The nonstructural protein NSs induces a variable antibody response in domestic ruminants naturally infected with Rift Valley fever virus.

Rift Valley fever (RVF) is an emerging zoonosis in Africa which has spread to Egypt, the Arabian Peninsula, Madagascar, and Comoros. RVF virus (RVFV) (Bunyaviridae family, Phlebovirus genus) causes a wide range of symptoms in humans, from benign fever to fatal hemorrhagic fever. Ruminants are severely affected by the disease, which leads to a high rate of mortality in young animals and to abortions and teratogenesis in pregnant females. Diagnostic tests include virus isolation and genome or antibody detection. During RVFV infection, the nucleoprotein encapsidating the tripartite RNA genome is expressed in large amounts and raises a robust antibody response, while the envelope glycoproteins elicit neutralizing antibodies which play a major role in protection. Much less is known about the antigenicity/immunogenicity of the nonstructural protein NSs, which is a major virulence factor. Here we have developed a competitive enzyme-linked immunosorbent assay (ELISA) enabling detection of low levels of NSs-specific antibodies in naturally infected or vaccinated ruminants. Detection of the NSs antibodies was validated by Western blotting. Altogether, our data showed that the NSs antibodies were detected in only 55% of animals naturally infected by RVFV, indicating that NSs does not induce a consistently high immune response. These results are discussed in light of differentiation between infected and vaccinated animals (DIVA) tests distinguishing naturally infected animals and those vaccinated with NSs-defective vaccines.

Tissue tropism and target cells of NSs-deleted rift valley fever virus in live immunodeficient mice.

BACKGROUND: Rift Valley fever virus (RVFV) causes disease in livestock and humans. It can be transmitted by mosquitoes, inhalation or physical contact with the body fluids of infected animals. Severe clinical cases are characterized by acute hepatitis with hemorrhage, meningoencephalitis and/or retinitis. The dynamics of RVFV infection and the cell types infected in vivo are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: RVFV strains expressing humanized Renilla luciferase (hRLuc) or green fluorescent protein (GFP) were generated and inoculated to susceptible Ifnar1-deficient mice. We investigated the tissue tropism in these mice and the nature of the target cells in vivo using whole-organ imaging and flow cytometry. After intraperitoneal inoculation, hRLuc signal was observed primarily in the thymus, spleen and liver. Macrophages infiltrating various tissues, in particular the adipose tissue surrounding the pancreas also expressed the virus. The liver rapidly turned into the major luminescent organ and the mice succumbed to severe hepatitis. The brain remained weakly luminescent throughout infection. FACS analysis in RVFV-GFP-infected mice showed that the macrophages, dendritic cells and granulocytes were main target cells for RVFV. The crucial role of cells of the monocyte/macrophage/dendritic lineage during RVFV infection was confirmed by the slower viral dissemination, decrease in RVFV titers in blood, and prolonged survival of macrophage- and dendritic cell-depleted mice following treatment with clodronate liposomes. Upon dermal and nasal inoculations, the viral dissemination was primarily observed in the lymph node draining the injected ear and in the lungs respectively, with a significant increase in survival time. CONCLUSIONS/SIGNIFICANCE: These findings reveal the high levels of phagocytic cells harboring RVFV during viral infection in Ifnar1-deficient mice. They demonstrate that bioluminescent and fluorescent viruses can shed new light into the pathogenesis of RVFV infection.

Characterization of wild-type and alternate transcription termination signals in the Rift Valley fever virus genome.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a phlebovirus of the family Bunyaviridae, which affects humans and ruminants in Africa and the Middle East. RFV virus (RVFV) possesses a single-stranded tripartite RNA genome of negative/ambisense polarity. The S segment utilizes the ambisense strategy and codes for two proteins, the N nucleoprotein and the nonstructural NSs protein, in opposite orientations. The two open reading frames (ORFs) are separated by an intergenic region (IGR) highly conserved among strains and containing a motif, 5'-GCUGC-3', present on the genome and antigenome, which was shown previously to play a role in transcription termination (C. G. Albarino, B. H. Bird, and S. T. Nichol, J. Virol. 81:5246-5256, 2007; T. Ikegami, S. Won, C. J. Peters, and S. Makino, J. Virol. 81:8421-8438, 2007). Here, we created recombinant RVFVs with mutations or deletions in the IGR and showed that the substitution of the motif sequence by a series of five A's inactivated transcription termination at the wild-type site but allowed the transcriptase to recognize another site with the consensus sequence present in the opposite ORF. Similar situations were observed for mutants in which the motif was still present in the IGR but located close to the stop codon of the translated ORF, supporting a model in which transcription is coupled to translation and translocating ribosomes abrogate transcription termination. Our data also showed that the signal tolerated some sequence variations, since mutation into 5'-GCAGC-3' was functional, and 5'-GUAGC-3' is likely the signal for the termination of the 3' end of the L mRNA.

A new mouse model reveals a critical role for host innate immunity in resistance to Rift Valley fever.

Rift Valley fever (RVF) is an arthropod-borne viral disease repeatedly reported in many African countries and, more recently, in Saudi Arabia and Yemen. RVF virus (RVFV) primarily infects domesticated ruminants, resulting in miscarriage in pregnant females and death for newborns and young animals. It also has the ability to infect humans, causing a feverish syndrome, meningoencephalitis, or hemorrhagic fever. The various outcomes of RVFV infection in animals and humans argue for the existence of host genetic determinants controlling the disease. We investigated the susceptibility of inbred mouse strains to infection with the virulent RVFV ZH548 strain. Compared with classical BALB/cByJ mice, wild-derived Mus m. musculus MBT/Pas mice exhibited earlier and greater viremia and died sooner, a result in sharp contrast with their resistance to infection with West Nile virus and influenza A. Infection of mouse embryonic fibroblasts (MEFs) from MBT/Pas mice with RVFV also resulted in higher viral production. Microarray and quantitative RT-PCR experiments showed that BALB/cByJ MEFs displayed a significant activation of the type I IFN pathway. In contrast, MBT/Pas MEFs elicited a delayed and partial type I IFN response to RVFV infection. RNA interference-mediated inhibition of genes that were not induced by RVFV in MBT/Pas MEFs increased viral production in BALB/cByJ MEFs, thus demonstrating their functional importance in limiting viral replication. We conclude that the failure of MBT/Pas murine strain to induce, in due course, a complete innate immune response is instrumental in the selective susceptibility to RVF.

Evaluation of a commercial competitive ELISA for the detection of antibodies to Rift Valley fever virus in sera of domestic ruminants in France.

This paper describes the sensitivity and specificity of a commercial competitive ELISA for the detection of antibodies to Rift Valley fever virus (RVFV) from sera of French domestic ruminants. Field samples were collected from mainland France for the known-negative sera (cattle=191, goats=119, sheep=192) and from ruminants of a French overseas territory (Mayotte) for the known-positive sera. A cut-off value of 43% was determined for all species, achieving a sensitivity and specificity of 100% and a concordance of 100% with the species-specific threshold recommended by the manufacturer. Our results demonstrate that this ELISA may be a suitable diagnostic tool for disease surveillance programmes and import/export veterinary certification of French cattle, goats and sheep.

RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses.

Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.

Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs.

DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR reaction, DNA is obtained in which the natural hydrogen bonding rule is inversed. When PCR is performed at limiting denaturation temperatures, it is possible to recover GC-rich viral genomes and inverted Alu elements embedded in cellular mRNAs resulting from editing by dsRNA dependent host cell adenosine deaminases. The editing of Alu elements in cellular mRNAs was strongly enhanced by type I interferon induction indicating a novel link mRNA metabolism and innate immunity.

A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells.

Rift Valley fever virus (RVFV) nonstructural protein NSs acts as the major determinant of virulence by antagonizing interferon beta (IFN-beta) gene expression. We demonstrate here that NSs interacts with the host protein SAP30, which belongs to Sin3A/NCoR/HDACs repressor complexes and interacts with the transcription factor YY1 that regulates IFN-beta gene expression. Using confocal microscopy and chromatin immunoprecipitation, we show that SAP30, YY1, and Sin3A-associated corepressor factors strongly colocalize with nuclear NSs filaments and that NSs, SAP30 and Sin3A-associated factors are recruited on the IFN-beta promoter through YY1, inhibiting CBP recruitment, histone acetylation, and transcriptional activation. To ascertain the role of SAP30, we produced, by reverse genetics, a recombinant RVFV in which the interacting domain in NSs was deleted. The virus was unable to inhibit the IFN response and was avirulent for mice. We discuss here the strategy developed by the highly pathogenic RVFV to evade the host antiviral response, affecting nuclear organization and IFN-beta promoter chromatin structure.

Viral suppressors of RNA interference impair RNA silencing induced by a Semliki Forest virus replicon in tick cells.

It was recently shown that infection of ISE6 tick cells by a recombinant Semliki Forest virus (SFV) expressing a heterologous gene induced small interfering RNAs (siRNAs) and silencing of the gene. To gain information on RNA interference (RNAi) in ticks, three known viral inhibitors that act in different ways, the NS1 protein of Influenza virus, NSs of Tospovirus Tomato spotted wilt virus and HC-Pro of Zucchini yellow mosaic virus were expressed and investigated to determine if they antagonize induced RNAi. Using the recombinant SFV replicon expressing firefly luciferase, silencing was induced and the suppressor activity of these inhibitors during or after initiation of siRNA synthesis was tested, to determine which step of the RNAi pathway is impaired. It was found that these proteins, identified in mammalian or plant systems, also display activity in tick cells. These data suggest that ticks utilize a mechanism similar to the one found in other eukaryotes.

Rift Valley fever virus noncoding regions of L, M and S segments regulate RNA synthesis.

Rift Valley fever virus (RVFV) (Phlebovirus, Bunyaviridae) possesses a genome composed of three negative-stranded RNA molecules. Each segment contains 3' and 5' noncoding regions with terminal complementary sequences forming a panhandle structure. We showed that transcription-replication of the L, M and S segments is regulated, and we established a minigenome rescue system expressing a CAT reporter to investigate the role of the noncoding regions in this process. The L, M and S segment-based minigenomes were shown to drive bona fide transcription and replication and to express variable levels of CAT reporter, indicating differential promoter activities within the noncoding sequences. In addition, we found a good correlation between the relative promoter strength and the abundance of viral RNA species in RVFV-infected cells. Altogether, these results show that RVFV minigenomes are powerful tools to study transcription and replication and constitute a valuable basis to rescue infectious virus from cDNAs.

The N terminus of Rift Valley fever virus nucleoprotein is essential for dimerization.

Rift Valley fever virus (RVFV) is a Phlebovirus in the Bunyaviridae family. The nucleoprotein N is the most abundant component of the virion; numerous copies of N associate with the viral RNA genome and form pseudohelicoidal ribonucleoproteins (RNPs) circularized by a panhandle structure formed by the base-paired RNA sequences at the 3' and 5' termini. These structures play a central role in transcription and replication. We investigated the intermolecular interactions of the RVFV N protein and found that after chemical cross-linking treatment, the nucleoprotein from purified RNPs migrates mainly as dimers. The N-N interaction was studied using the yeast two-hybrid system, the GST pull-down method, and mutational analysis. We demonstrated that the N terminus from residue 1 to 71, and particularly Tyr 4 and Phe 11, which are conserved among phlebovirus N sequences, are involved in the interaction. The C-terminal region did not seem to be essential for the N-N interaction. Moreover, we showed that N(TOS), the N protein of the related Toscana phlebovirus, interacts with itself and forms heterodimers with N(RVF), suggesting that the dimeric form of N may be a conserved feature in phlebovirus RNPs.

Nairovirus RNA sequences expressed by a Semliki Forest virus replicon induce RNA interference in tick cells.

We report the successful infection of the cell line ISE6 derived from Ixodes scapularis tick embryos by the tick-borne Hazara virus (HAZV), a nairovirus in the family Bunyaviridae. Using a recombinant Semliki Forest alphavirus replicon that replicates in these cells, we were able to inhibit replication of HAZV, and we showed that this blockage is mediated by the replication of the Semliki Forest alphavirus replicon; the vector containing the HAZV nucleoprotein gene in sense or antisense orientation efficiently inhibited HAZV replication. Moreover, expression of a distantly related nucleoprotein gene from Crimean-Congo hemorrhagic fever nairovirus failed to induce HAZV silencing, indicating that the inhibition is sequence specific. The resistance of these cells to replicate HAZV correlated with the detection of specific RNase activity and 21- to 24-nucleotide-long small interfering RNAs. Altogether, these results strongly suggest that pathogen-derived resistance can be established in the tick cells via a mechanism of RNA interference.

NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription.

Rift Valley fever virus (RVFV) is an important cause of epizootics and epidemics in Africa and a potential agent of bioterrorism. A better understanding of the factors that govern RVFV virulence and pathogenicity is required, given the urgent need for antiviral therapies and safe vaccines. We have previously shown that RVFV strains with mutations in the NSs gene are excellent inducers of alpha/beta interferon (IFN-alpha/beta) and are highly attenuated in mice. Here, we demonstrate that NSs is sufficient to block IFN-beta gene expression at the transcriptional level. In cells transiently expressing NSs, IFN-beta transcripts were not inducible by viral infection or by transfection of poly(I:C). NSs with anti-IFN activity accumulated in the nucleus. In contrast, mutant forms of NSs that had lost their IFN-inhibiting activity remained in the cytoplasm, indicating that nuclear localization plays a role. IFN synthesis is regulated by specific transcription factors, including interferon regulatory factor (IRF-3), NF-kappaB, and AP-1. In the presence of NSs, IRF-3 was still activated and moved to the nucleus. Likewise, NF-kappaB and AP-1 were activated normally, as shown in electrophoretic mobility shift assays. Moreover, NSs was found to inhibit transcriptional activity of a constitutive promoter, in agreement with recent findings showing that NSs targets the basal cellular transcription factor TFIIH. The present results suggest that NSs, unlike other viral IFN antagonists, does not inhibit IFN-specific transcription factors but blocks IFN gene expression at a subsequent step.

TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus.

The Rift Valley fever virus (RVFV) is the causative agent of fatal hemorrhagic fever in humans and acute hepatitis in ruminants. We found that infection by RVFV leads to a rapid and drastic suppression of host cellular RNA synthesis that parallels a decrease of the TFIIH transcription factor cellular concentration. Using yeast two hybrid system, recombinant technology, and confocal microscopy, we further demonstrated that the nonstructural viral NSs protein interacts with the p44 component of TFIIH to form nuclear filamentous structures that also contain XPB subunit of TFIIH. By competing with XPD, the natural partner of p44 within TFIIH, and sequestering p44 and XPB subunits, NSs prevents the assembly of TFIIH subunits, thus destabilizing the normal host cell life. These observations shed light on the mechanism utilized by RVFV to evade the host response.