Distribuição dos genótipos do vírus GB-C (HGV) em indivíduos da cidade de São Paulo, Brasil

Há na literatura vários estudos filogenéticos e de distribuição de genótipos do chamado "Vírus GB-C" ou da "Hepatite G", mais conhecido pela dupla sigla "GBV-C/HGV". Ocorre que, em sua grande maioria, estas pesquisas foram realizadas com amostras de grupos ligados epidemiologicamente e não com indivíduos representativos da população geral. O presente estudo é uma continuação do primeiro trabalho no Brasil feito com este tipo de amostragem. Trata-se de análise filogenética e distribuição genotípica do GBV-C/HGV a partir de amostras isoladas dentre mais de 1.000 indivíduos da cidade de São Paulo. Para tanto, um fragmento de 728 pares de base da região 5' não-codificadora (5´NCR) do genoma viral, de 24 amostras, foi sequenciado e submetido à analise filogenética. Foram identificados os genótipos 1, 2a e 2b nas respectivas freqüências: 8,3% (2/24), 50% (12/24) e 41,7% (10/24). Concluindo, São Paulo apresenta uma distribuição de genótipos semelhante à publicada para outros estados e regiões do Brasil, endossando a idéia de que os tipos 1 e 2 teriam vindo com os africanos e europeus, respectivamente, e portanto estariam na população do país desde a sua formação.There has been several studies worldwide on phylogenetics and genotype distribution of the GB-virus C / Hepatitis G virus (GBV-C/HGV). However, in their great majority, those investigations were based on some epidemiologically linked group, rather than on a representative sampling of the general population. The present is a continuation of the first study in Brazil with such a population; it addresses the GBV-C/HGV phylogenetics and genotype distribution based on samples identified among more than 1,000 individuals of the city of São Paulo. For this purpose, a 728 bp fragment of the 5´non-coding region (5´NCR) of the viral genome, from 24 isolates, was sequenced and subjected to phylogenetic analysis. Genotypes 1, 2a and 2b were found at 8.3% (2/24), 50% (12/24) and 41.7% (10/24), respectively. In conclusion São Paulo displays a genotype distribution similar to the published data for other States and Regions of Brazil, endorsing the notion that types 1 and 2 would have entered the country with African and European people, respectively, since its earliest formation

Effect of swab pooling on the Accula point-of-care RT-PCR for SARS-CoV-2 detection

IntroductionSwab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described.MethodsWe tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens.ResultsWe observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit.ConclusionClinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized

Visualization of positive and negative sense viral RNA for probing the mechanism of direct-acting antivirals against hepatitis C virus

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action

Nova estimativa da prevalência da infecção pelo vírus "TT" (TTV) em populações de baixo e alto risco de São Paulo, Brasil

A prevalência da infecção pelo vírus "TT" (TTV) foi investigada pela técnica da Reação da Polimerase em Cadeia (PCR) em grupos considerados de baixo risco (doadores de sangue e crianças/adolescentes saudáveis) e de alto risco de exposição parenteral (hemofílicos); todos provenientes da cidade de São Paulo. Oligonucleotídeos empregados como primers, homólogos à região não traduzível (UTR) do genoma viral, mostraram-se muito mais universais, revelando frequências muito mais altas em ambos os grupos ( >; ou = 81%) do que os primers anteriormente utilizados, baseados na região genômica traduzível "N22" (doadores de sangue, 5,5%, e hemofílicos, 42,3%). O "PCR-UTR" também revelou um perfil interessante em crianças/adolescentes saudáveis: alta prevalência nos primeiros anos de vida e queda significativa em meninos adolescentes. O "PCR-N22", por sua vez, apresentou alta frequência em hemofílicos que receberam derivados de sangue fresco (58%) relativa àqueles que foram tratados com fatores de coagulação submetidos à inativação viral (9,4%) e doadores de sangue (5,5%).The prevalence of TT virus (TTV) infection was investigated by Polymerase Chain Reaction (PCR) in low- (blood donors and healthy children/adolescents) and high-risk (hemophiliacs) groups from São Paulo, Brazil. Primers based on the untranslated region (UTR) of the viral genome proved to be much more ubiquitous, leading to much higher frequencies for both groups ( >; or = 81%) than the earlier N22-PCR directed to the open reading frame 1 (blood donors, 5.5%, and hemophiliacs, 42.3%). The UTR-PCR also revealed an interesting profile for healthy children/adolescents: very high prevalence at the early years and significant decrease in male teenagers. The N22-PCR, in turn, demonstrated higher frequency in hemophiliacs treated with fresh blood products (58%), than in those treated with virus-inactivated clotting factors (9.4%) and blood donors (5.5%)

Hepatitis B virus infection in Haemodialysis Centres from Santa Catarina State, Southern Brazil. Predictive risk factors for infection and molecular epidemiology.

BACKGROUND: Patients under haemodialysis are considered at high risk to acquire hepatitis B virus (HBV) infection. Since few data are reported from Brazil, our aim was to assess the frequency and risk factors for HBV infection in haemodialysis patients from 22 Dialysis Centres from Santa Catarina State, south of Brazil. METHODS: This study includes 813 patients, 149 haemodialysis workers and 772 healthy controls matched by sex and age. Serum samples were assayed for HBV markers and viraemia was detected by nested PCR. HBV was genotyped by partial S gene sequencing. Univariate and multivariate statistical analyses with stepwise logistic regression analysis were carried out to analyse the relationship between HBV infection and the characteristics of patients and their Dialysis Units. RESULTS: Frequency of HBV infection was 10.0%, 2.7% and 2.7% among patients, haemodialysis workers and controls, respectively. Amidst patients, the most frequent HBV genotypes were A (30.6%), D (57.1%) and F (12.2%). Univariate analysis showed association between HBV infection and total time in haemodialysis, type of dialysis equipment, hygiene and sterilization of equipment, number of times reusing the dialysis lines and filters, number of patients per care-worker and current HCV infection. The logistic regression model showed that total time in haemodialysis, number of times of reusing the dialysis lines and filters, and number of patients per worker were significantly related to HBV infection. CONCLUSIONS: Frequency of HBV infection among haemodialysis patients at Santa Catarina state is very high. The most frequent HBV genotypes were A, D and F. The risk for a patient to become HBV positive increase 1.47 times each month of haemodialysis; 1.96 times if the dialysis unit reuses the lines and filters > or = 10 times compared with haemodialysis units which reuse < 10 times; 3.42 times if the number of patients per worker is more than five. Sequence similarity among the HBV S gene from isolates of different patients pointed out to nosocomial transmission

Approaches for the development of antiviral compounds : the case of hepatitis C virus

Traditional methods for general drug discovery typically include evaluating random compound libraries for activity in relevant cell-free or cell-based assays. Success in antiviral development has emerged from the discovery of more focused libraries that provide clues about structure activity relationships. Combining these with more recent approaches including structural biology and computational modeling can work efficiently to hasten discovery of active molecules, but that is not enough. There are issues related to biology, toxicology, pharmacology, and metabolism that have to be addressed before a hit compound becomes nominated for clinical development. The objective of gaining early preclinical knowledge is to reduce the risk of failure in Phases 1, 2, and 3, leading to the goal of approved drugs that benefit the infected individual. This review uses hepatitis C virus (HCV), for which we still do not have an ideal therapeutic modality, as an example of the multidisciplinary efforts needed to discover new antiviral drugs for the benefit of humanity

New prevalence estimate of TT virus (TTV) infection in low- and high-risk population from São Paulo, Brazil

The prevalence of TT virus (TTV) infection was investigated by Polymerase Chain Reaction (PCR) in low- (blood donors and healthy children/adolescents) and high-risk (hemophiliacs) groups from São Paulo, Brazil. Primers based on the untranslated region (UTR) of the viral genome proved to be much more ubiquitous, leading to much higher frequencies for both groups ( > or = 81%) than the earlier N22-PCR directed to the open reading frame 1 (blood donors, 5.5%, and hemophiliacs, 42.3%). The UTR-PCR also revealed an interesting profile for healthy children/adolescents: very high prevalence at the early years and significant decrease in male teenagers. The N22-PCR, in turn, demonstrated higher frequency in hemophiliacs treated with fresh blood products (58%), than in those treated with virus-inactivated clotting factors (9.4%) and blood donors (5.5%)

Biophysics-Guided Lead Discovery of HBV Capsid Assembly Modifiers

Hepatitis B virus (HBV) is the leading cause of chronic liver pathologies worldwide. HBV nucleocapsid, a key structural component, is formed through the self-assembly of the capsid protein units. Therefore, interfering with the self-assembly process is a promising approach for the development of novel antiviral agents. Applied to HBV, this approach has led to several classes of capsid assembly modulators (CAMs). Here, we report structurally novel CAMs with moderate activity and low toxicity, discovered through a biophysics-guided approach combining docking, molecular dynamics simulations, and a series of assays with a particular emphasis on biophysical experiments. Several of the identified compounds induce the formation of aberrant capsids and inhibit HBV DNA replication in vitro, suggesting that they possess modest capsid assembly modulation effects. The synergistic computational and experimental approaches provided key insights that facilitated the identification of compounds with promising activities. The discovery of preclinical CAMs presents opportunities for subsequent optimization efforts, thereby opening new avenues for HBV inhibition