Epidemic history of major genotypes of hepatitis C virus in Uruguay

Infection, Genetics and Evolution 32 (2015) 231–238

Contents lists available at ScienceDirect

Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid

Epidemic history of major genotypes of hepatitis C virus in Uruguay M. Castells a, G. Bello b, S. Ifrán c, S. Pereyra c, S. Boschi c, R. Uriarte c, J. Cristina d, R. Colina a,⇑ a

Laboratorio de Virología Molecular, Regional Norte, Centro Universitario de la Regional Noroeste, Universidad de la República, Rivera 1350, Salto, Uruguay Laboratorio de AIDS & Inmunología Molecular, Instituto Oswaldo Cruz – FIOCRUZ, Avenida Brasil 4365, Río de Janeiro, Brazil c Laboratorio de Biología Molecular, Asociación Española Primera de Socorros Mutuos, Boulevard Artigas 1515, Montevideo, Uruguay d Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo, Uruguay b

a r t i c l e

i n f o

Article history: Received 27 November 2014 Received in revised form 10 March 2015 Accepted 13 March 2015 Available online 20 March 2015 Keywords: Hepatitis C History Reconstruction Clades Evolution Uruguay

a b s t r a c t Worldwide, more than 170 million people are chronically infected with the hepatitis C virus (HCV) and every year die more than 350,000 people from HCV-related liver diseases. Recently, HCV was reclassified into seven major genotypes and 67 subtypes. Some subtypes as 1a, 1b and 3a, have become epidemic as a result of the new parenteral transmission routes and are responsible for most HCV infections in Western countries. HCV 1a subtype have been sub-categorized into two separate sub clades. Recent studies based on the analysis of NS5B genome region, reveal that HCV epidemics in Argentina and Brazil are characterized by multiple introductions events of subtypes 1a, 1b and 3a, followed by subsequent local dispersion. There is no data about HCV genotypes circulating in Uruguay and their evolutionary and demographic history. To this end, a total of 153 HCV NS5B gene sequences were obtained from Uruguayan patients between 2005 and 2011. 86 (56%) sequences grouped with subtype 1a, 40 (26%) with subtype 3a and 27 (18%) with subtype 1b. Furthermore, subtype 1a sequences were distributed among both clades, 1 (n = 62, 72%) and 2 (n = 24, 28%). Four local HCV clades were found: UY-1a(I), UY-1a(II), UY-1a(III) and UY-3a; comprising a 39% of all HCV viruses analyzed in this study. HCV epidemic in Uruguay has been driving by multiple introductions of subtypes 1a, 1b and 3a and by local dissemination of a few country-specific strains. The evolutionary and demographic history of the major Uruguayan HCV clade UY-1a(I) was reconstructed under two different molecular clock rate models and displayed an epidemic history characterized by an initial phase of rapid expansion followed by a more recent reduction of growth rate since 2000–2005. This is the first comprehensive study about the molecular epidemiology and epidemic history of HCV in Uruguay. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Worldwide, more than 170 million people are chronically infected with the hepatitis C virus (HCV) (Lavanchy, 2011) and every year die more than 350,000 people from HCV-related liver diseases (WHO, 2003). HCV is the major cause of fibrosis, cirrhosis and hepatocellular carcinoma (El-Serag, 2002; Poynard et al., 1997), and also the main indicator for liver transplantation (Sanyal, 2010; Baldo et al., 2008). HCV is mainly transmitted by parenteral route, being blood transfusion and intravenous drug use the most frequent risk factors (Alter, 2007; Shepard et al., 2005). Recently, HCV was reclassified into seven major genotypes and 67 subtypes (Smith et al., 2014). Moreover, HCV 1a subtype ⇑ Corresponding author at: Laboratorio de Virología Molecular, Regional Norte, Universidad de la República, Gral. Rivera 1350, 50000 Salto, Uruguay. Tel.: +598 473 34816; fax: +598 473 22154. E-mail address: [email protected] (R. Colina). http://dx.doi.org/10.1016/j.meegid.2015.03.021 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.

isolates have been sub-categorized into two separate sub clades (Pickett et al., 2011). Some subtypes as 1a, 1b and 3a, have become epidemic as a result of the new parenteral transmission routes and are responsible for most HCV infections in Western countries (Simmonds, 2013). A different pattern of sequence diversity is observed in Africa and South-East Asia where HCV has remained endemic for a long time and a much larger number of highly divergent HCV genotypes circulate at high prevalence (Simmonds, 2004, 2013). Latin American, with about eight million HCV-infected persons, has a relative low HCV prevalence (1.3%) in comparison with others geographic regions around the world; although the precise prevalence of this virus greatly vary between countries and between country regions (Szabo et al., 2012; Méndez-Sánchez et al., 2010). Genotypes 1 and 3 were the most prevalent HCV genotypes in Latin America (Kershenobich et al., 2011), similar to the pattern observed in most Western countries. Recent studies based mainly on the analysis of NS5B genome region, reveal that

232

M. Castells et al. / Infection, Genetics and Evolution 32 (2015) 231–238

HCV RNA extraction was carried out with TRIzol Reagent (Invitrogen) and High Pure RNA Isolation Kit (Roche Applied Science) as indicated by the manufacturers, starting from a volume of 140 ll of serum sample for each extraction protocol. Random hexamers, SuperScript II reverse transcriptase enzyme (Invitrogen) and 10 ll of total RNA were used to obtain 50 ll of cDNA. A fragment of 386 nucleotides of the NS5B gene was amplified by hemi-nested PCR as described elsewhere (Cantaloube et al., 2005), by adding 5 ll of cDNA to the first PCR round reaction mix and then from this, 1 ll of PCR product was added to the second PCR round reaction mix, with a final volume 25 ll in both PCR rounds. Direct sequencing was performed in both DNA senses by using second round PCR primers. Consensus sequences were obtained by alignment of both sequenced strands (sense and antisense) with SeqMan program (Swindell and Plasterer, 1997). 2.2. Worldwide HCV reference sequences

Fig. 1. Maximum Likelihood phylogenetic tree for the genotyping of the three major subtypes circulating in Uruguayan HCV positive samples 1a, 1b and 3a is shown. Subtype 1a, 1b and 3a sequences are represented in blue, red and green respectively. All the reference genomes are represented in black lines. The aLRT values for the critical nodes are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

HCV epidemics in Argentina and Brazil are characterized by multiple introductions events of subtypes 1a, 1b and 3a, followed by subsequent local dispersion of some of those viral strains (Culasso et al., 2012; del Pino et al., 2013; Lampe et al., 2013; Ré et al., 2011). Uruguay is a small country from the Southern cone, bordered by Argentina and Brazil, with a population of about 3.2 million inhabitants (Instituto Nacional de Estadística, 2011). It has been estimated that around 0.4% of Uruguayan blood bank donors population is seropositive for HCV (DLSP, 2002) but it can increase up to 8.8% in non-injecting drug users (Caiaffa et al., 2011) and 10.1% in intravenous drug users (Osimani et al., 2005). There is no data published about the most prevalent HCV genotypes circulating in Uruguay and their evolutionary and demographic history. To this end, we obtained a total of 153 HCV NS5B gene sequences from serologically HCV positives Uruguayan patients between 2005 and 2011. These sequences were subjected to phylogenetic analyses together with reference HCV sequences isolated worldwide to identify Uruguayan-specific HCV clusters and a Bayesian coalescent-based method was then used to investigate the evolutionary and population history of the largest Uruguayan-specific HCV lineage identified. 2. Materials and methods 2.1. Uruguayan HCV sequences Serum samples were collected from 2005 to 2011 from serologically HCV positives Uruguayan patients. All patients were followed by the Asociación Española Primera de Socorros Mutuos (AEPSM) at Montevideo city, and provided an informed consent in accordance with national ethical regulations and the Declaration of Helsinki. AESPM is a reference center for HCV viral load measurement, receiving samples from patients that live all across the country. It was not possible to include a fully information about the group of patients involved in this study such as age, gender, exact localization, treatment or risk groups.

HCV NS5B gene sequences from different genotypes and subtypes were obtained from the Los Alamos HCV database (Kuiken et al., 2005, 2008). In addition, NS5B sequences of HCV subtype 1a (n = 582), 1b (n = 330), and 3a (n = 306) that were representative of different geographic regions around the world (Lampe et al., 2013) were also included. 2.3. Phylogenetic analysis HCV sequences were aligned with Clustal program (Tamura et al., 2011), generating a sequence alignment of 331 nucleotides covering positions 8297–8627 of the reference sequence H77 (GenBank accession number AF009606). A sliding window analysis of distances approach implemented in the SimPlot program (Lole et al., 1999) was used to detected inter-genotype or inter-subtype recombination. No recombinant HCV strains were found among sequences here included (not shown). The model of nucleotide substitution that best fit the different datasets (GTR + G4 + I) was selected using the JModelTest program (Posada, 2008) according to the Akaike Information Criterion (AIC; Akaike, 1974). Maximum Likelihood (ML) phylogenetic trees were reconstructed with PhyML program (Guindon and Gascuel, 2003) using an online web server (Guindon et al., 2010). The branches support was estimated with the approximate likelihood-ratio test (aLRT) (Anisimova and Gascuel, 2006). 2.4. Evolutionary and demographic reconstructions The time of most recent common ancestor (tMRCA) and the demography history of the major Uruguayan HCV clade were jointly estimated using BEAST v1.7.5 package (Drummond et al., 2012). Bayesian analyses were conducted using a Bayesian Skyline coalescent tree prior (Drummond et al., 2005), the GTR + G4 + I nucleotide substitution model and a lognormal relaxed (uncorrelated) molecular clock model (Ho et al., 2005; Drummond et al., 2006). Two substitution rates previously estimated for the NS5B gene (5  10 4–7  10 4 substitutions/site/ year (Pybus et al., 2001; Tanaka et al., 2002) and 7  10 4– 2  10 3 substitutions/site/year (Magiorkinis et al., 2009)) were used as priors. A Markov Chain Monte Carlo (MCMC) was run for 10  107 generations and the results were visualized with Tracer v1.5.0 program (available from http://beast.bio.ed.ac.uk/Tracer) discarding the initial 10% of the run as burn in. The effective sample size (ESS) values were checked to evaluate the convergence of the analysis, accepting only values higher than 200 for all the parameters. The effective number of infections was represented graphically with Tracer v1.5.0.

M. Castells et al. / Infection, Genetics and Evolution 32 (2015) 231–238

233

Fig. 2a. Maximum Likelihood phylogenetic tree of subtype 1a performed with the Uruguayan sequences and the dataset with the world representative sequences. Black points and numbers 1 and 2 indicate the nodes that separate the clades identified by Pickett et al., 2011, respectively. With red are represented the Uruguayan sequences belonging to the clade 1 and in blue the Uruguayan sequences belonging to the clade 2. The curved line named UY-1a(I) indicates the monophyletic Uruguayan cluster. With curved line red and blue named UY-1a(II) and UY-1a(III) respectively are indicated the minority Uruguayan monophyletic clusters belonging to the clade 1 and 2 respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3. Results 3.1. Subtypes 1a, 3a and 1b are the predominant HCV clades in Uruguay Of 209 serum samples analyzed at this study, 153 were successfully amplified, sequenced and determined as subtype 1a, 1b or 3a. According to the ML phylogenetic analysis with reference sequences for all the genotypes and subtypes currently available at the Los Alamos Database, 86 (56%) of HCV Uruguayan samples grouped with the subtype 1a, 40 (26%) grouped with subtype 3a and 27 (18%) grouped with the subtype 1b (Fig. 1).

3.2. Multiple introductions of HCV subtypes 1a, 1b and 3a into Uruguay In order to assess whether the Uruguayan HCV epidemic mainly resulted from local dispersion of few founder strains or have resulted from multiple independent entries, Uruguayan HCV sequences here obtained were combined with subtype 1a, 1b and 3a references sequences from different geographic origins and that were previously described (Lampe et al., 2013). The ML analyses revealed multiple independent introductions (n > 15) of each major epidemic subtype into the local population (Fig. 2). Subtype 1a Uruguayan sequences were distributed among both

234

M. Castells et al. / Infection, Genetics and Evolution 32 (2015) 231–238

Fig. 2b. Maximum Likelihood phylogenetic tree of subtype 3a performed with the Uruguayan sequences and the dataset with the world representative sequences. With red are represented the Uruguayan sequences. The curved line named UY-3a indicates the monophyletic Uruguayan cluster. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

clades 1 (n = 62, 72%) and 2 (n = 24, 28%) described by Pickett et al. (2011). A large fraction of Uruguayan subtype 1a sequences (n = 49, 57%) branched in three highly supported (aLRT > 0.90) country-specific monophyletic clusters here called UY-1a(I) (n = 35, 41%), UY-1a(II) (n = 8, 9%) and UY-1a(III) (n = 6, 7%) (Fig. 2a). Clusters UY-1a(I) and UY-1a(II) branched within clade 1, whereas cluster UY-1a(III) was nested within clade 2 (Fig. 2a). The remaining Uruguayan subtype 1a sequences (n = 37, 43%) were dispersed among multiple independent lineages of small size (n 6 3) that were intermixed among sequences of nonUruguayan origin (Fig. 2a). An important fraction of Uruguayan

subtype 3a sequences (n = 10, 25%) also branched within a single country-specific monophyletic clade here called UY-3a (aLRT = 0.76), whereas the remaining lineages (n = 30, 75%) were distributed among multiple independent lineages of small size (n 6 2) (Fig. 2b). All Uruguayan subtype 1b sequences were dispersed among multiple lineages of small size (n 6 3) (Fig. 2c). 3.3. Evolutionary and demographic history of local cluster UY-1a(I) In order to better characterize the HCV epidemic in Uruguay, we reconstructed the evolutionary and demographic history of major

M. Castells et al. / Infection, Genetics and Evolution 32 (2015) 231–238

235

Fig. 2c. Maximum Likelihood phylogenetic tree of subtype 1b performed with the Uruguayan sequences and the dataset with the world representative sequences. With red are represented the Uruguayan sequences. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

cluster UY-1a(I) using the Bayesian Skyline plot method implemented in BEAST package. Evolutionary and demographic parameters was reconstructed under two different priors intervals for NS5B gene substitution rate: a ‘‘slow’’ molecular clock rate (5  10 4–7  10 4 substitutions/site/year) estimated by Pybus et al. (2001), Tanaka et al. (2002), and a ‘‘fast’’ clock rate (7  10 4–2  10 3 substitutions/site/year) estimated by Magiorkinis et al. (2009) (Table 1). The analysis under the ‘‘slow rate’’ model indicates that cluster UY-1a(I) began to spread among Uruguayan population in 1989 (1979–1997, 95% HPD) and showed a period of exponential growth until the early 2000s, after which

growth rate seems to stabilize (Fig. 3a). According to the ‘‘fast rate’’ model, cluster UY-1a(I) began to spread among Uruguayan population in 2000 (1993–2005, 95% HPD) and showed a period of exponential growth until 2005, after epidemic which growth rate stabilize (Fig. 3b). 4. Discussion This is the first comprehensive study about the molecular epidemiology and epidemic history HCV in Uruguay. The most prevalent HCV genotypes and subtypes circulating in Uruguay were 1a

236

M. Castells et al. / Infection, Genetics and Evolution 32 (2015) 231–238

Table 1 Bayesian coalescent inference of cluster UY-1a isolated in Uruguay. The years to the MRCA and the mean rate for the two runs performed at this study are shown. Rate (0.7–2.0) E (5–7) E

4

3

Parameter

Value (mean)

Value (median)

HPD

ESS

Years to the MRCA Mean rate

10.92 1.40 E 3

10.06 1.42 E 3

5.99–17.82 7.46 E 4–2.00 E 3

2,226.44 3,837.00

Years to the MRCA Mean rate

21.92 6.08 E 4

21.14 6.09 E 4

13.92–32.04 4.84 E 4–7.33 E 4

3,691.72 8,319.30

(56%), 3a (26%) and 1b (18%), a pattern similar to that described in other countries from South America (Culasso et al., 2012; di Filippo et al., 2012; Di Lello et al., 2009; Jaspe et al., 2012; Lampe et al., 2010, 2013). When prevalence of subtype 1a subclades described by Pickett et al. (2011) was assessed, however, some important differences emerge among countries. Whereas nearly all (98%) Brazilian HCV subtype 1a viruses belong to clade 1 (Lampe et al., 2013), Uruguayan subtype 1a viruses were more evenly distributed among clades 1 (72%) and 2 (28%). This supports a greater genetic diversity of HCV subtype 1a epidemic in Uruguay than that observed in Brazil, which could have important consequence for treatment because potential differences in the response to treatment between both subtype 1a clades has been described (Peresda-Silva et al., 2012).

Fig. 3. Bayesian Skyline plots showing the epidemic history of HCV Uruguayan cluster Uru-1a, reconstructed using ‘‘slow’’ (a) and ‘‘fast’’ (b) rate estimates as a prior on the substitution rate for NS5B. Median (dark line) and upper and lower 95% HPD (light lines) estimates of effective population size (Y-axis) through time in years (X-axis) are shown in each graphic.

The molecular complexity of the HCV epidemic in Uruguay is related to the existence of multiple introductions of this virus in the country, as has been previously observed in Argentina and Brazil (Culasso et al., 2012; del Pino et al., 2013; Lampe et al., 2010, 2013; Ré et al., 2011) and this could be an intrinsic characteristic of the HCV epidemics in most Western countries. Despite multiple subtype 1a, 1b and 3a strains has been introduced in the Uruguayan population, a few local HCV clades were detected. The four largest Uruguayan lineages, here called UY-1a(I), UY-1a(II), UY-1a(III) and UY-3a comprise together 39% of all HCV viruses analyzed in this study, thus indicating that an important proportion of HCV infections in Uruguay resulted from the local dispersion of a few subtype 1a and 3a viral strains. It is interesting to note that the proportion of HCV sequences belonging to those major lineages have been increasing over time from 3.9% in 2005/2006 to 17.0% in 2010/2011. Analysis of the risk factors for HCV infection in those patients belonging to major Uruguayan-specific HCV clades may be of paramount importance to stop or at less decrease the dispersion of the virus in the country. The evolutionary and demographic history of the major Uruguayan HCV clade UY-1a(I) was reconstructed under two different molecular clock rate models. The ‘‘slow rate model’’ indicates that cluster UY-1a(I) began to spread among Uruguayan population around 1989, thus preceding the introduction of antiHCV screening tests in Uruguayan blood banks at 1995 (Galzerano, 2002). According to this model, the HCV clade UY1a(I) continuous to growth exponentially until the early 2000s, after which growth rate seems to stabilize. Of note, such stabilization coincides with a restructuration of the National Drug Board and the National Drug Secretary (Junta Nacional de Drogas, JND, 2000) that since 2000 onwards has being working in the implementations of prevention campaigns against injecting drugs use, public awareness of the risks of acquire HCV and other viral infections, and started the treatment of HCV in people affected by drug use. The ‘‘fast rate model’’, as expected, supports a much more recent onset date of clade UY-1a(I) at around 2000 and further suggests that this Uruguayan clade continuous to growth exponentially until 2005. According to this model, the clade UY-1a(I) started to be disseminated among Uruguayan population several years later than the implementation of the HCV screening system in blood banks. The stabilization of the effective number of infections of UY-1a(I) clade around 2005 is also consistent with the notion that the restructuration of the National JND could have been decisive for the control of expansion of this clade. Although restructuration of the JND in Uruguay began in the 2000, the effects of the information and prevention campaigns should appear some years later (Junta Nacional de Drogas, 2001). Thus, both molecular clock rates models supports the hypothesis that expansion of the major subtype 1a clade may have been mainly driven by transmission through injecting drug use networks. This hypothesis could not be formally tested because we have no epidemiological information about HCV-infected patients included in this study; but could provide an important framework for implementation of prevention campaigns to reduce the number of new HCV infections; particularly for subtype 1a.

M. Castells et al. / Infection, Genetics and Evolution 32 (2015) 231–238

In addition to the limitation about the lack of epidemiological data, it is important to mention that PCR protocols used in the present work was designed and tested, mostly with samples from Europe. It is possible that in South American region, HCV can accumulate mutations on the primers region and as a consequence decrease the sensitivity of the test. Further studies will needed in order to have the full evolutionary picture of HCV in Uruguay, nevertheless, the present work answer important questions about the molecular evolution of HCV in this country. Furthermore, it is important to mention a study where the authors describe a possible confusion in using Bayesian Skyline plot (BSP) to infer demographic history (Heller et al., 2013). Nevertheless, viruses may be outside of the type of population structure mentioned as problematic in that study, taking into accounts the biological properties of the viral replication cycles. In summary, this study showed that HCV epidemic in Uruguay has been driving by multiple introductions of subtypes 1a, 1b and 3a and by local dissemination of a few country-specific strains. The major Uruguayan HCV clade here detected belongs to subtype 1a and displayed an epidemic history characterized by an initial phase of rapid expansion followed by a more recent reduction of growth rate since 2000–2005. These results suggest that public health actions for diagnosis, treatment and prevention of HCV infections may have had a transcendental impact in the transmission dynamics of major local HCV clades circulating in Uruguay. The information gathered at this work offer important insights for understanding the dynamic of the HCV epidemic in a region were no previous studies were performed previously. Acknowledgments This work was supported by projects from: Program ‘‘Polo de Desarrollo Universitario’’ (PDU), Universidad de República (UdelaR), Uruguay. Project CSIC I + D 2010, Universidad de la República (UdelaR). ANII-ALI 1603, from the Agencia Nacional de Investigación e Innovación (ANII), Uruguay. M.C. acknowledges financial support for Master studies from the Sistema Nacional de Becas, Agencia Nacional de Investigación e Innovación (ANII), Uruguay. References Akaike, H., 1974. A new look at the statistical model identification. IEEE Trans. Autom. Control 19, 716–723. Alter, M.J., 2007. Epidemiology of hepatitis C virus infection. World J. Gastroenterol. 14 (17), 2436–2441. Anisimova, M., Gascuel, O., 2006. Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Syst. Biol. 55 (4), 539–552. Baldo, V., Baldovin, T., Trivello, R., Floreani, A., 2008. Epidemiology of HCV infection. Curr. Pharm. Des. 14 (17), 1646–1654. Caiaffa, W.T., Zocratto, K.F., Osimani, M.L., Martínez, P.L., Radulich, G., Latorre, L., Muzzio, E., Segura, M., Chiparelli, H., Russi, J., Rey, J., Vazquez, E., Cuchi, P., SosaEstani, S., Rossi, D., Weissenbacher, M., 2011. Hepatitis C virus among noninjecting cocaine users (NICUs) in South America: can injectors be a bridge? Addiction. 106 (1), 143–151. Cantaloube, J.F., Gallian, P., Attoui, H., Biagini, P., De Micco, P., de Lamballerie, X., 2005. Genotype distribution and molecular epidemiology of hepatitis C virus in blood donors from southeast France. J. Clin. Microbiol. 43 (8), 3624–3629. Culasso, A.C., Elizalde, M., Campos, R.H., Barbini, L., 2012. Molecular survey of hepatitis C virus in the touristic city of Mar del Plata, Argentina. PLoS ONE 7 (9), e44757. del Pino, N., Oubiña, J.R., Rodríguez-Frías, F., Esteban, J.I., Buti, M., Otero, T., Gregori, J., García-Cehic, D., Camos, S., Cubero, M., Casillas, R., Guàrdia, J., Esteban, R., Quer, J., 2013. Molecular epidemiology and putative origin of hepatitis C virus in random volunteers from Argentina. World J. Gastroenterol. 19 (35), 5813–5827. di Filippo, D., Cortes-Mancera, F., Beltran, M., Arbelaez, M.P., Jaramillo, S., Restrepo, J.C., Correa, G., Navas, M.C., 2012. Molecular characterization of hepatitis c virus in multi-transfused Colombian patients. Virol. J. 23 (9), 242. Di Lello, F.A., Piñeiro, Y., Leone, F.G., Muñoz, G., Campos, R.H., 2009. Diversity of hepatitis B and C viruses in Chile. J. Med. Virol. 81 (11), 1887–1894. Departamento de laboratorio de salud pública (DLSP). Boletín epidemiológico. Uruguay. Ministerio de Salud Pública, 2002.

237

Drummond, A.J., Ho, S.Y., Phillips, M.J., Rambaut, A., 2006. Relaxed phylogenetics and dating with confidence. PLoS Biol. 4 (5), e88. Drummond, A.J., Rambaut, A., Shapiro, B., Pybus, O.G., 2005. Bayesian coalescent inference of past population dynamics from molecular sequences. Mol. Biol. Evol. 22 (5), 1185–1192. Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29 (8), 1969–1973. El-Serag, H.B., 2002. Hepatocellular carcinoma: an epidemiologic view. J. Clin. Gastroenterol. 35 (5 Suppl 2), S72–S78. Galzerano J. 2002. Available at: . Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59 (3), 307–321. Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52 (5), 696–704. Heller, R., Chikhi, L., Siegismund, H.R., 2013. The confounding effect of population structure on Bayesian skyline plot inferences of demographic history. PLoS ONE 8 (5), e62992. Ho, S.Y., Phillips, M.J., Drummond, A.J., Cooper, A., 2005. Accuracy of rate estimation using relaxed-clock models with a critical focus on the early metazoan radiation. Mol. Biol. Evol. 22 (5), 1355–1363. Instituto Nacional de Estadística. 2011. Available at: . Jaspe, R.C., Sulbarán, Y.F., Sulbarán, M.Z., Loureiro, C.L., Rangel, H.R., Pujol, F.H., 2012. Prevalence of amino acid mutations in hepatitis C virus core and NS5B regions among Venezuelan viral isolates and comparison with worldwide isolates. Virol. J. 21 (9), 214. Junta Nacional de Drogas. 2000. Available at: . Junta Nacional de Drogas. Evaluación del progreso de control de drogas. 2001. Available at: . Kershenobich, D., Razavi, H.A., Sánchez-Avila, J.F., Bessone, F., Coelho, H.S., Dagher, L., Gonçales, F.L., Quiroz, J.F., Rodriguez-Perez, F., Rosado, B., Wallace, C., Negro, F., Silva, M., 2011. Trends and projections of hepatitis C virus epidemiology in Latin America. Liver Int. 31 (Suppl 2), 18–29. Kuiken, C., Hraber, P., Thurmond, J., Yusim, K., 2008. The hepatitis C sequence database in Los Alamos. Nucleic Acids Res., 36 Kuiken, C., Yusim, K., Boykin, L., Richardson, R., 2005. The Los Alamos hepatitis C sequence database. Bioinformatics 21 (3), 379–384. Lampe, E., Espirito-Santo, M.P., Martins, R.M., Bello, G., 2010. Epidemic history of Hepatitis C virus in Brazil. Infect Genet Evol. 10 (7), 886–895. Lampe, E., Lewis-Ximenez, L., Espírito-Santo, M.P., Delvaux, N.M., Pereira, S.A., Peres-da-Silva, A., Martins, R.M., Soares, M.A., Santos, A.F., Vidal, L.L., Germano, F.N., de Martinez, A.M., Basso, R., Pinho, J.R., Malta, F.M., Gomes-Gouvêa, M., Moliterno, R.A., Bertolini, D.A., Fujishima, M.A., Bello, G., 2013. Genetic diversity of HCV in Brazil. Antivir. Ther. 18 (3 Pt B), 435–444. Lavanchy, D., 2011. Evolving epidemiology of hepatitis C virus. Clin. Microbiol. Infect. 17 (2), 107–115. Lole, K.S., Bollinger, R.C., Parnjape, R.S., Gadkari, D., Kulkarni, S.S., 1999. Full length human immunodeficiency virus type I genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol. 73, 152–160. Magiorkinis, G., Magiorkinis, E., Paraskevis, D., Ho, S.Y., Shapiro, B., Pybus, O.G., Allain, J.P., Hatzakis, A., 2009. The global spread of hepatitis C virus 1a and 1b: a phylodynamic and phylogeographic analysis. PLoS Med. 6 (12), e1000198. Méndez-Sánchez, N., Gutiérrez-Grobe, Y., Kobashi-Margáin, R.A., 2010. Epidemiology of HCV infection in Latin America. Ann. Hepatol. 9 (Suppl), 27–29. Osimani, M.L., Latorre, L., Garibotto, G., Scarlatta, L., Chiparelli, H., Vidal, J., 2005. VIH, Hepatitis B, Hepatitis C y VDRL en usuarios de cocaína no inyectable en Uruguay. Adicciones: Revista de socidrogalcohol 17 (2), 157–162. Peres-da-Silva, A., Almeida, A.J., Lampe, E., 2012. Genetic diversity of NS3 protease from Brazilian HCV isolates and possible implications for therapy with directacting antiviral drugs. Mem. Inst. Oswaldo Cruz 107 (2), 254–261. Pickett, B.E., Striker, R., Lefkowitz, E.J., 2011. Evidence for separation of HCV subtype 1a into two distinct clades. J. Viral Hepat. 18 (9), 608–618. Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25 (7), 1253–1256. Poynard, T., Bedossa, P., Opolon, P., 1997. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 349 (9055), 825–832. Pybus, O.G., Charleston, M.A., Gupta, S., Rambaut, A., Holmes, E.C., Harvey, P.H., 2001. The epidemic behavior of the hepatitis C virus. Science 292 (5525), 2323– 2325. Ré, V.E., Culasso, A.C., Mengarelli, S., Farías, A.A., Fay, F., Pisano, M.B., Elbarcha, O., Contigiani, M.S., Campos, R.H., 2011. Phylodynamics of hepatitis C virus subtype 2c in the province of Córdoba, Argentina. PLoS ONE 6 (5), e19471. Sanyal, A.J., 2010. The Institute of Medicine report on viral hepatitis: a call to action. Hepatology 51, 727–728. Shepard, C.W., Finelli, L., Alter, M.J., 2005. Global epidemiology of hepatitis C virus infection. Lancet Infect. Dis. 5 (9), 558–567. Simmonds, P., 2004. Genetic diversity and evolution of hepatitis C virus–15 years on. J. Gen. Virol. 85 (Pt 11), 3173–3188. Simmonds, P., 2013. The origin of hepatitis C virus. Curr. Top. Microbiol. Immunol. 369, 1–15.

238

M. Castells et al. / Infection, Genetics and Evolution 32 (2015) 231–238

Smith, D.B., Bukh, J., Kuiken, C., Muerhoff, A.S., Rice, C.M., Stapleton, J.T., Simmonds, P., 2014. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 59 (1), 318–327. Swindell, S.R., Plasterer, T.N., 1997. SEQMAN. Contig assembly. Methods Mol. Biol. 70, 75–89. Szabo, S.M., Bibby, M., Yuan, Y., Donato, B.M., Jiménez-Mendez, R., CastañedaHernández, G., Rodríguez-Torres, M., Levy, A.R., 2012. The epidemiologic burden of hepatitis C virus infection in Latin America. Ann. Hepatol. 11 (5), 623–635.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28 (10), 2731–2739. Tanaka, Y., Hanada, K., Mizokami, M., Yeo, A.E., Shih, J.W., Gojobori, T., Alter, H.J., 2002. A comparison of the molecular clock of hepatitis C virus in the United States and Japan predicts that hepatocellular carcinoma incidence in the United States will increase over the next two decades. Proc. Natl. Acad. Sci. U.S.A. 99 (24), 15584–15589. WHO. Hepatitis C. 2003.