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.

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.

Complete nucleotide sequences of two blaKPC-2-bearing IncN Plasmids isolated from sequence type 442 Klebsiella pneumoniae clinical strains four years apart.

We sequenced the oldest blaKPC-2-bearing plasmid isolated in Brazil and another plasmid also carried by a Klebsiella pneumoniae strain of sequence type 442 (ST442), isolated 52 months later. Both plasmids present an IncN backbone and few acquired regions. Because the 2005 plasmid presented deletions and a truncated gene within Tn4401b compared to the 2009 plasmid, we can thus infer that IncN blaKPC-2-bearing plasmids pFCF1305 and pFCF3SP had a common ancestor circulating in Brazil prior to May 2005.

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.

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.

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.

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.

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.

Characterization of mitochondrial genome of Haemagogus janthinomys (Diptera: Culicidae).

Haemagogus janthinomys is a mosquito of high importance in public health due its involvement on natural wild cycles of two important arboviruses in the Brazilian Amazon region: Yellow Fever virus (Flaviviridae, Flavivirus) and Mayaro virus (Togaviridae, Alphavirus). Here, we have sequenced and described all the mitochondrial genes for the Hg. janthinomys species. The complete coding sequence is14 937 bp long and includes 37 functional genes, of which 13 codes for proteins, 22 for tRNA and 2 for ribosomal subunits. Region A + T (control region) is not presented here. The data should be helpful on further taxonomic and evolutionary studies of this important arbovirus vector.

Draft Genome Sequence of Limnobacter sp. Strain CACIAM 66H1, a Heterotrophic Bacterium Associated with Cyanobacteria.

Ecological interactions between cyanobacteria and heterotrophic prokaryotes are poorly known. To improve the genomic studies of heterotrophic bacterium-cyanobacterium associations, the draft genome sequence (3.2 Mbp) of Limnobacter sp. strain CACIAM 66H1, found in a nonaxenic culture of Synechococcus sp. (cyanobacteria), is presented here.

Draft Genome Sequence of Flavihumibacter sp. Strain CACIAM 22H1, a Heterotrophic Bacterium Associated with Cyanobacteria.

Here, we present a draft genome and annotation of Flavihumibacter sp. CACIAM 22H1, isolated from Bolonha Lake, Brazil, which will provide further insight into the production of substances of biotechnological interest.

Draft Genome Sequence of Microcystis aeruginosa CACIAM 03, a Cyanobacterium Isolated from an Amazonian Freshwater Environment.

Given its toxigenic potential, Microcystis aeruginosa is an important bloom-forming cyanobacterium. Here, we present a draft genome and annotation of the strain CACIAM 03, which was isolated from an Amazonian freshwater environment.

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.

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.

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.

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.

Genome-wide study of the defective sucrose fermenter strain of Vibrio cholerae from the Latin American cholera epidemic.

The 7th cholera pandemic reached Latin America in 1991, spreading from Peru to virtually all Latin American countries. During the late epidemic period, a strain that failed to ferment sucrose dominated cholera outbreaks in the Northern Brazilian Amazon region. In order to understand the genomic characteristics and the determinants of this altered sucrose fermenting phenotype, the genome of the strain IEC224 was sequenced. This paper reports a broad genomic study of this strain, showing its correlation with the major epidemic lineage. The potentially mobile genomic regions are shown to possess GC content deviation, and harbor the main V. cholera virulence genes. A novel bioinformatic approach was applied in order to identify the putative functions of hypothetical proteins, and was compared with the automatic annotation by RAST. The genome of a large bacteriophage was found to be integrated to the IEC224's alanine aminopeptidase gene. The presence of this phage is shown to be a common characteristic of the El Tor strains from the Latin American epidemic, as well as its putative ancestor from Angola. The defective sucrose fermenting phenotype is shown to be due to a single nucleotide insertion in the V. cholerae sucrose-specific transportation gene. This frame-shift mutation truncated a membrane protein, altering its structural pore-like conformation. Further, the identification of a common bacteriophage reinforces both the monophyletic and African-Origin hypotheses for the main causative agent of the 1991 Latin America cholera epidemics.