{"paper_id":"2dc2ce3c-de9d-44d2-be24-433ed70db7c1","body_text":"1 \n \nLow risk of transmission of prototype and newly emerged Oropouche 1 \nvirus strains by European Culex pipiens, Aedes albopictus, and 2 \nAnopheles atroparvus mosquitoes 3 \nAna Rosales-Rosas*1, Edith Janssens*2, Sam Verwimp1, Koen Bartholomeeusen2, Cédric Van Dun1, 4 \nKatrien Trappeniers1, Kevin K. Ariën 2,3, Ruth Müller 4, Leen Delang#1, Marco Brustolin#4 5 \n1Virus-Host Interactions & Therapeutic Approaches (VITA) Research Group, Department of 6 \nMicrobiology, Immunology and Transplantation, Rega Institute for Medical Research, University of 7 \nLeuven, Belgium 8 \n2 Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine Antwerp, Antwerp, 9 \nBelgium  10 \n3Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium 11 \n4Entomology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine Antwerp, 12 \nAntwerp, Belgium 13 \n 14 \n*Shared first author 15 \n#Shared corresponding author: Dr. Marco Brustolin, mbrustolin@itg.be; Prof. Leen Delang, 16 \nleen.delang@kuleuven.be 17 \nAbstract 18 \nOropouche virus (OROV) is an emerging arbovirus of growing public health concern, with increasing 19 \nincidence and geographic spread. Since its discovery in 1955, OROV has caused multiple outbreaks 20 \nin South and Central America, with a new introduction in Cuba since May 2024. Recent travel-related 21 \ncases in Europe and the Americas underscore its potential for global dissemination. Assessing 22 \nvector competence outside endemic regions is critical in the context of global travel and climate 23 \nchange. 24 \nWe evaluated the vector competence of three mosquito species commonly found in Europe—Culex 25 \n(Cx.) pipiens, Aedes (Ae.) albopictus, and Anopheles (An.) atroparvus—using two OROV strains: the 26 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n2 \n \nprototype TRVL9760 (1955, Trinidad and Tobago) and a recent isolate OROV-IRCCS-SCDC_1/2024 27 \n(2024, imported from Cuba to Italy). Mosquitoes were orally infected and examined at 7- and 14-days 28 \npost-infection. We assessed infection (body), dissemination (peripheral tissues), and transmission 29 \npotential (saliva) by measuring infectious virus particles using the gold standard focus-forming 30 \nassays. 31 \nOur findings show that Cx. pipiens and An. atroparvus were not susceptible to infection or did not 32 \nallow transmission with OROV. In Ae. albopictus , low infection rates were observed: 6.7% of  33 \nmosquitoes showed infection at day 7 with the prototype strain, and 3.1% at day 14 with OROV-34 \nIRCCS-SCDC_1/2024. All infected mosquitoes showed viral dissemination, but none had infectious 35 \nvirus in their saliva, indicating low risk for transmission. 36 \nThese results confirm limited vector competence of European mosquito species for OROV and 37 \nemphasize the importance of continued entomological surveillance to inform future risk 38 \nassessments. 39 \nKeywords: Oropouche virus; Europe; vector competence; transmission;  reassortant strain; 40 \nemergent arbovirus 41 \nIntroduction 42 \nOropouche virus (OROV) is an emerging arbovirus and a growing public health concern, marked by 43 \nits recent increased incidence and geographic expansion. First identified in 1955 in Trinidad and 44 \nTobago, OROV was the second most common arboviral disease in South America after dengue, until 45 \nthe emergence of chikungunya (2013) and Zika virus (2015)  (1,2). Nevertheless, only nine vector 46 \ncompetence studies have been published to date, highlighting the limited research focus of this 47 \nneglected arbovirus for which no antiviral treatments or vaccines are currently available (3–5).  48 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n3 \n \nTraditionally confined to areas near the Amazon rainforest and the Caribbean, OROV propagation 49 \nand clinical severity have intensified since December 2023. OROV disease is typically mild and self-50 \nlimiting in immunocompetent individuals with symptoms such as fever, headache, muscle and joint 51 \npain, sensitivity to light, and occasionally more severe complications like meningitis or encephalitis. 52 \n(6,7).   However, newly introduced epidemic waves in Cuba since May 2024, along with two fatal 53 \ncases in healthy Brazilian women, and multiple reports of OROV-linked miscarriages, microcephaly, 54 \nand fetal deaths, have brought renewed attention to this historically neglected arbovirus  (1,8). 55 \nAdditionally, the report of several travel -related OROV cases in countries such as Italy, Spain, 56 \nGermany, France, the United States, and Canada further underscores the virus' capacity for 57 \ninternational spread (9–11). These developments mark a notable shift in the epidemiological pattern 58 \nand clinical profile of OROV fever, raising questions on evolutionary changes of the pathogen, the 59 \nvectors involved in ongoing outbreaks, as well as potential vectors in currently non-endemic areas.  60 \nIdentifying biological adaptations of established versus newly emerged  strains is key in 61 \nunderstanding altered pathogen behavior. OROV belongs to the Orthobunyavirus genus (Simbu 62 \nserogroup), characterized by a negative -sense tri-segmented (small [S], medium [M], and large [L]) 63 \nRNA genome, known to exchange segments with other Simbu strains when co -infecting the same 64 \ncell. This reassortment drives genetic diversity and viral evolution, leading to potential changes in 65 \nvector competence and transmission dynamics  (12–14). Deiana et al. characterized the complete 66 \ngenome of a newly emerged OROV strain (OROV-IRCCS-SCDC_1/2024) isolated from a patient with 67 \nOROV-induced fever returning from Cuba to Italy in May 2024. This isolate has shown to be a 68 \nreassortant virus, containing sequences of the Brazil ‘22 -‘24 outbreak and older sequences, likely 69 \nreflecting the strain currently circulating in Cuba and Latin America  (15). Whether the observed 70 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n4 \n \ngeographic expansion and disease severity are linked to enhanced virulence in this adapted variant 71 \nis a current research priority. 72 \nFurthermore, a substantial knowledge gap remains regarding our understanding of the  reservoirs 73 \nand vectors involved in OROV transmission  (3). Several mammals, including sloths, non -human 74 \nprimates and birds, are considered potential reservoirs for sylvatic OROV maintenance, while no 75 \nvertebrates other than humans have been identified in urban transmission so far (16). The primary 76 \nvector of OROV is  considered the Culicoides midge (family Ceratopogonidae), with C. paraensis 77 \nstrongly implicated in both sylvatic and urban transmission, given its frequent presence in outbreak 78 \nzones, which is further supported by experimental data . According to the findings of Pinheiro and 79 \ncolleagues, C. paraensis midges are capable of transmitting OROV to hamsters within 6 to 12 days 80 \nfollowing a blood meal from infected individuals, with the minimum viral titer required for successful 81 \ntransmission and infection of around 5.2 log₁₀ SMLD₅₀/mL (median lethal dose of OROV in suckling 82 \nmice/mL blood) (17). In addition to biting midges, mosquitoes from the Culicidae family could also 83 \npose an important vector for OROV transmission . Although OROV has been isolated from various 84 \nanthropophilic mosquito species since its discovery , the consistently low detection rates and the 85 \nhigh experimentally determined threshold of infection (≥9.5 log10 SMLD 50/mL), suggested that 86 \nmosquitoes may not be efficient vectors  for human -to-human cycle maintenance  (2,3,18–21). 87 \nHowever, given the ongoing shifts in vector patterns driven by climate change and global mobility, it 88 \nis crucial to investigate whether vectors in non -endemic areas can support the transmission cycle 89 \nof newly emerged OROV strains. 90 \nTo address this, we evaluated the vector competence of three mosquito species commonly found 91 \nacross Europe: Ae. albopictus, Cx. pipiens, and An. atroparvus. These species were exposed to a 92 \nnewly emerged OROV strain ( OROV-IRCCS-SCDC_1/2024), isolated from a traveler returning from 93 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n5 \n \nCuba to Italy in 2024, or the prototype strain (TRVL9760) originally isolated in 1955. Mosquitoes were 94 \nexamined at 7 - and 14 -days post  infection (dpi) for evidence of infection, dissemination, and 95 \ntransmission potential using the gold standard focus-forming assay (FFA) on Vero E6 cells.  96 \nThis study expands current knowledge of OROV transmission potential in European mosquito 97 \nspecies through the comparison of a historical and an emergent strain. By generating new data on 98 \ninfection, dissemination and transmission, it provides an evidence base to guide surveillance and 99 \npreparedness in the context of OROV’s recent expansion and its relevance for international public 100 \nhealth. 101 \nMaterial and Methods 102 \nMosquito species  103 \nIn this study, three distinct mosquito species were used: Anopheles atroparvus (strain Ebre delta, 104 \ninitially collected in Catalonia, Spain 2020) (22), Culex pipiens (strain 20CPip.BE-ITM collected in 105 \nAntwerp, Belgium 2020 ) (23), and Aedes albopictus (strain 21AAlb.IT-TER collected in Terni, Italy, 106 \n2021) (24). Established Aedes and Culex laboratory strains were achieved from the initial field -107 \ncollected specimens at the Merian Insectarium of the Institute of Tropical Medicine Antwerp 108 \n(Belgium).   109 \nMosquito rearing and maintenance 110 \nEggs were placed in trays containing 1.75 L of softened water, supplemented with Novo Fect (Jbl, 111 \nNeuhoken, Germany) for Anopheles- or Koi mini-Sticks (Tetra, Melle, Germany) for Aedes and Culex 112 \nlarvae development. Colonies were kept in 30  cm × 30 cm × 30 cm cages (BugDorm-1, Megaview, 113 \nTaichung, Taiwan) with access to 10% glucose solution with 0.1% of Methylparaben (Sigma-Aldrich, 114 \nDarmstadt, Germany). All mosquito strains were reared and maintained under controlled 115 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n6 \n \nenvironmental conditions, 27  °C temperature, 80% relative humidity, in an 11.5/11.5h light/dark 116 \ncycle with 0.5h of twilight between each cycle.  117 \nOROV stock preparation 118 \nThe OROV strains used for experimental infections were provided as following: OROV -IRCCS-119 \nSCDC_1/2024, a recent isolate from a traveler returning from Cuba to Italy (2024), was kindly 120 \nprovided by Prof.  Castilleti (Department of Infectious -Tropical Diseases and Microbiology, IRCCS 121 \nSacro Cuore Don Calabria Hospital, Verona), and the prototype strain 122 \nIP/OROV/TRL9760/12/08/2009, first identified in Trinidad and Tobago (1955) and isolated in France 123 \nkindly provided by the European Virus Archive Global (EVAg, Marseille, France ). Both virus strains 124 \nwere propagated in Vero E6 cells (African Green monkey kidney cells, CRL-1586, ATCC) in Minimum 125 \nEssential Medium (MEM, Biowest, Nuaillé, France), supplemented with 2% fetal bovine serum (FBS, 126 \nSigma-Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin, 1% L -Glutamine and 1% Sodium 127 \nPyruvate (Sigma-Aldrich, St. Louis, MO, USA). In short, cells were infected with virus at a multiplicity 128 \nof infection (MOI) of 0.01 for 1h at 37°C and 5% CO 2. Medium was replaced and the culture was 129 \nfurther incubated for 24h at 37°C and 5% CO2. Supernatant was collected, aliquoted and stored at -130 \n80°C until use. Viral titer was determined by a focus forming assay (FFA) using Vero E6 cells. 131 \nPairwise distances comparison of the OROV-IRCCS-SCDC_1/2024 strain to the OROV TRVL9760 132 \nThe sequences for the strains OROV TRVL9760 (accession numbers KC759122-24) and OROV-133 \nIRCCS-SCDC_1/2024 (accession numbers PP952117-19) were retrieved from  the National Center 134 \nfor Biotechnology Information public database. Using MEGA11 (25), an alignment was built for each 135 \nOROV segment including both strains, and the pairwise distances were calculated. The percentage 136 \nof similarity at nucleotide and amino acid level was determined by using the obtained distance value 137 \nin the formula:  138 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n7 \n \n𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑖𝑑𝑒𝑛𝑡𝑖𝑡𝑦 = (1 − 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒) × 100. 139 \nVector competence assay 140 \nFive- to seven-days-old female mosquitoes were caged in 450 mL cardboard cups (~60 per species 141 \nper experiment) and starved for 18h to stimulate feeding. Mosquitoes were exposed to OROV-spiked 142 \nhuman blood (final titer of 1 x 10 6 FFU/mL) via a Hemotek feeding system (SP6W1 -3, Hemotek Ltd, 143 \nBlackburn, UK) through collagen membrane (MEM5, Hemotek Ltd, Blackburn, UK) feeding at 38°C. 144 \nAfter 45 minutes of feeding, mosquitoes were anesthetized at 4°C. Fully engorged females were 145 \nselected on ice, divided in two sub -groups and placed in clean cardboard cups. Mosquitoes were 146 \nmaintained in climatic cupboards under the same controlled environmental conditions used for the 147 \nrearing with access to cotton soaked in 10% glucose solution. At 7 and 14  dpi, mosquitoes were 148 \nanesthetized with triethylamine (Sigma-Aldrich, Darmstadt, Germany) prior to mosquito dissection. 149 \nFor each mosquito, only legs (for the OROV-IRCCS-SCDC_1/2024 strain) or legs, wings and head (for 150 \nthe  TRVL9760 strain) were dissected first and placed in a 2 ml Eppendorf tube containing 1 mL of 151 \nmosquito medium (20% FBS in Dulbecco’s phosphate -buffered saline, 50 mg/mL 152 \npenicillin/streptomycin, 50 mg/mL gentamicin, and 2.5 mg/mL fungizone) containing a zinc -plated 153 \nsteel bead (4.5 mm). Next, mosquitoes were forced to salivate into a 20 µ L pipette tip filled with 10 154 \nµl of a sucrose:FBS (1:1) solution for 30 minutes, after which saliva was ejected into a 2 m L 155 \nEppendorf tube containing 90 µ L of mosquito medium. The remaining bodies were placed in 2  mL 156 \nEppendorf tubes containing 1 m L of mosquito medium with a zinc -plated steel bead. Body - and 157 \nperipheral organs-samples were homogenized at 30 Hz for 2 minutes using a TissueLyser II (Qiagen 158 \nGmbH, Hilden, Germany) and centrifuged for 30 s at 11,000 rpm. All samples were stored at -80 °C 159 \nuntil further analysis. Each mosquito species was subjected to two technical replicates for both 160 \nOROV strains. 161 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n8 \n \nFocus forming assay  162 \nTo determine vector competence status, the infectious viral load in body, legs and saliva of individual 163 \nmosquitoes was evaluated by focus forming assay (FFA), as previously described (26–28). In short, 164 \nsamples were serially diluted tenfold and applied to a confluent monolayer of Vero E6 cells, followed 165 \nby incubation for 24 hours at 37  °C with 5% CO₂ in the presence of 1% carboxymethyl cellulose 166 \n(CMC) in MEM, sup plemented with 5% FBS . After incubation, cells  were fixed using 4% 167 \nparaformaldehyde (Sigma-Aldrich, Darmstadt, Germany) in cold 1× PBS and subsequently blocked 168 \nwith blocking buffer (3% bovine serum albumin and 0.05% Tween -20 in cold 1× PBS). 169 \nImmunostaining was conducted using a monoclonal antibody against Oropouche virus (Oropouche 170 \nvirus immune ascitic fluid, VR-1228AF, ATCC) diluted 1:1000 in blocking buffer. Following four 171 \nwashes with cold 1× PBS, an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody 172 \n(Invitrogen, Life Science, Eugene , OR, USA ) was used for detection  (1:1000 in blocking buffer ). 173 \nFluorescent foci were visualized and counted using  a Leica DMi8 fluorescence microscope (Leica 174 \nMicrosystems, Wetzlar, Germany). Viral titer was expressed as focus -forming units per sample 175 \n(FFU/sample). Infection and transmission metrics were calculated as follows: the infection rate (IR) 176 \nwas defined as the proportion of mosquitoes with virus -positive bodies among the total number of 177 \nmosquitoes analyzed following exposure to an infectious bloodmeal; the dissemination rate (DIR) 178 \nwas the proportion of mosquitoes with virus -positive legs  (for the OROV-IRCCS-SCDC_1/2024 179 \nstrain) or virus -positive legs, wings and head (for the TRVL9760 strain)  among those with virus -180 \npositive bodies; and the transmission efficiency (TE) was the proportion of mosquitoes with virus -181 \npositive saliva among the total number of mosquitoes analyzed. 182 \nStatistical analysis 183 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n9 \n \nData analysis was performed using GraphPad Prism version 10.0.2. Differences in infection rate (IR), 184 \ndissemination rate (DIR), and transmission efficiency (TE) among mosquito species and across time 185 \npoints (7 and 14 dpi). To evaluate differences in viral titers in the body, peripheral organs, and saliva 186 \nbetween time points within the same OROV strain, two-tailed Mann-Whitney U tests were employed. 187 \nStatistical significance was defined as P < 0.05. 188 \nResults 189 \nTo assess the percentage of similarity of the new isolate to the prototype  OROV strain, sequences 190 \nfor both strains corresponding to the S, M, and L segments were aligned respectively, and pairwise 191 \ncomparisons were performed to assess the percentage of divergence at nucleotide and amino acid 192 \nlevel. The  newly emerged  OROV-IRCCS-SCDC_1/2024 isolate (Accession No. PP95117.3, 193 \nPP95118.3, PP95119.3) was 5.8% and 0% divergent at nucleotide and amino acid level, respectively, 194 \nin the short (S) segment. The medium (M) segment displayed 5.2% divergence at the nucleotide level 195 \nand 1.9% at the amino acid level , while the large (L) segment was 10% divergent at the nucleotide 196 \nlevel and 5% at the amino acid level (Table 1). 197 \n 198 \nTable 1.  Nucleotide and amino acid sequence pairwise comparisons of the OROV -IRCCS-199 \nSCDC_1/2024 strain with the prototype OROV TRVL9760 strain.  Segments are presented as S 200 \n(small), M (medium), and L (large), with the corresponding protein in square brackets. RdRp: RNA -201 \ndependent RNA polymerase. 202 \n  Divergence (%) \nStrain  S [Nucleoprotein] M [Membrane] L [RdRp] \nOROV-IRCCS-\nSCDC_1/2024 \nNucleotide 5.8 5.2 10.0 \nAmino acid 0.0 1.9 5.0 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n10 \n \nWith the prototype OROV strain (TRVL9760), Ae. albopictus mosquitoes showed an infection rate of 203 \n6.67% (3/45 mosquitoes) at 7 dpi. All three TRVL9760-infected mosquitoes showed a disseminated 204 \ninfection, while no infectious virus could be detected in their saliva  (Table 2, Figure 1) . The same 205 \nTRVL9760-infected Ae. albopictus mosquitoes had a mean viral titer of 45.6 FFU/sample in the body 206 \n7 days post infection , and a mean viral titer of 1.22×102 FFU/sample in the head, wings, and legs  207 \n(Figure 2). However, no infected mosquitoes could be detected at 14 dpi with this virus strain.  208 \n 209 \nTable 2.  Experimental infection, dissemination, and transmission outcomes in Aedes 210 \nalbopictus, Culex pipiens, and Anopheles atroparvus mosquitoes following an artificial OROV-211 \ninfectious blood meal. IR= Infection rate; DIR= Dissemination rate; TE= Transmission efficiency; n= 212 \nnumber of mosquitoes tested 213 \nStrain Input \nPFU/ml Species \n7 dpi 14 dpi \nIR \n(n) \nDIR  \n(n) \nTE \n (n) \nIR \n(n) \nDIR  \n(n) \nTE  \n(n) \nOROV-IRCCS-\nSCDC_1/2024  1×106 \nAe. albopictus 0 (76) 0 (76) 0 (76) 3.12 (64) 100 (2) 0 (64) \nCx. pipiens 0 (21) 0 (21) 0 (21) 0 (31) 0 (31) 0 (31) \nAn. atroparvus 0 (22) 0 (22) 0 (22) 0 (19) 0 (19) 0 (19) \n  Ae. albopictus 6.67 (45)  100 (3)  0 (45)  0 (22)   0 (22)  0 (22)  \nTRVL9760 \n(prototype) 1×106 Cx. pipiens 0 (40)  0 (40)  0 (40)  0 (42)  0 (42)  0 (42)  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n11 \n \nOn the contrary, infection with the newly emerged OROV strain (OROV-IRCCS-SCDC_1/2024) in Ae. 214 \nalbopictus could only be detected at 14 dpi, with an infection rate of 3.12% (2/64 mosquitoes). The 215 \ntwo OROV-IRCCS-SCDC_1/2024-infected Ae. albopictus mosquitoes show ed a disseminated 216 \ninfection, but no transmission of the virus (Table 2, Figure 1). These infected mosquitoes had a mean 217 \nviral titer of  15.1 FFU/sample in the body  at 14 days post infection , and a mean viral titer of 3.15 218 \nFFU/sample in the legs (Figure 2). None of the Cx. pipiens mosquito bodies or peripheral organs were 219 \npositive for the TRVL9760 strain, nor for the OROV-IRCCS-SCDC_1/2024 strain at either 7 or 14 dpi. 220 \nLikewise, An. atroparvus exhibited a lack of vector competence for the newly emerged OROV-IRCCS-221 \nSCDC_1/2024 strain.  222 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n12 \n \nFigure 1. Vector competence of Culex pipiens, Aedes albopictus, and Anopheles atroparvus for 223 \nOROV strains (A) TRVL9760  and (B) OROV-IRCCS-SCDC_1/2024. The bars indicate the infection 224 \nrate corresponding to mosquito bodies (blue), dissemination to peripheral organs (red), and 225 \ntransmission potential in the saliva (green) by means of focus forming assay.  The labels above the 226 \nbars represent the number of positive mosquitoes over the total number of mosquitoes. 227 \n 228 \n229 \nFigure 2. Viral titer (FFU/sample) in the bodies, peripheral organs ((head, wings, and) legs) of Aedes 230 \nalbopictus mosquitoes infected with the OROV TRVL -9760 and OROV-IRCCS-SCDC_1/2024 strains 231 \nat 7 and 14 dpi. For the OROV TRVL9760 strain, head, wings, and legs were included as proxy for 232 \ndissemination in peripheral organs, while only legs were included for the OROV -IRCCS-SCDC_1/2024 233 \nstrain. Each symbol represents an individual mosquito sample tested by means of a focus forming assay. 234 \nThe bars represent the mean with SD. To evaluate differences in viral titers in the body, peripheral 235 \norgans, and saliva between time points within the same OROV strain, two -tailed Mann-Whitney U 236 \ntests were employed. Statistical significance was defined as P < 0.05. 237 \n 238 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n13 \n \nDiscussion 239 \nOur findings demonstrate a low vector competence of Cx. pipiens, Ae. albopictus and An. atroparvus 240 \npresent in Europe for both the prototype and newly emerged OROV strains. Such results are relevant 241 \nfrom an epidemiological perspective given their wide dispersal across Southern and more temperate 242 \nNorthern Europe , incl.  in Belgium. Culex pipiens  mosquitoes are native to Belgium and often 243 \ncompose the most abundant species in entomological surveys (29,30). Likewise, An. atroparvus is 244 \nalso a Belgian native mosquito ; however, its sightings have been scarce in the last nationwide 245 \nentomological survey carried out (30). Additionally, Ae. albopictus mosquitoes have drawn more 246 \nattention in recent years as an invasive species in Belgium, with dedicated yearly efforts to monitor 247 \ntheir presence, as they are known vectors of clinically important arboviruses, including dengue and 248 \nchikungunya virus  (30,31).   249 \nUnlike molecular methods detecting viral RNA without indicating infectivity, we employed the gold-250 \nstandard focus-forming assay (FFA) on Vero E6 cells, directly measuring infectious virus particles, 251 \nhence providing accurate assessment of the transmission potential. 252 \nOur results align with a previous study assessing the vector competence of Cx. pipiens mosquitoes 253 \nfrom the USA, where no infection, dissemination or transmission of the prototype OROV strain was 254 \nreported 14 days post infection  (32). Nevertheless, these mosquitoes were able to transmit OROV 255 \n240023, a strain originally isolated from a febrile patient from Cuba, albeit to a low extent ( 1 out of 256 \n50 mosquitoes).  On the contrary, Cx. pipiens  mosquito populations from the UK were not 257 \nsusceptible to the infection with th is OROV 240023 strain (33). Such outcomes hint not only at a 258 \nstrain-specific variability in vector competence, but also at a geographic variation. Furthermore, a 259 \nrecent study reported a lack of vector competence of Italian Cx. pipiens mosquitoes (derived from 260 \nfield populations collected in Rome ) for the OROV-IRCCS-SCDC_1/2024 strain. These mosquitoes 261 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n14 \n \ndid not show infection or dissemination at 7 nor 14 days post infection (34), which concurs with our 262 \nfindings.  263 \nVariations in the kinetics of infection in Ae. albopictus mosquitoes were noticed in our study between 264 \nthe employed OROV strains. The mosquitoes that received the prototype OROV strain in the blood 265 \nmeal exhibited infection (6.67%, n= 45) and dissemination (100%, n= 3) already at 7 days post 266 \ninfection, whereas those that received  the newly emerged OROV-IRCCS-SCDC_1/2024 strain 267 \nshowed infection (3.12%, n= 64) and dissemination (100%, n= 2) only at 14 days post infection. Payne 268 \net al. reported a general low vector competence of Ae. albopictus (US population) for the prototype 269 \nOROV strain, with only 1 out of 50 mosquitoes (2%)  having a disseminated infection, but no 270 \ntransmission at 14 days post infection (32). On the other hand , Jansen et al.  have described Ae. 271 \nalbopictus mosquitoes (population from Heidelberg, Germany) transmitting the prototype OROV 272 \nstrain at 14 and 21  days post infection, when incubated at both 24 °C and 27 °C  (35). Our findings 273 \nregarding the prototype OROV infection kinetics in Ae. albopictus differ from what is reported by 274 \nPayne et al. (32) and Jansen et al. (35), as infection and dissemination were detected earlier in our 275 \nstudy (7 dpi), and there was no virus transmission. However, the Ae. albopictus mosquitoes from our 276 \nstudy have originated in Italy ; therefore,  we should consider that vector competence might be 277 \ninfluenced by the possible genetic heterogeneity presented among these mosquito populations, as 278 \nit has been observed previously for Ae. aegypti populations (36).  279 \nConcerning the newly emerged OROV-IRCCS-SCDC_1/2024 strain, Mancuso et al. found that Italian 280 \nAe. albopictus (derived from field populations collected in Rome)  were infected with OROV when 281 \nsampled at day s 7 and 21 post infection  (one infected mosquito out of 20 for each time point) ; 282 \nhowever, no dissemination, nor transmission was detected (37). Likewise, our study employed Ae. 283 \nalbopictus mosquitoes derived from an Italian population (collected in Terni) and we did not detect 284 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n15 \n \ntransmission for the newly emerged OROV strain, yet we did observe dissemination at 14 days post 285 \ninfection.  286 \nPrevious studies have highlighted the need to reconsider the role of Anopheles mosquitoes as 287 \npotential vectors of emerging arboviruses, such as Mayaro virus (38). Payne and colleagues reported 288 \na low infection rate (IR = 4%) in An. quadrimaculatus from the United States with Oropouche virus 289 \n(OROV), with no evidence of viral dissemination or transmission. In our study, we did not detect 290 \ninfection or dissemination in a European population of An. atroparvus, suggesting that this species 291 \nis unlikely to play a role in the transmission cycle of OROV.  292 \nRegardless of the time point post infection, the viral titer s of OROV in the bodies of Ae. albopictus 293 \nmosquitoes were similar between the two strains (Figure 2). Our study also reports on the amount of 294 \ninfectious virus particles detected in Ae. albopictus  bodies and peripheral organs following an 295 \nOROV-infectious blood meal. Other vector competence studies have described that the average 296 \nOROV viral titer detected in Cx. tarsalis bodies 10 days post infection was 31 PFU/mL, whereas the 297 \nviral titers for Cx. quinquefasciatus bodies and leg tissues were 128 and 37 PFU/mL, respectively. At 298 \n14 days post infection,  Cx. quinquefasciatus bodies displayed an average viral titer of 88 PFU/m L, 299 \nwhile leg tissues showed an average titer of 100 PFU/mL (39).  300 \nAll mosquito species tested showed a poor competence for either the prototype TRVL9760 or the 301 \nOROV-IRCCS-SCDC_1/2024 isolate. When comparing the newly emerged OROV-IRCCS-302 \nSCDC_1/2024 strain to the prototype, we found that the highest divergence at the nucleotide level 303 \n(10%) presented in the L segment, corresponding to the RdRp. This OROV-IRCCS-SCDC_1/2024 304 \nstrain has been described as a reassortant virus, with the S and L segments sharing high similarity 305 \nwith an emerging cluster of sequences that are most likely related to the  recent outbreaks in South 306 \nAmerica (40). Despite the genetic variations with the prototype OROV strain and the newly emerged 307 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n16 \n \nstrain belonging to a divergent OROV cluster, we did not observe  a different outcome in vector 308 \ncompetence for the OROV-IRCCS-SCDC_1/2024 strain in our study, for both Ae. albopictus and Cx. 309 \npipiens mosquitoes. Although OROV infectious virions were detected in the bodies and peripheral 310 \norgans of Ae. albopictus mosquitoes, virus was not found in the saliva of any of these mosquitoes, 311 \nsuggesting the presence of a strong barrier to OROV transmission  for the tested strains. 312 \nNevertheless, the observed viral replication of OROV in certain mosquitoes should not be neglected 313 \nand underscores the potential for arboviral adaptation and emergence, highlighting the importance 314 \nof continued surveillance. 315 \nDepending on the mosquito -virus combination, typically a higher viral titer in the blood meal can 316 \nyield a higher percentage of infected mosquitoes (41). While the infectious blood meal in our study 317 \ncontained 1×10⁶ PFU/mL of OROV —a dose within the range typically used in other studies  318 \n(32,33,42)—we cannot exclude the possibility that a higher viral dose might have resulted in greater 319 \nviral titers in mosquito bodies and, consequently, altered the transmission potential of Ae. 320 \nalbopictus. Further resea rch assessing the effect of several OROV doses on mosquito vector 321 \ncompetence could give a more comprehensive understanding of these infection dynamics. However, 322 \nthe viral input utilized falls on the upper end of the range of viremia detected in OROV -infected 323 \nhumans (6×10^3 and 7×10^5 PFU/ml  (43,44)), and therefore, the mosquitoes were exposed to an 324 \nepidemiologically relevant dose of the virus through the blood meal in this study. 325 \nA discrepancy in  our study is that dissemination was determined based on different  mosquito 326 \ntissues per strain. The legs were used as a proxy for dissemination for the OROV-IRCCS-327 \nSCDC_1/2024 infected mosquitoes, while head, wings, and legs  were employed for the prototype 328 \nTRVL-9760. Consequently, the viral titer measured in the OROV-IRCCS-SCDC_1/2024 infected 329 \nmosquito legs was lower compared to when using the head, wings, and legs together  for the 330 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n17 \n \nprototype infected mosquitoes (Figure 2). Both tissue types are valid approaches for the assessment 331 \nof virus dissemination within the mosquito body, as they represent secondary organs that become 332 \ninfected once the viral infection overcomes the midgut barrier. W e could observe that  the 333 \npercentage of virus dissemination between both OROV strains remained comparable. As such, this 334 \nfluctuation in viral titers resulted most likely due to the quantity of tissue tested , and it was further 335 \ndeemed not statistically significant; however, we should remark the low number of mosquitoes that 336 \ngot infected  (OROV-IRCCS-SCDC_1/2024, n=2; TRVL-9760, n=3), and thus take this into 337 \nconsideration during comparison.  338 \nSimilarly to mosquitoes, OROV infection in Culicoides paraensis midges has been shown to be dose 339 \ndependent. However, their threshold for infection is reportedly lower than for mosquitoes, with C. 340 \nparaensis midges exposed to OROV doses higher than  5.2 log 10 SMLD50/mL becoming infected  341 \n(≥13%) and capable of transmitting the virus (≥40%) (45).  342 \nMonitoring of these vector populations in Europe has been triggered during the past decade due to 343 \nbluetongue and Schmallenberg virus causing considerable economic consequences for European 344 \nfarmers and livestock  (46). Culicoides midge species are widely distributed across Belgium and 345 \nEurope with important variation observed in abundance and species diversity between both 346 \ncollection site and sampling period  (46,47). While Culicoides species are primarily monitored on 347 \nfarms and focused on identification of species related to infections of food-producing animal s, 348 \nfurther ecological studies mapping the species’ broader distribution in light of OROV transmission 349 \nin (peri-)urban settings, is crucial in identifying  potential risks of OROV transmission by Culicoides 350 \nto humans.  351 \nWhile the mosquito species tested in this study appear unlikely to be efficient vectors for  both the 352 \nprototype and a currently circulating OROV strain, ongoing surveillance of both mosquito and 353 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n18 \n \nCulicoides species remains a research priority . Vector- and viral evolution , together with climate 354 \nchange, are expected to alter transmission dynamics, highlighting the value of proactive monitoring 355 \nto stay ahead of the risks related to the ongoing global spread of this re-emerging virus. 356 \nEthical statement 357 \nThe study respects the Directive 2010/63/EU of the European Parliament  and of the Council of 22 358 \nSeptember 2010 on the protection of animals used for scientific  purposes. All animals used are 359 \ninvertebrates (three mosquito species: Aedes albopictus, Anopheles atroparvus, and Culex pipiens). 360 \nEthical approval is NOT required. 361 \nFunding statement 362 \nThis work was supported by the Belgian Directorate General for Development (DGD) (FA5). 363 \nThe insectaries at ITM are partially funded through the  Department of Economy, Science and 364 \nInnovation (EWI) of the Flemish Government. ARR is supported by a Baekeland Mandate fellowship 365 \n(HBC.2022.0144) from VLAIO O&O. SV is supported by a PhD fellowship from the Research 366 \nFoundation – Flanders (FWO) (11D5923N). 367 \nData availability 368 \nAll data are fully available. 369 \nAcknowledgements 370 \nThe authors would like to thank Prof. Joana-Rocha Pereira for kindly providing the prototype OROV 371 \nstock. We also acknowledged Dr. Concetta Castilletti for providing the OROV-IRCCS-SCDC_1/2024 372 \nstrain. The authors thank Dr. Nuria Busquets Martí (IRTA-CReSA, Bellaterra, Spain) for providing the 373 \nauthors with the colony of Anopheles atroparvus strain Ebre. We thank Maïlis Darmuzey and Martin 374 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint \n\n   \n \n19 \n \nFerrié for their advice regarding the foci forming assay for OROV. We also thank our laboratory 375 \ntechnicians Jacobus De Witte,  Lotte Wauters, Caroline Simons, and Kristien Minner for their help  376 \nrearing the mosquito colonies. We thank Winston Chiu and Joost Schepers at the Caps -It for their 377 \nassistance on the imaging and analysis of the foci forming assay plates.  378 \nConflict of interest 379 \nThe authors declare there is no conflict of interest 380 \nContribution 381 \nConceptualization: MB, LD. Methodology: MB, LD, ARR, EJ, KT. Investigation: ARR, EJ, MB, SV, CVD, 382 \nKT. Formal analysis: ARR, EJ, SV. Visualization: ARR, EJ. Writing – first draft: ARR, EJ, KT. Writing – 383 \nreview and editing: All authors. Funding acquisition: LD, KA, RM. All authors read and approved the  384 \nfinal version of the manuscript. 385 \nReferences 386 \n1. Oropouche virus disease [Internet]. [cited 2025 Jun 6]. Available from: 387 \nhttps://www.who.int/news-room/fact-sheets/detail/oropouche-virus-disease 388 \n2. ANDERSON CR, SPENCE L, DOWNS WG, AITKEN TH. Oropouche Virus: a New 389 \nHuman Disease Agent from Trinidad, West Indies. Am J Trop Med Hyg [Internet]. 390 \n1961 Jul 1 [cited 2025 Jun 12];10(4):574–8. 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It is made \nThe copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}