Isolation of Flaviviruses and Alphaviruses with Encephalitogenic Potential Diagnosed by Evandro Chagas Institute (Pará, Brazil) in the Period of 1954-2022: Six Decades of Discoveries.

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.

Negeviruses isolated from mosquitoes in the Brazilian Amazon.

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.

Oropouche orthobunyavirus: Genetic characterization of full-length genomes and development of molecular methods to discriminate natural reassortments.

Oropouche orthobunyavirus (OROV) has significant impact in public health in Amazon region. This arbovirus is one of the most common causes of febrile illness in Brazil, and is responsible for several epidemics since 1960's. In this study, we sequenced and characterized the complete coding sequences (S-, M- and L-RNA) of 35 OROV isolates from Brazil. Here, we classified 20 strains in genotype I from Pará and Maranhão states, nine as genotype II from Pará and Rondônia states confirmed, four classified into genotype III from Acre, Maranhão, Minas Gerais and Rondônia states and two genotype IV from Amazonas State. Also, we did not observe reassortment events involving the OROV isolates. In addition, we developed novel RT-PCR tools to identify reassortment events among OROV strains. These data will be useful to better understand the molecular epidemiology and diagnostic of OROV infections.

Characterization of the Gamboa Virus Serogroup (Orthobunyavirus Genus, Peribunyaviridae Family).

Comprehensive comparative phylogenetic analyses were performed on 17 Gamboa serogroup viruses (GAMSVs) from distinct geographic regions in the Americas and other representative members of the genus Orthobunyavirus (Peribunyaviridae), based on small (S), medium (M), and large (L) open reading frame full-length and partial sequences. Genome characterization showed that the GAMSVs divide into four clades or genotypes. The GAMSVs have a genetic organization similar to other orthobunyaviruses, except that they have a larger NSm protein than other orthobunyaviruses. A serosurvey for Gamboa virus antibodies was performed in plasma from birds, other wild animals, and humans living around the Tucuruí hydroelectric dam in Pará state, northern Brazil, a known focus of GAMSV activity. Newborn chicks (Gallus gallus domesticus) were experimentally infected with a GAMSV, and the pathogenesis is described. Histopathological changes were primarily in the lungs and liver. Also, a review of the ecology of the GAMSVs in the Americas is included. In sum, this study presents the genomic and evolutionary characterization of the Gamboa group and the potential model of pathogenesis, which would be helpful for diagnostic purposes, epidemiology, and immunopathogenesis studies.

New Virus Genome Sequences of the Guama Serogroup (Genus Orthobunyavirus, Family Bunyaviridae), Isolated in the Brazilian Amazon Region.

This is the first announcement of two nearly complete viral genome sequences belonging to the Guama serogroup (genus Orthobunyavirus, family Bunyaviridae) isolated in the Brazilian Amazon region: Mirim virus (MIRV; BEAN7722) and Ananindeua virus (ANUV; BEAN109303).

Guillain-Barré Syndrome After Zika Virus Infection in Brazil.

Zika virus (ZIKV) is an emerging flavivirus, which has caused a widespread outbreak in the Americas. Shortly after its introduction in 2015, a cluster of cases with Guillain-Barré syndrome was detected in Brazil. Herein, we describe two cases from the city of Salvador, who developed ascending paresis after an acute exanthematous illness. The patients were admitted to the intensive care unit with tetraparesis and cranial nerve palsy, which resolved after intravenous administration of human immunoglobulin. Serological evaluation detected IgM-specific ZIKV antibodies. In regions of Zika virus transmission, health-care workers must be aware of the potential severe neurological complications associated with ZIKV infection and be prepared to provide prompt diagnosis, treatment, and supportive care.

Zika virus complete genome from Salvador, Bahia, Brazil.

In May 2015 the first autochthonous Zika virus infection was reported in Brazil. Rapid and urgent measures are needed to contain the ongoing outbreak. Here we report the full-length ZIKV coding sequence from Bahia. Genetic analysis of outbreak sequences will be essential for characterizing the diversity of circulating strains, identifying hotspots of virus transmission and guiding public health control. Rapid and urgent measures are needed to contain the ongoing outbreak.

Zika virus in the Americas: Early epidemiological and genetic findings.

Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

Emergence and potential for spread of Chikungunya virus in Brazil.

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.

Pacui Virus, Rio Preto da Eva Virus, and Tapirape Virus, Three Distinct Viruses within the Family Bunyaviridae.

Nearly complete genome sequences for three ungrouped viruses, Pacui virus (BEAN27326), Rio Preto da Eva virus (BEAR540870), and Tapirape virus (BEAN767592) isolated in the Amazon region are reported here. All three genomic segments (small, medium and large RNA) were recovered and were similar to members of the genus Orthobunyavirus.

Diagnosis of arboviruses using indirect sandwich IgG ELISA in horses from the Brazilian Amazon.

BACKGROUND: The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family. FINDINGS: Sensitivity varied between 40.85 and 100%; the specificity was low and ranged from 39.71 to 67.0%; and the accuracy varied between 41 and 65.2%. The test developed in this study yielded a large number of serological cross-reactions. CONCLUSIONS: The test can be employed to detect IgG antibodies within one arbovirus family; however, the hemagglutination test or other more specific techniques, such as the serum neutralization test in mice or the plaque-reduction neutralization test, are essential complementary methods for positive cases.

Polymorphism of DC-SIGN (CD209) promoter in association with clinical symptoms of dengue fever.

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.

Diagnosis of arboviruses using indirect sandwich IgG ELISA in horses from the Brazilian Amazon

The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family.

Diagnosis of arboviruses using indirect sandwich IgG ELISA in horses from the Brazilian Amazon

The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family.

Detection of arboviruses of public health interest in free-living New World primates (Sapajus spp.; Alouatta caraya) captured in Mato Grosso do Sul, Brazil

Introduction A sero-epidemiological survey was undertaken to detect the circulation of arboviruses in free-living non-human primates. Methods 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). Results 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. Conclusions 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. .

Detection of arboviruses of public health interest in free-living New World primates (Sapajus spp.; Alouatta caraya) captured in Mato Grosso do Sul, Brazil.

INTRODUCTION: A sero-epidemiological survey was undertaken to detect the circulation of arboviruses in free-living non-human primates. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Phylogeography of dengue virus serotype 4, Brazil, 2010-2011.

Dengue virus serotype 4 (DENV-4) reemerged in Roraima State, Brazil, 28 years after it was last detected in the country in 1982. To study the origin and evolution of this reemergence, full-length sequences were obtained for 16 DENV-4 isolates from northern (Roraima, Amazonas, Pará States) and northeastern (Bahia State) Brazil during the 2010 and 2011 dengue virus seasons and for an isolate from the 1982 epidemic in Roraima. Spatiotemporal dynamics of DENV-4 introductions in Brazil were applied to envelope genes and full genomes by using Bayesian phylogeographic analyses. An introduction of genotype I into Brazil from Southeast Asia was confirmed, and full genome phylogeographic analyses revealed multiple introductions of DENV-4 genotype II in Brazil, providing evidence for >3 introductions of this genotype within the last decade: 2 from Venezuela to Roraima and 1 from Colombia to Amazonas. The phylogeographic analysis of full genome data has demonstrated the origins of DENV-4 throughout Brazil.