Genomic Characterization of a Bataï Orthobunyavirus, Previously Classified as Ilesha Virus, from Field-Caught Mosquitoes in Senegal, Bandia 1969.

Bataï virus (BATV), belonging to the Orthobunyavirus genus, is an emerging mosquito-borne virus with documented cases in Asia, Europe, and Africa. It causes various symptoms in humans and ruminants. Another related virus is Ilesha virus (ILEV), which causes a range of diseases in humans and is mainly found in African countries. This study aimed to genetically identify and characterize a BATV strain previously misclassified as ILEV in Senegal. The strain was reactivated and subjected to whole genome sequencing using an Illumina-based approach. Genetic analyses and phylogeny were performed to assess the evolutionary relationships. Genomic analyses revealed a close similarity between the Senegal strain and the BATV strains UgMP-6830 from Uganda. The genetic distances indicated high homology. Phylogenetic analysis confirmed the Senegal strain's clustering with BATV. This study corrects the misclassification, confirming the presence of BATV in West Africa. This research represents the first evidence of BATV circulation in West Africa, underscoring the importance of genomic approaches in virus classification. Retrospective sequencing is crucial for reevaluating strains and identifying potential public health threats among neglected viruses.

Outbreak of Oropouche virus in frontier regions in western Amazon.

Oropouche virus (OROV) is characterized as a re-emerging arbovirus of great concern for public health, being responsible for several outbreaks of acute fever identified in Latin American countries, registering more than half a million reported cases. The incidence of reports of this virus is intrinsically favored by environmental conditions, in which such characteristics are related to the increase and distribution of the vector population to areas of human traffic. Moreover, there is a problem regarding the lack of diagnosis in Brazil that aggregates the success of the etiologic agent. Thus, by means of molecular techniques, we identified 27 positive cases of the OROV circulating in border locations in western Amazon, with 44.44% (12/27) of the cohort characterized as infected individuals with reported symptoms, mainly ranging from fever, myalgia, and back pain. Among the positive samples, it was possible to obtain a total of 48.14% (13/27) samples to analyze the S and M segments of Oropouche, which showed similarities among the Brazilian sequences. Thus, it was possible to verify the circulation of the OROV in Rondonia and border areas, in which the tracking of neglected arboviruses is necessary for the genomic surveillance of emerging and re-emerging viruses.IMPORTANCEThe western Amazon region is known for outbreaks of acute febrile illnesses, to which the lack of specific diagnostics for different pathogens hinders the management of patients in healthcare units. The Oropouche virus has already been recorded in the region in the 1990s. However, this is the first study, after this record, to perform the detection of individuals with acute febrile illness using a screening test to exclude Zika, dengue, and chikungunya, confirmed by sequencing the circulation of the virus in the state of Rondonia and border areas. We emphasize the importance of including diagnostics for viruses such as Oropouche, which suffers underreporting for years and is related to seasonal periods in Western Amazon locations, a factor that has a direct influence on public health in the region. In addition, we emphasize the importance of genomic surveillance in the elucidation of outbreaks that affect the resident population of these locations.

Oropouche virus: A neglected global arboviral threat.

The Oropouche virus is an important arthropod-borne virus in the Peribunyaviridae family that can cause febrile illnesses, and it is widely distributed in tropical regions such as Central and South America. Since the virus was first identified, a large number of related cases are reported every year. No deaths have been reported to date, however, the virus can cause systemic infections, including the nervous and blood systems, leading to serious complications. The transmission of Oropouche virus occurs through both urban and sylvatic cycles, with the anthropophilic biting midge Culicoides paraensis serving as the primary vector in urban areas. Direct human-to-human transmission of Oropouche virus has not been observed. Oropouche virus consists of three segments, and the proteins encoded by the different segments enables the virus to replicate efficiently in the host and to resist the host's immune response. Phylogenetic analyses showed that Oropouche virus sequences are geographically distinct and have closer homologies with Iquitos virus and Perdoes virus, which belong to the family Peribunyaviridae. Despite the enormous threat it poses to public health, there are currently no licensed vaccines or specific antiviral treatments for the disease it causes. Recent studies have utilised imJatobal virusmunoinformatics approaches to develop epitope-based peptide vaccines, which have laid the groundwork for the clinical use of vaccines. The present review focuses on the structure, epidemiology, immunity and phylogeny of Oropouche virus, as well as the progress of vaccine development, thereby attracting wider attention and research, particularly with regard to potential vaccine programs.

Differential role of NSs genes in the neurovirulence of two genogroups of Akabane virus causing postnatal encephalomyelitis.

Akabane virus (AKAV) is a member of the genus Orthobunyavirus, family Peribunyaviridae. In addition to AKAV strains that cause fetal Akabane disease, which is characterized by abortion in ruminants, some AKAV strains cause postnatal infection characterized by nonsuppurative encephalomyelitis in ruminants. Here, we focused on the NSs protein, a virulence factor for most viruses belonging to the genus Orthobunyavirus, and we hypothesized that this protein would act as a neurovirulence factor in AKAV strains causing postnatal encephalomyelitis. We generated AKAV strains that were unable to produce the NSs protein, derived from two different genogroups, genogroups I and II, and then examined the role of their NSs proteins by inoculating mice intracerebrally with these modified viruses. Our results revealed that the neurovirulence of genogroup II strains is dependent on the NSs protein, whereas that of genogroup I strains is independent of this protein. Notably, infection of primary cultured bovine cells with these viruses suggested that the NSs proteins of both genogroups suppress innate immune-related gene expression with equal efficiency. These results indicate differences in the determinants of virulence of orthobunyaviruses.

The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population.

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

Isolation and identification of Tete virus group (Peribunyaviridae: Orthobunyavirus) from Culicoides biting midges collected in Lichuan County, China.

In July 2018, a virus (JXLC1806-2) was isolated from Culicoides biting midges collected in Lichuan County, Jiangxi Province, China. The virus isolate showed significant cytopathic effects within 48 hours after inoculation with mammalian cells (BHK-21). JXLC1806-2 virus could form plaques in BHK-21 cells, and the virus titer was 1×105.6 pfu/mL. After inoculation with the virus, suckling mice developed disease and died. The nucleotide and amino sequence analysis showed that the JXLC1806-2 virus genome was composed of S, M and L segments. Phylogenetic analysis showed that the S, M and L genes of JXLC1806-2 virus belonged to the Tete serogroup, Orthobunyavirus, but formed an independent evolutionary branch from the other members of the Tete serogroup. The results showed that the JXLC1806-2 virus, which was named as Lichuan virus, is a new member of Tete serogroup, and this is the first time that a Tete serogroup virus has been isolated in China.

Expanding diversity of bunyaviruses identified in mosquitoes.

Mosquitoes interact with various organisms in the environment, and female mosquitoes in particular serve as vectors that directly transmit a number of microorganisms to humans and animals by blood-sucking. Comprehensive analysis of mosquito-borne viruses has led to the understanding of the existence of diverse viral species and to the identification of zoonotic arboviruses responsible for significant outbreaks and epidemics. In the present study on mosquito-borne bunyaviruses we employed a broad-spectrum RT-PCR approach and identified eighteen different additional species in the Phenuiviridae family and also a number of related but unclassified bunyaviruses in mosquitoes collected in Zambia. The entire RNA genome segments of the newly identified viruses were further analyzed by RNA sequencing with a ribonuclease R (RNase R) treatment to reduce host-derived RNAs and enrich viral RNAs, taking advantage of the dsRNA panhandle structure of the bunyavirus genome. All three or four genome segments were identified in eight bunyavirus species. Furthermore, L segments of three different novel viruses related to the Leishbunyaviridae were found in mosquitoes together with genes from the suspected host, the Crithidia parasite. In summary, our virus detection approach using a combination of broad-spectrum RT-PCR and RNA sequencing analysis with a simple virus enrichment method allowed the discovery of novel bunyaviruses. The diversity of bunyaviruses is still expanding and studies on this will allow a better understanding of the ecology of hematophagous mosquitoes.

In vivo and in vitro characterization of a new Oya virus isolate from Culicoides spp. and its seroprevalence in domestic animals in Yunnan, China.

Biting midges are one of the most common hematophagous insects. They are capable of transmitting a wide range of arboviruses and have a significant impact on public health and veterinary medicine. Herein, from midge samples collected in 2013 in Yunnan, China, one sample induced a cell cytopathic effect (CPE) in BHK-21, MA104, and PK15 cell lines. Next-generation sequencing data, RACE and PCR determined the genome sequence of the sample and designated as an Oya virus (OYAV) isolate SZC50. Phylogenetic analysis of the sample revealed that it was cluster into viruses from species Orthobunyavirus catqueense. The open reading frames of S, M, and L segment of OYAV SZC50 were closest to those of OYAV SC0806. Moreover, 831 serum samples (736 pigs, 45 cattle, and 50 sheep) were gathered from 13 cities in Yunnan Province to detect neutralizing antibody of OYAV SZC50. A significant proportion of OYAV SZC50 antibody (more than 30%) was found in Yunnan pig populations, with the positive rate of OYAV SZC50 antibody in pigs from Malipo reaching 95%. To determine the pathogenicity of OYAV SZC50, we chose three animal models: specific pathogen-free Kunming mice, C57BL/6 mice lacking the interferon α/ß receptor, and chicken embryos. At 5, 6, and 7 days post-infection, all adult and suckling C57BL/6 mice, and specific pathogen-free suckling Kunming mice were dead. Our finding was expanding the knowledge about the infection and pathogenic risk of the neglected virus in the Orthobunyavirus.

Diversity and Genetic Reassortment of Keystone Virus in Mosquito Populations in Florida.

Keystone orthobunyavirus (KEYV), a member of the genus Orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. Although data on human infections are limited, the virus has been linked to a fever/rash syndrome and, possibly, encephalitis, with early studies suggesting that 20% of persons in the Tampa, Florida, region had antibodies to KEYV. To assess the distribution and diversity of KEYV in other regions of Florida, we collected > 6,000 mosquitoes from 43 sampling sites in St. Johns County between June 2019 and April 2020. Mosquitoes were separated into pools by species and collection date and site. All pools with Aedes spp. (293 pools, 2,171 mosquitoes) were screened with a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay that identifies KEYV and other closely related virus species of what was previously designated as the California encephalitis serogroup. In 2020, screening for KEYV was expanded to include 211 pools of Culex mosquitoes from sites where KEYV-positive Aedes spp. had been identified. rRT-PCR-positive samples were inoculated into cell cultures, and five KEYV isolates from Aedes atlanticus pools were isolated and sequenced. Analyses of the KEYV large genome segment sequences revealed two distinct KEYV clades, whereas analyses of the medium and small genome segments uncovered past reassortment events. Our data documented the ongoing seasonal circulation of multiple KEYV clades within Ae. atlanticus mosquito populations along the east coast of Florida, highlighting the need for further studies of the impact of this virus on human health.

Mechanisms of bunyavirus morphogenesis and egress.

Unlike many segmented negative-sense RNA viruses, most members of the Bunyavirales bud at Golgi membranes, as opposed to the plasma membrane. Central players in this assembly process are the envelope glycoproteins, Gn and Gc, which upon translation undergo proteolytic processing, glycosylation and trafficking to the Golgi, where they interact with ribonucleoprotein genome segments and bud into Golgi-derived compartments. The processes involved in genome packaging during virion assembly can lead to the generation of reassorted viruses, if a cell is co-infected with two different bunyaviruses, due to mismatching of viral genome segment packaging. This can lead to viruses with high pathogenic potential, as demonstrated by the emergence of Schmallenberg virus. This review focuses on the assembly pathways of tri-segmented bunyaviruses, highlighting some areas in need of further research to understand these important pathogens with zoonotic potential.

Immunization with a small fragment of the Schmallenberg virus nucleoprotein highly conserved across the Orthobunyaviruses of the Simbu serogroup reduces viremia in SBV challenged IFNAR-/- mice.

Schmallenberg Virus (SBV), an arbovirus from the Peribunyaviridae family and Orthobunyavirus genus, was discovered in late 2011 in Germany and has been circulating in Europe, Asia and Africa ever since. The virus causes a disease associated with ruminants that includes fever, fetal malformation, drop in milk production, diarrhoea and stillbirths, becoming a burden for small and large farms. Building on previous studies on SBV nucleoprotein (SBV-N) as a promising vaccine candidate, we have investigated the possible protein regions responsible for protection. Based on selective truncation of domains designed from the available crystal structure of the SBV-N, we identified both the N-terminal domain (N-term; Met1 - Thr133) and a smaller fragment within (C4; Met1 - Ala58) as vaccine prototypes. Two injections of the N-term and C4 polypeptides protected mice knockout for type I interferon (IFN) receptors (IFNAR-/-) challenged with virulent SBV, opposite to control groups that presented severe signs of morbidity and weight loss. Viremia analyses along with the presence of IFN-γ secreted from splenocytes re-stimulated with the N-terminal region of the protein corroborate that these two portions of SBV-N can be employed as subunit vaccines. Apart from both proteinaceous fragments being easily produced in bacterial cells, the C4 polypeptide shares a high sequence homology (∼87.1 %) with the corresponding region of nucleoproteins of several viruses of the Simbu serogroup, a group of Orthobunyaviruses that comprises SBV and veterinary pathogens like Akabane virus and human infecting viruses like Oropouche. Thus, we propose that this smaller fragment is better suited for vaccine nanoparticle formulation, and it paves the way to further research with other related Orthobunyaviruses.

Genetic characterization of the rare Bruconha virus (Bunyavirales: Orthobunyavirus) isolated in Vale do Ribeira (Atlantic Forest biome), Southeastern Brazil.

Brazil is a great source of arbovirus diversity, mainly in the Amazon region. However, other biomes, especially the Atlantic Forest, may also be a hotspot for emerging viruses, including Bunyaviruses (Negarnaviricota: Bunyavirales). For instance, Vale do Ribeira, located in the Southeastern region, has been widely studied for virus surveillance, where Flavivirus, Alphavirus and Bunyaviruses were isolated during the last decades, including Bruconha virus (BRCV), a member of Orthobunyavirus genus Group C, in 1976. Recently, a new isolate of BRCV named Span321532 was obtained from an adult sentinel mouse placed in Iguape city in 2011, and a full-length genome was generated with nucleotide differences ranging between 1.5%, 5.3% and 5% (L, M and S segments, respectively) from the prototype isolated 35 years earlier. In addition, each segment placed BRCV into different clusters, showing the high variety within Bunyavirales. Although no evidence for reassortants was detected, this finding reiterates the need for new surveillance and genomic studies in the area considering the high mutation rates of arbovirus, and also to identify the hosts capable of supporting the continuous circulation of Orthobunyavirus.

Characterization of experimental Shuni virus infection in the mouse.

Shuni virus (SHUV), an orthobunyavirus of the Simbu serogroup, was initially isolated in Nigeria in the 1960s, further detected in other African countries and in the Middle East, and is now endemic in Israel. Transmitted by blood-sucking insects, SHUV infection is associated with neurological disease in cattle and horses, and with abortion, stillbirth, or the birth of malformed offspring in ruminants. Surveillance studies also indicated a zoonotic potential. This study aimed to test the susceptibility of the well-characterized interferon (IFN)-α/ß receptor knock-out mouse model (Ifnar-/-), to identify target cells, and to describe the neuropathological features. Ifnar-/-mice were subcutaneously infected with two different SHUV strains, including a strain isolated from the brain of a heifer showing neurological signs. The second strain represented a natural deletion mutant exhibiting a loss of function of the S-segment-encoded nonstructural protein NSs, which counteracts the host's IFN response. Here it is shown that Ifnar-/-mice are susceptible to both SHUV strains and can develop fatal disease. Histological examination confirmed meningoencephalomyelitis in mice as described in cattle with natural and experimental infections. RNA in situ hybridization was applied using RNA Scope™ for SHUV detection. Target cells identified included neurons and astrocytes, as well as macrophages in the spleen and gut-associated lymphoid tissue. Thus, this mouse model is particularly beneficial for the evaluation of virulence determinants in the pathogenesis of SHUV infection in animals.

Structure, function, and evolution of the Orthobunyavirus membrane fusion glycoprotein.

La Crosse virus, responsible for pediatric encephalitis in the United States, and Schmallenberg virus, a highly teratogenic veterinary virus in Europe, belong to the large Orthobunyavirus genus of zoonotic arthropod-borne pathogens distributed worldwide. Viruses in this under-studied genus cause CNS infections or fever with debilitating arthralgia/myalgia syndromes, with no effective treatment. The main surface antigen, glycoprotein Gc (∼1,000 residues), has a variable N-terminal half (GcS) targeted by the patients' antibody response and a conserved C-terminal moiety (GcF) responsible for membrane fusion during cell entry. Here, we report the X-ray structure of post-fusion La Crosse and Schmallenberg virus GcF, revealing the molecular determinants for hairpin formation and trimerization required to drive membrane fusion. We further experimentally confirm the role of residues in the fusion loops and in a vestigial endoplasmic reticulum (ER) translocation sequence at the GcS-GcF junction. The resulting knowledge provides essential molecular underpinnings for future development of potential therapeutic treatments and vaccines.

Whole Genome Sequencing Analysis of African Orthobunyavirus Isolates Reveals Naturally Interspecies Segments Recombinations between Bunyamwera and Ngari Viruses.

Bunyamwera virus is the prototype of the Bunyamwera serogroup, which belongs to the order Bunyavirales of the Orthobunyavirus genus in the Peribunyaviridae family. Bunyamwera is a negative-sense RNA virus composed of three segments S, M, and L. Genetic recombination is possible between members of this order as it is already documented. Additionally, it can lead to pathogenic or host range improvement, if it occurs with viruses of public health and agricultural importance such as Rift Valley fever virus and Crimea-Congo hemorrhagic fever virus. Here, we characterize five African Orthobunyavirus viruses from different geographical regions. Our results suggest that the five newly characterized strains are identified as Bunyamwera virus strains. Furthermore, two of the five strains sequenced in this study are recombinant strains, as fragments of their segments are carried by Ngari and Bunyamwera strains. Further investigations are needed to understand the functional impact of these recombinations.

The mechanism of genome replication and transcription in bunyaviruses.

Bunyaviruses are negative sense, single-strand RNA viruses that infect a wide range of vertebrate, invertebrate and plant hosts. WHO lists three bunyavirus diseases as priority diseases requiring urgent development of medical countermeasures highlighting their high epidemic potential. While the viral large (L) protein containing the RNA-dependent RNA polymerase is a key enzyme in the viral replication cycle and therefore a suitable drug target, our knowledge on the structure and activities of this multifunctional protein has, until recently, been very limited. However, in the last few years, facilitated by the technical advances in the field of cryogenic electron microscopy, many structures of bunyavirus L proteins have been solved. These structures significantly enhance our mechanistic understanding of bunyavirus genome replication and transcription processes and highlight differences and commonalities between the L proteins of different bunyavirus families. Here, we provide a review of our current understanding of genome replication and transcription in bunyaviruses with a focus on the viral L protein. Further, we compare within bunyaviruses and with the related influenza virus polymerase complex and highlight open questions.

Sequencing and description of the complete mitochondrial genome of Limatus durhamii (Diptera: Culicidae).

The genus Limatus (Diptera: Culicidae) are wild mosquitoes belonging to the Sabethini tribe that occurs in tropical countries and is related to transmission cycles of Orthobunyavirus (Bunyaviridae), particularly in the Amazon region. Given the unavailability of information related to evolutionary biology and molecular taxonomy aspects of this genus, we report here the first complete sequencing of the mitochondrial genome of Limatus durhamii Theobald, 1901. The NextSeq 500 platform was used for sample sequencing, and the mitochondrial sequence obtained was 14,875 bp long, comprising 37 functional subunits (13 PCGs, 22 tRNA and 02 rRNA). The phylogeny reconstructed by maximum likelihood based on the concatenation of all 13 PCGs corroborated the known taxonomic classification based most on aspects of the external morphology and few molecular studies. The data and information produced here may be useful in the future development of taxonomic and evolutionary studies for the genus, as well as the Culicidae family itself.

Oropouche Virus Glycoprotein Topology and Cellular Requirements for Glycoprotein Secretion.

Oropouche virus (OROV; genus Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, which encodes the surface glycoproteins Gn and Gc plus the nonstructural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion, and unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibits glycoprotein secretion. Infection studies have previously shown that the cellular endosomal sorting complexes required for transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant-negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly colocalizes with OROV glycoproteins and membranes costained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant-negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlight differences in M polyprotein processing across orthobunyaviruses, indicate that Golgi and ESCRT function are required for glycoprotein secretion, and identify CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins. IMPORTANCE Oropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterized by high fever, headache, myalgia, and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment, and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a nonstructural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular endosomal sorting complexes required for transport (ESCRT) membrane-remodeling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6.

Retrospective molecular investigation of Mayaro and Oropouche viruses at the human-animal interface in West-central Brazil, 2016-2018.

Mayaro virus (MAYV, Togaviridae) and Oropouche orthobunyavirus (OROV, Peribunyaviridae) are emerging enzootic arboviruses in Latin America. Outbreaks of febrile illness associated with MAYV and OROV have been reported among humans mainly in the northern region of Brazil since the 1980s, and recent data suggest these viruses have circulated also in more populated areas of western Brazil. MAYV shares mosquito vectors with yellow fever virus and it has been historically detected during yellow fever epidemics. Aiming to investigate the transmission of OROV and MAYV at the human-animal interface during a yellow fever, chikungunya and Zika outbreaks in Brazil, we conducted a retrospective molecular investigation in 810 wild and domestic animals, 106 febrile patients, and 22.931 vectors collected from 2016 to 2018 in Cuiaba and Campo Grande metropolitan regions, western Brazil. All samples tested negative for OROV and MAYV RNA by RT-qPCR. Findings presented here suggest no active circulation of MAYV and OROV in the sampled hosts. Active surveillance and retrospective investigations are instrumental approaches for the detection of cryptic and subclinical activity of enzootic arboviruses and together serve as a warning system to implement appropriate actions to prevent outbreaks.