Serological and Molecular Evidence of the Circulation of the Venezuelan Equine Encephalitis Virus Subtype IIIA in Humans, Wild Vertebrates and Mosquitos in the Brazilian Amazon.

Understanding the interaction between viruses and ecosystems in areas with or without anthropic interference can contribute to the organization of public health services, as well as prevention and disease control. An arbovirus survey was conducted at Caxiuanã National Forest, Pará, Brazil, where 632 local residents, 338 vertebrates and 15,774 pools of hematophagous arthropods were investigated. Neutralization antibodies of the Venezuelan Equine Encephalitis virus, subtype IIIA, Mucambo virus (MUCV) were detected in 57.3% and 61.5% of humans and wild vertebrates, respectively; in addition, genomic fragments of MUCV were detected in pool of Uranotaenia (Ura.) geometrica. The obtained data suggest an enzootic circulation of MUCV in the area. Understanding the circulation of endemic and neglected arboviruses, such as MUCV, represents an important health problem for the local residents and for the people living in the nearby urban centers.

Emergence of new insect-restrictive viruses in the Amazon region.

The complete genome was determined for 12 viruses isolated from 8 different pools of mosquitoes (Culex sp. and Psorophora ferox) collected at Brejeira farm, Canaan dos Carajas, Para state in northern Brazil. Eight of the viruses were distantly related to Piura virus, hereafter designated as Brejeira virus; the other 4 were similar to Wallerfield virus.

Characterization of the Candiru antigenic complex (Bunyaviridae: Phlebovirus), a highly diverse and reassorting group of viruses affecting humans in tropical America.

The genus Phlebovirus of the family Bunyaviridae consists of approximately 70 named viruses, currently assigned to nine serocomplexes (species) based on antigenic similarities. Sixteen other named viruses that show little serologic relationship to the nine recognized groups are also classified as tentative species in the genus. In an effort to develop a more precise classification system for phleboviruses, we are attempting to sequence most of the named viruses in the genus with the goal of clarifying their phylogenetic relationships. In this report, we describe the serologic and phylogenetic relationships of 13 viruses that were found to be members of the Candiru serocomplex; 6 of them cause disease in humans. Analysis of full genome sequences revealed branching inconsistencies that suggest five reassortment events, all involving the M segment, and thus appear to be natural reassortants. This high rate of reassortment illustrates the inaccuracy of a classification system based solely on antigenic relationships.

Evaluation of an immunoglobulin M-specific capture enzyme-linked immunosorbent assay for rapid diagnosis of dengue infection.

Various assays have been developed to diagnose dengue virus infection, relying on techniques from the fields of serology and molecular biology. Many of these assays have been successful, but there is still an urgent need for accurate, simple and rapid diagnostic assays to diagnose dengue virus infection and to assist in patient management. Using a panel of well-characterized sera and a collection of retrospective samples obtained during the dengue epidemics that occurred in Belém, Brazil, between 2002 and 2009, a modified immunoglobulin M-specific capture enzyme-linked immunosorbent assay (Rapid-MAC-ELISA) was evaluated and compared with the "gold standard" MAC-ELISA, in order to assess the specificity, sensitivity, stability, reproducibility and cost-effectiveness of this new assay. These results demonstrated that the Rapid-MAC-ELISA is comparable to the MAC-ELISA in terms of sensitivity and specificity and is highly reproducible; additionally, it is easily performed, less expensive than other available formats and can be completed within three hours. Furthermore, the Rapid-MAC-ELISA can be used for the diagnosis of dengue virus infections in resource-limited areas where dengue is endemic.

Yellow fever virus in Haemagogus leucocelaenus and Aedes serratus mosquitoes, southern Brazil, 2008.

Yellow fever virus (YFV) was isolated from Haemagogus leucocelaenus mosquitoes during an epizootic in 2001 in the Rio Grande do Sul State in southern Brazil. In October 2008, a yellow fever outbreak was reported there, with nonhuman primate deaths and human cases. This latter outbreak led to intensification of surveillance measures for early detection of YFV and support for vaccination programs. We report entomologic surveillance in 2 municipalities that recorded nonhuman primate deaths. Mosquitoes were collected at ground level, identified, and processed for virus isolation and molecular analyses. Eight YFV strains were isolated (7 from pools of Hg. leucocelaenus mosquitoes and another from Aedes serratus mosquitoes); 6 were sequenced, and they grouped in the YFV South American genotype I. The results confirmed the role of Hg. leucocelaenus mosquitoes as the main YFV vector in southern Brazil and suggest that Ae. serratus mosquitoes may have a potential role as a secondary vector.

Mayaro fever virus, Brazilian Amazon.

In February 2008, a Mayaro fever virus (MAYV) outbreak occurred in a settlement in Santa Barbara municipality, northern Brazil. Patients had rash, fever, and severe arthralgia lasting up to 7 days. Immunoglobulin M against MAYV was detected by ELISA in 36 persons; 3 MAYV isolates sequenced were characterized as genotype D.

Oropouche fever epidemic in Northern Brazil: epidemiology and molecular characterization of isolates.

BACKGROUND: Oropouche fever virus is an important arbovirus associated with febrile disease that re-emerged in 2006 in several municipalities of Pará State, Bragantina region, Amazon, Brazil, 26 years after the last epidemic. OBJECTIVE: To investigate an Oropouche fever outbreak in this region. STUDY DESIGN: A serologic survey and prospective study of acute febrile cases were performed in Magalhães Barata (urban and rural areas) and Maracanã (rural area) municipalities. Serology (IgM-ELISA and hemagglutination-inhibition [HI]), virus isolation, RT-PCR and real-time-PCR were used to confirm Oropouche virus (OROV) as responsible for the febrile outbreaks. RESULTS: Real-time-PCR showed high titers of OROV in acute-phase serum samples from febrile patients. From 113 of 119 acutely febrile patients with paired serum samples, OROV infections was confirmed by serologic conversion (n=76) or high titers (n=37) for both HI and IgM-ELISA. Patients had a febrile disease characterized by headache, chills, dizziness, photophobia, myalgia, nausea, and vomiting. Females and children under 15 years of age were most affected. Nucleotide sequencing of six OROV isolates identified that genotype II was associated with the human disease epidemic. CONCLUSIONS: Oropouche fever, which has re-emerged in the Bragantina region in eastern Amazon 26 years after the last epidemic, is caused by genotype II, a lineage previously found only in Peru and western Brazil.

Genetic characterization of orthobunyavirus Melao, strains BE AR633512 and BE AR8033, and experimental infection in golden hamsters (Mesocricetus auratus).

Melao virus (MELV) strains BE AR8033 and BE AR633512 were isolated from pools of Ochlerotatus scapularis mosquitoes in Belém, Pará State (1955), and Alta Floresta, Rondônia State (2000), Brazil, respectively. The aim of the present study was to molecularly characterize these strains and to describe the histopathological, biochemical and immunological changes in golden hamsters (Mesocricetus auratus) following intraperitoneal injection of MELV strains. Hamsters were susceptible to both of the MELV strains studied. Viraemia was observed 3-6 days post-infection (p.i.) for BE AR633512 and only on the second day p.i. for BE AR8033. Neutralizing antibodies against both strains were detected in blood samples obtained at 5 days p.i. and persisted up to 30 days p.i. Aspartate aminotransferase, alanine aminotransferase and blood urea nitrogen were significantly altered in animals infected with the two MELV strains, while creatinine was only altered in animals inoculated with BE AR633512. Histopathological changes were observed in the central nervous system, liver, kidney and spleen of hamsters, and infection was confirmed by detection of specific MELV antigens by immunohistochemistry. Strain BE AR633512 caused more severe tissue damage than strain BE AR8033, showing increased neurovirulence and pathogenicity. Genetic analysis based on the full-length sequences of the glycoprotein (Gn and Gc) and nucleocapsid protein (N) genes revealed high levels of homology between the MELV strains. Interestingly, the greatest genetic divergence was found for the Gn gene of strain BE AR633512, in which several synonymous and non-synonymous mutations causing changes in RNA secondary structure were observed. Further studies will be necessary to investigate the role of Gn and Gc mutations in the MELV pathogenicity.