Screening of febrile patients with suspected malaria from the Brazilian Amazon for virus infection.

Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential.

Fatal Outcome of Chikungunya Virus Infection in Brazil.

BACKGROUND: Chikungunya virus (CHIKV) emerged in the Americas in 2013 and has caused approximately 2.1 million cases and >600 deaths. A retrospective investigation was undertaken to describe clinical, epidemiological, and viral genomic features associated with deaths caused by CHIKV in Ceará state, northeast Brazil. METHODS: Sera, cerebrospinal fluid (CSF), and tissue samples from 100 fatal cases with suspected arbovirus infection were tested for CHIKV, dengue virus (DENV), and Zika virus (ZIKV). Clinical, epidemiological, and death reports were obtained for patients with confirmed CHIKV infection. Logistic regression analysis was undertaken to identify independent factors associated with risk of death during CHIKV infection. Phylogenetic analysis was conducted using whole genomes from a subset of cases. RESULTS: Sixty-eight fatal cases had CHIKV infection confirmed by reverse-transcription quantitative polymerase chain reaction (52.9%), viral antigen (41.1%), and/or specific immunoglobulin M (63.2%). Co-detection of CHIKV with DENV was found in 22% of fatal cases, ZIKV in 2.9%, and DENV and ZIKV in 1.5%. A total of 39 CHIKV deaths presented with neurological signs and symptoms, and CHIKV-RNA was found in the CSF of 92.3% of these patients. Fatal outcomes were associated with irreversible multiple organ dysfunction syndrome. Patients with diabetes appear to die at a higher frequency during the subacute phase. Genetic analysis showed circulation of 2 CHIKV East-Central-South African (ECSA) lineages in Ceará and revealed no unique virus genomic mutation associated with fatal outcome. CONCLUSIONS: The investigation of the largest cross-sectional cohort of CHIKV deaths to date reveals that CHIKV-ECSA strains can cause death in individuals from both risk and nonrisk groups, including young adults.

Insect-specific viruses and arboviruses in adult male culicids from Midwestern Brazil.

Viruses were identified from male anthropophilic mosquitoes from Mato Grosso (MT) State, Midwest Brazil from February 2017 to January 2018. Mosquitoes tested included Aedes (Stegomyia) aegypti (1139 males; 84 pools), Culex quinquefasciatus (9426 males; 179 pools), Culex sp. (3 males; 3 pools) and Psorophora albigenu (1 male; 1 pool) collected from four cities of MT. Pools were subjected to viral RNA extraction followed by RT-PCRs specific for ten flaviviruses, five alphaviruses and Simbu serogroup of orthobunyaviruses. Positive pools were passaged three times in VERO cells (alphavirus and orthobunyavirus) or C6/36 cells (flavivirus), with isolates confirmed through RT-PCR and nucleotide sequencing. We detected pools positive for Ilhéus (1 pool), dengue serotype 4 (1), Mayaro (12), equine encephalitis virus (1) yellow fever (1), Oropouche (2), Zika (4) and chikungunya (12) viruses. High throughput sequencing of arbovirus positive pools identified 35 insect-specific viruses (ISVs) from the families Circoviridae (2), Parvoviridae (2), Totiviridae (1), Flaviviridae (1), Iflaviridae (2), Mesoniviridae (4), Nodaviridae (2), Luteoviridae (1), Phasmaviridae (1) Phenuiviridae (2), Rhabdoviridae (2), Orthomyxoviridae (1), Xinmoviridae (1), and unclassified Bunyavirales (1), unclassified Picornavirales (3), unclassified Riboviria (4) and taxon Negevirus (5). From these, five novel viruses were tentatively named Mojica circovirus, Kuia iflavirus, Muxirum negevirus, Lambada picorna-like virus and Tacuru picorna-like virus. Our findings underscore the diversity and wide geographical distribution of ISVs and arboviruses infecting male culicids.

Evolutionary and Molecular Analysis of Complete Genome Sequences of Norovirus From Brazil: Emerging Recombinant Strain GII.P16/GII.4.

Noroviruses (NoVs) are enteric viruses that cause acute gastroenteritis, and the pandemic GII.4 genotype is spreading and evolving rapidly. The recombinant GII.P16/GII.4_Sydney strain emerged in 2016, replacing GII.P31/GII.4_Sydney (GII.P31 formerly known as GII.Pe) in some countries. We analyzed the complete genome of 20 NoV strains (17 GII.P31/GII.4_ Sydney and 3 GII.P16/GII.4_Sydney) from Belém and Manaus, Brazil, collected from 2012 to 2016. Phylogenetic trees were constructed by maximum likelihood method from 191 full NoV-VP1 sequences, demonstrated segregation of the Sydney lineage in two larger clades, suggesting that GII.4 strains associated with GII.P16 already have modifications compared with GII.P31/GII.4. Additionally, the Bayesian Markov Chain Monte Carlo method was used to reconstruct a time-scaled phylogenetic tree formed by GII.P16 ORF1 sequences (n = 117) and three complete GII.P16 sequences from Belém. The phylogenetic tree indicated the presence of six clades classified into different capsid genotypes and locations. Evolutionary rates of the ORF1 gene of GII.P16 strains was estimated at 2.01 × 10-3 substitutions/site/year, and the most recent common ancestors were estimated in 2011 (2011-2012, 95% HPD). Comparing the amino acid (AA) sequence coding for ORF1 with the prototype strain GII.P16/GII.4, 36 AA changes were observed, mainly in the non-structural proteins p48, p22, and RdRp. GII.P16/GII.4 strains of this study presented changes in amino acids 310, 333, 373, and 393 of the antigenic sites in the P2 subdomain, and ML tree indicating the division within the Sydney lineage according to the GII.P16 and GII.P31 polymerases. Notably, as noroviruses have high recombination rates and the GII.4 genotype was prevalent for a long time in several locations, additional and continuous evolutionary analyses of this new genotype should be needed in the future.

Whole-Genome Sequencing of an Unusual Human Papillomavirus (HPV71) from Latin America (Brazil).

We report the complete genome sequencing of human papillomavirus 71 from Latin America (Brazil).

Complete genome sequence of human T-cell lymphotropic type 1 from patients with different clinical profiles, including infective dermatitis.

The HTLV-1 is the first human retrovirus and is associated with several clinical syndromes, however, the pathogenesis of these clinical manifestations is still not fully understood. Furthermore, there are few complete genomes publicly available, about 0.12 complete genomes per 10,000 infected individuals and the databases have a major deficiency of sequences information. This study generated and characterized 31 HTLV-1 complete genomes sequences derived from individuals with Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (TSP/HAM), Adult T-cell leukemia/lymphoma (ATL), infective dermatitis associated to HTLV-1 (IDH) and asymptomatic patients. These sequences are associated to clinical and epidemiological information about the patients. The sequencing data generated on Ion Torrent PGM platform were assembled and mapped against the reference HTLV-1 genome. These sequences were genotyped as Cosmopolitan subtype, Transcontinental subgroup. We identified the variants in the coding regions of the genome of the different clinical profiles, however, no statistical relation was detected. This study contributed to increase of HTLV-1 complete genomes in the world. Furthermore, to better investigate the contribution of HTLV-1 mutations for the disease outcome it is necessary to evaluate the interaction of the viral genome and characteristics of the human host.

Comparação da resistência ao cisalhamento após colagem com dois cimentos ortodônticos ­ estudo in vitro / A comparison of shear bond strength after bonding with two orthodontic cements ­ an in vitro study

O objetivo deste trabalho foi avaliar a resistência ao cisalhamento de bandas ortodônticas cimentadas com um novo compósito Orthobite, comparando-as ao cimento ionômero de vidro convencional em esmalte bovino e analisar o Índice de Remanescente Adesivo (IRA). A amostra consistiu de 30 dentes bovinos divididos em 2 grupos: Grupo I ­ Orthobite (FGM) e Grupo II ­ CIV MERON (VOCO). Foi realizada profilaxia com pedra-pomes e água, seguida da cimentação conforme instruções dos fabricantes. Logo após, os mesmos foram submetidos ao ensaio de cisalhamento com velocidade de 0,5 mm/minutos. Os dados foram submetidos aos testes de T Student e Kruskal-Wallis. Foram encontradas diferenças estatísticas significantes entre os grupos, tanto nos valores de resistência ao cisalhamento (p>0,05), quanto na avaliação do IRA (p>0,05). O cimento Orthobite mostrou-se estatisticamente superior ao MERON, ou seja, apresentou um melhor vedamento das margens e uma maior resistência ao deslocamento, dessa forma essa união estável entre o dente e a banda é fundamental para o sucesso do tratamento ortodôntico (AU)

The objective of this study was to evaluate the shear strength of orthodontic bands cemented with a new Orthobite composite, comparing them to the conventional glass ionomer cement in bovine enamel and to analyze the Adhesive Remnant Index (ARI). The sample consisted of 30 bovine teeth divided into 2 groups: Group I ­ Orthobite (FGM) and Group II ­ CIV MERON (VOCO). Prophylaxis has been made with pumice and water followed by cementation according to the manufacturer's instructions. Afterwards, they were subjected to the shear test at a speed of 0.5 mm/minute speed. Data were submitted to the Student T and Kruskal-Wallis tests. Significant statistical differences were found between the groups both in the shear strength values (p> 0.05) and in the ARI assessment (p> 0.05). The Orthobite cement proved to be statistically superior to MERON, that is, it presented a better margins seal and a greater resistance to displacement, so this stable union between the tooth and the band is fundamental for the success of the orthodontic treatment. (AU)

Genomic characterization of orthobunyavirus of veterinary importance in America.

During 2013, in Argentina, three new isolates of serogroup Bunyamwera virus (genus Orthobunyavirus, family Peribunyaviridae) were recovered from two horses with encephalitis, and from an aborted equine fetus. In the present study, we report the complete genome sequence, genetic characterization, and phylogenetic analysis of three new strains isolated in Argentina to clarifying their relationship within the Bunyamwera serogroup virus and to investigate the evolutionary history of viruses with segmented genomes.

Comparative analysis of the transcriptome of the Amazonian fish species Colossoma macropomum (tambaqui) and hybrid tambacu by next generation sequencing.

BACKGROUND: The C. macropomum is a characiform fish from the Amazon basin that has been hybridized with other pacu species to produce commercial hybrids, such as the tambacu. However, little is known of the functional genomics of the parental species or these hybrid forms. The transcriptome of C. macropomum and tambacu were sequenced using 454 Roche platform (pyrosequencing) techniques to characterize the domains of Gene Ontology (GO) and to evaluate the levels of gene expression in the two organisms. RESULTS: The 8,188,945 reads were assembled into 400,845 contigs. A total of 58,322 contigs were annotated with a predominance of biological processes for both organisms, as determined by Gene Ontology (GO). Similar numbers of metabolic pathways were identified in both the C. macropomum and the tambacu, with the metabolism category presenting the largest number of transcripts. The BUSCO analysis indicated that our assembly was more than 40% complete. We identified 21,986 genes for the two fishes. The P and Log2FC values indicated significant differences in the levels of gene expression, with a total of 600 up-regulated genes. CONCLUSION: In spite of the lack of a reference genome, the functional annotation was successful, and confirmed a considerable difference in the specificity and levels of gene expression between the two organisms. This report provides a comprehensive baseline for the genetic management of these commercially important fishes, in particular for the identification of specific genes that may represent markers involved in the immunity, growth, and fertility of these organisms, with potential practical applications in aquaculture management.

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.

Insights Into Limnothrix sp. Metabolism Based on Comparative Genomics.

Currently only four genome sequences for Limnothrix spp. are publicly available, and information on the genetic properties of cyanobacteria belonging to this genus is limited. In this study, we report the draft genome of Limnothrix sp. CACIAM 69d, isolated from the reservoir of a hydroelectric dam located in the Amazon ecosystem, from where cyanobacterial genomic data are still scarce. Comparative genomic analysis of Limnothrix revealed the presence of key enzymes in the cyanobacterial central carbon metabolism and how it is well equipped for environmental sulfur and nitrogen acquisition. Additionally, this work covered the analysis of Limnothrix CRISPR-Cas systems, pathways related to biosynthesis of secondary metabolites and assembly of extracellular polymeric substances and their exportation. A trans-AT PKS gene cluster was identified in two strains, possibly related to the novel toxin Limnothrixin biosynthesis. Overall, the draft genome of Limnothrix sp. CACIAM 69d adds new data to the small Limnothrix genome library and contributes to a growing representativeness of cyanobacterial genomes from the Amazon region. The comparative genomic analysis of Limnothrix made it possible to highlight unique genes for each strain and understand the overall features of their metabolism.

De novo transcriptome based on next-generation sequencing reveals candidate genes with sex-specific expression in Arapaima gigas (Schinz, 1822), an ancient Amazonian freshwater fish.

BACKGROUND: The Arapaima (Arapaima gigas) is one of the world's largest freshwater bony fish, and is found in the rivers of the Amazon basin. This species is a potential aquaculture resource, although reproductive management in captivity is limited in particular due to the lack of external sexual dimorphism. In this study, using the 454 Roche platform (pyrosequencing) techniques, we evaluated a major portion of the transcriptome of this important Amazonian species. RESULTS: Four libraries obtained from the liver and skin tissue of juvenile specimens (representing males and females separately) were sequenced, yielding 5,453,919 high-quality reads. The de novo transcriptome assembly resulted in 175,792 contigs, with 51,057 significant blast hits. A total of 38,586 transcripts were mapped by Gene Ontology using Blast2GO. We identified 20,219 genes in the total transcriptome (9,551 in the liver and 16,818 in the skin). The gene expression analyses indicated 105 genes in the liver and 204 in the skin with differentiated expression profiles, with 95 being over-expressed in the females and 214 in the males. The log2 Fold Change and heatmap based on Reads Per Kilobase per Million mapped reads (RPKM) revealed that the gene expression in the skin is highly differentiated between male and female arapaima, while the levels of expression in the liver are similar between the sexes. CONCLUSION: Transcriptome analysis based on pyrosequencing proved to be a reliable tool for the identification of genes with differentiated expression profiles between male and female arapaima. These results provide useful insights into the molecular pathways of sexual dimorphism in this important Amazonian species, and for comparative analyses with other teleosts.

Opsoclonus-myoclonus-ataxia syndrome associated with chikungunya and dengue virus co-infection.

Opsoclonus-myoclonus-ataxia syndrome (OMAS) is a rare neurological disorder characterized by irregular multidirectional eye movements, myoclonus, cerebellar ataxia, sleep disturbances, and cognitive dysfunction. Although most commonly related to paraneoplastic syndrome, this condition has occasionally been described following infectious illnesses. This article reports the first case of OMAS in association with chikungunya and dengue virus co-infection. The genetic analysis identified chikungunya virus of East/Central/South African genotype and dengue serotype 4 virus of genotype II. This report represents an unusual clinical syndrome associated with viral co-infection and reinforces the need for clinical vigilance with regard to neurological syndromes in the context of emergent arboviruses.

Descrição da anatomia radiográfica do crânio do Cebus Apella (Linnaeus, 1758) / Anatomic-radiographic description of cebus apella (linnaeus, 1758) skull

Introdução: as semelhanças filogenéticas entre primatas não humanos e seres humanos estimulam estudos de seu sistema estomatognático, visando seu uso em pesquisas. Objetivo: descrever as estruturas anatômicas da maxila e da mandíbula do Cebus apella, comparando às características humanas. Material e Método: foram utilizados dois animais adultos. Após a remoção do tecido orgânico, os crânios e maxilas foram radiografados e fotografados, sendo analisados com a lupa. Resultado e discussão: as estruturas anatômicas da maxila e da mandíbula apresentavam características semelhantes ao ser humano, mas com algumas peculiaridades, tais como: a presença do terceiro pré-molar; proeminências caninas evidentes em ambos os arcos; a colocação mental do majestoso; largura do ramo ascendente do maxilar; a presença do forame mandibular; Formato V do maxilar; o tamanho do forame incisivo; presença da sutura incisiva. Com relção à interpretação radiográfica da câmara pulpar e canal radicular desta espécie, os dentes incisivos central e lateral superiores, caninos superiores e inferiores e 1º, 2º, 3º Pré-molares inferiores são dentes com canais únicos, amplos, de fácil acesso e, desse modo, ideais para experimentos endodônticos. Conclusão: o Cebus apella pode ser usado como modelo de estudo em tratamento endodôntico.

Introduction: the phylogenetic similarities between non-human primates and humans stimulate studies of their stomatognathic system, aiming their use in research. Objective: the objective of this study was to describe the anatomical structures of the maxilla and mandible of Cebus apella comparing to human characteristics. Material and Method: two adult animals were used. After removing the organic tissue, the skulls and jaws were x-rayed and photographed, being analyzed with the magnifying glass. Results: the results showed that the anatomical structures of the maxilla and the mandible had similar characteristics to the human being, but with some peculiarities, such as: the presence of the third premolar; prominent canine prominences in both arches; the mental setting of the majestic; width of ascending branch of maxilla; the presence of the mandibular foramen; V shape of the jaw; incisor foramen size; presence of the incisive suture. With respect to the radiographic interpretation of the pulp chamber and root canal of this species, the maxillary and mandibular maxillary teeth, maxillary and mandibular maxillary teeth are teeth with single, wide, easily accessible ideal for endodontic experiments. Conclusion: thus, the authors conclude that Cebus apella can be used as a study model in endodontic treatment.

Complete Genome Sequence of Porphyrobacter sp. Strain CACIAM 03H1, a Proteobacterium Obtained from a Nonaxenic Culture of Microcystis aeruginosa.

Here, we present a complete genome sequence and annotation of Porphyrobacter sp. strain CACIAM 03H1, which was obtained from a nonaxenic culture of Microcystis aeruginosa, a cyanobacterium isolated from an Amazonian freshwater environment.

Draft Genome Sequence of Alkalinema sp. Strain CACIAM 70d, a Cyanobacterium Isolated from an Amazonian Freshwater Environment.

In order to increase the genomic data of cyanobacterial strains isolated in Brazil, we hereby present the draft genome sequence of the Alkalinema sp. strain CACIAM 70d, isolated from an Amazonian freshwater environment. This report describes the first genome available for this genus.

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).

Characterization of the Bujaru, frijoles and Tapara antigenic complexes into the sandfly fever group and two unclassified phleboviruses from Brazil.

The genus Phlebovirus includes the sandfly fever viruses and tick-transmitted uukuviruses. Sandfly fever group viruses have been isolated from various vertebrate species and from phlebotomines and occasionally alternative arthropods, e.g. mosquitoes, or ceratopogonids of the genus Culicoides. Uukuniemi serogroup viruses have been isolated from various vertebrate species and from ticks. Despite the public health importance of some viruses of the genus, the genomic diversity of phleboviruses that could be incriminated as causative of human or veterinary diseases remains underestimated. Here we describe the nearly complete sequences and genomic characterization of two phleboviruses belonging to the Bujaru antigenic complex: the prototype species and the Munguba virus. Furthermore, six previously unclassified phleboviruses isolated in Brazil were also sequenced and characterized: Ambe, Anhanga, Joa, Uriurana, Urucuri and Tapara viruses. The results of the phylogenetic analysis indicated that these viruses group with viruses of three antigenic complexes (Bujaru, Tapara and frijoles clades), with two unclassified phleboviruses. We also performed genomic reassortment analysis and confirmed that there were no events for the viruses described in this study, but we found a new potential reassortment in Medjerda Valley virus, which contains S and L segments of Arbia virus, and probably a unique M segment, both viruses circulate in the same geographic region, indicating these two isolates represent two distinct viruses. This study provides insights into the genetic diversity, classification and evolution of phleboviruses.