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Vector competence for Oropouche virus: A systematic review of pre-2024 experiments [1]

['Emily N. Gallichotte', 'Department Of Microbiology', 'Immunology', 'Pathology', 'Colorado State University', 'Fort Collins', 'Colorado', 'United States Of America', 'Gregory D. Ebel', 'Colin J. Carlson']

Date: 2025-05 

Abstract The 2023–24 epidemic of Oropouche fever in the Americas and the associated ongoing outbreak in Cuba suggests a potential state shift in the epidemiology of the disease, raising questions about which vectors are driving transmission. In this study, we conduct a systematic review of vector competence experiments with Oropouche virus (OROV, Orthobunyavirus) that were published prior to the 2023–24 epidemic season. Only seven studies were published by September 2024, highlighting the chronic neglect that Oropouche virus (like many other orthobunyaviruses) has been subjected to since its discovery in 1954. Two species of midge (Culicoides paraensis and C. sonorensis) consistently demonstrate a high competence to transmit OROV (~30%), while mosquitoes (including both Aedes and Culex spp.) exhibited an infection rate consistently below ~20%, and showed limited OROV transmission. Further research is needed to establish which vectors are involved in the ongoing outbreak in Cuba, and whether local vectors and wildlife communities create any risk of establishment in non-endemic regions.

Abstract Oropouche virus has recently become an urgent threat to public health in Central and South America. OROV is mainly transmitted by biting midges; however, some public health agencies and scientific sources note that some mosquito species transmit the virus. We conducted a systematic review of literature prior to the current epidemic, and identified seven studies that experimentally tested the ability of vectors to become infected with, and transmit OROV (i.e., that assessed their vector competence). These studies have consistently found that biting midges become infected at higher rates than mosquitoes, which rarely transmit the virus. It is unclear which vectors are responsible for transmitting OROV in the current outbreak. Existing published data support the observation that biting midges are likely to be significant vectors compared to mosquitoes, which are comparatively incompetent. However, increased vector surveillance and pathogen testing, and additional vector competence experiments using current OROV strains, are urgently needed.

Citation: Gallichotte EN, Ebel GD, Carlson CJ (2025) Vector competence for Oropouche virus: A systematic review of pre-2024 experiments. PLoS Negl Trop Dis 19(4): e0013014. https://doi.org/10.1371/journal.pntd.0013014 Editor: Olaf Horstick, University of Heidelberg, GERMANY Received: October 25, 2024; Accepted: March 27, 2025; Published: April 30, 2025 Copyright: © 2025 Gallichotte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All data are publicly available on Figshare (10.6084/M9.FIGSHARE.27157029.V1; https://figshare.com/articles/dataset/Oropouche_vector_competence_data/27157029?file=49855260) Funding: This work was supported by National Science Foundation DBI 2021909, 2213854, and 2515340 (CJC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Oropouche virus (OROV) is a Simbu serogroup orthobunyavirus endemic to South America and parts of the Caribbean. Historically, OROV has been neglected compared to other arboviral diseases, such as yellow fever, dengue, chikungunya, or Zika. However, an ongoing epidemic of Oropouche fever has brought new attention to the virus. In the first seven months of 2024, there were more than 8,000 cases across the Americas, mostly concentrated in Brazil; at the time of writing, a second epidemic wave is ongoing in Cuba, with over 11,000 suspected cases. The scale of this outbreak may be connected to evolutionary changes in the pathogen: recent genetic analyses revealed that the OROV lineage currently circulating in Brazil is a novel reassortant containing M segment from viruses detected in the eastern Amazon from 2009-2018, and L and S segments from viruses detected in Peru from 2008-2021 [1]. In vitro characterization of the novel reassortant virus recently demonstrated that it replicates to higher levels than the prototypic strain in mammalian cells, and is less sensitive to neutralization by human OROV immune sera collected prior to 2016 [2]. This reassortment event may also result in changes in the clinical presentation of Oropouche fever. Prior to 2024, symptoms were generally considered similar to other febrile illnesses, and no deaths had been reported [3]; in the 2024 outbreak, however, two deaths caused by OROV were reported in healthy young women, and there have been multiple reports of miscarriage, fetal deaths, and microcephaly associated with OROV infection [4]. Surprisingly little is known about the vectors involved in the current epidemic. Current evidence suggests that, unlike many other arthropod-borne orthobunyaviruses, OROV is primarily transmitted by culicoid midges (Ceratopogoidae: Culicoides) rather than mosquitoes or ticks [5]. C. paraensis are considered the principal vector of epidemic urban OROV transmission due to their high abundance in locations of previous OROV outbreaks, and OROV isolation from C. paraensis multiple times during a 1975 outbreak in Brazil [6–8]. Culicoides are efficient vectors for many arboviruses, including other Simbu serogroup orthobunyaviruses in South America [9]. They feed on a variety of vertebrates, and three-toed sloths, birds, and non-human primates (capuchin and howler-monkeys) are thought to be the primary hosts of sylvatic OROV [10]. When OROV was first detected in 1955 in Trinidad & Tobago, over 700 mosquitoes (Aedes, Wyeomyias, Psorophora, Mansonia, Culex, Anopheles, Haemogogous, and other unidentified sabethines) were collected from the same area as the infected patient, but OROV was only detected in Coquillettidia venezuelensis (referred to as Mansonia venezuelensis in the paper) [11]. In 1961, OROV was isolated from a pool of Aedes serratus in Brazil [12]. Because Cq. venezuelensis and Ae. serratus are hematophagous mosquitoes that inhabit sylvatic environments, they were suggested as potential sylvatic vectors. Cx. quinquefasciatus have also been proposed as a secondary, urban, anthropophilic vector because OROV has been isolated from them multiple times [7,13]. However, in all instances of virus isolation from mosquitoes, the resulting detection rates were very low, suggesting poor susceptibility of the vector to infection. Overall, these suggest that many blood-feeding arthropods are exposed to the virus in nature, but mosquitoes may not be meaningfully involved in transmission. The scale of the current epidemic, particularly in Cuba, has prompted speculation about a potential shift to mosquito vectors, especially since no Culicoides species have been reported there, but so far no observational data supports this idea [14,15]. To define the extent and outcomes of previously published vector competence experiments using OROV, we developed a standardized dataset of all pre-2024 records of vector competence experiments that studied OROV, following a previously-developed data standard [16]. The data standard contains fields for vector, virus, exposure, experimental and infection conditions, and experimental outcomes, allowing us to standardize variables, making it easier to compare across studies. Despite the large number of outbreaks over the last 50 years, there is significant uncertainty regarding which vectors are responsible for OROV transmission, including in the current epidemic. Because the virus is spreading to locations where it has not previously been detected (e.g., Cuba [17]), and could continue to spread to new locations (e.g., the United States), it is critical to understand transmission risk posed by a wide range of potential vectors. Unfortunately, we found that before 2024 there was limited experimental research testing the ability of different vectors to become infected with, and transmit OROV. The small number of studies that have been conducted demonstrate that Culex spp. can infrequently be infected with OROV, but transmit virus at low rates compared to Culicoides midges. It thus seems unlikely that mosquitoes have been a major vector of OROV thus far.

Materials and methods Systematic search A systematic search was conducted on September 17, 2024 on PubMed using the search term “Oropouche virus” (no other databases were used, nor were additional spellings or abbreviations included as search terms). No filters or limits were used. The search returned 168 publications, which were imported into Rayyan for manual screening [18]. Exclusion criteria included: reviews, news articles, commentaries, surveillance studies, experimental studies in systems other than vectors (e.g., cells, mice, etc.), etc.). The following inclusion criteria were required: full text available in English; experimental OROV infections in vectors (mosquitoes or midges), and raw data must be available (e.g., number of individual vectors positive and total number tested, not derived rates) (no papers were identified that did not have raw data available). A single reviewer screened all publications. Two additional publications were identified from citation searching. One publication was frequently cited in multiple reviews stating susceptibility of multiple vectors to OROV infection [11]. This paper was identified in our original search, but had been excluded because there was no mention of experimental vector competence in the title or abstract. A second publication did not show up in our original search, but was also identified due to frequent reference in other publications [19]. There were seven publications that met all criteria and were used in our analyses. Data collection Information was extracted from publications into a standardized template, following a previously-published minimum data and metadata standard [16]. Data sections include information on vectors, viruses, experimental conditions, infection conditions, and infection outcomes (specifically sample type tested, assay used to detect infection, number tested, and number positive). Information not provided in publications were left blank, and no assumptions were made about any missing or unclear information. Experiments evaluating the ability of OROV to be mechanically transmitted by vectors were not included. Authors of de Mendonça et al. [20] were contacted for the vector origin year which was provided. Risk of bias assessment, effect measures, reporting bias assessment and certainty assessment were not determined. Data analysis and statistics All data were analyzed in GraphPad Prism Version 10.2.3.

Discussion Our systematic review revealed that, in the six decades between the discovery of Oropouche virus and the 2023–24 epidemic in the Americas, only seven vector competence studies were published. Even accounting for the possibility that more experiments were conducted but never published, our findings reveal that OROV has been subject to chronic neglect – a pattern that is true more broadly of most orthobunyaviruses, particularly compared to well-studied flaviviruses (e.g., yellow fever virus and dengue virus) and alphaviruses (e.g., chikungunya virus and Mayaro virus) [25]. Nevertheless, the seven studies we identified – and the nine arthropod species they examined – provide a useful starting point for establishing the vectors involved in sylvatic and urban OROV transmission, as well as the basic biology of OROV-vector interactions. Despite variation in study design, methods, and materials (e.g., geographic origin of vectors or time since collection from the field), results consistently showed that Aedes and Culex mosquitoes rarely become infected with OROV following an infectious bloodmeal, and have a limited ability to transmit the virus. Intrathoracic injections, which bypass the initial midgut infection and escape barriers and deliver virus directly into the hemocoel, revealed that OROV can replicate in mosquitoes, suggesting lack of infection following oral exposure, is likely not molecular incompatibility between the virus and vector, but instead limited ability to infect the midgut. While studies based on intrathoracic injection are useful to understand fundamental questions of virus-vector interactions, we also caution that they are not representative of natural infection and transmission risk. In nature, vectors will only be exposed to virus orally, and therefore, transmission when these barriers are bypassed (e.g., intrathoracic injection), are not relevant to transmission. Midges (Culicoides spp.) are the primary vectors of many viruses of medical and veterinary importance, including bluetongue virus (BTV), Schmallenberg virus, and OROV-related viruses within the Simbu serogroup of the genus Orthobunyavirus [9]. However, they remain dramatically under-studied and under-surveyed in comparison to mosquitoes and ticks. Despite the limited number of publications, all experimental results support the observation that Culicoides midges are highly competent vectors for OROV. However, there are few studies experimentally evaluating midge vector competence for any virus due to challenges surrounding lab colonization and experimental manipulation of these arthropods [26]. Epidemiological evidence implicates C. parensis as the primary urban vector [7], but detection and isolation rates in wild C. paraensis pools have been low (4 positive of 31,555 tested, ~ 0.01% positivity) [6,7]. Importantly, while there are over 1,300 species of Culicoides, OROV has only been detected in C. paraensis during outbreak vector surveillance, and only two species (C. paraensis and C. sonorensis) have been experimentally tested and demonstrated to transmit OROV [7,9]. Additionally, while they are abundant globally, there is far less surveillance of Culicoides compared to other vectors (e.g., mosquitoes), leaving incomplete distribution maps, and there are no reported occurrences of any Culicoides spp. in Cuba, where OROV transmission is endemic [27]. Further research is needed to confirm which Culicoides species are actively involved in the current OROV outbreaks, or could someday pose a risk, especially in new locations. Based on current evidence, mosquitoes are unlikely to be a primary vector of OROV in natural settings. Despite extensive efforts, OROV has rarely been isolated from mosquito vectors of other arboviruses, even during outbreaks when it has been estimated >15% of the population is infected [7]. Of > 28,000 Cx. quinquefasciatus mosquitoes tested during outbreaks over 14 years, only three have tested positive (~0.01%), including a mosquito engorged with blood caught in a hospital ward at the bedside of a viremic patient 1975 in Pará, Brazil [6,7]. Low infection and transmission rates seen in experimental vector competence studies, and incredibly low rates of virus isolation during outbreaks, suggest that Cx. quinquefasciatus are likely a comparatively poor vector for OROV. However, vector competence is only one component of vectorial capacity, which also incorporates factors such as vector density and bloodfeeding behavior [28]; therefore, a vector with low competence may still be important at maintaining and transmitting the virus in nature, as has been demonstrated with Ae. albopictus and dengue virus [29]. It remains unknown whether shifts in vector-virus interactions have contributed to the current epidemic. It is possible that genetic changes to the virus have improved vector competence, similar to what has been seen with chikungunya virus and Ae. albopictus, or West Nile virus and Culex spp. mosquitoes [30–32]. New experiments should focus on establishing whether the novel reassortant is more transmissible by Culicoides midges, or can be newly maintained by common urban vectors such as Ae. aegypti or Cx. quinquefasciatus. Importantly, a recent paper using OROV isolated from a febrile patient from Cuba in 2024, demonstrated experimental infection rates in mosquitoes (Ae. albopictus, Anopheles quadrimaculatus, Cx. quinquefasciatus and Cx. pipiens) were low (<4%), and comparable to rates of mosquitoes infected with the 1955 prototypic strain (<2%), suggesting the currently circulating virus is not adapted to, nor improved at infection of and transmission by mosquitoes [33]. However, other factors could also contribute to the unusual intensity of recent and ongoing outbreaks, including climate change, urbanization, deforestation, and human mobility [10,29,34,35]. Field studies on the epidemiology and drivers of these outbreaks will be an important complement to experimental work, particularly to establish whether Oropouche virus will continue to emerge as a threat to public health in the coming decades.

Acknowledgments We thank Silvana de Mendonça and colleagues for providing additional data, and thank Doug Brackney and Nate Grubaugh for helpful discussion.

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