Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake Virus

Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake Virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake Virus Siyuan Liu, Xiaoyu Wang, Fei Wang, Wahid Zaman, Cihan Yang, Doudou Huang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3969850/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 May, 2024 Read the published version in Parasites & Vectors → Version 1 posted 7 You are reading this latest preprint version Abstract Background: Mosquito-borne viruses cause various infectious diseases in humans and animals. Oya virus (OYAV) and Ebinur lake virus (EBIV), belonging to the genus Orthobunyavirus within the Peribunyaviridae family, are recognized as neglected viruses with the potential to pose threats to animal or public health. The evaluation of vector competence is essential for predicting the arbovirus transmission risk. Methods: To investigate the range of mosquito vectors for OYAV and EBIV, four medically significant mosquitoes — Culex pipiens pallens , Culex quinquefasciatus , Aedes albopictus , and Aedes aegypti — were selected to measure their vector susceptibility through blood-feeding infection. The RNA copies and infection rates of various mosquito species were determined using RT-qPCR. Subsequently, susceptible mosquito species were examined to determine their vector competence. Results: The results revealed that Cx. pipiens pallens can support in vivo infection and multiplication of OYAV, while Cx. quinquefasciatus , Ae. albopictus, and Ae. aegypti exhibit lower susceptibility. Furthermore, Cx. pipiens pallens can be infected by EBIV, posing a potential risk of virus transmission (with a transmission rate of up to 15.4%) at 7 days post-infection. In contrast, Cx. quinquefasciatus and Ae. albopictus exhibited poor vector competence. Conclusions: This study evaluated the possible transmission risk of OYAV and EBIV using four mosquito species. The findings indicate a potential risk of EBIV transmission in Cx. pipiens pallens . Mosquito vector arbovirus orthobunyavirus Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Mosquitoes, being important vectors, can carry and transmit a diverse range of pathogens, including arboviruses, primarily from the Peribunyaviridae , Togaviridae , Flaviviridae , Phenuiviridae , and Reoviridae families, along with several types of parasites [1]. The family Peribunyaviridae , genus Orthobunyavirus , contains three-segmented, negative-stranded RNA viruses transmitted exclusively by arthropods [2]. So far, nearly 200 viruses of the genus Orthobunyavirus have been identified and grouped into 20 serogroups based on their serological relatedness [3]. Multiple viruses belonging to the Simbu and Bunyawera serogroups have been found in various mosquito species, including Culex spp., Aedes spp., and Anopheles spp [4, 5, 6]. Members of the family Peribunyaviridae can introduce illnesses in vertebrates, with encephalitis caused by the Oropouche virus (OROV) and La Crosse virus (LACV) affecting humans [7, 8]. Additionally, ruminants are at risk of abortions and teratogenic effects from Bunyamwera virus (BUNV) and Cache Valley virus (CVV) [9, 10]. Oya virus (OYAV) and Ebinur lake virus (EBIV) belong to the Orthobunyavirus genus. OYAV is a member of the Simbu serogroup and shares the same virus species as the Cat Que virus (CQV), which is the Orthobunyavirus catqueense [11]. OYAV was first isolated from cell cultures from pig lungs in Malaysia in 2000 [13]. Subsequent studies found several viral isolates of this species in China, Vietnam, and India from invertebrate and vertebrate hosts [12][14, 15, 16, 17, 18]. EBIV, a member of the Bunyamwera serogroup, was initially isolated from the pools of Culex modestus in Xinjiang Province, China, and was later been detected in various samples from Russia and Kenya [19, 20]. Experimental investigations have demonstrated that these viruses efficiently invade and replicate within various cell lines derived from vertebrates and mosquitoes [16, 21]. In addition, OYAV is lethal in suckling and C57BL/6 adult mice [16], while EBIV has been proven to be fatal in BALB/c mice [22]. Although no confirmed cases of OYAV and EBIV have been recorded, a serological survey revealed that 93% of pigs within pig farming zones in Malaysia tested positive for OYAV [13], and the seroprevalence of OYAV in pigs in Yunnan, China, exceeded 30 percent [16]. Moreover, the antibodies against EBIV have previously been detected in residents of Xinjiang [21]. These findings indicate that OYAV and EBIV may potentially threaten animal or public health. The prevalent mosquito genera in China are Aedes and Culex [6]. Culex pipiens pallens and Cx. quinquefasciatus, both belonging to the Cx. pipiens complex, are predominantly distributed in northern and southern parts of China, respectively [23]. The Cx. pipiens complex plays a crucial role in transmitting arboviruses, particularly the West Nile virus [24, 25]. Aedes albopictus and Ae. aegypti are recognized as the chief vectors of DENV [26, 27]. Aedes albopictus is a widespread species in the southern regions of China [28]. In China, Ae. aegypti has also been found in parts of Yunnan, Hainan, and Guangdong, and the geographical distribution for Ae. aegypti is expending in recent years [29]. Our group's previous work evaluated the vector competence of Ae. aegypti for EBIV [30]. Meanwhile, the susceptibility of Culex and Ae. aegypti mosquitoes to CQV has now been reported from India [31]. Nevertheless, there is a need for more relevant mosquito studies on EBIV and OYAV isolates in China, where different genotypes/isolates of the virus, mosquito species, and geographical strains may have an impact on vector competence. This study aimed to assess the infection, replication, and transmission ability of OYAV and EBIV in Cx. pipiens pallens , Cx. quinquefasciatus , Ae. albopictus (common mosquito species in China), and Ae. aegypti by the oral blood-feeding route (natural infection simulation) to explore their abilities to contribute to potential outbreak preparedness by informing local mosquito-control organizations. Methods Mosquito species and rearing In this study, different mosquito species were utilized. The reared Cx. pipiens pallens (Beijing strain, kindly provided by China CDC), Cx. quinquefasciatus (Wuhan strain), Ae. albopictus (Jiangsu strain, kindly provided by China CDC), and Ae . aegypti (Rockefeller strain, kindly provided by Qian Han from Hainan University). The feeding methods and processes were the same as described earlier [30]. Cell lines and Viral stock BHK cells (golden hamster kidney cells) and PK 15 cells (pig kidney cells) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% FBS, 1% penicillin/streptomycin (Gibco) at 37 ℃ and 5% CO 2 . The viral strains included OYAV (strain SZC50) and EBIV (strain Cu20-XJ). The titers of OYAV and EBIV were determined using the plaque formation assay, and their working stocks were found to be 1.4×10 7 plaque-forming units mL−1 (PFU/mL) and 7.0×10 7 PFU/mL, respectively. Mosquito adult infection through blood feeding For infections through an artificial mosquito feeding system (Hemotek), 3 to 5-day-old adult females were starved for 12 to 24 h and fed with a mixture of blood and virus supernatant. The titer of the virus-blood mixture was determined at the same time by plaque assay. Refer to previously published articles for detailed procedures on oral infection and because our group's previous study described the results of EBIV infection in Ae. aegypti [30], this study no longer uses EBIV infection in Ae. aegypti . To establish the appropriate concentration of OYAV and EBIV in different mosquito species, Cx. pipiens pallens , Cx. quinquefasciatus , Ae. albopictus, and Ae. aegypti infected with different virus-blood concentrations (OYAV: two serial titers ranging from 10 6 -10 5 PFU/mL; EBIV: three serial titers ranging from 10 7 -10 5 PFU/mL) were subsequently collected at 4 days post-infection (dpi) for viral RNA determination. A previous study showed that adult female BALB/c mice infected with 10 PFU of EBIV reached 10 6 PFU/mL viremia at 2 dpi. Also, in conjunction with the preliminary findings from this study, viruses with a titer of 10 6 PFU/mL were chosen for infection. Infected mosquitoes were collected at 4, 7, 10, and 14 dpi for viral RNA determination. To determine OYAV and EBIV distribution in infected Cx. pipiens pallens , Cx.quinquefasciatus , Ae. albopictus, and Ae. aegypti via artificial blood feeding (final viral titer of 10 6 PFU/mL), viral RNA in mosquito bodies, heads, and saliva at 4, 7, 10, and 14 dpi were examined. The detailed procedures for collecting and preserving mosquito saliva can be found in prior published articles [30]. All mosquito tissues and saliva were stored in 250 μL RPMI 1640 supplemented with 2% Penicillin/Streptomycin/Gentamicin Solution. Prior to processing, all samples were kept at -80°C. Vector competence of the mosquitoes was evaluated by calculating the infection rate (IR; number of positive bodies/the total number of mosquitoes tested), dissemination rate (DR; number of positive heads/the number of the positive abdomen), transmission rate (TR; number of positive saliva/the number of positive abdomen). Evaluation of viral replication by RT-qPCR All samples were initially homogenized using a Low Temperature Tissue Homogenizer Grinding Machine (Servicebio) (operating frequency = 60Hz, operation time = 10 s, pause time = 10 s, cycles = 3, and setting temperature = 4˚C), followed by centrifugation for 5 min at 10, 000×g and 4˚C. The total RNA of each sample was extracted using an automated nucleic acid extraction system following the manufacturer’s instructions (NanoMagBio). Using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad), viral RNA copies of each sample were quantified. For OYAV, the Luna ® Universal Probe One-Step RT-qPCR Kit (NEB) was used, and for EBIV, the HiScript II One Step qRT-PCR Probe Kit (Vazyme) was used. The primer and probe sets used are presented in previous studies [18]. The cutoff for positive samples determined via RT-qPCR was Ct (OYAV) < 36 and Ct (EBIV) < 35. The positive cutoff value was evaluated by comparing paired serial ten-fold dilutions either inoculated on cells or assayed via RT-qPCR (Table S1) [32]. The equation for the standard curve was shown in Table S2 and used to calculate the viral genome copies in each sample. Statistical Analysis GraphPad Prism 8.0.2 (GraphPad Software Inc) was used to analyze all data. Fisher’s exact test was used to compare the mosquito’s infection, dissemination, and transmission rates between different treatments. One-way ANOVA with Tukey’s multiple comparison was used to compare the mean of the genome copies among more than two data sets. P ≤ 0.05 were considered statistically significant. The figure of graphical abstract was created with BioRender.com (https://www.biorender.com/) and authorization for publication had been granted. Results The susceptibility of adult Culex and Aedes mosquitoes to different titers of OYAV and EBIV The infectivity of mosquitoes consuming artificial blood meals with varying virus titers was investigated. Four mosquito species — Cx. pipiens pallens , Cx. quinquefasciatus , Ae. albopictus, and Ae. aegypti —were fed with blood meals containing 3-4.3×10 5 PFU/mL and 3-7 × 10 6 PFU/mL of OYAV, respectively. The infection rates (IR) and viral replication were assessed at 4 dpi (Fig. 1A and B). No infection was detected in Cx. pipiens pallens at 10 5 PFU/mL OYAV among 40 tested mosquitoes. However, at a titer of 10 6 PFU/mL, the IR increased to 42.0%, with an average viral RNA concentration of 10 4.44 copies/μL and a peak of 10 6.33 copies/μL, marking a significant increase in IR compared to the lower titer ( P < 0.0001). For Cx. quinquefasciatus , Ae. albopictus and Ae. aegypti , IR was not significantly affected by blood meal infections at different titers, with IR remaining below 18%. Meanwhile, at the OYAV infection titer of 10 6 PFU/mL, the IR of Cx. pipiens pallens was significantly higher than that of Cx. quinquefasciatus , Ae. albopictus and Ae. aegypti ( P = 0.0211; 0.0025; 0.0006, respectively). There was no significant difference in the mean viral RNA copies among different species of infected mosquitoes at varying infection titers. Three mosquito species — Cx. pipiens pallens , Cx. quinquefasciatus and Ae. albopictus —were exposed to EBIV in blood meals with titers ranging from 1.8-6.0×10 5 PFU/mL, 3.8-5.7×10 6 PFU/mL, and 1.3-2.5 × 10 7 PFU/mL, respectively. Their IR and viral replication were assessed at 4 dpi (Fig. 1C and D). In Cx. pipiens pallens , a positive correlation was observed between blood meal titers and IR, with an IR of 80% at the highest titer of 10 7 PFU/mL, significantly surpassing the rates at lower titers (all P < 0.0001). The mean viral RNA copies in positive mosquitoes were 10 5.55 copies/μL (10 5 PFU/mL: 10 4.51 copies/μL; 10 6 PFU/mL: 10 3.61 copies/μL), peaking at 10 7.99 copies/μL, notably higher than in lower titer infections. For Cx. quinquefasciatus , EBIV RNA was not detected at a blood meal titer of 10 5 PFU/mL. However, at 10 6 PFU/mL and 10 7 PFU/mL, the IR increased to 17.5% and 16.7%, respectively, with mean viral RNA copies of 10 4.81 copies/µL and 10 3.99 copies/µL. In Ae. albopictus , there were no infections at 10 5 PFU/mL and 10 6 PFU/mL. Nevertheless, at a 10 7 PFU/mL titer, the IR rose to 35%, with mean viral RNA copies of 10 3.90 copies/μL and a maximum of 10 5.03 copies/μL. At the highest titer of 10 7 PFU/mL, Cx. pipiens pallens exhibited a significantly higher IR than other mosquito species ( P < 0.0001). At a titer of 10 6 PFU/mL, Cx. pipiens pallens and Cx. quinquefasciatus displayed similar IRs, both higher than that of Ae. albopictus ( P = 0.0173). Dynamic variations in viral titers in adult mosquitoes infected with OYAV or EBIV via oral blood feeding To assess adult mosquitoes' susceptibility to OYAV and EBIV, they were infected through oral blood feeding at an average titer of 3.75 × 10 6 PFU/mL, with samples collected at 4, 7, 10, and 14 dpi for RNA analysis (Fig. 2 A-H). For Cx. pipiens pallens (Fig. 2 A and B), the OYAV IR remained relatively stable from 4 to 14 dpi, fluctuating between 35.7% and 40.0%. The mean viral RNA copies in positive mosquitoes varied slightly, ranging from 10 4.00 to 10 4.33 copies/μL. In contrast, EBIV infection resulted in significantly lower IRs at 4 ( P = 0.0003), 10 ( P = 0.0003), and 14 ( P < 0.0001) dpi compared to OYAV. The peak IR for EBIV was observed at 7 dpi (26.7%), notably higher than the rates at 4, 10 (both P = 0.0148) and 14 dpi ( P = 0.0060). The mean viral RNA copies in EBIV-positive mosquitoes was 10 4.59 copies/μL, with some exceeding 10 8 copies/μL. For Cx. quinquefasciatus (Fig. 2 C and D), the OYAV IR decreased from 18.3% at 4 dpi to 1.6% at 14 dpi. The mean viral RNA copies remained relatively constant, with a maximum of 10 3.35 copies/μL at 4 dpi. EBIV infection results in higher IRs at 4 and 10 dpi (20.0% and 21.2%, respectively) than those at 7 and 14 dpi (13.6% and 9.1%, respectively). The mean viral RNA copies were highest at 14 dpi, with one positive mosquito having a viral RNA copy of 10 7.73 copies/μL and a mean of 10 5.13 copies/μL. The mean viral RNA copies of EBIV-positive mosquitoes at 4 and 10 dpi were significantly higher than those infected with OYAV ( P = 0.0366 and 0.0120, respectively). For Ae. albopictus (Fig. 2 E and F), the IR decreased over time following OYAV infection, with the highest IR of 16.7% at 4 dpi and the lowest at 6.3% at 14 dpi. At 7 dpi, positive mosquitoes' mean viral RNA copies was 10 5.25 copies/μL, with one mosquito having viral RNA copies of 10 7.26 copies/μL. EBIV showed low infectivity in Ae. albopictus , with only one positive detection at 4, 7, and 10 dpi (IR of 1.6%, 1.6%, and 1.7%, respectively), whereas no positive detections were achieved among 60 mosquitoes at 14 dpi. For Ae. aegypti (Fig. 2 G and H), the OYAV IR did not significantly change from 4 to 14 days, remaining between 6.7% and 10%. The mean viral RNA copies in positive mosquitoes also showed no significant variation, with the highest mean viral RNA copies observed at 14 dpi (10 5.92 copies/μL). Some mosquitoes exhibited higher viral RNA copies, reaching 10 8.31 copies/μL. Dissemination and transmission dynamics of OYAV and EBIV in mosquitoes Based on the above results, it was found that the OYAV IR of Cx. pipiens pallens remained stable at 4-14 dpi, significantly higher than other mosquito species. Consequently, a titer of 3.75×10 6 PFU/mL of OYAV was used for further infection studies in Cx. pipiens pallens . Viral replication was monitored in the mosquito’s bodies, heads, and saliva at 4, 7, 10, and 14 dpi (Fig. 3 A-F). The IR and mean viral RNA copies in positive mosquitoes were consistent with earlier observations. Notably, OYAV was almost undetectable in the heads of approximately twenty positive mosquitoes. However, OYAV was detected in the head of one positive mosquito at 4 and 10 dpi, corresponding to the DR of 3.8% and 4.8%, with viral RNA copies of 10 7.33 copies/μL and 10 4.45 copies/μL, respectively. OYAV was not detected in saliva samples at 4-10 dpi. In contrast, the above results showed both Cx. pipiens pallens and Cx. quinquefasciatus supports the multiplication of EBIV in vivo, except for Ae. albopictus . While Cx. pipiens pallens were infected using a titer of 3.75×10 6 PFU/mL of EBIV, and viral replication was observed in the bodies, heads, and saliva of 60 mosquitoes at 4, 7, 10, and 14 dpi (Fig. 4 A-F). The IR and mean viral RNA copies in positive mosquitoes were similar to previous results. Some mosquito bodies exhibited viral RNA copies exceeding 10 7 copies/μL (7 dpi: 6), with a few individuals reaching 10 8 copies/μL or higher. EBIV was detected in the heads of positive mosquitoes at 7 and 10 dpi, with mean viral RNA copies of 10 7.32 copies/μL and 10 5.74 copies/μL, respectively, corresponding to DRs of 46.2% and 50.0%. At 7 dpi, the virus was detected in the saliva of two positive mosquitoes, with mean viral RNA copies of 10 4.03 copies/μL (equivalent to an EBIV titer of 10 1.38 PFU/mL) and a TR of 15.4%. Furthermore, Cx. quinquefasciatus was infected with a 5.7×10 6 PFU/mL titer of EBIV (Fig. 4 G-L). The IR and mean viral RNA copies in positive mosquitoes were consistent with prior findings. A few positive mosquitoes had viral RNA copies exceeding 10 6 copies/μL (8 mosquitoes). EBIV was detected in the heads of positive mosquito samples at 4, 7, 10, and 14 dpi. The mean viral RNA copies in the heads were 10 5.27 copies/μL, 10 5.32 copies/μL, 10 6.85 copies/μL, and 10 6.14 copies/μL, respectively, corresponding to DRs of 44.4%, 100.0%, 55.6%, and 60.0%. EBIV was not detected in samples from 4 dpi to 10 dpi. Discussion Previous research demonstrated that CQV, classified within the species Orthobunyavirus catqueense alongside OYAV, could be detected in Ae. aegypti , Cx. quinquefasciatus and Cx. tritaeniorhynchus at 12 dpi through intrathoracic and artificial membrane/oral feeding routes [31]. Our findings further indicated that Cx. pipiens pallens had a notably higher IR to OYAV (35.7%-40.0%) compared to Cx. quinquefasciatus , Ae. albopictus , and Ae. aegypti (6.3%-18.3%) at 4-14 dpi. Cx. pipiens pallens might be highly susceptible to OYAV, potentially supporting its multiplication. Interestingly, OYAV was detected in the head of only one positive mosquito at 4 and 10 dpi. The viral load in the head (10 7.33 copies/μL) at 4 dpi was higher than the highest viral load in the mosquito’s body (10 6.64 copies/μL), yet OYAV was not detected in the saliva. Arboviruses, during their invasion process, must navigate the innate immune responses of mosquitoes and overcome several barriers, including the midgut infection barrier (MIB), midgut escape barrier (MEB), salivary gland infection barrier (SGIB), and salivary gland escape barrier (SGEB) [33]. Among these barriers, MEB might be a substantial barrier to OYAV’s hemolymph circulation entry. However, factors influencing arbovirus midgut escape are complex, such as the necessity for a virus to reach a threshold level for escape and viral dose considerations [34, 35]. The role of MEB in OYAV transmission by Cx. pipiens pallens requires further experimental evidence. Our team’s earlier studies documented that Ae. aegypti mosquitoes could be infected by EBIV, implying a potential threat of virus transmission. It was further evidenced by the detection of EBIV in the saliva of these mosquitoes at 14 dpi, with an average viral load exceeding 6.3 PFU per mosquito [30]. The current research also uncovered that Cx. pipiens pallens and Cx. quinquefasciatus exhibited the highest IR of 26.7% and 21.2%, respectively, in contrast to the notably lower IR of 1.7% in Ae. albopictus . Specifically, after EBIV infection via oral route in Cx. pipiens pallens , the DR was found to be 46.2%, with a TR of 15.4%, and the mean viral RNA copies in saliva of 10 4.03 copies/μL (equivalent to a mean viral dose of 6.00 PFU per mosquito). The severity of EBIV’s impact is further highlighted by previous studies on BALB/c mice, where more than 90% succumbed to infection with extremely low doses of EBIV (1-10 PFU), indicating a greater pathogenicity of this virus compared to other orthobunyaviruses [22]. This suggests a potential risk of EBIV transmission from Cx. pipiens pallens to vertebrate hosts. In Cx. quinquefasciatus , the DR for oral EBIV infection ranged from 44.4% to 100%, with high levels of viral RNA in the heads of infected mosquitoes. However, EBIV was not detected in the saliva of positive mosquitoes. Thus, the current observation leads to speculation that the salivary gland barrier may be crucial in limiting the transmission of EBIV by Cx. quinquefasciatus . This hypothesis is supported by the fact that salivary proteins can alter the trajectory of viral infection in mosquitoes [36] and that SGIB and SGEB act as modulators of arbovirus transmission [37]. In essence, this study underscores the variable susceptibility of different mosquito species to EBIV and highlights the complex interplay of physiological barriers during the transmission of this virus. Within our analyzable range, Cx. pipiens pallens exhibits the highest susceptibility to OYAV, while Ae. aegypti is the most susceptible to EBIV. Both Ae. aegypti and Cx. pipiens pallens can be infected by EBIV, presenting a potential risk of virus transmission. However, it is critical to note that vectorial capacity can be affected by different genotypes/isolates of the virus and different mosquito species and geographic strains. For example, experimental studies of mosquito infections through the Oropouche virus (OROV) of the same Simbu serogroup have shown different IR and TR of Cx. quinquefasciatus infected by different OROV isolates from different geographic strains [38, 39]. Meanwhile, researchers have demonstrated that the genetic background of the vector significantly influences the virus’s susceptibility [40, 41]. The EBIV (Cu20-XJ) used in this study was isolated from Cx . modestus [20], and Cx. tritaeniorhynchus is a known vector for transmission of Japanese encephalitis in China [42]. However, relevant experiments were not conducted due to the unavailability of these two mosquito species during the study duration. Therefore, the vector competence of these significant mosquito species in China for EBIV and OYAV remains to be uncovered. Additionally, as the OYAV (SZC50) was isolated from midge samples [16], it is worth considering midges as potential vectors for SZC50. Therefore, further studies are necessary to clarify the risk of virus transmission by examining different virus isolates infecting various geographical strains of mosquito species. Finally, it is noteworthy to investigate if mosquitoes that test positive for the virus in their saliva may infect vertebrate hosts through their bites. Additional studies on the transmission cycle involving the virus, mosquitoes, and vertebrates are needed. Conclusions Our study focused on examining the susceptibility of Cx. pipiens pallens , Cx. quinquefasciatus , Ae. albopictus , and Ae. aegypti to OYAV, while Cx. pipiens pallens , Cx. quinquefasciatus , and Ae. albopictus to EBIV. It also evaluated the vectorial capacity for OYAV transmission in Cx. pipiens pallens and the EBIV transmission in both Cx. pipiens pallens and Cx. quinquefasciatus . This investigation enhances our understanding of the transmission potential of neglected orthobunyaviruses and contributes to the defense against their potential emergence globally, including in China. Abbreviations DR: Dissemination rate; EBIV: Ebinur lake virus; IR: Infection rate; OYAV: Oya virus; PFU: Plaque-forming unit; TR: Transmission rate; Declarations Acknowledgement The authors would like to thank the Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, China CDC for kindly provided the Ae. albopictus and Cx. pipiens pallens mosquitos. Furthermore, we would like to thank the Institutional Center for Shared Technologies and Facilities of Wuhan Institute of Virology. Funding This work was supported by the National Key Research and Development Program of China (2022YFC2302700), the National Natural Science Foundation of China (U22A20363), and the Youth Program of Wuhan Institute of Virology (2023QNTJ-03). Availability of data and materials The corresponding author can provide the datasets used and/or analyzed during the current investigation upon reasonable request. Author contributions ZY and HX conceived the idea and coordinated the project. SL, XW, and CY performed experiments. SL,XW, and CY analyzed data. JW, QL, ZY, and HX provided resources. SL, XW, CY, DH and HM performed the maintenance of mosquitoes. SL, FW, WZ, and HX drew the figures and drafted the manuscript. SL, FW, JW, QL, ZY and HX revised the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable. Consent for publication Participants made aware during the consenting process that their views will be shared widely including publication in peer review journals. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Supplementary Files GraphicalAbstract.png TableS1.xlsx TableS2.xlsx Cite Share Download PDF Status: Published Journal Publication published 06 May, 2024 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 17 Mar, 2024 Reviews received at journal 11 Mar, 2024 Reviewers agreed at journal 22 Feb, 2024 Reviewers invited by journal 22 Feb, 2024 Editor assigned by journal 21 Feb, 2024 Submission checks completed at journal 21 Feb, 2024 First submitted to journal 19 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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11:33:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3969850/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3969850/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-024-06295-5","type":"published","date":"2024-05-07T03:58:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51535303,"identity":"13896b3a-c495-4d47-b17f-83e32239b877","added_by":"auto","created_at":"2024-02-23 09:21:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":596686,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent viral titer of OYAV (Oya virus) and EBIV (Ebinur Lake Virus) infection rates of adult different mosquitoes species through blood feeding.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViral RNA copies (A) and infection rates (B) of \u003cem\u003eCulex. pipiens pallens\u003c/em\u003e, \u003cem\u003eCulex. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAedes. albopictus,\u003c/em\u003e and \u003cem\u003eAedes. aegypti\u003c/em\u003e at 4 days after feeding on blood meals with continuous dilution of OYAV from 10\u003csup\u003e6\u003c/sup\u003e to 10\u003csup\u003e5\u003c/sup\u003e PFU/mL. Viral RNA copies (C) and infection rates (D) of \u003cem\u003eCx. pipiens pallens\u003c/em\u003e, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, and \u003cem\u003eAe. albopictus\u003c/em\u003e were evaluated after 4 days of feeding on blood meals with a continuous dilution of EBIV from 10\u003csup\u003e7\u003c/sup\u003e to 10\u003csup\u003e5\u003c/sup\u003e PFU/mL. Each dot represents an individual mosquito, and the grey dots indicate samples with Ct values \u0026gt; 36 (OYAV) and \u0026gt; 35 (EBIV). Infection rates were analyzed with Fisher’s exact test, and mean viral RNA copies/μL were analyzed with One-way ANOVA with Tukey’s multiple comparison (*: \u003cem\u003eP\u003c/em\u003e ≤ 0.05, **: \u003cem\u003eP\u003c/em\u003e ≤ 0.01, ***: \u003cem\u003eP\u003c/em\u003e ≤ 0.005, ****: \u003cem\u003eP\u003c/em\u003e ≤ 0.0001). The “ns” indicates that no significant difference was observed.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3969850/v1/109d0086349fde36af3f339f.png"},{"id":51534817,"identity":"8b00fc8b-cd86-428e-906f-dc22aac3969d","added_by":"auto","created_at":"2024-02-23 09:13:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1847731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOYAV and EBIV infection rates of adult different mosquito species through blood feeding.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViral RNA copies in \u003cem\u003eCx. pipiens pallens\u003c/em\u003e (A), \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e (C), \u003cem\u003eAe. albopictus\u003c/em\u003e (E), and \u003cem\u003eAe. aegypti\u003c/em\u003e (G) and infection rates in \u003cem\u003eCx. pipiens pallens\u003c/em\u003e (B), \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e (D), \u003cem\u003eAe. albopictus\u003c/em\u003e (F), and \u003cem\u003eAe. aegypti\u003c/em\u003e (H) at 4, 7, 10, and 14 days after feeding on a blood meal containing 10\u003csup\u003e6\u003c/sup\u003e PFU/mL OYAV and EBIV. Each dot represents an individual mosquito, and the grey dots indicate samples with Ct values \u0026gt; 36 (OYAV) and \u0026gt; 35 (EBIV). The infection rates were analyzed with Fisher’s exact test, and mean viral RNA copies/μL were analyzed with One-way ANOVA with Tukey’s multiple comparison (*: \u003cem\u003eP\u003c/em\u003e ≤ 0.05, **: \u003cem\u003eP\u003c/em\u003e ≤ 0.01, ***: \u003cem\u003eP\u003c/em\u003e ≤ 0.005, ****: \u003cem\u003eP\u003c/em\u003e ≤ 0.0001).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3969850/v1/f43ea70fb12141f1caaf4a12.png"},{"id":51535304,"identity":"0d261571-52a8-444b-95bb-ffd719ac9765","added_by":"auto","created_at":"2024-02-23 09:21:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":756445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOYAV infection, dissemination and transmission in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCx. pipiens pallens.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eViral RNA copies in the bodies (A), heads (C), and saliva (E) samples and Infection rates (B), dissemination rates (D), and transmission rates (F) of mosquitoes at 4, 7, 10, and 14 days after feeding on blood meal containing 3.2 × 10\u003csup\u003e6\u003c/sup\u003e PFU/mL OYAV. Each dot represents an individual mosquito, and the grey dots indicate samples with Ct value \u0026gt; 36. The rates were analyzed with Fisher’s exact test, and mean viral RNA copies/μL were analyzed with One-way ANOVA with Tukey’s multiple comparison (*: \u003cem\u003eP\u003c/em\u003e ≤ 0.05, **: \u003cem\u003eP\u003c/em\u003e ≤ 0.01, ***: \u003cem\u003eP\u003c/em\u003e ≤ 0.005, ****: \u003cem\u003eP\u003c/em\u003e ≤ 0.0001).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3969850/v1/9a63deb0b882d4526b4dfeea.png"},{"id":51534822,"identity":"f45d408b-0f12-4d70-b9ba-2b08b36f65d9","added_by":"auto","created_at":"2024-02-23 09:13:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1771528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEBIV infection, dissemination and transmission in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCx. pipiens pallens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCx. quinquefasciatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViral RNA copies in the bodies (A), heads (C), and saliva (E) samples and Infection rates (B), dissemination rates (D), and transmission rates (F) of \u003cem\u003eCx. pipiens pallens\u003c/em\u003e at 4, 7, 10, and 14 days after feeding on blood meal containing 3.8 × 10\u003csup\u003e6\u003c/sup\u003e PFU/mL EBIV. Viral RNA copies in the body (G), head (I), and saliva (K) samples and Infection rates (H), dissemination rates (J), and transmission rates (L) of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e at 4, 7, 10, and 14 days after feeding on a blood meal containing 5.7 × 10\u003csup\u003e6\u003c/sup\u003e PFU/mL EBIV. Each dot represents an individual mosquito, and the grey dots indicate samples with Ct value \u0026gt; 35. The infection rates were analyzed with Fisher’s exact test, and mean viral RNA copies/μL were analyzed with One-way ANOVA with Tukey’s multiple comparison (*: \u003cem\u003eP\u003c/em\u003e ≤ 0.05, **: \u003cem\u003eP\u003c/em\u003e ≤ 0.01, ***: \u003cem\u003eP\u003c/em\u003e ≤ 0.005, ****: \u003cem\u003eP\u003c/em\u003e ≤ 0.0001).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3969850/v1/4985054a907a8264aa267f54.png"},{"id":56140314,"identity":"962bf629-8bda-40af-aee7-00cc08fb35e2","added_by":"auto","created_at":"2024-05-09 04:11:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1694576,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3969850/v1/e3e8f634-8144-425a-a8e9-0a20bfc2c0c7.pdf"},{"id":51534820,"identity":"a26bc7b1-71ca-4b01-8dec-e4e58862694f","added_by":"auto","created_at":"2024-02-23 09:13:55","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3462558,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-3969850/v1/197988f45f778997523ceba2.png"},{"id":51534818,"identity":"e3a3746d-fc05-416a-b0cc-fca6bfca516d","added_by":"auto","created_at":"2024-02-23 09:13:55","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11590,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3969850/v1/02d28022961c9a21a21b757f.xlsx"},{"id":51534821,"identity":"2e9a9ddc-74d9-45d8-b8b7-29cc26459964","added_by":"auto","created_at":"2024-02-23 09:13:55","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10888,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3969850/v1/e9d0776909eac3efb0a24a35.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake Virus","fulltext":[{"header":"Background","content":"\u003cp\u003eMosquitoes, being important vectors, can carry and transmit a diverse range of pathogens, including arboviruses, primarily from the \u003cem\u003ePeribunyaviridae\u003c/em\u003e, \u003cem\u003eTogaviridae\u003c/em\u003e, \u003cem\u003eFlaviviridae\u003c/em\u003e, \u003cem\u003ePhenuiviridae\u003c/em\u003e, and \u003cem\u003eReoviridae\u003c/em\u003e families, along with several types of parasites [1].\u003c/p\u003e\n\u003cp\u003eThe family \u003cem\u003ePeribunyaviridae\u003c/em\u003e, genus \u003cem\u003eOrthobunyavirus\u003c/em\u003e, contains three-segmented, negative-stranded RNA viruses transmitted exclusively by arthropods [2]. So far, nearly 200 viruses of the genus \u003cem\u003eOrthobunyavirus\u003c/em\u003e have been identified and grouped into 20 serogroups based on their serological relatedness [3]. Multiple viruses belonging to the Simbu and Bunyawera serogroups have been found in various mosquito species, including \u003cem\u003eCulex\u003c/em\u003e spp., \u003cem\u003eAedes\u003c/em\u003e spp., and \u003cem\u003eAnopheles\u003c/em\u003e spp [4, 5, 6]. Members of the family \u003cem\u003ePeribunyaviridae\u003c/em\u003e can introduce illnesses in vertebrates, with encephalitis caused by the Oropouche virus (OROV) and La Crosse virus (LACV) affecting humans [7, 8]. Additionally, ruminants are at risk of abortions and teratogenic effects from Bunyamwera virus (BUNV) and Cache Valley virus (CVV) [9, 10].\u003c/p\u003e\n\u003cp\u003eOya virus (OYAV) and Ebinur lake virus (EBIV) belong to the \u003cem\u003eOrthobunyavirus\u003c/em\u003e genus. OYAV is a member of the Simbu serogroup and shares the same virus species as the Cat Que virus (CQV), which is the\u0026nbsp;\u003cem\u003eOrthobunyavirus catqueense\u003c/em\u003e [11].\u0026nbsp;OYAV was first isolated from cell cultures from pig lungs in Malaysia in 2000 [13]. Subsequent studies found several viral isolates of this species in China, Vietnam, and India from invertebrate and vertebrate hosts [12][14, 15, 16, 17, 18]. EBIV, a member of the Bunyamwera serogroup, was initially isolated from the pools of \u003cem\u003eCulex modestus\u003c/em\u003e in Xinjiang Province, China, and was later been detected in various samples from Russia and Kenya [19, 20]. Experimental investigations have demonstrated that these viruses efficiently invade and replicate within various cell lines derived from vertebrates and mosquitoes [16, 21]. In addition, OYAV is lethal in suckling and C57BL/6 adult mice [16], while EBIV has been proven to be fatal in BALB/c mice [22]. Although no confirmed cases of OYAV and EBIV have been recorded, a serological survey revealed that 93% of pigs within pig farming zones in Malaysia tested positive for OYAV [13], and the seroprevalence of OYAV in pigs in Yunnan, China, exceeded 30 percent [16]. Moreover, the antibodies against EBIV have previously been detected in residents of Xinjiang [21]. These findings indicate that OYAV and EBIV may potentially threaten animal or public health.\u003c/p\u003e\n\u003cp\u003eThe prevalent mosquito genera in China are \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e [6]. \u003cem\u003eCulex pipiens pallens\u003c/em\u003e and \u003cem\u003eCx.\u003c/em\u003e\u003cem\u003e\u0026nbsp;quinquefasciatus,\u003c/em\u003e both belonging to the \u003cem\u003eCx. pipiens\u003c/em\u003e complex, are predominantly distributed in northern and southern parts of China, respectively [23]. The \u003cem\u003eCx. pipiens\u003c/em\u003e complex plays a crucial role in transmitting arboviruses, particularly the West Nile virus [24, 25]. \u003cem\u003eAedes albopictus\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e are recognized as the chief vectors of DENV [26, 27]. \u003cem\u003eAedes albopictus\u003c/em\u003e is a widespread species in the southern regions of China [28]. In China, \u003cem\u003eAe. aegypti\u003c/em\u003e has also been found in parts of Yunnan, Hainan, and Guangdong, and the geographical distribution for \u003cem\u003eAe. aegypti\u003c/em\u003e is expending in recent years [29]. Our group\u0026apos;s previous work evaluated the vector competence of \u003cem\u003eAe. aegypti\u003c/em\u003e for EBIV [30]. Meanwhile, the susceptibility of \u003cem\u003eCulex\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes to CQV has now been reported from India [31]. Nevertheless, there is a need for more relevant mosquito studies on EBIV and OYAV isolates in China, where different genotypes/isolates of the virus, mosquito species, and geographical strains may have an impact on vector competence.\u003c/p\u003e\n\u003cp\u003eThis study aimed to assess the infection, replication, and transmission ability of OYAV and EBIV in \u003cem\u003eCx. pipiens pallens\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Cx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus\u003c/em\u003e (common mosquito species in China), and \u003cem\u003eAe. aegypti\u003c/em\u003e by the oral blood-feeding route (natural infection simulation) to explore their abilities to contribute to potential outbreak preparedness by informing local mosquito-control organizations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMosquito species and rearing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, different mosquito species were utilized. The reared \u003cem\u003eCx. pipiens pallens\u003c/em\u003e (Beijing strain, kindly provided by China CDC), \u003cem\u003eCx. quinquefasciatus\u0026nbsp;\u003c/em\u003e(Wuhan strain), \u003cem\u003eAe. albopictus\u003c/em\u003e (Jiangsu strain, kindly provided by China CDC), and \u003cem\u003eAe\u003c/em\u003e.\u003cem\u003e\u0026nbsp;aegypti\u003c/em\u003e (Rockefeller strain, kindly provided by Qian Han from Hainan University). The feeding methods and processes were the same as described earlier [30].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and Viral stock\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBHK cells (golden hamster kidney cells) and PK 15 cells (pig kidney cells) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Gibco) containing 10% FBS, 1% penicillin/streptomycin (Gibco) at 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe viral strains included OYAV (strain SZC50) and EBIV (strain Cu20-XJ). The titers of OYAV and EBIV were determined using the plaque formation assay, and their working stocks were found to be 1.4\u0026times;10\u003csup\u003e7\u003c/sup\u003e plaque-forming units mL\u0026minus;1 (PFU/mL) and 7.0\u0026times;10\u003csup\u003e7\u003c/sup\u003e PFU/mL, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMosquito adult infection through blood feeding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor infections through an artificial mosquito feeding system (Hemotek), 3 to 5-day-old adult females were starved for 12 to 24 h and fed with a mixture of blood and virus supernatant. The titer of the virus-blood mixture was determined at the same time by plaque assay. Refer to previously published articles for detailed procedures on oral infection and because our group\u0026apos;s previous study described the results of EBIV infection in \u003cem\u003eAe. aegypti\u003c/em\u003e [30], this study no longer uses EBIV infection in \u003cem\u003eAe. aegypti\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo establish the appropriate concentration of OYAV and EBIV in different mosquito species,\u0026nbsp;\u003cem\u003eCx. pipiens pallens\u003c/em\u003e, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus,\u003c/em\u003e and\u0026nbsp;\u003cem\u003eAe. aegypti\u003c/em\u003e infected with different virus-blood concentrations (OYAV: two serial titers ranging from\u0026nbsp;10\u003csup\u003e6\u003c/sup\u003e-10\u003csup\u003e5\u003c/sup\u003e PFU/mL; EBIV: three serial titers ranging from 10\u003csup\u003e7\u003c/sup\u003e-10\u003csup\u003e5\u003c/sup\u003e PFU/mL) were subsequently collected at 4 days post-infection (dpi) for viral RNA determination.\u003c/p\u003e\n\u003cp\u003eA previous study showed that adult female BALB/c mice infected with 10 PFU of EBIV reached 10\u003csup\u003e6\u003c/sup\u003e PFU/mL viremia at 2 dpi. Also, in conjunction with the preliminary findings from this study, viruses with a titer of 10\u003csup\u003e6\u003c/sup\u003e PFU/mL were chosen for infection. Infected mosquitoes were collected at 4, 7, 10, and 14 dpi for viral RNA determination.\u003c/p\u003e\n\u003cp\u003eTo determine OYAV and EBIV distribution in infected \u003cem\u003eCx. pipiens pallens\u003c/em\u003e, \u003cem\u003eCx.quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus,\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e via artificial blood feeding (final viral titer of 10\u003csup\u003e6\u003c/sup\u003e PFU/mL), viral RNA in mosquito bodies, heads, and saliva at 4, 7, 10, and 14 dpi were examined. The detailed procedures for collecting and preserving mosquito saliva can be found in prior published articles [30]. All mosquito tissues and saliva were stored in 250 \u0026mu;L RPMI 1640 supplemented with 2% Penicillin/Streptomycin/Gentamicin Solution. Prior to processing, all samples were kept at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eVector competence of the mosquitoes was evaluated by calculating the infection rate (IR; number of positive\u0026nbsp;bodies/the total number of mosquitoes tested), dissemination rate (DR; number of positive heads/the number of the positive abdomen), transmission rate (TR; number of positive saliva/the number of positive abdomen).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of viral replication by RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll samples were initially homogenized using a Low Temperature Tissue Homogenizer Grinding Machine (Servicebio) (operating frequency = 60Hz, operation time = 10 s, pause time = 10 s, cycles = 3, and setting temperature = 4˚C), followed by centrifugation for 5 min at 10, 000\u0026times;g and 4˚C. The total RNA of each sample was extracted using an automated nucleic acid extraction system following the manufacturer\u0026rsquo;s instructions (NanoMagBio).\u003c/p\u003e\n\u003cp\u003eUsing the CFX96 Touch Real-Time PCR Detection System (Bio-Rad), viral RNA copies of each sample were quantified. For OYAV, the Luna\u003csup\u003e\u0026reg;\u003c/sup\u003e Universal Probe One-Step RT-qPCR Kit (NEB) was used, and for EBIV, the HiScript II One Step qRT-PCR Probe Kit (Vazyme) was used. The primer and probe sets used are presented in previous studies [18]. The cutoff for positive samples determined via RT-qPCR was Ct (OYAV) \u0026lt; 36 and Ct (EBIV) \u0026lt; 35. The positive cutoff value was evaluated by comparing paired serial ten-fold dilutions either inoculated on cells or assayed via RT-qPCR (Table\u0026nbsp;S1) [32]. The equation for the standard curve was shown in Table S2 and used to calculate the viral genome copies in each sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism 8.0.2 (GraphPad Software Inc) was used to analyze all data. Fisher\u0026rsquo;s exact test was used to compare the mosquito\u0026rsquo;s infection, dissemination, and transmission rates between different treatments. One-way ANOVA with Tukey\u0026rsquo;s multiple comparison was used to compare the mean of the genome copies among more than two data sets. \u003cem\u003eP\u003c/em\u003e \u0026le; 0.05 were considered statistically significant.\u003c/p\u003e\n\u003cp\u003eThe figure of graphical abstract was created with BioRender.com (https://www.biorender.com/) and authorization for publication had been granted.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe susceptibility of adult \u003cem\u003eCulex\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e mosquitoes to different titers of OYAV and EBIV\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe infectivity of mosquitoes consuming artificial blood meals with varying virus titers was investigated.\u0026nbsp;Four mosquito species\u0026nbsp;\u0026mdash;\u003cem\u003eCx. pipiens pallens\u003c/em\u003e, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus,\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e \u0026mdash;were fed with blood meals containing 3-4.3\u0026times;10\u003csup\u003e5\u003c/sup\u003e PFU/mL and 3-7 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e PFU/mL of OYAV, respectively. The infection rates (IR) and viral replication were assessed at 4 dpi (Fig. 1A and B). No infection was detected in \u003cem\u003eCx. pipiens pallens\u003c/em\u003e at 10\u003csup\u003e5\u003c/sup\u003e PFU/mL OYAV among 40 tested mosquitoes. However, at a titer of 10\u003csup\u003e6\u003c/sup\u003e PFU/mL, the IR increased to 42.0%, with an average viral RNA concentration of 10\u003csup\u003e4.44\u0026nbsp;\u003c/sup\u003ecopies/\u0026mu;L and a peak of 10\u003csup\u003e6.33\u0026nbsp;\u003c/sup\u003ecopies/\u0026mu;L, marking a significant increase in IR compared to the lower titer (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). For \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e, IR was not significantly affected by blood meal infections at different titers, with IR remaining below 18%. Meanwhile, at the OYAV infection titer of 10\u003csup\u003e6\u003c/sup\u003e PFU/mL, the IR of \u003cem\u003eCx. pipiens pallens\u003c/em\u003e was significantly higher than that of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.0211; 0.0025; 0.0006, respectively). There was no significant difference in the mean viral RNA copies among different species of infected mosquitoes at varying infection titers.\u003c/p\u003e\n\u003cp\u003eThree mosquito species\u0026nbsp;\u0026mdash;\u003cem\u003eCx. pipiens pallens\u003c/em\u003e, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e \u0026mdash;were exposed to EBIV in blood meals with titers ranging from 1.8-6.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e PFU/mL, 3.8-5.7\u0026times;10\u003csup\u003e6\u003c/sup\u003e PFU/mL, and 1.3-2.5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e PFU/mL, respectively. Their IR and viral replication were assessed at 4 dpi (Fig. 1C and D). In \u003cem\u003eCx. pipiens pallens\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003ea positive correlation was observed between blood meal titers and IR, with an IR of 80% at the highest titer of 10\u003csup\u003e7\u003c/sup\u003e PFU/mL, significantly surpassing the rates at lower titers (all \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). The mean viral RNA copies in positive mosquitoes were 10\u003csup\u003e5.55\u003c/sup\u003e copies/\u0026mu;L (10\u003csup\u003e5\u003c/sup\u003e PFU/mL: 10\u003csup\u003e4.51\u003c/sup\u003e copies/\u0026mu;L; 10\u003csup\u003e6\u003c/sup\u003e PFU/mL: 10\u003csup\u003e3.61\u003c/sup\u003e copies/\u0026mu;L), peaking at 10\u003csup\u003e7.99\u003c/sup\u003e copies/\u0026mu;L, notably higher than in lower titer infections. For \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, EBIV RNA was not detected at a blood meal titer of 10\u003csup\u003e5\u003c/sup\u003e PFU/mL. However, at 10\u003csup\u003e6\u003c/sup\u003e PFU/mL and 10\u003csup\u003e7\u003c/sup\u003e PFU/mL, the IR increased to 17.5% and 16.7%, respectively, with mean viral RNA copies of 10\u003csup\u003e4.81\u003c/sup\u003e copies/\u0026micro;L and 10\u003csup\u003e3.99\u003c/sup\u003e copies/\u0026micro;L. In \u003cem\u003eAe. albopictus\u003c/em\u003e, there were no infections at 10\u003csup\u003e5\u003c/sup\u003e PFU/mL and 10\u003csup\u003e6\u003c/sup\u003e PFU/mL. Nevertheless, at a 10\u003csup\u003e7\u003c/sup\u003e PFU/mL titer, the IR rose to 35%, with mean viral RNA copies of 10\u003csup\u003e3.90\u003c/sup\u003e copies/\u0026mu;L and a maximum of 10\u003csup\u003e5.03\u003c/sup\u003e copies/\u0026mu;L. At the highest titer of 10\u003csup\u003e7\u003c/sup\u003e PFU/mL, \u003cem\u003eCx. pipiens pallens\u003c/em\u003e exhibited a significantly higher IR than other mosquito species (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). At a titer of 10\u003csup\u003e6\u003c/sup\u003e PFU/mL, \u003cem\u003eCx. pipiens pallens\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e displayed similar IRs, both higher than that of \u003cem\u003eAe. albopictus\u0026nbsp;\u003c/em\u003e(\u003cem\u003eP\u003c/em\u003e = 0.0173).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDynamic variations in viral titers in adult mosquitoes infected with OYAV or EBIV via oral blood feeding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess adult mosquitoes\u0026apos; susceptibility to OYAV and EBIV, they were infected through oral blood feeding at an average titer of 3.75 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e PFU/mL, with samples collected at 4, 7, 10, and 14 dpi for RNA analysis (Fig. 2 A-H).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eCx. pipiens pallens\u0026nbsp;\u003c/em\u003e(Fig. 2 A and B), the OYAV IR remained relatively stable from 4 to 14 dpi, fluctuating between 35.7% and 40.0%. The mean viral RNA copies in positive mosquitoes varied slightly, ranging from 10\u003csup\u003e4.00\u003c/sup\u003e to 10\u003csup\u003e4.33\u003c/sup\u003e copies/\u0026mu;L. In contrast, EBIV infection resulted in significantly lower IRs at 4 (\u003cem\u003eP\u003c/em\u003e = 0.0003), 10 (\u003cem\u003eP\u003c/em\u003e = 0.0003), and 14 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001) dpi compared to OYAV. The peak IR for EBIV was observed at 7 dpi (26.7%), notably higher than the rates at 4, 10 (both \u003cem\u003eP\u003c/em\u003e = 0.0148) and 14 dpi (\u003cem\u003eP\u003c/em\u003e = 0.0060). The mean viral RNA copies in EBIV-positive mosquitoes was 10\u003csup\u003e4.59\u003c/sup\u003e copies/\u0026mu;L, with some exceeding 10\u003csup\u003e8\u003c/sup\u003e copies/\u0026mu;L.\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eCx. quinquefasciatus\u0026nbsp;\u003c/em\u003e(Fig. 2 C and D), the OYAV IR decreased from 18.3% at 4 dpi to 1.6% at 14 dpi. The mean viral RNA copies remained relatively constant, with a maximum of 10\u003csup\u003e3.35\u003c/sup\u003e copies/\u0026mu;L at 4 dpi. EBIV infection results in higher IRs at 4 and 10 dpi (20.0% and 21.2%, respectively) than those at 7 and 14 dpi (13.6% and 9.1%, respectively). The mean viral RNA copies were highest at 14 dpi, with one positive mosquito having a viral RNA copy of 10\u003csup\u003e7.73\u003c/sup\u003e copies/\u0026mu;L and a mean of 10\u003csup\u003e5.13\u003c/sup\u003e copies/\u0026mu;L. The mean viral RNA copies of EBIV-positive mosquitoes at 4 and 10 dpi were significantly higher than those infected with OYAV (\u003cem\u003eP\u003c/em\u003e = 0.0366 and 0.0120, respectively).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eAe. albopictus\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Fig. 2 E and F), the IR decreased over time following OYAV infection, with the highest IR of 16.7% at 4 dpi and the lowest at 6.3% at 14 dpi. At 7 dpi, positive mosquitoes\u0026apos; mean viral RNA copies was 10\u003csup\u003e5.25\u003c/sup\u003e copies/\u0026mu;L, with one mosquito having viral RNA copies of 10\u003csup\u003e7.26\u003c/sup\u003e copies/\u0026mu;L. EBIV showed low infectivity in \u003cem\u003eAe. albopictus\u003c/em\u003e, with only one positive detection at 4, 7, and 10 dpi (IR of 1.6%, 1.6%, and 1.7%, respectively), whereas no positive detections were achieved among 60 mosquitoes at 14 dpi.\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eAe. aegypti\u0026nbsp;\u003c/em\u003e(Fig. 2 G and H), the OYAV IR did not significantly change from 4 to 14 days, remaining between 6.7% and 10%. The mean viral RNA copies in positive mosquitoes also showed no significant variation, with the highest mean viral RNA copies observed at 14 dpi (10\u003csup\u003e5.92\u003c/sup\u003e copies/\u0026mu;L). Some mosquitoes exhibited higher viral RNA copies, reaching 10\u003csup\u003e8.31\u003c/sup\u003e copies/\u0026mu;L.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDissemination and transmission dynamics of OYAV and EBIV in mosquitoes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the above results, it was found that\u0026nbsp;the OYAV IR of \u003cem\u003eCx. pipiens pallens\u003c/em\u003e remained stable at 4-14 dpi, significantly higher than other mosquito species. Consequently, a titer of 3.75\u0026times;10\u003csup\u003e6\u003c/sup\u003e PFU/mL of OYAV was used for further infection studies in \u003cem\u003eCx. pipiens pallens\u003c/em\u003e. Viral replication was monitored in the mosquito\u0026rsquo;s bodies, heads, and saliva at 4, 7, 10, and 14 dpi (Fig. 3 A-F). The IR and mean viral RNA copies in positive mosquitoes were consistent with earlier observations. Notably, OYAV was almost undetectable in the heads of approximately twenty positive mosquitoes. However, OYAV was detected in the head of one positive mosquito at 4 and 10 dpi, corresponding to the DR of 3.8% and 4.8%, with viral RNA copies of 10\u003csup\u003e7.33\u003c/sup\u003e copies/\u0026mu;L and 10\u003csup\u003e4.45\u003c/sup\u003e copies/\u0026mu;L, respectively. OYAV was not detected in saliva samples at 4-10 dpi.\u003c/p\u003e\n\u003cp\u003eIn contrast, the above results showed both \u003cem\u003eCx. pipiens pallens\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e supports the multiplication of EBIV in vivo, except for \u003cem\u003eAe. albopictus\u003c/em\u003e. While \u003cem\u003eCx. pipiens pallens\u003c/em\u003e were infected using a titer of 3.75\u0026times;10\u003csup\u003e6\u003c/sup\u003e PFU/mL of EBIV, and viral replication was observed in the bodies, heads, and saliva of 60 mosquitoes at 4, 7, 10, and 14 dpi (Fig. 4 A-F). The IR and mean viral RNA copies in positive mosquitoes were similar to previous results. Some mosquito bodies exhibited viral RNA copies exceeding 10\u003csup\u003e7\u003c/sup\u003e copies/\u0026mu;L (7 dpi: 6), with a few individuals reaching 10\u003csup\u003e8\u003c/sup\u003e copies/\u0026mu;L or higher. EBIV was detected in the heads of positive mosquitoes at 7 and 10 dpi, with mean viral RNA copies of 10\u003csup\u003e7.32\u003c/sup\u003e copies/\u0026mu;L and 10\u003csup\u003e5.74\u003c/sup\u003e copies/\u0026mu;L, respectively, corresponding to DRs of 46.2% and 50.0%. At 7 dpi, the virus was detected in the saliva of two positive mosquitoes, with mean viral RNA copies of 10\u003csup\u003e4.03\u003c/sup\u003e copies/\u0026mu;L (equivalent to an EBIV titer of 10\u003csup\u003e1.38\u003c/sup\u003e PFU/mL) and a TR of 15.4%.\u003c/p\u003e\n\u003cp\u003eFurthermore, \u003cem\u003eCx. quinquefasciatus\u0026nbsp;\u003c/em\u003ewas\u003cem\u003e\u0026nbsp;\u003c/em\u003einfected\u003cem\u003e\u0026nbsp;\u003c/em\u003ewith a 5.7\u0026times;10\u003csup\u003e6\u003c/sup\u003e PFU/mL titer of EBIV (Fig. 4 G-L). The IR and mean viral RNA copies in positive mosquitoes were consistent with prior findings. A few positive mosquitoes had viral RNA copies exceeding 10\u003csup\u003e6\u003c/sup\u003e copies/\u0026mu;L (8 mosquitoes). EBIV was detected in the heads of positive mosquito samples at 4, 7, 10, and 14 dpi. The mean viral RNA copies in the heads were 10\u003csup\u003e5.27\u003c/sup\u003e copies/\u0026mu;L, 10\u003csup\u003e5.32\u003c/sup\u003e copies/\u0026mu;L, 10\u003csup\u003e6.85\u003c/sup\u003e copies/\u0026mu;L, and 10\u003csup\u003e6.14\u003c/sup\u003e copies/\u0026mu;L, respectively, corresponding to DRs of 44.4%, 100.0%, 55.6%, and 60.0%. EBIV was not detected in samples from 4 dpi to 10 dpi.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious research demonstrated that CQV, classified within the species \u003cem\u003eOrthobunyavirus catqueense\u003c/em\u003e alongside OYAV, could be detected in \u003cem\u003eAe. aegypti\u003c/em\u003e, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e and \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e at 12 dpi through intrathoracic and artificial membrane/oral feeding routes [31]. Our findings further indicated that \u003cem\u003eCx. pipiens pallens\u003c/em\u003e had a notably higher IR to OYAV (35.7%-40.0%) compared to \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus\u003c/em\u003e, and \u003cem\u003eAe. aegypti\u003c/em\u003e (6.3%-18.3%) at 4-14 dpi. \u003cem\u003eCx. pipiens pallens\u003c/em\u003e might be highly susceptible to OYAV, potentially supporting its multiplication. Interestingly, OYAV was detected in the head of only one positive mosquito at 4 and 10 dpi. The viral load in the head (10\u003csup\u003e7.33\u003c/sup\u003e copies/\u0026mu;L) at 4 dpi was higher than the highest viral load in the mosquito\u0026rsquo;s body (10\u003csup\u003e6.64\u0026nbsp;\u003c/sup\u003ecopies/\u0026mu;L), yet OYAV was not detected in the saliva. Arboviruses, during their invasion process, must navigate the innate immune responses of mosquitoes and overcome several barriers, including the midgut infection barrier (MIB), midgut escape barrier (MEB), salivary gland infection barrier (SGIB), and salivary gland escape barrier (SGEB) [33].\u0026nbsp;Among these barriers, MEB might be a substantial barrier to OYAV\u0026rsquo;s hemolymph circulation entry. However, factors influencing arbovirus midgut escape are complex, such as the necessity for a virus to reach a threshold level for escape and viral dose considerations [34, 35]. The role of MEB in OYAV transmission by \u003cem\u003eCx. pipiens pallens\u003c/em\u003e requires further experimental evidence.\u003c/p\u003e\n\u003cp\u003eOur team\u0026rsquo;s earlier studies documented that \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes could be infected by EBIV, implying a potential threat of virus transmission. It was further evidenced by the detection of EBIV in the saliva of these mosquitoes at 14 dpi, with an average viral load exceeding 6.3 PFU per mosquito [30]. The current research also uncovered\u0026nbsp;that \u003cem\u003eCx. pipiens pallens\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e exhibited the highest IR of 26.7% and 21.2%, respectively, in contrast to the notably lower IR of 1.7% in \u003cem\u003eAe. albopictus\u003c/em\u003e. Specifically, after EBIV infection via oral route in \u003cem\u003eCx. pipiens pallens\u003c/em\u003e, the DR was found to be 46.2%, with a TR of 15.4%, and the mean viral RNA copies in saliva of 10\u003csup\u003e4.03\u003c/sup\u003e copies/\u0026mu;L (equivalent to a mean viral dose of 6.00 PFU per mosquito). The severity of EBIV\u0026rsquo;s impact is further highlighted by previous studies on BALB/c mice, where more than 90% succumbed to infection with extremely low doses of EBIV (1-10 PFU), indicating a greater pathogenicity of this virus compared to other orthobunyaviruses [22]. This suggests a potential risk of EBIV transmission from \u003cem\u003eCx. pipiens pallens\u0026nbsp;\u003c/em\u003eto vertebrate hosts. In \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e,\u0026nbsp;the DR for oral EBIV infection ranged from 44.4% to 100%, with high levels of viral RNA in the heads of infected mosquitoes. However, EBIV was not detected in the saliva of positive mosquitoes. Thus, the current observation leads to speculation that the salivary gland barrier may be crucial in limiting the transmission of EBIV by \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e. This hypothesis is supported by the fact that salivary proteins can alter the trajectory of viral infection in mosquitoes [36] and that SGIB and SGEB act as modulators of arbovirus transmission [37]. In essence, this study underscores the variable susceptibility of different mosquito species to EBIV and highlights the complex interplay of physiological barriers during the transmission of this virus.\u003c/p\u003e\n\u003cp\u003eWithin our analyzable range, \u003cem\u003eCx. pipiens pallens\u003c/em\u003e exhibits the highest susceptibility to OYAV, while \u003cem\u003eAe. aegypti\u003c/em\u003e is the most susceptible to EBIV. Both \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eCx. pipiens pallens\u003c/em\u003e can be infected by EBIV, presenting a potential risk of virus transmission. However, it is critical to note that vectorial capacity can be affected by different genotypes/isolates of the virus and different mosquito species and geographic strains. For example, experimental studies of mosquito infections through the Oropouche virus (OROV) of the same Simbu serogroup have shown different IR and TR of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e infected by different OROV isolates from different geographic strains [38, 39].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMeanwhile, researchers have demonstrated that the genetic background of the vector significantly influences the virus\u0026rsquo;s susceptibility [40, 41]. The EBIV (Cu20-XJ) used in this study was isolated from\u0026nbsp;\u003cem\u003eCx\u003c/em\u003e.\u003cem\u003e\u0026nbsp;modestus\u0026nbsp;\u003c/em\u003e[20], and\u0026nbsp;\u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e is a known vector for transmission of Japanese encephalitis in China [42]. However, relevant experiments were not conducted due to the unavailability of these two mosquito species during the study duration. Therefore, the\u0026nbsp;vector competence\u0026nbsp;of these significant mosquito species in China for EBIV and OYAV remains to be uncovered. Additionally, as the OYAV (SZC50) was isolated from midge samples [16], it is worth considering midges as potential vectors for SZC50. Therefore, further studies are necessary to clarify the risk of virus transmission by examining different virus isolates infecting various geographical strains of mosquito species.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, it is noteworthy to investigate if mosquitoes that test positive for the virus in their saliva may infect vertebrate hosts through their bites. Additional studies on the transmission cycle involving the virus, mosquitoes, and vertebrates are needed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study focused on examining the susceptibility of \u003cem\u003eCx. pipiens pallens\u003c/em\u003e, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus\u003c/em\u003e, and \u003cem\u003eAe. aegypti\u003c/em\u003e to OYAV, while \u003cem\u003eCx. pipiens pallens\u003c/em\u003e, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, and \u003cem\u003eAe. albopictus\u003c/em\u003e to EBIV. It also evaluated the vectorial capacity for OYAV transmission in \u003cem\u003eCx. pipiens pallens\u003c/em\u003e and the EBIV transmission in both \u003cem\u003eCx. pipiens pallens\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e. This investigation enhances our understanding of the transmission potential of neglected orthobunyaviruses and contributes to the defense against their potential emergence globally, including in China.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDR: Dissemination rate; EBIV: Ebinur lake virus; IR: Infection rate; OYAV: Oya virus; PFU: Plaque-forming unit; TR: Transmission rate;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, China CDC for kindly provided the \u003cem\u003eAe. albopictus\u003c/em\u003e and \u003cem\u003eCx. pipiens pallens\u003c/em\u003e mosquitos. Furthermore, we would like to thank the Institutional Center for Shared Technologies and Facilities of Wuhan Institute of Virology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2022YFC2302700), the National Natural Science Foundation of China (U22A20363), and the Youth Program of Wuhan Institute of Virology (2023QNTJ-03).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author can provide the datasets used and/or analyzed during the current investigation upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZY and HX conceived the idea and coordinated the project. SL,\u0026nbsp;XW,\u0026nbsp;and\u0026nbsp;CY performed experiments. SL,XW, and CY analyzed data. JW, QL, ZY, and HX provided resources. SL, XW, CY, DH and HM performed the maintenance of mosquitoes. SL, FW, WZ, and HX drew the figures and drafted the manuscript. SL, FW, JW, QL, ZY and HX revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants made aware during the consenting process that their views will be shared widely including publication in peer review journals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOnen H, Luzala MM, Kigozi S, Sikumbili RM, Muanga C-JK, Zola EN, et al. 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Sci Rep.\u003cem\u003e \u003c/em\u003e2014;4:4908; doi: 10.1038/srep04908.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mosquito, vector, arbovirus, orthobunyavirus","lastPublishedDoi":"10.21203/rs.3.rs-3969850/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3969850/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eMosquito-borne viruses cause various infectious diseases in humans and animals. Oya virus (OYAV) and Ebinur lake virus (EBIV), belonging to the genus \u003cem\u003eOrthobunyavirus\u003c/em\u003e within the \u003cem\u003ePeribunyaviridae\u003c/em\u003e family, are recognized as neglected viruses with the potential to pose threats to animal or public health. The evaluation of vector competence is essential for predicting the arbovirus transmission risk.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e To investigate the range of mosquito vectors for OYAV and EBIV, four medically significant mosquitoes —\u003cem\u003eCulex pipiens pallens\u003c/em\u003e, \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e, \u003cem\u003eAedes albopictus\u003c/em\u003e, and \u003cem\u003eAedes aegypti\u003c/em\u003e \u003cem\u003e—\u003c/em\u003ewere selected to measure their vector susceptibility through blood-feeding infection. The RNA copies and infection rates of various mosquito species were determined using RT-qPCR. Subsequently, susceptible mosquito species were examined to determine their vector competence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The results revealed that \u003cem\u003eCx. pipiens pallens \u003c/em\u003ecan support in vivo infection and multiplication of OYAV, while \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eAe. albopictus,\u003c/em\u003eand \u003cem\u003eAe. aegypti\u003c/em\u003e exhibit lower susceptibility. Furthermore, \u003cem\u003eCx. pipiens pallens\u003c/em\u003ecan be infected by EBIV, posing a potential risk of virus transmission (with a transmission rate of up to 15.4%) at 7 days post-infection. In contrast, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e and \u003cem\u003eAe. albopictus \u003c/em\u003eexhibited poor vector competence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This study evaluated the possible transmission risk of OYAV and EBIV using four mosquito species. The findings indicate a potential risk of EBIV transmission in \u003cem\u003eCx. pipiens pallens\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake Virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-23 09:13:50","doi":"10.21203/rs.3.rs-3969850/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-17T13:46:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-11T14:46:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32253cb5-9643-4677-88d1-44c93420af45","date":"2024-02-22T13:02:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-22T12:37:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-21T16:37:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-21T16:25:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-02-19T11:32:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3df48a0-4ab1-46d7-bea8-e4430fed4b81","owner":[],"postedDate":"February 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-09T03:58:39+00:00","versionOfRecord":{"articleIdentity":"rs-3969850","link":"https://doi.org/10.1186/s13071-024-06295-5","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2024-05-07 03:58:39","publishedOnDateReadable":"May 7th, 2024"},"versionCreatedAt":"2024-02-23 09:13:50","video":"","vorDoi":"10.1186/s13071-024-06295-5","vorDoiUrl":"https://doi.org/10.1186/s13071-024-06295-5","workflowStages":[]},"version":"v1","identity":"rs-3969850","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3969850","identity":"rs-3969850","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}