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Diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction [1]

['Giulia Gallo', 'Institut Pasteur', 'Université Paris Cité', 'Département De Virologie', 'Unité Des Stratégies Antivirales', 'Paris', 'Sorbonne Université', 'Ecole Doctorale Complexité Du Vivant', 'Petr Kotlik', 'Laboratory Of Molecular Ecology']

Date: 2022-11 

Orthohantaviruses are rodent-borne emerging viruses that may cause severe diseases in humans but no apparent pathology in their small mammal reservoirs. However, the mechanisms leading to tolerance or pathogenicity in humans and persistence in rodent reservoirs are poorly understood, as is the manner in which they spread within and between organisms. Here, we used a range of cellular and molecular approaches to investigate the interactions of three different orthohantaviruses–Puumala virus (PUUV), responsible for a mild to moderate form of hemorrhagic fever with renal syndrome in humans, Tula virus (TULV) with low pathogenicity, and non-pathogenic Prospect Hill virus (PHV)–with human and rodent host cell lines. Besides the fact that cell susceptibility to virus infection was shown to depend on the cell type and virus strain, the three orthohantaviruses were able to infect Vero E6 and HuH7 human cells, but only the former secreted infectious particles. In cells derived from PUUV reservoir, the bank vole (Myodes glareolus), PUUV achieved a complete viral cycle, while TULV did not enter the cells and PHV infected them but did not produce infectious particles, reflecting differences in host specificity. A search for mature virions by electron microscopy (EM) revealed that TULV assembly occurred in part at the plasma membrane, whereas PHV particles were trapped in autophagic vacuoles in cells of the heterologous rodent host. We described differential interactions of orthohantaviruses with cellular factors, as supported by the cellular distribution of viral nucleocapsid protein with cell compartments, and proteomics identification of cellular partners. Our results also showed that interferon (IFN) dependent gene expression was regulated in a cell and virus species dependent manner. Overall, our study highlighted the complexity of the host-virus relationship and demonstrated that orthohantaviruses are restricted at different levels of the viral cycle. In addition, the study opens new avenues to further investigate how these viruses differ in their interactions with cells to evade innate immunity and how it depends on tissue type and host species.

Orthohantaviruses are zoonotic RNA viruses found all over the world in association with small mammal reservoirs. When occasionally transmitted to humans by aerosol they can cause two main types of diseases: hemorrhagic fever with renal syndrome (HFRS) mainly in Europe and Asia and hantavirus cardiopulmonary syndrome (HCPS) in North and South America with a case fatality rate of up to 15% and 40% respectively. An increasing number of outbreaks are recorded in endemic areas and, with disturbance of rodent habitats, climate change and the risk of virus genome reassortment, the emergence of new orthohantaviruses is of concern. With no treatment, no vaccines and few molecular tools, little is known about the pathophysiology of these viruses. Our comparative study of the viral cycle and interaction of different pathogenic and low or non-pathogenic orthohantaviruses in cells derived from human or rodent hosts reveals differences in entry, RNA replication or release of infectious particles, concurrently to regulation of host genes. This study illustrates how the development of a rodent host cell model together with the availability of an annotated bank vole genome may contribute to a better understanding of mechanisms of the interactions between orthohantaviruses and their hosts.

Funding: GG was funded by ED CDV (Sorbonne Université) and by the Flash maturation program of Institut Pasteur, ME and NT benefited from ANTIGONE fundings, a European FFP7 program. MM received the support of France Génomique (ANR-10-INBS-09). R.G.U. acknowledges support by the Helmholtz Association within the Initiative and Networking Fund for Infection Research Greifswald (project HANTadapt-022021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository ( https://www.ebi.ac.uk/pride/ ) with the dataset identifier PXD032365 The RNA-seq data have been deposited to NCBI-GEO (accession GSE198751) and are freely available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198751 .

Copyright: © 2022 Gallo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We hypothesized that characterizing differences in the capacity of orthohantaviruses to infect cells, interact with cellular factors, regulate cellular functions and propagate, will provide insight into different outcomes of orthohantavirus infection. Therefore, our objectives were to compare the interactions of pathogenic (PUUV) and low and non-pathogenic (TULV and PHV) orthohantaviruses with cells derived from the human host, and the bank vole, with a particular effort in studying rodent cells-virus relationships, to date poorly described. Moreover, due to the apparent conflicting results present in the literature (usually focused on one virus or one cell line), we thought it would be important to address these questions by designing a comprehensive study (multiple cell lines, multiple viruses) and by including different omics approaches, scarcely reported for orthohantaviruses in the literature. The ability of the viruses to replicate and produce infectious particles was assessed by monitoring the intracellular expression of N protein during primary and secondary infections and by quantifying the copy number of viral genomes produced by infected cells. EM was used to detect mature viral particles in infected cells and immunofluorescence staining to follow viral interaction and remodeling of cellular compartments. This prompted us to investigate cellular interactors of viral proteins by mass-spectrometric identification and search for transcription signatures in bank vole cells infected by orthohantaviruses, as compared to human cells, performing RNA-sequencing (RNA-Seq) and real time-quantitative PCR (RT-qPCR). Altogether our data highlight different levels of cellular restriction to orthohantaviral infection, which can be linked with the observed regulation of gene expression depending on the virus, the cell type and its host origin raising questions about innate immune responses of infected cells. These differential interactions are discussed in the context of the outcomes of orthohantavirus infections.

In order to bring information on mechanisms of pathogenicity and persistence of orthohantaviruses in their different hosts, we performed comparative studies of the interactions carried out in human and rodent cell lines by three orthohantaviruses: human pathogenic Puumala virus (PUUV), low or non-pathogenic Tula virus (TULV) and Prospect Hill virus (PHV). PUUV is one of the most important European orthohantavirus, which is responsible for nephropathia epidemica, a mild to moderate form of HFRS, and is hosted by the bank vole (M. glareolus syn. Clethrionomys glareolus). PHV is considered as a viral model of apathogenic orthohantaviruses and is associated to the meadow vole (Microtus pennsylvanicus), from North America. TULV is found in Europe and is hosted by the common vole (Microtus arvalis), although it was also molecularly detected in other related vole species [ 36 ]. It is considered to be of low pathogenicity due to the rare report of human cases [ 37 , 38 ].

Due to the lack of molecular tools, including reverse genetics systems and, annotated genome, transcriptome and proteome libraries for each animal reservoir, few studies have been conducted on the viral life cycle during orthohantavirus infection. Therefore, only fragmentary information is available [ 32 ], especially on the interactions of orthohantaviruses with the immune system of their rodent reservoirs [ 9 , 33 ]. Furthermore, due to the fact that orthohantaviruses cause persistent asymptomatic infection in rodent hosts, relevant animal models of pathogenicity are missing, in particular for Old World orthohantaviruses [ 34 ]. Information on the role of endothelial and immune cells in pathogenesis comes mainly from studies on the activated factors found in infected cells and in patients with HFRS or HCPS [ 35 ].

As suggested by their low number, structural hantaviral proteins may harbor multiple functions to successfully accomplish a viral cycle. The viral N protein not only protects the viral RNA and acts in viral replication and transcription, but also interacts with multiple cellular factors [ 21 ], is associated to the endoplasmic reticulum-Golgi intermediate compartment, ERGIC [ 22 ] and regulates cellular processes such as mRNA post-transcription decay [ 23 ], apoptosis [ 24 – 26 ] and interacts with the cytoskeleton network [ 27 ]. In the case of envelope glycoproteins, in addition to their roles in entry, trafficking and maturation of viral particles, they could also contribute to innate immunity through interaction of their long cytosolic tails with cellular factors. The GnCT has indeed been shown to activate cellular kinases implicated in the regulation of endothelial and immune functions [ 28 , 29 ]. An antagonistic role to interferon antiviral activity has also been demonstrated, which could be linked to the pathogenicity of orthohantaviruses [ 30 , 31 ].

In human and rodent hosts, orthohantaviruses primarily infect epithelial and endothelial cells and also macrophages and dendritic cells, which could promote the propagation of viruses in the organism [ 9 ]. It is thought that the first step for cellular susceptibility to viral infection could rely on interaction with different integrins, used as primary receptors, depending on the virus origin and its pathogenicity [ 10 , 11 ]. However, a recent study has shown that protocadherin-1 is crucial for infection of endothelial cells by the pathogenic New World orthohantaviruses, Andes virus (ANDV) and Sin Nombre virus (SNV), and suggests that integrins probably only play minor roles in cellular infection [ 12 ]. In support, bank vole-derived immortalized cells express low levels of integrin-β3 [ 13 ] and virions can be internalized by clathrin-dependent [ 14 ] and independent [ 15 ] mechanisms, as well as by macropinocytosis [ 16 ]. The replication cycle of the three RNA segments of negative polarity, S, M and L, of orthohantaviruses then entirely takes place in the cytoplasm. These three genome segments encode the nucleocapsid (N), the RNA polymerase and the two envelope glycoproteins, Gn and Gc, generated by cleavage of a glycoprotein precursor, GPC. Maturation and assembly of Gn/Gc complexes take place in the secretory pathway [ 17 ]. Then, the cytoplasmic ribonucleoproteins (RNP), consisting of viral RNA segments wrapped into N protein oligomers, interact with the Gn glycoprotein cytosolic tails (GnCT) at the Golgi site of viral particle assembly and budding [ 18 , 19 ]. However, in contrast to Old World orthohantaviruses, some orthohantaviruses from the Americas could bud directly at the plasma membrane [ 20 ].

In contrast to the human host, which can be occasionally infected, animal reservoirs are persistently infected by orthohantaviruses throughout their life span without any obvious sign of pathology [ 6 ]. In these natural hosts, viruses are transmitted through inhalation of virus contaminated aerosols and wound contact with infected saliva during aggressive behavior associated with mating. Viral RNA can be found in different organs of rodents and has been detected in lungs, kidneys, liver, brain and gallbladder, but importantly is excreted in urine and feces of infected animals [ 7 , 8 ].

Orthohantaviruses are emerging viruses hosted by small mammals, such as rodents and shrews, with which they have co-evolved for a long time [ 1 ]. Because of the global distribution of their animal reservoirs, orthohantaviruses are found on all inhabited continents of our planet. To date, only orthohantaviruses specifically infecting rodents have been identified as causing human diseases upon viral transmission through inhalation when humans come into contact with aerosolized excreta of infected animal reservoirs. Recently, orthohantaviruses have been involved in recurrent human epidemics [ 2 ]. According to the orthohantavirus species and affected organs in humans, two different diseases have been described: the hantavirus cardiopulmonary syndrome (HCPS) exclusively in the Americas, and the so-called hemorrhagic fever with renal syndrome (HFRS) mainly in Eurasia. First, viruses enter the respiratory tract and then propagate to different organs, in particular lung, kidney and liver, whose physiology is altered in infected patients. Even though the precise mechanism of propagation is not completely understood, it is known that vascular leakage and thrombocytopenia contribute to both HCPS and HFRS, probably due to bursts of cytokines produced by infected endothelial cells and by immune cells recruited at the site of infection [ 3 – 5 ].

Groups of data are represented as the mean of biological triplicates and the standard deviation to the mean was calculated. For RT-qPCR analysis of cellular gene expression, the mean of each virus infected cells was compared to the mean of non-infected cells by standard One-way ANOVA using Dunnett method for multiple comparisons. For quantification of viral genome copies in lysates and supernatants, as well as for infections calculated as the percentage of N+ cells, standard Two-way ANOVA using Šidák method for multiple comparisons was applied. Finally, statistical analysis of neutralization assay was performed using standard Two-way ANOVA with Tukey method. Only data with 95% confidence interval were considered significant with * p< 0.0332, ** p<0.021, *** p<0.0002 and **** p<0.0001, while “ns” indicates non-significant variation.

To evaluate the reliability and reproducibility of the results obtained in the RNA-Seq analyses, we used RT-qPCR to validate the gene expression patterns of selected genes. Among the differentially expressed genes (DEG) of bank vole identified by RNA-Seq, 11 genes were selected to be validated by RT-qPCR. To design primers for DEG, which could only be detected by using the mouse reference genome, transcripts of putative bank vole orthologs were identified in the bank vole transcriptome [ 44 ] as described above.

RNA preparation was used to construct strand-specific single-end cDNA libraries according to the manufacturer’s instructions (TruSeq Stranded mRNA sample prep kit, Illumina). Illumina NextSeq 500 sequencer was used to sequence libraries. The RNA-seq analysis was performed with the Sequana framework [ 46 ]. First, the viral infection has been verified using the mapper pipeline and specific viral RNA sequences are detected in the infected samples. Then, we used the RNA-seq pipeline (v0.13.0), which is built on top of Snakemake 5.8.1 [ 47 ] and is available online ( https://github.com/sequana/sequana_rnaseq ). Reads were trimmed from adapters using Cutadapt 2.10 [ 48 ] and then mapped to the M. musculus GRCm38/mm10 genome and M. glareolus draft genome using STAR 2.7.3a [ 49 ]. Sequencing of the bank vole genome was recently performed using a combination of shotgun, Chicago, and Dovetail HiC library reads [ 50 ]. The assembly yielded a total of over 4300 scaffolds, 39 of which were larger than 50 Kb and covered 99% of the genome, and was annotated with the GAWN pipeline ( https://github.com/enormandeau/gawn ) using a BLASTX search [ 43 ] against the Swissprot database (UniProt Consortium 2019). FeatureCounts 2.0.0 [ 51 ] was used to create the count matrix, assigning reads to features with strand-specificity information. Quality control statistics were summarized using MultiQC 1.8 [ 52 ]. Statistical analysis on the count matrix was performed to identify differentially regulated genes by comparing infected to non-infected samples. Clustering of transcriptomic profiles was assessed using a principal component analysis (PCA). Tests for differential expression were performed using DESeq2 library 1.24.0 [ 53 ] scripts based on SARTools 1.7.0 [ 54 ] indicating the significance (Benjamini–Hochberg-adjusted P-values, FDR < 0.05) and the effect size (fold change) for each comparison. Finally, enrichment analysis was performed using modules from Sequana. The GO enrichment module uses the PantherDB [ 55 ] and QuickGO [ 56 ] services; the KEGG pathways enrichment uses the gseapy ( https://github.com/zqfang/GSEApy/ ), EnrichR [ 57 ], KEGG [ 58 ], and BioMart services. All programmatic access to the online web services was performed via BioServices [ 59 ].

MyglaSWRecB cells were plated at 2.5x10 5 cells per well of 12-well microplates and infected 24h later. Four wells per condition were incubated either with PUUV or PHV at MOI of 0.5 or remained non-infected. RNA was prepared from cells at dpi 5. Cells were washed with PBS and incubated for 5 min at room temperature with 500 μL of Trizol (Tri Reagent, Sigma Aldrich) before addition of 150 μL of chloroform for 10 min and centrifugation at 4°C for 10 min at 11,000 rpm. The aqueous phase was recovered and precipitated with isopropanol. After centrifugation, the pellet was washed in 70% ethanol and then suspended in 50 μL of RNase free water.

MS raw files were processed using PEAKS Online X (build 1.5, Bioinformatics Solutions Inc.). Data were searched against the Human Uniprot release 2021_03 database consisting of reviewed-only sequences including 20387 total entries. The sequences of hantaviral N proteins were added for the search, as internal control. Parent mass tolerance was set to 20 ppm, with fragment mass tolerance of 0.05 Da. Specific tryptic cleavage was selected and a maximum of 2 missed cleavages was authorized. For identification, the following post-translational modifications were included: acetyl (Protein N-term), oxidation (M), deamidation (NQ) as variables and half of a disulfide bridge (C) as fixed. Identifications were filtered based on a 1% FDR (False Discovery Rate) threshold at both peptide and protein group levels. Label free quantification was performed using the PEAKS Online X quantification module, allowing a mass tolerance of 20 ppm for match between runs and auto-detection of retention time shift tolerance. Protein abundance was inferred using the top 3-peptide method and TIC was used for normalization. Multivariate statistics on protein measurements were performed using Qlucore Omics Explorer 3.7 (Qlucore AB, Lund, Sweden). A two-group comparison was used to compare sequentially proteins identified in each viral N protein samples and in the empty plasmid control sample, a p-value lower than 0.05 was used to filter differential candidates considered as potential N protein interaction partners. The PRIDE database, dedicated to mass spectrometry-based proteomics data [ 45 ], was chosen to deposit our LC/MS/MS data.

Proteins on beads in association with viral N protein of PUUV, TULV or PHV, or beads which have been incubated with the lysate of cells transfected with the empty plasmid as a control, were incubated overnight at 37°C with 20 μL of trypsin (sequencing grade, Promega) at 25 μg/mL in 25 mM NH 4 HCO 3 . Peptides were desalted using ZipTip μ-C18 Pipette Tips (Millipore). Peptide mixtures were analyzed by a Q-Exactive Plus coupled to a Nano-LC Proxeon 1000 both from ThermoFisher Scientific. Peptides were separated by chromatography with the following settings: Acclaim PepMap100 C18 pre-column (0.075 x 20 mm, 3 μm, 100 Å), Pepmap-RSLC Proxeon C18 column (0.075 x 500 mm, 2 μm, 100 Å), 300 nl/min flow rate, a 98 min acetonitrile (ACN) gradient from 95% solvent A (H 2 O/0.1% FA) to 35% solvent B (100% ACN/0.1% FA) followed by column regeneration, giving a total acquisition time of 145 minutes. Peptides were analyzed in the Orbitrap cell in positive mode, at a resolution of 70,000, with a mass range of m/z 200–2000 and an AGC target of 3.10 6 . MS/MS data were acquired in the Orbitrap cell in a Top20 mode. Peptides were selected for fragmentation by Higher-energy C-trap Dissociation (HCD) with a Normalized Collisional Energy of 27%, a dynamic exclusion of 60 seconds, a quadrupole isolation window of 1.4 Da and an AGC target of 2.10 5 . Peptides with unassigned charge states or monocharged were excluded from the MS/MS acquisition. The maximum ion accumulation times were set to 50 msec for MS and 45 msec for MS/MS acquisitions.

Transfection, pull down and LC/MS/MS were performed on sample triplicates, as previously described [ 30 ]. In brief, 10 6 HEK293T cells were transfected, using the JetPRIME reagent (Polyplus), with 1 μg of plasmid pCiNeo-streptag encoding PUUV-N, TULV-N or PHV-N or pCiNeo-streptag empty plasmid as a control, and incubated for 24 h at 37°C. For each condition, 1 mg of proteins was pulled down for 1 h, at 4°C, on 25 μL of packed sepharose beads linked to Strep-Tactin (IBA). After washing in lysis buffer (NET/1% TX100), the beads were suspended in 25 mM NH 4 HCO 3 , pelleted by centrifugation and kept on ice covered by a film of bicarbonate ready to be processed for mass spectrometry analysis.

Samples were washed in PBS and post-fixed by incubation for 1 h with 2% osmium tetroxide (Agar Scientific). Cells were then fully dehydrated in a graded series of ethanol solutions and propylene oxide. They were impregnated with a 1∶1 mixture of propylene oxide/Epon resin (Sigma) and left overnight in pure resin. Samples were then embedded in Epon resin (Sigma), which was allowed to polymerize for 48 hours at 60°C. Ultra-thin sections (90 nm) of these blocks were obtained with a Leica EM UC7 ultramicrotome (Leica Microsystem). Sections were stained with 2% uranyl acetate (Agar Scientific), 5% lead citrate (Sigma), and observations were made with a transmission electron microscope (JEOL 1011).

To visualize the cellular compartments following infection, as well as the presence of viral particles both inside the cells and in the extracellular space, cellular pellets were prepared as follows for EM analysis. Vero E6, HuH7 and MyglaSWRecB cells were seeded in culture flasks and infected 24 h later with PUUV, TULV or PHV at a MOI of 2. After 5 days, cells were washed in PBS before being recovered by trypsinization, centrifuged for 5 min at 1000 rpm, then pelleted cells were suspended in a fixation buffer (4% FA, 1% glutaraldehyde, pH 7.3). Pellets were kept at least 48 h at 4°C and then a few days at room temperature before being treated for EM.

In designing primers for amplification of mRNA from the bank vole cell line, transcripts corresponding to putative M. glareolus orthologs of mouse IFNα, β and λ2/λ3 genes were screened and found by BLAST [ 43 ] in a recently published, unannotated bank vole transcriptome [ 44 ]. Data were normalized to the actin mRNA and presented as relative expression compared to non-infected cells (2 -ΔΔCt ).

RT-qPCR assays were performed as previously described [ 30 ]. In brief, 5×10 4 cells seeded in 12-well plates were infected 24 h later at a MOI of 1, with virus diluted in DMEM/5% FBS. RNA from lysates of infected cells was recovered at different time points using TRI Reagent (Sigma), according to the manufacturer’s instructions. RNA from cells treated for 24 h with poly-IC at 10μg/mL was used as a control of IFNs activation. RNAs were quantified with a Nanodrop spectrophotometer (ND1000, ThermoFisher Scientific) and then stored at −80°C. After treatment of the samples with DNase, reverse-transcription was performed using High Capacity cDNA Transcription kit (Applied Biosystems) with random primers. The reactions were carried out in a thermocycler as follows: 10 min at 25°C, 2 h at 37°C, 5 min at 85°C. Quantification of cDNAs was then performed by RT-qPCR using SYBR Green technology (EurobioGreen Mix qPCR 2X Lo-Rox, Eurobio) with gene-specific primers. Primers for amplification of human mRNAs ( Table 1 ) were either devised from online website PrimerBank-MGA-PGA ( https://pga.mgh.harvard.edu/primerbank/ ) or manually designed.

To quantify the copy number of viral genomes, internal standard curves specific of each virus were set up and included to all qPCR runs. Short sequences corresponding to viral S segments (nucleotides 73–295) of each virus were inserted into the pSP72 vector (Promega). Reactions for in vitro transcription were carried out following manufacturer’s instruction and led to the synthesis of in vitro transcribed viral RNA by use of the T7 polymerase of Riboprobe Combination SP6/T7 kit (Promega). Plasmid DNA was then removed by DNaseI treatment (Promega). RNA quantification and residual DNA contamination were determined using a Qubit fluorimeter (ThermoFisher Scientific). Standard curves were determined by serial dilution of in vitro transcribed RNA combined to RT-qPCR (see below transcriptomic section) using primers amplifying an S–segment specific internal sequence ( Table 1 ).

Vero E6, HuH7 and MyglaSWRecB cells were plated at a density of 2.5x10 4 in 24-well plates and infected the next day at a MOI of 1. Infection and viral RNA quantification within cells and in the supernatant were performed as described [ 30 ]. Briefly, cells were incubated for 1 h at 37°C with 150 μL of virus diluted in DMEM/5% FBS, then 1 mL of the same medium was added for further incubation for 3 or 7 days. Recovered supernatants were centrifugated at 1500 rpm for 5 min and kept frozen at -80°C. In parallel, cell layers were lysed in NET/1% TX-100 (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.5, 0.5 mM with 1% Triton X100) supplemented with a cocktail of protease inhibitor (cOmplete, Roche), and of phosphatase inhibitors (PhosSTOP, Sigma). The post nuclear cytoplasmic fraction was recovered by centrifugation of the cell lysates at 13,000 rpm for 15 min at 4° C and stored at -80°C. RNA from both lysates and supernatants was extracted using QIAmp Viral RNA mini kit (Qiagen) and quantified using a Nanodrop spectrophotometer (ND1000, ThermoFisher Scientific). Reverse-transcription of viral RNA was performed using SuperScript III Reverse Transcriptase (Invitrogen) with virus-specific S segment primers for 1 h at 55°C: 5’-ACCCGCCATGAACAGCAAC-3’ for PUUV, 5’-ACCCGCCATGAACAGCAAA-3’ for TULV and 5’-ACTCGCCATGAGCAGCAGC-3’ for PHV.

Infected cells were fixed, permeabilized and then incubated with primary and secondary antibodies as described above for immunofluorescence staining of N protein. Cells were then treated for 5 min at -20°C with 100% of cold ethanol, washed once with PBS and dehydrated in 70% RNase-free ethanol overnight at 4°C. The next day, cells were rehydrated in saline sodium citrate (SSC) buffer at 0.3 M sodium chloride and 30 mM sodium citrate (Invitrogen) for 1 h at room temperature before incubation for 1 h at 60°C in hybridization buffer containing 50% formamide, 10% dextran sodium sulfate salt (Fluka) and 20 μg/mL of salmon sperm DNA (Invitrogen) diluted in SSC. One hundred pmol of a single stranded DNA probe (5’-ACATCAAGGACATTTCCATATCGAAGGCTTGATCTCTCCTT-3’), tagged with Alexa Fluor 488 at its 5’ end, were then added to the hybridization buffer for 5 min at 60°C followed by 4 h at 37°C to detect viral antigenomic (+)RNA and a DNA probe (5’-ACTTATATATATGCACGTAGCATATATATAAGT-3’) tagged with Alexa Fluor 555 (Sigma Aldrich) complementary to the viral genomic (-)RNA. Cells were washed three times with SSC pre-heated at 40°C and then mounted on microscopy slides as described above.

For lipid droplet detection, infected cells were washed with DMEM and 150 μL of BODIPY 558/568 C12 diluted in DMEM, at a final concentration of 0.1 μg/mL, were added to each well. Cells were incubated with this solution for 10h at 37°C, washed with PBS and then fixed for immunostaining of viral-N proteins.

To enrich cells in recycling endosomes, infected cells were washed with DMEM then treated with brefeldin A (BFA, ThermoFisher Scientific) diluted to a final concentration of 10 μg/mL. BFA disrupts the trans-Golgi network and blocks recycling vesicles, enhancing their visualization after staining. Cells were incubated with the drug for 30 min at 37°C. Cy3-Tf at a concentration of 1.5 μg/mL in DMEM was then incubated with living cells for 30 min. After washing with PBS, cells were fixed for immunostaining of viral-N proteins.

In order to detect cell surface antigens, living cells were incubated with primary and thereafter secondary antibodies, both diluted in PBS containing 0.1% sodium azide and complemented with 2% FBS and washings were also performed in PBS with 0.1% sodium azide. Cells were fixed at the end of the reactions with PBS /FA 3.7%, then processed as above for immunostaining.

Cells were plated on glass coverslip (Marienfeld) in 24-well plates at 2x10 4 cells per well, then, N-protein transfected cells or orthohantavirus-infected cells were treated at the indicated times. For the detection of intracellular components, cells were fixed for 15 min at room temperature with 3.7% formaldehyde (FA, Sigma-Aldrich). Fixed cells were blocked with glycine at 20 mM (Sigma-Aldrich) in phosphate buffered saline (PBS) for 15 min. Cells were permeabilized with 0.5% Triton X100 in PBS for 5 min and then washed with PBS + 0.05% Tween20 (PBS-T). Cells were then incubated with the primary antibodies diluted in PBS-T containing 1% bovine serum albumin (BSA, Cell Signaling), for 1 h. After washing in PBS-T, primary antibodies were detected with Ig species-specific secondary antibodies conjugated either to Alexa Fluor 488 or Alexa Fluor 555 dyes (ThermoFisher Scientific) diluted in PBS-T-BSA 1%, for 1 h. Cells were washed with PBS-T and mounted in Fluoromount DAPI-G (Southern Biotechnology).

For the IFN-λ neutralization assay, orthohantaviruses were pre-incubated with the goat anti-IFNλ1 or λ2 antibodies at 10 μg/mL for 30 min at room temperature according to Prescott et al. [ 42 ], then 150 μL of pre-treated viruses were incubated at 37°C for 1h with cell cultures. The viral input was discarded before addition of 1mL of medium/5% FBS. The effect of anti-IFNλ1/λ2 antibodies on virus infectivity was determined at dpi 3.

viral titer = (N x IC) / V x 100, by taking into account the number of plated cells (N), the % of infected cells (IC), the volume of virus (V) in mL. Of note, viruses replicated differently in VeroE6 cells, with titers being around 1–2.5x10 5 IU/mL for PUUV, 2-5x10 6 IU/mL for PHV and 5x10 7 - 10 8 IU/mL for TULV. The indicated MOI used in infection experiments was calculated from the infectious titers of the viral stocks.

Infectious particles present in virus stocks were titrated on Vero E6 cells, according to Barriga et al [ 41 ], performing intracellular fluorescence at 48h-72h post infection. In brief, cells seeded at 1.5x10 4 cells in 24-well plates, were adhered on glass coverslips for 20-24h, then incubated with 150 μL of different dilutions of virus supernatant for 1 h before addition of 1 mL of medium for further incubation. The percentage of infected Vero E6 cells was calculated by counting the cells positively stained by the A1C5 monoclonal antibody, specific to an epitope expressed by N protein of PUUV-CG18-20 strain, and conserved at the N terminus of the N protein of the different orthohantavirus strains used here. The percentage of N protein positive cells (N+) was recorded as a function of the dilution of the supernatants as illustrated in S1 Fig . The viral titers in infectious units per mL (IU/mL) were then calculated from the linear part of the curve as follow:

Viral N protein was detected using a commercial mouse monoclonal antibody (A1C5, antibodies-online) raised against the N protein of PUUV-CG18-20 strain, while a home-made rabbit polyclonal antibody raised against the ectodomain of PUUV-Gn was used to detect PUUV, TULV and PHV glycoproteins [ 30 ]. The following rabbit polyclonal antibodies were used to detect cellular compartments: anti-calnexin for the endoplasmic reticulum, anti-LMAN1 for the ERGIC, anti-giantin for the Golgi, and Golgin 97 for the Trans Golgi Network, all provided by Novus Bio, while anti-EEA1 from Cell Signaling and anti-Rab7 from Abcam were used to detect early and late endosomes, respectively. Anti-DDX6 (Novus Bio) was used to detect P-bodies. Cytoskeleton filaments were detected with anti-tubulin (Novus Bio) and anti-vimentin (Abcam) and actin filaments were visualized using Alexa Fluor 555 phalloidin (ThermoFisher Scientific). The rabbit anti-NCBP2 antibody was obtained from Sigma and the rabbit anti-NKRF antibody from Life Technology. The rabbit anti-ribophorin I polyclonal antibody was a kind gift from Ewin Ivessa (Center of Biomedical Chemistry, University of Vienna). A goat anti-human IL29 (IFN-λ1) and a goat anti-human IL28A (IFN-λ2), both from Biotechne were used in neutralization assay. Goat anti-rabbit or -mouse immunoglobulins conjugated to Alexa Fluor 488 or Alexa Fluor 555 were purchased from Invitrogen.

PUUV strain Sotkamo and PHV strain 3571, as well as Vero E6 cells, were kindly provided by Andreas Rang (Charité Berlin, Germany). TULV strain Moravia was obtained from Alexander Plyusnin (Helsinki University, Finland). Virus stocks were prepared on Vero E6 cells seeded in 75 cm 2 culture flasks, the day before infection to let cells adhere to the surface. Virus was added for 1 h on the cell layer at a multiplicity of infection (MOI) around 0.1 in 2 ml of DMEM 5% FBS. Twenty mL of DMEM/5% FBS were then added. Culture supernatants were recovered 6- (TULV) or 7- (PUUV, PHV) days post infection (dpi) and kept frozen in aliquot at -80°C.

Except for HUVEC, the different media used to maintain human and vole cells were supplemented with 10% of heat-inactivated fetal bovine serum (FBS), purchased from Biosera. All cell lines were kept growing at 37°C, 5% CO 2 in humid atmosphere. Cells were mycoplasma free, as determined by Mycoalert test (Lonza)

Bank vole cells derived from different organs of M. glareolus were immortalized by the large T antigen of Simian virus 40 (SV40), as described [ 39 ]. The different bank vole cell lines, generated within the EVAg project by Charité (Berlin, Germany) were kindly provided by Isabella Eckerle and Marcel Müller (Bonn University, Germany): MyglaSWRecB and MyglaSWTrach cells are epithelial cells from kidneys and from the tracheal apparatus respectively and MyglaAECcl2 cell clone is derived from alveolar lung epithelial cells. BVK168 is a spontaneously immortalized bank vole kidney cell line [ 40 ], kindly provided by Sandra Essbauer (Bundeswehr Institute of Microbiology, Munich, Germany). All these cells were cultured in DMEM. MH-S, a house mouse (Mus musculus) alveolar macrophage cell line (ATCC-CRL-2019), was grown in RPMI 1640.

Vero E6 cells, kidney epithelial cells from African green monkey, were grown in Dulbecco’s Modified Eagle’s Medium (DMEM). HuH7 (human hepatocarcinoma), A549 (human lung carcinoma, ATCC-CCL-185) and Caco-2 (human colorectal adenocarcinoma) cell lines were maintained in DMEM supplemented with non-essential amino acids and 1 mM sodium pyruvate. The human monocyte THP-1 cell line was maintained in suspension in RPMI 1640 Medium. Cells were treated with phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) at 100 nM in Macrophage-SFM medium for 24 or 48 hours, in order to induce their differentiation in macrophages. Human umbilical vascular endothelial cells, HUVEC (a kind gift from Philippe Afonso, Institut Pasteur, Paris) were grown in EndoGRO-MV complete media kit (Merck). Media and additives were purchased from GIBCO (Invitrogen) except when otherwise indicated.

Results

Susceptibility to infection of human and rodent host-derived cell lines differs with orthohantavirus species In order to provide cellular models to study orthohantavirus interactions with their hosts, we first compared the permissiveness of epithelial and endothelial cell lines derived from different organs of human or rodent origin known to be targeted by orthohantaviruses, i.e. lung, kidney, macrophages, as well as additional cell types. In order to determine if cells could be infected by pathogenic PUUV, low-pathogenic TULV and non-pathogenic PHV, using a MOI of 1, we quantified by immunostaining the percentage of infected cells expressing the viral N protein at dpi 3 and 7 (Fig 1). These two time points were chosen since orthohantaviruses replicate slowly, allowing then the visualization of the N protein of PUUV and PHV at day 3 (dpi 2 for TULV) to titrate viruses, while day 7 post infection corresponded to the time used for the preparation of viral stocks and to evaluate whether the virus was amplified. Simian Vero E6 cells, which are susceptible to infection, due to the lack of expression of type-I IFN, were used to produce all three viral stocks and as a reference. As shown in the histogram in Fig 1A, the sole human cell lines to be infected by the three orthohantaviruses were the hepatocyte HuH7 cell line, for which the percentage of N+ cells increased through time reaching 33%, 90% and 36% for PUUV, TULV and PHV, respectively, and the THP1 cell line differentiated into macrophages, reaching, at dpi 7, 10 to 20% of infection depending on the virus. At this late time point of infection (dpi 7), the lung alveolar epithelial A549 cells were susceptible to PUUV (30% of N+ cells), while TULV and PHV could barely infect them (less than 5% of N+ cells). In contrast, the intestinal epithelial cell line, Caco-2, was mainly susceptible to TULV, reaching 75% of infected cells, while PUUV and PHV at most, gave rise to 10% of N+ cells. Interestingly, around 50% of the HUVEC were infected by PUUV and PHV but this percentage did not increase with time and these cells were scarcely susceptible to TULV infection. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Susceptibility of human and rodent cells to orthohantavirus infection. Cells grown on glass coverslips were inoculated with a MOI of 1 with the different orthohantaviruses, then used at dpi3 and dpi7 for intracellular immunofluorescence staining of the viral N protein using the A1C5 monoclonal antibody to evaluate the infectivity of PUUV (orange bars), TULV (blue bars), and PHV (green bars). The histogram in (A) shows the percentage of infected cells (N+) of human origin derived from liver (HuH7), lung (A549), intestine (Caco2), as well as differentiated monocytes (THP1dif) and vascular endothelial cells (HUVEC). The non-human primate Vero E6 cells used to prepare virus stocks were added as a control. Histogram in (B) shows the susceptibility of rodent cell lines to orthohantaviruses, including renal cells (MyglaSWRecB and BVK168) and airway epithelia (MyglaSWTrach and MyglaAECcl2) from bank vole. The results were obtained from at least three independent experiments and the error bars correspond to standard deviation to the mean. Statistical analysis between dpi3 and dpi7 was performed using Two-way ANOVA and are shown according to p-values represented by *p-value <0.0332, **p-value<0.0021, ***p-value<0.0002, ****p-value<0.0001. Non-significant variations are not marked and are further detailed in S1 Fig. Pictures in (C) show N immunofluorescence detection at dpi 3 in Vero E6, HuH7 and MyglaSWRecB cells infected by PUUV, TULV or PHV. Viral N protein is stained in green and the nucleus in blue with DAPI (4′,6-diamidino-2-phenylindole). The scale bar corresponds to 100 μm. https://doi.org/10.1371/journal.pntd.0010844.g001 In parallel, we evaluated the susceptibility to orthohantavirus infection of cell lines derived from the animal reservoir of PUUV (Fig 1B). Four cell lines have been derived from lung (MyglaAECcl2 and MyglaSWTrach) or kidney (MyglaSWRecB and BVK) of the bank vole, the natural host of PUUV. Regardless of whether they originated from the respiratory tract or the kidney, bank vole cell lines could be infected with PUUV and PHV. However, BVK168 cells were poorly infected by PUUV, while in similar conditions–at dpi7- around 30% of MyglaSWRecB cells and 40% of MyglaAECcl2 cells were found to be N+. Infection of MyglaSWTrach cells was obtained but was higher with PHV (30%) than with PUUV (10%). Importantly, TULV infection was not successful in these M. glareolus cell lines. A M. musculus derived cell line, MHS, was not infected by any of the three orthohantaviruses. Remarkably, statistical analysis revealed that the N+ proportion of the different Mygla cells only increased significantly from dpi3 to dpi7 for PUUV. Our results show that, even using an in vitro model of infection, orthohantaviruses exhibit differences in cell specificity depending on the cell type and the host from which they originated.

Bank vole cells, in contrast to HuH7 cells, release infectious PUUV particles Cell susceptibility analyses (Fig 1) revealed differences in entry and replication of orthohantaviruses in different cell lines. To further investigate these results, we compared the viral life cycle of PUUV, TULV and PHV in human and bank vole cells as compared to permissive Vero E6 cells by looking whether infection led to production of mature infectious particles. A schematic representation of the procedure used is shown in Fig 2A. We chose the human HuH7 cell line, which could be infected by the three orthohantaviruses, and the rodent MyglaSWRecB cell line, which proved to be a suitable model exhibiting a species-specific restriction of TULV infection, while being efficiently infected with PUUV and PHV. We first determined infectious titers on Vero E6 cells of the human and bank vole cell supernatants, recovered at dpi 7 following infection by each one of the three viruses as compared to Vero E6 cells (Fig 2B–2D, left panels, and S1A-C, right panels). The supernatants of HuH7 and MyglaSWRecB infected cells were also tested in secondary infections on these same human and rodent cell lines by evaluating the percentage of N+ cells at dpi 3 (Fig 2B–2D middle panels) and extrapolating an infectious titer. In order to compare the infectivity of the supernatants, the same dilutions were used in primary and secondary infections for each orthohantavirus. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Ability of human and bank vole cells to produce infectious viruses. A schematic representation of the workflow of experiments performed to evaluate the capacity of production of infectious virus by the cell lines further used in the study is shown in (A). Supernatants of Vero E6, HuH7 and MyglaSWRecB cells primarily infected (1st) at MOI 0.5 by PUUV (B), TULV (C) or PHV (D) were recovered at dpi 7 for determination of their infectious titers on Vero E6 cells (left panels) and then for their ability to secondary (2nd) infect HuH7 and MyglaSWRecB cells (middle panels). Infectious titers were calculated from the percentage of N+ cells determined by immunofluorescence staining at dpi 3 as described in the Materials and Methods section. In parallel, copies of viral RNA produced in the three cell lines infected by PUUV (B), TULV (C) and PHV (D) were quantified at dpi 3 and dpi 7 (right panels) both, from the cellular fraction (intracellular viral RNA) and from their supernatants (released viral particles). The histograms show the copy numbers of viral RNA per sample calculated based on RNA standard curves corresponding to S segments specific of each orthohantavirus. The same viral preparation was used for each virus to infect the different cell lines (input). The error bars represent the standard deviation to the mean of technical replicate of biological triplicate for each sample. In (E), an IFN-λ neutralization assay was performed. PUUV, TULV or PHV were either left untreated (NT) or were pretreated with anti-human IFN-λ1 or with anti-human IFN-λ2 antibodies prior to infection at a MOI of 0.5, then their infectious titers on Vero E6 and HuH7 human cells were compared at dpi 3. Statistical analyses were performed to compare the amount of RNA between day 3 and day 7 in the cell lysates, as well as the amount of RNA among day 3, day 7 and the input in the supernatant. For neutralization assay, infection in absence of neutralizing antibodies was compared to those in presence of either anti-IFNλ1 or anti-IFNλ2. “ns” indicates not significant differences, while the stars (*) mark significant variations as explained in Fig 1. RecB was used as an abbreviation of MyglaSWRecB in the middle B-D panels. https://doi.org/10.1371/journal.pntd.0010844.g002 While PUUV efficiently infected and produced infectious particles in Vero E6 cells with a titer of 105 IU/mL, its titer in infected HuH7 cells (Fig 2B left panel) was at the limit of detection (<102 IU/mL) and as a result did not infect the three cell lines. Interestingly MyglaSWRecB cells infected with PUUV produced infectious particles at similar level to Vero E6 with a viral titer of 1.2x105 IU/mL and could re-infect these three cell lines (middle panel). Concerning TULV, high viral titers were produced in the supernatant of infected Vero E6 cells (108 IU/mL) and TULV was infectious for HuH7 cells, but not for MyglaSWRecB cells (Figs 2C, left panel and S1), consistent with the host specificity of bank voles for PUUV and not TULV. However, although HuH7 cells were highly susceptible to TULV infection (Fig 1A) the amount of infectious virus produced in HuH7 supernatant was considerably reduced, as compared to Vero E6 cells, with a titer reduction by 2 log (106 IU/mL) and this supernatant was not able to significantly re-infect the same cell line (Fig 2C, middle panel). In the case of PHV, and similarly to what was observed with TULV the supernatant from PHV infected HuH7 cells was less infectious than the one from Vero E6 with a viral titer of 2x105 IU/mL versus 2x106 IU/mL in Vero E6 (Fig 2D, left panel). Moreover, this supernatant was barely infectious when used in secondary infection of HuH7 cells. Surprisingly, although MyglaSWRecB cells were susceptible to PHV infection (Fig 1B) its supernatant contained almost no infectious particles exhibiting a titer of 102 IU/mL as compared to 2x106 IU/mL in PHV-infected Vero E6 cells. In addition, this bank vole cells’ supernatant was not able to re-infect the three cell lines (Fig 2D, middle panel). The differences in the production of infectious particles, depending on the cell lines and virus species, highlight the different levels of cellular restriction to the achievement of the hantaviral cycles, complementing the results on the susceptibility of cells to infection. Importantly, these results altogether show that the production of infectious particles on human HuH7 cells was restricted for all three viruses, whereas three distinct outcomes were observed on bank vole cells: production of infectious PUUV particles, restriction at an early stage of TULV infection, and restriction at a final stage impairing PHV particle egress.

Quantification of viral RNA copy number in infected cells confirms the differences in orthohantavirus production associated with cell susceptibility Since the supernatants of infected cells exhibited variable infectivity, depending on the orthohantavirus used, and to validate the above observations, we tested whether viral genomes were replicating intracellularly and could be detected in the supernatant, reflecting the release of viral particles. Indeed, it could be that viral particles would be produced without being infectious due to the production of immature virions, or due to the presence in the supernatant of cytokines with antiviral activity. In this regard the virus stocks produced on Vero E6 could be associated to IFN-λ induced by orthohantavirus infection as described by Prescott et al. [42]. In order to test whether the presence of some IFN-λ secreted by Vero E6 cells could impact orthohantavirus infectivity in our experimental conditions, we performed a neutralization assay. Pre-incubation of the three viruses with anti-IFNλ1 or anti-IFNλ2 anitbodies did not significantly impact the viral growth since titers on Vero E6 cells, as well as on human HuH7 and A549 cells were not improving by such treatment (Fig 2E). Beside the determination of the viral titers produced in the supernatants of HuH7 and MyglaSWRecB cells infected either by PUUV, TULV or PHV as compared to Vero E6 (Fig 2B–2D, left panels, and S1), we quantified by RT-qPCR the number of copies of viral genomes in cell lysates, as well as those released in the cell supernatants, at dpi 3 and dpi 7 (Fig 2A and 2B–2D, right panels). The RNA copy numbers were determined by referring to standard curves of in vitro transcribed RNA corresponding to a specific sequence of each viral S segment. PUUV was replicating from dpi 3 to dpi 7 in Vero E6, HuH7 and MyglaSWRecB cells, as shown by amplification of the amount of intracellular copies of viral genomes (Fig 2B, right panel). In parallel, viral RNA was not present at day 3 in the supernatants, but clearly detectable at dpi 7 in Vero E6 (3x105 copies/mL) and in MyglaSWRecB (6.7x105 copies/mL) cells. Of note, the copy number of viral genomes in the supernatant of HuH7 cells remained low (around 105 copies/mL) and was not statistically significant, not exceeding the viral input. As expected from the much higher titers of TULV (around 108 IU/mL), as compared to PUUV (around 105 IU/mL), a large and significant number of intracellular copies of TULV genome was measured in Vero E6 (3x107 copies/mL) and HuH7 (2x107 copies/mL) cells at dpi 7 as compared to dpi 3, while viral RNA load did not increase intracellularly and was not detected in MyglaSWRecB cell supernatant (Fig 2C, right panel), confirming that these cells were not permissive to TULV. Besides, similarly to PUUV infected HuH7 cells, a low amount of TULV RNA copies was detected at dpi 7 (1.5x106 copies/mL) in HuH7 supernatant, correlating with its lower infectivity (Fig 2C, left panel) as compared to a higher copy number of TULV genome (7.5x106 copies/mL) found in Vero E6 supernatant. In the case of PHV (Fig 2D, right panel), the number of RNA copies, which was already high at dpi 3 in Vero E6 cell lysate (1.1x106 copies /mL), did not significantly increase intracellularly at dpi 7 (1.4x106 copies/mL). In contrast, the amount of viral RNA in HuH7 and MyglaSWRecB cells was significantly lower and did not increase either with time from dpi 3 to dpi 7 (around 3x105 copies/mL). Correlating with this low level of replication in HuH7 and MyglaSWRecB cells, the amount of copies of viral RNA released in the supernatant of these two cell lines was low, not exceeding the viral input (around 3x105 copies/mL) at both time points, as compared to the amount of PHV genome (106 copies/mL) in Vero E6 supernatant. This confirms the fact that PHV replication in bank vole cells did not lead to production of infectious viral particles.

Interferon response is differentially affected by orthohantavirus infection in human and bank vole cells Because viruses elicit antiviral responses that they must counteract in order to propagate, we wondered whether differential regulation of interferon might contribute to the observed differences in the life cycle of orthohantaviruses. Therefore, we quantified the production of IFNα, IFNβ and IFNλ (λ1 and λ2/λ3) mRNA by performing RT-qPCR in human (HuH7 and A549) and bank vole (MyglaSWRecB) cell lines infected with PUUV, TULV or PHV. The experiment was conducted at dpi 5 (Fig 8), which corresponds to the peak of innate immune response as shown in our previous study exploring the regulation of genes expressed in A549 human cells infected with PUUV [30]. We included this cell line in the present study for comparison. Poly-IC was used to control the level of IFN activities in human and bank vole cell lines. Although IFNα was poorly activated, the two other IFN families, IFNβ and IFNλ, were activated in human A549 cells treated with poly-IC, showing around 103-fold change in relative mRNA expression levels as compared to untreated cells. However, none of the three viruses induced IFNα, whereas PUUV and PHV, but not TULV, significantly activated IFNβ, IFNλ1 and IFNλ2/λ3 (Fig 8A). In contrast, none of these orthohantaviruses induced a significant amount of IFNs in HuH7 cells, despite the fact that activation of IFNβ, IFNλ1 and IFNλ2/λ3 occurred when poly-IC was used (Fig 8B). PPT PowerPoint slide

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TIFF original image Download: Fig 8. Regulation of IFN expression in infected human and bank vole cells. Human A549 (A) and HuH7 (B) cells and rodent MyglaSWRecB cells (C) were left non-infected (grey bars) or were infected with PUUV (orange bars), TULV (blue bars) or PHV (green bars) at a MOI of 0.5. As positive control, a condition where cells were treated with poly-IC was included (yellow bars). For each condition, the mRNA expression of the different IFN type I (IFNα and β) and type III (IFNλ1, λ2 and λ3), was compared with non-treated cells (2-ΔΔCT) by RT-qPCR. Error bars correspond to the standard deviation from the mean determined from at least three replicates of three independent samples. Statistical analysis showed non-significant (ns) and significant variations relative to non-infected cells with p-values p<0.0332 (*), p<0.0021(**), p<0.0002 (***) and p<0.0001(****). https://doi.org/10.1371/journal.pntd.0010844.g008 Little is known about bank vole transcriptome and proteome, but interestingly, a bank vole transcriptome assembly has recently been produced by a high throughput sequencing experiment [44]. We used it to search for IFNα, IFNβ and IFNλ2/λ3 mouse orthologs to design primers for amplification of these genes in the M. glareolus cell line, MyglaSWRecB. It is noteworthy that no equivalent of human λ1 (IL29) can be found in house mouse genomes. Poly-IC induced high levels of IFNβ mRNA (>103-fold change) in this cell line, whereas significant but lower levels of IFNα and IFNλ2/λ3 were activated. Therefore, the very low fold change (5 times) in IFNβ RNA expression found in MyglaSWRecB cells infected with the three orthohantaviruses (Fig 8C), could be compared to the low activation level in A549 cells. However, statistical analysis revealed that this low level of IFNβ appeared to be not significantly different from the IFNβ level in non-infected cells. This suggests that PUUV and PHV, replicating in MyglaSWRecB cells, do not induce appreciable levels of type I and type III IFNs at dpi 5, whether they release infectious particles (PUUV) or not (PHV). This is also the case of TULV, even though it does not replicate efficiently in this cell line.

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