Genotype distribution and molecular epidemiology of hepatitis C virus in blood donors from southeast France.

Abstract

The genotype distribution of hepatitis C virus (HCV) in blood donors from southeast France was tracked for a period of 13 years (1991 to 2003). Virus genomes from 321 samples were analyzed by amplification and sequencing of the NS5b and E1 regions. The most frequent genotypes were 1b (30.2%), 1a (27.7%), and 3a (22.4%). Although it was less common, genotype 2 was characterized by the presence of strains belonging to 11 different subtypes, including 5 that had never been characterized. Genotypes 1a, 1b, 3a, and 4a presented typical "epidemic" profiles, with a large number of isolates per subtype and short mean genetic distances between isolates. Type 2 isolates displayed a typical "endemic" profile, with a large number of subtypes and very few isolates in each subtype. The epidemiology of HCV infection in southeast France changed radically during the study period in relation to modifications in the etiology of infection. We observed the emergence of new epidemic subtypes (subtypes 1a and 3) linked to intravenous drug use and a decrease in the types linked to blood transfusion and nosocomial infection (epidemic subtype 1b and endemic type 2). Comparison of strains from blood donors with strains from a cohort of inpatients in the same region during 2001 and 2002 demonstrated for the first time that the monitoring of blood donors is a generally valid indicator of HCV epidemiology in terms of genotype distribution.

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JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2005, p. 3624–3629
0095-1137/05/$08.00?0doi:10.1128/JCM.43.8.3624–3629.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 8
Genotype Distribution and Molecular Epidemiology of Hepatitis C
Virus in Blood Donors from Southeast France
Jean-Franc ¸ois Cantaloube,1* Pierre Gallian,1Houssam Attoui,1Philippe Biagini,1
Philippe De Micco,1and Xavier de Lamballerie2
Unite ´ des Virus Emergents EA3292, EFS Alpes-Me ´diterrane ´e,1and Faculte ´ de Me ´decine, Universite ´
de la Me ´diterrane ´e, 27 Bd. Jean Moulin, 13005 Marseille,2France
Received 2 March 2005/Returned for modification 11 April 2005/Accepted 26 April 2005
The genotype distribution of hepatitis C virus (HCV) in blood donors from southeast France was tracked for
a period of 13 years (1991 to 2003). Virus genomes from 321 samples were analyzed by amplification and
sequencing of the NS5b and E1 regions. The most frequent genotypes were 1b (30.2%), 1a (27.7%), and 3a
(22.4%). Although it was less common, genotype 2 was characterized by the presence of strains belonging to 11
different subtypes, including 5 that had never been characterized. Genotypes 1a, 1b, 3a, and 4a presented
typical “epidemic” profiles, with a large number of isolates per subtype and short mean genetic distances
between isolates. Type 2 isolates displayed a typical “endemic” profile, with a large number of subtypes and
very few isolates in each subtype. The epidemiology of HCV infection in southeast France changed radically
during the study period in relation to modifications in the etiology of infection. We observed the emergence of
new epidemic subtypes (subtypes 1a and 3) linked to intravenous drug use and a decrease in the types linked
to blood transfusion and nosocomial infection (epidemic subtype 1b and endemic type 2). Comparison of
strains from blood donors with strains from a cohort of inpatients in the same region during 2001 and 2002
demonstrated for the first time that the monitoring of blood donors is a generally valid indicator of HCV
epidemiology in terms of genotype distribution.
More than 170 million people are currently infected by the
hepatitis C virus (HCV), which represents a serious cause of
chronic liver disease that may progress to cirrhosis liver and
hepatocarcinoma. It is an enveloped virus with a single-
stranded, positive-sense, nonsegmented RNA genome of ap-
proximately 9,500 nucleotides that encodes a polyprotein of
approximately 3,000 amino acids (8, 12). Analysis of the HCV
genome has demonstrated extremely high heterogeneity in
both structural and nonstructural coding regions and has iden-
tified at least six different genotypes that have generally been
divided into several subtypes (2, 25, 32).
The prevalence and distribution of HCV genotypes depend
on geographical location (19). Three broad patterns of geno-
type distribution have been identified to date (30). One pat-
tern, characterized by high genetic diversity, involves geo-
graphically discrete areas of West Africa with types 1 and 2 (4,
13, 27), Central Africa with type 4 (11, 20), and Asia with types
3 and 6 (1, 29, 37, 38). This pattern is suggestive of a long
period of endemic infection (19, 30). Another pattern involves
areas with a few subtypes circulating in specific risk groups,
e.g., subtype 3a in drug addicts (21). The third pattern involves
areas where a single subtype is present, such as in Egypt with
subtype 4a (6, 11, 24) and South Africa with subtype 5a (7, 31).
Previous blood donor studies in France have suggested that
genotypes 1, 2, 3, 4, and 5 were the most prevalent. However,
genotyping in those studies was performed by analysis of the 5?
noncoding region, which cannot discriminate subtypes (34, 35).
The reference method for determination of the HCV subtype
is phylogenetic analysis, based on sequencing of the NS5b and
E1 genome regions (32). The aim of this study was to analyze
the genotype distribution of HCV by NS5b or E1 sequencing of
the viruses in 321 blood samples collected from donors be-
tween 1991 and 2003. The epidemiology of the virus in our
population was analyzed as a function of sex, collection date,
and year of birth. The distribution of HCV strains identified in
blood donors was compared with the distributions previously
reported in patients in southeast France (35).
MATERIALS AND METHODS
Blood donors. In France, blood donation practice concerns nonremunerated
voluntary donors between the ages of 18 and 65 years. These donors undergo a
preliminary inquiry which allows the identification and exclusion of individuals
with parenteral risk factors. A total of 321 plasma samples collected from HCV-
infected blood donors in southeast France between 1991 and 2003 were analyzed.
There were 181 men and 140 women, with their ages ranging from 18 to 65 years
(mean, 38.5 ? 11.4 years). All tested positive for anti-HCV antibody by a
third-generation immunoenzymatic assay (Ortho Diagnostic Systems, Raritan,
N.J.). Positive results were confirmed by the RIBA HCV 3.0 SIA (Chiron,
Emeryville, Calif.). Detection of HCV RNA in plasma was performed with a
commercially available Amplicor HCV kit (Roche Diagnostic Systems, Meylan,
France). All tests were carried out in accordance with the manufacturers’ in-
structions.
Genotyping. Subtyping was accomplished by amplifying and sequencing a
339-bp amplicon of the NS5b region. A seminested PCR was performed with
primers NS5-1 (sense; 5?-TAT-GAY-ACC-CGY-TGC-TTT-GAC-3?) and NS5-2
(reverse; 5?-GAG-GAG-CAA-GAT-GTT-ATC-AGC-TC-3?) for primary ampli-
fication and primers NS5-1 and NS5-3 (reverse; 5?-GAA-TAC-CTG-GTC-AT
A-GCC-TCC-G-3?) for secondary amplification. Subtyping was also carried out
with type-specific primers, as described previously (5).
Extraction of RNA from 200 ?l of plasma, reverse transcription with random
hexaprimers, and PCR of the NS5b region with generic primers and the E1-
encoding region with type-specific primers were performed as reported previ-
ously (5). Amplicons in the E1 and NS5 regions were sequenced directly with the
* Corresponding author. Mailing address: Unite ´ des Virus Emer-
gents. Etablissement de Transfusion Sanguine Alpes-Me ´diterrane ´e,
149 bd. Baille, 13005 Marseille, France. Phone: 4.91.25.59.74. Fax:
4.91.18.95.98. E-mail: jfc-ets-ap@gulliver.fr.
3624

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amplification primers, a D-rhodamine DNA sequencing kit, and an ABI Prism
377 sequencer (Perkin-Elmer).
Phylogenetic analysis. The nucleotide sequences of the HCV strains from
infected patients in Marseilles, France, and a panel of sequences retrieved from
the GenBank database (see the phylogenetic trees in the Results section) were
aligned by using the ClustalW 1.8 software package (36). Phylogenetic analysis
for strain typing was focused on two genomic regions, i.e., a 357-nucleotide
sequence (positions 688 to 1044) in the E1 region and a 339-nucleotide sequence
(positions 8002 to 8340) in the NS5b region. The nucleotide positions refer to the
subtype 2a sequence with GenBank accession number D00944. Phylogenetic
analysis was performed with the MEGA software package (version 2.1, 2001;
Pennsylvania State University, University Park) (16) by using the p distance for
distance determination and the neighbor-joining method for tree drawing. The
reliability of phylogenetic analysis was evaluated by a bootstrap test with 1,000
replications.
Statistical analysis. Statistical analysis was performed with the Systat v10.2
software package (Systat Software Inc.). The results are expressed as means ?
standard deviations or as percentages, with 95% confidence intervals calculated
according to the normal distribution or the binomial distribution, as applicable.
Means between groups were compared by using the t test or the Student test, and
group frequency was compared by the chi-square test or Fisher’s exact test. The
frequency distributions of the different genotypes within groups (birth year or
collection year) were analyzed by the extended Mantel-Haenszel chi-square test.
RESULTS
Distribution of HCV genotypes. All 321 HCV RNA-positive
samples collected between 1991 and 2003 were amplified and
sequenced. The type-specific strategy in the E1 region was used
for four samples for which sequencing of the NS5b region
failed (because of the absence of PCR amplification). The
results demonstrated the presence of subtype 4a in all four
cases. Table 1 details the distributions of the different types
together with demographic data. The distributions of the HCV
subtypes can be summarized as follows: type 1b, 30.2%; type
1a, 27.7%; type 3a, 22.4%; type 4d, 5%; type 4a, 2.5%; and type
5a, 1.3%.
Genotype 2, with an overall prevalence of 10.9%, presented
a special subtype distribution. As shown in Fig. 1, phylogenetic
analysis of the NS5b region identified a total of 35 type 2
strains. Twenty-nine strains clustered within six previously de-
scribed subtypes, i.e., 2a, 4 strains (11.4%); 2b, 4 strains
(11.4%); 2c, 10 strains (28.6%); 2i, 7 strains (20%); 2k, 3
strains (8.6%); and 2l, 1 strain (2.9%). The remaining six
strains, i.e., MRS40, MRS123, MRS41, MRS50, MRS117, and
MRS121, were unassignable to any previously described sub-
type and represented five putative new subtypes.
Epidemiology. Determination of the frequency distribution
of HCV genotype according to the year of collection showed a
significant increase in the prevalence of subtype 3a from 0% in
1990 to 28% in 1999 (?2? 16.05; P ? 0.01). The prevalence of
subtype 1b dropped significantly from 50% in 1990, when it was
predominant, to 23% in 1998 (?2? 6.34; P ? 0.05). A slight
evolution of the distribution of subtype 1a has been observed
since 2000 (Fig. 2); however, this was not found to be statisti-
cally significant.
The sex ratios for the different subtypes were also deter-
mined and were compared with the sex ratio of the entire
population. Significant differences were found for subtype 1a
strains, which showed an overrepresentation among males (?2
? 7.5; P ? 0.005), and for subtype 1b strains, which showed an
overrepresentation among females (?2? 10.8; P ? 0.002). The
other subtypes showed no significant differences with regard to
sex ratio, but it is noteworthy that subtype 4a was identified
only in males (n ? 8).
Analysis of the strain distribution according to age showed
that donors infected by types 2 (mean age, 43.3 ? 12.0 years)
and 1b (41.4 ? 13.4 years) were significantly older than donors
infected by types 1a (35.6 ? 9.5 years), 3a (35.4 ? 9.5 years),
and 4d (34.4 ? 5.8 years) (1a versus 1b, P ? 0.001; 1a versus 2,
P ? 0.001; 3a versus 1b, P ? 0.001; 3a versus 2, P ? 0.001; 4d
versus 1b, P ? 0.01; and 4d versus 2, P ? 0.01). The differences
between types 1a, 3a, and 4d and between types 1b and 2 were
not significant. The paucity of genetic data regarding subtype
5a strains (four strains of which were characterized) does not
permit judgment of the statistical significance of the differences
in the mean age of the donors infected with subtype 5a (45.5 ?
18.1 years) compared to the mean age of the donors infected
with the other subtypes.
The mean age of men infected with subtype 1a was statisti-
cally higher than that of women (37.1 ? 9.2 years versus 32.0
? 9.3 years; P ? 0.025). Indeed, subtype 1a was identified in
approximately 35% of men born between 1941 and 1970 and
increased from 8 to 38% among women born during the same
period. The opposite results were obtained for subtypes 1b and
2, for which the mean female age was higher than mean male
age (43.9 ? 12.7 years and 38.9 ? 13.8 years, respectively, and
46.1 ? 7.5 years and 40.2 ? 15.0 years, respectively), However,
statistical significance was low for subtype 1b (P ? 0.071) and
null for type 2.
TABLE 1. Epidemiological features of the different subtypes
GenotypeNo. of blood donors Sex ratioa
Mean age (yr)c
All blood donors MenWomen
1a
1b
2
3a
4a
4d
5a
89
97
35
72
2.5
0.9
0.9
1.3
NDb
1
0.33
1.3
35.6 ? 9.5
43.9 ? 12.7
43.3 ? 12.0
35.4 ? 9.5
39.0 ? 7.7
34.4 ? 5.8
45.5 ? 18.1
38.3 ? 11.5
37.1 ? 9.2
38.9 ? 13.8
40.2 ? 15.0
34.2 ? 9.4
45.3 ? 8.3
34.3 ? 6.6
ND
37.3 ? 11.3
32.0 ? 9.2
41.4 ? 13.4
46.1 ? 7.5
36.9 ? 9.7
ND
34.6 ? 5.4
ND
39.7 ? 11.6
8
16
4
Total321
aNumber of males to number of females.
bND, not determined.
cValues are 95% confidence intervals.
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Comparison of strains in blood donors and patients from
the same region. The distribution of the 96 strains identified in
our plasma samples collected from blood donors between 2001
and 2002 was compared with the distribution of strains iden-
tified in samples collected from 184 patients living in the same
region during the same period (35). The distributions were
similar for subtypes 1 (56.2% versus 61.4%), 2 (6% versus
8.3%), and 3 (24.5% versus 28.1%). Conversely, subtype 4 was
significantly more prevalent in patients than in donors (13.6%
versus 7.3%; ?2? 8.32; P ? 0.01). It should be noted that the
sex ratio was significantly different in the two populations (1.3
in blood donors versus 2.2 in patients; P ? 0.001).
Analysis of HCV strain clustering in a phylogenetic tree built
with sequences identified in donors and patients showed no
striking pattern. No typical subtype pattern could be associated
with the two populations.
The distribution of the strains and the mean genetic dis-
tances between subtypes were compared between donors and
patients. The mean genetic distances were significantly lower
for subtypes 1a (0.041 ? 0.008 versus 0.047 ? 0.009) and 1b
(0.053 ? 0.007 versus 0.063 ? 0.007) in patients than in donors
(P ? 0.01). This was not the case for subtype 3a (0.050 ? 0.009
versus 0.049 ? 0.006), which showed comparable mean genetic
distances in donors and patients.
DISCUSSION
The first donor samples in this study were collected in 1991
during the first year after implementation of serological
screening for HCV in French blood banks. At that time the
epidemiological distribution featured a large predominance of
subtype 1b strains (?50%), followed by types 1a and 2 (?20%
each), and a low prevalence of other types (?5% each). In
2003 a radically different distribution was observed, with a
predominance of subtype 1a (?40%), followed by subtypes 3a
and 1b (?30% each). Over the 12-year study period, genotypes
1a and 3a emerged while genotypes 1b and 2 regressed.
To our knowledge, this is the first study which has analyzed
the evolution of the HCV subtype distribution as a function of
the year of blood collection. To date, previous studies have
analyzed the subtype distribution as a function of the age of the
patients. For example, a shift in the distribution was observed
in northeast Italy, where subtype 1b and 2 were practically
replaced by subtypes 1a and 3a (9). Another study with Ger-
man patients showed that subtype 1b was predominant among
elderly patients, while subtype 1a was predominant among the
younger population (26). A third study with Russian patients
showed that subtype 1b was progressively replaced by subtype
3a (14).
FIG. 1. Phylogenetic tree of partial NS5b nucleotide sequences of
33 HCV strains isolated from blood donors (??) and a panel of
reference strains identified by their GenBank accession numbers. New
subtypes are indicated by arrowheads. The numbers at the right cor-
respond to prototype strain subtypes as previously published by
Stuyver et al. (33) for subtypes 2a, 2b, 2c, 2d, 2e, 2f, 2k, and 2l and
Tokita et al. (39) for subtypes 2e* and 2f*. The numbers at the nodes
are the percentages of 1,000 bootstrap replicates higher than 50%. The
scale bar indicates the p distances. The vertical dotted line represents
separation between subtypes.
3626 CANTALOUBE ET AL.J. CLIN. MICROBIOL.

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Past studies have shown that the main risk factor for infec-
tion by genotypes 1a and 3a is intravenous drug use (10, 18, 35)
and that the main risk factor for infection by genotypes 1b and
2 is blood transfusion (10, 18). This epidemiological scenario
was corroborated by analysis of the age data for the different
groups. Patients infected by genotypes 1b and 2 were older
than those infected by genotypes 3a and 1a. This finding sug-
gests that at least two successive changes occurred. The first,
implicating genotypes 1b and 2, was linked to blood transfu-
sion; and the second, implicating genotypes 3a and 1a, was
linked to intravenous drug use.
Access to the database previously described by Tamalet et
al. (35) gave us a unique opportunity to compare HCV infec-
tion in blood donors and hospitalized patients from the same
region during the same time period (2001 and 2002). Despite
the differences in ages, sex ratios, and clinical presentations,
the distribution of genotypes in the two populations was glo-
bally similar for strains belonging to types 1, 2, and 3 (account-
ing for 86.4 and 92.7% of HCV strains in patients and donors,
respectively). This finding suggests that the monitoring of
blood donors provides a relatively unbiased picture of the
epidemiological situation. The logical exception to this rule
involves donor population underrepresentation of genotype 4,
which was associated with human immunodeficiency virus in-
fection in the study of Tamalet et al. (35).
Comparison of the donor and the patient data provided two
other important insights. The first is that the mean genetic
distances between subtype 1a and subtype 1b isolates were
shorter in the patient population. A probable explanation for
this difference in subtype 1a strains is that drug addicts are
often treated in public hospitals but rarely donate blood. Re-
garding subtype 1b, the most likely explanation is association
with nosocomial transmission, in particular, in hemodialysis
patients (35). Another important insight obtained by analysis
of the phylogenetic reconstructions was the absence of cluster-
ing between the patient and the donor strains. This finding
refutes the hypothesis of a phylogenetic basis for pathogenicity
in each subtype.
Analysis of the HCV genotypes within a defined popula-
tion is a useful epidemiological tool for the study of the
evolution of HCV infection in different geographical regions
and risk groups. However, the utility of genotype analysis
depends on the technique used. Until now, most data have
been obtained by analysis of the viral 5? noncoding region.
This method allows good discrimination between the six
HCV types but not differentiation between subtypes due to
the high degree of conservation of the 5? noncoding region.
Prior comparison of the typing results based on the 5? non-
coding region (34) or the NS5b region (data not shown) in
our population showed that ?30% of the strains assigned to
subtype 1b by using the 5? noncoding region were in fact
subtype 1a strains. Based on that preliminary finding, sub-
typing in the current study was based on analysis of the
NS5b region or the envelope sequence.
The epidemiological patterns of the different viral subtypes
were of special interest. Genotypes 1a, 3a, 4a, and 1b presented
typical “epidemic” profiles, with a large number of isolates for
a given subtype and short mean genetic distances between
isolates. All four of these genotypes are related to parenteral
transmission, associated with the use of blood products and
intravenous drug abuse over the last 50 years in Europe, North
America, Japan, and Australia (19, 21, 30). The most likely
explanations for this pattern insofar as subtypes 1a, 3a, and 4a
are concerned are the explosive epidemiological spread of
HCV through intravenous drug use and the recent nature of
their emergence. The slightly greater mean distances between
subtype 1b isolates were probably due to slower spread
through blood transfusion and to the fact that transmission
peaked prior to 1990, meaning that this subtype has been
evolving longer than subtypes 1a, 3a, and 4a.
Genotype 2 isolates from donors and patients displayed a
typical “endemic” profile, with a large number of subtypes
(11 genotype 2 subtypes) and few isolates in each subtype.
Infection by type 2 was also associated with the older age
bracket in our study. Remarkably, type 2 accounted for
more than 20% of strains collected before 1995 but was
never found between 2001 and 2002. This finding suggests
that this endemic genotype is progressively being replaced
by epidemic genotypes. The most prevalent subtypes were
2a, 2b, 2c, 2i, and 2k, all of which presented more than three
strains each. Among the various subtype 2 isolates found in
our study, two, i.e., subtypes 2i and 2l, were exclusive to
FIG. 2. Distribution of hepatitis C virus types according to date of collection. The number of cases is indicated in parentheses.
VOL. 43, 2005HCV GENOTYPE DISTRIBUTION IN FRENCH BLOOD DONORS 3627

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France (23). Subtype 2k has previously been observed in
Moldavia and Russia (28) and is one of the two subtypes
responsiblefortherecombinantstrainidentifiedinSt.Peters-
burg, Russia (15). Subtype 2a, 2b, and 2c are common in
East Asia, North America, and Europe (mainly Italy) (3, 17,
22). Six type 2 strains could not be assigned to any previ-
ously described subtypes and belonged to five newly identi-
fied subtypes (Fig. 1).
Overall, this study provides strong evidence that the molec-
ular epidemiology of HCV infection in southeast France has
changed radically in relation to the changing etiology of infec-
tion over the last 15 years. The main change involved the
emergence of new epidemic genotypes and regression of en-
demic type 2 genotypes. This study also demonstrated for the
first time that monitoring of blood donors is a globally valid
epidemiological indicator of HCV genotype distribution and
demography markers.
ACKNOWLEDGMENTS
We thank C. Tamalet for providing the sequences of strains isolated
from patients.
This study was supported by grant 2003-09 from the “Conseil Sci-
entifique de l’Etablissement Franc ¸ais du Sang.”
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