Archives of Virology (2018) 163:1981–1984

https://doi.org/10.1007/s00705-018-3813-y

ANNOTATED SEQUENCE RECORD

Full genome and phylogenetic analysis of hepatitis B virus genotype F

in Mexican isolates
David Esaú Fragoso‑Fonseca1,2 · Noé Escobar‑Escamilla1,2 · Lourdes Teresa Lloret y Sánchez1 ·
Claudia Elena Wong‑Arámbula1 · Lucía Hernández‑Rivas1 · José Alberto Díaz‑Quiñonez1,3 ·
José Ernesto Ramírez‑González1

Received: 30 August 2017 / Accepted: 8 February 2018 / Published online: 16 March 2018
© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Abstract
Hepatitis B virus genotype F (HBV/F) is endemic in Central and South America with a minor proportion in Mexico and
North America. HBV/F is divided into subgenotypes and subtypes with particular geographic circulation patterns. Here, we
report the complete genome sequence and molecular characterization of HBV/F from three isolates. Phylogenetic analysis
with all available HBV/F sequences showed that our sequences belonged to the F1b subtype and, in addition, the absence
of the previously reported F1a subtype in Mexican isolates. Our findings suggest the circulation of HBV/F1b, the first phy-
logenomic study of HBV/F in Mexico.

In Latin America, approximately 7 to 12 million people
are chronically infected with hepatitis B virus (HBV) [1].
Based on an intergroup divergence of > 7.5 % in its com-
plete genome sequence, ten HBV genotypes (A-J) have been
described. These are divided into subgenotypes with genetic
differences of between 4 - 7.5%. For this reason, obtaining
and analyzing the full-length genome, rather than a partial
sequence, is the most accurate tool and the “gold standard”
for subgenotype assignment. [2]. In particular, HBV geno-
type F (HBV/F) is a highly divergent genotype: F1 to F4
Handling Editor: Michael Carpenter.
GenBank accession numbers  KY458061, KM998715 and
KY458062.
* José Ernesto Ramírez‑González
ernesto.ramirez@salud.gob.mx
1
Instituto de Diagnóstico y Referencia Epidemiológicos
(InDRE) “Dr. Manuel Martínez Báez”, Secretaría de
Salud, Francisco de P. Miranda 177, Lomas de Plateros,
01480 Álvaro Obregón, Mexico City, Mexico
2 2010 (InDRE 1008), 2011 (InDRE 1103) and 2012 (InDRE
Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional (IPN), Prolongación de Carpio y Plan
de Ayala, Casco de Santo Tomás, S/N, Casco de Santo
Tomás, 11340 Miguel Hidalgo, Mexico City, Mexico
3
División de Estudios de Posgrado, Facultad de Medicina,
Universidad Nacional Autónoma de México (UNAM),
Paseo de las Facultades S/N, Ciudad Universitaria,
04510 Mexico City, Mexico
subgenotypes have been proposed with F1, F2 and F4 being
divided into subtypes “a” and “b” [3].
HBV genotypes and subgenotypes have different ethnic
and geographic distributions, as well as distinct disease
development and treatment response features. It has been
proposed that HBV/F originated in the American continent
and is the most prevalent genotype in South and Central
America as well as in native populations of Alaska [4]. In
Mexico, HBV/F is unusual with a frequency of identifica-
tion ranging from 3 - 9% in those reports investigating the
circulating genotypes in different Mexican study popula-
tions [5–7]; the endemic genotypes being recognised as
G and H [8]. HBV/F subgenotype prevalence in Mexican
populations remains poorly understood. Recently, the first
identification of the F1b subgenotype in low-income risk
patients from Oaxaca was reported [9]. Here, we describe
the whole genome sequence analysis of HBV/F isolates from
Mexico City. In order to determine the subgenotype and to
analyze the known molecular biomarkers associated with
disease progression, persistence and drug resistance [10],
three plasma samples with HBsAg positive results from
1471), were chosen from a retrospective study of HBV geno-
types performed by our research group (not published).
Universal primer sets, described in Table  1, were
designed to amplify five fragments covering the complete
genome of HBV genotypes A-H as well as subgenotypes.
PCR products were obtained using the AmpliTaq Gold™

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1982 D. E. Fragoso-Fonseca et al.

Table 1  Primers used in this Primer Sequence 5’- 3’ Position (nt)a Product size (bp)
study
HBV58F TGC​TGG​TGG​CTC​CAG​TTC​ 58-75 423
HBV479R GAG​GAC​AMACG​GGC​AAC​ATA​ 479-460
HBV409F CAT​CCT​GCT​GCT​ATG​CCT​CA 409-428 788
HBV1196R GTT​GCR​TCA​GCA​AAC​ACT​TGG​ 1196-1176
HBV1100F CGC​CAA​CTT​AYA​AGG​CCT​TT 1100-1119 842
HBV1941R TCC​ACA​GWA​GCT​CCA​AAT​TCT  1941-1921
HBV1814F ATG​YAA​CTT​TTT​CAC​CTC​TGCC​ 1814-1835 666
HBV2479R TTT​CCC​ACC​TTR​TGA​GTC​CAA​ 2479-2459
HBV2371F AAG​AAG​AAC​TCC​CTC​GCC​TC 2371-2390 1009
HBV199R TAA​CAC​GAGMAGG​GGT​CCTA​ 199-180
a
 According to HBV reference sequence NC_003977

DNA Polymerase LD kit (Applied Biosystems, Foster City,
CA, USA) according to the manufacturer´s instructions. Fur-
ther, amplicons were sequenced on both strands by conven-
tional Sanger methods using the BigDye Terminator Cycle
3.1 sequencing kit and the 3500 Genetic Analyzer instru-
ment (Applied Biosystems).
Amplicon sequences were assembled to determine the
subgenotype through a phylogenomic analysis. Evolutionary
relationships were established using a dataset of 120 HBV/F
complete genome sequences available in the GenBank data-
base obtained from Alaskan, and Central and South Ameri-
can isolates (Fig 1). Complete genome sequences from this
study were deposited in GenBank under accession numbers
KY458061, KY458062 and KM998715.
Phylogenetic analysis identified the three isolates from
Mexico belonged to the F1b subgenotype with the surface
protein (S) amino acid sequence showing a Lysine at posi-
tions 122 and 160, corresponding to the adw serotype [11].
Sequence alignment showed three amino acid changes exclu-
sively present in the InDRE 1008, InDRE 1103 and InDRE
1471 isolates that had not been previously been reported:
K228R in the gene P and H13L in the domain RT (cor-
responding to T179S in the HBsAg). Of note, molecular
biomarkers related to pathogenesis were not found in any of
the three isolates.
Our findings contrast with previous reports where the cir-
culation of F1a in Mexico is demonstrated [4, 12]. In fact,
of the 16 sequences previously submitted to GenBank as
HBV/F, 11 of them were later reported as misclassified and
were actually genotype H [8]. The remaining 5 sequences,
correspond to 4 partial sequences not useful for determining
the subgenotype [2], while the 1 complete genome (strain
RVLA; accession number KF199901), without subtyp-
ing data actually belongs, according to our phylogenomic
analysis, to F1b (Figure 1). At this moment, the available
sequence data, the recent report on F1b circulation in at risk
populations, and the subtyping findings presented here, sug-
gest that F1b is an endemic HBV/F subtype found in Mexico,
in addition to genotypes HBV/G and HBV/H. However, the
presence of others subtypes needs to be determined, as well
as more in-depth clinical information on HBV/F infections.
Finally, correct identification of HBV subgenotypes, through
use of accurate methodologies, is important to increase our
understanding of the evolutionary history and origins of the
HBV/F in the Americas region.

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Characterization of HBV in Mexican isolates 1983

Fig. 1  Phylogenomic analysis
of HBV/F1b InDRE 1008,
InDRE 1103 and InDRE 1471.
Sequences are labelled using
accession numbers followed
by country code and strain
name. The described HBV/F
subgenotypes and HBV/H (used
as an outgroup) were included.
Phylogenomic inference was
conducted with MEGA6 soft-
ware [13] and the evolutionary
history was inferred through
use of the maximum likelihood
method based on the Tamura-
Nei model

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1984 D. E. Fragoso-Fonseca et al.

Funding  Not applicable.
Compliance with ethical standards  interferon alfa in patients coinfected with HIV and HBV genotype
Conflict of interest  We declare that we have no conflict of interest.
Ethical approval  This article does not contain any studies with human
participants or animals performed by any of the authors.
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