Modulating Chikungunya and Mayaro virus induced disease severity in mice using low concentrations of anti-IFNAR1 antibodies

Modulating Chikungunya and Mayaro virus induced disease severity in mice using low concentrations of anti-IFNAR1 antibodies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Modulating Chikungunya and Mayaro virus induced disease severity in mice using low concentrations of anti-IFNAR1 antibodies Konrad Matthias Wesselmann, Léa Luciani, Gregory Moureau, Jean-Selim Driouich, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6812821/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Infectious disease research relies in large parts on in vivo models particularly for studying pathogenesis and preclinical studies. The laboratory mouse (Mus musculus) is the most widely used animal model, with a wide range of wild-type and genetically modified mouse strains available, though their accessibility and suitability may vary depending on the specific research objectives. Chikungunya virus (CHIKV), a mosquito-borne arthritogenic alphavirus, has emerged in various new regions and caused several millions of cases within the last decade. Mayaro virus (MAYV) and o’nyong-nyong virus (ONNV) are arthritogenic alphaviruses closely related to CHIKV, but each remains geographically restricted to a single region of the world. Mouse models for these viruses use either genetically modified immunodeficient mice resulting in lethal illness or footpad injection resulting in locally induced arthropathy. Here, we present a proof-of-concept study demonstrating how disease severity in mice can be modeled using sub-neutralizing concentrations of an interferon 1 receptor (IFNAR1) blocking monoclonal antibody (mAb). Seven weeks-old female C57BL/6 mice were intraperitoneally injected with varying anti-IFNAR1 antibody doses 24 or 48 hours before intraperitoneally infection with CHIKV, MAYV or ONNV. Viral loads in blood and clinical disease were evaluated the days following infection. For both CHIKV and MAYV, we observed a dose-dependent increase in disease severity with the administration of anti-IFNAR1 mAb. While a 1mg dose induced severe disease, a lower dose of 0.1mg resulted in moderate symptoms in mice, mainly facial pain expression signs, accompanied by detectable viremia in the days preceding symptom onset. We also demonstrated that viral loads in organs and serum concentrations of inflammatory cytokines and chemokines were increased for both viruses when animal received anti-IFNAR1 mAb. Finally, we observed that ONNV infection did not induce symptoms in mice, yet seroconversion occurred only in those receiving anti-IFNAR1 mAb, suggesting the antibody may increase susceptibility despite the low susceptibility of mice to ONNV. In conclusion, we provided the proof of concept that disease severity can be modulated using low concentrations of anti-IFNAR1 mAb. We employed this approach to develop a new mouse model for mild systemic CHIKV and MAYV disease. Using a widely accessible mouse strain, a commercial antibody and a common injection method this model can be easily implemented. Health sciences/Diseases/Infectious diseases Health sciences/Diseases/Infectious diseases/Viral infection Chikungunya virus Mayaro virus Mice anti-IFNAR1 immunocompromised Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Alphaviruses are positive-sense single-stranded enveloped RNA viruses of the family Togaviridae . Depending on their pathogenicity in humans, they are classified into two groups: encephalitic and arthritogenic (1). Chikungunya virus (CHIKV), Mayaro virus (MAYV) and o’nyong-nyong virus (ONNV) are antigenically related arthritogenic alphaviruses belonging to the Semliki Forest complex (2). CHIKV is of particular concern because of its recurrent re-emergence and its recent geographic expansion. Originally causing few outbreaks in Africa and Asia, CHIKV re-emerged in 2005 causing major outbreaks in the Indian Ocean, then in Southeast Asia (3). Between 2013 and 2015 several CHIKV lineages were introduced to the Americas causing large-scale epidemics (4). The adaptation of the East-Central-South African genotype (ECSA) to the Aedes albopictus mosquito vector, enabled further spread of the virus into more temperate climate zones, notably in Europe (3). Currently millions of people live in endemic areas (5). Unlike most other arboviral diseases, CHIKV infection is symptomatic in most cases (between 70 and 80%), and almost half of these people go on to develop persistent, debilitating forms of arthralgia (6). There is currently no specific anti-viral treatment for this debilitating disease. Though, the first vaccine against CHIKV received FDA approval in 2023. MAYV and ONNV also cause potentially persistent arthralgia, but their circulation is currently restricted by the geographical distribution of their respective vectors: South America for MAYV (7) and sub-Saharan Africa for ONNV (8). Thus, in these regions there is co-circulation with CHIKV. Experimental animal models are crucial for studying these viruses, particularly for analyzing their pathogenesis and evaluating the efficacy of new antiviral therapies. Murine models of arthritogenic alphaviruses are difficult to set-up because wild-type mice generally do not develop clinical signs. Several murine models for acute arthritogenic alphavirus infections have been developed. They can be attributed to three categories: footpad injection models, neonatal models and knockout mice models (9). Infection of mice using the footpad model is performed by intradermal or subcutaneous injection of the virus into the footpad of one of the hindlegs. Wilde type (WT) mice infected with CHIKV using this model show a biphasic footpad swelling in the injected foot and measurable viremia but are somewhat variable between different virus strains (10). Infection with MAYV has been reported to also induce biphasic swelling by some groups (11) and but not by others (12). ONNV does not appear to cause footpad swelling in WT-mice (12). In contrast, infection of newborn mice with CHIKV is uniformly fatal (9). Commonly used knockout (KO) mouse models include type I interferon (α/β) receptor-deficient mice (A129) and mice lacking both type I and type II interferon (γ) receptors (AG129). ONNV infection results in approximately 50% lethality in A129 mice (13), whereas CHIKV and MAYV infections lead to 100% mortality (9,14). AG129 mouse infection with any of the three viruses is lethal. KO mouse strains however are not widely available and more costly than WT mice. As an alternative approach, the blocking of mice type I interferon receptor using the monoclonal antibody (mAb) MAR1-5A3 targeting the IFNAR1 subunit of the receptor has been explored for a couple of other arboviruses such as Zika, dengue, Crimean-Congo hemorrhagic fever viruses as well as CHIKV (15–18). The antibody has a short half-life of 5 days when administered at saturating doses. This duration is reduced to 1.5 days when given at sub-saturating doses, presumably due to substantial intracellular reservoirs of IFNAR1 cycling to the surface (19). Herein, we explore the use of non-saturating doses of this anti-IFNAR1 mAb in WT mice to propose a simple and cost-effective non-severe infection model for CHIKV, MAYV, or ONNV which causes mild systemic disease. Material and methods Viruses CHIKV LR2006_OPY1 (CHIKV OPY1), MAYV UVE/MAYV/1954/TT/TC625 and ONNV UVE/ONNV/UNK/SN/Dakar-234, were provided by the European Virus Archive GLOBAL (EVAg; https://www.european-virusarchive.com/). Virus working stocks were prepared by inoculation of the virus at a multiplicity on infection (MOI) of 0.001 (CHIKV, ONNV) and 0.01 (MAYV) of a 75cm2 culture flask of confluent Vero E6 cells with MEM medium supplemented with 2.5% FBS. Viruses were harvested at first apparition of cytopathic effects, filtered (45µm), supplemented with 25mM HEPES (Sigma-Aldrich), aliquoted and stored at-80°C. To determine infectious titers of the virus solutions ten-fold serial dilutions of two unfrozen aliquots were prepared for the inoculation of confluent VeroE6 cells on two 96-well culture plates. Each dilution was performed in sextuplicate. Plates were incubated for 7 days and the absence or presence of a cytopathic effect was determined in each well. 50% tissue-culture infectious dose titers (TCID 50 ) were determined using the method described by Reed and Muench (20). All experiments with infectious virus were performed in a biosafety level 3 (BSL3) laboratory. In vivo experiments In vivo experiments were approved by the local ethical committee (C2EA—14) and the French ‘Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation’ (APAFIS #39524) and performed in accordance with the French national guidelines and the European legislation covering the use of animals for scientific purposes. This study was conducted according to ARRIVE guidelines (https://arriveguidelines.org/). Animal handling: Six weeks-old female C57BL/6 mice (strain code 027) were provided by Charles-River Laboratories. Animals were maintained in ISOcage P - Bioexclusion System (Techniplast) with unlimited access to water/food and a 14h/10h light/dark cycle. A wooden gnawing block and extra bedding material was provided as cage enrichment. Experiments started after one week of acclimatization. Animals were weighed and monitored daily for the duration of the study to detect the appearance of any clinical signs of illness and suffering. A cumulative score with points for specific symptoms was used to assess disease severity for each animal. Points were given for pilo-erection (1 point), hunched posture (1 point), orbital tightening (1 point), ear positions facing outwards away from the indicating pain (1 point), hypo-reactivity (2 points), lethargy (5 points), pale mucous membranes (5 points), moribund, generalized trembling or loss of movement control (10 points). We noticed that the weight of the mice was quite variable and small weight losses did not correlate with clinical disease or viral loads. Thus, only significant weight losses (weight losses >15% or >20%) were considered as signs of clinical disease (5 and 10 points respectively). Weight curves of individual mice are displayed in Figures S1-7. Mice with a cumulative score of 10 or more were euthanized. Injections, blood sampling and euthanasia by cervical dislocation were performed under general anesthesia obtained with isoflurane (Isoflurin®, Axience). Study design: We decided to use the simplest possible animal model: intraperitoneal injection, clinical/weight monitoring and tail blood sampling. To find an animal model representative of human alphavirus infection, we varied three parameters: the dose of virus injected (from 10 3 to 10 5 TCID 50 ), the dose of anti-IFNAR injected (from 0.1mg to 1mg initial plus 0.5mg follow-up) and the time between IFNAR injection and viral infection (24 or 48 h). Group size was calculated with an effect size of 2 and a power of 80%, resulting in 5-6 animals/group. Groups of 6 animals per group were used in all experiments, except for the groups 10 3 TCID 50 , 1mg, 48h in the pilot study on CHIKV (Figure1) and the group 10 3 TCID 50 , 0.1mg in the experiment investigating CHIKV infection with varying infectious doses and varying preceding doses of anti-IFNAR1 mAb (Figure 2) which had grouped sizes of 4 and 5 respectively. Animals of the same group were held together in a single cage. A total of 261 animals were used in this study: 34 animals were used for a pilot study on CHIKV (Figure 1); 47 animals were used for investigating CHIKV infection with varying infectious doses and varying preceding doses of anti-IFNAR1 mAb (Figure 2); 12 animals were used investigating CHIKV infection in young and mature adult mice (Figure 3); 30 animals were used for a pilot study on MAYV (Figure 4); 24 animals were used for the establishment of a MAYV infection model and for investigating MAYV infection in young and mature adult mice (Figure 5); 30 animals were used for investigating the susceptibility of mice for ONNV infection (Figure 6); and 84 animals were used for investigating viral loads in various organs and serum cytokine/chemokine concentrations after infection with the established model (Figures 7 and 8).Animals were randomly assigned to groups but confounders were not controlled. Since, the same experimenters carried out infection/treatment/clinical follow-up, it was impossible to perform a blind trial. Inexplicable death was set as exclusion criteria. No animals were excluded from the study. Anti-IFNAR1 mAb injection: Mouse anti-IFNAR1 mAb (MAR1-5A3) was purchased from Leinco Technologies Inc. Upon reception the antibody was aliquoted and stored at -80°C. The antibody was administered intraperitoneally (100µl diluted in 0.9% sodium chloride solution). Mice that received 0mg of anti-IFNAR mAb were injected with saline solution only. Infection: Seven weeks-old anesthetized animals were intraperitoneally infected with 100µL containing different doses of virus in 0.9% sodium chloride solution. Mock-infected groups were inoculated with 100µL of 0.9% sodium chloride solution. Blood sampling on live infected mice: Anesthetized animals were bled by caudal artery puncturing. Approximately 10µl of blood were diluted in 90µl of 0.9% sodium chloride solution containing 0.01M EDTA. When we monitored viremia kinetics daily for the first 4 days post-infection (dpi), for animal welfare reasons, we limited the number of blood collections per mouse. For each group, half of the mice were sampled at 1 and 3 dpi, while the other half were sampled at 2 and 4 dpi. This approach allowed for daily monitoring of viremia for each group while ensuring a 48-hour interval between blood collections for each animal. Quantitative real-time RT-PCR (RT-qPCR) assays All experiments were conducted in a molecular biology laboratory that is specifically devoted to molecular clinical diagnosis and which includes separate laboratories dedicated to each step of the procedure. Prior to PCR amplification, RNA extraction was carried out using the QIAamp 96 DNA kit and the Qiacube HT kit and the Qiacube HT (both from Qiagen) following the manufacturer's instructions. Shortly, 100µl of tissue clarified homogenates prepared as previously described (18), spiked with 10µL of internal control (bacteriophage MS2), or 50µl of 10-fold diluted blood were transferred into an S-block containing the recommended volumes of VXL, proteinase K and RNA carrier. RT-qPCR (CHIKV, MAYV or ONNV, and MS2 viral genome detection) using SuperScript® III Platinum® One-Step RT-qPCR Kit with ROX (#11732-088, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA). CHIKV (21), MAYV (under review) and ONNV (22) assay primer and probe sequences and concentrations were used as described in the literature. Quantification was provided by four log serial dilutions of an appropriate T7-generated synthetic RNA standard of known quantities (10 7 to 10 4 copies/reaction). Amplification was performed with the QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) using standard cycling parameters: 15min at 50°C, 2 min at 95°C, and 40 amplification cycles (95°C for 15 sec followed by 45sec at 60°C). Results were analyzed using QuantStudio 12K Flex Applied Biosystems software v1.2.3. Positive controls, primers and probes were provided by the European virus archive-Marseille (EVAM) under the label technological platforms of Aix-Marseille (https://evam.european-virus-archive.com/). ELISA assay Measurement of serum levels of specific IgG was performed for CHIKV and ONNV using the kit ‘Anti-Chikungunya Virus ELISA (IgG)’ (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) according to the manufacturer’s instructions. Measurement of serum levels of specific IgG was performed for MAYV using the kit ‘Anti-Mayaro Virus ELISA (IgG)’ (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) according to the manufacturer’s instructions. With both kits, and in order to detect mouse IgG, secondary antibody was replaced by Goat anti-Mouse IgG (H+L) secondary Antibody, HRP (#32430, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 in HBSS, 1% BSA. Optical density (OD) at 450nm was measured. Samples with an OD 450 >0.1 were considered positive. ELISA results are displayed in Figure S8 and Table S1. Quantification of serum Cytokine levels Serum cytokine levels were measured using Proquantum immunoassays (#A41150, #A43656, #A43658, #A44837 and #A46736, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The PCR step of the assay was performed on a BioRad CFX96TM thermal cycler, software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA). Graphical representations and statistical analysis Graphical representations and statistical analyses were performed with Graphpad Prism 9.4.1 (Graphpad software). Statistical analysis was performed using Log-Rank test and Mann-Whitney test. P-values lower than 0.05 were considered statistically significant. Statistical details for each experiment are described in the figure legends. Experimental timelines were created on biorender.com. Results Reducing anti-IFNAR1 mAb doses modulates CHIKV infection severity in mice To investigate the potential for modulating infection severity in mice following the administration of anti-IFNAR1 mAbs, groups of 7week-old female C57BL/6 mice received intraperitoneally one dose on anti-IFNAR1 mAb one or two days prior the intraperitoneal infection with doses of CHIKV strain OPY1 (genotype ECSA) ranging from 10 2 to 10 5 TCID 50 . Blood viral loads were measured in the days following infection by collecting blood samples from some animals. The mice were monitored daily for 15 days in the initial experiment and later for 21 days for signs of viral disease. The monitoring period was extended after the initial experiments due to persisting symptoms. On the basis of a clinical assessment grid detailed in the materials and methods section, survival curves for (i) mortality (based on human endpoints), (ii) onset of severe disease (score >4 at least on day) and (iii) symptomatic disease (moderate or severe) (score >1 at least on day or =1 two consecutive days) were generated. In an initial experiment, we aimed to define the two extreme conditions of our model (severe disease and mild to no disease) based on the literature (23,24) and the manufacturer’s guidelines for the anti-IFNAR1 mAb (25). The extreme doses of anti-IFNAR1 mAb were set at 0.1 and 1 mg per mouse and associated with viral doses of 10² or 10³ TCID₅₀ respectively. Both conditions were evaluated with a 24h or 48h interval between injections (Figure 1a). We also had two control groups, one of uninfected animals receiving the highest dose of anti-IFNAR alone (1 mg) and a second of animals infected with 10 3 TCID 50 but receiving no anti-IFNAR mAb. Overall, infection in mice receiving 1 mg of anti-IFNAR1 mAb 48h before the infection was more severe (66% mortality, 83% severe disease) compared to those receiving the same dose of mAb 24h prior infection (50% mortality and severe disease) (Figure 1b and c). Some mice receiving the lowest dose of anti-IFNAR1 mAb developed mild disease characterised mainly by facial expression of pain. The proportion of symptomatic mice was also higher when anti-IFNAR1 mAb was administered 48 hours before infection (66% versus 33%) (Figure 1d). Symptom onset occurred between 4 and 10 dpi. None of the mice of both control groups exhibited any clinical signs. Peak blood viral loads were observed between 2 and 4 dpi in all groups. However, viral loads exhibited important variability across all groups and time points (Figure 1e). Based on the results of this pilot experiment, we decided to retain a 48-hour interval between the injection of anti-IFNAR1 mAb and infection, while also using higher viral doses for the further development of the model. In a second experiment, we evaluated the effect of three doses of anti-IFNAR1 mAb (0.1, 0.3 and 1.0 mg per mouse) in combination with three viral doses (10 3 , 10 4 and 10 5 TCID 50 ); not all combinations have been tested, Figure 2a). We also had a group receiving no anti-IFNAR1 mAb and the highest viral dose (10 5 TCID 50 ). Overall, symptom onset occurred between 4 and 10 dpi. The two groups receiving the highest dose of anti-IFNAR1 mAb presented the highest rates of mortality (66% in both conditions) (Figure 2b). Mice receiving a viral dose of 10 3 and 10 4 TCID 50 and 0.3 mg of anti-IFNAR1 mAb presented lower rates of mortality, 33% and 50% respectively, with an additional 16% and 33% suffering from mild disease. No death or severe disease could be observed in mice receiving the lowest dose of anti-IFNAR1 (0.1 mg). However, mild disease occurred in 83%, 100% and 83% with viral doses of 10 3 , 10 4 and 10 5 TCID 50 respectively (Figure 2d). None of the mice receiving no anti-IFNAR1 mAb and the highest viral dose exhibited any clinical signs. Positive viral loads were observed in most infected animals, with a tendency for the proportion of infected animals to rise as the viral dose increased. All animals that received no anti-IFNAR1 mAbs and the highest viral dose were viremic but with viral loads not exceeding 10 8 copies/mL. Viral loads were highly variable across all groups, but this variability decreased in groups receiving higher initial viral doses. (Figure 2e). At this stage, it appeared that the administration of 0.1 mg per mouse of anti-IFNAR1 mAb, followed 48h later by an intraperitoneal infection with 10 4 or 10 5 TCID 50 of CHIKV represents the optimal experimental conditions. This regimen induced high viremia in the days following infection, resulted in moderate and transient illness in almost all mice (100% and 83.3%) (Figure 2d), and leads to seroconversion in 100% of the animals within 3 weeks (Figure 8b and Table S1). In a final experiment, we assessed the optimal condition (0.1 mg/mouse of anti-IFNAR1 monoclonal antibody administered 48 hours prior to intraperitoneal infection with 10⁵ TCID₅₀ of CHIKV) in mature adult mice, specifically 11-week-old female C57BL/6 mice (Figure 3a). A control group consisting of 7-week-old mice was included. Animals were bled at 3 days post-infection (dpi) and monitored daily for 21 days for signs of viral disease. No mortality or severe disease was observed in the 7-week-old group, although 66% of the animals exhibited mild symptoms, in line with results from the second experiment (Figure 3b). In contrast, among the 11-week-old mice, 33% developed severe disease and an additional 50% showed mild symptoms, resulting in a total of 83% of symptomatic animals (Figure 3b). Similar viral loads were detected in both groups at 3 dpi (Figure 3c). Reducing anti-IFNAR1 mAb doses modulates MAYV infection severity in mice To study the potential for modulating the severity of MAYV infection in mice after administration of anti-IFNAR1 mAbs, we used the same approach applied to CHIKV. Briefly, groups of 7 weeks-old female C57BL/6 mice received intraperitoneally anti-IFNAR1 mAb one or two days prior the intraperitoneal infection with doses of MAYV strain MAYV/1954/TT/TC625 (genotype D) ranging from 10 3 to 10 5 TCID 50 . Blood viral loads were measured in the days following infection and mice were monitored daily for 21 days. On the basis of a clinical assessment grid, survival curves were generated. Similar to the CHIKV infection model, we aimed to define the two extreme conditions of our model (severe disease and mild to no disease) in a first experiment. Doses of 0.1 and 1mg of anti-IFNAR1 mAb per mouse were associated with viral doses of 10 3 or 10 4 TCID₅₀ respectively (Figure 4a). Both conditions were evaluated with a 24h or 48h interval between injections. We also had a group receiving no anti-IFNAR1 mAb and a viral dose of 10 4 TCID 50 . A pattern similar to that of CHIKV has been observed (Figure 4). Indeed, there were more diseased mice among those receiving the anti-IFNAR antibody 48h before infection. A dose of 1 mg per mouse of anti-IFNAR1 mAb followed by a viral dose of 10 4 TCID 50 elicited death in 83% and 67% and a symptomatic disease in 100% and 83% of mice when the mAb was administrated 48h or 24h before infection respectively. A symptomatic disease (always mild, with the exception of one mouse) was induced in 67% and 50% of mice receiving 0.1mg of anti-IFNAR1 mAb 48h or 24h prior to infection, respectively. Of note, 17% of animals of the group that received no anti-IFNAR1 mAb developed a mild disease. These results suggest a higher intrinsic pathogenicity of our strain of MAYV in this model compared to CHIKV. Viral loads in animals that received anti-IFNAR1 mAb at any concentration or time point prior to infection were generally variable, but higher than in animals that received no mAb (Figure 4e). All animals that received no anti-IFNAR1 mAbs were viremic but with viral loads not exceeding 10 9 copies/mL. In a second experiment, we evaluated the effect of 0.1 mg per mouse of anti-IFNAR1 mAb with two viral doses (10 3 , and 10 5 TCID 50 ) (Figure 5a). Blood was sampled at two and four dpi. No death or severe disease was observed and symptom onset occurred between 2 and 7 dpi (Figure 5b). All the mice receiving the higher viral dose developed mild disease, in contrast to only 33% of those receiving the lower dose. Positive viral loads were observed in most infected animals with a variability which reduced in mice receiving the higher viral dose (Figure 5c). At this point, the administration of 0.1 mg per mouse of anti-IFNAR1 monoclonal antibody followed 48h later by intraperitoneal infection with 10⁵ TCID₅₀ of MAYV emerged as the optimal condition. This protocol consistently induced high viremia in the days post-infection, caused moderate and transient clinical signs in all animals, and led to seroconversion in 100% of mice within three weeks (Figure S8e and Table S1). As for CHIKV we assessed in a final experiment this optimal condition in mature adult mice (Figure 5d). A control group consisting of 7-week-old mice was included. Animals were bled at 3 dpi and monitored daily for 21 days for signs of viral disease. In contrast to our observations with CHIKV, differences were noted between young and adult mice following MAYV infection (Figure 5e and 5f). Adult mice exhibited significantly lower viral loads and a smaller proportion displayed mild clinical symptoms (33% versus 100%), although this difference did not reach statistical significance. Low susceptibility of mice to ONNV ONNV has been reported to be less virulent than CHIKV and MAYV in murine models (13,26), exhibiting lower viral loads compared to those observed with CHIKV and MAYV (12). We therefore infected animal with a high viral dose (10 5 TCID 50 ) and tested three anti-IFNAR1 mAb dosing regimens: 0.1mg, 1mg administered 48 hours prior to infection, and 1mg administered 48 hours prior to infection followed by an additional 0.5mg dose at 1dpi. An additional group with infected with 10 5 TCID 50 and 0.1mg was tested as well (Figure 6a). We also had a group of animals receiving no anti-IFNAR1 mAb and the same viral dose. We used the same experimental approach applied to CHIKV and MAYV. Groups of 7 weeks-old female C57BL/6 mice received intraperitoneally anti-IFNAR1 mAb prior the intraperitoneal infection with ONNV strain ONNV/UNK/SN/Dakar-234. Blood viral loads were measured at 2 and 4 dpi and mice were monitored daily for 21 days. On the basis of a clinical assessment grid, survival curves were generated (Figure 6b). Even with high mAb doses, the results confirmed the limited susceptibility of mice to this virus under the tested conditions. Specifically, only one mouse developed symptoms, and low and variable viremia were detected only when anti-IFNAR1 mAb were administrated regardless the dose (Figure 6c). The only notable outcome was that 100% of the mice treated with anti-IFNAR seroconverted, in contrast to only 16% in the group that received the virus alone (Figure S8g). Viral replication in organs and blood cytokine profiles following CHIKV, MAYV, and ONNV infection To evaluate our final model, we measured in a last experiment the viral replication in various organs (brain, heart, liver, spleen, small intestine and large intestine) and the serum concentrations of inflammatory cytokines and chemokines (IFN-α, IFN-γ, MCP-1, IL-6 and TNF-α) at 3 and 4 dpi (i.e., peak of viremia). We used the optimal conditions previously defined: 7 weeks-old female C57BL/6 mice received 0.1mg anti-IFNAR1 mAb followed by an intraperitoneal infection with 10 5 TCID 50 of CHIKV, MAYV or ONNV 48h later. For each virus, we had a group of animals receiving no anti-IFNAR1 mAb. We also had a control group of uninfected animals receiving no anti-IFNAR1 mAb (mock). Groups of 6 mice were euthanized at 3 and 4dpi (Figure 7a). For CHIKV and MAYV, mice that received the anti-IFNAR1 mAb exhibited increased viral loads in brain, heart, liver and spleen relative to untreated controls (Figure 7b-g). For CHIKV, significant differences were observed in all tested organs at both, 3 and 4dpi. For MAYV, significant differences could be observed in all tested organs except the small intestine at 3 dpi and in brain and liver at 4 dpi. Undetectable or very low viral loads were observed in all ONNV-infected mice, with a slight non-significant trend toward higher levels with anti-IFNAR1 mAb-treated animals (Figure 7f and g). Serum concentration of cytokines following infection were measured and compared to mock group, as well as between groups receiving anti-IFNAR1 mAb or not. For CHIKV all animals exhibited significant elevation of IFN-α, IFN-γ and MCP-1 concentrations compared to mock group at both 3 and 4dpi (Figure 8). Similarly, for MAYV concentrations of these three cytokines were elevated at least one of the two days with significant increase of concentrations observed at 4dpi for IFN-α and 3dpi for IFN-γ and MCP-1. With ONNV, a more moderate increase in IFN-α, IFN-γ and MCP-1 levels was observed. The only significant increase of cytokine concentrations after ONNV infection was IFN-α at 3dpi. For all viruses, almost all concentrations of IL-6 and TNF-α measured were below the limit of detection (Figure S10). Discussion This work proposes a simple and affordable approach to refine the pathology of mouse models of arthritogenic alphaviruses. Partial immunosuppression through low-dose administration of anti-IFNAR1 mAb was employed to modulate alphavirus-induced disease. This approach, based on a widely available mouse strain, a commercially accessible mAb, and a straightforward protocol, is easily adaptable and could be valuable for studying other viral pathogens. An interferon response, primarily involving type I IFNs, has been shown to enhance survival in vivo and effectively limit alphaviral RNA persistence, which is associated with chronic arthralgia (27). The used mAb recognizes an epitope of the IFNAR1 subunit of the receptor recognizing type I interferons. Type IFNs activate various immune cells like dendritic cells or macrophages. In other cells, a signaling cascade is initiated leading to the expression of a variety of interferon-stimulated genes promoting an antiviral state (28). In vivo , the anti-IFNAR1 mAb MAR1-5A3 induces immunosuppression by blocking type I IFN receptors rendering mice more susceptible to infection. Various models of viral infection has been described using this mAb (15,16,29,30). The dose recommended by the manufacturer to fully saturate the receptor is 2.5mg per mouse as a loading dose, with a maintenance dose of 0.5mg per week. However, most studies reported that a loading dose of 1mg is sufficient to obtain immunosuppression. Lower doses have also been described with Zika virus (0.5mg) (31) and lymphocytic choriomeningitis virus (0.25mg) (32). The pharmacokinetics of this mAb are not fully characterized, with the notable feature that its half-life is shorter at lower doses (≈ 1.5 days) compared to higher doses (≈ 7 days). According to the manufacturer, this may be due to large intracellular pools of IFNAR-1 in certain cells that cycle to the surface and bind the antibody, thereby accelerating its clearance at lower concentrations (19). In this study we used different sub-saturating doses of an IFNAR1 mAb to explore the effect on disease severity of different alphaviruses. Typically, CHIKV and MAYV do not induce any symptoms in WT mice after intraperitoneal infection (33). For both CHIKV and MAYV, we were able to induce mild disease with an antibody dose as low as 0.1 mg. Increasing the dose to 0.3 mg or 1 mg led to more severe disease, with mortality rates of 66% and 83%, respectively, indicating a dose-dependent effect. Additionally, higher viral inocula reduce intra-group variability in terms of blood viral load and clinical signs. We performed a more detailed characterization of the model using the following parameters: administration of 0.1 mg per mouse of anti-IFNAR1 mAb, followed 48 hours later by infection with CHIKV, MAYV, or ONNV at a dose of 10⁵ TCID₅₀. For CHIKV and MAYV, this model closely resembles the acute phase of infection observed in humans and non-human primates (NHPs)(34,35). It induces moderate but measurable clinical illness lasting several days. As in humans and NHP models, viremia is high and constant, and viral loads are detected in various organ. Seroconversion is observed in all animals between 2- and 3-weeks post-infection. ONNV has been described to be less pathogenic than CHIKV and MAYV in mice. A129 mice carrying null mutations in the IFN-α/β receptor present mortality rates from 50–100% depending on virus strain and dose (13,26) while CHIKV and MAYV quickly induce death (36). In this study, mice did not develop any symptoms after infection regardless of dose of mAb. Nevertheless, all mice infected with the highest dose of ONNV and receiving the anti-IFNAR1 mAb seroconverted, whereas only 16% of mice that did not receive the mAb seroconverted, indicating a potential role of the antibody in enhancing viral susceptibility. During alphavirus infections a wide range of cytokines and chemokines are upregulated including IFNs, CXC and CC chemokines, interleukins, colony stimulating factors and cytokines of the TNF superfamily(37–40). In our model, we measured various blood cytokines and chemokines at 3 and 4 dpi. We detected elevated serum concentrations of IFN-α, IFN-γ and MCP-1 in CHIKV and MAYV infected mice. IL-6 and TNF-α could not be detected. The observed levels of IFN-α, IFN-γ, and MCP-1 are largely consistent with findings from infected patients (37–40), NHP models (35,41–43), and the footpad injection mouse model (10). However, reduced MCP-1 levels have also been reported in some CHIKV-infected patients (44). Concerning IL-6 and TNF-α however, studies are discordant. Several studies detected elevated levels of one or both cytokines shortly after infection or symptom onset (37,38,40,43–46), while others did not detect significant increases of one or both of these cytokines (41–45). In our experiments, blood sampling was performed at 3 and 4 dpi, which corresponds to the period just before the onset of clinical symptoms. It is therefore possible that IL-6 and TNF-α levels increase only after symptom onset. While they are mostly known for causing arthralgia and fever, CHIKV and MAYV can cause a wide range of other clinical manifestations. In our final model, we detected high CHIKV and MAYV loads at 3 and 4 dpi in almost all sampled organs (brain, heart, liver, spleen, large- and small-intestine). This broad tropism is helped by the widespread expression of MXRA8, the receptor for arthritogenic alphaviruses (47). Studies exploring the pathophysiology for CHIKV patients with fatal outcomes found CHIKV to cause multi-organ failure affecting all tested organs (brain, heart, lung, liver, spleen and kidneys)(44,48). Though not considered to be a neurotropic virus, various articles describe neurological complications in CHIKV patients (49,50). A MCP-1 mediated trojan horse mechanism was proposed as explanation for CHIKV to cross the blood brain barrier (44). MAYV has been shown to be able to infect human astrocytes (51) and cause brain damage in rhesus macaques (42). Cardiovascular manifestations have been documented in CHIKV-infected patients most notably myocarditis (52). In rhesus macaques, MAYV has been shown to affect the heart, leading to tissue damage (42). Gastro-intestinal manifestations are common among CHIKV-infect patients (49) and in rhesus macaques, CHIKV was shown to infect several gastro-intestinal tissues and affect the gastro-intestinal microbiome (53). This study has several limitations. Although clear clinical distress is evident in the infected mice, weight loss is either minimal or not statistically significant. Pathology in joint and muscle tissues in animals infected with this model were not studied. Anti-IFNAR1 mAb and virus doses were not normalized by weight resulting in a slight variation of doses between individuals, especially in older mice. However, various mice presented facial pain signs up to the day of euthanasia at 21dpi. Even though they did not present joint swellings as impressive as observed in footpad injection mouse models, it is possible that these mice developed arthralgia. Indeed, IFN-α whose receptor is blocked by the anti-IFNAR1 mAb has been found to act early to prevent long-term persistence of arthralgia (27). Treatment with anti-INFAR1 mAb may thus not only induce more severe but also longer persisting symptoms in mice infected with arthritogenic alphaviruses. Declarations Acknowledgements We thank the technical staff of the Unit of Emerging Viruses for providing viruses and molecular biology reagents. Disclosure statement No potential conflict of interest was reported by the author(s). Funding This work was funded by Agence Nationale de la Recherche project COALITION (project number ANR-20-CE92-0054). KW PhD is funded by Agence Nationale de la Recherche project COALITION (project number ANR-20-CE92-0054) and European Union project ZOE (Grant agreement 101135094). Contributions KW, LL, AN and XdL conceived and designed the studies. KW, LL, GM, JSD and OB conducted the experiments. KW and LL performed data analysis and figure conceptualization and realization. MG provided essential resources. KW, LL and AN wrote the manuscript. All authors reviewed and approved the manuscript. Data availability Data is provided within the manuscript, supplementary information files or can be obtained upon request. References Zaid A, Burt FJ, Liu X, Poo YS, Zandi K, Suhrbier A, et al. Arthritogenic alphaviruses: epidemiological and clinical perspective on emerging arboviruses. 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Cardiology. 2021;146(3):324–34. Chen H, Shi J, Tang C, Xu J, Li B, Wang J, et al. CHIKV infection drives shifts in the gastrointestinal microbiome and metabolites in rhesus monkeys. Microbiome. 2024 Aug 30;12(1):161. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables20250530.xlsx SupplementaryFigures20250530.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6812821","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":493542657,"identity":"1a8df206-221f-4406-bab3-3e0af2fa47bc","order_by":0,"name":"Konrad Matthias Wesselmann","email":"data:image/png;base64,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","orcid":"","institution":"Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRBA)","correspondingAuthor":true,"prefix":"","firstName":"Konrad","middleName":"Matthias","lastName":"Wesselmann","suffix":""},{"id":493542658,"identity":"20b0dc8d-a0f3-4594-bd77-4aaa1e6dd0c3","order_by":1,"name":"Léa Luciani","email":"","orcid":"","institution":"Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRBA)","correspondingAuthor":false,"prefix":"","firstName":"Léa","middleName":"","lastName":"Luciani","suffix":""},{"id":493542659,"identity":"b96267f4-cb3e-4c8a-bd2e-9bec8bda6a31","order_by":2,"name":"Gregory Moureau","email":"","orcid":"","institution":"Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRBA)","correspondingAuthor":false,"prefix":"","firstName":"Gregory","middleName":"","lastName":"Moureau","suffix":""},{"id":493542663,"identity":"c899c1cb-5939-44b8-b88d-6970fc389780","order_by":3,"name":"Jean-Selim Driouich","email":"","orcid":"","institution":"Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRBA)","correspondingAuthor":false,"prefix":"","firstName":"Jean-Selim","middleName":"","lastName":"Driouich","suffix":""},{"id":493542666,"identity":"8cd8b957-bc5d-464c-960a-7759a0d3609c","order_by":4,"name":"Ornellie Bernadin","email":"","orcid":"","institution":"Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRBA)","correspondingAuthor":false,"prefix":"","firstName":"Ornellie","middleName":"","lastName":"Bernadin","suffix":""},{"id":493542669,"identity":"c365105a-8cf0-44d3-88a5-81f55ce1d83e","order_by":5,"name":"Magali Gilles","email":"","orcid":"","institution":"Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRBA)","correspondingAuthor":false,"prefix":"","firstName":"Magali","middleName":"","lastName":"Gilles","suffix":""},{"id":493542670,"identity":"264aca05-af79-49d9-8478-df313950c86f","order_by":6,"name":"Xavier de Lamballerie","email":"","orcid":"","institution":"Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRBA)","correspondingAuthor":false,"prefix":"","firstName":"Xavier","middleName":"","lastName":"de Lamballerie","suffix":""},{"id":493542671,"identity":"c4588961-11c2-4a03-a81c-18793b06e31e","order_by":7,"name":"Antoine Nougairède","email":"","orcid":"","institution":"Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRBA)","correspondingAuthor":false,"prefix":"","firstName":"Antoine","middleName":"","lastName":"Nougairède","suffix":""}],"badges":[],"createdAt":"2025-06-03 15:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6812821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6812821/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88890469,"identity":"5571782e-bcf9-4cef-9a6f-72a3cde0d6f9","added_by":"auto","created_at":"2025-08-12 12:46:04","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":223338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePilot study on CHIKV.\u003c/strong\u003e 7 weeks old C57BL/6J mice were injected high (1mg) or low (0.1mg) doses of anti-IFNAR1 mAb 24h or 48h before infection with 10\u003csup\u003e3\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e or 10\u003csup\u003e2\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e of CHIKV (\u003cstrong\u003ea\u003c/strong\u003e). Two control groups received 1mg anti-INAR1 mAb and a mock infection or 0mg anti-IFNAR1 mAb and 10\u003csup\u003e3\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e of CHIKV. Survival (\u003cstrong\u003eb\u003c/strong\u003e), appearance of severe disease (\u003cstrong\u003ec\u003c/strong\u003e) and symptomatic disease (\u003cstrong\u003ed\u003c/strong\u003e) were monitored daily after infection for 15 days. Viral loads were measured at days post-infection one and three or two and four in half the mice respectively (\u003cstrong\u003ee\u003c/strong\u003e). Corresponding serology results are displayed in Figure\u0026nbsp;S8a and Table\u0026nbsp;S1a. Data are represented as Kaplan-Meier curves (\u003cstrong\u003eb-d\u003c/strong\u003e) or mean±SD (\u003cstrong\u003ee\u003c/strong\u003e). Two-sided statistical analysis was performed using Log-rank (Mantel-Cox) test (\u003cstrong\u003eb-d\u003c/strong\u003e) (details in Table S2). *, ** and *** mean p-value ranging between 0.01−0.05, 0.05–0.001 and 0.001–0.0001 respectively. ****: p-value ≤0.0001. ns: not significant. If not otherwise indicated comparisons were made with the #-marked group.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/e292bac657e250a1b71e9559.jpeg"},{"id":88892707,"identity":"42569810-5281-4858-b35a-6ce3179cc9c6","added_by":"auto","created_at":"2025-08-12 12:54:05","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCHIKV infection with varying infectious doses and varying preceding doses of anti-IFNAR1 mAb.\u003c/strong\u003e 7 weeks old C57BL/6J mice were injected anti-IFNAR1 mAb (doses varying 0.1-1mg) 48h before infection with varying CHIKV doses (10\u003csup\u003e3\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e or 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e) (\u003cstrong\u003ea\u003c/strong\u003e). A control group received 0mg anti-IFNAR1 mAb and 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e CHIKV. Survival (\u003cstrong\u003eb\u003c/strong\u003e), appearance of severe disease (\u003cstrong\u003ec\u003c/strong\u003e) and symptomatic disease (\u003cstrong\u003ed\u003c/strong\u003e) were monitored daily after infection for 21 days. Viral loads were measured at days post-infection two and four (\u003cstrong\u003ee\u003c/strong\u003e). Corresponding serology results are displayed in Figure\u0026nbsp;S8b and Table\u0026nbsp;S1b. Data are represented as Kaplan-Meier curves (\u003cstrong\u003eb-d\u003c/strong\u003e) or mean±SD (d). Two-sided statistical analysis was performed using Log-rank (Mantel-Cox) test (\u003cstrong\u003eb-d\u003c/strong\u003e) or Shapiro–Wilk normality test followed by Student t-test, Student t-test with Welch correction or Mann-Whitney test (\u003cstrong\u003ee\u003c/strong\u003e) (details in Table S3). *, ** and *** mean p-value ranging between 0.01−0.05, 0.05–0.001 and 0.001–0.0001 respectively. ****: p-value ≤0.0001. ns: not significant. If not otherwise indicated comparisons were made with the #-marked group.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/c9904587cf3fbc21bc4270aa.jpeg"},{"id":88892717,"identity":"a97cfbd4-45b9-4128-96d4-19880bfee514","added_by":"auto","created_at":"2025-08-12 12:54:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCHIKV infection in young and mature adult mice. \u003c/strong\u003e7 or 11 weeks old C57BL/6J mice were injected with anti-IFNAR1 mAb (0.1mg) 48h before infection CHIKV (10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e) (\u003cstrong\u003ea\u003c/strong\u003e). Survival (\u003cstrong\u003eb\u003c/strong\u003e), appearance of severe disease (\u003cstrong\u003ec\u003c/strong\u003e) and symptomatic disease (\u003cstrong\u003ed\u003c/strong\u003e) were monitored daily after infection for 21 days. Viral loads were measured at day post-infection three (\u003cstrong\u003ed\u003c/strong\u003e). Corresponding serology results are displayed in Figure\u0026nbsp;S8c and Table\u0026nbsp;S1c. Data are represented as Kaplan-Meier curves (\u003cstrong\u003eb\u003c/strong\u003e) or mean±SD (\u003cstrong\u003ec\u003c/strong\u003e). Two-sided statistical analysis was performed using Log-rank (Mantel-Cox) test (\u003cstrong\u003eb\u003c/strong\u003e) or Shapiro–Wilk normality test followed by Mann-Whitney test (\u003cstrong\u003ec\u003c/strong\u003e) (details in Table S4). *, ** and *** mean p-value ranging between 0.01−0.05, 0.05–0.001 and 0.001–0.0001 respectively. ****: p-value ≤0.0001. ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/e6da87f9c135b65a941de0a5.jpeg"},{"id":88892708,"identity":"1db88b95-1dbe-46d0-99fa-47928a80f105","added_by":"auto","created_at":"2025-08-12 12:54:05","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":220435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePilot study on MAYV\u003c/strong\u003e. 7 weeks old C57BL/6J mice were injected very high (1mg) or very low (0.1mg) doses of anti-IFNAR1 mAb 24h or 48h before infection with 10\u003csup\u003e3\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e or 10\u003csup\u003e4\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e of CHIKV (\u003cstrong\u003ea\u003c/strong\u003e). A control group received 0mg anti-IFNAR1 mAb and 10\u003csup\u003e4\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e of MAYV. Survival (\u003cstrong\u003eb\u003c/strong\u003e), appearance of severe disease (\u003cstrong\u003ec\u003c/strong\u003e) and symptomatic disease (\u003cstrong\u003ed\u003c/strong\u003e) were monitored daily after infection for 17 days. Viral loads were measured at days post-infection one and three or two and four in half the mice respectively (\u003cstrong\u003ee\u003c/strong\u003e). Corresponding serology results are displayed in Figure\u0026nbsp;S8d and Table\u0026nbsp;S1d. Data are represented as Kaplan-Meier curves (\u003cstrong\u003eb-d\u003c/strong\u003e) or mean±SD (\u003cstrong\u003ee\u003c/strong\u003e). Two-sided statistical analysis was performed using Log-rank (Mantel-Cox) test (\u003cstrong\u003eb-d\u003c/strong\u003e) (details in Table S5). *, ** and *** mean p-value ranging between 0.01−0.05, 0.05–0.001 and 0.001–0.0001 respectively. ****: p-value ≤0.0001. ns: not significant. If not otherwise indicated comparisons were made with the #-marked group.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/d1bed72a0979976b6f8714c3.jpeg"},{"id":88890477,"identity":"42c6ca79-2210-402e-b2e2-8996980315f6","added_by":"auto","created_at":"2025-08-12 12:46:05","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":316209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of MAYV infection model.\u003c/strong\u003e Infection with 10\u003csup\u003e3\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e or 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e and a preceding dose of 0.1mg anti-IFNAR1 mAb (\u003cstrong\u003ea-c\u003c/strong\u003e) and MAYV infection in young and mature adult mice with a preceding dose of 0.1mg anti-IFNAR1 mAb and a high viral titer of 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e (\u003cstrong\u003ed-f\u003c/strong\u003e). 7 weeks old C57BL/6J mice were injected anti-IFNAR1 mAb (0.1mg) 48h before infection with varying MAYV doses (10\u003csup\u003e3\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e or 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e) (\u003cstrong\u003ea\u003c/strong\u003e). Survival, appearance of severe disease and symptomatic disease (\u003cstrong\u003eb\u003c/strong\u003e) were monitored daily after infection for 21 days. Viral loads were measured at days post-infection two and four (\u003cstrong\u003ec\u003c/strong\u003e). Corresponding serology results are displayed in Table\u003cem\u003e\u0026nbsp;\u003c/em\u003eS1e. 7 or 11 weeks old C57BL/6J mice were injected with anti-IFNAR1 mAb (0.1mg) 48h before infection CHIKV (10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e) (\u003cstrong\u003ed\u003c/strong\u003e). Survival, appearance of severe disease and symptomatic disease (\u003cstrong\u003ee\u003c/strong\u003e) were monitored daily after infection for 21 days. Viral loads were measured at day post-infection three (\u003cstrong\u003ef\u003c/strong\u003e). Corresponding serology results are displayed in Figure\u0026nbsp;S8f and Table\u003cem\u003e\u0026nbsp;\u003c/em\u003eS1f. Data are represented as Kaplan-Meier curves (\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e) or mean±SD (\u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e). Two-sided statistical analysis was performed using Log-rank (Mantel-Cox) test (\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e) or Shapiro–Wilk normality test followed by Mann-Whitney test (\u003cstrong\u003ef\u003c/strong\u003e) (details in Table S6). *, ** and *** mean p-value ranging between 0.01−0.05, 0.05–0.001 and 0.001–0.0001 respectively. ****: p-value ≤0.0001. ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/596bdacbf30e6d8da30fff31.jpeg"},{"id":88892709,"identity":"e587dda0-44b0-4ee5-b01c-a8b8232cc76b","added_by":"auto","created_at":"2025-08-12 12:54:05","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":165255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow susceptibility of mice for ONNV infection.\u003c/strong\u003e 7 weeks old C57BL/6J mice were injected anti-IFNAR1 mAb (doses varying 0.1-1mg) 48h before infection with ONNV (10\u003csup\u003e4\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e or 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e) (\u003cstrong\u003ea\u003c/strong\u003e). One group received 1mg anti-IFNAR1 mAb 48h pre-infection with 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e and 0.5mg 24h post-infection. Survival, appearance of severe disease and symptomatic disease were monitored daily after infection for 21 days (\u003cstrong\u003eb\u003c/strong\u003e). Viral loads were measured at days post-infection two and four (\u003cstrong\u003ec\u003c/strong\u003e). Corresponding serology results are displayed in Figure\u0026nbsp;S8g and Table\u0026nbsp;S1g. Data are represented as Kaplan-Meier curves (\u003cstrong\u003eb\u003c/strong\u003e) or mean±SD (\u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/79693a948ad2d24cdb5e2fec.jpeg"},{"id":88892715,"identity":"086efeda-decd-4378-9520-343c31858518","added_by":"auto","created_at":"2025-08-12 12:54:05","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":430808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViral loads in various organs after infection with the established model.\u003c/strong\u003e 7 weeks old C57BL/6J mice were injected with anti-IFNAR1 mAb (0.1mg) 48h before infection with CHIKV, MAYV or ONNV (10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e) (\u003cstrong\u003ea\u003c/strong\u003e). Mice were euthanized at 3 (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e) or 4 (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e) dpi and viral loads in brain, heart, liver, spleen, small intestine and large intestine were determined by RT-qPCR. Data are represented as mean±SD. Two-sided statistical analysis was performed using Shapiro–Wilk normality test followed by Student t-test, Student t-test with Welch correction or Mann-Whitney test (details in Table S7). *, ** and *** mean p-value ranging between 0.01−0.05, 0.05–0.001 and 0.001–0.0001 respectively. ****: p-value ≤0.0001. ns: not significant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/7f68735b0b2da8b0599d9e98.jpeg"},{"id":88890488,"identity":"6935138d-af72-4d49-aa41-5793c1870ece","added_by":"auto","created_at":"2025-08-12 12:46:05","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":296103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum cytokine/chemokine concentrations after infection with the established model.\u003c/strong\u003e 7 weeks old C57BL/6J mice were injected with anti-IFNAR1 mAb (0.1mg) 48h before infection with CHIKV, MAYV or ONNV (10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e). Mice were euthanized at 3 (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e) or 4 (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e) dpi and serum cytokine concentrations were determined using ProQuantum high-sensitivity immunoassays. Data are represented as mean±SD. Two-sided statistical analysis was performed using Shapiro–Wilk normality test followed by Student t-test, Student t-test with Welch correction or Mann-Whitney test (details in Table S9). *, ** and *** mean p-value ranging between 0.01−0.05, 0.05–0.001 and 0.001–0.0001 respectively. ****: p-value ≤0.0001. ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/57f6b8dca3f445d046372968.jpeg"},{"id":89375791,"identity":"22fefe82-2a5d-47b4-8d72-99b3923b00fc","added_by":"auto","created_at":"2025-08-19 11:02:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3032303,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/9350b8ee-bc4b-4b01-8f09-18d5f670731a.pdf"},{"id":88890470,"identity":"703fbe9c-22db-4394-8471-fb2b7ea460d8","added_by":"auto","created_at":"2025-08-12 12:46:05","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":61585,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables20250530.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/4eefed9018436ee9843cbce8.xlsx"},{"id":88896860,"identity":"8e3751b3-3189-4c66-81b9-a8f5a1999885","added_by":"auto","created_at":"2025-08-12 13:10:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1831484,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures20250530.docx","url":"https://assets-eu.researchsquare.com/files/rs-6812821/v1/469605c335480916f21f9ca2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modulating Chikungunya and Mayaro virus induced disease severity in mice using low concentrations of anti-IFNAR1 antibodies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlphaviruses are positive-sense single-stranded enveloped RNA viruses of the family \u003cem\u003eTogaviridae\u003c/em\u003e. Depending on their pathogenicity in humans, they are classified into two groups: encephalitic and arthritogenic (1). Chikungunya virus (CHIKV), Mayaro virus (MAYV) and o\u0026rsquo;nyong-nyong virus (ONNV) are antigenically related arthritogenic alphaviruses belonging to the Semliki Forest complex (2). CHIKV is of particular concern because of its recurrent re-emergence and its recent geographic expansion. Originally causing few outbreaks in Africa and Asia, CHIKV re-emerged in 2005 causing major outbreaks in the Indian Ocean, then in Southeast Asia (3). Between 2013 and 2015 several CHIKV lineages were introduced to the Americas causing large-scale epidemics (4). The adaptation of the East-Central-South African genotype (ECSA) to the \u003cem\u003eAedes albopictus\u003c/em\u003e mosquito vector, enabled further spread of the virus into more temperate climate zones, notably in Europe (3). Currently millions of people live in endemic areas (5). Unlike most other arboviral diseases, CHIKV infection is symptomatic in most cases (between 70 and 80%), and almost half of these people go on to develop persistent, debilitating forms of arthralgia (6). There is currently no specific anti-viral treatment for this debilitating disease. Though, the first vaccine against CHIKV received FDA approval in 2023. MAYV and ONNV also cause potentially persistent arthralgia, but their circulation is currently restricted by the geographical distribution of their respective vectors: South America for MAYV (7) and sub-Saharan Africa for ONNV (8). Thus, in these regions there is co-circulation with CHIKV.\u003c/p\u003e \u003cp\u003eExperimental animal models are crucial for studying these viruses, particularly for analyzing their pathogenesis and evaluating the efficacy of new antiviral therapies. Murine models of arthritogenic alphaviruses are difficult to set-up because wild-type mice generally do not develop clinical signs. Several murine models for acute arthritogenic alphavirus infections have been developed. They can be attributed to three categories: footpad injection models, neonatal models and knockout mice models (9). Infection of mice using the footpad model is performed by intradermal or subcutaneous injection of the virus into the footpad of one of the hindlegs. Wilde type (WT) mice infected with CHIKV using this model show a biphasic footpad swelling in the injected foot and measurable viremia but are somewhat variable between different virus strains (10). Infection with MAYV has been reported to also induce biphasic swelling by some groups (11) and but not by others (12). ONNV does not appear to cause footpad swelling in WT-mice (12). In contrast, infection of newborn mice with CHIKV is uniformly fatal (9). Commonly used knockout (KO) mouse models include type I interferon (α/β) receptor-deficient mice (A129) and mice lacking both type I and type II interferon (γ) receptors (AG129). ONNV infection results in approximately 50% lethality in A129 mice (13), whereas CHIKV and MAYV infections lead to 100% mortality (9,14). AG129 mouse infection with any of the three viruses is lethal. KO mouse strains however are not widely available and more costly than WT mice. As an alternative approach, the blocking of mice type I interferon receptor using the monoclonal antibody (mAb) MAR1-5A3 targeting the IFNAR1 subunit of the receptor has been explored for a couple of other arboviruses such as Zika, dengue, Crimean-Congo hemorrhagic fever viruses as well as CHIKV (15\u0026ndash;18). The antibody has a short half-life of 5 days when administered at saturating doses. This duration is reduced to 1.5 days when given at sub-saturating doses, presumably due to substantial intracellular reservoirs of IFNAR1 cycling to the surface (19).\u003c/p\u003e \u003cp\u003eHerein, we explore the use of non-saturating doses of this anti-IFNAR1 mAb in WT mice to propose a simple and cost-effective non-severe infection model for CHIKV, MAYV, or ONNV which causes mild systemic disease.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003ch4\u003eViruses\u003c/h4\u003e\n\u003cp\u003eCHIKV LR2006_OPY1 (CHIKV OPY1), MAYV UVE/MAYV/1954/TT/TC625 and ONNV UVE/ONNV/UNK/SN/Dakar-234, were provided by the European Virus Archive GLOBAL (EVAg; https://www.european-virusarchive.com/). Virus working stocks were prepared by inoculation of the virus at a multiplicity on infection (MOI) of 0.001 (CHIKV, ONNV) and 0.01 (MAYV) of a 75cm2 culture flask of confluent Vero E6 cells with MEM medium supplemented with 2.5% FBS. Viruses were harvested at first apparition of cytopathic effects, filtered (45\u0026micro;m), supplemented with 25mM HEPES (Sigma-Aldrich), aliquoted and stored at-80\u0026deg;C. To determine infectious titers of the virus solutions ten-fold serial dilutions of two unfrozen aliquots were prepared for the inoculation of confluent VeroE6 cells on two 96-well culture plates. Each dilution was performed in sextuplicate. Plates were incubated for 7 days and the absence or presence of a cytopathic effect was determined in each well. 50% tissue-culture infectious dose titers (TCID\u003csub\u003e50\u003c/sub\u003e) were determined using the method described by Reed and Muench (20). All experiments with infectious virus were performed in a biosafety level 3 (BSL3) laboratory.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo experiments\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e experiments were approved by the local ethical committee (C2EA\u0026mdash;14) and the French \u0026lsquo;Minist\u0026egrave;re de l\u0026rsquo;Enseignement Sup\u0026eacute;rieur, de la Recherche et de l\u0026rsquo;Innovation\u0026rsquo; (APAFIS #39524) and performed in accordance with the French national guidelines and the European legislation covering the use of animals for scientific purposes. This study was conducted according to ARRIVE guidelines (https://arriveguidelines.org/).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAnimal handling:\u003c/u\u003e Six weeks-old female C57BL/6 mice (strain code 027) were provided by Charles-River Laboratories. Animals were maintained in ISOcage P - Bioexclusion System (Techniplast) with unlimited access to water/food and a 14h/10h light/dark cycle. A wooden gnawing block and extra bedding material was provided as cage enrichment. Experiments started after one week of acclimatization. Animals were weighed and monitored daily for the duration of the study to detect the appearance of any clinical signs of illness and suffering. A cumulative score with points for specific symptoms was used to assess disease severity for each animal. Points were given for pilo-erection (1 point), hunched posture (1 point), orbital tightening (1 point), ear positions facing outwards away from the indicating pain (1 point), hypo-reactivity (2 points), lethargy (5 points), pale mucous membranes (5 points), moribund, generalized trembling or loss of movement control (10 points). We noticed that the weight of the mice was quite variable and small weight losses did not correlate with clinical disease or viral loads. Thus, only significant weight losses (weight losses \u0026gt;15% or \u0026gt;20%) were considered as signs of clinical disease (5 and 10 points respectively). Weight curves of individual mice are displayed in Figures S1-7. Mice with a cumulative score of 10 or more were euthanized. Injections, blood sampling and euthanasia by cervical dislocation were performed under general anesthesia obtained with isoflurane (Isoflurin\u0026reg;, Axience).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eStudy design:\u003c/u\u003e We decided to use the simplest possible animal model: intraperitoneal injection, clinical/weight monitoring and tail blood sampling. To find an animal model representative of human alphavirus infection, we varied three parameters: the dose of virus injected (from 10\u003csup\u003e3\u003c/sup\u003e to 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e), the dose of anti-IFNAR injected (from 0.1mg to 1mg initial plus 0.5mg follow-up) and the time between IFNAR injection and viral infection (24 or 48 h). Group size was calculated with an effect size of 2 and a power of 80%, resulting in 5-6 animals/group. Groups of 6 animals per group were used in all experiments, except for the groups 10\u003csup\u003e3\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e, 1mg, 48h in the pilot study on CHIKV (Figure1) and the group 10\u003csup\u003e3\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e, 0.1mg in the experiment investigating CHIKV infection with varying infectious doses and varying preceding doses of anti-IFNAR1 mAb (Figure 2) which had grouped sizes of 4 and 5 respectively. Animals of the same group were held together in a single cage. A total of 261 animals were used in this study: 34 animals were used for a pilot study on CHIKV (Figure 1); 47 animals were used for investigating CHIKV infection with varying infectious doses and varying preceding doses of anti-IFNAR1 mAb (Figure 2); 12 animals were used investigating CHIKV infection in young and mature adult mice (Figure 3); 30 animals were used for a pilot study on MAYV (Figure 4); 24 animals were used for the establishment of a MAYV infection model and for investigating MAYV infection in young and mature adult mice (Figure 5); 30 animals were used for investigating the susceptibility of mice for ONNV infection (Figure 6); and 84 animals were used for investigating viral loads in various organs and serum cytokine/chemokine concentrations after infection with the established model (Figures 7 and 8).Animals were randomly assigned to groups but confounders were not controlled. Since, the same experimenters carried out infection/treatment/clinical follow-up, it was impossible to perform a blind trial. Inexplicable death was set as exclusion criteria. No animals were excluded from the study.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAnti-IFNAR1 mAb injection:\u003c/u\u003e Mouse anti-IFNAR1 mAb (MAR1-5A3) was purchased from Leinco Technologies Inc. Upon reception the antibody was aliquoted and stored at -80\u0026deg;C. The antibody was administered intraperitoneally (100\u0026micro;l diluted in 0.9% sodium chloride solution). Mice that received 0mg of anti-IFNAR mAb were injected with saline solution only.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eInfection:\u003c/u\u003e Seven weeks-old anesthetized animals were intraperitoneally infected with 100\u0026micro;L containing different doses of virus in 0.9% sodium chloride solution. Mock-infected groups were inoculated with 100\u0026micro;L of 0.9% sodium chloride solution.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eBlood sampling on live infected mice:\u003c/u\u003e Anesthetized animals were bled by caudal artery puncturing. Approximately 10\u0026micro;l of blood were diluted in 90\u0026micro;l of 0.9% sodium chloride solution containing 0.01M EDTA. When we monitored viremia kinetics daily for the first 4 days post-infection (dpi), for animal welfare reasons, we limited the number of blood collections per mouse. For each group, half of the mice were sampled at 1 and 3 dpi, while the other half were sampled at 2 and 4 dpi. This approach allowed for daily monitoring of viremia for each group while ensuring a 48-hour interval between blood collections for each animal.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eQuantitative real-time RT-PCR (RT-qPCR) assays\u003c/h4\u003e\n\u003cp\u003eAll experiments were conducted in a molecular biology laboratory that is specifically devoted to molecular clinical diagnosis and which includes separate laboratories dedicated to each step of the procedure. Prior to PCR amplification, RNA extraction was carried out using the QIAamp 96 DNA kit and the Qiacube HT kit and the Qiacube HT (both from Qiagen) following the manufacturer\u0026apos;s instructions. Shortly, 100\u0026micro;l of tissue clarified homogenates prepared as previously described\u0026nbsp;(18), spiked with 10\u0026micro;L of internal control (bacteriophage MS2), or 50\u0026micro;l of 10-fold diluted blood were transferred into an S-block containing the recommended volumes of VXL, proteinase K and RNA carrier. RT-qPCR (CHIKV, MAYV or ONNV, and MS2 viral genome detection) using SuperScript\u0026reg; III Platinum\u0026reg; One-Step RT-qPCR Kit with ROX (#11732-088, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA). CHIKV\u0026nbsp;(21), MAYV (under review) and ONNV\u0026nbsp;(22)\u0026nbsp;assay primer and probe sequences and concentrations were used as described in the literature. Quantification was provided by four log serial dilutions of an appropriate T7-generated synthetic RNA standard of known quantities (10\u003csup\u003e7\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e copies/reaction). Amplification was performed with the QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) using standard cycling parameters: 15min at 50\u0026deg;C, 2 min at 95\u0026deg;C, and 40 amplification cycles (95\u0026deg;C for 15 sec followed by 45sec at 60\u0026deg;C). Results were analyzed using QuantStudio 12K Flex Applied Biosystems software v1.2.3. Positive controls, primers and probes were provided by the European virus archive-Marseille (EVAM) under the label technological platforms of Aix-Marseille (https://evam.european-virus-archive.com/).\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eELISA assay\u003c/h4\u003e\n\u003cp\u003eMeasurement of serum levels of specific IgG was performed for CHIKV and ONNV using the kit \u0026lsquo;Anti-Chikungunya Virus ELISA (IgG)\u0026rsquo; (EUROIMMUN Medizinische Labordiagnostika AG, L\u0026uuml;beck, Germany) according to the manufacturer\u0026rsquo;s instructions. Measurement of serum levels of specific IgG was performed for MAYV using the kit \u0026lsquo;Anti-Mayaro Virus ELISA (IgG)\u0026rsquo; (EUROIMMUN Medizinische Labordiagnostika AG, L\u0026uuml;beck, Germany) according to the manufacturer\u0026rsquo;s instructions. With both kits, and in order to detect mouse IgG, secondary antibody was replaced by Goat anti-Mouse IgG (H+L) secondary Antibody, HRP (#32430, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 in HBSS, 1% BSA. Optical density (OD) at 450nm was measured. Samples with an OD\u003csub\u003e450\u003c/sub\u003e\u0026gt;0.1 were considered positive. ELISA results are displayed in Figure S8 and Table S1.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eQuantification of serum Cytokine levels\u003c/h4\u003e\n\u003cp\u003eSerum cytokine levels were measured using Proquantum immunoassays (#A41150, #A43656, #A43658, #A44837 and #A46736, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s instructions. The PCR step of the assay was performed on a BioRad CFX96TM thermal cycler, software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eGraphical representations and statistical analysis\u003c/h4\u003e\n\u003cp\u003eGraphical representations and statistical analyses were performed with Graphpad Prism 9.4.1 (Graphpad software). Statistical analysis was performed using Log-Rank test and Mann-Whitney test. P-values lower than 0.05 were considered statistically significant. Statistical details for each experiment are described in the figure legends. Experimental timelines were created on biorender.com.\u003c/p\u003e"},{"header":"Results","content":"\u003ch4\u003eReducing anti-IFNAR1 mAb doses modulates CHIKV infection severity in mice\u003c/h4\u003e\n\u003cp\u003eTo investigate the potential for modulating infection severity in mice following the administration of anti-IFNAR1 mAbs, groups of 7week-old female C57BL/6 mice\u0026nbsp;received intraperitoneally one dose on\u0026nbsp;anti-IFNAR1 mAb one or two days prior the intraperitoneal infection with doses of CHIKV strain OPY1 (genotype ECSA) ranging from 10\u003csup\u003e2\u003c/sup\u003e to 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e. Blood viral loads were measured in the days following infection by collecting blood samples from some animals. The mice were monitored daily for 15 days in the initial experiment and later for 21 days for signs of viral disease. The monitoring period was extended after the initial experiments due to persisting symptoms. On the basis of a clinical assessment grid detailed in the materials and methods section, survival curves for (i) mortality (based on human endpoints), (ii) onset of severe disease (score \u0026gt;4 at least on day) and (iii) symptomatic disease (moderate or severe) (score \u0026gt;1 at least on day or =1 two consecutive days) were generated.\u003c/p\u003e\n\u003cp\u003eIn an initial experiment, we aimed to define the two extreme conditions of our model (severe disease and mild to no disease) based on the literature\u0026nbsp;(23,24)\u0026nbsp;and the manufacturer\u0026rsquo;s guidelines for the anti-IFNAR1 mAb\u0026nbsp;(25). The extreme doses of anti-IFNAR1 mAb were set at 0.1 and 1 mg per mouse and associated with viral doses of 10\u0026sup2; or 10\u0026sup3; TCID₅₀ respectively. Both conditions were evaluated with a 24h or 48h interval between injections (Figure 1a). We also had two control groups, one of uninfected animals receiving the highest dose of anti-IFNAR alone (1 mg) and a second of animals infected with 10\u003csup\u003e3\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e but receiving no anti-IFNAR mAb. Overall, infection in mice receiving 1 mg of anti-IFNAR1 mAb 48h before the infection was more severe (66% mortality, 83% severe disease) compared to those receiving the same dose of mAb 24h prior infection (50% mortality and severe disease) (Figure 1b and c). Some mice receiving the lowest dose of anti-IFNAR1 mAb developed mild disease characterised mainly by facial expression of pain. The proportion of symptomatic mice was also higher when anti-IFNAR1 mAb was administered 48 hours before infection (66% versus 33%) (Figure 1d). Symptom onset occurred between 4 and 10 dpi. None of the mice of both control groups exhibited any clinical signs. Peak blood viral loads were observed between 2 and 4 dpi in all groups. However, viral loads exhibited important variability across all groups and time points (Figure 1e). Based on the results of this pilot experiment, we decided to retain a 48-hour interval between the injection of anti-IFNAR1 mAb and infection, while also using higher viral doses for the further development of the model.\u003c/p\u003e\n\u003cp\u003eIn a second experiment, we evaluated the effect of three doses of anti-IFNAR1 mAb (0.1, 0.3 and 1.0 mg per mouse) in combination with three viral doses (10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e); not all combinations have been tested, Figure 2a). We also had a group receiving no anti-IFNAR1 mAb and the highest viral dose (10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e). Overall, symptom onset occurred between 4 and 10 dpi. The two groups receiving the highest dose of anti-IFNAR1 mAb presented the highest rates of mortality (66% in both conditions) (Figure\u0026nbsp;2b). Mice receiving a viral dose of 10\u003csup\u003e3\u003c/sup\u003e and 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e and 0.3 mg of anti-IFNAR1 mAb presented lower rates of mortality, 33% and 50% respectively, with an additional 16% and 33% suffering from mild disease. No death or severe disease could be observed in mice receiving the lowest dose of anti-IFNAR1 (0.1 mg). However, mild disease occurred in 83%, 100% and 83% with viral doses of 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e respectively (Figure 2d). None of the mice receiving no anti-IFNAR1 mAb and the highest viral dose exhibited any clinical signs. Positive viral loads were observed in most infected animals, with a tendency for the proportion of infected animals to rise as the viral dose increased. All animals that received no anti-IFNAR1 mAbs and the highest viral dose were viremic but with viral loads not exceeding 10\u003csup\u003e8\u003c/sup\u003e copies/mL. Viral loads were highly variable across all groups, but this variability decreased in groups receiving higher initial viral doses. (Figure 2e).\u003c/p\u003e\n\u003cp\u003eAt this stage, it appeared that the administration of 0.1 mg per mouse of anti-IFNAR1 mAb, followed 48h later by an intraperitoneal infection with 10\u003csup\u003e4\u003c/sup\u003e or 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of CHIKV represents the optimal experimental conditions. This regimen induced high viremia in the days following infection, resulted in moderate and transient illness in almost all mice (100% and 83.3%) (Figure 2d), and leads to seroconversion in 100% of the animals within 3 weeks (Figure 8b and Table S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn a final experiment, we assessed the optimal condition (0.1 mg/mouse of anti-IFNAR1 monoclonal antibody administered 48 hours prior to intraperitoneal infection with 10⁵ TCID₅₀ of CHIKV) in mature adult mice, specifically 11-week-old female C57BL/6 mice (Figure 3a). A control group consisting of 7-week-old mice was included. Animals were bled at 3 days post-infection (dpi) and monitored daily for 21 days for signs of viral disease. No mortality or severe disease was observed in the 7-week-old group, although 66% of the animals exhibited mild symptoms, in line with results from the second experiment (Figure\u0026nbsp;3b). In contrast, among the 11-week-old mice, 33% developed severe disease and an additional 50% showed mild symptoms, resulting in a total of 83% of symptomatic animals (Figure\u0026nbsp;3b). Similar viral loads were detected in both groups at 3 dpi (Figure\u0026nbsp;3c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReducing anti-IFNAR1 mAb doses modulates MAYV infection severity in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the potential for modulating the severity of MAYV infection in mice after administration of anti-IFNAR1 mAbs, we used the same approach applied to CHIKV. Briefly, groups of 7 weeks-old female C57BL/6 mice\u0026nbsp;received intraperitoneally\u0026nbsp;anti-IFNAR1 mAb one or two days prior the intraperitoneal infection with doses of MAYV strain MAYV/1954/TT/TC625 (genotype D) ranging from 10\u003csup\u003e3\u003c/sup\u003e to 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e. Blood viral loads were measured in the days following infection and mice were monitored daily for 21 days. On the basis of a clinical assessment grid, survival curves were generated.\u003c/p\u003e\n\u003cp\u003eSimilar to the CHIKV infection model, we aimed to define the two extreme conditions of our model (severe disease and mild to no disease) in a first experiment. Doses of 0.1 and 1mg of anti-IFNAR1 mAb per mouse were associated with viral doses of 10\u003csup\u003e3\u003c/sup\u003e or 10\u003csup\u003e4\u003c/sup\u003eTCID₅₀ respectively (Figure 4a). Both conditions were evaluated with a 24h or 48h interval between injections. We also had a group receiving no anti-IFNAR1 mAb and a viral dose of 10\u003csup\u003e4\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e. A pattern similar to that of CHIKV has been observed (Figure\u0026nbsp;4). Indeed, there were more diseased mice among those receiving the anti-IFNAR antibody 48h before infection. A dose of 1 mg per mouse of anti-IFNAR1 mAb followed by a viral dose of 10\u003csup\u003e4\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e elicited death in 83% and 67% and a symptomatic disease in 100% and 83% of mice when the mAb was administrated 48h or 24h before infection respectively. A symptomatic disease (always mild, with the exception of one mouse) was induced in 67% and 50% of mice receiving 0.1mg of anti-IFNAR1 mAb 48h or 24h prior to infection, respectively. Of note, 17% of animals of the group that received no anti-IFNAR1 mAb developed a mild disease. These results suggest a higher intrinsic pathogenicity of our strain of MAYV in this model compared to CHIKV. Viral loads in animals that received anti-IFNAR1 mAb at any concentration or time point prior to infection were generally variable, but higher than in animals that received no mAb (Figure 4e). All animals that received no anti-IFNAR1 mAbs were viremic but with viral loads not exceeding 10\u003csup\u003e9\u003c/sup\u003ecopies/mL.\u003c/p\u003e\n\u003cp\u003eIn a second experiment, we evaluated the effect of 0.1 mg per mouse of anti-IFNAR1 mAb with two viral doses (10\u003csup\u003e3\u003c/sup\u003e, and 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e) (Figure 5a). Blood was sampled at two and four dpi. No death or severe disease was observed and symptom onset occurred between 2 and 7 dpi (Figure\u0026nbsp;5b). All the mice receiving the higher viral dose developed mild disease, in contrast to only 33% of those receiving the lower dose. Positive viral loads were observed in most infected animals with a variability which reduced in mice receiving the higher viral dose (Figure\u0026nbsp;5c).\u003c/p\u003e\n\u003cp\u003eAt this point, the administration of 0.1 mg per mouse of anti-IFNAR1 monoclonal antibody followed 48h later by intraperitoneal infection with 10⁵ TCID₅₀ of MAYV emerged as the optimal condition. This protocol consistently induced high viremia in the days post-infection, caused moderate and transient clinical signs in all animals, and led to seroconversion in 100% of mice within three weeks (Figure S8e and Table S1).\u003c/p\u003e\n\u003cp\u003eAs for CHIKV we assessed in a final experiment this optimal condition in mature adult mice (Figure 5d). A control group consisting of 7-week-old mice was included. Animals were bled at 3 dpi and monitored daily for 21 days for signs of viral disease. In contrast to our observations with CHIKV, differences were noted between young and adult mice following MAYV infection (Figure 5e and 5f). Adult mice exhibited significantly lower viral loads and a smaller proportion displayed mild clinical symptoms (33% versus 100%), although this difference did not reach statistical significance.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eLow susceptibility of mice to ONNV\u003c/h4\u003e\n\u003cp\u003eONNV has been reported to be less virulent than CHIKV and MAYV in murine models\u0026nbsp;(13,26), exhibiting lower viral loads compared to those observed with CHIKV and MAYV\u0026nbsp;(12). We therefore infected animal with a high viral dose (10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e) and tested three anti-IFNAR1 mAb dosing regimens: 0.1mg, 1mg administered 48 hours prior to infection, and 1mg administered 48 hours prior to infection followed by an additional 0.5mg dose at 1dpi. An additional group with infected with 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e and 0.1mg was tested as well (Figure 6a). We also had a group of animals receiving no anti-IFNAR1 mAb and the same viral dose. We used the same experimental approach applied to CHIKV and MAYV. Groups of 7 weeks-old female C57BL/6 mice received intraperitoneally anti-IFNAR1 mAb prior the intraperitoneal infection with ONNV strain ONNV/UNK/SN/Dakar-234. Blood viral loads were measured at 2 and 4 dpi and mice were monitored daily for 21 days. On the basis of a clinical assessment grid, survival curves were generated (Figure 6b). Even with high mAb doses, the results confirmed the limited susceptibility of mice to this virus under the tested conditions. Specifically, only one mouse developed symptoms, and low and variable viremia were detected only when anti-IFNAR1 mAb were administrated regardless the dose (Figure 6c). The only notable outcome was that 100% of the mice treated with anti-IFNAR seroconverted, in contrast to only 16% in the group that received the virus alone (Figure S8g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eViral replication in organs and blood cytokine profiles following CHIKV, MAYV, and ONNV infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate our final model, we measured in a last experiment the viral replication in various organs (brain, heart, liver, spleen, small intestine and large intestine) and the serum concentrations of inflammatory cytokines and chemokines (IFN-\u0026alpha;, IFN-\u0026gamma;, MCP-1, IL-6 and TNF-\u0026alpha;)\u0026nbsp;at 3 and 4 dpi (i.e., peak of viremia). We used the optimal conditions previously defined: 7 weeks-old female C57BL/6 mice received 0.1mg anti-IFNAR1 mAb followed by an intraperitoneal infection with 10\u003csup\u003e5\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e of CHIKV, MAYV or ONNV 48h later. For each virus, we had a group of animals receiving no anti-IFNAR1 mAb. We also had a control group of uninfected animals receiving no anti-IFNAR1 mAb (mock). Groups of 6 mice were euthanized at 3 and 4dpi (Figure 7a).\u003c/p\u003e\n\u003cp\u003eFor CHIKV and MAYV, mice that received the anti-IFNAR1 mAb exhibited increased viral loads in brain, heart, liver and spleen relative to untreated controls (Figure\u0026nbsp;7b-g). For CHIKV, significant differences were observed in all tested organs at both, 3 and 4dpi. For MAYV, significant differences could be observed in all tested organs except the small intestine at 3 dpi and in brain and liver at 4 dpi. Undetectable or very low viral loads were observed in all ONNV-infected mice, with a slight non-significant trend toward higher levels with anti-IFNAR1 mAb-treated animals (Figure\u0026nbsp;7f and g).\u003c/p\u003e\n\u003cp\u003eSerum concentration of cytokines following infection were measured and compared to mock group, as well as between groups receiving anti-IFNAR1 mAb or not. For CHIKV all animals exhibited significant elevation of IFN-\u0026alpha;, IFN-\u0026gamma; and MCP-1 concentrations compared to mock group at both 3 and 4dpi (Figure 8). Similarly, for MAYV concentrations of these three cytokines were elevated at least one of the two days with significant increase of concentrations observed at 4dpi for IFN-\u0026alpha; and 3dpi for IFN-\u0026gamma; and MCP-1. With ONNV, a more moderate increase in IFN-\u0026alpha;, IFN-\u0026gamma; and MCP-1 levels was observed. The only significant increase of cytokine concentrations after ONNV infection was IFN-\u0026alpha; at 3dpi. For all viruses, almost all concentrations of IL-6 and TNF-\u0026alpha; measured were below the limit of detection (Figure S10).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis work proposes a simple and affordable approach to refine the pathology of mouse models of arthritogenic alphaviruses. Partial immunosuppression through low-dose administration of anti-IFNAR1 mAb was employed to modulate alphavirus-induced disease. This approach, based on a widely available mouse strain, a commercially accessible mAb, and a straightforward protocol, is easily adaptable and could be valuable for studying other viral pathogens.\u003c/p\u003e \u003cp\u003eAn interferon response, primarily involving type I IFNs, has been shown to enhance survival \u003cem\u003ein vivo\u003c/em\u003e and effectively limit alphaviral RNA persistence, which is associated with chronic arthralgia (27). The used mAb recognizes an epitope of the IFNAR1 subunit of the receptor recognizing type I interferons. Type IFNs activate various immune cells like dendritic cells or macrophages. In other cells, a signaling cascade is initiated leading to the expression of a variety of interferon-stimulated genes promoting an antiviral state (28). \u003cem\u003eIn vivo\u003c/em\u003e, the anti-IFNAR1 mAb MAR1-5A3 induces immunosuppression by blocking type I IFN receptors rendering mice more susceptible to infection. Various models of viral infection has been described using this mAb (15,16,29,30). The dose recommended by the manufacturer to fully saturate the receptor is 2.5mg per mouse as a loading dose, with a maintenance dose of 0.5mg per week. However, most studies reported that a loading dose of 1mg is sufficient to obtain immunosuppression. Lower doses have also been described with Zika virus (0.5mg) (31) and lymphocytic choriomeningitis virus (0.25mg) (32). The pharmacokinetics of this mAb are not fully characterized, with the notable feature that its half-life is shorter at lower doses (\u0026asymp;\u0026thinsp;1.5 days) compared to higher doses (\u0026asymp;\u0026thinsp;7 days). According to the manufacturer, this may be due to large intracellular pools of IFNAR-1 in certain cells that cycle to the surface and bind the antibody, thereby accelerating its clearance at lower concentrations (19).\u003c/p\u003e \u003cp\u003eIn this study we used different sub-saturating doses of an IFNAR1 mAb to explore the effect on disease severity of different alphaviruses. Typically, CHIKV and MAYV do not induce any symptoms in WT mice after intraperitoneal infection (33). For both CHIKV and MAYV, we were able to induce mild disease with an antibody dose as low as 0.1 mg. Increasing the dose to 0.3 mg or 1 mg led to more severe disease, with mortality rates of 66% and 83%, respectively, indicating a dose-dependent effect. Additionally, higher viral inocula reduce intra-group variability in terms of blood viral load and clinical signs.\u003c/p\u003e \u003cp\u003eWe performed a more detailed characterization of the model using the following parameters: administration of 0.1 mg per mouse of anti-IFNAR1 mAb, followed 48 hours later by infection with CHIKV, MAYV, or ONNV at a dose of 10⁵ TCID₅₀. For CHIKV and MAYV, this model closely resembles the acute phase of infection observed in humans and non-human primates (NHPs)(34,35). It induces moderate but measurable clinical illness lasting several days. As in humans and NHP models, viremia is high and constant, and viral loads are detected in various organ. Seroconversion is observed in all animals between 2- and 3-weeks post-infection. ONNV has been described to be less pathogenic than CHIKV and MAYV in mice. A129 mice carrying null mutations in the IFN-α/β receptor present mortality rates from 50\u0026ndash;100% depending on virus strain and dose (13,26) while CHIKV and MAYV quickly induce death (36). In this study, mice did not develop any symptoms after infection regardless of dose of mAb. Nevertheless, all mice infected with the highest dose of ONNV and receiving the anti-IFNAR1 mAb seroconverted, whereas only 16% of mice that did not receive the mAb seroconverted, indicating a potential role of the antibody in enhancing viral susceptibility.\u003c/p\u003e \u003cp\u003eDuring alphavirus infections a wide range of cytokines and chemokines are upregulated including IFNs, CXC and CC chemokines, interleukins, colony stimulating factors and cytokines of the TNF superfamily(37\u0026ndash;40). In our model, we measured various blood cytokines and chemokines at 3 and 4 dpi. We detected elevated serum concentrations of IFN-α, IFN-γ and MCP-1 in CHIKV and MAYV infected mice. IL-6 and TNF-α could not be detected. The observed levels of IFN-α, IFN-γ, and MCP-1 are largely consistent with findings from infected patients (37\u0026ndash;40), NHP models (35,41\u0026ndash;43), and the footpad injection mouse model (10). However, reduced MCP-1 levels have also been reported in some CHIKV-infected patients (44). Concerning IL-6 and TNF-α however, studies are discordant. Several studies detected elevated levels of one or both cytokines shortly after infection or symptom onset (37,38,40,43\u0026ndash;46), while others did not detect significant increases of one or both of these cytokines (41\u0026ndash;45). In our experiments, blood sampling was performed at 3 and 4 dpi, which corresponds to the period just before the onset of clinical symptoms. It is therefore possible that IL-6 and TNF-α levels increase only after symptom onset.\u003c/p\u003e \u003cp\u003eWhile they are mostly known for causing arthralgia and fever, CHIKV and MAYV can cause a wide range of other clinical manifestations. In our final model, we detected high CHIKV and MAYV loads at 3 and 4 dpi in almost all sampled organs (brain, heart, liver, spleen, large- and small-intestine). This broad tropism is helped by the widespread expression of MXRA8, the receptor for arthritogenic alphaviruses (47). Studies exploring the pathophysiology for CHIKV patients with fatal outcomes found CHIKV to cause multi-organ failure affecting all tested organs (brain, heart, lung, liver, spleen and kidneys)(44,48). Though not considered to be a neurotropic virus, various articles describe neurological complications in CHIKV patients (49,50). A MCP-1 mediated trojan horse mechanism was proposed as explanation for CHIKV to cross the blood brain barrier (44). MAYV has been shown to be able to infect human astrocytes (51) and cause brain damage in rhesus macaques (42). Cardiovascular manifestations have been documented in CHIKV-infected patients most notably myocarditis (52). In rhesus macaques, MAYV has been shown to affect the heart, leading to tissue damage (42). Gastro-intestinal manifestations are common among CHIKV-infect patients (49) and in rhesus macaques, CHIKV was shown to infect several gastro-intestinal tissues and affect the gastro-intestinal microbiome (53).\u003c/p\u003e \u003cp\u003eThis study has several limitations. Although clear clinical distress is evident in the infected mice, weight loss is either minimal or not statistically significant. Pathology in joint and muscle tissues in animals infected with this model were not studied. Anti-IFNAR1 mAb and virus doses were not normalized by weight resulting in a slight variation of doses between individuals, especially in older mice. However, various mice presented facial pain signs up to the day of euthanasia at 21dpi. Even though they did not present joint swellings as impressive as observed in footpad injection mouse models, it is possible that these mice developed arthralgia. Indeed, IFN-α whose receptor is blocked by the anti-IFNAR1 mAb has been found to act early to prevent long-term persistence of arthralgia (27). Treatment with anti-INFAR1 mAb may thus not only induce more severe but also longer persisting symptoms in mice infected with arthritogenic alphaviruses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eWe thank the technical staff of the Unit of Emerging Viruses for providing viruses and molecular biology reagents.\u003c/p\u003e\n\u003ch3\u003eDisclosure statement\u003c/h3\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the author(s).\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work was funded by Agence Nationale de la Recherche project COALITION (project number ANR-20-CE92-0054). KW PhD is funded by Agence Nationale de la Recherche project COALITION (project number ANR-20-CE92-0054) and European Union project ZOE (Grant agreement 101135094).\u003c/p\u003e\n\u003ch3\u003eContributions\u003c/h3\u003e\n\u003cp\u003eKW, LL, AN and XdL conceived and designed the studies. KW, LL, GM, JSD and OB conducted the experiments. KW and LL performed data analysis and figure conceptualization and realization. MG provided essential resources. KW, LL and AN wrote the manuscript. All authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eData is provided within the manuscript, supplementary information files or can be obtained upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZaid A, Burt FJ, Liu X, Poo YS, Zandi K, Suhrbier A, et al. Arthritogenic alphaviruses: epidemiological and clinical perspective on emerging arboviruses. The Lancet Infectious Diseases. 2021 May;21(5):e123\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eth ICTV report, Negative-sense RNA Viruses, Perybunyaviridae, Genus: Orthobunyavirus [Internet]. [cited 2022 Apr 7]. 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Microbiome. 2024 Aug 30;12(1):161. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chikungunya virus, Mayaro virus, Mice, anti-IFNAR1, immunocompromised","lastPublishedDoi":"10.21203/rs.3.rs-6812821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6812821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInfectious disease research relies in large parts on \u003cem\u003ein vivo\u003c/em\u003e models particularly for studying pathogenesis and preclinical studies. The laboratory mouse (Mus musculus) is the most widely used animal model, with a wide range of wild-type and genetically modified mouse strains available, though their accessibility and suitability may vary depending on the specific research objectives. Chikungunya virus (CHIKV), a mosquito-borne arthritogenic alphavirus, has emerged in various new regions and caused several millions of cases within the last decade. Mayaro virus (MAYV) and o\u0026rsquo;nyong-nyong virus (ONNV) are arthritogenic alphaviruses closely related to CHIKV, but each remains geographically restricted to a single region of the world. Mouse models for these viruses use either genetically modified immunodeficient mice resulting in lethal illness or footpad injection resulting in locally induced arthropathy. Here, we present a proof-of-concept study demonstrating how disease severity in mice can be modeled using sub-neutralizing concentrations of an interferon 1 receptor (IFNAR1) blocking monoclonal antibody (mAb). Seven weeks-old female C57BL/6 mice were intraperitoneally injected with varying anti-IFNAR1 antibody doses 24 or 48 hours before intraperitoneally infection with CHIKV, MAYV or ONNV. Viral loads in blood and clinical disease were evaluated the days following infection. For both CHIKV and MAYV, we observed a dose-dependent increase in disease severity with the administration of anti-IFNAR1 mAb. While a 1mg dose induced severe disease, a lower dose of 0.1mg resulted in moderate symptoms in mice, mainly facial pain expression signs, accompanied by detectable viremia in the days preceding symptom onset. We also demonstrated that viral loads in organs and serum concentrations of inflammatory cytokines and chemokines were increased for both viruses when animal received anti-IFNAR1 mAb. Finally, we observed that ONNV infection did not induce symptoms in mice, yet seroconversion occurred only in those receiving anti-IFNAR1 mAb, suggesting the antibody may increase susceptibility despite the low susceptibility of mice to ONNV. In conclusion, we provided the proof of concept that disease severity can be modulated using low concentrations of anti-IFNAR1 mAb. We employed this approach to develop a new mouse model for mild systemic CHIKV and MAYV disease. Using a widely accessible mouse strain, a commercial antibody and a common injection method this model can be easily implemented.\u003c/p\u003e","manuscriptTitle":"Modulating Chikungunya and Mayaro virus induced disease severity in mice using low concentrations of anti-IFNAR1 antibodies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 12:46:00","doi":"10.21203/rs.3.rs-6812821/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2f2f30a-4e7c-4004-a3f8-55ed0a6f8d80","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52410646,"name":"Health sciences/Diseases/Infectious diseases"},{"id":52410647,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"}],"tags":[],"updatedAt":"2025-08-19T10:53:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-12 12:46:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6812821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6812821","identity":"rs-6812821","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}