GENOMIC CHARACTERIZATION OF HCV
Genomic and phylogenetic
analysis of Hepatitis C virus strains from Argentina
F. Quarleri1 , Betty H. Robertson2, Verónica Mathet1, Saswati D.
Sinha2, Isabel Badía3, Bernardo Frider4, Amalia Ferro3 , Cristina
Galoppo3, Silvia Sookoian4, Gustavo Castaño4 , José R. Oubiña1
Microbiología, Parasitología e Inmunología, Facultad de Medicina,
Universidad de Buenos Aires; 3Unidad 4 Hepatología, Hospital de
Niños Ricardo Gutiérrez; 4Unidad de Hepatología, Hospital Cosme
Argerich, Buenos Aires; 2Hepatitis Branch, Centers for Disease Control
and Prevention, Atlanta, USA.
Key words: hepatitis C virus, HCV genotyping, HCV nucleotide
genomic characterization was performed by nucleotide sequence analysis
(n=50) combined with restriction fragment length polymorphism (RFLP)
of the 5’ UTR region in 82 isolates corresponding to different
Argentine groups. Genotype 1 was detected in 70.7 % of the samples (58
out of 82), genotype 2 in 21.9% (18 of 82) and genotype 3 in the
remaining 6 sera (7.3%). HCV 1b subtype contributed with 35.3 % to the
whole population studied (29 of 82) and was detected in 6 out of 21
sporadic cases. Besides their epidemiological significance, these
results should be taken into account when future vaccines are
considered on the basis of geographical HCV genotypic prevalence.
genómico y filogenético de cepas del virus de Hepatitis C de
Argentina. El análisis del RNA del virus de la hepatitis C (HCV)
permite clasificar diferentes aislamientos por lo menos en seis
genotipos que a su vez abarcan diverso número de subtipos. Algunos de
ellos se asocian a diferencias en el curso evolutivo de la infección
y a una distinta sensibilidad al tratamiento antiviral. Este estudio
muestra el análisis mediante secuenciación nucleotídica combinado
con el del polimorfismo del tamaño de fragmentos de restricción de
la región 5’ UTR de 82 cepas de HCV de Argentina provenientes de
múltiples grupos poblacionales. El genotipo 1 fue detectado en el
70,7% de las muestras (58 / 82), el tipo 2 en el 21,9% (18 / 82) y el
genotipo 3 en los restantes 6 sueros (7,3%). El subtipo 1b contribuyó
con un 35,3 % al total de la población estudiada (29 / 82) y fue
detectado en 6 de 21 casos esporádicos (28,5%). Además de aportar
nuevos datos a la epidemiología molecular regional del HCV, la
prevalencia de los genotipos aquí descriptos deberá ser considerada
al momento de evaluarse futuros ensayos de vacuna.
Postal address: Dr. José Raúl Oubiña, Departamento de
Microbiología, Facultad de Medicina, UBA, Paraguay 2155, 1121 Buenos
Fax: 54-1-962-5404; E-mail: email@example.com
Hepatitis C virus (HCV) is the etiologic agent of most parentally
transmitted non A - non B hepatitis. A chronic course is observed in
more than 50% of infected patients, who may develop cirrhosis and even
HCV is a single strand RNA virus with a positive polarity having a
nucleotide extension of approximately 10,000 bases. Its viral genome
shows two non-coding regions (5’UTR and 3’UTR) which flank a
central region coding for a polyprotein, from which derive after
post-translational cleavage structural (envelope and core) and
non-structural proteins (NS2 - NS5). Most conserved regions are those
located at the 5’ end and within a subregion of the 3’ end2. In
contrast, regions showing greater variability are present within
coding regions for gp1 and gp2 glycoproteins3.
HCV presents a hierarchical distribution mainly based on a dissimilar
degree of nucleotide sequence homology, which enables its
classification in various groups (types) and subtypes. Each group
consists of one or multiple HCV nucleotide sequences obtained from
infected individuals (isolates). Within each infected individual,
multiple related viable viral genomes (quasispecies), cocirculate,
which may be differentiated by only a few nucleotides. This is a
feature of RNA viruses and is mainly associated to their genomic
replication. Such genomic variability is also related to both the
possibility to induce viral persistence and the appearance of drug
resistance, as well as posing a major difficulty to obtain a vaccine.
The high degree of HCV nucleotide heterogeneity has led to a
considerable controversy in nomenclature and classification of this
A convenient system has been proposed4 which includes 6 different
types (namely 1,2,3, etc.) with a sequence homology below 69% inter
se. Each type includes one or several subtypes (named a, b, c, etc.
according to their date of discovery). Among subtypes belonging to the
same type roughly 79% sequence homology is observed. Finally, such
subtypes include a variable number of isolates, which exhibit more
than 88% nucleotide sequence homology5.
Among non-commercial techniques for HCV genotyping, analysis by
restriction fragment length polymorphism (RFLP) of amplicons obtained
by reverse transcription coupled to Nested PCR of the 5’ UTR
region6,7 and subtype specific core-based PCR amplification8, 9, 10
have been widely used. However, none of these HCV typing methods -as
well as others recently manufactured- are as conclusive as the
complete genomic sequencing of each isolate. However, despite being
the gold standard, such method cannot be usually applied due to its
high complexity, raised costs, and time requirements which are
incompatible with diagnostic needs.
HCV genomic characterization is relevant not only for molecular
epidemiological studies11 but also for a proper interpretation of
diagnostic tests currently marketed to detect specific antibodies or
viral genomes6, 12, 13.
Viremia levels14 and HCV genotypes15 have been implicated among
predictive factors of the severity of chronic infection. Moreover,
dissimilar incidence of hepatocellular carcinoma among American and
Japanese patients despite a similar seroprevalence for HCV, has been
associated to differences in genotype prevalence in both countries. In
Japan a greater prevalence of 1b subtype is observed in such tumors,
where cirrhosis does not appear to be a mandatory step16 as usually
observed in Western countries. Thus, 1b overexpression in Italian
patients has not been attributed to a mere genotypic difference
related to older infected individuals. Moreover, such subtype is also
associated to tumor development in cirrhotic patients, independently
of age or sex17. On the other hand, 1b subtype18, quasispecies
complexity distribution within a given patient19 and the existence of
point mutations20 have been reported as predictive factors of viral
resistance to interferon therapy.
Traditionally, viruses have been classified according to their
antigenic properties, but within the last few years - and due to
advances in molecular biology- genotypic classification has also been
feasible. The potential significance of such findings lies in the
possibility to investigate virus-host interactions, as well as viral
factors associated to both infection severity and treatment response.
The aim of the present study was to perform HCV genotyping in chronic
carriers by means of nucleotide sequencing combined with RFLP in
Material and methods
Population: 82 HCV chronically infected patients, 71 adults and 11
children (47 male; mean age 27.5 yr-old, range 2 - 71 yr) were
These subjects were classified within 3 groups: those who had
parenteral risk of infection (n=56: 28 who received blood
transfusions, 17 intravenous drug users [IVDU], 8 dialyzed -referred
to the Faculty of Medicine from a Center located at Buenos Aires
Province- 2 health workers, and 1 patient who reported a past
surgery); those with non-parenteral risk (n=5) and sporadic cases
Sample collection. All blood samples were obtained under appropriate
conditions for RNA handling. Three to 5 ml of whole blood were
collected by vein puncture. Sera were separated by centrifugation
within 3 hs, aliquoted and kept at -70ºC until further processing.
RNA extraction, reverse transcription and amplification by Nested PCR
of the 5’ UTR: 200 µl of serum were treated with guanidinium
isothiocyanate and acidic phenol following a protocol previously
The equivalent to 100 µl of serum was processed for reverse
transcription at 70ºC using the thermostable enzyme Tth (Promega, WI,
USA) or the recombinant rTth (Perkin Elmer, Roche Molecular Systems,
Branchburg, NJ, USA). Amplification of the initial cDNA was carried
out by using the same enzyme. Nested PCR was performed using Taq
polymerase (Promega, Perkin Elmer or Boheringer Mannheim, Germany).
Primers used were HCV1 (outer antisense) HCV2, (outer sense) HCV3
(inner antisense) and HCV4 (inner sense) as described22, thus allowing
the synthesis of 210 bp amplicons.
Throughout the whole procedure, Kwok and Higuchi rules23 were strictly
followed, except that from serum collection to agarose gel loading,
different sets of micropipettes and special aerosol resistant tips
(ART, Molecular Bio-Products, Inc.) were used. To validate results a
negative control was included from the extraction step every four
samples and another negative control was also added from reverse
transcription. A positive control was included from RNA extraction.
RFLP genotyping: This procedure was developed following the
methodology proposed by Davidson et al.7 slightly modified by the
authors. Briefly, 10 µl of products obtained by RT - Nested PCR were
digested with the following sets of endonucleases: Hae III / Rsa I,
Hinf I / BstN I. According to the result obtained, further digestions
were carried out with BstU I (for type 1) or with ScrF I (for types 2
or 3). Enzymatic treatment was carried out at 37ºC for 2 hs, except
for Hinf I/BstN I which was later treated for 2 hs at 60ºC, as
performed with BstU I. This methodology has been reported to allow
discrimination between subtypes “a” and “b” for types 1, 2 or
37. RFLP was carried out in 32 out of the 82 samples, which have not
Sequencing of 5’ UTR amplicons: 100 µl of Nested PCR products were
purified from 6% polyacrylamide gel. Bands were eluted using a
solution with 0.5% ammonium acetate, 0.01 M magnesium acetate, 1mM
EDTA and 0.1% SDS with slow agitation for 12 hs. After centrifugation,
supernatants were collected and a phenol - chloroform extraction was
carried out followed by ethanol precipitation in presence of ammonium
DNA was resuspended in 5 - 20 µl of sterilized bidistilled water,
according to product yield, as measured by spectrophotometric reading
at 260 nm.
Sequencing was carried out according to the method of Sanger24,
partially modified by the cycle sequencing procedure, alternately
using for each sample both internal primers (HCV2 and HCV4) with 5’
end fluorescein-labelled dideoxynucleotides in an automatic sequencer
(ABI 373A, Applied Bioosystems, Foster City, CA, USA). To avoid
misinterpretations, each template was obtained at least from 2
different aliquots of RNA and sequenced bidirectionally, using HCV3
and HCV4 alternately.
In turn, when nucleotide sequences at positions corresponding to
endonuclease recognition sites were established, computarized
predictive mode RFLP analysis (Lassergene Program for Windows,
MAPDRAW) was carried out. In addition multiple and pairwise sequence
alignment (MEGALIGN Program) allowed strain classification within HCV
genomic types using Clustal method25. Fifty HCV isolates were analyzed
by this methodology.
Type 1 was detected in 58 out of 82 samples (70.7%), type 2 in a
further 18 (21.9%) and type 3 in the remaining 6 sera (7.3%) (Fig.1).
Within the RFLP discrimination ranges, various HCV subtypes - except
for 3b- were observed, as shown in Fig. 2 and Table 1. However, no
samples belonging to types 4, 5 or 6 were detected.
In sporadic cases genotype 1 accounted for 71.4%, with 42.8% for
subtype 1a (9/21) and 28.6% for 1b (6/21), and subtype 2a/c for the
remaining 28.6% (n=6).
Among the 56 cases with parenteral transmission risk (hemodialyzed,
polytransfused and IVDU), 38 (67.8%) belonged to genotype 1 (18 to
subtype 1a - 32.1%- and 20 to 1b -35.7%-), 12 (21.4%) to type 2 (10 to
2a/c -17.8%- and 2 to 2b -3.6%-) and the remaining 6 to type 3 (10.7%,
all subtype 3a).
Among the 5 patients with non-parenteral transmission risk, only
genotype 1 was demonstrated (1a in 2 and 1b in 3).
Partial nucleotide sequence alignment is observed in Figure 3, while a
phylogenetic tree is depicted in Figure 4.
HCV genomic characterization was performed with serum samples from
chronically infected Argentine patients by means of nucleotide
sequencing -with predicted type-specific restriction patterns- and
RFLP analysis of amplicons obtained from the 5’ UTR region. Although
HCV genotype distribution was not apparently different among children
and adults, it must be taken into account that an appropriate
comparison according to age cannot be performed, due to the small
number of children studied.
All samples were classified as belonging to types 1, 2 or 3. These
genotypes exhibit a world-wide distribution, in contrast to genotypes
4, 5 and 6 whose location has so far been restricted to Middle East,
South Africa and Hong Kong, respectively.
This study demonstrates that genotype 1 exhibits the greatest
prevalence, as it was detected in 58 out of 82 samples analyzed
(70.7%), irrespectively of the group studied. Within genotype 1,
subtypes 1a and 1b accounted for identical percentages, corresponding
each to 35.4% of the whole population (Fig.1).
Comparative genotypic analysis of diverse groups showed interesting
features (Fig.2). For example, despite of their common origin from a
dialysis unit, these patients exhibited dissimilar and evenly
distributed HCV genotypes, suggesting a different source of infection.
In contrast, among transfused patients (n=28) subtype 1b was
predominant, since it was detected in 12 sera (42.8%) followed by
subtypes 1a and 2a/c (n=6, each). The IVDU group showed also genotype
1 high prevalence (13 out of 17 sera, 76.4%), but in this case both
subtypes 1a and 1b contributed with similar percentages (41.1 and
HCV genotyping of sporadic cases deserves special consideration, since
it might truly represent circulating local strains, without external
influences which promote their association with specific (sub)types,
as can be observed when concentrates of coagulation factors obtained
in Western countries are administered to haemophiliacs (only 1 patient
in our study). Since type 1 was detected in 15 out of 21 sporadic
cases (71.4%) it is suggested that this type may account for a vast
majority of community acquired HCV Argentine patients.
HCV subtyping by RFLP may eventually lead to erroneous conclusions.
With regard to genotype 1, 1a or 1b subtypes are ascribed according to
nucleotide located at position 243 (G or A). However, other subtypes
may exhibit an indistinguishible restriction pattern: i.e. 1c is
identical to 1a7, 27. Furthermore, it has been documented that
approximately 10% of genuine 1a strains possess a G at position 243,
while 2% of 1b strains show an A at the same location26.
Type 2 was detected in 18 patients (21.9%), showing a strong
predominance of subtype 2a/c -indistinguishible at 5’ UTR- (16/18)
over 2b subtype. Genotype 2 was not detected in IVDU. Subtype 2c has
been recently documented in Argentina by sequence analysis in the core
and NS5 regions from a sample showing discrepancies using 2 different
methodologies (unpublished authors’ data)
Type 3 was found in 6 patients (8.3% of the whole population), 3 of
them among IVDU, as reported in other studies27. RFLP subtyping
demonstrated only 3a strains. As previously mentioned for types 1 and
2, limitations of this method are also pertinent to genotype 3, since
subtypes 3c, 3d and 3e cannot be distinguished from 3a at the 5’ UTR
region, while those classified as 3b show the same restriction pattern
exhibited by 3f7, 28.
From these considerations, it is concluded that for subtype assignment
it is mandatory to perform simultaneous sequencing of coding genomic
regions which exhibit a greater degree of nucleotide heterogeneity
within a given type (i.e. E1, core or NS5). However, 5’UTR amplicons
analyzed by RFLP are still one of the most widely used methods.
Significant nucleotide conservation at such location among different
strains, which allows the use of universal primers, and therefore
maximal sensitivity for detection and subsequent typing, explains RFLP
current acceptance. Paradoxically, such conservation is at the same
time a hindrance for conclusive subtyping.
Computer analysis allowed nucleotide sequence alignment (Fig. 3) as
well as a comparison of local strains with subtype-specific
prototypes. As observed in the cladogram depicted in Figure 4, all
local strains were ascribed to type 1, 2 or 3, while a closer
relationship between genotypes 1 and 3 has been confirmed.
Although the high complexity of c-DNA sequencing precludes large-scale
HCV typing, it allowed us to strengthen RFLP usefulness. Whereas
genomic information is limited with the latter, it is extremely
valuable for massive typing.
Results shown in this study, together with our previous observations
of local HCV strains 21, show a partial view of the molecular
epidemiology of this agent in Argentina. This is the first study of
multiple nucleotide sequence and phylogenetic analysis carried out
with Latin American samples.
HCV mixed infections are an interesting field of current research.We
have previously shown a high proportion of HCV mixed infections
(45.4%) in Argentine samples21, although this rate was not observed in
the present study. At least two factors might explain this
discrepancy. On one hand, it is known that the core-based PCR
amplification method8 -10 may readily detect different subtypes but
produces a certain degree of mispriming34, while RFLP was reported to
need roughly equimolar concentrations for detecting HCV mixed
infections6, 7. On the other hand, since 50 out of the 82
characterized genomes were analyzed by predicted RFLP from direct
sequencing of PCR products, it seems plausible that only predominant
genomes would have been detected. Therefore, possible HCV minor
populations -i.e. contributing to mixed infections- within each of the
directly sequenced isolates cannot be entirely ruled out. Thus,
diverse sensitivity and specificity for each methodology should be
taken into account when comparing our two studies.
In contrast with recent HCV molecular epidemiologic observations from
South Brazil, where genotype 1> 3> 2 prevalence has been shown
-although without subtyping-32, our data depict a pattern similar to
that recently reported in Venezuela33, while strengthen our initial
Our findings may provide useful information for diagnostic detection
of genomic HCV13 and for a better interpretation of genotype-dependent
serology6, 12. Bearing in mind that HCV superinfection has been
documented in non-human primates infected with different genotypes29,
experimental vaccines currently in preparation30, 31 should consider
not only the efficacy of protection against challenge with a genotype
identical to the immunogen, but also to heterologous genotypes, on
occasion genetically distant as 1b vs 2a/c.
The proper knowledge of prevalent HCV genotypes in different world
areas will contribute to develop an adequate prophylaxis to avoid
infection by this agent.
Acknowledgments: This study was supported partly by Pan
American Health Organization, Centers for Disease Control and
Prevention (USA), CONICET (Argentina), University of Buenos Aires
(Argentina), University of El Salvador (Argentina), Roemmers
Foundation, and Polar Foundation (Argentina).
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Table 1. HCV types and subtypes distribution among Argentine
Genotype Parenteral Non-parenteral Sporadic Total
risk risk cases
1 a / c 18 2 9 29
1b 20 3 6 29
2 a / c 10 0 6 16
2 b / c 2 0 0 2
3 a / c / d / e 6 0 0 6
Total 56 5 21 82
Fig. 1.- HCV genotypes in
chronically infected subjects.
Fig. 2.- HCV subtype prevalence within different Argentine groups.
Fig. 3.- Sequence alignment
of 50 Argentine HCV isolates corresponding to positions 205 to 224
from the 5’UTR region of genomic RNA. At the top of the figure
consensus sequence is shown. Boxes represent polymorphic sites.
Fig. 4.- Phylogenetic tree of 5’ UTR region from HCV genomes, using
Clustal method with weighted residue weight table. Fifty Argentine
sequences are named solely with a number: samples # 498, 594,
611,614,618, 619, 630, 660, 668, 726, 745, 751, 760, 768, 782, 784,
785, 789, 791, 793, 794, 803, 804, 812, 813, 818, 824, 828, 905, 906,
959 - 963, 965 - 969, 971, 973, 974, 976, 980, 982, 985, 986, 989 and
990, correspond to Accession numbers from GenBank AF041264 to
AF041313, respectively; those sequences obtained from GenBank used as
references for phylogenetic tree construction are identified by their
accession number (preceded with a letter).