse Transcriptase Real Time PCR Detection of Rift Valley Fever Virus RNA in Formalin‐Fixed, Paraffin‐Embedded Tissues

Rift Valley Fever Virus (RVFV) is a mosquito‐borne zoonotic pathogen endemic in parts of Africa and the Arabian Peninsula. RVFV causes acute febrile disease with mass abortion and death in ruminants and occasional encephalitis and death in humans. In the US, RVFV is a BSL‐3Ag Select Agent and a foreign animal disease, requiring special containment laboratories for research work. Research goals include the study of RVFV pathogenesis and the development of novel diagnostic tools and disease prevention methods such as vaccines. Ongoing surveillance plays a critical role in prevention and control of outbreaks and virus spread. Formalin‐fixed, paraffin‐embedded tissues (FFPET) are non‐infectious diagnostic specimens, routinely used for histopathology and immunohistochemistry (IHC), however use of this diagnostic specimen for molecular detection of pathogen RNA is underinvestigated. The objective of this work is to develop and validate a protocol for use of FFPET for the sensitive and specific detection of RVFV RNA.Study samples were fresh‐frozen tissues (FFT) and FFPET (liver, spleen, lung, kidney) collected from sheep and calves experimentally infected with two RVFV strains (Kenya 2006 and Saudi Arabia 2001). Deparaffinization of paraffin whorls (5‐5μm thick), tissue lysis with proteinase K, and reversal of nucleic acid cross linking in heated alkaline buffer was performed in a single tube using commercial buffers (Qiagen). RNA was purified using automated magnetic bead extraction on the Taco mini (GeneReach) for FFPET or the Applied Biosystems reagents on the Kingfisher (FFT). Multiplex Reverse Transcriptase Real Time PCR (RT‐qPCR) for RVFV L, M and S gene segments was used to detect RVFV RNA using Quanta qScript XLT One‐Step RT‐qPCR ToughMix (FFPET) or AgPath RT‐qPCR mix (FFT). IHC for RVFV antigen was done with a mouse monoclonal anti‐RVFV nucleoprotein antibody and avidin‐biotin complex detection.To date, 18 FFPET samples have been tested. For samples that were both IHC and FFT RT‐qPCR positive (n=13), FFPET results were 100% positive. For FFT samples (n=5) that were not tested by RT‐qPCR but were IHC positive, the FFPET RVFV RT‐qPCR results were also positive. Testing of an additional 46 FFPE samples is presently underway.Using this newly developed single tube FFPET technique for nucleic acid recovery, followed by a rapid, low cost magnetic bead nucleic acid extraction allows for highly sensitive detection of RNA specific for RVFV. This novel method could strengthen routine diagnostics, surveillance and molecular epidemiological studies that are currently limited by the strict biosecurity requirements for RVFV samples. Moreover, this methodology could also be expanded for detection of RNA targets for additional high impact pathogens or for transcriptional studies that must relie on archival FFPET.Support or Funding InformationThis work was funded by startup funds provided to Dr. Davis by the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University and uses archival animal study materials from research funded by grants from the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), Grant No. 2010‐ST061‐AG0001, USDA Agricultural Services project 3020‐32000‐005‐00D and the Kansas Bioscience Authority.

143 Rift Valley fever virus

The capacity of Rift Valley fever virus (RVFV) to spread into new territories by crossing significant natural geographic barriers, re-emerge in endemic regions after long periods of silence to cause large outbreaks in human and animal populations constitute a formidable challenge for public and veterinary health authorities as well as for scientific communities worldwide. In spite of recent advances in research on RVFV pathogenesis, molecular epidemiology, outbreak prediction, development of new diagnostic tools and vaccines, some fundamental aspects of the epidemiology and ecology of the virus remain elusive. Large outbreaks of RVF are associated with anomalous high rainfalls leading to massive flooding and the resultant swarms of competent mosquito vectors transmitting the virus to a wide range of susceptible vertebrate species. However, the exact mechanism of RVFV natural transmission during interepizootic periods remains largely unknown, including the postulated long-term virus persistence in transovarially infected eggs of floodwater Aedes mosquito species, and the role of wild mammals as reservoirs. The presence of competent mosquito vectors in countries free of RVF, the wide range of mammals susceptible to the virus, the global changes in climate, and increased animal trade and travel are some of the factors which might contribute to international spread. This presentation provides the background to the major outbreaks, molecular biology and epidemiology of RVFV, and overviews aspects of ecology, host and vector range which make the virus a potential global emerging threat.

Chemotactic and inflammatory responses in the liver and brain are associated with pathogenesis of Rift Valley fever virus infection in the mouse.

Rift Valley fever virus (RVFV) is a major human and animal pathogen associated with severe disease including hemorrhagic fever or encephalitis. RVFV is endemic to parts of Africa and the Arabian Peninsula, but there is significant concern regarding its introduction into non-endemic regions and the potentially devastating effect to livestock populations with concurrent infections of humans. To date, there is little detailed data directly comparing the host response to infection with wild-type or vaccine strains of RVFV and correlation with viral pathogenesis. Here we characterized clinical and systemic immune responses to infection with wild-type strain ZH501 or IND vaccine strain MP-12 in the C57BL/6 mouse. Animals infected with live-attenuated MP-12 survived productive viral infection with little evidence of clinical disease and minimal cytokine response in evaluated tissues. In contrast, ZH501 infection was lethal, caused depletion of lymphocytes and platelets and elicited a strong, systemic cytokine response which correlated with high virus titers and significant tissue pathology. Lymphopenia and platelet depletion were indicators of disease onset with indications of lymphocyte recovery correlating with increases in G-CSF production. RVFV is hepatotropic and in these studies significant clinical and histological data supported these findings; however, significant evidence of a pro-inflammatory response in the liver was not apparent. Rather, viral infection resulted in a chemokine response indicating infiltration of immunoreactive cells, such as neutrophils, which was supported by histological data. In brains of ZH501 infected mice, a significant chemokine and pro-inflammatory cytokine response was evident, but with little pathology indicating meningoencephalitis. These data suggest that RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis yet the long-term course of disease for those that might survive the initial hepatitis is neurologic in nature which is supported by observations of human disease and the BALB/c mouse model.

The Pathogenesis of Rift Valley Fever

Rift Valley fever (RVF) is an emerging zoonotic disease distributed in sub-Saharan African countries and the Arabian Peninsula. The disease is caused by the Rift Valley fever virus (RVFV) of the family Bunyaviridae and the genus Phlebovirus. The virus is transmitted by mosquitoes, and virus replication in domestic ruminant results in high rates of mortality and abortion. RVFV infection in humans usually causes a self-limiting, acute and febrile illness; however, a small number of cases progress to neurological disorders, partial or complete blindness, hemorrhagic fever, or thrombosis. This review describes the pathology of RVF in human patients and several animal models, and summarizes the role of viral virulence factors and host factors that affect RVFV pathogenesis.

Apoptosis of Hepatocytes Caused by Punta Toro Virus ( Bunyaviridae: Phlebovirus) and Its Implication for Phlebovirus Pathogenesis

Experimental infection of hamsters with Punta Toro virus (PTV) produces a disease with clinical and pathological similarities to the severe human hemorrhagic fever caused by Rift Valley fever virus (RVFV), thus providing an animal model for RVFV pathogenesis. In this model, hepatocytic apoptosis is the main pathological component of liver injuries that are responsible for severe hemorrhagic manifestations. To further elucidate whether viral replication in hepatocytes directly causes apoptosis, we studied the morphological and biochemical changes of apoptosis in HepG2 cells at different time points after PTV infection. Cellular viability began to decrease 12 hours after infection compared with controls. Caspases 3/7 were activated significantly at 48 and 72 hours after infection, and phosphatidylserine translocation and DNA fragmentation were also detected at 48 and 72 hours. Cell cycle analysis by flow cytometry showed that infected HepG2 cells were arrested at G(0)/G(1) phase. Furthermore, virus titer increased with apoptosis progression, suggesting that viral replication is necessary for the apoptotic process. These results indicate that PTV infection alone, without a secondary inflammatory cellular reaction, induces hepatocytic apoptosis and suggest that future therapeutics for RVFV hemorrhagic disease might target inhibition of cellular apoptotic pathways during the acute infection.