Oropouche Virus Research Articles

First outbreak of Oropouche Fever reported in a non-endemic western region of the Peruvian Amazon: Molecular diagnosis and clinical characteristics

IntroductionOropouche fever is an under-reported and emerging infectious disease caused by Oropouche virus (OROV). Its incidence is under-estimated mainly due to clinical similarities with other endemic arboviral diseases and the lack of specific diagnostic tests. We report the first outbreak of Oropouche fever in a western region of the Peruvian Amazon in Huanuco, Peru. MethodsA transversal study was carried out during an outbreak in the western Region of Huanuco, Peru between January and July of 2016. Blood samples of 268 patients with acute febrile syndrome were collected and analyzed for OROV via RT- PCR and genetic sequencing. ResultsOf all 268 patients, 46 (17%) cases tested positive for OROV. The most common symptoms reported were headache with a frequency of 87% (n = 40) followed by myalgia with 76% (n = 35), arthralgia with 65.2% (n = 30), retro-ocular pain 60.8% (n = 28) and hyporexia with 50% (n = 23). Some patients showed a clinical presentation suggestive of severe OROV infection, of which 4.3% (n = 2) had low platelet count, 8.6% (n = 4) had intense abdominal pain, and 2.1% (n = 1) had a presentation with thoracic pain. ConclusionThis study reports an outbreak of OROV in a region where this virus was not previously identified. The disease caused by OROV is an emerging, underdiagnosed infection that requires further research to determine its virulence, pathogenesis, host range and vectors involved in the urban and sylvatic cycles as well as identifying new genotypes to implement sensitive and specific diagnostic tools that can be applied to endemic regions.

Lack of Evidence of Sylvatic Transmission of Dengue Viruses in the Amazon Rainforest Near Iquitos, Peru.

Dengue viruses (DENV) are currently responsible for more human morbidity and mortality than any other known arbovirus, and all four DENV are known to exist in sylvatic cycles that might allow these viruses to persist if the urban (Aedes aegypti) cycle could be controlled. To determine whether DENV were being maintained in a sylvatic cycle in a forested area about 14 km southwest of Iquitos, Peru, a city in which all 4 serotypes of DENV circulate, we placed 20 DENV seronegative Aotus monkeys in cages either in the canopy or near ground level for a total of 125.6 months. Despite capturing >66,000 mosquitoes in traps that collected some of the mosquitoes attracted to these monkeys, blood samples obtained once a month from each animal were tested and found to be negative by an enzyme-linked immunosorbent assay for IgM and IgG antibodies to dengue, yellow fever, Venezuelan equine encephalitis, Oropouche, and Mayaro viruses. Although all four DENV serotypes were endemic in nearby Iquitos, the findings of this study did not support a DENV sylvatic maintenance and transmission cycle in a selected area of the Amazon rainforest in northeastern Peru.

Molecular detection and clinical characteristics of Bartonella bacilliformis, Leptospira spp., and Rickettsia spp. in the Southeastern Peruvian Amazon basin

BackgroundAcute febrile illness (AFI) represent a significant health challenge in the Peruvian Amazon basin population due to their diverse etiologies and the unavailability of specific on-site diagnostic methods, resulting in underreporting of cases. In Peru, one of the most endemic regions to dengue and leptospirosis is Madre de Dios, a region also endemic to emergent bacterial etiologic agents of AFI, such as bartonellosis and rickettsiosis, whose prevalence is usually underreported.We aimed to molecularly identify the presence of Leptospira spp., Bartonella bacilliformis, and Rickettsia spp. by Polymerase Chain Reaction in serum samples from patients with AFI from Puerto Maldonado-Madre de Dios in Peru.MethodsSerum samples from patients with acute febrile illness were analyzed by real-time PCR for detecting the presence of Bartonella bacilliformis, Leptospira spp. and Rickettsia spp.ResultsBartonella bacilliformis was the most prevalent bacteria identified in 21.6% (30/139) of the samples, followed by Leptospira spp. in 11.5% (16/139) and Rickettsia spp. in 6.5% (9/139) of the samples. No co-infections were observed between these bacteria. The most frequent symptoms associated with fever among all groups, were headaches, myalgias, and arthralgias. We found no statistically significant differences in the clinical presentation between patients infected with each bacterium.ConclusionsIn a previous study, we shown the presence of dengue, chikungunya, Zika and oropouche virus. We were able to identify these pathogens in 29.5% of all the samples, with chikungunya and OROV as the most frequently found in 9.4 and 8.6% of all the samples, respectively. In this study we show that B. bacilliformis (21.6%), Leptospira spp. (11.5%) and Rickettsia spp. (6.5%) accounted for the main etiologies of AFI in samples from Puerto Maldonado-Madre de Dios, Perú. Our analysis of their clinical presentation, further shows the importance of implementing more sensitive and specific on-site diagnostic tools in the national surveillance programs.This study confirms that the un-specificity of signs and symptoms is not only associated with arboviral infections, but also with the clinical presentation of endemic bacterial infections.

Immunoinformatics Approach for Epitope-Based Peptide Vaccine Design and Active Site Prediction against Polyprotein of Emerging Oropouche Virus.

Oropouche virus (OROV) is an emerging pathogen which causes Oropouche fever and meningitis in humans. Several outbreaks of OROV in South America, especially in Brazil, have changed its status as an emerging disease, but no vaccine or specific drug target is available yet. Our approach was to identify the epitope-based vaccine candidates as well as the ligand-binding pockets through the use of immunoinformatics. In this report, we identified both T-cell and B-cell epitopes of the most antigenic OROV polyprotein with the potential to induce both humoral and cell-mediated immunity. Eighteen highly antigenic and immunogenic CD8+ T-cell epitopes were identified, including three 100% conserved epitopes (TSSWGCEEY, CSMCGLIHY, and LAIDTGCLY) as the potential vaccine candidates. The selected epitopes showed 95.77% coverage for the mixed Brazilian population. The docking simulation ensured the binding interaction with high affinity. A total of five highly conserved and nontoxic linear B-cell epitopes “NQKIDLSQL,” “HPLSTSQIGDRC,” “SHCNLEFTAITADKIMSL,” “PEKIPAKEGWLTFSKEHTSSW,” and “HHYKPTKNLPHVVPRYH” were selected as potential vaccine candidates. The predicted eight conformational B-cell epitopes represent the accessibility for the entered virus. In the posttherapeutic strategy, ten ligand-binding pockets were identified for effective inhibitor design against emerging OROV infection. Collectively, this research provides novel candidates for epitope-based peptide vaccine design against OROV.

First outbreak of Oropouche Fever reported in a non-endemic central-western region of the Peruvian Amazon. Molecular diagnosis and clinical characteristics

Background: Oropouche virus (OROV) is an underreported and emerging infectious disease. Its incidence is underestimated mainly due to clinical similarities with other diseases that are also caused by arboviruses present in endemic areas. We report the first outbreak of OROV in the western region of the Peruvian Amazon in the department of Huanuco, Peru. Methods & Materials: Transversal studie about a outbreak occurred in the region of Huanuco, Peru during July of 2016. Blood samples were taken from 268 patients who presented acute febrile syndrome to be later analyzed for Oropouche Virus via Polymerase Chain Reaction. Results: Of all 268 patients, 46 (17%) cases tested were positive for OROV. the most common symptom reported was headaches with a frequency of 87% (n = 40) followed by myalgias with 76% (n = 35), arthralgias with 65.2% (n = 30), retro-ocular pain 60.8% (n = 28) and hyporexia with 50% (n = 23). Concerning signs and symptoms that may suggest severe OROV infection, 4.3% (n = 2) had low platelet count, 8.6% (n = 4) had intense abdominal pain, and only 2.1% (n = 1) had a presentation with thoracic pain. Conclusion: The disease caused by the OROV is an emerging, underdiagnosed infection that requires ongoing research to determine its virulence, pathogenesis, host range and vectors involved in the urban and sylvatic cycles as well as identifying new genotypes to implement diagnostic tools with appropriate sensitivity, that can be applied to endemic regions. This study reports an outbreak of OROV in a region where it was not previously identified. Even though there have not been any mortalities reported since the initial identification of OROV, recent outbreaks demonstrate a pattern that highlights the emerging potential of the virus and shows the need to prioritize local epidemiological surveillance and the implementation of new public health policies to decrease the health implications that future outbreaks may cause in the population.

Emergent viruses in America: The case of Oropouche virus

Emergent viruses in America: The case of Oropouche virus

MicroRNA and cellular targets profiling reveal miR-217 and miR-576-3p as proviral factors during Oropouche infection.

Oropouche Virus is the etiological agent of an arbovirus febrile disease that affects thousands of people and is widespread throughout Central and South American countries. Although isolated in 1950’s, still there is scarce information regarding the virus biology and its prevalence is likely underestimated. In order to identify and elucidate interactions with host cells factors and increase the understanding about the Oropouche Virus biology, we performed microRNA (miRNA) and target genes screening in human hepatocarcinoma cell line HuH-7. Cellular miRNAs are short non-coding RNAs that regulates gene expression post-transcriptionally and play key roles in several steps of viral infections. The large scale RT-qPCR based screening found 13 differentially expressed miRNAs in Oropouche infected cells. Further validation confirmed that miR-217 and miR-576-3p were 5.5 fold up-regulated at early stages of virus infection (6 hours post-infection). Using bioinformatics and pathway enrichment analysis, we predicted the cellular targets genes for miR-217 and miR-576-3p. Differential expression analysis of RNA from 95 selected targets revealed genes involved in innate immunity modulation, viral release and neurological disorder outcomes. Further analysis revealed the gene of decapping protein 2 (DCP2), a previous known restriction factor for bunyaviruses transcription, as a miR-217 candidate target that is progressively down-regulated during Oropouche infection. Our analysis also showed that activators genes involved in innate immune response through IFN-β pathway, as STING (Stimulator of Interferon Genes) and TRAF3 (TNF-Receptor Associated Factor 3), were down-regulated as the infection progress. Inhibition of miR-217 or miR-576-3p restricts OROV replication, decreasing viral RNA (up to 8.3 fold) and virus titer (3 fold). Finally, we showed that virus escape IFN-β mediated immune response increasing the levels of cellular miR-576-3p resulting in a decreasing of its partners STING and TRAF3. We concluded stating that the present study, the first for a Peribunyaviridae member, gives insights in its prospective pathways that could help to understand virus biology, interactions with host cells and pathogenesis, suggesting that the virus escapes the antiviral cellular pathways increasing the expression of cognates miRNAs.

Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro

Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV-rNP). OROV-rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV-rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV-rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV-rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses.

Oropouche Fever: A Review.

Oropouche fever is an emerging zoonotic disease caused by Oropouche virus (OROV), an arthropod transmitted Orthobunyavirus circulating in South and Central America. During the last 60 years, more than 30 epidemics and over half a million clinical cases attributed to OROV infection have been reported in Brazil, Peru, Panama, Trinidad and Tobago. OROV fever is considered the second most frequent arboviral febrile disease in Brazil after dengue fever. OROV is transmitted through both urban and sylvatic transmission cycles, with the primary vector in the urban cycle being the anthropophilic biting midge Culicoides paraensis. Currently, there is no evidence of direct human-to-human OROV transmission. OROV fever is usually either undiagnosed due to its mild, self-limited manifestations or misdiagnosed because its clinical characteristics are similar to dengue, chikungunya, Zika and yellow fever, including malaria as well. At present, there is no specific antiviral treatment, and in the absence of a vaccine for effective prophylaxis of human populations in endemic areas, the disease prevention relies solely on vector control strategies and personal protection measures. OROV fever is considered to have the potential to spread across the American continent and under favorable climatic conditions may expand its geographic distribution to other continents. In view of OROV’s emergence, increased interest for formerly neglected tropical diseases and within the One Health concept, the existing knowledge and gaps of knowledge on OROV fever are reviewed.

Diagnosis of Zika Virus Infection by Peptide Array and Enzyme-Linked Immunosorbent Assay.

ABSTRACTZika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection).

Human Orthobunyavirus Infections, Tefé, Amazonas, Brazil.

Introduction:Several orthobunyaviruses are important arthropod-borne pathogens, responsible for a variety of diseases in humans, from acute febrile illness to encephalitis.Methods:We collected serum samples from a series of dengue suspected cases in Tefé, a mid-size city located in the interior of the Amazonas state, Brazil. Viral RNA extraction was performed, and specimens were tested for dengue virus using RT-PCR. Thirty dengue negative samples were further tested for Mayaro virus (MAYV) and Oropouche virus (OROV) using an RT-qPCR protocol previously described. Positive samples were characterized by MegaBLAST analysis over the entire nucleotide collection of the main public databases, and also by maximum likelihood phylogenetic reconstruction of the S genome segment.Results:We detected nine OROV or OROV-like positive cases among 30 patients reporting fever and headache, as the most common symptoms. The closest nucleotide sequence returned from the MegaBLAST analysis belongs to an OROV isolated in Peru 2008. Moreover, all Tefé samples grouped in the same clade with the OROV reference sequence and other closely-related OROV-like viruses.Discussion:Dengue viruses are still the most important arbovirus worldwide, causing hundreds of millions of infections every year. Nonetheless, other arboviruses like chikungunya virus, Zika virus, and yellow fever virus have emerged in the last few years and are now a public health concern in several countries. OROV is believed to have caused more than 500,000 febrile infections in Brazil over recent decades. Therefore, the results described in this study strengthen that this arbovirus, and its closely-related recombinants, should be under continuous surveillance, at least in the endemic countries of Latin America.

Oropouche fever, an emergent disease from the Americas

Oropouche virus is the aetiological agent of Oropouche fever, a zoonotic disease mainly transmitted by midges of the species Culicoides paraensis. Although the virus was discovered in 1955, more attention has been given recently to both the virus and the disease due to outbreaks of Oropouche fever in different areas of Brazil and Peru. Serological studies in human and wild mammals have also found Oropouche virus in Argentina, Bolivia, Colombia, and Ecuador. Several mammals act as reservoirs of the disease, although the sylvatic cycle of Oropouche virus remains to be assessed properly. Oropouche fever lacks key symptoms to be differentiated from other arboviral febrile illnesses from the Americas. Sporadic cases of aseptic meningitis have also been described with good prognosis. Habitat loss can increase the likelihood of Oropouche virus emergence in the short-term in South America.

Emerging and reemerging arboviruses: A new threat in Eastern Peru.

BackgroundArboviral diseases are one of the most common causes of acute febrile illness (AFI) and a significant health problem in South America. In Peru, laboratory etiologic identification of these infections occurs in less than 50% of cases, leading to underdiagnoses of important emerging arboviruses.AimTo assess the prevalence of the Dengue (DENV), Oropouche (OROV), Chikungunya (CHIKV), Mayaro (MAYV) and Zika (ZIKV) viruses in patients with acute febrile illness from Puerto Maldonado (Peru).MethodologySerum samples were obtained from patients with AFI during January 2016 to March 2016. A total of 139 specimens were analyzed for the presence of DENV, OROV, CHIKV, MAYV, and ZIKV using polymerase chain reaction (PCR).ResultsCHIKV in 9.4% and OROV in 8.6% were the most prevalent arboviruses, followed by DENV and ZIKV, with a prevalence of 6.5% and 5%, respectively. Among all patients, the most common symptoms accompanying fever were headaches 79.9%, muscle pain 65.5% and joint pain 63.3%.ConclusionsDuring this short 3-month period, 4 arboviruses were detected by PCR, CHIKV and OROV being the most common arboviruses in Puerto Maldonado (Peru). Thus, it is crucial to include OROV detection in the national health surveillance. Furthermore, the etiologic clinical diagnosis of arboviral infections is not possible due to the low specificity of symptoms; therefore an increase of cases confirmed by molecular diagnostic methods will enhance arboviral surveillance in Peru.

Neutralizing antibodies for orthobunyaviruses in Pantanal, Brazil.

The Pantanal is a hotspot for arbovirus studies in South America. Various medically important flaviviruses and alphaviruses have been reported in domestic and wild animals in the region. To expand the knowledge of local arbovirus circulation, a serosurvey for 14 Brazilian orthobunyaviruses was conducted with equines, sheep and free-ranging caimans. Sera were tested for specific viral antibodies using plaque-reduction neutralization test (PRNT). Monotypic reactions were detected for Maguari, Xingu, Apeu, Guaroa, Murutucu, Oriboca, Oropouche and Nepuyo viruses. Despite the low titers for most of the orthobunyaviruses tested, the detection of monotypic reactions for eight orthobunyaviruses suggests the Pantanal as a region of great orthobunyavirus diversity. The present data, in conjunction with previous studies that detected a high diversity of other arboviruses, ratify the Pantanal as an important natural reservoir for sylvatic and medically important arboviruses in Brazil.

Sera of Peruvians with fever of unknown origins include viral nucleic acids from non-vertebrate hosts.

Serum samples collected from 88 Peruvians with unexplained fever were analyzed for viral sequences using metagenomics. Nucleic acids of anelloviruses, pegivirus A (GBV-C), HIV, Dengue virus, and Oropouche virus were detected. We also characterized from two sera the RNA genomes of new species of partitivirus and dicistrovirus belonging to viral families known to infect fungi or arthropod, respectively. Genomic DNA of a putative fungal cellularhost could be PCR amplified from the partitivirus-containing serum sample. The detection in human serum of nucleic acids from viral families not known to infect vertebrates may indicate contamination during sample collection and aliquoting or human infection by their presumed cellular host, here a fungus. The role, if any, of the non-vertebrate infecting viruses detected in serum in inducing fever is unknown.

The Potential for Reassortment between Oropouche and Schmallenberg Orthobunyaviruses.

A number of viruses within the Peribunyaviridae family are naturally occurring reassortants, a common phenomenon for segmented viruses. Using a minigenome-reporter and virus-like particle (VLP) production assay, we have accessed the potential of Oropouche virus (OROV), Schmallenberg virus (SBV), and other orthobunyaviruses within the Simbu serogroup to reassort. We found that the untranslated region (UTR) in the medium segment is a potential contributing factor for reassortment by the tested viruses. We demonstrate that for promoter activity to occur it was essential that the viral RNA polymerase (L) and nucleocapsid (N) proteins were from the same virus, reinforcing the hypothesis that the large and small segments that encode these proteins segregate together during genome reassortment. Our results indicate that, given the right epidemiological setting, reassortment between SBV and OROV would potentially be feasible and could contribute to the emergence of a new Simbu virus.

Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses.

ABSTRACTWe describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed.

Sensitivity to BST-2 restriction correlates with Orthobunyavirus host range

Orthobunyaviruses include several recently emerging viruses of significant medical and veterinary importance. There is currently very limited understanding on what determines the host species range of these pathogens. In this study we discovered that BST-2/tetherin restricts orthobunyavirus replication in a host-specific manner. We show that viruses with human tropism (Oropouche virus and La Crosse virus) are restricted by sheep BST-2 but not by the human orthologue, while viruses with ruminant tropism (Schmallenberg virus and others) are restricted by human BST-2 but not by the sheep orthologue. We also show that BST-2 blocks orthobunyaviruses replication by reducing the amount of envelope glycoprotein into viral particles egressing from infected cells. This is the first study identifying a restriction factor that correlates with species susceptibility to orthobunyavirus infection. This work provides insight to help us dissect the adaptive changes that bunyaviruses require to cross the species barrier and emerge into new species.

Oropouche virus: poised for headlines?

Oropouche virus (OROV) is a midge-borne human pathogen that causes periodic outbreaks of a flulike illness predominately in the Northern parts of Brazil. However, despite its significant public health importance in Central and South America the virus remains rather obscure to the rest of the world.

Vegetation loss and the 2016 Oropouche fever outbreak in Peru.

BACKGROUND Oropouche virus causes Oropouche fever, an arboviral disease transmitted mainly by midges of the genus Culicoides and Culex mosquitoes. Clinical presentation of Oropouche fever in humans includes fever, headache, rash, myalgia, and in rare cases spontaneous bleeding and aseptic meningitis. Landscape change has been proposed as a driver of Oropouche fever emergence.OBJECTIVE To investigate the landscape epidemiology of the Oropouche fever outbreak that began in April 2016 in Cusco, Peru.METHODS We used information of vegetation and multivariate spatial analyses including ecological niche modeling. Vegetation was characterised using16-day composite enhanced vegetation index (EVI) images at 500 m spatial resolution from the MODIS sensor carried by the Terra satellite.FINDINGS Cases were distributed across seven Peruvian districts in two provinces. La Concepcion was the province with most of the affected districts. EVI time series across 2000 to 2016 suggested a decline in the vegetation in sites with Oropouche fever cases before the epidemic. Our ecological niche modeling suggests that other areas in Junin, Apurimac, and Madre de Dios departments are at risk of Oropouche fever occurrence.MAIN CONCLUSIONS Our results may provide a guide for future fieldwork to test hypotheses regarding Oropouche fever emergence and habitat loss in tropical Latin America.