Introduction and Objectives It is estimated that chronic hepatitis B virus (HBV) infection accounts for one million deaths/year due to cirrhosis and liver cancer. Currently, several drugs are used in the treatment of HBV; however, a complete cure is still controversial. The major challenge is the persistence of viral covalently closed circular DNA (cccDNA), as well as the ability of HBV to integrate into the host genome, which enables the infection's reactivation. Interfering RNA (RNAi) is a post-transcriptional mechanism of gene silencing and is a promising alternative for the treatment of chronic hepatitis B. We aimed to construct effective RNAi lentiviral vector to silencing HBV proteins (HBsAg, HBcAg, HBeAg) and pre-genomic RNA (pgRNA), via RNAi. Materials and Methods The silencing vector candidates targets overlapped Open Reading Frames (ORFs), allowing different viral proteins and the pgRNA to be silenced with a single RNAi. The efficiency of silencing by lentiviral vectors candidates used individually or in combination, have been assessed by quantification of HBV proteins by eletroquimioluminescence and quantification of HBV DNA during the post-transfection period by quantitative PCR. Results Three silencing vectors candidates were constructed and tested in silico to prevent off-target effects. Stability and secondary structures have also been tested. Huh7 cells were transfected with 1ug of purified HBV genome circular monomers (genotype A1) and 3 days later, infected with the first lentiviral candidate (siHBV-1), targeting S/Pol genes of HBV (108 TU/mL). From the third day post-infection, HBsAg became undetectable on cells infected by the lentiviral vectors, while untreated controls maintained viral protein expression (p<0.002). HBV DNA were also undetectable by PCR. Conclusions siHBV-1was able to silence HBV in vitro. This approach allows long- term, sustained knockdown of HBV replication and gene expression, which can effectively promote HBV clearance in chronic carriers.

Conflict of interest: No Introduction and Objectives: INTRODUCTION: Chronic hepatitis B represents a significant global health challenge. Current treatments often fail due to the persistence of HBV DNA as covalently closed circular DNA or integrated into the host genome, leading to viral reactivation. RNA interference (RNAi) emerges as a promising approach for treating chronic hepatitis through post-transcriptional gene silencing. OBJECTIVE: To design and evaluate the efficacy of RNAi lentiviral vectors targeting key HBV proteins (HBsAg, HBcAg, HBeAg) and pre-genomic RNA (pgRNA) for silencing. Patients / Materials and Methods: Silencing vectors were designed to target overlapping open reading frames, allowing simultaneous silencing of multiple viral proteins and pgRNA with a single RNAi construct. Three vector candidates (siHBV-1 to 3) underwent rigorous in silico testing to minimize off-target effects, evaluating stability and secondary structures. Lentiviral vector production was assessed using flow cytometry to detect green fluorescent protein expression in cell culture. Huh7 cells were transfected with 1 μg of purified HBV genotype A circular monomers, followed by infection with the first lentiviral candidate (siHBV-1), targeting S/Pol genes of HBV (108 TU/mL), three days later. Quantification of HBV proteins using chemiluminescence and HBV DNA using quantitative PCR (qPCR) was performed throughout the post-transfection period. Results and Discussion: Effective silencing of HBsAg expression was observed in cells infected with siHBV-1, with undetectable levels from the third day post-infection compared to untreated controls (p<0.002). Furthermore, HBV DNA was undetectable by qPCR, indicating successful silencing of HBV genotype A in vitro. The production of lentiviral candidates siHBV-2 and siHBV-3 is currently under evaluation. Flow cytometry will be used to determine the transduction rates in Huh7 cells once the tests are completed. Future investigations will explore combinations of these three siHBV vectors to optimize HBV silencing. Conclusions: Lentiviral vector-mediated RNAi offers a promising approach for sustained suppression of HBV replication and gene expression, potentially promoting HBV clearance in chronic carriers.

Considered a neglected pathogen until the last 2 decades, an increase in the availability of genetic data of hepatitis D virus (HDV) strains around the world has been noticed recently, resulting in the proposition of different classifications. Our study aimed to determine the molecular epidemiological profile of HDV isolates circulating in areas of endemicity and non-areas of endemicity in Brazil.

INTRODUCTION: Hepatitis B virus (HBV) infection is a public health problem; therefore, we aimed to report HBV genotypes in Ceará, Brazil. METHODS: A total of 103 HBsAg-positive samples were subjected to HBV genotyping and subgenotyping. RESULTS: The following genetic compositions of samples were found: F-54% (F2-83.33%), A-40% (A1-65%), D-6%, C2-1%, E-1%, and G-1%. CONCLUSIONS: Some genotypes are only prevalent in certain parts of the world; however, the State of Ceará is a hub for migration and has one of the most important liver transplantation centers in Brazil, which can explain the prevalence of the F genotype.

Hepatitis B virus (HBV) is an enveloped virus that induces chronic liver disease. HBV has been classified into eight genotypes (A-H) according to its genome sequence by using Sanger sequencing or reverse hybridization. Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants, whereas reverse hybridization detects only known mutations. Next-generation sequencing (NGS) is a robust tool for clinical virology with different protocols available. The objective of this study was to develop a new method for the study of viral genetic polymorphisms or more accurate genotyping using genome amplification followed by NGS. Plasma obtained from five chronically infected HBV individuals was used for viral DNA isolation. HBV full-genome PCR amplification was the enrichment method for NGS. Primers were used to amplify all HBV genotypes in three overlapping amplicons, following a tagmentation step and Illumina NGS. For phylogenetic analysis, sequences were extracted from the HBVdb database. We were able to amplify a full HBV genome; further, NGS was shown to be a robust method and allowed better genotyping, mainly in patients carrying mixed genotypes, classified according to other techniques. This new method may be significant for whole genome analyses, including other viruses.

Hepatitis A virus (HAV) outbreaks among men who have sex with men (MSM) have been reported worldwide and associated primarily with sexual transmission through oral-anal sex. Here, we provide the molecular and evolutionary description of a European strain, linked to HAV outbreaks among MSM, detected in a Brazilian homosexual couple. Bayesian analysis provided evidence that the viral isolates were introduced in Brazil from Spain between the end of 2016 and the beginning of 2017.

Hepatitis C virus genotype 1a (HCV-1a) comprises clades I and II. The Q80K polymorphism is found predominantly in clade I but rarely in clade II. Here, we investigated whether natural polymorphisms in HCV-1a clade II entailed structural protein changes when occurrence of the Q80K variant was simulated. Based on HCV-1a clade I and II protein sequences, the structure of the HCV-1a Q80K mutant NS3-4A was obtained by comparative modeling. Its physicochemical properties were studied by molecular dynamics simulations and network analysis. Results demonstrate that, in the presence of the K80 variant, clade II protease polymorphisms A91 and S/G174 led to variations in hydrogen bond occupancies. Structural analyses revealed differences in (i) flexibility of the H57 catalytic residue on the NS3 protease and (ii) correlations between amino acids on the NS3 protease and the NS4A cofactor. The latter indicated possible destabilization of interactions, resulting in increased separation of these proteins. The present findings describe how the relationships between different HCV-1a NS3 protease amino acid residues could affect the appearance of viral variants and the existence of distinct genetic barriers to HCV-1a isolates.

Hepatitis B virus (HBV) subgenotypes may be related to clinical outcomes and response to antiviral therapy. Most Brazilian studies on HBV subgenotypes are restricted to some regions and to specific population groups. Here, we provide an insight about genetic diversity of HBV subgenotypes in 321 serum samples from all five geographical regions, providing a representative overview of their circulation among chronic carriers. Overall, HBV/A1 was the most prevalent subgenotype, being found as the major one in all regions except in South Brazil. Among HBV/D samples, subgenotype D3 was the most prevalent, found in 51.5%, followed by D2 (27.3%) and D4 (21.2%). D2 and D3 were the most prevalent subgenotypes in South region, with high similarity with European strains. D4 was found in North and Northeast region and clustered with strains from Cape Verde and India. For HBV/F, the most frequent subgenotype was F2 (84.1%), followed by F4 (10.1%) and F1 (5.8%), closely related with strains from Venezuela, Argentina and Chile, respectively. Phylogeographic analyses were performed using an HBV full-length genome obtained from samples infected with genotypes rarely found in Brazil (B, C, and E). According to Bayesian inference, HBV/B2 and HBV/C2 were probably introduced in Brazil through China, and HBV/E from Guinea, all of them mostly linked to recent events of human migration. In conclusion, this study provided a comprehensive overview of the current circulation of HBV subgenotypes in Brazil. Our findings might contribute to a better understand of the dynamics of viral variants, to establish a permanent molecular surveillance on the introduction and dispersion patterns of new strains and, thus, to support public policies to control HBV dissemination in Brazil.

Background and aims: Treatment for hepatitis C has evolved significantly with the licensing of direct-acting antiviral drugs (DAAs). However, one of the limiting factors of the effectiveness of antiviral therapy with protease inhibitors (PIs) is the emergence of resistance caused by point mutations. The aim of this study was to determine the prevalence of resistance-associated substitutions (RASs) in HCV NS3 gene in patients inected with genotype 1 before therapy with simeprevir. Methods: A total of 73 serum samples from 15 treatment-experienced patients with boceprevir/telaprevir and 58 DAA-naive patients were collected before therapy with DAAs simeprevir, daclatasvir and/or sofosbuvir. Presence of baseline resistance-associated substitutions (RAS) in the serine protease domain of HCV NS3 was analyzed by nucleotide sequencing followed by amino acid deduction. Results: Overall RAS prevalence in this study was 13.7% (10/73). RAS prevalence for HCV subtype 1b was 17.4% (4/23) while for HCV subtype 1a was 12% (6/50). Primary mutations V36M/L and R155K were observed only in HCV subtype 1a, whereas T545 and Q80K were identified only in HCV subtype 1b. RAS V36M, which is related to reduction of susceptibility to second-generation Pls, was the most frequent in the study (6.9%; 5/73). Conclusions: Our results indicated that Brazilian isolates of HCV present a distinct pattern of RAS depending on the infecting viral subtype. In contrast to data from other countries, RAS Q80K prevalence in Brazil is low in HCV subtype la. This study improves the knowledge of genetic barrier for resistance to PIs involving RASs in chronically infected patients and its possible impact on an unsuccessful treatment outcome, information that might be crucial to upcoming decisions of incorporation of new DAAs in Brazilian guidelines of antiviral therapy against HCV infection. (C) 2019 Elsevier Masson SAS. All rights reserved.

Diabetes mellitus type 2 (DM2) patients have higher risk to be infected with parenterally transmitted viruses, like hepatitis B or C virus. This study aims to determine HBV and HCV infection prevalence in DM2 patients from Northeast and Southeast Brazil. A total of 537 DM2 patients were included, 194 (36.12%) males and 343 (63.87%) females, with mean age of 57.13±11.49 years. HBV and HCV markers were determined using serological and molecular analysis, and risk factors were evaluated in a subgroup from Southeast (n = 84). Two HBV acute (HBsAg+/anti-HBc -) and one HBV chronic case (HBsAg+/anti-HBc+) were found. Six individuals (1.1%) were isolated anti-HBc, 37 (6.9%) had HBV infection resolved (anti-HBc+/anti-HBs+), 40 (7.4%) were considered HBV vaccinated (anti-HBc-/anti-HBs+). Thirteen patients (2.42%) had anti-HCV and 7 of them were HCV RNA+. In the subgroup, anti-HBc positivity was associated to age and anti-HCV positivity was associated to age, time of diabetes diagnosis, total bilirubin, indirect bilirubin, alkaline phosphatase at bivariate analysis, but none of them was statistically significant at multivariate analysis. As conclusion, low prevalence of HBV and high prevalence HCV was found in DM2 patients.

The selection of viral strains with resistance-associated substitutions at hepatitis C virus (HCV) NS5A and NS5B genes is considered one of the limiting factors for achieving sustained virologic response (SVR) to combination of direct-acting antivirals daclatasvir (DCV) and sofosbuvir (SOF). Since 2015, this interferon-free regimen has been available in Brazilian clinical routine for treating mono- and HCV/HIV-coinfected patients chronically infected with genotypes 1 and 3. Our aim was to assess SVR rate for Brazilian patients chronically infected with genotypes 1 and 3 after DCV/SOF therapy and the frequency of baseline RASs in HCV NS5A and NS5B genes. Serum samples were collected from 107 monoinfected patients and 25 HCV/HIV co-infected patients before antiviral therapy with DCV/SOF. Genetic diversity of NS5A and NS5B genes was assessed by direct nucleotide sequencing. Overall, SVR rate was 95.4% (126/132), and treatment failure occurred in five monoinfected and one HCV/HIV co-infected patient. NS5A RASs frequency was higher for HCV/HIV patients (28%) than monoinfected patients (16.8%). No difference was evidenced between mono- and HCV/HIV-coinfected groups (15% vs. 16%) regarding NS5B gene. Genotype (GT) 1b strains had significantly more baseline substitutions in NS5A (31.6%) than GT 1a and 3a. At least one primary NS5A RAS described in literature at loci 28, 30, 31 or 93 was identified in HCV GTs 1 strains for both groups. As for NS5B, RASs at positions 159 and 316 was observed only in GT 1b strains. This study highlighted that SVR rate in clinical routine in Brazil was similar to randomized clinical trials (89-98%). Our research provided genetic data about the circulation of resistant variants in Brazil. Despite its presence, most of identified baseline mutations did not negatively impact treatment outcome. Genetic diversity of circulating strains suggested that most of the Brazilian HCV chronic carriers are susceptible to new therapeutic regimens including recently approved DAAs.

In Brazil, hepatitis C treatment has been evolving significantly with the licensing of direct-acting antivirals (DAAs). However, viral determinants (amino acid substitutions in hepatitis C virus (HCV) genome and infective genotype) associated with host factors (hepatic condition and prior HCV therapy) might limit the achievement of sustained virologic response (SVR). Here, we described two case reports in which the occurrence of HCV NS5A mutations A30K (subtype 3a) and Y93N (subtype 1a) might have influenced daclatasvir (DCV)/sofosbuvir (SOF) combined therapy non-response. Despite high response rates for DAA combined therapies in Brazil, these case reports stated the importance of an investigation about how to manage a DAA treatment failure since a combination of factors, especially the occurrence of resistance substitutions, could impact a rescue therapy with new available antivirals in clinical routine.

Hepatitis B virus (HBV) testing in oral fluid samples may provide advantages in diagnosis, screening or prevalence studies, especially among individuals with venous access difficulties. This study aims to optimize one commercially available assay for detecting total anti-HBc marker in oral fluid samples and to evaluate its utility under real life conditions in different settings for the purposes of prevalence and diagnostic studies. Oral fluid was collected using a Salivette device and some parameters were initially evaluated: type of elution buffer and sample volume. Thereafter, the utility of oral fluid samples for detection of anti-HBc was evaluated in real life conditions in which, 1296 individuals gave serum and oral fluid samples. All serum samples were submitted to commercial EIAs to detect total anti-HBc, according to the manufacturer’s instructions and oral fluid samples according to previous optimization. In optimization evaluation, PBS/BSA 0.5% and 100 μL of oral fluid (volume was two-fold increased compared to serum in EIA) were chosen as transport buffer and sample volume. In the field study, anti-HBc was detected in 211 out of 1296 serum samples giving overall oral fluid sensitivity of 52.6% and specificity of 96%. Concordance was higher in ambulatory setting (67.7) compared to general population (31.8). Mean ± standard deviation values of optical density/cutoff (OD/CO) in serum samples were higher in false-negative oral fluid samples than those seen in true positive samples. Sensitivity was higher in those presenting active infection compared to anti-HBc isolate and past infection. Sensitivity also increased in the ambulatory group when HCV individuals were excluded. It was possible to optimize a commercial EIA for detecting anti-HBc in oral fluid samples and where the highest concordance was found in ambulatory settings and among individuals with active infection.

This study aims to evaluate the utility of an optimized enzyme immunoassay (EIA) to detect and quantify antibodies against hepatitis B surface antibody (anti‐HBs) in dried blood spots (DBSs) within the context of human immunodeficiency virus (HIV) status. Serum and DBS samples were obtained from 56 HIV+ and 99 HIV− patients and subjected to EIA for the detection of anti‐HBs, where sample volume and cut off value were modified for DBS testing. Sensitivities of anti‐HBs detection in DBS were 79.8% and 76.8% in HIV− and HIV+ subjects, respectively. Concordant results for anti‐HBs in serum and DBS presented high mean CD8 cell counts, HIV viral load and optical density (OD) values of anti‐HBs. Anti‐HBs titers were significantly higher in serum, whether or not anti‐HBs titers were detected in DBS. It was possible to detect anti‐HBs in DBS as low as 17.4 and 27.3 IU/mL among HIV+ and HIV− subjects, respectively. In conclusion, DBS can be used to detect and quantify anti‐HBs in HIV‐infected individuals, which could increase access to diagnosis and vaccination.