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Hantavirus Pulmonary Syndrome is an, often fatal, emerging zoonotic disease in the Americas caused by hantaviruses (family: Hantaviridae). In Brazil, hantavirus routine diagnosis is based on serology (IgM-ELISA) while RT-PCR is often used to confirm acute infection. A Semi-nested RT-PCR and an internally controlled RT-qPCR assays were developed for detection and quantification of four hantaviruses strains circulating in the Brazilian Amazon: Anajatuba (ANAJV) and Castelo dos Sonhos (CASV) strains of Andes virus (ANDV) species; and Rio Mamoré (RIOMV) and Laguna Negra (LNV) strains of LNV species. A consensus region in the N gene of these hantaviruses was used to design the primer sets and a hydrolysis probe. In vitro transcribed RNA was diluted in standards with known concentration. MS2 bacteriophage RNA was detected together with hantavirus RNA as an exogenous control in a duplex reaction. RT-qPCR efficiency was around 100% and the limit of detection was 0.9 copies/μL of RNA for RT-qPCR and 10 copies/μL of RNA for Semi-nested RT-PCR. There was no amplification of either negative samples or samples positive to other pathogens. To assess the protocol for clinical sensitivity, specificity and general accuracy values, both assays were used to test two groups of samples: one comprising patients with disease (n = 50) and other containing samples from healthy individuals (n = 50), according to IgM-ELISA results. A third group of samples (n = 27) infected with other pathogens were tested for specificity analysis. RT-qPCR was more sensitive than semi-nested RT-PCR, being able to detect three samples undetected by conventional RT-PCR. RT-qPCR clinical sensitivity, specificity and general accuracy values were 92.5%, 100% and 97.63%, respectively. Thus, the assays developed in this study were able to detect the four Brazilian Amazon hantaviruses with good specificity and sensitivity, and may become powerful tools in diagnostic, surveillance and research applications of these and possibly other hantaviruses.

Background There are several groups of viruses including Insect Specific Viruses (ISV) such as the taxon Negevirus , a group of viruses phylogenetically related to plant viruses. Negeviruses replicate in mosquito cells, but not in vertebrate cells. Methods Pools of hematophagous arthropods were inoculated in Vero and C6/36 cells. The cells were observed to detect possible cytopathic effect. Then, indirect immunofluorescence, RT-PCR, and nucleotide sequencing were performed. Results Seven samples which presented negative results for flaviviruses, alphaviruses and bunyaviruses, but showed cytopathic effect in C6/36 cells were sequenced. We identified the occurrence of a variety of ISVs, most of them belonging to the taxon Negevirus : The Brejeira, Negev, Cordoba and Wallerfield viruses, including a new virus for science, tentatively named Feitosa virus. Conclusions We detected negeviruses in the Amazon region, including two viruses that were isolated for the first time in Brazil: Cordoba virus and the Negev virus and, a new virus for science: the Feitosa virus.

A sero-epidemiological survey was undertaken to detect the circulation of arboviruses in free-living non-human primates. Blood samples were obtained from 16 non-human primates (13 Sapajus spp. and three Alouatta caraya) that were captured using terrestrial traps and anesthetic darts in woodland regions in the municipalities of Campo Grande, Aquidauana, Jardim, Miranda and Corumbá in the State of Mato Grosso do Sul, Brazil. The samples were sent to the Instituto Evandro Chagas (IEC) in Ananindeua, Pará, Brazil, to detect antibodies against 19 species of arboviruses using a hemagglutination inhibition test (HI). Of the 16 primates investigated in the present study, five (31.2%) were serologically positive for an arbovirus. Of these five, two (12.5%) exhibited antibodies to the Flavivirus genus, one (6.2%) exhibited a monotypic reaction to Cacipacoré virus, one (6.2%) was associated with Mayaro virus, and one (6.2%) was positive for Oropouche virus. Based on the positive serology observed in the present study, it was possible to conclude that arboviruses circulate among free-living primates. The viruses in the areas studied might have been introduced by infected humans or by primates from endemic or enzootic areas. Studies of this nature, as well as efficient and continuous surveillance programs, are needed to monitor viral activities in endemic and enzootic regions.

Neurological symptoms of approximately two thirds of patients with Guillain-Barré syndrome (GBS) begin a few weeks after an apparently benign febrile infection with respiratory or gastrointestinal manifestations1. Using serological methods, several studies identified as major infectious agents involved in GBS: Campylobacter jejuni, Mycoplasma pneumoniae, Cytomegalovirus, Epstein-Barr virus and Haemophilus influenzae2-5, but some flaviviruses with a documented circulation in Brazil were identified as triggers of GBS in the last two decades, especially dengue virus (DENV), West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV)4,6-9.

Background In December 2013, an outbreak of Chikungunya virus (CHIKV) caused by the Asian genotype was notified in the Caribbean. The outbreak has since spread to 38 regions in the Americas. By September 2014, the first autochthonous CHIKV infections were confirmed in Oiapoque, North Brazil, and in Feira de Santana, Northeast Brazil. Methods We compiled epidemiological and clinical data on suspected CHIKV cases in Brazil and polymerase-chain-reaction-based diagnostic was conducted on 68 serum samples from patients with symptom onset between April and September 2014. Two imported and four autochthonous cases were selected for virus propagation, RNA isolation, full-length genome sequencing, and phylogenetic analysis. We then followed CDC/PAHO guidelines to estimate the risk of establishment of CHIKV in Brazilian municipalities. Results We detected 41 CHIKV importations and 27 autochthonous cases in Brazil. Epidemiological and phylogenetic analyses indicated local transmission of the Asian CHIKV genotype in Oiapoque. Unexpectedly, we also discovered that the ECSA genotype is circulating in Feira de Santana. The presumed index case of the ECSA genotype was an individual who had recently returned from Angola and developed symptoms in Feira de Santana. We estimate that, if CHIKV becomes established in Brazil, transmission could occur in 94% of municipalities in the country and provide maps of the risk of importation of each strain of CHIKV in Brazil. Conclusions The etiological strains associated with the early-phase CHIKV outbreaks in Brazil belong to the Asian and ECSA genotypes. Continued surveillance and vector mitigation strategies are needed to reduce the future public health impact of CHIKV in the Americas.

In order to investigate the pathogenicity of the virus strain GOI 4191 that was isolated from a fatal adverse event after yellow fever virus (YFV) vaccination, an experimental assay using hamsters (Mesocricetus auratus) as animal model and YFV 17DD vaccine strain as virus reference was accomplished. The two virus strains were inoculated by intracerebral, intrahepatic and subcutaneous routes. The levels of viremia, antibody response, and aminotransferases were determined in sera; while virus, antigen and histopathological changes were determined in the viscera. No viremia was detected for either strain following infection; the immune response was demonstrated to be more effective to strain GOI 4191; and no significant aminotransferase levels alterations were detected. Strain GOI 4191 was recovered only from the brain of animals inoculated by the IC route. Viral antigens were detected in liver and brain by immunohistochemical assay. Histothological changes in the viscera were characterized by inflammatory infiltrate, hepatocellular necrosis, and viral encephalitis. Histological alterations and detection of viral antigen were observed in the liver of animals inoculated by the intrahepatic route. These findings were similar for both strains used in the experiment; however, significant differences were observed from those results previously reported for wild type YFV strains. Visando investigar a possível patogenicidade do vírus isolado (GOI 4191) de um evento adverso fatal pela vacinação antiamarílica, realizou-se um ensaio experimental em Syrian hamsters (Mesocricetus auratus), usando-se a cepa vacinal 17DD como parâmetro. As amostras virais foram inoculadas por via intracerebral, intra-hepática e subcutânea. Nos soros foram determinados níveis de viremia, resposta imune e aminotransferases, e nas vísceras a presença de vírus, antígeno e lesões teciduais. Não se detectou viremia para as duas amostras, a resposta imune foi maior para GOI 4191, e as aminotransferases não apresentaram alterações compatíveis com danos hepáticos. Nos animais inoculados por via intracerebral o vírus foi recuperado somente a partir do cérebro, sendo o antígeno viral detectado, por imuno-histoquímica, no cérebro e fígado. Infiltrado inflamatório e corpúsculos acidófilos foram observados no fígado e lesões tipo encefalite viral no sistema nervoso central. Alterações histológicas e antígeno viral foram observados, também, no fígado dos animais infectados por via intra-hepática, e ausentes naqueles inoculados por via subcutânea. Os resultados foram similares para as duas amostras testadas, entretanto distintos daqueles relatados na literatura para cepas silvestres do vírus amarílico.

Em amostra da população da cidade de Rio Branco (Acre), foi pesquisada a freqüência de portadores de anticorpos contra os arbovírus, mais prevalentes na região amazônica, e o vírus vacinal da febre amarela, antes e após a imunização com a vacina 17D. Das 390 pessoas incluídas na primeira fase do estudo (agosto de 1999), somente 190 compareceram em janeiro de 2000, três meses após a aplicação da vacina 17D (outubro de 1999). Nas amostras da primeira fase, as freqüências de soropositivos (IH) para os vírus estudados foram: 17D (27,2%); Dengue-1 (0,3%); Dengue-2 (4,1%); Dengue-3 (0%); Dengue-4 (0%), entre outros 8 vírus. Nas amostras séricas de janeiro (2000), a soroconversão para o 17D foi de 89,7% (130/145) e 3,2% (6/190) passaram a ter anticorpos contra o sorotipo 3 (DEN-3). Em conclusão, por conta da elevada taxa de cobertura vacinal e de soroconversão há redução significativa do risco de urbanização do vírus da febre amarela na cidade de Rio Branco, apesar de não ser desprezível a possibilidade de uma nova epidemia de dengue, pelo DEN-3, a semelhança da registrada em 2000 e 2001 pelos sorotipos 1 e 2. During a yellow fever vaccination campaign among residents of Rio Branco (Acre State), the frequency of HI antibodies to the most prevalent arboviruses in the Amazon region and to yellow fever virus was determined before and three months after immunization with YF 17D vaccine. From 390 inhabitants included in the first phase of serologic survey (August 1999), only 190 provided a second serum sample, after the use of 17D vaccine (January 2000). Among first phase samples, the frequency of HI antibodies was: 17D (27.2%); Ilheus (5.9%); Mayaro (5.4%); Caraparu (4.9%); Dengue-2 (4.1%); Oropouche (2.3%); and Dengue-1 (0.3%). In the second study phase, the serologic conversion to YF reached 89.7% among previously negative persons. Serologic conversions were also observed to Ilheus (6.2%); Dengue-3 (3.2%); Mayaro (1.1%); and Oropouche (1.1%) viruses. In conclusion, considering the high YF antibody rate after vaccination, the risk of urban yellow fever seems insignificant, although the lower prevalence of HI antibodies to dengue viruses, is of concern and inhabitants are under high risk of dengue outbreaks, especially to DEN-3 recently introduced in Brazil, as was observed in 2000 and 2001 with DEN-1 and DEN-2.

Viruses with encephalitogenic potential can cause neurological conditions of clinical and epidemiological importance, such as Saint Louis encephalitis virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Dengue virus, Zika virus, Chikungunya virus, Mayaro virus and West Nile virus. The objective of the present study was to determine the number of arboviruses with neuroinvasive potential isolated in Brazil that corresponds to the collection of viral samples belonging to the Department of Arbovirology and Hemorrhagic Fevers, Evandro Chagas Institute (SAARB/IEC) of the Laboratory Network of National Reference for Arbovirus Diagnosis from 1954 to 2022. In the analyzed period, a total of 1,347 arbovirus samples with encephalitogenic potential were isolated from mice; 5,065 human samples were isolated exclusively by cell culture; and 676 viruses were isolated from mosquitoes. The emergence of new arboviruses may be responsible for diseases still unknown to humans, making the Amazon region a hotspot for infectious diseases due to its fauna and flora species characteristics. The detection of circulating arboviruses with the potential to cause neuroinvasive diseases is constant, which justifies the continuation of active epidemiological surveillance work that offers adequate support to the public health system regarding the virological diagnosis of circulating arboviruses in Brazil.

De junho a dezembro de 1999, foram coletadas 785 amostras de soro de pacientes com suspeita clínica de dengue e/ou febre amarela. Os pacientes foram atendidos nas unidades de saúde distribuídas pelas seis mesorregiões do Estado do Pará, Brasil. As amostras de soro foram testadas pelo método de inibição da hemaglutinação para detecção de anticorpos para Flavivirus e pelo ensaio imunoenzimático para detecção de imunoglobulina M para dengue e febre amarela. Das amostras coletadas, 563 (71,7%) foram positivas pelo IH, e dentre estas 150 (26,6%) foram positivas pelo ELISA-IgM. O vírus dengue foi responsável pela maioria das infecções recentes em todas as mesorregiões e os casos de febre amarela detectados neste estudo foram restritos às mesorregiões Marajó e Sudeste.

C-type lectin DC-SIGN receptor, encoded by CD209, plays a key role in the infection of dendritic cells by dengue virus (DENV). Because the -336A/G SNP (rs4804803) polymorphism in the promoter of CD209 modulates DC-SIGN expression, we investigated the putative association of this polymorphism with DENV infection and its pathogenesis. A control sample of 72 individuals, rigorously selected through a clinical investigation for absence of past dengue fever (DF) was compared to a sample of 168 patients (156 classical DF; 12 dengue hemorrhagic fever), all residents from Pará, Brazil. However, the prevalence of symptoms showed a trend higher in the AA genotype (Wilcoxon test; Z=2.02; p=0.04). Hence, our findings indicate that the G allele downregulates the spectrum of symptoms during the early acute phase of DENV infection, putatively decreasing the viremia, as suggested in the literature.