Artur Trancoso Lopo de Queiroz

The presence of resistance mutations to protease and polymerase inhibitors in Hepatitis C virus sequences from the Los Alamos databank

Several new direct‐acting antiviral (DAA) drugs are in development for chronic hepatitis C viral (HCV) infection, and NS3‐NS4A serine protease and the NS5B RNA‐dependent RNA polymerase have been the major targets. HCV variants displaying drug‐resistant phenotypes have been observed both in vitro and during clinical trials. Our aim was to characterize amino acid changes at positions previously associated with resistance in protease (NS3) and polymerase (NS5B) regions from treatment‐naïve HCV patients infected with genotypes 1a, 1b and 3a. All 1383 NS3 protease sequences (genotype 1a = 680, 1b = 498 and 3a = 205) and 806 NS5B polymerase sequences (genotypes 1a = 471, 1b = 329, 3a = 6) were collected from Los Alamos databank. Genotype 3a protease sequences showed the typical low‐level resistance mutation V36L. NS3 sequences from other genotypes presented mutations on positions 36, 39, 41, 43, 54, 80, 109, 155 and 168 in a frequency lower than 2%, except for the mutation Q80R found in 35% of genotype 1a isolates. Polymerase sequences from genotype 3a patients showed five typical mutations: L419I, I424V, I482L, V499A and S556G. Two positions presented high polymorphism in the NS5B region from genotype 1a (V499A) and genotype 1b (C316N) subjects. Our results demonstrated a natural profile of genotype 3a that can be associated with the pre‐existence of HCV variants resistant to first‐generation protease inhibitors and to non‐nucleoside polymerase inhibitors. Likewise, genotype 1b isolates and genotype 1a sequences exhibited pre‐existing mutations associated with resistance to Palm II and Thumb I polymerase inhibitors, respectively.

Rates of and Reasons for Failure of Commercial Human Immunodeficiency Virus Type 1 Viral Load Assays in Brazil

ABSTRACT We examined failures of commercial human immunodeficiency virus type 1 (HIV-1) viral load assays of 1,195 plasma samples from Brazilian patients. Assay failure was assumed for samples in which the virus was undetectable by commercial assay but which tested positive by real-time reverse transcription-PCR of the HIV-1 long terminal repeat (LTR) region or if the viral load differed by >2 log10 from that determined by LTR assay. Failure rates for Bayer Versant bDNA 3.0, Roche Amplicor Monitor v1.5, and bioMerieux NucliSens QT were 0.68, 0.47, and 4.33%, respectively. NucliSens may be inadequate for use in Brazil.

Protease inhibitor resistance mutations in untreated Brazilian patients infected with HCV: Novel insights about targeted genotyping approaches

Several new direct‐acting antiviral (DAA) drugs are being developed or are already approved for the treatment of chronic hepatitis C virus (HCV) infection. HCV variants presenting drug‐resistant phenotypes were observed both in vitro and during clinical trials. The aim of this study was to characterize amino acid changes at positions previously associated with resistance in the NS3 protease in untreated Brazilian patients infected with HCV genotypes 1a and 1b. Plasma samples from 171 untreated Brazilian patients infected with HCV were obtained from the Department of Gastroenterology of Clinics Hospital (HCFMUSP) in São Paulo, Brazil. Nested PCR and Sanger sequencing were used to obtain genetic information on the NS3 protein. Bioinformatics was used to confirm subtype information and analyze frequencies of resistance mutations. The results from the genotype analysis using non‐NS3 targeted methods were at variance with those obtained from the NS3 protease phylogenetic analyses. It was found that 7.4% of patients infected with HCV genotype 1a showed the resistance‐associated mutations V36L, T54S, Q80K, and R155K, while 5.1% of patients infected with HCV genotype 1b had the resistance‐associated mutations V36L, Q41R, T54S, and D168S. Notably, codons at positions 80 and 155 differed between samples from Brazilian patient used in this study and global isolates. The present study demonstrates that genotyping methods targeting the NS3 protein showed a difference of results when compared to mainstream methodologies (INNO‐LiPA and polymerase sequencing). The resistance mutations present in untreated patients infected with HCV and codon composition bias by geographical location warrant closer examination. J. Med. Virol. 86: 1714–1721, 2014.

Re-mapping the molecular features of the human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1 Brazilian sequences using a bioinformatics unit established in Salvador, Bahia, Brazil, to give support to the viral epidemiology studies

The analysis of genetic data for human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type 1 (HTLV-1) is essential to improve treatment and public health strategies as well as to select strains for vaccine programs. However, the analysis of large quantities of genetic data requires collaborative efforts in bioinformatics, computer biology, molecular biology, evolution, and medical science. The objective of this study was to review and improve the molecular epidemiology of HIV-1 and HTLV-1 viruses isolated in Brazil using bioinformatic tools available in the Laboratório Avançado de Sáude Pública (Lasp) bioinformatics unit. The analysis of HIV-1 isolates confirmed a heterogeneous distribution of the viral genotypes circulating in the country. The Brazilian HIV-1 epidemic is characterized by the presence of multiple subtypes (B, F1, C) and B/F1 recombinant virus while, on the other hand, most of the HTLV-1 sequences were classified as Transcontinental subgroup of the Cosmopolitan subtype. Despite the high variation among HIV-1 subtypes, protein glycosylation and phosphorylation domains were conserved in the pol, gag, and env genes of the Brazilian HIV-1 strains suggesting constraints in the HIV-1 evolution process. As expected, the functional protein sites were highly conservative in the HTLV-1 env gene sequences. Furthermore, the presence of these functional sites in HIV-1 and HTLV-1 strains could help in the development of vaccines that pre-empt the viral escape process.

The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

Prediction of CD8+ Epitopes in Leishmania braziliensis Proteins Using EPIBOT: In Silico Search and In Vivo Validation.

Background Leishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4+ and CD8+ T cell Line 1Lineepitopes. Identification of CD8+ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms. Methodology/Principal Findings We developed a two-phase algorithm and implemented in an open source software called EPIBOT, that consolidates the results obtained with single prediction algorithms, generating a final output in which epitopes are ranked. EPIBOT was initially trained using a set of 831 known epitopes from 397 proteins from IEDB. We then screened 63 Leishmania braziliensis vaccine candidates with the EPIBOT trained tool to search for CD8+ T cell epitopes. A proof-of-concept experiment was conducted with the top eight CD8+ epitopes, elected by EPIBOT. To do this, the elected peptides were synthesized and validated for their in vivo cytotoxicity. Among the tested epitopes, three were able to induce lysis of pulsed-target cells. Conclusion Our results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8+ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.

10-valent pneumococcal conjugate vaccine (PCV10) decreases metabolic activity but not nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae

BACKGROUND The effect of pneumococcal vaccination is widely variable when measured by nasopharyngeal carriage of vaccine and non-vaccine targets. The aim of this study was to compare the carriage rates and metabolic activity of Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Moraxella catarrhalis among children who were or were not vaccinated with PCV10. METHODS We included children with acute respiratory infection aged 6-23months from a cross-sectional study (CHIADO-IVAS). Nasopharyngeal aspirates were collected and respiratory pathogens were quantified by nCounter digital transcriptomics (Nanostring) and metagenomic sequencing of 16S ribosomal RNA (Illumina). The metabolic rate was calculated by the ratio between RNA transcripts and 16S DNA reads. RESULTS Out of the 80 patients in this study, 53 were vaccinated with PCV10 and 27 were unvaccinated. There was no difference in nasopharyngeal carriage rates of S. pneumoniae, S. aureus, H. influenzae or M. catarrhalis by either transcriptomic analysis or 16S metagenomics. However, unvaccinated children presented a higher metabolic rate for S. pneumoniae compared to PCV10-vaccinated children (Median [25-75th percentiles]: 126 [22.75-218.41] vs. 0[0-47.83], p=0.004). Furthermore, unvaccinated children presented a positive correlation between mRNA counts and 16S DNA reads for S. pneumoniae (r=0.707; p<0.001) and H. influenzae (r=0.525; p=0.005), in contrast to vaccinated children. No such effect was observed for S. aureus and M. catarrhalis. CONCLUSIONS Vaccination by PCV10 exerts a pathogen-specific effect on pneumococcal metabolic rate. Pathogen RNA/DNA ratio might represent a more sensitive readout for vaccine follow-up, as compared to nasopharyngeal carriage.

Meta-Analysis of Aedes aegypti Expression Datasets: Comparing Virus Infection and Blood-Fed Transcriptomes to Identify Markers of Virus Presence

The mosquito Aedes aegypti (L.) is vector of several arboviruses including dengue, yellow fever, chikungunya, and more recently zika. Previous transcriptomic studies have been performed to elucidate altered pathways in response to viral infection. However, the intrinsic coupling between alimentation and infection were unappreciated in these studies. Feeding is required for the initial mosquito contact with the virus and these events are highly dependent. Addressing this relationship, we reinterrogated datasets of virus-infected mosquitoes with two different diet schemes (fed and unfed mosquitoes), evaluating the metabolic cross-talk during both processes. We constructed coexpression networks with the differentially expressed genes of these comparison: virus-infected versus blood-fed mosquitoes and virus-infected versus unfed mosquitoes. Our analysis identified one module with 110 genes that correlated with infection status (representing ~0.7% of the A. aegypti genome). Furthermore, we performed a machine-learning approach and summarized the infection status using only four genes (AAEL012128, AAEL014210, AAEL002477, and AAEL005350). While three of the four genes were annotated as hypothetical proteins, AAEL012128 gene is a membrane amino acid transporter correlated with viral envelope binding. This gene alone is able to discriminate all infected samples and thus should have a key role to discriminate viral infection in the A. aegypti mosquito. Moreover, validation using external datasets found this gene as differentially expressed in four transcriptomic experiments. Therefore, these genes may serve as a proxy of viral infection in the mosquito and the others 106 identified genes provides a framework to future studies.

High-Quality Draft Genome Sequence of Bacillus amyloliquefaciens Strain 629, an Endophyte from Theobroma cacao

ABSTRACT Bacillus amyloliquefaciens strain 629 is an endophyte isolated from Theobroma cacao L. Here, we report the draft genome sequence (3.9 Mb) of B. amyloliquefaciens strain 629 containing 16 contigs (3,903,367 bp), 3,912 coding sequences, and an average 46.5% G+C content.

LeishDB: a database of coding gene annotation and non-coding RNAs in Leishmania braziliensis

Abstract Leishmania braziliensis is the etiological agent of cutaneous leishmaniasis, a disease with high public health importance, affecting 12 million people worldwide. Although its genome sequence was originally published in 2007, the two reference public annotations still presents at least 80% of the genes simply classified as hypothetical or putative proteins. Furthermore, it is notable the absence of non-coding RNA (ncRNA) sequences from Leishmania species in public databases. These poorly annotated coding genes and ncRNAs could be important players for the understanding of this protozoan biology, the mechanisms behind host-parasite interactions and disease control. Herein, we performed a new prediction and annotation of L. braziliensis protein-coding genes and non-coding RNAs, using recently developed predictive algorithms and updated databases. In summary, we identified 11 491 ORFs, with 5263 (45.80%) of them associated with proteins available in public databases. Moreover, we identified for the first time the repertoire of 11 243 ncRNAs belonging to different classes distributed along the genome. The accuracy of our predictions was verified by transcriptional evidence using RNA-seq, confirming that they are actually generating real transcripts. These data were organized in a public repository named LeishDB (www.leishdb.com), which represents an improvement on the publicly available data related to genomic annotation for L. braziliensis. This updated information can be useful for future genomics, transcriptomics and metabolomics studies; being an additional tool for genome annotation pipelines and novel studies associated with the understanding of this protozoan genome complexity, organization, biology, and development of innovative methodologies for disease control and diagnostics. Database URL: www.leishdb.com