Hepatitis B virus (HBV) is a globally distributed human pathogen that causes acute and chronic infections. HBV shows a high genetic heterogeneity and has been classified into at least eight genotypes (A-H), based on an intergroup nucleotide divergence greater than 7.5% in the complete genome sequences. Moreover, “subgenotypes” are defined as groups characterized by a divergence between 4% to 7.5%, and “clades” are subgroups showing less than 4% nucleotide divergence (Kramvis et al., 2008; Shi et al., 2013).Fil: Mojsiejczuk, Laura Noelia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres, Carolina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pisano, María Belén. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ré, Viviana Elizabeth. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Campos, Rodolfo Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Flichman, Diego Martin. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Campos, Rodolfo Hector

Flichman, Diego Martin

Mojsiejczuk, Laura Noelia

Pisano, María Belén

Ré, Viviana Elizabeth

Torres, Carolina

Infection Genetics and Evolution

English

Laura Noelia Mojsiejczuk

Carolina Torres

María Belén Pisano

Viviana Re

Rodolfo Héctor Campos

Diego Martín Flichman

Crossref

New pieces on genetic diversity and evolutionary history of hepatitis B virus: Characterization of the novel subgenotype F6

CONICET Digital

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Infection, Genetics and Evolution 47 (2017) 140–142Contents lists available at ScienceDirectInfection, Genetics and Evolutionj ourna l homepage: www.e lsev ie r .com/ locate /meeg idNew pieces on genetic diversity andevolutionary history of hepatitis B virus:Characterization of the novel subgenotype F6Dear Editor,Hepatitis B virus (HBV) is a globally distributed human pathogenthat causes acute and chronic infections. HBV shows a high genetic het-erogeneity and has been classified into at least eight genotypes (A-H),based on an intergroup nucleotide divergence greater than 7.5% in thecomplete genome sequences. Moreover, “subgenotypes” are definedas groups characterized by a divergence between 4% to 7.5%, andFig. 1. Phylogenetic analysis of HBV genotype F.MaximumLikelihood tree constructed on compRaxML v8.2.8. The new complete genome sequences reported in this work are shown in italicvalues obtained with 1000 pseudoreplicates). The scale bar indicates genetic distances.http://dx.doi.org/10.1016/j.meegid.2016.11.0231567-1348/© 2016 Elsevier B.V. All rights reserved.“clades” are subgroups showing less than 4% nucleotide divergence(Kramvis et al., 2008; Shi et al., 2013).It iswidely accepted that genotype F is native from theAmericas and itis believed that it might have been endemic in the continent during thepre-Columbian times. At present, it has been found in native populationsfrom Alaska, Central and South America, and it is the most prevalent inadmixed populations with Native American ancestry (Arauz-Ruiz et al.,1997; Campos et al., 2005; Devesa et al., 2008;Huy et al., 2006; Livingstonet al., 2007; Piñeiro y Leone et al., 2008). This is one of themost divergentHBV lineages, showing a high intra-genotype diversity. Therefore, it iscurrently classified into five different subgenotypes: F1 to F5 (Kramviset al., 2008; Martínez et al., 2014; Shi et al., 2013).In a previous study published in Infection, Genetic and Evolution,wehave proposed a division of subgenotype F4 into two clades: F4a andlete genome sequences of genotype F (n=216) and genotype H as outgroup (n=6)withs. Statistical support for the main groups is shown in the corresponding nodes (bootstrapTable 1Estimates of mean divergence (p-distance, % ± SE) between groups of HBV genotype F.The analysis was conducted inMEGA6 and involved 216 genotype F complete genome se-quences (F1 = 109, F2 = 21, F3 = 26, F4 = 51, F5= 2, F6= 7). Divergence between F6and the closest group is shown in bold.F1 F2 F3 F4 F5F2 5.79 ± 0.38F3 5.37 ± 0.36 4.39 ± 0.31F4 5.69 ± 0.41 4.54 ± 0.28 4.23 ± 0.34F5 6.61 ± 0.40 5.39 ± 0.32 5.34 ± 0.37 5.19 ± 0.36F6 6.24 ± 0.41 5.14 ± 0.27 4.94 ± 0.35 4.11 ± 0.27 5.66 ± 0.37141New pieces on genetic diversity and evolutionary history of hepatitis B virus: Characterization of the novel subgenotype F6F4b. The latter was defined based on the report and analysis of threecomplete genome sequences isolated from patients from the city ofBuenos Aires that confo\rmed a separate monophyletic cluster andshowed 3.8% divergence in genomic sequence with the previouslyknown F4 strains (Mojsiejczuk et al., 2015). While these featuresallowed establishing a new cladewithin the genotype F, the genetic dis-tance value is close to the lower limit conventionally accepted to consid-er a group as a new subgenotype (4.0%). Recently, four new strains fromArgentina that grouped with the previously reported F4b sequenceswere detected. One of them was obtained from a patient with chronicHBV infection resident in the city of Buenos Aires. The other three addi-tional sequences were identified in patients from the province ofCórdoba, in the central region of the country, and corresponded to par-tial genomic sequences previously reported by Gallego et al. (2014).Hence, we characterized these four strains and analyzed them in thecontext of the genotype F evolutionary history. Complete genomeFig. 2. Time-scaled phylogeny of HBV genotype F. Reconstructed on complete genome sequenceskyline demographic model and a relaxed clock model (UCLN) by calibration with a mean fixeancestor heights” algorithm. Posterior clade probabilities of main groups are shown on the novalue) are displayed and high posterior density interval of the nodal dates are represented breader is referred to the web version of this article.)sequences were obtained by PCR and direct sequencing, and recombina-tion signals were discarded with RDP4 v4.71 software package (Martinet al., 2015). Then, a Maximum Likelihood phylogenetic analysis andgenetic distances calculation were conducted as previously described(Mojsiejczuk et al., 2015). Datasets were updated for genotype Fcomplete genomes newly reported in GenBank, including Panamaniansequences from the recently described groups: F1c and F5 (Martínezet al., 2014).The phylogenetic tree showed that the four new complete genomes(D42_BsAs, CbaArg16, CbaArg25, CbaArg42) clustered together withthe previous named F4b sequences with maximal bootstrap support(Fig. 1). Subsequently, the mean nucleotide divergence (mean ± stan-dard error) between groups was calculated. After the addition of thefour novel strains, the nucleotide divergence over the complete genomesequence between “F4a” and the previously named “F4b” cluster was4.11 ± 0.27 (Table 1). The highly supported monophyletic group andthe genetic distance over 4% suggest that these two lineages should beclassified as separate subgenotypes (hereafter subgenotypes F4 andF6). Moreover, the novel subgenotype F6, formed by the four novelstrains and the former “F4b” sequences, had distinctive nucleotide andamino acid signature patterns (Supplementary Fig. Fig. S1).To date, several recommendations were introduced for an accuratedesignation of HBV genotypes and subgenotypes, including monophy-letic origin, adequate bootstrap support and minimum sequence diver-gences among themost relevant (Kramvis et al., 2008; Pourkarim et al.,2010). Even the existence of specific nucleotide and amino acid patternsand theminimum sequence number have been discussed to suggest theexistence of a new group (Pourkarim et al., 2010). Notwithstanding, thes of genotype F (n=216) in BEAST v1.8.2. The analysiswas performed under the Bayesiand on 1 × 10−5 s/s/y. Maximum clade credibility tree was summarized using the “commondes (*constrained group). For selected nodes, the ancestor date (based on tMRCA mediany the blue bars. (For interpretation of the references to colour in this figure legend, the142 New pieces on genetic diversity and evolutionary history of hepatitis B virus: Characterization of the novel subgenotype F6suitability of an “appropriate sampling” cannot be judged a priori andthere is always the possibility that the incorporation of new sequencesmay change the hierarchical status of the group. Thereby, increasingthe amount of data from different geographical locations might be par-ticularly relevant in the case of genotype F, considering that it shows ahigh intragroup divergence and probably it evolved in isolated nativepopulations scattered throughout the continent. In this context, a per-manent review of classifications will keep being of utmost importancein the future.Additionally, a Bayesian coalescent analysiswas performed in order toestimate the time to the most recent common ancestor (tMRCA) for F6and to reevaluate the genotype F origin and divergence times. The datasetincluded all non-recombinant American genotype F complete genomesequences available at GenBank (n = 216). We previously reportedthat Maximum Likelihood and Bayesian coalescent analyses of genotypeF suggest dissimilar hypotheses about subgenotypes diversification,being the first better supported (Mojsiejczuk et al., 2015). Accordingly,topological constraints were applied to the Bayesian analysis, to forcethe best supported hypothesis, grouping subgenotypes F2, F4, F5 andF6. A more detailed description of analytical conditions is provided inFig. 2 legend. The analysis showed that the starting point of F6 diversifi-cation dates from approximately 930 CE [HPD95% = 251–1466 CE] andlasted until around 1600 CE. The MRCAs for each subgenotype, includingF6, dates from1250 to 700 years ago, suggesting that the expansion of ge-notype F strains that are currently circulating mainly took place duringthe post-Columbian era (Fig. 2). The genotype F tMRCA was similar tothose previously calculated using the samemedian evolutionary rate, es-timating an age of around 6600 years [HPD95% = 3280–12,856] (Melloet al., 2013; Mojsiejczuk et al., 2015; Torres et al., 2011).In summary, new complete genome sequences of HBV genotype Ffrom Argentina were described and, based on the genetic divergenceand phylogenetic evidences, it is proposed to classify these new isolatesalong with the sequences previously reported as a novel “subgenotypeF6”. Accordingly, it is considered that the accurate classification ofHBV subgenotypes and a more exhaustive sampling might providenew insights for understanding the evolutionary history of HBV geno-type F and proposing new hypotheses addressing its diversificationprocess.Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.meegid.2016.11.023.AcknowledgmentsThis work was supported by grants from the Consejo Nacionalde Investigaciones Científicas y Técnicas (CONICET; PIP2012-11220110100215), and the Agencia Nacional de Promoción Científicay Tecnológica (ANPCyT; PICT2011-0705).The following is the supplementary data related to this article.ReferencesArauz-Ruiz, P., Norder, H., Visoná, K.A., Magnius, L.O., 1997. Molecular epidemiology ofhepatitis B virus in Central America reflected in the genetic variability of the smallS gene. J. Infect. Dis. 176:851–858. http://dx.doi.org/10.1086/516507.Campos, R.H., Mbayed, V.A., Piñeiro y Leone, F.G., 2005. 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Phylogenet.Evol. 59:114–122. http://dx.doi.org/10.1016/j.ympev.2011.01.010.Laura Noelia MojsiejczukCarolina TorresUniversidad de Buenos Aires, Facultad de Farmacia y Bioquímica,Departamento de Microbiología, Inmunología y Biotecnología, Cátedra deVirología, Junín 956 4to piso, Ciudad Autónoma de Buenos Aires, Argentina.Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),ArgentinaMaría Belén PisanoViviana ReUniversidad Nacional de Córdoba, Facultad de Ciencias Médicas, Institutode Virología “Dr. Vanella”, Enfermera Gordillo Gómez s/n, CiudadUniversitaria, Córdoba, Argentina.Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),ArgentinaRodolfo Héctor CamposDiego Martín Flichman*Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica,Departamento de Microbiología, Inmunología y Biotecnología, Cátedra deVirología, Junín 956 4to piso, Ciudad Autónoma de Buenos Aires, Argentina.Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),Argentina*Corresponding author at: Universidad de Buenos Aires, Facultad deFarmacia y Bioquímica, Departamento de Microbiología, Inmunología yBiotecnología, Cátedra de Virología, Junín 956 4to piso, (C1113AAB),Ciudad Autónoma de Buenos Aires, Argentina.E-mail address: dflichman@ffyb.uba.ar.8 September 2016Available online 26 November 2016

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New pieces on genetic diversity and evolutionary history of hepatitis B virus: Characterization of the novel subgenotype F6

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