70f36828-f03f-486a-bb62-b4be3cb59d06 https://cloud.gbif.org/lac/resource?r=ipk_orov Monica Sanchez Gonzalez IPK

Havana Havana CU

monica.sanchez@ipk.sld.cu Ariamys Companioni IPK

Havana Havana CU

ariamys@ipk.sld.cu Gladys Gutierrez-Bugallo IPK

Havana Havana CU

ggutierrezbugallo@gmail.com https://orcid.org/0000-0003-4415-7045 Eric Camacho IPK

Havana Havana CU

Silvia Serrano IPK

Havana Havana CU

Henry Rodriguez-Potrony Centro Provincial de Higiene y Epidemiologia

Santiago de Cuba Santiago de Cuba CU

Yuneisy Alfonso Centro Provincial de Higiene y Epidemiologia

Santiago de Cuba Santiago de Cuba CU

Barbara Liberty Centro Provincial de Higiene, Epidemiologia y Microbiologia

Cienfuegos Cienfuegos CU

Javier Varens Centro Provincial de Higiene, Epidemiologia y Microbiologia

Cienfuegos Cienfuegos CU

Yanet Martínez IPK

Havana Havana CU

Zulema Menendez IPK

Havana Havana CU

Gladys Gutierrez-Bugallo IPK

Havana Havana CU

ggutierrezbugallo@gmail.com https://orcid.org/0000-0003-4415-7045 2025-10-30 eng From May to October 2024, Cuba experienced a significant outbreak of Oropouche virus (OROV), an Orthobunyavirus historically confined to the Amazon region, where it circulates between sylvatic vectors and vertebrate hosts. Prior to this event, no documented circulation of Orthobunyaviruses had been reported in Cuba, leaving the role of local vectors in transmission largely unknown. To investigate potential vectors, we conducted entomo-virological surveys in areas of active transmission across three Cuban provinces during the outbreak period. Adult insects were collected using both traps and manual aspirators, and tested for OROV by real-time RT-PCR. A total of 2,180 specimens, representing six dipteran species or families, were collected. Culex quinquefasciatus and Aedes aegypti were the only species captured across all three provinces. At active OROV transmission sites, Cx. quinquefasciatus was the most frequently sampled species (n = 1,785), followed by Ae. aegypti (n = 285) and members of the Ceratopogonidae family (n = 49). Eleven pools, comprising Cx. quinquefasciatus, Ae. aegypti, and members of the Ceratopogonidae family, tested positive for OROV. These findings suggest the possible involvement of multiple vector species in the Cuban outbreak. Further studies are needed to assess the vector competence of these species and to better understand their role in OROV transmission dynamics within the Caribbean context. Sampling event GBIF Dataset Type Vocabulary: http://rs.gbif.org/vocabulary/gbif/dataset_type_2015-07-10.xml Aedes aegypti Culex quinquefasciatus Ceratopogonidae Orthobunyavirus. n/a To the extent possible under law, the publisher has waived all rights to these data and has dedicated them to the Public Domain (CC0 1.0). Users may copy, modify, distribute and use the work, including for commercial purposes, without restriction. Creative Commons Zero v1.0 Universal https://spdx.org/licenses/CC0-1.0.html CC0-1.0 https://cloud.gbif.org/lac/archive.do?r=ipk_orov Insect collection was conducted at 14 active OROV transmission areas across three Cuban provinces between May and October 2024: 1. Santiago de Cuba (Armando García, Distrito José Martí, Caney, 30 de Noviembre, 28 de Septiembre, Finlay, Distrito Josué País, Julian Grimau and Ernesto Che Guevara localities), 2. Cienfuegos ( III and VIII localities), and 3. Havana (Pulido Humaran, Grimau, and Puentes Grandes localities). Specimens were collected using adult traps (BG-Sentinel traps with BG-Lure cartridges and New Jersey light traps), which were deployed for 24 h starting at 8:00 a.m., and with insect aspirators (Prokopack) used once at each site, both outdoors and indoors, primarily in homes with confirmed or suspected OROV cases. Collection sites were categorized based on vegetation cover as follows: low (≤30%), moderate (30–70%), and high (≥70%) vegetation, following the criteria described by Vázquez et al. (2017). -82.487043 -75.773223 23.103557 20.012536 2024-05-23 2024-10-14 The dataset comprises adult insect specimens collected during entomo-virological surveys in Cuba between May and October 2024, in the context of an Oropouche virus (Orthobunyavirus) outbreak. The taxonomic focus is on Diptera (true flies), including both mosquito species and biting midges potentially involved in virus transmission. phylum Arthropoda family Culicidae class Insecta order Diptera family Ceratopogonidae kingdom Animalia From May to October 2024, Cuba experienced a significant outbreak of Oropouche virus (OROV), an Orthobunyavirus historically confined to the Amazon region, where it circulates between sylvatic vectors and vertebrate hosts. Prior to this event, no documented circulation of Orthobunyaviruses had been reported in Cuba, leaving the role of local vectors in transmission largely unknown. To investigate potential vectors, we conducted entomo-virological surveys in areas of active transmission across three Cuban provinces during the outbreak period. Adult insects were collected using both traps and manual aspirators, and tested for OROV by real-time RT-PCR. A total of 2180 specimens, representing six dipteran species or families, were collected. Culex quinquefasciatus and Aedes aegypti were the only species captured across all three provinces. At active OROV transmission sites, Cx. quinquefasciatus was the most frequently sampled species (n = 1785), followed by Ae. aegypti (n = 285) and members of the Ceratopogonidae family (n = 49). Eleven pools, comprising Cx. quinquefasciatus, Ae. aegypti, and members of the Ceratopogonidae family, tested positive for OROV. These findings suggest the possible involvement of multiple vector species in the Cuban outbreak. Further studies are needed to assess the vector competence of these species and to better understand their role in OROV transmission dynamics within the Caribbean context. Oropouche virus (OROV) (order Bunyavirales, family Peribunyaviridae, genus Orthobunyavirus, Simbu serogroup) is an emerging arbovirus in South and Central America (Romero-Alvarez and Escobar, 2018). Initially detected in Trinidad and Tobago (Anderson et al., 1961), OROV transmission has historically been confined to the Amazon Basin (Sakkas et al., 2018; Scachetti et al., 2025), where it circulates primarily through a sylvatic cycle involving dipteran vectors such as biting midges and mosquitoes, and vertebrate hosts including sloths, non-human primates, and other mammals (OPS/OMS, 2024). Due to its increasing geographic spread and potential for urban transmission, the Pan American Health Organization (PAHO) issued an alert in February 2024 highlighting the virus’s potential to emerge beyond its traditional range (OPS/OMS, 2024a). This alert followed outbreaks reported in cities near the Amazon region, where human cases typically coincided with the rainy season, when vector populations increase (Mohapatra et al., 2024). The midge Culicoides paraensis (Goeldi, 1905) (Diptera: Ceratopogonidae) has been identified as the principal vector of OROV in various settings, including sylvatic, peri-urban, and urban outbreaks (Pinheiro et al., 1982; Santamaría et al., 2008; Sakkas et al., 2018). Among mosquitoes, Culex quinquefasciatus (Say, 1823) has been suggested as a secondary vector due to its high abundance in endemic regions (Cardoso et al., 2015; Riccò et al., 2024) and limited but demonstrated vector competence in laboratory conditions (Hoch et al., 1987; de Mendonça et al., 2021). Other species, such as Coquillettidia venezuelensis (Theobald, 1912) and Aedes serratus (Theobald, 1901), have also been proposed as potential vectors (Anderson et al., 1961; Sakkas et al., 2018). The first documented outbreak of OROV beyond its typical geographic zone occurred in Cuba on May 27, 2024 (Benítez et al., 2024). Human cases of Oropouche fever were initially confirmed in Santiago de Cuba province, later expanding to Cienfuegos and subsequently to other parts of the country, totaling 506 confirmed cases by September 2024 (OPS/OMS, 2024b). Given the lack of prior Orthobunyavirus circulation on the island, knowledge about potential OROV vectors in Cuba is extremely limited. In response, entomo-virological surveillance was implemented to identify insect species involved in the outbreak. Vector identification during an emerging arboviral event is essential for informing targeted control measures and building effective response strategies (Diallo and Diallo, 2017). Recognizing the vectors responsible for OROV transmission in Cuba is not only critical for national public health planning but also holds global relevance, as it represents the virus’s first known establishment in a Caribbean island and its occurrence in both urban and rural environments. Here, we present the results of the initial entomological investigations conducted in three Cuban provinces between May and October 2024. A total of 2180 specimens were collected from 14 localities with active OROV transmission. These specimens belonged to six distinct species across two mosquito genera: Aedes (Ae. aegypti, Ae. albopictus, Ae. serratus, and Ae. taeniorhynchus) and Culex (Cx. quinquefasciatus and Cx. nigripalpus), as well as members of the family Ceratopogonidae. Overall, the most frequently captured species was Cx. quinquefasciatus (n = 1785), followed by Ae. aegypti (n = 285), Ceratopogonidae spp. (n = 49), Ae. serratus (n = 21), Cx. nigripalpus (n = 18), Ae. albopictus (n = 14), and Ae. taeniorhynchus (n = 8). Provincial distribution showed that Cx. quinquefasciatus accounted for the majority of captures in Santiago de Cuba (87%) and Cienfuegos (88%), while Ae. aegypti was the predominant sampled species in Havana, comprising 55% of the total specimens collected there. Overall, vegetation cover analysis at the sampled locations showed that the majority of captures occurred in areas with moderate vegetation cover. In Santiago de Cuba, Cx. quinquefasciatus was most frequently captured in areas with moderate vegetation cover (44%), followed by low (29%) and high (28%) cover. Ceratopogonidae were equally split between moderate cover (50%) and the combined high and low categories (25% each). Ae. aegypti occurred mainly in low vegetation areas (45%), with moderate (33%) and high (26%) cover less represented. In Cienfuegos, captures for all species were highest in areas with moderate vegetation cover. Cx. quinquefasciatus was recorded at 52% in moderate, 33% in high, and 15% in low cover areas. Cx. nigripalpus occurred predominantly in moderate cover (60%), with the remainder in high cover (40%). All Ae. aegypti specimens were found in moderate cover areas. In Havana, most captures occurred in sites with high vegetation cover. The proportion of specimens collected in high vegetation cover ranged from 100% for Ae. albopictus and Ae. serratus, to 70% for Ae. aegypti, and 65% for Cx. quinquefasciatus. A total of 1238 insects were processed by RT-PCR, of which 1213 were apparently non-engorged females and 25 were males. Specimens were grouped into 81 female pools and one male pool, distributed as follows: 57 pools of Cx. quinquefasciatus, 17 of Ae. aegypti, 2 of Ae. albopictus, 2 of Ceratopogonidae spp., 2 of Cx nigripalpus, 1 of Ae. serratus, and 1 of Ae. taeniorhynchus. The single male pool consisted of Culex quinquefasciatus. In total, 11 pools (14%) tested positive for OROV by RT-PCR, nine from Santiago de Cuba and two from Havana. Among these, 64% were composed of Cx. quinquefasciatus, 27% of Ae. aegypti, and the remainder of Ceratopogonidae spp. All positive pools corresponded to specimens collected from May to August. The highest MIR was estimated for Ae. aegypti (30.3), followed by Ceratopogonidae spp. (20.4), and Cx. quinquefasciatus (7.1). unknown Gladys Gutierrez-Bugallo IPK

Havana Havana CU

ggutierrezbugallo@gmail.com https://orcid.org/0000-0003-4415-7045 Ariamys Companioni IPK

Havana Havana CU

ariamys@ipk.sld.cu Identified areas of active OROV transmission within three Cuban provinces based on epidemiological data. Deployed traps and conducted manual aspiration to collect adult insects at selected sites. Sorted specimens to species or family level using morphological identification. Pooled specimens by species, location, and date of collection. Tested pools for OROV using real-time RT-PCR. Recorded taxonomic, spatial, and temporal data for each specimen or pool. Entomo-virological surveys were conducted from May to October 2024 in areas of active Oropouche virus (OROV) transmission across three Cuban provinces. The study area included urban and peri-urban sites where confirmed human cases had been reported. Sampling focused on adult Diptera, particularly mosquito species and other potential vectors, to investigate their involvement in local OROV transmission. Adult insects were collected using a combination of trapping methods and manual aspiration in locations with active OROV circulation. Sampling aimed to cover sites in all three provinces and to document both the diversity and abundance of potential vector species. Monica Sánchez González author Here, we present the results of the initial entomological investigations conducted in three Cuban provinces between May and October 2024. Insect collection was conducted at 14 active OROV transmission areas across three Cuban provinces between May and October 2024: 1. Santiago de Cuba (Armando García, Distrito José Martí, Caney, 30 de Noviembre, 28 de Septiembre, Finlay, Distrito Josué País, Julian Grimau and Ernesto Che Guevara localities), 2. Cienfuegos ( III and VIII localities), and 3. Havana (Pulido Humaran, Grimau, and Puentes Grandes localities). Specimens were collected using adult traps (BG-Sentinel traps with BG-Lure cartridges and New Jersey light traps), which were deployed for 24 h starting at 8:00 a.m., and with insect aspirators (Prokopack) used once at each site, both outdoors and indoors, primarily in homes with confirmed or suspected OROV cases. Collection sites were categorized based on vegetation cover as follows: low (≤30%), moderate (30–70%), and high (≥70%) vegetation, following the criteria described by Vázquez et al. (2017). Collected specimens were stored at 4 °C during transportation and handling. Taxonomic identification was performed using established morphological keys (González, 2008) at the Entomology Reference Laboratory of the Pedro Kourí Institute of Tropical Medicine. Insects were sorted into pools of 5 to 25 individuals based on species, sex, collection date, and location. For female insects, only those that were visibly non-engorged were included in the pools. In addition, specimens that did not maintain the cold chain after collection were excluded from molecular analysis. Insect pools were homogenized in 500 µL of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. After centrifugation at 13,000 ×g for 15 minutes at 4 °C, 140 µL of the supernatant was used for viral RNA extraction using the QIAamp Viral RNA Mini Kit (QIAGEN, Germany), following the manufacturer’s instructions. Detection of Oropouche virus (OROV) RNA targeted a fragment of the S gene using a one-step real-time reverse transcription PCR (RT-qPCR) protocol, as described by Naveca et al. (2017). The minimum infection rate (MIR) is an indicator of virus activity within a vector population (Chatterjee et al., 2021). 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Emerg Infect Dis. 2021;27 10:2711-4. doi:10.3201/eid2710.204760. 70f36828-f03f-486a-bb62-b4be3cb59d06/v1.5.xml