Sequencing and Description of the Mitochondrial Genome of Orthopodomyia fascipes (Diptera: Culicidae).

The genus Orthopodomyia Theobald, 1904 (Diptera: Culicidae) comprises 36 wild mosquito species, with distribution largely restricted to tropical and temperate areas, most of which are not recognized as vectors of epidemiological importance due to the lack of information related to their bionomy and involvement in the cycle transmission of infectious agents. Furthermore, their evolutionary relationships are not completely understood, reflecting the scarcity of genetic information about the genus. Therefore, in this study, we report the first complete description of the mitochondrial genome of a Neotropical species representing the genus, Orthopodomyia fascipes Coquillet, 1906, collected in the Brazilian Amazon region. Using High Throughput Sequencing, we obtained a mitochondrial sequence of 15,598 bp, with an average coverage of 418.5×, comprising 37 functional subunits and a final portion rich in A + T, corresponding to the control region. The phylogenetic analysis, using Maximum Likelihood and Bayesian Inference based on the 13 protein-coding genes, corroborated the monophyly of Culicidae and its two subfamilies, supporting the proximity between the tribes Orthopodomyiini and Mansoniini, partially disagreeing with previous studies based on the use of molecular and morphological markers. The information generated in this study contributes to a better understanding of the taxonomy and evolutionary history of the genus and other groups of Culicidae.

Evaluation of Multiple RNA Extraction Protocols for Chikungunya Virus Screening in Aedes aegypti Mosquitoes.

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning.

Isolation and sequencing of Orthoflavivirus ilheusense from mosquitoes collected in the Brazilian cerrado, West-Central region, Brazil.

The Orthoflavivirus ilheusense (ILHV) is an arbovirus that was first isolated in Brazil in 1944 during an epidemiologic investigation of yellow fever. Is a member of the Flaviviridae family and it belongs to the antigenic complex of the Ntaya virus group. Psorophora ferox is the primary vector of ILHV and this study presents the isolation and phylogenetic analysis of ILHV in a pool of Ps. ferox collected in the state of Goiás in 2021. Viral isolation tests were performed on Vero cells and C6/36 clones. The indirect immunofluorescence test (IFI) was used to confirm the positivity of the sample. The positive sample underwent RT-qPCR, sequencing, and phylogenetic analysis. This is the first report of ILHV circulation in this municipality and presented close relationship between this isolate and another ILHV isolate collected in the city of Belém (PA).

Genomic characterization of a novel Hepatovirus identified in Maranhão state, Brazil.

Bats are efficient reservoirs of a number of viruses with zoonotic potential, and are involved directly in the transmission cycle of many zoonoses. In the present study, which is part of a larger project that is documenting the viromes of the bat species found in the Mid-North states of Maranhão and Piauí, we analyzed 16 pooled samples obtained from four species of bat of the genus Artibeus-Artibeus obscurus, Artibeus cinereus, Artibeus lituratus and Artibeus planirostris. We describe and identify a Hepatovirus, denominated Hepatovirus H isolate sotense, which was found in a pool of internal organs (liver and lungs) extracted from a specimen of A. planirostris, a frugivorous bat, collected in the Cerrado biome of Maranhão state. This material was analyzed using new generation sequencing, which produced a contig of 7390 nucleotides and presented a degree of identity with a number of existing Hepatovirus sequences available for bats (amino acid identity of 61.5% with Bat hepatovirus C of Miniopterus cf. manavi, 66.6% with Bat hepatovirus G of Coleura afra, 67.4% with Hepatovirus G2 of Rhinolophus landeri, and 75.3% with Hepatovirus H2 of Rhinolophus landeri). The analysis of the functional domains of this contig confirmed a pattern consistent with the characteristics of the genus Hepatovirus (Picornaviridae). In the phylogenetic tree with several other Hepatovirus species, this genome also grouped in a monophyletic clade with Hepatovirus H (HepV-H1; HepV-H2, and HepV-H3) albeit on an external branch, which suggests that it may be a distinct genotype within this species. This is the first isolate of Hepatovirus H identified in bats from South America, and represents an important discovery, given that most studies of viruses associated with bats in the state of Maranhão have focused on the family Rhabdoviridae.

Identification of Viruses in Molossus Bats from the Brazilian Amazon: A Descriptive Metagenomic Analysis.

Bats are widely distributed in Brazil, including the Amazon region, and their association with viral pathogens is well-known. This work aimed to evaluate the metavirome in samples of Molossus sp. bats captured in the Brazilian Amazon from 2019 to 2021. Lung samples from 58 bats were divided into 13 pools for RNA isolation and sequencing followed by bioinformatic analysis. The Retroviridae family showed the highest abundance of viral reads. Although no complete genome could be recovered, the Paramyxoviridae and Dicistroviridae families showed the formation of contigs with satisfactory identity and size characteristics for further analysis. One contig of the Paramyxoviridae family was characterized as belonging to the genus Morbillivirus, being grouped most closely phylogenetically to Porcine morbillivirus. The contig related to the Dicistroviridae family was identified within the Cripavirus genus, with 94%, 91%, and 42% amino acid identity with Culex dicistrovirus 2, Rhopalosiphum padi, and Aphid lethal paralysis, respectively. The presence of viruses in bats needs constant updating since the study was able to identify viral sequences related to families or genera still poorly described in the literature in association with bats.

Detection of a Multiple Circulation Event of Dengue Virus 2 Strains in the Northern Region of Brazil.

Dengue virus serotype 2 (DENV-2) is responsible for dengue epidemics on a global scale and is associated with severe cases of the disease. This study conducted a phylogenetic investigation of DENV-2 isolates from 2017 to 2021 originating from the northern states of Brazil. A total of 32 samples from DENV-2 isolates were analyzed, including 12 from Acre, 19 from Roraima, and one from Tocantins. Only one lineage of the Asian-American genotype and one lineage of the cosmopolitan genotype were observed: Lineage 1, Asian-American genotype (connection to Puerto Rico); Lineage 5, cosmopolitan genotype (connection to Peru). Our results provide important data regarding the study of DENV genotypes and lineage distribution and open up possibilities for probable introduction and dissemination routes.

Sequencing and Analysis of the Mitochondrial Genome of Aedes aegypti (Diptera: Culicidae) from the Brazilian Amazon Region.

Aedes aegypti is a mosquito native to the African continent, which is now widespread in the tropical and subtropical regions of the world. In many regions, it represents a major challenge to public health, given its role in the cycle of transmission of important arboviruses, such as Dengue, Zika, and Chikungunya. Considering the epidemiological importance of Ae. aegypti, the present study sequenced the partial mitochondrial genome of a sample collected in the municipality of Balsas, in the Brazilian state of Maranhão, followed by High Throughput Sequencing and phylogenetic analyses. The mitochondrial sequence obtained here was 15,863 bp long, and contained 37 functional subunits (thirteen PCGs, twenty-two tRNAs and two rRNAs) in addition to a partial final portion rich in A+T. The data obtained here contribute to the enrichment of our knowledge of the taxonomy and evolutionary biology of this prominent disease vector. These findings represent an important advancement in the understanding of the characteristics of the populations of northeastern Brazil and provide valuable insights into the taxonomy and evolutionary biology of this prominent disease vector.

Multiple introductions and country-wide spread of DENV-2 genotype II (Cosmopolitan) in Brazil.

Dengue virus serotype 2, genotype Cosmopolitan (DENV-2-GII), is one of the most widespread DENV strains globally. In the USA, DENV-2 epidemics have been dominated by DENV-2 genotype Asian-American (DENV-2-GIII), and the first cases of DENV-2-GII were only described in 2019, in Peru, and in 2021 in Brazil. To gain new information about the circulation of DENV-2-GII in Brazil, we sequenced 237 DENV-2 confirmed cases sampled between March 2021 and March 2023 and revealed that DENV-2-GII is already present in all geographic regions of Brazil. The phylogeographic analysis inferred that DENV-2-GII was introduced at least four times in Brazil, between May 2020 and August 2022, generating multiple clades that spread throughout the country with different success. Despite multiple introductions of DENV-2-GII, analysis of the country-wide laboratory surveillance data showed that the Brazilian dengue epidemic in 2022 was dominated by DENV-1 in most states. We hypothesize that massive circulation of DENV-2-GIII in previous years in Brazil might have created a population immune barrier against symptomatic homotypic reinfections by DENV-2-GII, leading to sustained cryptic circulation in asymptomatic cases and localized outbreaks of this new genotype. In summary, our study stresses the importance of arboviral genomic surveillance to close monitoring and better understanding the potential impact of DENV-2-GII in the coming years.

First isolation of West Nile virus in Brazil

BACKGROUND Serological evidence of West Nile virus (WNV) infection has been reported in different regions of Brazil from equine and human hosts but the virus had never been isolated in the country. OBJECTIVES We sought to identify the viral etiology of equine encephalitis in Espírito Santo state. METHODS We performed viral culture in C6/36 cells, molecular detection of WNV genome, histopathology and immunohistochemistry from horse cerebral tissue. We also carried out sequencing, phylogenetic analysis and molecular clock. FINDINGS Histopathologic analysis from horse cerebral tissue showed injury related to encephalitis and WNV infection was confirmed by immunohistochemistry. The virus was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) from brain tissue and subsequently isolated in C6/36 cells. WNV full-length genome was sequenced showing the isolated strain belongs to lineage 1a. The molecular clock indicated that Brazilian WNV strain share the same common ancestor that were circulating in US during 2002-2005. MAIN CONCLUSIONS Here we report the first isolation of WNV in Brazil from a horse with neurologic disease, which was clustered into lineage 1a with others US WNV strains isolated in beginning of 2000's decade.

Potential role of dengue virus, chikungunya virus and Zika virus in neurological diseases

This study showed that laboratory markers of recent infection by dengue, Zika or chikungunya arboviruses were detected in the biological samples of approximately one-third of patients with encephalitis, myelitis, encephalomyelitis or Guillain-Barré syndrome, in a surveillance programme in Piauí state, Brazil, between 2015-2016. Fever and myalgia had been associated with these cases. Since in non-tropical countries most infections or parainfectious diseases associated with the nervous system are attributed to herpesviruses, enteroviruses, and Campylobacter jejuni, the present findings indicate that in tropical countries, arboviruses may now play a more important role and reinforce the need for their surveillance and systematic investigation in the tropics.

Molecular characterisation of dengue virus type 1 reveals lineage replacement during circulation in Brazilian territory

Dengue fever is the most important arbovirus infection found in tropical regions around the world. Dispersal of the vector and an increase in migratory flow between countries have led to large epidemics and severe clinical outcomes, such as dengue haemorrhagic fever and dengue shock syndrome. This study analysed the genetic variability of the dengue virus serotype 1 (DENV-1) in Brazil with regard to the full-length structural genes C/prM/M/E among 34 strains isolated during epidemics that occurred in the country between 1994-2011. Virus phylogeny and time of divergence were also evaluated with only the E gene of the strains isolated from 1994-2008. An analysis of amino acid differences between these strains and the French Guiana strain (FGA/89) revealed the presence of important nonsynonymous substitutions in the amino acid sequences, including residues E297 (Met→Thr) and E338 (Ser→Leu). A phylogenetic analysis of E proteins comparing the studied isolates and other strains selected from the GenBank database showed that the Brazilian DENV-1 strains since 1982 belonged to genotype V. This analysis also showed that different introductions of strains from the 1990s represented lineage replacement, with the identification of three lineages that cluster all isolates from the Americas. An analysis of the divergence time of DENV-1 indicated that the lineage circulating in Brazil emerged from an ancestral lineage that originated approximately 44.35 years ago.

Estudo experimental sobre a patogenicidade do Vírus Ilhéus em hamsters dourados (Mesocricetus auratus) / Pathogenesis of the Ilheus virus in golden hamsters (Mesocricetus auratus)

Visando investigar a patogenicidade do Flavivirus Ilhéus (VILH) foi inoculada, via intraperitoneal, 9,8 DL de suspensão 50 viral em hamsters dourados jovens (Mesocricetus auratus) e, diariamente, soros e vísceras (cérebro, fígado, coração, baço, rins e pulmões) de animais infectados e de controles não-infectados foram obtidos sob anestesia. Durante o experimento foi determinado o título viral do VILH em soros e vísceras infectados, em camundongos recém-nascidos. Ademais, a detecção de antígeno e os níveis de anticorpos por testes de fixação do complemento e inibição da hemaglutinação foram realizados nos soros. Exame histopatológico por HE e a detecção de antígenos virais por Imunohistoquímica (IHQ) foram realizados nos tecidos dos animais. A dose inoculada ocasionou a morte dos animais por encefalite no sétimo dia pós inoculação. Todos os órgãos estudados apresentaram alterações teciduais detectáveis por histopatologia. Volumosa presença de antígeno viral foi detectada por IHQ no cérebro, e, em menor quantidade, no fígado, baço e rins; porém, nestes órgãos, a presença de antígeno viral foi transitória e de leve intensidade, o que corroborou com os títulos virais obtidos nesses órgãos. Não foram encontrados antígenos virais em coração e pulmões, sugerindo que os títulos (DL ) 50 observados nesses órgãos, durante a titulação em camundongos, decorreram da presença do VILH na corrente sanguínea (viremia). Os achados deste estudo reforçam o importante e conhecido neurotropismo do VILH.

The pathogenesis of the Ilheus flavivirus (Flaviviridae) was investigated in golden hamsters (Mesocricetus auratus) using an inoculum of 9.8 LD50 via intraperitoneal (IP). For ten days, two infected and one control animals were anesthetized, and blood and viscera fragments (brain, liver, heart, lung, spleen and kidneys) were collected on a daily basis for determination of viral titers in newborn mice and antigens/antibody by complement fixation and hemagglutination inhibition tests. Additionally, the pathology of animal tissues was studied by the the hematoxylin and eosin method, viral antigens were detected by immunochemistry, and all collected viscera showed histopathological changes. Large amounts of ILHV antigens were detected by immunohistochemistry in the brain, and in lower quantities in the liver, spleen and kidneys, corroborating with newborn viral titers in them. This inoculum resulted in a fatal outcome of all infected animals seven days after experimentation. Viral antigens were not found in the heart and lungs, suggesting that the viral titers obtained were caused by viremia and not by viral damage. The information in this study confirms the neurotropism and neuropathogenicity of ILHV.

Molecular epidemiology of dengue virus serotypes 2 and 3 isolated in Brazil from 1991 to 2008 / Epidemiologia molecular dos sorotipos 2 e 3 do vírus dengue, isolados no Brasil de 1991 a 2008

The dengue virus (DENV1-4) causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in tropical and subtropical areas. The aim of this study was to evaluate the circulating genotypes of DENV. This was accomplished by sequencing the PrM and E genes of Brazilian isolates of DENV2 and DENV3 that were obtained between 1991 and 2008 from various geographic regions. Phylogenetic analyses of DENV2 demonstrated that the genotype III (Southeast Asian/American), in spite of several nucleotide and amino acid changes, was the only one that circulated over the past 19 years. Since its introduction in 2000, the DENV3 isolates that have been analyzed have all grouped intogenotype III (Indian subcontinent) and there has been no evidence of DENV3 belonging to other genotypes in this study.

O vírus dengue (DENV1-4) causa a dengue clássica e a febre hemorrágica da dengue / síndrome de choque da dengue (FHD/SCD) em regiões tropicais e subtropicais. O objetivo deste estudo foi avaliar os genótipos circulantes de DENV2 e DENV3 obtidos em distintas regiões geográficas no período de 1991 a 2008. As análises filogenéticas de DENV2 demonstraram que o genótipo III (Sudeste da Ásia/América), apesar das diversas alterações nucleotídicas e de aminoácidos, foi o único a circular durante os últimos 19 anos. Desde a sua introdução no estudo, em 2000, todas as amostras isoladas de DENV3 analisadas foram agrupadas no genótipo III (subcontinente indiano). Não foram encontradas evidências de que o DENV3 pertença a outros genótipos investigados.

Vigilância sorológica para arbovírus em Juruti, Pará, Brasil / Serological survey for arboviruses in Juruti, Pará State, Brazil

O objetivo foi avaliar a prevalência de anticorpos para arbovírus na área de influência do Projeto Juruti antes da exploração mineral local. Foram examinados 1.597 soros humanos e 85 de animais silvestres. A pesquisa de anticorpos foi realizada pelo teste de inibição da hemaglutinação contra antígenos dos arbovírus mais prevalentes na Amazônia brasileira e IgM-ELISA para dengue e febre amarela. Soros humanos inibidores da hemaglutinação positivos para Mayaro e Oropouche foram também testados por IgM-ELISA. Anticorpos inibidores da hemaglutinação para alfavírus, orthobunyavirus e/ou flavivírus foram detectados, sendo 28,7 por cento reações monotípicas. Infecções recentes para Oropouche (n = 23), Mayaro (n = 5), e dengue (n = 20) foram confirmadas por IgM-ELISA. A prevalência de anticorpos inibidores da hemaglutinação em soros de animais silvestres foi de 8,2 por cento para flavivírus. Os resultados obtidos mostram que houve circulação ativa dos vírus Mayaro, Oropouche e dengue no período do estudo em humanos em Juruti, Pará, Brasil, e de diversos arbovírus em animais silvestres, indicando presença desses vírus em áreas do Município de Juruti.

This study aimed to measure the prevalence of antibodies to arboviruses in the area affected by the Juruti Project before local mining operations. A total of 1,597 human and 85 wild animal sera were examined, using the hemagglutination inhibition test against 19 antigens from the most prevalent arboviruses in the Brazilian Amazon and IgM-ELISA for dengue and yellow fever. Positive sera for Mayaro and Oropouche viruses were also tested by IgM-ELISA. Prevalence of hemagglutination inhibition antibodies to alphaviruses, flaviviruses, and orthobunyaviruses in humans was 28.7 percent, while for wild animals it was 8.2 percent for flaviviruses. Recent infections based on presence of IgM was confirmed for Mayaro (n = 5), Oropouche (n = 23), and dengue (n = 20). The results showed active circulation of the dengue, Mayaro, and Oropouche viruses in Juruti, as well as flaviviruses in wild animals, suggesting the circulation of these viruses in the Municipality of Juruti.

Caracterização molecular e biológica do vírus dengue circulante no Brasil / Molecular and biological characterization of dengue virus circulating in Brazil

A febre do dengue está amplamente distribuída por todas as áreas tropicais do mundo. Em certas áreas endêmicas, as formas graves da doença, febre hemorrágica do dengue (FHD) e síndrome de choque do dengue (SCD), ocorrem com frequência. Outras manifestações clínicas, como, por exemplo, encefalopatias, encefalites, mielite transversa, também tem sido observadas. O nosso objetivo foi a caracterização genética e biológica de amostras de vírus VDEN2 e 3, com ênfase no caráter de neurovirulência do vóirus VDEN2. As amostras de VDEN 2 e 3 utilizadas foram isoladas na Serviço de Arbovírus do Instituto Evandro Chagas a partir de soro de pacientes com quadro clínico de dengue (FD e FHD) e manifestações neurológicas. Os vírus padrão foram o VDEN2 Nova Guiné C e 44-2, VDEN3 H87. As 11 sequências do vírus VDEN2 quando comparados entre si e com 16 outras sequências do VDN2 apresentaram percentual médio de identidade nucleotídica e aminoácido de 89,5 e 99,8 por cento respectivamente. Os estudos filogenéticos realizados mostram os isolados brasileiros pertencem ao genótipo III de origem Asiática. As 10 sequências do vírus VDEN3 quando comparados entre si e com 16 outras sequências do VDEN3 apresentaram percentual médio identidade nucleotídica e aminoácido de 89,6 e 100 por cento respectivamente. Os estudos filogenéticos realizados mostram que estão agrupados ao genótipo III também de origem Asiática. O vírus VDEN2 isolado a partir de caso mielite transversa (Bel61082) foi caracterizado pelo sequenciamento nucleotidico da região estrutural assim como em testes de neurovirulência em camundongos recém-nascidos. A susceptibilidade de culturas primárias de neurônios e astrócitos, células de glioma humano, também foi estudada para fins de caracterização do caráter neurovirulento do vírus. O estudo de neurovirulência em infecção de camundongo mostrou sinais aparentes de doença apenas após a inoculação da cepa NGC, mas a replicação do vírus Bel 61082 pode ser detectada...pró-apoptótica.