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A New Mouse Model Reveals a Critical Role for Innate in Resistance to Rift Valley

This information is current as Tânia Zaverucha do Valle, Agnès Billecocq, Laurent of September 25, 2021. Guillemot, Rudi Alberts, Céline Gommet, Robert Geffers, Kátia Calabrese, Klaus Schughart, Michèle Bouloy, Xavier Montagutelli and Jean-Jacques Panthier J Immunol 2010; 185:6146-6156; Prepublished online 11

October 2010; Downloaded from doi: 10.4049/jimmunol.1000949 http://www.jimmunol.org/content/185/10/6146

Supplementary http://www.jimmunol.org/content/suppl/2010/10/12/jimmunol.100094 http://www.jimmunol.org/ Material 9.DC1 References This article cites 46 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/185/10/6146.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A New Mouse Model Reveals a Critical Role for Host Innate Immunity in Resistance to

Taˆnia Zaverucha do Valle,*,† Agne`s Billecocq,‡ Laurent Guillemot,*,† Rudi Alberts,x,{ Ce´line Gommet,‖ Robert Geffers,# Ka´tia Calabrese,** Klaus Schughart,x,{ Miche`le Bouloy,‡ Xavier Montagutelli,*,† and Jean-Jacques Panthier*,†

Rift Valley fever (RVF) is an arthropod-borne viral repeatedly reported in many African countries and, more recently, in Saudi Arabia and Yemen. RVF (RVFV) primarily infects domesticated ruminants, resulting in miscarriage in pregnant females and for newborns and young animals. It also has the ability to infect humans, causing a feverish , , or hemorrhagic fever. The various outcomes of RVFV 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 Downloaded from MBT/Pas mice exhibited earlier and greater and died sooner, a result in sharp contrast with their resistance to infection with 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 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 http://www.jimmunol.org/ that the failure of MBT/Pas murine strain to induce, in due course, a complete innate is instrumental in the selective susceptibility to RVF. The Journal of Immunology, 2010, 185: 6146–6156.

ift Valley fever (RVF) is an arthropod-borne disease mosquitoes of the genera Aedes and , although other arthro- caused by a virus of the Bunyaviridae family, genus pods may play a role in its spread (1). In natural ecosystems, RVF R Phlebovirus. RVF virus (RVFV) is transmitted mostly by affects mainly sheep, cattle, goats, and humans, but other , such as camels and buffaloes, may exhibit the disease. RVF out- breaks represent a threat for humans in endemic areas, where by guest on September 25, 2021 *Unite´ Ge´ne´tique Fonctionnelle de la Souris, ‡Unite´ Ge´ne´tique Mole´culaire des people may be infected by mosquitoes or by direct contact with ‖ Bunyavirus, and Animalerie Centrale, Institut Pasteur; †Unite´ de Recherche Asso- animals or possibly raw milk (1, 2). They inflict severe economic cie´e 2578, Centre National de la Recherche Scientifique, Paris, France; xDepartment of Infection Genetics, Helmholtz Centre for Infection Research, Braunschweig; losses, especially upon trade activities (1). Originally present in {University of Veterinary Medicine, Hannover; #Department of Cell Biology, Ar- Eastern and Southern Africa, RVFV has spread in recent years to ray Facility, Helmholtz Centre for Infection Research, Braunschweig, Germany Western Africa, Madagascar, and even outside Africa to Saudi **Fundac¸a˜o Oswaldo Cruz, Laborato´rio de Imunomodulac¸a˜o e Protozoologia, Rio de Janeiro, Brazil Arabia and Yemen (3). Received for publication March 25, 2010. Accepted for publication September 8, The RVFV has a tripartite ssRNA genome, consisting of large, 2010. medium, and small segments. The small segment encodes the N This work was supported in part by “ResistFever” Grant 07-GANI-007 from the nucleocapsid and the small segment nonstructural (NSs) (4), Agence Nationale de la Recherche. It was also supported by a Programme Trans- which is a main factor of virulence. Indeed, the deletion of NSs is versal de Recherche grant awarded to K.S., M.B., and J.-J.P. from the Helmholtz Centre for Infection Research and the Institut Pasteur. Part of this work was facili- responsible for RVFV avirulence in mice (5). NSs protein acts tated through the International Network for Capacity Building for the Control of through several independent mechanisms. Early postinfection (p.i.), Emerging Viral Borne Zoonotic (Arbo-Zoonet) supported by the NSs interacts with Sin3A associated protein 30, a subunit of histone European Union under Grant Agreement 211757 and the Arbo-Zoonet Coordination Action Project 211757 of the European Commission. The Unite´ Fonctionnelle de la deacetylase complex, and maintains the promoter of IFN-b (Ifnb1) Souris at the Institut Pasteur is supported by Merck Serono. T.Z.V. was awarded in a transcriptionally silent state, blocking the production of fellowships from Conselho Nacional de Desenvolvimento Cientifico e Technologico, Brazil (210215/2006-2) and the Agence Nationale de la Recherche. R.A. is supported IFN-b as early as 3–4 h p.i. (6). NSs also induces the specific de- by the Helmholtz Virtual Centre “GeNeSys–German Network for Systems Genetics.” gradation of the dsRNA-dependent protein kinase PKR/EIF2AK2 The microarray data presented in this article have been submitted to the National and facilitates efficient viral (7, 8). Moreover, NSs se- Center for Biotechnology Information’s Gene Expression Omnibus under accession questers p44 and XPB, subunits of the RNA polymerase II TFIIH number GSE18064. factor. This sequestration was correlated with the general inhibition Address correspondence and reprint requests to Prof. J.-J. Panthier and Prof. M. of cellular transcription in RVFV-infected cells, which occurs at Bouloy, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex, France. E-mail addresses: [email protected] and [email protected] late stages of infection (.8–9 h p.i) (9). The online version of this article contains supplemental material. Genetic determinants seem to play an important role in modeling Abbreviations used in this paper: ISG, IFN-stimulated gene; MEF, mouse embryonic RVF outcomes. In humans, infection may lead to a great variety of fibroblast; MOI, multiplicity of infection; NS, small segment nonstructural protein; clinical manifestations that range from a febrile influenza-like ill- p.i., postinfection; qRT-PCR, quantitative RT-PCR; RVF, Rift Valley fever; RVFV, ness to retinitis, , and with fatal hemorrhagic Rift Valley fever virus; siRNA, small interfering RNA. fever (1). Similarly, a wide variation in susceptibility to RVF is Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 observed in different animal breeds, ranging from unapparent or www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000949 The Journal of Immunology 6147 moderate febrile reactions to high , severe prostration, and Materials and Methods death in the most susceptible animals (2). Experiments with the rat Ethics statement model confirmed the existence of genetic determinants in RVF (10– All studies on animals followed the guidelines on the ethical use of animals 13). However, surprisingly, an influence of genetic factors could not from the European Communities Council Directive of November 24, 1986 be demonstrated in a large survey of 34 classical inbred laboratory (86/609/EEC). All animal experiments were approved and conducted in mouse strains (11). To further decipher the host–pathogen in- accordance with the Institut Pasteur Biosafety Committee. teraction in mice, we tested the susceptibility of additional inbred Mice, cells, and virus strains of mice by including mouse strains derived from mice of various Mus subspecies recently trapped in the wild. This collection BALB/cByJ and C57BL/6J inbred mice were purchased from Charles River of wild-derived inbred strains encompasses genetic variation accu- Laboratories (L’Arbresle, France). 129/Sv/Pas and MBT/Pas mice were ∼ bred in the Institut Pasteur facilities. Mouse embryonic fibroblasts (MEFs) mulated over 1 million years (14), offering a much larger genetic were generated from embryos at day 13.5 of gestation. Cultures were variation than classical laboratory strains. The latter originated from genotyped by PCR for sex determination using the Smcx and Smcy genes to only a small number of founder animals and shows a remarkably identify cells from male embryos (18). Only MEFs from male embryos high level of shared ancestry, largely contributed by the M. m. were used for further experiments. RVFV strain ZH548 was isolated from D domesticus a human case in (19). Strain rec-ZH NSs was produced by reverse subspecies (15, 16). The MBT/Pas inbred strain was genetics from the ZH548 genome by deleting the complete NSs gene (6). derived from M. m. musculus animals trapped in Bulgaria in 1980; RVFV strain Kenya 98 (0523) was isolated from a human case in Kenya the mouse colony was later propagated by sib-mating at the Institut (20). West Nile virus strain IS-98-ST1 was isolated from a in Pasteur (17). ; it is highly neuroinvasive in adult mice (21). A/Puerto Rico/8/34

In this article, we report that MBT/Pas mice exhibit an extreme (H1N1) PR8 FR 0807 influenza virus is a mouse-adapted strain (22, 23). Downloaded from Viral stocks were produced under 3 conditions. susceptibility to experimental infection with the virulent RVFV ZH548 and Kenya 98 strains compared with BALB/cByJ mice, Mice infection and monitoring demonstrating for the first time, to the best of our knowledge, Groups of age-matched 9–12-wk-old males were inoculated with 102 PFU natural variation in the severity of susceptibility of the host to RVF RVFV, 103 PFU West Nile virus i.p., or 2 3 103 PFU influenza A virus among inbred strains of mice. To investigate the underlying intranasally. Infected mice were monitored for mortality daily. Experi-

mechanism, we compared gene expression profiles of BALB/cByJ ments were performed in biosafety level 3 isolators. For analysis of vire- http://www.jimmunol.org/ and MBT/Pas cells following infection with RVFV. Our data show mia, blood samples were collected by retro-orbital puncture from 10 mice at days 1 and 3 p.i. Virus was titered using plaque assays on Vero cells. that MBT/Pas cells exhibit an impaired induction of type I IFN response compared with BALB/cByJ cells, although MBT/ IFN dosage Pas cells were capable of producing type I IFNs. Interestingly, this IFN-a and IFN-b were performed on BALB/cByJ and MBT/Pas poor response results from the inability of MBT/Pas cells to induce sera, 24 and 48 h p.i., using the Mouse IFN-a and -b ELISA kits, according a complete set of IFN-stimulated genes (ISGs) p.i. with RVFV. to the manufacturer’s instructions (PBL Biomedical Laboratories, by guest on September 25, 2021

FIGURE 1. Survival and virological analysis of inbred mice after viral . A, Fifteen males of the BALB/cByJ, C57BL/6J, 129/Sv/Pas, and MBT/ Pas strains were inoculated with 102 PFU of RVFV ZH548 strain i.p. and monitored for mortality daily. B, Viremia in RVFV-infected BALB/cByJ and MBT/Pas mice p.i. Eight males of the BALB/cByJ and MBT/Pas strains were inoculated with the West Nile virus IS-98-ST1 i.p. (C) or with the mouse- adapted influenza A virus PR8-Fre intranasally (D) and monitored for mortality daily. Statistical differences were evaluated using the Kaplan–Meier test for mortality and Mann–Whitney U test for viremia. pp , 0.05; ppp , 0.01; pppp , 0.001. 6148 HOST GENETICS CONTROL OF RIFT VALLEY FEVER

were carried in triplicates. After 1 h, cells were washed in PBS twice and grown in DMEM supplemented with 2% FCS. Supernatants were collected at 15 and 20 h p.i. Titration was performed by plaque assays in Vero cells. For the microarray experiments, MEFs were infected using an MOI of 5. Cell monolayers were harvested 9 h later, and total were extracted. For the quantitative RT-PCR (qRT-PCR) experiments, MEFs from three BALB/cByJ and MBT/Pas male embryos were plated at identical cell densities. Twenty-four hours later, they were infected using an MOI of 5 with RVFV strain ZH548 or rec-ZHDNSs or with sterile medium (mock infected). Cell monolayers were harvested 3, 6, and 9 h later, and total RNAs were extracted. Immunocytology For the measurement of the infectivity efficiency, BALB/cByJ and MBT/Pas MEFs were infected with ZH548 RVFV at an MOI of 0.1, 1, and 5, as determined by plaque assays on Vero cells. Nine hours p.i., cells were fixed and labeled with specific Abs against the nucleoprotein N and the non- FIGURE 2. Replication of RVFV in BALB/cByJ and MBT/Pas fibroblasts. structural protein NSs of RVFV, followed by fluorescent secondary Abs. Virus production by BALB/cByJ and MBT/Pas MEFs at 15 and 20 h p.i. with Infectivity efficiency was calculated by counting the number of RVFV ZH548 strain at MOIs of 1, 5, and 10. Statistical analyses were performed infected cells against the total number of Hoechst-stained nuclei. pp , ppp , with the Student t test on log10-transformed data. p 0.01; p 0.001. RNA extraction

Total RNAs from infected and mock-infected MEF monolayers were Downloaded from Piscataway, NJ). The kits’ lower limits of quantification were 12.5 and 15.6 extracted using TRIzol reagent (Roche, Basel Switzerland), according to a b pg/ml for IFN- and IFN- , respectively. the manufacturer’s instructions. DNA was digested by DNAse treatment Cell infection using a DNA-free kit (Ambion, Austin, TX). RNA quality was assessed by electrophoresis and OD. MEFs from BALB/cByJ and MBT/Pas male embryos were plated at Expression microarrays and data analysis identical densities in culture dishes 24 h prior to infection. To determine the efficacy of virus replication in MEFs, cells were infected using a multi- Gene-expression profiling was performed using Affymetrix GeneChip http://www.jimmunol.org/ plicity of infection (MOI) of 1, 5, or 10 for the ZH548 strain. Experiments Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA). The 430 2.0 by guest on September 25, 2021

FIGURE 3. Microarray analysis of BALB/cByJ and MBT/Pas fibroblasts at 9 h following RVFV infection. A, Total number of genes whose expression was differentially modulated in ZH548-infected BALB/cByJ and MBT/Pas MEFs compared with mock-infected cultures. Numbers for up- and downregulated genes are in the gray and black area, respectively. B, Venn diagram of the number of genes upregulated (↑)ordownregulated(↓) in BALB/cByJ and MBT/Pas MEFs and their overlap. C, Enrichment of gene functions by upregulated or downregulated genes in BALB/cByJ MEFs. D, The enrichment for gene functions in MBT/Pas cells. The Journal of Immunology 6149 chip contains .27,000 unique transcripts. Experiments were performed cByJ, C57BL/6J, and 129/Sv/Pas) and the wild-derived MBT/Pas according to the manufacturer’s protocol. Raw data files were background 2 inbred strain were infected i.p. with 10 PFU (= 10 LD50)of corrected, quantile normalized, and summarized using the robust multi- RVFVZH548strain.Mortalitywasmonitoreddailyfor2wk.In array averaging method (24) and transformed in log2 values. Principal- component analysis of all regulated genes confirmed that the expression agreement with an earlier report (11), classical inbred strains showed changes were true biological variations and were not caused by variations little phenotypic variation in susceptibility to RVF, with a mean time in experimental conditions. Differentially expressed genes were identified to death of 7.19 6 0.21, 6.06 6 0.37, and 6.06 6 0.45dforBALB/ , using dChip software (25). A false discovery rate 0.05 using 100 per- cByJ, C57BL/6J, and 129/Sv/Pas strains, respectively. C57BL/6J mutations was applied. Genes were further analyzed using the Functions and Disease tool from Ingenuity Pathways Analysis (IngenuitySystems; mice were only slightly more susceptible than BALB/cByJ mice http://www.ingenuity.com/). (p = 0.046). In contrast, wild-derived MBT/Pas mice were highly susceptible to RVFV: all MBT/Pas mice were dead as early as day 4 qRT-PCR p.i., with a mean time to death of 3.19 6 0.10 d (p , 0.001) (Fig. 1A). To test whether the earlier susceptibility of MBT/Pas mice was Equal amounts of total RNAs from infected and mock-infected MEFs 0, 3, 6, and 9 h p.i. were used in a two-step qRT-PCR. RT-PCR was performed using associated with increased viral load in peripheral tissues, BALB/ randomprimers (p[dN]6,RocheDiagnostic, Mannheim, Germany)and avian cByJ and MBT/Pas mice (n = 10) were infected i.p. with 102 PFU myeloblastosis virus reverse transcriptase (Promega, Madison, WI). Then, of RVFV ZH548 and bled at days 1 and 3 p.i. Infectious RVFV was quantitative PCR was done using SYBR Green Master Mix (Applied Bio- detected in sera of BALB/cByJ mice starting from day 3 p.i. In systems, Warrington, U.K.) and previously described specific primers (Primer bank, http://pga.mgh.harvard.edu/primerbank/). To avoid in- contrast, RVFV was already detected from day 1 onward in sera of terference due to polymorphisms between the sequences of the BALB/cByJ all MBT/Pas mice. In addition, at day3p.i.,thelevelsofRVFVin and MBT/Pas genomes, the hybridization site of each primer was sequenced the sera of MBT/Pas mice were more than three orders of magnitude Downloaded from in the BALB/cByJ and MBT/Pas genomic DNA. When polymorphisms higher than the levels detected in BALB/cByJ mice (107.7 6 0.3 and were identified, the corresponding primers were substituted with a new pair. 104.2 6 0.5 PFU, respectively; p =0.010;Fig.1B). Thus, MBT/Pas Supplemental Table I shows the list of the primers used. Data were analyzed by the 22DDCT method, whereby changes in expression of target genes are mice produced virus earlier, and their viral loads were higher calculated relative to an internal control gene (26). Five internal control compared with BALB/cByJ mice. We also challenged BALB/cByJ genes, Gapdh, Rpl7, Rrm2, Tbp, and Tubb5, were tested in mock- and RVFV-infected MEFs from both backgrounds at each time point p.i. Ex- pression of the Tbp gene was similar in BALB/cByJ and MBT/Pas MEFs http://www.jimmunol.org/ and was unaffected by infection with RVFV until 9 h p.i. Hence, Tbp was selected as internal control gene for the quantitative PCR experiments.

RNA interference experiments The sequences of the three individual stealth RNAi small interfering RNA (siRNA; Invitrogen, Calrsbad, CA) used to target Irf7, Isg15, Oasl2, and Rig-I are presented in Supplemental Table II. Briefly, BALB/cByJ MEFs (2 3 105 cells) were plated onto 35-mm plates. Transfections were carried out according to the manufacturer’s instructions using a final concentration by guest on September 25, 2021 of 10 nM siRNA and 1.7 ml/ml Lipofectamine RNAiMAX (Invitrogen). Twenty-four hours after siRNA transfection, the MEFs were infected with RVFV strain rec-ZHDNSs at an MOI of 5 or were treated with 100 IU/ml recombinant mouse IFN-aA/IFN-a3 (PBL Biomedical Laboratories, Pis- cataway, NJ). The RNAs were extracted 6 h p.i. or 4 h post–IFN-a addi- tion. The levels of mRNA specific for each target gene were measured by qRT-PCR. Experiments were done in triplicates. The induction of Ifnb1 gene by siRNA was tested in MEFs transfected with the specific or the scrambled siRNA compared with nontransfected cells. The effect of transient expression of siRNA on viral production was measured on transfected BALB/cByJ MEFs. Twenty-four hours posttransfection, the MEFs were infected with the ZH548 RVFV strain or mock infected. At 20 h p.i., supernatants were harvested from the culture, and virus titers were determined using plaque assay on Vero cells.

Statistical analysis and microarray data The survival curves were compared using Kaplan–Meier tests (27). For viral burden in mice, the nonparametric Mann–Whitney U test was used. For qRT-PCR data and viral production in cells, Student t tests were performed on log10-transformed data. All data were analyzed using Stat- View software (SAS Institute, Cary, NC). Data are given as mean 6 SEM. The complete microarray data have been deposited in National Center for biotechnology Information’s Gene Expression Omnibus (28) and are available for download (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE18064) through GEO Series accession number GSE18064.

Results FIGURE 4. Expression profiles of RVFV-responsive genes 9 h p.i. A, Increased susceptibility of MBT/Pas mice to RVFV infection Heat maps showing 29 genes related to the IFN innate-immune response that were upregulated p.i. in BALB/cByJ MEFs. B, Expression of the same To identify genetic factors that may influence susceptibility to RVFV genes in MBT/Pas MEFs. Green and red squares indicate reduced and among inbred strains of mice, we used the virulent strain ZH548, increased levels of expression, respectively. Black squares indicate no a human isolate from the Egyptian outbreak in 1977–1978 (19). change in expression level. The color scale at the bottom indicates the

Groups of 9–12-wk-old, age-matched male mice of various genetic magnitude of changes. Values are in log2. Data from three biological backgrounds, including classical laboratory inbred strains (BALB/ replicates are shown for each experimental condition. 6150 HOST GENETICS CONTROL OF RIFT VALLEY FEVER and MBT/Pas mice with 102 PFU RVFV Kenya 98 strain. Survival mice survived (Fig. 1C) (30). Similar results were obtained p.i. with curves obtained with ZH548 or Kenya 98 strains were similar, in- the mouse-adapted influenza virus PR8 FR0708. Influenza dicating that the susceptible phenotype of MBT/Pas mice is not A-infected MBT/Pas mice had a higher survival than did BALB/ specific to the ZH548 viral strain. To test whether these mice may cByJ mice (p =0.015;Fig.1D). Together, these results suggest that exhibit a generalized immunodeficiency, MBT/Pas and Ifnar12/2 MBT/Pas mice are able to produce and respond to type I IFN and mice were infected i.p. with 102 PFURVFVrec-ZHDNSs, which that they carry a selective defect that does not enable this strain to lacks a functional NSs gene and, therefore, is avirulent in immu- successfully combat an RVFV infection. nocompetent mice (6). Rec-ZHDNSs–infected MBT/Pas mice We further investigated this difference in susceptibility by remained alive, whereas Ifnar12/2 mice, which are nonresponsive to in vitro infection of MEFs. BALB/cByJ and MBT/Pas MEFs were typeIIFN,diedwithin2dp.i.(datanotshown).Weassessedthe infected at an MOI of 1, 5, and 10. Supernatants were analyzed by ability of MBT/Pas mice to express type I IFN . The levels plaque assay for the production of infectious RVFV at 15 and 20 h of IFN-a in sera were significantly higher 48 h after ZH548 infection p.i. Notably, RVFV titers were significantly higher in MBT/Pas in MBT/Pas mice (151–883 pg/ml) than in BALB/cByJ mice (,12.5 MEF supernatants than in BALB/cByJ MEF supernatants at dif- pg/ml). IFN-b remained below ELISA’s detection levels in sera ferent MOIs and times p.i. (Fig. 2). To test whether the higher viral from ZH548-infected MBT/Pas and BALB/cByJ mice. This may be production in MBT/Pas MEFs resulted from increased infection due to the fact that, at low concentrations, IFN-b rapidly binds to its rate, BALB/cByJ and MBT/Pas MEFs were infected with RVFV , and concentrations of the free IFN-b decrease rap- ZH548 at an MOI of 0.1, 1, and 5. The percentage of nucleocapsid idly (29). In addition, we tested the susceptibility of MBT/Pas mice (N) protein+ and NSs+ (i.e., infected) cells was 1.5, 17.6, and 30.4 to other . BALB/cByJ and MBT/Pas mice were infected i.p. for MOIs of 0.1, 1, and 5, respectively, in BALB/cByJ MEFs. The Downloaded from 3 with 10 PFU (= 100 LD50) West Nile virus strain IS-98-ST1 (21). infection efficiency was similar in MBT/Pas MEFs (p . 0.5 for West Nile virus-infected BALB/cByJ mice died, whereas MBT/Pas each MOI). Thus, increased cell infectivity is unlikely to explain

Table I. Upregulation of the innate-immune response genes in RVFV-infected BALB/cByJ and MBT/Pas MEFs, as determined by microarray experiments http://www.jimmunol.org/

Transcript Accession Fold Change in p Value for Fold Change in p Value for Gene Gene Name Number BALB/cByJ Cellsa BALB/cByJ Cellsb MBT/Pas Cellsa MBT/Pas Cellsb Dai/Zbp1 Z-DNA–binding protein 1 NM_021394 1.11 0.02* 0.01 0.9 Gvin1 GTPase, very large IFN inducible 1 NM_029000 2.72 0.0003*** 20.09 0.3 Ifi202b IFN-activated gene 202B NM_011940 1.90 0.02* 0.24 0.3 Ifi203 IFN-activated gene 203 NM_008328 2.63 0.0007*** 0.64 0.1 Ifi204 IFN-activated gene 204 NM_008329 1.81 0.02* 0.21 0.2 Ifi205 IFN-activated gene 205 NM_172648 2.11 0.003** 0.94 0.01*

Ifi35 IFN-induced protein 35 NM_027320 1.36 0.0003*** 0.59 0.001** by guest on September 25, 2021 Ifi44 IFN-induced protein 44 NM_133871 3.75 0.001*** 1.48 0.007** Ifit1/Isg56 IFN-induced protein NM_008331 4.87 0.0004*** 3.95 0.0009*** with tetratricopeptide repeats 1 Ifit3/Ifi49 IFN-induced protein NM_010501 1.99 0.0005*** 0.88 0.01* with tetratricopeptide repeats 3 Iigp1 IFN-inducible GTPase 1 AF194871 2.41 0.00002*** 0.39 0.05* Iigp2/Irgm2 IFN-inducible GTPase 2 NM_019440 2.51 0.003** 0.23 0.3 Il6 IL 6 NM_031168 1.75 0.03* 1.53 0.05* Ip10/Cxcl10 Chemokine (C-X-C motif) ligand 10 NM_021274 2.13 0.00002*** 2.09 0.0002*** Irf7 IFN regulatory factor 7 NM_016850 1.97 0.002** 0.39 0.1 Isg12/ IFN, a-inducible protein 27 NM_029803 1.74 0.04* 20.19 0.3 Ifi27l2a Isg15 & ISG15 ubiquitin-like modifier NM_015783 3.41 0.001** 1.54 0.01* Gm9706c Isg20 IFN-stimulated protein NM_020583 2.11 0.0003*** 3.49 0.0002*** Irf9/Isgf3g IFN-dependent positive-acting NM_008394 1.51 0.0001*** 0.73 0.03* transcription factor 3g Lpg2/Dhx58 DEXH (Asp-Glu-X-His) box NM_030150 2.30 0.0004*** 1.02 0.003** polypeptide 58 Mda5/Ifih1 IFN induced with C domain 1 NM_027835 2.78 0.0003*** 1.71 0.02* Oas1a 29-59 oligoadenylate synthetase 1A NM_145211 1.79 0.001** 0.26 0.04* Oasl1 2’9-59 oligoadenylate synthetase- NM_145209 1.65 0.002** 0.81 0.006** like 1 Oasl2 29-59 oligoadenylate synthetase-like 2 NM_011854 4.63 0.003** 0.14 0.4 Pkr/Eif2ak2 Eukaryotic translation initiation NM_011163 1.92 0.04* 0.51 0.1 factor 2-a kinase 2 Rig-I/Ddx58 DEAD (Asp-Glu-Ala-Asp) box NM_172689 1.38 0.002** 0.35 0.03* polypeptide 58 Stat1 Signal transducer and activator of NM_009283 1.81 0.005** 0.36 0.2 transcription 1 Stat2 Signal transducer and activator of NM_019963 2.56 0.00006*** 0.33 0.2 transcription 2 Trim25 Tripartite motif protein 25 NM_009546 1.06 0.005** 0.71 0.1

a The fold changes in mRNA levels in RVFV-infected cells relative to mock-infected cells are displayed in log2 values. bThe p values were calculated by dChip Software, based on 100 random permutations. cThe microarray probe for Isg15 also hybridizes with the hypothetical Gm9708 (see Results). pp , 0.05; ppp , 0.01; pppp , 0.001. The Journal of Immunology 6151 the higher production of RVFV by MBT/Pas MEFs. Altogether, upregulated, whereas 614 were downregulated (Fig. 3A,3B). these data further confirm the importance of the host genotype in Downregulation of this high number of genes is likely a reflection controlling RVFV spreading and strongly suggest that one of the of the high amount of NSs that is able to inhibit the transcriptional mechanisms could be the efficiency of RVFV replication in activity of constitutive promoters (31). infected cells. To identify the main biological functions that were regulated by RVFV, we used the Ingenuity Pathway Analysis database. In MBT/Pas MEFs exhibit a weak IFN-dependent response BALB/cByJ MEFs, the upregulated genes were primarily related to against RVFV ZH548 infection compared with BALB/cByJ “viral functions” and “immune response” categories, whereas the MEFs downregulated genes were mostly related to the “cell death” To examine the global effects of RVFV infection and the associated category (Fig. 3C). “Viral functions” describes functions associ- host response, we compared the gene-expression profiles of RVFV- ated with the normal activity of viruses after they have infected the infected BALB/cByJ and MBT/Pas MEFs using microarrays. An host cell. Examples include activation, replication, and transcrip- MOI of 5 was used to ensure that a significant percentage of cells tion of viruses. “Immune response” includes functions such as Ab would be infected. Total RNA from three culture dishes of mock- or response, engulfment, and proliferation of immune cells and tis- ZH548-infected MEFs from BALB/cByJ and MBT/Pas embryos sues, as well as functions specific to an immune response, such as were extracted at 9 h p.i., a time at which NSs-induced inhibition of phagocytosis, fever, and Th1 immune response. “Cell death” transcription is still low (9). Total RNAs were hybridized to describes functions associated with cellular death and survival. Affymetrix Mouse Genome 430 2.0 chips. Data were normalized Examples of functions included in this category are cytolysis, and transformed into log2 values, and the fold changes between necrosis, survival, and recovery of cells. In MBT/Pas MEFs, the Downloaded from ZH548- and mock-infected MEFs were calculated. A gene was upregulated genes also showed an overrepresentation of the im- considered regulated by RVFV if its expression in infected cells mune response and viral functions (Fig. 3D), suggesting that was $2-fold higher or lower than its expression in mock- MBT/Pas cells were able to elicit an innate immune response. infected cells of the same genetic background. Quantitative However, these categories are less significantly represented in analysis of the data obtained with BALB/cByJ MEFs showed that MBT/Pas than in BALB/cByJ response (compare Fig. 3C,3D).

229 unique genes (0.82% of cellular transcripts) fulfilled these Clear differences between strains became apparent when the dif- http://www.jimmunol.org/ criteria. Of these, 152 were upregulated, with a maximal 29.2-fold ferentially regulated genes in BALB/cByJ and MBT/Pas MEFs increase; 77 were downregulated, with a maximal 24.1-fold were compared. Seventy-eight percent of the genes that were decrease (Fig. 3A,3B, Supplemental Table III). In MBT/Pas downregulated in BALB/cByJ MEFs were also downregulated in MEFs, 819 genes were regulated by RVFV. Of these, 205 were MBT/Pas MEFs. In contrast, only 35% of the genes upregulated in by guest on September 25, 2021

FIGURE 5. Genes induced by RVFV infection in BALB/cByJ and MBT/Pas cells 9 h p.i. The IFN- a/b gene induction occurs in two steps. Early sig- naling events following virus infection (left panel). Viral components are sensed by cytoplasmic path- ogen recognition receptors, which trigger cascades that activate NF-kB and IRF3. These proteins enter the nucleus and stimulate the transcription of Ifnb1 and a first set of ISGs, such as Isg15 and Ifit1. The produced IFN-b binds the type I IFNAR and acti- vates the JAK/STAT pathway (late signaling events; right panel). Phosphorylated STAT1 and STAT2 bind IRF9 to form ISGF3. ISGF3 enters the nucleus and stimulates the transcription of ISGs, including Irf7. IRF7, together with IRF3, activates Ifna and Ifnb1 genes, thus creating a positive-feedback loop. 6152 HOST GENETICS CONTROL OF RIFT VALLEY FEVER

BALB/cByJ MEFs were also upregulated in MBT/Pas MEFs (Fig. Infection of BALB/cByJ and MBT/Pas MEFs with virulent 3B). These observations suggest that MBT/Pas MEFs were able to RVFV ZH548 stimulates IFN-b and IFN-a4 expression elicit only a restricted range of antiviral responses compared with The representative virulent isolate ZH548 expresses the NSs BALB/cByJ MEFs. protein, which was shown previously to block IFN-b production (6, Thus, among the genes that were upregulated in BALB/cByJ 31). To our surprise, we observed an upregulation of many ISGs in MEFs, we considered those that are typically induced after type response to ZH548 infection. Therefore, we decided to assess the I IFN stimulation (Fig. 4A). Table I shows a list of 29 of these effect of NSs on type I IFN expression in MEFs. We compared the genes and their fold changes in BALB/cByJ and MBT/Pas MEFs. expression of Ifnb1 and Ifna4 genes p.i. with ZH548 or rec- Many of these genes were not upregulated by RVFV in MBT/ ZHDNSs, a strain derived from ZH548 in which the NSs gene is Pas cells (also compare Fig. 4A,4B). Indeed, only seven genes totally deleted (6). BALB/cByJ MEFs were infected with ZH548 encoding RNA MDA5 (Mda5/Ifih1) and LGP2 (Lgp2/ and rec-ZHDNSs strains using an MOI of 5, and the expression of Dhx58), IFN-stimulated exonuclease ISG20 (32), ubiquitin-like Ifnb1 and Ifna4 genes was examined 0, 3, 6, and 9 h later by qRT- modifier protein ISG15 (Isg15), IFN-induced protein with tetra- PCR. Because NSs inhibits RNA polymerase II tricopeptide repeats 1 (Ifit1/Isg56) (33), IFN-induced protein 44 TFIIH factor beyond ∼8–9 h p.i (9), gene expression was not (Ifi44), and chemokine CXCL10 (Cxcl10/Ip10) were upregulated analyzed at later times. Markedly higher Ifnb1 and Ifna4 mRNA in MBT/Pas MEFs. Of note, the genes for RNA helicase RIG-I levels (76- and 1,034-fold, respectively) were observed at 6 h p.i. (Rig-I/Ddx58), dsRNA-dependent protein kinase PKR (Eif1ak2), when BALB/cByJ MEFs were infected with rec-ZHDNSs com- the three ISGF3 subunits (i.e., IRF9, STAT1, STAT2), IRF7, and pared with ZH548 (Fig. 6). Similar increases in Ifnb1 and Ifna4 three 29-59-oligoadenylate synthetases (OAS1a, OASL1, OASL2) mRNA levels were obtained when MBT/Pas MEFs were infected Downloaded from (34) were stimulated at significantly lower levels in MBT/Pas with rec-ZHDNSs compared with ZH548 (data not shown). These MEFs than in BALB/cByJ MEFs. Fig. 5 provides an illustration experiments confirmed the role of NSs as an inhibitor of Ifnb1 of the two successive phases of the induction of ISG expression, in gene transcription. They revealed that Ifna4 expression was also which genes upregulated in BALB/c MEFs and/or MBT/Pas strongly inhibited when the RVFV genome carried NSs. Finally, MEFs are indicated. A number of genes in the early and late they showed that the expression of Ifnb1 and Ifna4 genes was still phases were stimulated by RVFV in BALB/cByJ MEFs but not in significantly stimulated by the virulent ZH548 strain in MEFs, http://www.jimmunol.org/ MBT/Pas MEFs. Altogether, these data suggest that the lack of an thus allowing a possible response to IFN-as or IFN-b. appropriate type I IFN response most likely accounts for the In general, the upregulation of ISGs was weak in MBT/Pas cells higher replication of RVF viral particles in MBT/Pas MEFs. compared with BALB/cByJ cells. To test whether this was due to a disparity in IFN production, we studied the induction of Ifnb1 and Ifna4 in MEFs of both genotypes. Ifnb1 and Ifna4 genes showed a congruent and significant difference in transcript levels (Fig. 7). by guest on September 25, 2021

FIGURE 6. Effect of NSs viral protein on the expression of Ifnb1 and FIGURE 7. Induction kinetics of Ifnb1 and Ifna4 genes in BALB/cByJ Ifna4 genes. Quantification of Ifnb1 (A) and Ifna4 (B) mRNA in BALB/ and MBT/Pas fibroblasts infected with RVFV. Quantification of Ifnb1 (A) cByJ MEFs infected with the virulent ZH548 or attenuated rec-ZHDNSs and Ifna4 (B) mRNA in BALB/cByJ and MBT/Pas MEFs infected with the strain of RVFV. Expression levels are displayed relative to the Tbp gene. virulent ZH548 strain of RVFV. Expression levels are displayed relative to

Statistical analysis was performed using the Student t test on log10-trans- the Tbp gene. Statistical analysis was performed using the Student t test on formed data. ppp , 0.01; pppp , 0.001. log10-transformed data. pp , 0.05. The Journal of Immunology 6153

At 6 h p.i., Ifnb1 and Ifna4 mRNA levels were similar in BALB/ into three groups, according to their induction profile. The first cByJ and MBT/Pas MEFs. At 9 h p.i., the mRNA levels for Ifnb1 profile was observed for Ifit3. This gene was expressed at higher and Ifna4 were higher in the MBT/Pas MEFs than in the BALB/ levels in MBT/Pas MEFs than in BALB/cByJ MEFs late in in- cByJ MEFs. Therefore, the weak upregulation of ISGs and higher fection (Fig. 8A). The second profile was observed for Ifit1, Stat2, viral production in MBT/Pas MEFs cannot simply be explained by and Rig-I. These genes had delayed kinetics of induction in MBT/ stronger inhibition of Ifnb1 by NSs nor by the defective induction Pas MEFs; their expression was higher in BALB/cByJ MEFs early of type I IFNs. in infection, but they reached similar levels in both cultures at 9 h p.i. (Fig. 8B–D). The last profile consisted of genes that were Incomplete and delayed innate immune response to RVFV induced weakly or not at all in MBT/Pas MEFs. The latter profile ZH548 infection in MBT/Pas MEFs was observed for the Irf7, Oasl2, and Isg15 genes (Fig. 8E–G). To further examine the response of MBT/Pas cells to virus in- Thus, the MBT/Pas cells elicited a delayed and only partial type I fection, we selected seven key genes from the IFN-a/b gene- IFN response to RVFV infection. induction pathways for kinetics analysis: Rig-I, Stat2, Ifit3 (Ifi49), Ifit1 (Isg56), Irf7, Oasl2, and Isg15. RNAs were extracted Knockdown of Isg15 and Oasl2 leads to increased virus at 0, 3, 6, and 9 h p.i. from mock- and ZH548-infected BALB/ production cByJ and MBT/Pas MEFs. Most selected genes showed congruent The functional importance of genes that were not fully induced in and significant differences in transcript levels (Fig. 8). Overall, the RVFV-infected MBT/Pas MEFs was evaluated further. We used results obtained by qRT-PCR were largely consistent with the siRNAs to inhibit their expression in BALB/cByJ MEFs and signal intensities of DNA microarray hybridization, except for measured the effect of this reduced expression on the viral pro- Downloaded from Isg15, which was highly upregulated by ZH548 in BALB/cByJ duction. MEFs but not in MBT/Pas MEFs, whereas the results obtained by We determined the ability of different stealth RNAi siRNAs to microarrays indicated that Isg15 was upregulated in BALB/cByJ downregulate Irf7, Isg15, Oasl2, and Rig-I gene expression. RVFV and MBT/Pas MEFs. We found that the microarray probes for rec-ZHDNSs strain was shown to be an efficient trigger for the Isg15 are not specific for this gene because they also hybridize expression of type I IFN (Fig. 6), which, in turn, induces ISG

with the hypothetical Gm9706 gene (Affymetrix database; data transcription. Therefore, rec-ZHDNSs was first used to stimulate http://www.jimmunol.org/ not shown). Sequencing of the amplicon confirmed the specificity the expression of type I IFN, which eventually induces Irf7, Isg15, of Isg15 primers used in qRT-PCR and validated data of the Oasl2, and Rig-I gene expression. Three siRNAs for Irf7, Isg15, quantitative PCR (data not shown). Oasl2, and Rig-I were independently transfected in BALB/ByJ Comparison of the levels and kinetics of expression for BALB/ MEFs; 24 h later, the transfected MEFs were infected with cByJ and MBT/Pas MEFs allowed classification of the seven genes RVFV rec-ZHDNSs. At 6 h p.i., total RNAs were extracted, and by guest on September 25, 2021

FIGURE 8. Induction kinetics of immune-response genes in BALB/cByJ and MBT/Pas fibroblasts infected with RVFV. Expression of Ifit3 (A), Ifit1 (B), Rig-I (C), Stat2 (D), Irf7 (E), Isg15 (F), and Oasl2 (G) was measured by qRT-PCR. Expression levels are displayed relative to the Tbp gene. Statistical analysis was performed with the Student t test on log10-transformed data. pp , 0.05; ppp , 0.01; pppp , 0.001. 6154 HOST GENETICS CONTROL OF RIFT VALLEY FEVER

Irf7, Isg15, Oasl2, and Rig-I mRNA levels were examined by gene expression. Treatment of BALB/ByJ MEFs with 100 IU/ml qRT-PCR. As shown in Fig. 9A–D, the mRNA levels for Irf7, IFN-a for 4 h increased the levels of expression of Irf7, Isg15, Isg15, Oasl2, and Rig-I were significantly higher in the rec- Oasl2, and Rig-I by 2.5-, 5.1-, 6.1-, and 3.0-fold, respectively. ZHDNSs–infected MEFs than in the mock-infected MEFs, dem- Moreover, the mRNA levels for Irf7, Isg15, Oasl2, and Rig-I were onstrating that genes had been activated. Moreover, the mRNA significantly lower when the IFN-a–treated MEFs were trans- levels for Irf7, Isg15, Oasl2, and Rig-I were lower in at least one fected with the specific siRNA compared with the MEFs that were of the three specific siRNA-treated cells compared with cells transfected with scrambled siRNA. Indeed, the most efficient transfected with scrambled siRNA. The most efficient siRNAs siRNA resulted in 57.1, 94.5, 79.6, and 85.1% reduction in the resulted in 80, 85, and 92% reduction in the target RNAs for target RNAs for Irf7, Isg15, Oasl2, and Rig-I, respectively (data Oasl2, Rig-I, and Isg15, respectively. Inhibition of Irf7 expression not shown). These experiments showed that siRNAs were able to by the siRNAs was less efficient, and the best siRNA only reduced downregulate Irf7, Isg15, Oasl2, and Rig-I gene expression in the target RNA to 52% of the control. We also used mouse IFN-a, MEFs that were infected with an RVFVor treated with type I IFN. rather than rec-ZHDNSs, to stimulate Irf7, Isg15, Oasl2, and Rig-I siRNAs can also trigger the IFN response under certain con- ditions. Therefore, it was important to exclude this possibility. BALB/cByJ MEFs were transfected with the most efficient siRNAs for Irf7, Isg15, Oasl2, and Rig-I. Thirty hours after transfection, total RNAs were extracted, and Ifnb1 mRNA levels were mea- sured by qRT-PCR. The specific siRNA-treated MEFs did not

express higher Ifnb1 mRNA levels than MEFs treated with Downloaded from scrambled siRNA or control (no siRNA), indicating that the siR- NAs did not stimulate Ifnb1 expression (data not shown). Finally, to determine whether siRNAs targeting Irf7, Isg15, Oasl2,orRig-I are able to increase RVFV production, specific and scrambled siRNAs were transfected into BALB/cByJ MEFs.

Twenty-four hours later, the transfected cells were infected with http://www.jimmunol.org/ RVFV strain ZH548 at an MOI of 5. The supernatants were har- vested 20 h p.i. and assayed for virus titers. Fig. 9E shows that virus titers were significantly increased in the supernatants of the MEFs transfected with specific siRNAs for Isg15 and Oasl2 compared with MEFs receiving scrambled siRNA (p = 0.0007 and p = 0.0262, respectively). Thus, these findings demonstrate the functional importance of both genes in controlling infection.

Discussion by guest on September 25, 2021 RVFV infection causes symptoms of various severities in several mammalian . In contrast to European breeds, indigenous African sheep, goats, and cattle may show no clinical signs of illness, despite exhibiting a brief period of viremia (2). Such observations suggest that genetic factors are crucial for an effec- tive host defense against RVFV infection. Previous investigations in the mouse did not describe reproducible differences in the susceptibility to RVF among various inbred mouse strains (11). Because this failure may be due to the limited genetic diversity that exists among common mouse laboratory strains, we tested mouse strains that were derived from mice trapped in the wild. The wild-derived MBT/Pas inbred strain belongs to the Mus m. musculus subspecies, whereas most laboratory strains carry a ge- netic make-up that is mainly derived from the subspecies Mus m. domesticus (16). The results presented in this article showed that FIGURE 9. Knockdown effects of Irf7, Isg15, Oasl2, and Rig-I genes on their expression and viral production. qRT-PCR analysis showed the in- MBT/Pas mice, in contrast to many common laboratory mouse hibition of Irf7 (A), Isg15 (B), Oasl2 (C), and Rig-I (D) gene expression strains, exhibited high susceptibility to RVF, which makes them after transfection with specific siRNA (siRNA-1, -2 or -3) or scrambled a useful model for further study of RVFV pathology. siRNA (scrambled). mRNA levels are calculated relative to the gene ex- After inoculation with the virulent RVFV ZH548 strain, mice pression in cells transfected with the scrambled siRNA. BALB/cByJ MEFs developed similar clinical and histological manifestations in MBT/ were transfected with the siRNAs; 24 h later, they were infected with the Pas and BALB/cByJ genetic backgrounds. In MBT/Pas mice, the rec-ZHDNSs strain of RVFV. Total RNAs were extracted 6 h p.i. Mock- virus was detected in the liver at day 2 p.i. High levels of liver en- infected MEFs (mock-inf.) were included as an additional control. Sta- zymes were observed (15- and 27-fold increase versus noninfected tistical analysis was performed with the Student t test on log10-transformed controls for aspartate aminotransferase and alanine transaminase, data. pp , 0.05; ppp , 0.01; pppp , 0.001. The most efficient siRNA, respectively), reflecting hepatocytes’ injury and death. A few hours namely siRNA-2, -1, -2, and -3 for Irf7, Isg15, Oasl2, and Rig-I re- spectively, was transfected in BALB/cByJ MEFs. Twenty-four hours later, before death, MBT/Pas mice were prostrated and adopted a hunched the transfected MEFs were infected with the virulent ZH548 strain of posture. At necropsy, the liver exhibited extensive damage. Histo- RVFV. E, The numbers of viral particles in the supernatant, displayed in pathologic examination confirmed hepatic disease as the fatal 6 log10 per 10 cells, were measured 20 h later. Statistical analysis was lesion. The same features were observed in ZH548-infected BALB/ performed with the Mann–Whitney U test. pp , 0.05; pppp , 0.001. cByJ mice but with a delay of 4 d. The Journal of Immunology 6155

The rapid death of MBT/Pas mice within 4 d p.i. and the 3000- MBT/Pas mice survived an infection with 102 PFU of an NSs- fold higher production of infectious viral particles in the serum at deficient RVFV, whereas the same viral dose killed mice deficient day 3 p.i. suggested that a less efficient innate intracellular antiviral in type I IFN signaling (35) (data not shown). Accordingly, MBT/ response contributes to the high susceptibility of MBT/Pas mice to Pas MEFs were able to produce type I IFNs after viral infection the RVFV ZH548 strain. The higher virus production in MBT/Pas and respond to them. Moreover, MBT/Pas mice are resistant to mice compared with BALB/cByJ mice in vivo could be reproduced infection with West Nile virus and influenza A (30). Interestingly, in cultured MEFs. Therefore, we compared the response to RVFV infection of BALB/cByJ or MBT/Pas MEFs induced a weak infection in MBT/Pas and BALB/cByJ MEFs. At a high MOI, ISGs stimulation of type I IFN genes compared with the 23- and 41-fold were among the main upregulated genes in BALB/cByJ cells, thus induction of genes for IFN-b and IFN-a4 seen in West Nile virus- implicating that the IFN-signaling pathway was significantly in- infected MEFs (46). This weak stimulation in virus-infected cells duced by RVFV. This was unexpected, given that the Ifnb1 gene was probably due to the synthesis of viral inhibitors of Ifnb1 gene, transcription was reported to be blocked by the RVFV NSs protein such as NSs protein. In this study, we focused on the immune (6). Our data show that, although the infection of MEFs with wild- response and investigated the role of ISGs differentially activated type ZH548 virus induced a 70-fold lower Ifnb1 expression in the two types of MEFs and we found that at least, some of them compared with an NSs-null virus, Ifnb1 and Ifna4 genes were still contribute to the antiviral defense. induced by ZH548 infection. Thus, despite the strategies de- In summary, our study shows that the inability of the MBT/ veloped by RVFV to escape host-defense mechanisms, the virus Pas cells to limit virus production is the result of several defects in was still able to activate the host innate-immune system and in- the early and late phases of the IFN response, although MBT/Pas duced ISGs. The essential role for type I IFN in RVFV patho- mice are capable of producing IFNs and responding to them. These Downloaded from genesis is corroborated by the fact that mice deficient for IFN-a/b defects result in a failure to control the spread of the fast-growing receptor subunit 1 (Ifnar12/2) exhibited enhanced viremia and RVFVin cultured cells and are likely to contribute to the early death earlier lethality than did wild-type mice p.i. with ZH548 (35) (data of RVFV-infected MBT/Pas mice. According to our current model, not shown). Moreover, treatment with an IFN inducer is effica- increased viremia leads to earlier infection and higher viral burden cious in preventing the disease in mice (36). in the liver, as well as to rapid acute hepatitis and death of infected

Our analyses further revealed that MBT/Pas MEFs elicited MTB/Pas mice. This hypothesis is supported by seminal experi- http://www.jimmunol.org/ a weak and only partial innate-immunity response to viral infection ments by Cedric Mims (47), who showed that histological changes compared with BALB/cByJ MEFs. Irf7 mRNA was only weakly in the mouse liver during RVFV infection correlated with viral induced by infection in MBT/Pas cells compared with BALB/ load in the blood. Interestingly, the defects seen in MBT/Pas mice cByJ cells. IRF7 plays a critical role within the IFN pathway. It is affect specifically the response against RVFV, because these mice required for Ifna4 gene induction, and its absence is associated were resistant to other viral infections. Finally, the MBT/Pas in- with increased susceptibility to various pathogens, such as en- bred strain seems an interesting model to study the selective cephalomyocarditis virus and vesicular stomatitis virus (37). susceptibility to RVF. This strain would also be appropriate to test However, we could not confirm a functional role for Irf7 down- RVFV candidates. regulation in RVFV production because it was not possible to by guest on September 25, 2021 abolish its expression by siRNA transfection. Therefore, we tested Acknowledgments the functional role of other genes that were also induced in BALB/ We thank Philippe Despre`s and Bernard Charley for West Nile virus strain cByJ MEFs but not induced in susceptible MBT/Pas MEFs. Isg15 IS-98-ST1 and recombinant mouse IFN-aA, respectively, and Sylvio Celso was differentially regulated by RVFV infection in both MEFs. Gonc¸alves da Costa and Jean-Louis Gue´net for valuable discussions. We . Isg15 encodes an ubiquitin-like protein that modifies 150 pro- thank Eliette Bonnefoy, Jean-Marc Cavaillon, and Mustapha Si-Tahar for teins through ISGylation (38). ISG15 inhibits the degradation of constructive comments and critical reading of the manuscript. We also IRF3, thus establishing a direct positive feedback loop to enhance thank Wenjia Zhu for technical help during her short stay in the laboratory. IFN-b expression (39). Furthermore, the increased susceptibility of Isg15-deficient mice to infection with Sinbis virus, influenza Disclosures virus, and HSV-1 suggests that Isg15 is critical for the host re- The authors have no financial conflicts of interest. sponse to viral infections (40). The antiviral effect of ISG15 seems to be virus specific, because Isg15-deficient mice exhibited no increase susceptibility to infection with vesicular stomatitis virus References or lymphocytic choriomeningitis virus compared with wild-type 1. Gerdes, G. H. 2004. Rift Valley fever. Rev. - Off. Int. Epizoot. 23: 613–623. mice (41). Our data point to a significant role for the ISG15 2. Food and Agriculture Organization. 2003. Recognizing Rift Valley Fever. In FAO Animal Health Manual. F. G. Davies and V. Martin, eds. Food and agri- ubiquitin-like protein in the antiviral host defense against RVFV culture organization of the United Nations, Rome, p. 45. infection. Similarly, the Oasl2 gene was upregulated 24-fold p.i. in 3. Flick, R., and M. Bouloy. 2005. Rift Valley fever virus. Curr. Mol. Med. 5: 827– BALB/cByJ MEFs, whereas its expression remained low in MBT/ 834. 4. Giorgi, C., L. Accardi, L. Nicoletti, M. C. Gro, K. Takehara, C. Hilditch, Pas MEFs. The OASL2 protein is active as a 29-59-oligoadenylate S. Morikawa, and D. H. Bishop. 1991. Sequences and coding strategies of the S synthetase (42), and the OAS family members represent potent RNAs of Toscana and Rift Valley fever viruses compared to those of Punta Toro, Sicilian Sandfly fever, and Uukuniemi viruses. Virology 180: 738–753. antiviral proteins. Indeed, Oas1b is involved in the innate response 5. Muller, R., J. F. Saluzzo, N. Lopez, T. Dreier, M. Turell, J. Smith, and of mice to West Nile virus infection (30, 43), and OAS1 is a ge- M. 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