Low risk of transmission of prototype and newly emerged Oropouche virus strains by European Culex pipiens, Aedes albopictus, and Anopheles atroparvus mosquitoes

18 Oropouche virus (OROV) is an emerging arbovirus of growing public health concern, with increasing 19 incidence and geographic spread. Since its discovery in 1955, OROV has caused multiple outbreaks 20 in South and Central America, with a new introduction in Cuba since May 2024. Recent travel-related 21 cases in Europe and the Americas underscore its potential for global dissemination. Assessing 22 vector competence outside endemic regions is critical in the context of global travel and climate 23 change. 24 We evaluated the vector competence of three mosquito species commonly found in Europe—Culex 25 (Cx.) pipiens, Aedes (Ae.) albopictus, and Anopheles (An.) atroparvus—using two OROV strains: the 26 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 2 prototype TRVL9760 (1955, Trinidad and Tobago) and a recent isolate OROV-IRCCS-SCDC_1/2024 27 (2024, imported from Cuba to Italy). Mosquitoes were orally infected and examined at 7- and 14-days 28 post-infection. We assessed infection (body), dissemination (peripheral tissues), and transmission 29 potential (saliva) by measuring infectious virus particles using the gold standard focus-forming 30 assays. 31 Our findings show that Cx. pipiens and An. atroparvus were not susceptible to infection or did not 32 allow transmission with OROV. In Ae. albopictus , low infection rates were observed: 6.7% of 33 mosquitoes showed infection at day 7 with the prototype strain, and 3.1% at day 14 with OROV-34 IRCCS-SCDC_1/2024. All infected mosquitoes showed viral dissemination, but none had infectious 35 virus in their saliva, indicating low risk for transmission. 36 These results confirm limited vector competence of European mosquito species for OROV and 37 emphasize the importance of continued entomological surveillance to inform future risk 38 assessments. 39

Oropouche virus; Europe; vector competence; transmission; reassortant strain; 40 emergent arbovirus 41

42 Oropouche virus (OROV) is an emerging arbovirus and a growing public health concern, marked by 43 its recent increased incidence and geographic expansion. First identified in 1955 in Trinidad and 44 Tobago, OROV was the second most common arboviral disease in South America after dengue, until 45 the emergence of chikungunya (2013) and Zika virus (2015) (1,2). Nevertheless, only nine vector 46 competence studies have been published to date, highlighting the limited research focus of this 47 neglected arbovirus for which no antiviral treatments or vaccines are currently available (3–5). 48 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 3 Traditionally confined to areas near the Amazon rainforest and the Caribbean, OROV propagation 49 and clinical severity have intensified since December 2023. OROV disease is typically mild and self-50 limiting in immunocompetent individuals with symptoms such as fever, headache, muscle and joint 51 pain, sensitivity to light, and occasionally more severe complications like meningitis or encephalitis. 52 (6,7). However, newly introduced epidemic waves in Cuba since May 2024, along with two fatal 53 cases in healthy Brazilian women, and multiple reports of OROV-linked miscarriages, microcephaly, 54 and fetal deaths, have brought renewed attention to this historically neglected arbovirus (1,8). 55 Additionally, the report of several travel -related OROV cases in countries such as Italy, Spain, 56 Germany, France, the United States, and Canada further underscores the virus' capacity for 57 international spread (9–11). These developments mark a notable shift in the epidemiological pattern 58 and clinical profile of OROV fever, raising questions on evolutionary changes of the pathogen, the 59 vectors involved in ongoing outbreaks, as well as potential vectors in currently non-endemic areas. 60 Identifying biological adaptations of established versus newly emerged strains is key in 61 understanding altered pathogen behavior. OROV belongs to the Orthobunyavirus genus (Simbu 62 serogroup), characterized by a negative -sense tri-segmented (small [S], medium [M], and large [L]) 63 RNA genome, known to exchange segments with other Simbu strains when co -infecting the same 64 cell. This reassortment drives genetic diversity and viral evolution, leading to potential changes in 65 vector competence and transmission dynamics (12–14). Deiana et al. characterized the complete 66 genome of a newly emerged OROV strain (OROV-IRCCS-SCDC_1/2024) isolated from a patient with 67 OROV-induced fever returning from Cuba to Italy in May 2024. This isolate has shown to be a 68 reassortant virus, containing sequences of the Brazil ‘22 -‘24 outbreak and older sequences, likely 69 reflecting the strain currently circulating in Cuba and Latin America (15). Whether the observed 70 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 4 geographic expansion and disease severity are linked to enhanced virulence in this adapted variant 71 is a current research priority. 72 Furthermore, a substantial knowledge gap remains regarding our understanding of the reservoirs 73 and vectors involved in OROV transmission (3). Several mammals, including sloths, non -human 74 primates and birds, are considered potential reservoirs for sylvatic OROV maintenance, while no 75 vertebrates other than humans have been identified in urban transmission so far (16). The primary 76 vector of OROV is considered the Culicoides midge (family Ceratopogonidae), with C. paraensis 77 strongly implicated in both sylvatic and urban transmission, given its frequent presence in outbreak 78 zones, which is further supported by experimental data . According to the findings of Pinheiro and 79 colleagues, C. paraensis midges are capable of transmitting OROV to hamsters within 6 to 12 days 80 following a blood meal from infected individuals, with the minimum viral titer required for successful 81 transmission and infection of around 5.2 log₁₀ SMLD₅₀/mL (median lethal dose of OROV in suckling 82 mice/mL blood) (17). In addition to biting midges, mosquitoes from the Culicidae family could also 83 pose an important vector for OROV transmission . Although OROV has been isolated from various 84 anthropophilic mosquito species since its discovery , the consistently low detection rates and the 85 high experimentally determined threshold of infection (≥9.5 log10 SMLD 50/mL), suggested that 86 mosquitoes may not be efficient vectors for human -to-human cycle maintenance (2,3,18–21). 87 However, given the ongoing shifts in vector patterns driven by climate change and global mobility, it 88 is crucial to investigate whether vectors in non -endemic areas can support the transmission cycle 89 of newly emerged OROV strains. 90 To address this, we evaluated the vector competence of three mosquito species commonly found 91 across Europe: Ae. albopictus, Cx. pipiens, and An. atroparvus. These species were exposed to a 92 newly emerged OROV strain ( OROV-IRCCS-SCDC_1/2024), isolated from a traveler returning from 93 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 5 Cuba to Italy in 2024, or the prototype strain (TRVL9760) originally isolated in 1955. Mosquitoes were 94 examined at 7 - and 14 -days post infection (dpi) for evidence of infection, dissemination, and 95 transmission potential using the gold standard focus-forming assay (FFA) on Vero E6 cells. 96 This study expands current knowledge of OROV transmission potential in European mosquito 97 species through the comparison of a historical and an emergent strain. By generating new data on 98 infection, dissemination and transmission, it provides an evidence base to guide surveillance and 99 preparedness in the context of OROV’s recent expansion and its relevance for international public 100 health. 101

102 Mosquito species 103 In this study, three distinct mosquito species were used: Anopheles atroparvus (strain Ebre delta, 104 initially collected in Catalonia, Spain 2020) (22), Culex pipiens (strain 20CPip.BE-ITM collected in 105 Antwerp, Belgium 2020 ) (23), and Aedes albopictus (strain 21AAlb.IT-TER collected in Terni, Italy, 106 2021) (24). Established Aedes and Culex laboratory strains were achieved from the initial field -107 collected specimens at the Merian Insectarium of the Institute of Tropical Medicine Antwerp 108 (Belgium). 109 Mosquito rearing and maintenance 110 Eggs were placed in trays containing 1.75 L of softened water, supplemented with Novo Fect (Jbl, 111 Neuhoken, Germany) for Anopheles- or Koi mini-Sticks (Tetra, Melle, Germany) for Aedes and Culex 112 larvae development. Colonies were kept in 30 cm × 30 cm × 30 cm cages (BugDorm-1, Megaview, 113 Taichung, Taiwan) with access to 10% glucose solution with 0.1% of Methylparaben (Sigma-Aldrich, 114 Darmstadt, Germany). All mosquito strains were reared and maintained under controlled 115 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 6 environmental conditions, 27 °C temperature, 80% relative humidity, in an 11.5/11.5h light/dark 116 cycle with 0.5h of twilight between each cycle. 117 OROV stock preparation 118 The OROV strains used for experimental infections were provided as following: OROV -IRCCS-119 SCDC_1/2024, a recent isolate from a traveler returning from Cuba to Italy (2024), was kindly 120 provided by Prof. Castilleti (Department of Infectious -Tropical Diseases and Microbiology, IRCCS 121 Sacro Cuore Don Calabria Hospital, Verona), and the prototype strain 122 IP/OROV/TRL9760/12/08/2009, first identified in Trinidad and Tobago (1955) and isolated in France 123 kindly provided by the European Virus Archive Global (EVAg, Marseille, France ). Both virus strains 124 were propagated in Vero E6 cells (African Green monkey kidney cells, CRL-1586, ATCC) in Minimum 125 Essential Medium (MEM, Biowest, Nuaillé, France), supplemented with 2% fetal bovine serum (FBS, 126 Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin, 1% L -Glutamine and 1% Sodium 127 Pyruvate (Sigma-Aldrich, St. Louis, MO, USA). In short, cells were infected with virus at a multiplicity 128 of infection (MOI) of 0.01 for 1h at 37°C and 5% CO 2. Medium was replaced and the culture was 129 further incubated for 24h at 37°C and 5% CO2. Supernatant was collected, aliquoted and stored at -130 80°C until use. Viral titer was determined by a focus forming assay (FFA) using Vero E6 cells. 131 Pairwise distances comparison of the OROV-IRCCS-SCDC_1/2024 strain to the OROV TRVL9760 132 The sequences for the strains OROV TRVL9760 (accession numbers KC759122-24) and OROV-133 IRCCS-SCDC_1/2024 (accession numbers PP952117-19) were retrieved from the National Center 134 for Biotechnology Information public database. Using MEGA11 (25), an alignment was built for each 135 OROV segment including both strains, and the pairwise distances were calculated. The percentage 136 of similarity at nucleotide and amino acid level was determined by using the obtained distance value 137 in the formula: 138 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 7 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑖𝑑𝑒𝑛𝑡𝑖𝑡𝑦 = (1 − 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒) × 100. 139 Vector competence assay 140 Five- to seven-days-old female mosquitoes were caged in 450 mL cardboard cups (~60 per species 141 per experiment) and starved for 18h to stimulate feeding. Mosquitoes were exposed to OROV-spiked 142 human blood (final titer of 1 x 10 6 FFU/mL) via a Hemotek feeding system (SP6W1 -3, Hemotek Ltd, 143 Blackburn, UK) through collagen membrane (MEM5, Hemotek Ltd, Blackburn, UK) feeding at 38°C. 144 After 45 minutes of feeding, mosquitoes were anesthetized at 4°C. Fully engorged females were 145 selected on ice, divided in two sub -groups and placed in clean cardboard cups. Mosquitoes were 146 maintained in climatic cupboards under the same controlled environmental conditions used for the 147 rearing with access to cotton soaked in 10% glucose solution. At 7 and 14 dpi, mosquitoes were 148 anesthetized with triethylamine (Sigma-Aldrich, Darmstadt, Germany) prior to mosquito dissection. 149 For each mosquito, only legs (for the OROV-IRCCS-SCDC_1/2024 strain) or legs, wings and head (for 150 the TRVL9760 strain) were dissected first and placed in a 2 ml Eppendorf tube containing 1 mL of 151 mosquito medium (20% FBS in Dulbecco’s phosphate -buffered saline, 50 mg/mL 152 penicillin/streptomycin, 50 mg/mL gentamicin, and 2.5 mg/mL fungizone) containing a zinc -plated 153 steel bead (4.5 mm). Next, mosquitoes were forced to salivate into a 20 µ L pipette tip filled with 10 154 µl of a sucrose:FBS (1:1) solution for 30 minutes, after which saliva was ejected into a 2 m L 155 Eppendorf tube containing 90 µ L of mosquito medium. The remaining bodies were placed in 2 mL 156 Eppendorf tubes containing 1 m L of mosquito medium with a zinc -plated steel bead. Body - and 157 peripheral organs-samples were homogenized at 30 Hz for 2 minutes using a TissueLyser II (Qiagen 158 GmbH, Hilden, Germany) and centrifuged for 30 s at 11,000 rpm. All samples were stored at -80 °C 159 until further analysis. Each mosquito species was subjected to two technical replicates for both 160 OROV strains. 161 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 8 Focus forming assay 162 To determine vector competence status, the infectious viral load in body, legs and saliva of individual 163 mosquitoes was evaluated by focus forming assay (FFA), as previously described (26–28). In short, 164 samples were serially diluted tenfold and applied to a confluent monolayer of Vero E6 cells, followed 165 by incubation for 24 hours at 37 °C with 5% CO₂ in the presence of 1% carboxymethyl cellulose 166 (CMC) in MEM, sup plemented with 5% FBS . After incubation, cells were fixed using 4% 167 paraformaldehyde (Sigma-Aldrich, Darmstadt, Germany) in cold 1× PBS and subsequently blocked 168 with blocking buffer (3% bovine serum albumin and 0.05% Tween -20 in cold 1× PBS). 169 Immunostaining was conducted using a monoclonal antibody against Oropouche virus (Oropouche 170 virus immune ascitic fluid, VR-1228AF, ATCC) diluted 1:1000 in blocking buffer. Following four 171 washes with cold 1× PBS, an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody 172 (Invitrogen, Life Science, Eugene , OR, USA ) was used for detection (1:1000 in blocking buffer ). 173 Fluorescent foci were visualized and counted using a Leica DMi8 fluorescence microscope (Leica 174 Microsystems, Wetzlar, Germany). Viral titer was expressed as focus -forming units per sample 175 (FFU/sample). Infection and transmission metrics were calculated as follows: the infection rate (IR) 176 was defined as the proportion of mosquitoes with virus -positive bodies among the total number of 177 mosquitoes analyzed following exposure to an infectious bloodmeal; the dissemination rate (DIR) 178 was the proportion of mosquitoes with virus -positive legs (for the OROV-IRCCS-SCDC_1/2024 179 strain) or virus -positive legs, wings and head (for the TRVL9760 strain) among those with virus -180 positive bodies; and the transmission efficiency (TE) was the proportion of mosquitoes with virus -181 positive saliva among the total number of mosquitoes analyzed. 182 Statistical analysis 183 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 9 Data analysis was performed using GraphPad Prism version 10.0.2. Differences in infection rate (IR), 184 dissemination rate (DIR), and transmission efficiency (TE) among mosquito species and across time 185 points (7 and 14 dpi). To evaluate differences in viral titers in the body, peripheral organs, and saliva 186 between time points within the same OROV strain, two-tailed Mann-Whitney U tests were employed. 187 Statistical significance was defined as P < 0.05. 188

189 To assess the percentage of similarity of the new isolate to the prototype OROV strain, sequences 190 for both strains corresponding to the S, M, and L segments were aligned respectively, and pairwise 191 comparisons were performed to assess the percentage of divergence at nucleotide and amino acid 192 level. The newly emerged OROV-IRCCS-SCDC_1/2024 isolate (Accession No. PP95117.3, 193 PP95118.3, PP95119.3) was 5.8% and 0% divergent at nucleotide and amino acid level, respectively, 194 in the short (S) segment. The medium (M) segment displayed 5.2% divergence at the nucleotide level 195 and 1.9% at the amino acid level , while the large (L) segment was 10% divergent at the nucleotide 196 level and 5% at the amino acid level (Table 1). 197 198 Table 1. Nucleotide and amino acid sequence pairwise comparisons of the OROV -IRCCS-199 SCDC_1/2024 strain with the prototype OROV TRVL9760 strain. Segments are presented as S 200 (small), M (medium), and L (large), with the corresponding protein in square brackets. RdRp: RNA -201 dependent RNA polymerase. 202 Divergence (%) Strain S [Nucleoprotein] M [Membrane] L [RdRp] OROV-IRCCS- SCDC_1/2024 Nucleotide 5.8 5.2 10.0 Amino acid 0.0 1.9 5.0 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 10 With the prototype OROV strain (TRVL9760), Ae. albopictus mosquitoes showed an infection rate of 203 6.67% (3/45 mosquitoes) at 7 dpi. All three TRVL9760-infected mosquitoes showed a disseminated 204 infection, while no infectious virus could be detected in their saliva (Table 2, Figure 1) . The same 205 TRVL9760-infected Ae. albopictus mosquitoes had a mean viral titer of 45.6 FFU/sample in the body 206 7 days post infection , and a mean viral titer of 1.22×102 FFU/sample in the head, wings, and legs 207 (Figure 2). However, no infected mosquitoes could be detected at 14 dpi with this virus strain. 208 209 Table 2. Experimental infection, dissemination, and transmission outcomes in Aedes 210 albopictus, Culex pipiens, and Anopheles atroparvus mosquitoes following an artificial OROV-211 infectious blood meal. IR= Infection rate; DIR= Dissemination rate; TE= Transmission efficiency; n= 212 number of mosquitoes tested 213 Strain Input PFU/ml Species 7 dpi 14 dpi IR (n) DIR (n) TE (n) IR (n) DIR (n) TE (n) OROV-IRCCS- SCDC_1/2024 1×106 Ae. albopictus 0 (76) 0 (76) 0 (76) 3.12 (64) 100 (2) 0 (64) Cx. pipiens 0 (21) 0 (21) 0 (21) 0 (31) 0 (31) 0 (31) An. atroparvus 0 (22) 0 (22) 0 (22) 0 (19) 0 (19) 0 (19) Ae. albopictus 6.67 (45) 100 (3) 0 (45) 0 (22) 0 (22) 0 (22) TRVL9760 (prototype) 1×106 Cx. pipiens 0 (40) 0 (40) 0 (40) 0 (42) 0 (42) 0 (42) .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 11 On the contrary, infection with the newly emerged OROV strain (OROV-IRCCS-SCDC_1/2024) in Ae. 214 albopictus could only be detected at 14 dpi, with an infection rate of 3.12% (2/64 mosquitoes). The 215 two OROV-IRCCS-SCDC_1/2024-infected Ae. albopictus mosquitoes show ed a disseminated 216 infection, but no transmission of the virus (Table 2, Figure 1). These infected mosquitoes had a mean 217 viral titer of 15.1 FFU/sample in the body at 14 days post infection , and a mean viral titer of 3.15 218 FFU/sample in the legs (Figure 2). None of the Cx. pipiens mosquito bodies or peripheral organs were 219 positive for the TRVL9760 strain, nor for the OROV-IRCCS-SCDC_1/2024 strain at either 7 or 14 dpi. 220 Likewise, An. atroparvus exhibited a lack of vector competence for the newly emerged OROV-IRCCS-221 SCDC_1/2024 strain. 222 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 12 Figure 1. Vector competence of Culex pipiens, Aedes albopictus, and Anopheles atroparvus for 223 OROV strains (A) TRVL9760 and (B) OROV-IRCCS-SCDC_1/2024. The bars indicate the infection 224 rate corresponding to mosquito bodies (blue), dissemination to peripheral organs (red), and 225 transmission potential in the saliva (green) by means of focus forming assay. The labels above the 226 bars represent the number of positive mosquitoes over the total number of mosquitoes. 227 228 229 Figure 2. Viral titer (FFU/sample) in the bodies, peripheral organs ((head, wings, and) legs) of Aedes 230 albopictus mosquitoes infected with the OROV TRVL -9760 and OROV-IRCCS-SCDC_1/2024 strains 231 at 7 and 14 dpi. For the OROV TRVL9760 strain, head, wings, and legs were included as proxy for 232 dissemination in peripheral organs, while only legs were included for the OROV -IRCCS-SCDC_1/2024 233 strain. Each symbol represents an individual mosquito sample tested by means of a focus forming assay. 234 The bars represent the mean with SD. To evaluate differences in viral titers in the body, peripheral 235 organs, and saliva between time points within the same OROV strain, two -tailed Mann-Whitney U 236 tests were employed. Statistical significance was defined as P < 0.05. 237 238 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 13

239 Our findings demonstrate a low vector competence of Cx. pipiens, Ae. albopictus and An. atroparvus 240 present in Europe for both the prototype and newly emerged OROV strains. Such results are relevant 241 from an epidemiological perspective given their wide dispersal across Southern and more temperate 242 Northern Europe , incl. in Belgium. Culex pipiens mosquitoes are native to Belgium and often 243 compose the most abundant species in entomological surveys (29,30). Likewise, An. atroparvus is 244 also a Belgian native mosquito ; however, its sightings have been scarce in the last nationwide 245 entomological survey carried out (30). Additionally, Ae. albopictus mosquitoes have drawn more 246 attention in recent years as an invasive species in Belgium, with dedicated yearly efforts to monitor 247 their presence, as they are known vectors of clinically important arboviruses, including dengue and 248 chikungunya virus (30,31). 249 Unlike molecular methods detecting viral RNA without indicating infectivity, we employed the gold-250 standard focus-forming assay (FFA) on Vero E6 cells, directly measuring infectious virus particles, 251 hence providing accurate assessment of the transmission potential. 252 Our results align with a previous study assessing the vector competence of Cx. pipiens mosquitoes 253 from the USA, where no infection, dissemination or transmission of the prototype OROV strain was 254 reported 14 days post infection (32). Nevertheless, these mosquitoes were able to transmit OROV 255 240023, a strain originally isolated from a febrile patient from Cuba, albeit to a low extent ( 1 out of 256 50 mosquitoes). On the contrary, Cx. pipiens mosquito populations from the UK were not 257 susceptible to the infection with th is OROV 240023 strain (33). Such outcomes hint not only at a 258 strain-specific variability in vector competence, but also at a geographic variation. Furthermore, a 259 recent study reported a lack of vector competence of Italian Cx. pipiens mosquitoes (derived from 260 field populations collected in Rome ) for the OROV-IRCCS-SCDC_1/2024 strain. These mosquitoes 261 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 14 did not show infection or dissemination at 7 nor 14 days post infection (34), which concurs with our 262 findings. 263 Variations in the kinetics of infection in Ae. albopictus mosquitoes were noticed in our study between 264 the employed OROV strains. The mosquitoes that received the prototype OROV strain in the blood 265 meal exhibited infection (6.67%, n= 45) and dissemination (100%, n= 3) already at 7 days post 266 infection, whereas those that received the newly emerged OROV-IRCCS-SCDC_1/2024 strain 267 showed infection (3.12%, n= 64) and dissemination (100%, n= 2) only at 14 days post infection. Payne 268 et al. reported a general low vector competence of Ae. albopictus (US population) for the prototype 269 OROV strain, with only 1 out of 50 mosquitoes (2%) having a disseminated infection, but no 270 transmission at 14 days post infection (32). On the other hand , Jansen et al. have described Ae. 271 albopictus mosquitoes (population from Heidelberg, Germany) transmitting the prototype OROV 272 strain at 14 and 21 days post infection, when incubated at both 24 °C and 27 °C (35). Our findings 273 regarding the prototype OROV infection kinetics in Ae. albopictus differ from what is reported by 274 Payne et al. (32) and Jansen et al. (35), as infection and dissemination were detected earlier in our 275 study (7 dpi), and there was no virus transmission. However, the Ae. albopictus mosquitoes from our 276 study have originated in Italy ; therefore, we should consider that vector competence might be 277 influenced by the possible genetic heterogeneity presented among these mosquito populations, as 278 it has been observed previously for Ae. aegypti populations (36). 279 Concerning the newly emerged OROV-IRCCS-SCDC_1/2024 strain, Mancuso et al. found that Italian 280 Ae. albopictus (derived from field populations collected in Rome) were infected with OROV when 281 sampled at day s 7 and 21 post infection (one infected mosquito out of 20 for each time point) ; 282 however, no dissemination, nor transmission was detected (37). Likewise, our study employed Ae. 283 albopictus mosquitoes derived from an Italian population (collected in Terni) and we did not detect 284 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 15 transmission for the newly emerged OROV strain, yet we did observe dissemination at 14 days post 285 infection. 286 Previous studies have highlighted the need to reconsider the role of Anopheles mosquitoes as 287 potential vectors of emerging arboviruses, such as Mayaro virus (38). Payne and colleagues reported 288 a low infection rate (IR = 4%) in An. quadrimaculatus from the United States with Oropouche virus 289 (OROV), with no evidence of viral dissemination or transmission. In our study, we did not detect 290 infection or dissemination in a European population of An. atroparvus, suggesting that this species 291 is unlikely to play a role in the transmission cycle of OROV. 292 Regardless of the time point post infection, the viral titer s of OROV in the bodies of Ae. albopictus 293 mosquitoes were similar between the two strains (Figure 2). Our study also reports on the amount of 294 infectious virus particles detected in Ae. albopictus bodies and peripheral organs following an 295 OROV-infectious blood meal. Other vector competence studies have described that the average 296 OROV viral titer detected in Cx. tarsalis bodies 10 days post infection was 31 PFU/mL, whereas the 297 viral titers for Cx. quinquefasciatus bodies and leg tissues were 128 and 37 PFU/mL, respectively. At 298 14 days post infection, Cx. quinquefasciatus bodies displayed an average viral titer of 88 PFU/m L, 299 while leg tissues showed an average titer of 100 PFU/mL (39). 300 All mosquito species tested showed a poor competence for either the prototype TRVL9760 or the 301 OROV-IRCCS-SCDC_1/2024 isolate. When comparing the newly emerged OROV-IRCCS-302 SCDC_1/2024 strain to the prototype, we found that the highest divergence at the nucleotide level 303 (10%) presented in the L segment, corresponding to the RdRp. This OROV-IRCCS-SCDC_1/2024 304 strain has been described as a reassortant virus, with the S and L segments sharing high similarity 305 with an emerging cluster of sequences that are most likely related to the recent outbreaks in South 306 America (40). Despite the genetic variations with the prototype OROV strain and the newly emerged 307 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 16 strain belonging to a divergent OROV cluster, we did not observe a different outcome in vector 308 competence for the OROV-IRCCS-SCDC_1/2024 strain in our study, for both Ae. albopictus and Cx. 309 pipiens mosquitoes. Although OROV infectious virions were detected in the bodies and peripheral 310 organs of Ae. albopictus mosquitoes, virus was not found in the saliva of any of these mosquitoes, 311 suggesting the presence of a strong barrier to OROV transmission for the tested strains. 312 Nevertheless, the observed viral replication of OROV in certain mosquitoes should not be neglected 313 and underscores the potential for arboviral adaptation and emergence, highlighting the importance 314 of continued surveillance. 315 Depending on the mosquito -virus combination, typically a higher viral titer in the blood meal can 316 yield a higher percentage of infected mosquitoes (41). While the infectious blood meal in our study 317 contained 1×10⁶ PFU/mL of OROV —a dose within the range typically used in other studies 318 (32,33,42)—we cannot exclude the possibility that a higher viral dose might have resulted in greater 319 viral titers in mosquito bodies and, consequently, altered the transmission potential of Ae. 320 albopictus. Further resea rch assessing the effect of several OROV doses on mosquito vector 321 competence could give a more comprehensive understanding of these infection dynamics. However, 322 the viral input utilized falls on the upper end of the range of viremia detected in OROV -infected 323 humans (6×10^3 and 7×10^5 PFU/ml (43,44)), and therefore, the mosquitoes were exposed to an 324 epidemiologically relevant dose of the virus through the blood meal in this study. 325 A discrepancy in our study is that dissemination was determined based on different mosquito 326 tissues per strain. The legs were used as a proxy for dissemination for the OROV-IRCCS-327 SCDC_1/2024 infected mosquitoes, while head, wings, and legs were employed for the prototype 328 TRVL-9760. Consequently, the viral titer measured in the OROV-IRCCS-SCDC_1/2024 infected 329 mosquito legs was lower compared to when using the head, wings, and legs together for the 330 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 17 prototype infected mosquitoes (Figure 2). Both tissue types are valid approaches for the assessment 331 of virus dissemination within the mosquito body, as they represent secondary organs that become 332 infected once the viral infection overcomes the midgut barrier. W e could observe that the 333 percentage of virus dissemination between both OROV strains remained comparable. As such, this 334 fluctuation in viral titers resulted most likely due to the quantity of tissue tested , and it was further 335 deemed not statistically significant; however, we should remark the low number of mosquitoes that 336 got infected (OROV-IRCCS-SCDC_1/2024, n=2; TRVL-9760, n=3), and thus take this into 337 consideration during comparison. 338 Similarly to mosquitoes, OROV infection in Culicoides paraensis midges has been shown to be dose 339 dependent. However, their threshold for infection is reportedly lower than for mosquitoes, with C. 340 paraensis midges exposed to OROV doses higher than 5.2 log 10 SMLD50/mL becoming infected 341 (≥13%) and capable of transmitting the virus (≥40%) (45). 342 Monitoring of these vector populations in Europe has been triggered during the past decade due to 343 bluetongue and Schmallenberg virus causing considerable economic consequences for European 344 farmers and livestock (46). Culicoides midge species are widely distributed across Belgium and 345 Europe with important variation observed in abundance and species diversity between both 346 collection site and sampling period (46,47). While Culicoides species are primarily monitored on 347 farms and focused on identification of species related to infections of food-producing animal s, 348 further ecological studies mapping the species’ broader distribution in light of OROV transmission 349 in (peri-)urban settings, is crucial in identifying potential risks of OROV transmission by Culicoides 350 to humans. 351 While the mosquito species tested in this study appear unlikely to be efficient vectors for both the 352 prototype and a currently circulating OROV strain, ongoing surveillance of both mosquito and 353 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 18 Culicoides species remains a research priority . Vector- and viral evolution , together with climate 354 change, are expected to alter transmission dynamics, highlighting the value of proactive monitoring 355 to stay ahead of the risks related to the ongoing global spread of this re-emerging virus. 356 Ethical statement 357 The study respects the Directive 2010/63/EU of the European Parliament and of the Council of 22 358 September 2010 on the protection of animals used for scientific purposes. All animals used are 359 invertebrates (three mosquito species: Aedes albopictus, Anopheles atroparvus, and Culex pipiens). 360 Ethical approval is NOT required. 361 Funding statement 362 This work was supported by the Belgian Directorate General for Development (DGD) (FA5). 363 The insectaries at ITM are partially funded through the Department of Economy, Science and 364 Innovation (EWI) of the Flemish Government. ARR is supported by a Baekeland Mandate fellowship 365 (HBC.2022.0144) from VLAIO O&O. SV is supported by a PhD fellowship from the Research 366 Foundation – Flanders (FWO) (11D5923N). 367 Data availability 368 All data are fully available. 369

370 The authors would like to thank Prof. Joana-Rocha Pereira for kindly providing the prototype OROV 371 stock. We also acknowledged Dr. Concetta Castilletti for providing the OROV-IRCCS-SCDC_1/2024 372 strain. The authors thank Dr. Nuria Busquets Martí (IRTA-CReSA, Bellaterra, Spain) for providing the 373 authors with the colony of Anopheles atroparvus strain Ebre. We thank Maïlis Darmuzey and Martin 374 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.27.672550doi: bioRxiv preprint 19 Ferrié for their advice regarding the foci forming assay for OROV. We also thank our laboratory 375 technicians Jacobus De Witte, Lotte Wauters, Caroline Simons, and Kristien Minner for their help 376 rearing the mosquito colonies. We thank Winston Chiu and Joost Schepers at the Caps -It for their 377 assistance on the imaging and analysis of the foci forming assay plates. 378 Conflict of interest 379 The authors declare there is no conflict of interest 380 Contribution 381 Conceptualization: MB, LD. Methodology: MB, LD, ARR, EJ, KT. Investigation: ARR, EJ, MB, SV, CVD, 382 KT. Formal analysis: ARR, EJ, SV. Visualization: ARR, EJ. Writing – first draft: ARR, EJ, KT. Writing – 383 review and editing: All authors. Funding acquisition: LD, KA, RM. All authors read and approved the 384 final version of the manuscript. 385

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