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PubMed2025

An Introduction to the Bunyaviruses.

The bunyaviruses are an ever-expanding group of RNA viruses that have been linked to a variety of different diseases around the world. First characterized nearly a century ago, over 500 different types of bunyaviruses have been characterized thus far, with hosts ranging from mammals to plants to single-celled organisms. As many of the currently described bunyaviruses have been found to be vector-borne, with transmission being mediated by either insects or rodents, these viruses have incorporated immune-evasive molecules into their relatively small genome. As these viruses have been implicated in a number of public health threats, there is an increased interest in performing experiments that could improve our understanding of these infectious agents. Therefore, the objectives of this book are outlined in this chapter, with a variety of techniques being described for the study of a variety of different bunyaviruses.

Animals↗

PubMedDate not supplied

Studies on transmission of Tataguine virus by Culex (pipens) fatigans mosquitoes.

Culex (pipens) fatigans mosquitoes were infected with Tataguine virus by pledget method. Virus was recovered from 40% of mosquitoes that fed on the infective blood meal after 21 days of incubation. Low titres of infective virus were found in the mosquitoes. Tataguine virus was not successfully transmitted to suckling hamsters exposed to the bites of infected mosquitoes.

Animals↗

PubMed2026

Oropouche virus: viral evolution, epidemiological trends, and challenges for control.

PURPOSE OF REVIEW: In recent years, OROV has emerged as a significant public health threat beyond the Amazon region. Here we review current epidemiological, virological, clinical and ecological knowledge of OROV to inform health practitioners, public health authorities and the scientific community and to facilitate the development of effective control strategies for OROV. RECENT FINDINGS: We describe the epidemiological, virological, ecological and clinical characteristics of OROV, focusing on lessons from the recent expansion, and highlighting needs for control and management of this emerging arbovirus. SUMMARY: This review aims to inform health practitioners, public health authorities and the scientific community of the recent reemergence and expansion of OROV beyond the Amazon Basin. The ecology, epidemiology, virology of OROV and clinical presentations of OROV infection are discussed, and knowledge gaps are identified.

Humans↗

PubMed2026

The Bunyamwera orthobunyavirus Gc glycoprotein head and stalk drives an infectious virion assembly pathway specific for the insect host.

The Orthobunyavirus genus of arthropod-borne segmented RNA viruses comprises important pathogens including the human-infecting Oropouche virus and ruminant-infecting Schmallenberg virus (SBV). The prototypical Bunyamwera orthobunyavirus (BUNV) possesses envelope-embedded glycoprotein Gn-Gc tripodal spikes, of which the ectodomains mediate virus entry, while endodomains interact with nucleoprotein (NP) enwrapped genome segments driving virion assembly. Interestingly, BUNV Gc head/stalk domains are redundant for virus growth in mammalian cells, consistent with isolations of SBV from ruminants bearing head/stalk deletions. However, these domains appear strictly maintained in orthobunyavirus isolations from arthropods in nature. To investigate the molecular mechanism that underlines this discrepancy, we compared the multiplication characteristics of wildtype BUNV (BUNV-WT) with a Gc head/stalk deleted BUNV (BUNV-∆7). In mammalian cells BUNV-WT and BUNV-∆7 grew to equivalent titres, whereas in insect cells BUNV-∆7 titres were 1000-fold lower and strikingly produced no virions following blood meal infection of Aedes mosquitoes. To understand this insect-specific restriction in virion production, we showed the intracellular abundance of BUNV-WT and BUNV-∆7 Gc and NP components were equivalent, suggesting the deletion impacted post-translational stages of the infection cycle. To explore this, we investigated Gc and ∆7-Gc interactions during BUNV-WT and BUNV-∆7 infections of both insect and mammalian cells by co-immunoprecipitation and multiplex mass spectrometry, revealing ∆7-Gc exhibited markedly reduced NP interactions in insect cells, potentially indicating reduced segment interactions during assembly. We hypothesize that the Gc head/stalk performs an insect cell-specific role in segment recruitment during virion formation, and that maintenance in nature of full-length Gc is due to this essential role in the insect host.

Animals↗

PubMed2025

Probing orthobunyavirus reassortment using Bunyamwera and Batai viruses as models.

Reassortment is a critical evolutionary mechanism for segmented viruses, enabling the exchange of intact genome segments during co-infection and driving orthobunyavirus evolution; however, the molecular mechanisms underpinning this process remain unclear. With over 100 orthobunyavirus species, many of which are significant human and veterinary pathogens, understanding how reassortment influences transmissibility and virulence is essential for preempting the emergence of novel pathogens. Here, we use Bunyamwera virus (BUNV) and Batai virus (BATV) as models to explore orthobunyavirus reassortment through reverse genetics. We established the first reverse genetics system for BATV, generated reassortants, and employed minigenome assays to assess replication machinery compatibility. Additionally, we developed a novel hybridization chain reaction assay for high-resolution visualization of viral RNA segments. Our findings revealed that all six reassortants between BUNV and BATV are viable, exhibiting notable phenotypic differences in interferon-deficient (IFNAR-/-) mice. This work introduces essential tools and new insights into orthobunyavirus reassortment and pathogenesis, laying the groundwork for understanding this critical evolutionary process.

Animals↗

PubMed2025

Clinical and laboratory profiles of Oropouche virus disease from the 2024 outbreak in Manaus, Brazilian Amazon.

BACKGROUND: The 2024 Oropouche virus (OROV) outbreak in Brazil raised public health concerns due to its unprecedented rapid spread, high incidence, and potential neurological complications. OROV symptoms overlap with locally endemic arbovirus diseases, like dengue virus (DENV), complicating diagnosis. The study aimed to compare clinical, laboratory, and immunological profiles in OROV and DENV cases, crucial for improving diagnosis and management. METHODS: This study analyzed 51 OROV and 78 of DENV cases consecutively enrolled in Manaus, Amazonas, Brazil, and monitored for 28 days. OROV diagnosis was performed by real-time PCR (RT-PCR) using serum and urine samples. OROV RT-PCR positive samples were genotyped. A paired Plaque Reduction Neutralization Test (PRNT) was conducted on samples collected at D1 and D28. Patients with a ≥ 4-fold increase in neutralizing antibody titer between D1 and D28 were considered OROV-positive. Clinical manifestations, hematology, biochemistry, and cytokine profiles were analyzed. Statistical analysis included comparison between OROV and DENV patients. RESULTS: Genome sequencing of OROV isolates confirmed presence of a previously reported novel reassortment event, consistent with ongoing localized transmission. Urine RT-PCR demonstrated low positivity compared to serum samples. The paired PRNT increased sensitivity in 45%. Clinically, OROV infection was associated with significantly higher frequencies of severe headache, myalgia, arthralgia, and rash compared to DENV infection (p < 0.001). Elevated alanine aminotransferase (ALT) levels were also observed in OROV patients (p < 0.001). Immunologically, OROV infection induced significantly increased levels of acute-phase CCL11 (eotaxin), CXCL10, IFN-γ, IL-1RA, and IL-10, which declined by day 28, while IL-5 increased during recovery. In contrast, DENV patients exhibited elevated levels of CCL2, G-CSF, and CCL3 in recovery phase. CONCLUSION: OROV symptoms overlap with DENV underscores the need for syndromic diagnostic approach in endemic regions. Continued genomic surveillance and expanded clinical studies are vital to assess long-term consequences. Given OROV's expanding geographic range, targeted public health measures are essential to mitigate future outbreaks and better understand its pathophysiology.

Humans↗

PubMed2025

Oropouche Virus Importation in Southern Brazil and Emerging Concern Calling for Enhanced Public Health Surveillance.

Oropouche virus (OROV), an arthropod-borne virus transmitted by Culicoides paraensis, is an endemic arbovirus that historically circulates mostly in the Amazon basin. Between 2022 and 2024, it reemerged as a more widespread public health concern in South America. We conducted a pooled-sample molecular surveillance study to understand the prevalence of Oropouche fever in Brazil's southernmost state. Over 18 months, we analyzed 4060 samples to monitor the virus emergence in the Rio Grande do Sul state. We detected the first human case of OROV in the state, and our phylogenetic reconstruction indicated a travel-related introduction from the Amazon region into Rio Grande do Sul. Despite the absence of local transmission, the invasion of Culicoides paraensis and enzootic circulation of the OROV in Rio Grande do Sul highlight the risk of Oropouche fever outbreaks in the region. We demonstrated that pooled-sample surveillance effectively monitors virus introduction during periods of low endemic circulation, serving as an essential active surveillance tool for the timely detection of virus emergence and enhancing public health preparedness. The multiple introductions of distinct OROV lineages into southern Brazil underscore the importance of genomic surveillance and public health strategies to monitor and mitigate arbovirus spread in the region.

Brazil↗

PubMed2025

Efficient Expression of Oropouche Virus Nonstructural Proteins NSs and NSm.

Oropouche fever, a mosquito- or midge-borne emerging zoonotic disease endemic to South and Central America, manifests as a dengue-like acute febrile illness with occasional occurrences of meningitis or meningoencephalitis. The causative agent, Oropouche virus (OROV), belongs to the genus Orthobunyavirus within the family Peribunyaviridae. Its tripartite negative-sense RNA genome comprises small (S), medium (M), and large (L) segments, encoding structural N, Gn/Gc, and L proteins, respectively. Additionally, the S- and M-segments encode nonstructural proteins: NSs and NSm, which may act as virulence factors. OROV NSs functions as an interferon antagonist with an unknown mechanism, while the roles of OROV NSm remain elusive. This chapter introduces efficient expression systems for OROV NSm and NSs proteins. Validating the presence of a signal peptide at the N-terminus of NSm protein is essential for its expression. Furthermore, expressing OROV NSs protein independently of an RNA polymerase II promoter is crucial to prevent restricted gene expression, potentially caused by NSs inhibiting cellular RNA polymerase II, as observed in closely related bunyavirus NSs proteins. These protein expression strategies offer insights into the molecular characterization of OROV NSm and NSs proteins, facilitating a deeper understanding of their virulence mechanisms.

Viral Nonstructural Proteins↗

PubMed1979

Comparative susceptibility of PS cells, XTC-2 cells, and suckling mice to infection with California group arboviruses (Bunyaviridae).

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Animals↗

PubMed2025

Genomic evidence of active circulation of Orthobunyavirus in Ecuador.

BACKGROUND: Between 2023 and 2025, the largest Oropouche fever epidemic recorded in history unfolded across Brazil seeding cases throughout Latin America. In 2024, three cases of Oropouche fever were reported in Ecuador. METHODS: An Oropouche fever case detected in Bolívar Province in June 2024 was preliminarily diagnosed as Oropouche virus (OROV) through RT-qPCR and was further processed using next-generation sequencing. RESULTS: Segments L and S of this virus forms a monophyly with another sequence circulating in Ecuador in April 2024 (i.e. PQ863772.1 isolate Ecuador traveler), with an uncertain province origin. Both sequences differ from previous OROV Ecuadorian sequences detected in 2016 and from the OROV strain driving the 2023-2025 epidemic in Brazil. CONCLUSIONS: Orthobunyavirus oropoucheense has an endemic circulation in Ecuador. Genomic surveillance of Orthobunyavirus in Ecuador and other regions should be actively pursued-independent of epidemics-to anticipate potential zoonotic outbreaks.

Adult↗

PubMed2024

Expansion of Oropouche virus in non-endemic Brazilian regions: analysis of genomic characterisation and ecological drivers.

BACKGROUND: Oropouche virus (OROV) is an arbovirus endemic in the Amazon region that closely resembles other arboviruses in terms of human disease, leading to potential misdiagnoses. The virus ecology has mostly restricted its occurrence to the Amazon biome; however, after a large 2023-24 OROV epidemic in the Brazilian Amazon region, outbreaks are being reported across Brazil and in other countries in Latin America. Here, we investigate the OROV spread outside Amazonia. METHODS: In this genomic and epidemiological study, OROV cases from January, 2023, to July, 2024, provided by the General Coordination of Public Health Laboratories of Brazil on Aug 1, 2024, were compared by geographical location (Amazon vs non-Amazon) and municipal population size, and a linear mixed model was employed to assess the relationship between agricultural area size and cases. OROV-positive samples from central laboratories of five non-Amazonian Brazilian states were sequenced using an amplicon-based approach. Bayesian phylogeographical analysis was performed with near full-length viral genomes, incorporating individual travel histories when relevant. The estimated dates of viral introductions in each sampled location were then contextualised with public epidemiological data. FINDINGS: Epidemic data show that outside the Amazon region, OROV cases frequency was 3·9-times higher in small municipalities than in large municipalities. The planted areas of some agricultural products, such as banana plantations, were positively correlated (r=0·39, p<0·0001) with OROV cases. The linear mixed model revealed that, besides banana, cassava also has larger (p<0·05) planted areas in municipalities with OROV cases when compared with those with no cases. The phylogenetic analysis of 32 new OROV genomes reconstructed multiple exportation events of the newly identified reassortant lineage from the Amazon to other Brazilian regions between January and March, 2024. At least three of the previously described OROV phylogenetic clades circulating in the Amazon were the source of viral introductions. Molecular clock analysis estimated that viral introductions happened from 50 days to 100 days before detecting the outbreaks in each state. INTERPRETATION: Our results confirm that the novel OROV reassortant lineage spread from the Amazon to other regions in early 2024, successfully establishing local transmission. The fact that outbreaks were observed in small municipalities, instead of large urban centres, suggests that local ecological conditions that are ideal for OROV vector occurrence, such as the banana plantation environment, might be important factors driving its spread in Brazil. FUNDING: DECIT, CNPq, FAPEAM, and Inova-Fiocruz. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Brazil↗

PubMed2001

Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis.

Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) contains a tripartite negative-sense RNA genome. The smallest RNA segment, S, encodes the nucleocapsid protein N and a nonstructural protein, NSs, in overlapping reading frames. We have generated a mutant virus lacking NSs, called BUNdelNSs, by reverse genetics. Compared with the wild-type (wt) virus, BUNdelNSs exhibited a smaller plaque size and generated titers of virus approximately 1 log lower. In mammalian cells, the mutant expressed greatly increased levels of N protein; significantly, the marked inhibition of host cell protein synthesis shown by wt virus was considerably impaired by BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed BALB/c mice with a slower time course than wt and exhibited a reduced cell-to-cell spread, and titers of virus in the brain were lower. In addition, the abrogation of NSs expression changed Bunyamwera virus from a noninducer to an inducer of an interferon-beta promoter. These results suggest that, although not essential for growth in tissue culture or in mice, the bunyavirus NSs protein has several functions in the virus life cycle and contributes to viral pathogenesis.

Aedes↗

PubMed2005

Attenuation of bunyavirus replication by rearrangement of viral coding and noncoding sequences.

Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.

Animals↗

PubMed1976

Morphology and morphogenesis of Crimean hemorrhagic fever virus.

Morphological characteristics of Crimean Hemorrhagic Fever Virus indicate that it is closely related to the Bunyaviridae family. However, there are some features (e.g. formation of crystal-like patterns in infected cells) demonstrating differences to the other known Bunyaviridae.

Animals↗

PubMed2002

Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon.

Production of alpha/beta interferons (IFN-alpha/beta) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-alpha/beta, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-kappaB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-alpha/beta system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-alpha/beta in order to increase the virulence of Bunyamwera virus.

Animals↗

PubMed1975

International arbovirus research.

International cooperation in arbovirus research became increasingly prominent shortly after it was found in the early 1940's, that antigenic relationships existed among certain viruses isolated in different areas of thw world. The interest of a number of scientists and of a private organization led to the establishment of an international information exchange and to the preparation of an international catalogue of arboviruses including certain other viruses of vertebrates. International cooperation was fostered by the interest taken in these agents by the World Health Organization and the creation by this agency of an international net of collaborating laboratories with assigned areas of the world. A number of recent problems have been revealed and solved, or partly solved by this international effort, among which are questions relating to: the Marburg agent disease, the existence of an antigenic relationship between rabies virus and some arboviruses, aspects of the ecology or viruses in the California, Uukuniemi and Kemerovo groups, as well as several unanticipated antigenic relationships among viruses. Numerous questions still remain the solution of which might be expedited by continued and increased cooperative international effort such as overwintering or survival of viruses under unfavorable circumstances, pathogenic potential for man and domestic animals of certain viruses, role on chronic illnesses, establishment of the etiology of certain diseases and investigations of the basic properties of viruses, particularly of the Bunyaviridae.

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PubMed1979

Pneumoviruses: the cell surface of lytically and persistently infected cells.

Human embryonic lung (MRC-5), feline embryo (FEA), mink lung (Mv1Lu) and monkey kidney (BSC-1) cells infected by respiratory syncytial virus showed characteristic morphological changes when viewed by scanning electron microscopy. The surfaces of respiratory syncytial virus-infected cells developed a profusion of slender filaments after 48 h incubation at 31 degrees C. Similar changes in surface morphology were observed in BSC-1 cells infected by murine pneumonia virus. Filament production therefore appears to be a common property of pneumo-viruses. Filaments were not observed in cells infected with either syncytial and non-syncytial herpes simplex virus, the cytocidal vesicular stomatitis and Batai (Bunyaviridae) viruses, or the focus-inducing rabbit fibroma virus. Filament production was not observed in cells infected with ts mutants of respiratory syncytial (RS) virus during incubation at the restrictive temperature, or in a persistently infected culture of BSC-1 cells at 37 degrees C. The persistently infected cells (the RS ts 1/BSC-1 line) had some of the characteristics of cells transformed by oncogenic viruses, namely ability to overlap adjacent cells and agglutination by a low concentration of concanavalin A. The pseudo-transformed phenotype was temperature-dependent, however, and suppressed by raising the temperature of incubation to 39 degrees C. The presence of virus antigen at the cell surface was similarly temperature-dependent in these cells, diminished at high temperature (39 degrees C) and enhanced at low temperature (31 degrees C), suggesting that the changes in the host cell were the result of insertion of virus protein into the cell membrane. Evidently, persistent infection by a cytoplasmic virus can produce alterations in the host cell usually associated with transformation by nuclear viruses.

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PubMed1979

Electron microscopy of Akabane virus.

Electron microscopy of negatively stained purified virus and of thin sections of infected cells and tissues showed Akabane virus being similar in morphology and morphogenesis to members of the family Bunyaviridae.

Animals↗