Any successful strategy to prevent and control HCV infection requires an understanding of the epidemic behaviour among the different genotypes. Here, we performed the first characterization of the epidemic history and transmission dynamics of HCV subtypes in Portugal. Direct sequencing of NS5B was performed on 230 direct-acting antiviral drugs (DAA)-treatment naïve patients in Lisbon. Phylogenetic analysis was used for subtyping and transmission cluster identification. Bayesian methods were used to reconstruct the epidemic history of HCV subtypes. Sequences were analysed for resistance-associated substitutions (RAS). The majority of strains were HCV-GT1 (62.6%), GT3 (18.3%, all subtype 3a) and GT4 (16.1%). Among GT1, the most frequent were subtypes 1a (75.5%) and 1b (24.5%). Polyphyletic patterns were found in all but 12 lineages suggesting multiple introductions of the different subtypes in this population. Five distinct epidemics were identified. The first significant HCV epidemic in Portugal occurred between 1930s and 1960s, was caused almost exclusively by GT1b and was likely associated with blood transfusions. Rapid expansion of GT3a occurred in the 1960s and GT1a in the 1980s, associated with intravenous drug use. The most recent epidemics were caused by GT4a and GT4d and seem to be associated with the resurgence of opioid use. The C316N substitution was found in 31.4% of GT1b-patients. Close surveillance of patients bearing this mutation and undergoing dasabuvir-based regimens will be important to determine its impact on treatment outcome.

This study is focused on the prevalent NS5 coding region resistance-associated substitutions (RASs) in DAA-naive genotype (GT)1 HCV-infected patients and their potential impact on success rates. Plasma RNA from 81 GT1 HCV-infected patients was extracted prior to an in-house nested RT-PCR of the NS5 coding region, which is followed by Sanger population sequencing. NS5A RASs were present in 28.4% (23/81) of all GT1-infected patients with 9.9% (8/81) having the Y93C/H mutation. NS5B RASs showed a prevalence of 14.8% (12/81) and were only detected in GT1b. Overall 38.3% (31/81) of all GT1 HCV-infected patients presented baseline RASs. The obtained data supports the usefulness of resistance testing prior to treatment since a statistically significant association was found between treatment failure and the baseline presence of specific NS5 RASs known as Y93C/H (p = 0.04).

In Portugal, the genetic diversity, origin of HBV and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A was the most prevalent (n = 54, 41.5%), followed by D (n = 44, 33.8%), and E (n = 32, 24.6%). Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV subgenotype D4 was the first to be introduced in Portugal around 1857 (HPD 95% 1699–1931) followed by D3 and A2 a few decades later. HBV genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results indicate a very important role of Portugal in the exportation of subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and provides new insights in the early and global epidemic history of this virus.

In 2015, 257 million people lived with chronic hepatitis B infection. In Portugal 1.4% of the population is chronically infected with hepatitis B virus. Limited information is available regarding the genetic diversity, origin of HBV in Portugal and its role in the dissemination of HBV worldwide. In this study, we aimed to investigate the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A (HBV/A) was the most prevalent genotype (n=54, 41.5%), followed by D [HBV/D; (n=44, 33.8%)], and E [HBV/E; (n=32, 24.6%)]. Spatio-temporal evolutionary dynamics were reconstructed using a Bayesian Markov Chain Monte Carlo method, as implemented in BEAST v1.10.4, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model under a Bayesian skyline plot and a continuous diffusion model. Our results indicate that HBV/D4 was the first subgenotype to be introduced in Portugal by the end of the XIX century, around 1857 (HPD 95% 1699 - 1931) followed by subgenotypes D3 and A2 a few decades later. Genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results also indicate a very important role of Portugal in the exportation of HBV subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and show that Portugal had an important role in the global spread of this virus.

This is a homemade protocol designed to amplify and sequence a 943bp fragment of the HBV pol gene. The polymerase region amplified here can be used to analyze drug resistance mutations. We use this protocol with HBV nucleic acid extracted from 200 µl of plasma sample, using the QIAamp DNA Blood Mini Kit (Qiagen, USA) following the manufacturer's procedure.