Tick-borne encephalitis virus’ (TBEV) geographic range and the human incidence are increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of tick-born encephalitis (TBE) in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurised raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and witness in real-time the seroconversion of recently exposed individuals and excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for the first alimentary-transmitted TBE in France, obtained its full-length genome sequence, and found that it belongs to the European subtype of TBEV. TBEV is now a notifiable human disease in France, which should facilitate surveillance of its incidence and distribution throughout France.

Bluetongue virus (BTV), an arbovirus transmitted by Culicoides biting midges, is a major concern of wild and domestic ruminants. While BTV induces type I interferon (alpha/beta interferon [IFN-α/β]) production in infected cells, several reports have described evasion strategies elaborated by this virus to dampen this intrinsic, innate response. In the present study, we suggest that BTV VP3 is a new viral antagonist of the IFN-β synthesis. Indeed, using split luciferase and coprecipitation assays, we report an interaction between VP3 and both the mitochondrial adapter protein MAVS and the IRF3-kinase IKKε. Overall, this study describes a putative role for the BTV structural protein VP3 in the control of the antiviral response.

26Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide 27 range of wild and domestic ruminants. In this report, we showed that BTV, through its virulence 28 non-structural protein NS3 (BTV-NS3), is able to activate the MAPK/ERK pathway. In 29 response to growth factors, the MAPK/ERK pathway activates cell survival, differentiation, 30 proliferation and protein translation but can also lead to the production of several inflammatory 31 cytokines. By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis 32 from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein 33 kinase B-Raf (BRAF), a crucial player of the MAPK/ERK pathway, as a new cellular interactor 34 of BTV-NS3. BRAF silencing led to a significant decrease of the MAPK/ERK activation by 35 BTV supporting a model where BTV-NS3 interacts with BRAF to activate this signaling 36 cascade. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the 37 MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be 38 specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK 39 pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its 40 own replication. Therefore, the activation of the MAPK/ERK pathway by BTV-NS3 could 41 benefit to BTV replication by promoting its own viral protein synthesis but could also explain 42 the deleterious inflammation associated with tissue damages as already observed in severe cases 43 of BT disease. Altogether, our data provide molecular mechanisms to explain the role of BTV-44 NS3 as a virulence factor and determinant of pathogenesis. 45 author/funder. All rights reserved. No reuse allowed without permission. Importance 46 47Bluetongue Virus (BTV) is responsible of the non-contagious arthropod-borne disease 48 Bluetongue (BT) transmitted to ruminants by blood-feeding midges. Despite the fact that BTV 49 has been extensively studied, we still have little understanding of the molecular determinants 50 of BTV virulence. In this report, we found that the virulence protein NS3 interacts with BRAF, 51a key component of the MAPK/ERK pathway. In response to growth factors, this pathway 52 promotes cell survival, increases protein translation but also contributes to the production of 53 inflammatory cytokines. We showed that BTV-NS3 enhances the MAPK/ERK pathway and 54 this activation is BRAF-dependent. Our results demonstrate, at the molecular level, how a 55 single virulence factor has evolved to target a cellular function to ensure its viral replication. 56On the other hand, our findings could also explain the deleterious inflammation associated with 57 tissue damages as already observed in severe cases of BT disease. 58 author/funder. All rights reserved. No reuse allowed without permission.

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection. IMPORTANCE Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of ruminants. Infections by this virus result in variable clinical outcomes depending on host and viral factors. We know that these variances largely rely on virus-host molecular interactions. To investigate these networks, our lab has developed since few years an interactomic research programme based on the yeast two-hybrid approach.To date, we have carried out the complete BTV interactome against two complementary DNA libraries, from bovine and culicoides cell lines, that allowed to the identification of about one hundred new putative cellular partners for BTV. We are now validating these interactions through biochemical and functional studies. To complete this interactomic programme, we now also combine affinity purification approach coupled to mass spectrometry. Upon immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified new cellular interactors including the serine/threonine kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway. We showed that BTV-NS3 enhances the MAPK/ERK pathway and this activation is BRAF-dependent. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate this pathway is conserved throughout multiple serotypes but appears to be specific to BTV compared to other orbiviruses. Inhibition of MAPK/ERK pathway activation with the pharmacological molecule U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.

Birds are one of the most species-diverse vertebrate groups and are susceptible to numerous hematophagous ectoparasites. Migratory birds likely contribute to the circulation of these ectoparasites and their associated pathogens. One of the many migration paths crosses the Mediterranean islands including Corsica and its wetlands, which are migration stopovers. In our study, we collected blood samples and hematophagous ectoparasites in migratory and sedentary bird populations in two coastal lagoons: Biguglia and Gradugine. A total of 1377 birds were captured from which 762 blood samples, 37 louse flies, and 44 ticks were collected. All the louse flies were identified as Ornithomya biloba and all the ticks were from the Ixodes genus: Ixodes sp. (8.5%), I. accuminatus/ventalloi (2.9%), I. arboricola/lividus (14.3%), I. frontalis (5.7%) and I. ricinus (68.6%). Five pathogens were detected: Anaplasma phagocytophilum, Erhlichia chaffeensis, and Rickettsia helvetica in ticks, and Trypanosoma sp. in louse flies. Ehrlichia chaffeensis and the West Nile virus were both detected in bird blood samples in Corsica. This is the first report of these tick, louse fly and pathogen species isolated on the bird population in Corsica. Our finding highlights the importance of bird populations in the presence of arthropod-borne pathogens in Corsican wetlands.

Tick borne encephalitis virus geographic range and human incidence is increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of TBE in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurized raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and to witness in real time the seroconversion of recently exposed individuals and the excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for as a source of dietary contamination in France, obtained its full-length genome sequence, and found that it does not cluster very closely neither with the isolate circulating in Alsace nor with any other isolate within the European lineage. TBEV is now a notifiable human disease in France, which should facilitate surveillance of TBEV incidence and distribution throughout France.