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.

Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso, Brazil.

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatus captured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatus pools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso, Brazil

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatuscaptured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatuspools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

Experimental infection of suckling mice by subcutaneous inoculation with Oropouche virus.

Oropouche virus, of the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu, is an important causative agent of arboviral febrile illness in Brazil. An estimated 500,000 cases of Oropouche fever have occurred in Brazil in the last 30 years, with recorded cases also in Panama, Peru, Suriname and Trinidad. We have developed an experimental model of Oropouche virus infection in neonatal BALB/c mouse by subcutaneous inoculation. The vast majority of infected animals developed disease on the 5th day post infection, characterized mainly by lethargy and paralysis, progressing to death within 10 days. Viral replication was documented in brain cells by in situ hybridization, immunohistochemistry and virus titration. Multi-step immunohistochemistry indicated neurons as the main target cells of OROV infection. Histopathology revealed glial reaction and astrocyte activation in the brain and spinal cord, with neuronal apoptosis. Spleen hyperplasia and mild meningitis were also found, without viable virus detected in liver and spleen. This is the first report of an experimental mouse model of OROV infection, with severe involvement of the central nervous system, and should become useful in pathogenesis studies, as well as in preclinical testing of therapeutic interventions for this emerging pathogen.

Reemergence of Oropouche fever, northern Brazil.

Oropouche fever has reemerged in Parauapebas and Porto de Moz municipalities, Pará State, Brazil. Serologic analysis (immunoglobulin M-ELISA) and virus isolation confirmed Oropouche virus (OROV) in both municipalities. Nucleotide sequencing of 2 OROV isolates from each location indicated genotypes I (Parauapebas) and II (Porto de Moz) in Brazil.

Changes in relative species compositions of biting midges (Diptera: Ceratopogonidae) and an outbreak of Oropouche virus in Iquitos, Peru.

Species compositions of Culicoides paraensis (Goeldi) (Diptera: Ceratopogonidae), the major vector of Oropouche virus to humans in Central and South American urban cycles, and Culicoides insinuatus Ortiz & Leon differed along a northeast-to-southwest transect across Iquitos, Department of Loreto, Peru. The relative distributions of the species were consistent with patterns of human outbreaks along the Amazon River. We resumed collection of biting midges between May 2000 and January 2004 at three sites previously sampled (1996 -1997) to determine whether the known vector was expanding its range relative to the earlier survey. C. paraensis did not replace C. insinuatus across the region surveyed. Instead, C. insinuatus dominated the more southern sites and significantly increased its relative proportion at all three sites. Apparently, microhabitat differences and not range expansion by C. paraensis were responsible for differences in species compositions across the sample sites.

Diagnosis of Oropouche virus infection by RT-nested-PCR.

Using the RT-PCR with primers that anneal to the 5' and the 3' extremities of the genome segments of bunyaviruses and internal primers that anneal to the S segment of Simbu serogroup viruses in a nested PCR it was possible to amplify the Oropouche virus (ORO) genome from the sera of three patients. These results show that this RT-nested-PCR is a useful tool for rapid diagnosis of Oropouche fever infections.

Nucleotide sequences and phylogeny of the nucleocapsid gene of Oropouche virus.

The nucleotide sequence of the S RNA segment of the Oropouche (ORO) virus prototype strain TRVL 9760 was determined and found to be 754 nucleotides in length. In the virion-complementary orientation, the RNA contained two overlapping open reading frames of 693 and 273 nucleotides that were predicted to encode proteins of 231 and 91 amino acids, respectively. Subsequently, the nucleotide sequences of the nucleocapsid genes of 27 additional ORO virus strains, representing a 42 year interval and a wide geographical range in South America, were determined. Phylogenetic analyses revealed that all the ORO virus strains formed a monophyletic group that comprised three distinct lineages. Lineage I contained the prototype strain from Trinidad and most of the Brazilian strains, lineage II contained six Peruvian strains isolated between 1992 and 1998, and two strains from western Brazil isolated in 1991, while lineage III comprised four strains isolated in Panama during 1989.

Epidemiology of endemic Oropouche virus transmission in upper Amazonian Peru.

A cross-sectional serosurvey of a rural community near Iquitos, Peru was conducted to determine Oropouche (ORO) virus antibody prevalence and risk factors for human infection. Venous blood samples, and demographic, social, and risk factor data were obtained from people age five years of age and older who lived in the village of Santa Clara on the Nanay River, a tributary of the Amazon River. Sera were tested for ORO viral antibody by an ELISA. The specificity of viral antibody reactivity was determined by a standard plaque-reduction neutralization test. Interview data were analyzed by univariate and multiple logistic regression to determine which variables were statistically associated with previous ORO viral infection, as indicated by the presence of IgG antibody. Final models were evaluated based on log-likelihood and Wald chi-square. Clustering of seropositive residents within houses was analyzed by the method of Walter. Among 1,227 persons sampled, 33.7% (n=414) were positive for ORO viral IgG antibody. Overall, antibody prevalence was similar for males (33.9%) and females (33.6%), and increased significantly with age for both sexes to include more than half of persons more than 25 years of age. The length of residence in the village was positively associated with serologic status; persons who had moved to the village within the past 15 years were less likely to be seropositive than life-long residents of the same age. Antibody prevalence among immigrants who had lived in Santa Clara more than 15 years was similar to that in life-long residents. The activity most predictive of previous ORO viral infection was travel to forest communities and travel to Iquitos. No evidence of spatial heterogeneity in ORO virus antibody distribution was observed. Results suggested that endemic transmission of ORO virus in this region has been ongoing during many decades, and that people are at considerable risk of infection.

Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru.

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.

Oropouche virus transmission in the Amazon River basin of Peru.

Seroepidemiologic studies were conducted to determine the prevalence of Oropouche (ORO) viral antibody, risk factors, and the incidence of infection among residents of the Amazon region of Peru. Blood samples, as well as demographic, cultural, and medical history data, were collected from residents in a sector of the city of Iquitos and in an adjacent rural and three neotropical rain forest communities. Blood specimens were obtained approximately one year later from a cohort of the same study subjects who were negative for ORO antibody on the initial cross-sectional survey. Sera were tested for ORO IgG antibody by an enzyme-linked immunosorbent assay. Antibody prevalences were 35% for residents of the urban population, 24-46% for the forest communities, and 18% for the rural community. Antibody prevalence increased with age, and subjects who were seropositive were significantly (P = 0.001) older (mean = 33 years) than the seronegative subjects (mean = 15 years). Multivariate analysis revealed that only age, urban and forest residence, and occupation as a farmer or housekeeper remained significantly associated with seropositivity. Seroconversion data for the same populations one year later demonstrated evidence of ORO viral infection among 28% of the residents in the rural community and 2% or less in the forest and urban communities. Oropouche virus infection was significantly associated with older age (P = 0.04) in the rural community (P < 0.001). These data support prior evidence of ORO viral infection among residents of Iquitos and surrounding villages and suggest that transmission of this virus occurs continuously in the population of this area of the Amazon basin.

Curso Internacional Enfermedades Virales Emergentes / Internatinal Course Emerging Viral Diseases

Contiene el resumen de las siguientes exposiciones: 1. Conceptos de infecciones emergentes y reemergentes/Robert E. Shope; 2. Red de laboratorios y programa de enfermedades emergentes del Instituto Nacional de Salud/Carlos Carillo; 3. Dengue y dengue hemorrágico en las Américas/Gary G. Clark; 4. Situación del dengue en el Perú/Daniel Neyra; 5. Fiebre amarilla en el Perú y riesgo de urbanización/César Cabezas; 6. Epidemiología y emergencia de la fiebre de oropouche/Francisco Pinheiro; 7. Bunyavirus en la amazonía peruana/Robert Shope; 8. Encefalitis equina venezolana y fiebre mayaro/Francisco Pinheiro; 9. Hepatitis virales B y delta en el Perú/César Cabezas; 10. Enfermedades por arenaviurs en las Américas/Robert B. Tesh; 11. Emergencia de hantavirus en las Américas/Charles Fulhorst; 12. Emergencia de virus transmitidos por artrópodos y roedores en el Perú/Douglas M. Watts; 13. HTLV I y II en el Perú/Eduardo Gotuzzo; 14. Diagnóstico de laboratorio de infecciones por arbovirus y hepatitis/Robert E. Shope; 15. Aplicación de técnicas moleculares en la detección y diagnóstico de infección por arbovirus/Laura J. Chandler; 16. Bioseguridad y riesgos en la manipulación de virus en el laboratorio/Alan Barrett; 17. Investigaciones en brotes/Paul Alfaro; 18. Intervención en brotes de enfermedades emergentes: nuevas vacunas candidatas/Robert E. Shope; 19. Intervención en brotes de enfermedades emergentes: control vectorial/Gary C. Clark

Epidemia de febre do Oropouche em Serra Pelada, município de Curionópolis, Pará, 1994 / Fever epidemic of Oropouche virus in Serra Pelada, municipality of Curionópolis, Pará State, 1994

In the final of November 1994, an outbreak of a febrile disease was observed in the Serra Pelada gold mine (5 degrees 35'S: 49 degrees 30'W) in the Southeast region of Pará State. Twenty samples were collected and sent to the laboratory of Arbovirus of Instituto Evandro Chagas. The tests showed that the disease was caused by Oropouche virus (Bunyaviridae, Bunyavirus, Simbu serological group). Between 8-22 December 296 serum samples were taken (54 from febrile patients, 16 paired samples and 242 from contacts and convalescent patients) of the 73 familiar groups. From febrile patients, ten Oropouche virus strains were obtained. From paired serum, six seroconversions were obtained and 242 other Oropouche infections were diagnosed by HI and MAC ELISA. The clinical-picture of febrile disease accompanied by severe bedache, chills, myalgia, photophobia retrobulbar pain and malaise was observed. Involvement of central nervous system was not observed. Based on the serological data, we estimated that in the outbreak of Serra Pelada around 5,000 cases occurred corresponding to a prevalence of 83 per cent.

[Outbreak of oropouche virus fever in Serra Pelada, municipality of Curionópolis, Pará, 1994]. / Epidemia de febre do Oropouche em Serra Pelada, município de Curionópolis, Pará, 1994.

In the final of November 1994, an outbreak of a febrile disease was observed in the Serra Pelada gold mine (5 degrees 35'S: 49 degrees 30'W) in the Southeast region of Pará State. Twenty samples were collected and sent to the laboratory of Arbovirus of Instituto Evandro Chagas. The tests showed that the disease was caused by Oropouche virus (Bunyaviridae, Bunyavirus, Simbu serological group). Between 8-22 December 296 serum samples were taken (54 from febrile patients, 16 paired samples and 242 from contacts and convalescent patients) of the 73 familiar groups. From febrile patients, ten Oropouche virus strains were obtained. From paired serum, six seroconversions were obtained and 242 other Oropouche infections were diagnosed by HI and MAC ELISA. The clinical-picture of febrile disease accompanied by severe bedache, chills, myalgia, photophobia retrobulbar pain and malaise was observed. Involvement of central nervous system was not observed. Based on the serological data, we estimated that in the outbreak of Serra Pelada around 5,000 cases occurred corresponding to a prevalence of 83%.

Primeiro registro de epidemias causadas pelo vírus Oropouche nos estados do Maranhäo e Goiás, Brasil / 1st register of an epidemic caused by Oropouche virus in the states of Maranhäo and Goiás, Brazil

Os autores descrevem a ocorrência de epidemias causadas pelo vírus Oropouche (Oro) nos Estados dpo Maranhäo (MA) e Goiás (GO) em 1988. 36 amostras de vírus foram obtidas a partir da inoculaçäo do sangue de 120 pacientes em camundongos recém nascidos. A doença foi caracterizada por febre, cefaléia, dores musculares, articulares, fotofobia, dor retro ocular, náuseas e tontura. 128 das 197 pessoas examinadas em Porto Franco, MA, tinham anticorpos inibidores da hemaglutinaçäo (IH) para o agente e, em 106 foram detectados anticorpos IGM por MACELISA. Todos os grupos etários foram infectados, embora a incidência tenha sido mais elevada entre aqueles com 10 a 19 anos de idade. Quanto ao sexo, a infecçäo ocorreu igualmente em ambos os sexos. Recorrência dos sintomas foi observada em 56% dos casos positivos estudados. A inoculaçäo em camundongos Swiss recém nascidos de 3.624 Culicoides paraensis (Ceratopogonidae) e 1.970 Culex (Culex) quinquefasciatus (Culicidae), coletados em Porto Francos-MA, resultou em um único isolamento do vírus ORO a partir dos Culicoides. Essa é a primeira descriçäo de casos confirmados de infecçäo pelo vírus Oropouche nos Estados do Maranhäo e Goiás, Brasil

[1st register of an epidemic caused by Oropouche virus in the states of Maranhão and Goiás, Brazil]. / Primeiro registro de epidemias causadas pelo vírus Oropouche nos estados do Maranhão e Goiás, Brasil.

The authors describe the occurrence of outbreaks caused by Oropouche virus (ORO) in the states of Maranhão and Goiás, Brazil in 1988. 36 strains of the virus were obtained from the intracerebral inoculation of the blood of 120 patients into 2-3 day-old infant mice. The illness was characterized by headache, fever, pain in the muscles, joints and back, photophobia, retrobulbar pain, nausea and dizziness. 128 of 197 people examined in Porto Franco, MA, had hemagglutination-inhibiting antibodies to the agent, while 106 of them had IgM antibodies by MAC ELISA test. All age groups were infected, although the incidence was higher among who had 10 to 19 years old. There was no difference, in relation to sex infections. Recurrence of symptoms was reported in 56% of sick people. Mice inoculated with 3624 Culicoides paraensis (Ceratopogonidae) and 1970 Culex (Cux.) quinquefasciatus (Culicidae) collected in Porto Franco resulted in one single isolation of ORO virus, from the Culicoides. These are the first confirmed cases of ORO infection in Maranhão and Goiás states.

Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture.

A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA) as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380). Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells.