Oropouche Virus: An Emerging Orthobunyavirus.

On 2 February 2024, the Pan American Health Organization/World Health Organization issued an epidemiological alert on rising Oropouche virus (OROV) infections in South America. By 3 August 2024, this alert level had escalated from medium to high. OROV has been a public health concern in Central and South America since its emergence in Brazil in the 1960s. However, the 2024 outbreak marks a turning point, with the sustained transmission in non-endemic regions of Brazil, local transmission in Cuba, two fatalities and several cases of vertical transmission. As of the end of August 2024, 9852 OROV cases have been confirmed. The 2024 OROV outbreak underscores critical gaps in our understanding of OROV pathogenesis and highlights the urgent need for antivirals and vaccines. This review aims to provide a concise overview of OROV, a neglected orthobunyavirus.

ddPCR for the Detection and Absolute Quantification of Oropouche Virus.

BACKGROUND: Oropouche virus (OROV) is a segmented RNA virus belonging to the genus Orthobunyavirus in the family Peribunyaviridae. Herein, an in-house droplet digital PCR (ddPCR) assay was used for the detection and quantification of OROV. METHODS: The ddPCR reaction was assessed as duplex assay using the human housekeeping gene RPP30. Limit of detection (LoD) analysis was performed in whole blood, serum, and urine. The assay was executed on a total of 28 clinical samples (whole blood n = 9, serum n = 11, and urine n = 8), of which 16 specimens were tested positive at the routine molecular diagnostics (endpoint and real-time PCRs). RESULTS: The LoD of the ddPCR performed using 10-fold serial dilution of OROV detected up to 1 cp/µL in all the biological matrices. Compared to the routine molecular diagnostics, the ddPCR assay showed 100% sensitivity for whole blood and serum and 75% for urine, highlighting higher positive rate of ddPCR. CONCLUSION: We have established a quantitative RNA detection method of OROV with high sensitivity and specificity based on ddPCR. This test is capable of quantitatively monitoring the viral load of OROV and can contribute, in addition to laboratory diagnosis, to shed light on the pathogenesis, filling in the knowledge gaps of this neglected disease and to the vector control programs.

Oropouche Virus (OROV) in Pregnancy: An Emerging Cause of Placental and Fetal Infection Associated with Stillbirth and Microcephaly following Vertical Transmission.

Oropouche virus (OROV) is an emerging arbovirus endemic in Latin America and the Caribbean that causes Oropouche fever, a febrile illness that clinically resembles some other arboviral infections. It is currently spreading through Brazil and surrounding countries, where, from 1 January to 1 August 2024, more than 8000 cases have been identified in Bolivia, Brazil, Columbia, and Peru and for the first time in Cuba. Travelers with Oropouche fever have been identified in the United States and Europe. A significant occurrence during this epidemic has been the report of pregnant women infected with OROV who have had miscarriages and stillborn fetuses with placental, umbilical blood and fetal somatic organ samples that were RT-PCR positive for OROV and negative for other arboviruses. In addition, there have been four cases of newborn infants having microcephaly, in which the cerebrospinal fluid tested positive for IgM antibodies to OROV and negative for other arboviruses. This communication examines the biology, epidemiology, and clinical features of OROV, summarizes the 2023-2024 Oropouche virus epidemic, and describes the reported cases of vertical transmission and congenital infection, fetal death, and microcephaly in pregnant women with Oropouche fever, addresses experimental animal infections and potential placental pathology findings of OROV, and reviews other bunyavirus agents that can cause vertical transmission. Recommendations are made for pregnant women travelling to the regions affected by the epidemic.

(Re-)Emergence of Oropouche Virus (OROV) Infections: Systematic Review and Meta-Analysis of Observational Studies.

Oropouche Virus (OROV; genus of Orthobunyavirus) is the causal agent of Oropouche Fever (OF). Due to the lack of specific signs and symptoms and the limited availability of diagnostic tests, the actual epidemiology of OROV infections and OF has been extensively disputed. In this systematic review with meta-analysis, a literature search was carried out in PubMed, Scopus, EMBASE, and MedRxiv in order to retrieve relevant articles on the documented occurrence of OROV infections. Pooled detection rates were then calculated for anti-OROV antibodies and virus detection (i.e., viral RNA detected by viral cultures and/or real-time polymerase chain reaction [RT-qPCR]). Where available, detection rates for other arboviruses (i.e., Dengue [DENV], Chikungunya [CHKV], and Zika Virus [ZIKV]) were calculated and compared to those for OROV. A total of 47 studies from South America and the Caribbean were retrieved. In individuals affected by febrile illness during OROV outbreaks, a documented prevalence of 0.45% (95% confidence interval [95%CI] 0.16 to 1.12) for virus isolation, 12.21% (95%CI 4.96 to 27.09) for seroprevalence (including both IgM and IgG class antibodies), and 12.45% (95%CI 3.28 to 37.39) for the detection of OROV-targeting IgM class antibodies were eventually documented. In the general population, seroprevalence was estimated to be 24.45% (95%CI 7.83 to 55.21) for IgG class antibodies. The OROV detection rate from the cerebrospinal fluids of suspected cases of viral encephalitis was estimated to be 2.40% (95%CI 1.17 to 5.03). The occurrence of OROV infections was consistently lower than that of DENV, CHKV, and ZIKV during outbreaks (Risk Ratio [RR] 24.82, 95%CI 21.12 to 29.16; RR 2.207, 95%CI 1.427 to 3.412; and RR 7.900, 95%CI 5.386 to 11.578, respectively) and in the general population (RR 23.614, 95%CI 20.584 to 27.129; RR 3.103, 95%CI 2.056 to 4.685; and RR 49.500, 95%CI 12.256 to 199.921, respectively). In conclusion, our study stresses the possibly high underestimation of OROV prevalence in the general population of South America, the potential global threat represented by this arbovirus infection, and the potential preventive role of a comprehensive "One Health approach".

Oropouche Fever, Cuba, May 2024.

Phylogenetic analyses showed that the virus responsible for a May 2024 Oropouche fever outbreak in Cuba was closely related to viruses from Brazil in 2023. Pools of Ceratopogonidae spp. biting midges and Culex quinquefasciatus mosquitoes were positive for Oropouche viral RNA. No cases were severe. Virus extension to new areas may increase case numbers and severity.

A reporter Oropouche virus expressing ZsGreen from the M segment enables pathogenesis studies in mice.

Oropouche fever caused by Oropouche virus (OROV) is a significant zoonosis in Central and South America. Despite its public health significance, we lack high-throughput diagnostics, therapeutics, and a comprehensive knowledge of OROV biology. Reporter viruses are valuable tools to rapidly study virus dynamics and develop neutralization and antiviral screening assays. OROV is a tri-segmented bunyavirus, which makes generating a reporter virus challenging, as introducing foreign elements into the viral genome typically affects fitness. We previously demonstrated that the non-structural gene NSm on the OROV medium (M) segment is non-essential for replication in vitro. Taking advantage of this, we have now generated a recombinant OROV expressing fluorescent protein ZsGreen in place of NSm. This reporter OROV is both stable and pathogenic in IFNAR-/- mice and provides a powerful tool for OROV pathogenesis studies and assay development.IMPORTANCEEmerging and reemerging infectious agents such as zoonotic bunyaviruses are of global health concern. Oropouche virus (OROV) causes recurring outbreaks of acute febrile illness in the Central and South American human populations. Biting midges are the primary transmission vectors, whereas sloths and non-human primates are their reservoir hosts. As global temperatures increase, we will likely see an expansion in arthropod-borne pathogens such as OROV. Therefore, developing reagents to study pathogen biology to aid in identifying druggable targets is essential. Here, we demonstrate the feasibility and use of a fluorescent OROV reporter in mice to study viral dynamics and pathogenesis. We show that this reporter OROV maintains characteristics such as growth and pathogenicity similar to the wild-type virus. Using this reporter virus, we can now develop methods to assist OROV studies and establish various high-throughput assays.

N-glycosylation of viral glycoprotein is a novel determinant for the tropism and virulence of highly pathogenic tick-borne bunyaviruses.

Severe fever with thrombocytopenia syndrome (SFTS) virus, a tick-borne bunyavirus, causes a severe/fatal disease termed SFTS; however, the viral virulence is not fully understood. The viral non-structural protein, NSs, is the sole known virulence factor. NSs disturbs host innate immune responses and an NSs-mutant SFTS virus causes no disease in an SFTS animal model. The present study reports a novel determinant of viral tropism as well as virulence in animal models, within the glycoprotein (GP) of SFTS virus and an SFTS-related tick-borne bunyavirus. Infection with mutant SFTS viruses lacking the N-linked glycosylation of GP resulted in negligible usage of calcium-dependent lectins in cells, less efficient infection, high susceptibility to a neutralizing antibody, low cytokine production in macrophage-like cells, and reduced virulence in Ifnar-/- mice, when compared with wildtype virus. Three SFTS virus-related bunyaviruses had N-glycosylation motifs at similar positions within their GP and a glycan-deficient mutant of Heartland virus showed in vitro and in vivo phenotypes like those of the SFTS virus. Thus, N-linked glycosylation of viral GP is a novel determinant for the tropism and virulence of SFTS virus and of a related virus. These findings will help us understand the process of severe/fatal diseases caused by tick-borne bunyaviruses.

Single-Molecule Visualization of Bunyavirus Genome Segments Using Fluorescence In Situ Hybridization.

The genome of most bunyaviruses is divided over three (S, M, and L) single-stranded RNA segments of negative polarity. The three viral RNA segments are essential to establish a productive infection. RNA fluorescence in situ hybridization (FISH) enables the detection, localization, and quantification of RNA molecules at single-molecule resolution. This chapter describes an RNA FISH method to directly visualize individual segment-specific bunyavirus RNAs in fixed infected cells and in mature virus particles, using Rift Valley fever virus as an example. Imaging of bunyavirus RNA segments is a valuable experimental tool to investigate fundamental aspects of the bunyavirus life cycle, such as virus replication, genome packaging, and virion assembly, among others.

Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

Replication of Akabane virus and related orthobunyaviruses in a fetal-bovine-brain-derived cell line.

Akabane virus (AKAV), Aino virus, Peaton virus, Sathuperi virus, and Shamonda virus are arthropod-borne viruses belonging to the order Elliovirales, family Peribunyaviridae, genus Orthobunyavirus. These viruses cause or may cause congenital malformations in ruminants, including hydranencephaly, poliomyelitis, and arthrogryposis, although their pathogenicity may vary among field cases. AKAV may cause relatively severe congenital lesions such as hydranencephaly in calves. Furthermore, strains of AKAV genogroups I and II exhibit different disease courses. Genogroup I strains predominantly cause postnatal viral encephalomyelitis, while genogroup II strains are primarily detected in cases of congenital malformation. However, the biological properties of AKAV and other orthobunyaviruses are insufficiently investigated in hosts in the field and in vitro. Here, we used an immortalized bovine brain cell line (FBBC-1) to investigate viral replication efficiency, cytopathogenicity, and host innate immune responses. AKAV genogroup II and Shamonda virus replicated to higher titers in FBBC-1 cells compared with the other viruses, and only AKAV caused cytopathic effects. These results may be associated with the severe congenital lesions in the brain caused by AKAV genogroup II. AKAV genogroup II strains replicated to higher titers in FBBC-1 cells than AKAV genogroup I strains, suggesting that genogroup II strains replicated more efficiently in fetal brain cells, accounting for the detection of the latter strains mainly in fetal infection cases. Therefore, FBBC-1 cells may serve as a valuable tool for investigating the virulence and tropism of the orthobunyaviruses for bovine neonatal brain tissues in vitro.

Bluetongue Virus Serotype 3 and Schmallenberg Virus in Culicoides Biting Midges, Western Germany, 2023.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

[Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody].

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

First case of Oropouche fever detected in the international border region of the Colombian Amazon: clinical characteristics and molecular diagnosis.

OBJECTIVES: We report the first case of Oropouche fever detected in the border region of Colombia. METHODS: Using a multiplex real-time polymerase chain reaction (PCR), genetic sequencing and clinical characteristics during the dengue epidemic in 2019, a total of 175 samples were analysed, from cases notified to the system epidemiological surveillance such as dengue. FINDINGS: The Oropouche virus (OROV) isolate from Leticia belongs to lineage 2 according to both M and S genome segments maximum likelihood (ML) analysis, shares a common ancestor with samples obtained in Esmeraldas, Ecuador and Turbaco, Colombia. The patient: a woman resident in the border neighbourhood of the municipality of Leticia had the following symptoms: fever, headache, retro-orbital pain and myalgias. MAIN CONCLUSION: This cross-border surveillance can be useful to give an alert about the entry or exit of arboviruses circulation in the region, which are often underreported in public health surveillance systems.

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

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 family Peribunyaviridae, 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 (strain SZC50) and EBIV (strain Cu20-XJ), the susceptibility of four mosquito species (Culex pipiens pallens, Cx. quinquefasciatus, Aedes albopictus, and Ae. aegypti) was measured through artificial oral infection. Then, mosquito species with a high infection rate (IR) were chosen to further evaluate the dissemination rate (DR), transmission rate (TR), and transmission efficiency. The viral RNA in each mosquito sample was determined by RT-qPCR. RESULTS: The results revealed that for OYAV, Cx. pipiens pallens had the highest IR (up to 40.0%) among the four species, but the DR and TR were 4.8% and 0.0%, respectively. For EBIV, Cx. pipiens pallens and Cx. quinquefasciatus had higher IR compared to Ae. albopictus (1.7%). However, the EBIV RNA and infectious virus were detected in Cx. pipiens pallens, with a TR of up to 15.4% and a transmission efficiency of 3.3%. CONCLUSIONS: The findings indicate that Cx. pipiens pallens was susceptible to OYAV but had an extremely low risk of transmitting the virus. Culex pipiens pallens and Cx. quinquefasciatus were susceptible to EBIV, and Cx. pipiens pallens had a higher transmission risk to EBIV than Cx. quinquefasciatus.

Virus discovery in cultured portunid crabs: Genomic, phylogenetic, histopathological and microscopic characterization of a reovirus and a new bunyavirus.

Portunid crabs are distributed worldwide and highly valued in aquaculture. Viral infections are the main limiting factor for the survival of these animals and, consequently, for the success of commercial-scale cultivation. However, there is still a lack of knowledge about the viruses that infect cultured portunid crabs worldwide. Herein, the genome sequence and phylogeny of Callinectes sapidus reovirus 2 (CsRV2) are described, and the discovery of a new bunyavirus in Callinectes danae cultured in southern Brazil is reported. The CsRV2 genome sequence consists of 12 dsRNA segments (20,909 nt) encode 13 proteins. The predicted RNA-dependent RNA polymerase (RdRp) shows a high level of similarity with that of Eriocheir sinensis reovirus 905, suggesting that CsRV2 belongs to the genus Cardoreovirus. The CsRV2 particles are icosahedral, measuring approximately 65 nm in diameter, and exhibit typical non-turreted reovirus morphology. High throughput sequencing data revealed the presence of an additional putative virus genome similar to bunyavirus, called Callinectes danae Portunibunyavirus 1 (CdPBV1). The CdPBV1 genome is tripartite, consisting of 6,654 nt, 3,120 nt and 1,656 nt single-stranded RNA segments that each encode a single protein. Each segment has a high identity with European shore crab virus 1, suggesting that CdPBV1 is a new representative of the family Cruliviridae. The putative spherical particles of CdPBV1 measure ∼120 nm in diameter and present a typical bunyavirus morphology. The results of the histopathological analysis suggest that these new viruses can affect the health and, consequently, the survival of C. danae in captivity. Therefore, the findings reported here should be used to improve prophylactic and pathogen control practices and contribute to the development and optimization of the production of soft-shell crabs on a commercial scale in Brazil.

Isolation and whole-genome sequence analysis of Balagodu virus in Japan.

Whole-genome sequencing of a virus isolated from Culicoides biting midges in southern Japan in 2020 revealed that it is a strain of Balagodu virus (BLGV; genus Orthobunyavirus; family Peribunyaviridae; order Bunyavirales). A solitary instance of BLGV isolation occurred in India in 1963. All assembled segments comprise complete protein-coding sequences that are similar to those of other orthobunyaviruses. The consensus 3'- and 5'-terminal sequences of orthobunyaviruses' genomic RNAs are also conserved in the Japanese BLGV strain. Here, we update the geographic distribution of BLGV and provide its complete sequence, contributing to the clarification of orthobunyavirus phylogeny.

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.

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.