Genotipagem do vírus da hepatite B de pacientes crônicos com resistência ao tratamento com lamivudina na Cidade de Ribeirão Preto, Estado de São Paulo

INTRODUCTION: Lamivudine is a nucleoside analogue that is used clinically for treating chronic hepatitis B infection. However, the main problem with prolonged use of lamivudine is the development of viral resistance to the treatment. Mutations in the YMDD motif of the hepatitis B virus DNA polymerase gene have been associated with resistance to drug therapy. So far, there have not been many studies in Brazil reporting on genotype-dependent development of resistance to lamivudine. Thus, the aim of the present study was to determine the possible correlation between a certain genotype and increased development of resistance to lamivudine among chronic hepatitis B patients. METHODS: HBV DNA in samples from 50 patients under lamivudine treatment was amplified by means of conventional PCR. Samples were collected at Hospital das Clínicas, FMRP-USP. The products were then sequenced and phylogenetic analysis was performed. RESULTS: Phylogenetic analysis revealed that 29 (58%) patients were infected with genotype D, 20 (40%) with genotype A and one (2%) with genotype F. Mutations in the YMDD motif occurred in 20% of the patients with genotype A and 27.6% of the patients with genotype D. CONCLUSIONS: Despite the small number of samples, our results indicated that mutations in the YMDD motif were 1.38 times more frequent in genotype D than in genotype A.INTRODUÇÃO: Lamivudina é um análogo de nucleosídeo clinicamente utilizado para o tratamento da infecção crônica pela hepatite B. Entretanto, o principal problema do uso prolongado da lamivudina é o desenvolvimento de resistência viral ao tratamento. Mutações no motivo YMDD no gene da DNA polimerase do vírus da hepatite B estão associados com a resistência a terapia medicamentosa. Até o presente momento, não há muitos estudos no Brasil que descrevem o desenvolvimento genótipo-dependente da resistência à lamivudina. Assim, o intuito do trabalho aqui descrito foi determinar a possível correlação entre um determinado genótipo e o desenvolvimento aumentado da resistência à lamivudina em pacientes com hepatite B crônica. MÉTODOS: O HBV DNA foi amplificado por PCR convencional a partir de 50 amostras coletadas de pacientes submetidos ao tratamento com lamivudina no Hospital das Clínicas- FMRP- USP. Posteriormente, os produtos foram seqüenciados e a análise filogenética foi realizada. RESULTADOS: A análise filogenética mostrou que 29 (58%) pacientes foram infectados com o genótipo D, 20 (40%) com o genótipo A e 1 (2%) com o genótipo F. Mutações no motivo YMDD ocorreu em 20% dos pacientes com genótipo A e 27,6% em pacientes do genótipo D. CONCLUSÕES: Apesar do baixo número de amostras, nossos resultados indicaram que mutações no motivo YMDD são 1,38 X mais frequentes no genótipo D em relação ao genótipo A.Universidade de São Paulo USP - FCFR

Efficient error correction for next-generation sequencing of viral amplicons

<p>Abstract</p> <p>Background</p> <p>Next-generation sequencing allows the analysis of an unprecedented number of viral sequence variants from infected patients, presenting a novel opportunity for understanding virus evolution, drug resistance and immune escape. However, sequencing in bulk is error prone. Thus, the generated data require error identification and correction. Most error-correction methods to date are not optimized for amplicon analysis and assume that the error rate is randomly distributed. Recent quality assessment of amplicon sequences obtained using 454-sequencing showed that the error rate is strongly linked to the presence and size of homopolymers, position in the sequence and length of the amplicon. All these parameters are strongly sequence specific and should be incorporated into the calibration of error-correction algorithms designed for amplicon sequencing.</p> <p>Results</p> <p>In this paper, we present two new efficient error correction algorithms optimized for viral amplicons: (i) k-mer-based error correction (KEC) and (ii) empirical frequency threshold (ET). Both were compared to a previously published clustering algorithm (SHORAH), in order to evaluate their relative performance on 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. All three algorithms show similar accuracy in finding true haplotypes. However, KEC and ET were significantly more efficient than SHORAH in removing false haplotypes and estimating the frequency of true ones.</p> <p>Conclusions</p> <p>Both algorithms, KEC and ET, are highly suitable for rapid recovery of error-free haplotypes obtained by 454-sequencing of amplicons from heterogeneous viruses.</p> <p>The implementations of the algorithms and data sets used for their testing are available at: <url>http://alan.cs.gsu.edu/NGS/?q=content/pyrosequencing-error-correction-algorithm</url></p

Molecular characterization of influenza viruses collected from young children in Uberlandia, Brazil - from 2001 to 2010

Submitted by sandra infurna ([email protected]) on 2016-04-18T15:54:13Z No. of bitstreams: 1 marilda2_siqueira_etal_IOC_2015.pdf: 469385 bytes, checksum: 073d8213a1c0800966464156fabb2e73 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-18T16:05:32Z (GMT) No. of bitstreams: 1 marilda2_siqueira_etal_IOC_2015.pdf: 469385 bytes, checksum: 073d8213a1c0800966464156fabb2e73 (MD5)Made available in DSpace on 2016-04-18T16:05:32Z (GMT). No. of bitstreams: 1 marilda2_siqueira_etal_IOC_2015.pdf: 469385 bytes, checksum: 073d8213a1c0800966464156fabb2e73 (MD5) Previous issue date: 2015Universidade Federal de Uberlândia (UFU). Instituto de Ciências Biomédicas. Laboratório de Virologia. Uberlândia, MG, Brasil.Universidade Federal de Uberlândia (UFU). Instituto de Ciências Biomédicas. Laboratório de Virologia. Uberlândia, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Vírus Respiratórios. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Vírus Respiratórios. Rio de Janeiro, RJ, Brasil.Universidade Federal de Uberlândia (UFU). Faculdade de Medicina. Uberlândia, MG, Brasil.Universidade Federal de Uberlândia (UFU). Instituto de Ciências Biomédicas. Laboratório de Virologia. Uberlândia, MG, Brasil.Background: Influenza remains a major health problem due to the seasonal epidemics that occur every year caused by the emergence of new influenza virus strains. Hemagglutinin (HA) and neuraminidase (NA) glycoproteins are under selective pressure and subjected to frequent changes by antigenic drift. Therefore, our main objective was to investigate the influenza cases in Uberlândia city, Midwestern Brazil, in order to monitor the appearance of new viral strains, despite the availability of a prophylactic vaccine. Methods: Nasopharyngeal samples were collected from 605 children less than five years of age presenting with acute respiratory disease and tested by immunofluorescence assay (IFA) for detection of adenovirus, respiratory syncytial virus, parainfluenza virus types 1, 2, and 3 and influenza virus types A and B. A reverse transcription-PCR (RT-PCR) for influenza viruses A and B was carried out to amplify partial segments of the HA and NA genes. The nucleotide sequences were analyzed and compared with sequences of the virus strains of the vaccine available in the same year of sample collection. Results: Forty samples (6.6%) were tested positive for influenza virus by IFA and RT-PCR, with 39 samples containing virus of type A and one of type B. By RT-PCR, the type A viruses were further characterized in subtypes H3N2, H1N2 and H1N1 (41.0%, 17.9%, and 2.6%, respectively). Deduced amino acid sequence analysis of the partial hemagglutinin sequence compared to sequences from vaccine strains, revealed that all strains found in Uberlândia had variations in the antigenic sites. The sequences of the receptor binding sites were preserved, although substitutions with similar amino acids were observed in few cases. The neuraminidase sequences did not show significant changes. All the H3 isolates detected in the 2001-2003 period had drifted from vaccine strain, unlike the isolates of the 2004-2007 period. Conclusions: These results suggest that the seasonal influenza vaccine effectiveness could be reduced because of A H3N2 variants that circulated in 2001-2003 years. Thus, an early monitoring of variants circulating in the country or in a region may provide important information about the probable efficacy of the vaccine that will be administered in an influenza season

Identification and molecular characterization of the infectious bursal disease virus (IBDV) from an outbreak in a broiler flock in midwestern Brazil Identificação e caracterização molecular do vírus da doença infecciosa da bolsa cloacal de um surto em lotes de frangos no centro-oeste do Brasil

In order to identify and characterize the agent of a suggestive clinical case of Gumboro disease (GD) that affected a 34-day-old broiler flock in Buriti Alegre (Goias State, Midwestern Brazil) in the year 2001, we carried out a combination of classic and modern virological methods. Histopathological analysis of the bursa revealed necrosis, presence of depleted follicles, some infiltration of heterophils, edema and formation of cystic cavities that are compatible with lesions observed in GD. Inoculation of embryonated eggs of specific pathogen-free (SPF) chickens with macerated bursa suspension resulted in embryo mortality and lesions which were also compatible with those caused by IBDV. A sample of bursa was submitted to a nested reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to amplify the hypervariable region of the VP2 gene. The amplicon that was obtained from this sample (BR-GO) was digested with the restriction enzymes TaqI, StyI and SspI, but not with SacI, a pattern similar to that observed with very virulent IBDV (vvIBDV) strains. Furthermore, nucleotide sequence analysis revealed alanine, isoleucine, and isoleucine at amino acid positions 222, 256, and 294, respectively, which are also found in vvIBDV strains. Finally, phylogenetic analysis grouped BR-GO isolate with other vvIBDV strains.<br>Para identificar e caracterizar o agente causador de um quadro clínico sugestivo de doença de Gumboro (DG) que afetou um plantel de frangos de corte com 34 dias de idade, em Buriti Alegre (estado de Goiás, centro-oeste do Brasil), no ano de 2001, procedeu-se uma combinação de métodos virológicos clássicos e modernos. Análises histopatológicas de bursas revelaram necrose, depleção de folículos linfóides, infiltração de heterófilos, edema e formação de cistos, lesões compatíveis com DG. A inoculação em ovos embrionados de galinhas SPF (specific pathogen-free) de uma suspensão de macerado de amostras de bursas resultou em mortalidade embrionária e lesões macroscópicas compatíveis com aquelas provocadas pelo VDIB. Amostras de bursas foram submetidas à técnica de transcrição reversa-reação em cadeia da polimerase (RT-PCR), utilizando-se oligonucleotídeos específicos para amplificação da região hipervariável do gene da VP2. Essa reação produziu um fragmento do tamanho esperado, que foi digerido pelas enzimas de restrição TaqI, StyI e SspI, mas não foi digerido com SacI. Este padrão foi o mesmo observado com cepas de VDIB hipervirulentas (vvVDIB). Análise da seqüência nucleotídica revelou a presença dos aminoácidos alanina, isoleucina e isoleucina nas posições 222, 256 e 294, respectivamente, característica dessas cepas. Além disso, análise filogenética realizada agrupou a cepa encontrada, denominada de BR-GO, com outras cepas de vvVDIB

Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent

Human rhinoviruses (HRV) are usually associated with mild respiratory symptoms in children. However, some studies have found that HRV can cause severe disease, especially when the patient is co-infected with a second virus. In this study, 532 nasopharyngeal aspirates (NPAs) were collected over a nine-year period from children at the Clinics Hospital of Uberlândia. The collected NPAs were then tested for HRV RNA using the reverse transcription-polymerase chain reaction. Eighty-three specimens from children diagnosed with lower respiratory tract illness (LRTI) were positive for HRV RNA and were then tested for the presence of eight other respiratory viruses. A second virus was detected in 37.3% (31/83) of the samples. The most frequent clinical diagnosis was bronchiolitis, followed by other LRTI and then pneumonia. The frequency of severe disease in children infected with more than one virus was not significantly different from the frequency of severe disease in children infected with HRV alone. Children infected with both HRV and parainfluenza virus (1.5 m.o.) were significantly younger than those infected by HRV alone (5.0 m.o.) (p = 0.0454). Overall, these results suggest that infection with a second virus does not lead to a higher frequency of severe syndromes in children presenting with LRTI