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Journal of Clinical Microbiology, October 2000, p. 3581-3584, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Genotyping of Hepatitis C Virus Isolates using
CLIP Sequencing
R. S.
Ross,1,*
S. O.
Viazov,1
C. D.
Holtzer,2
A.
Beyou,2
A.
Monnet,2
C.
Mazure,2 and
M.
Roggendorf1
Institute of Virology, National Reference
Centre for Hepatitis C, University of Essen, D-45122 Essen,
Germany,1 and Visible Genetics Europe,
Genopole, F-91035 Evry Cedex, France2
Received 6 April 2000/Returned for modification 22 June
2000/Accepted 27 July 2000
 |
ABSTRACT |
Determination of hepatitis C virus (HCV) genotypes and subtypes has
become increasingly important for the clinical management and prognosis
of HCV infections. The aim of the present study was to assess the
specificity and reliability of a newly developed, commercially
available HCV genotyping kit (TRUGENE HCV 5'NC
genotyping kit). This technique utilizes PCR fragments previously
generated by the diagnostic Roche AMPLICOR HCV test, which
are subsequently subjected to simultaneous PCR amplification and direct
sequencing (CLIP sequencing) of the 5' noncoding region (5'NCR). HCV
isolates from 100 randomly chosen patients were genotyped by both the
TRUGENE HCV 5'NC genotyping kit and DNA enzyme immunoassay
(DEIA). Typing results obtained by both methods were in complete
concordance in 91% of the cases. HCV RNA from the samples with
discordant genotype assignment in both assays was additionally
amplified with primers from the HCV core and NS5B regions. Phylogenetic analysis of the obtained sequences supported the results obtained from
DEIA in six cases and CLIP sequencing in two cases. In the former six
cases, the TRUGENE HCV 5'NC genotyping kit could not correctly differentiate between subtypes of genotypes 1 and 2 due to
the high conservation of the 5'NCR. However, since there was not any
misclassification between HCV genotypes 1 and non-1 types, the results
obtained with this system are, in general, reliable and can be used in
clinical practice. The TRUGENE HCV 5'NC genotyping kit in
our hands proved to be a fast and convenient technique that might be an
attractive option for HCV genotyping in laboratories already using the
Roche AMPLICOR HCV test for diagnostic reverse transcription-PCR.
| |
INTRODUCTION |
The hepatitis C virus (HCV) is a
positive-stranded RNA virus that was identified as the main cause of
posttransfusion non-A, non-B hepatitis (2). The genome of
this virus is highly variable. So far, six major genotypes and more
than 100 subtypes have been described (8, 19). Many studies
have indicated an association between HCV genotype and both the
responsiveness to alpha-interferon treatment and the degree of clinical
progression of chronic HCV infection (4, 19, 28). Therefore,
during the last few years, HCV genotyping has been implemented in
clinical laboratory settings and there is an ongoing demand for the
development of new, automated typing techniques that provide reliable
results within a reasonable time frame (8, 16, 19). The use
of a single amplification reaction for diagnostic HCV RNA detection
including the possibility of genotype determination from these PCR
products would be the most efficient way of HCV genotyping. In this
study, we utilized PCR fragments previously generated by the Roche
AMPLICOR HCV assay to assess the HCV subtype by subsequent PCR
amplification and direct sequencing of the 5' noncoding region (5'NCR)
with a recently developed, and now commercially available, automated
sequencing system.
| |
MATERIALS AND METHODS |
Specimens.
Randomly chosen sera from 100 HCV infected in-
and outpatients (72 men and 28 women; mean age, 46 years [range, 18 to
87 years]) attending Essen University Hospital were included in this
study. All patients tested positive for HCV antibodies by immunoassay (Sanofi Diagnostics Pasteur, Freiburg, Germany) and for HCV RNA by
diagnostic reverse transcription (RT)-PCR (AMPLICOR; Roche Diagnostics,
Mannheim, Germany). Sera were aliquoted and stored at 
80°C until
HCV typing.
TRUGENE HCV 5'NC genotyping kit.
The HCV subtype
of all samples was determined by the newly developed TRUGENE HCV
5'NC genotyping kit (Visible Genetics Europe, Evry, France). This
test uses the 244-bp fragment from the 5'NCR of HCV previously
amplified by the diagnostic Roche AMPLICOR HCV kit (27).
After purification with Chroma Spin 100 columns (Clontech Laboratories,
Palo Alto, Calif.), the Roche amplicons were subjected to simultaneous
PCR amplification and sequencing (CLIP sequencing), according to the
manufacturer's instructions (instruction manual for TRUGENE HCV
5'NC genotyping kit, Visible Genetics, Toronto, Canada). Two
characteristics of the CLIP reaction as a modification of the original
coupled amplification and sequencing method by Ruano and Kidd
(17) are noteworthy. (i) An engineered mutant of
thermostable DNA polymerase is used which lacks 5'-3' exonuclease activity and therefore produces uniform band intensities. (ii) Different far-red fluorescent dyes are linked to the two inward-facing CLIP primers, allowing a template to be sequenced in both directions in
a single run (26). Automated sequencing of the 183-bp
fragment (nucleotides [nt] 96 to 278 [numbering according to
reference 2]) resulting from the CLIP reaction was
performed on a MicroGene Clipper sequencer. This platform employs
ultrathin (50-µm-thick) disposable gels (26). Each pair of
forward and reverse sequences were combined and automatically aligned
with reference sequences stored in the GeneLibrarian database in order
to determine the HCV subtype and the closest isolate.
DEIA.
All samples were also typed by DNA enzyme immunoassay
(DEIA) (Gen-Eti-K DEIA; Sorin Biomedica, Saluggia, Italy), as described previously in full detail (24). In brief, a fragment from
the HCV core region was amplified by nested RT-PCR, and the resulting amplicons were hybridized to type- or subtype-specific oligonucleotide probes adsorbed to microwell plates. The double-stranded DNA hybrid thus formed was detected by DEIA.
Amplification and direct sequencing of HCV core and NS5B
fragments.
HCV RNA from the samples with discordant genotyping
results by CLIP sequencing and DEIA were subjected to additional RT-PCR with primers from the core and NS5B regions. The full details of both
procedures are given elsewhere (5, 25). The PCR products obtained were sequenced directly from both directions.
Phylogenetic analysis.
Sequences of the 183-bp HCV 5'NCR (nt
96 to 278 [numbering according to reference 2]),
216-bp core (nt 461 to 676 [numbering according to reference
2]), and 156-bp HCV NS5B (nt 8353 to 8508 [numbering according to reference 2]) DNA
fragments from those samples with discrepant typing results by the
TRUGENE HCV 5'NC genotyping kit and DEIA were subjected to
phylogenetic analysis using the PHYLIP software package, version 3.5c
(7). Distances between pairs of sequences were estimated
with the DNADIST program. Phylogenetic trees were constructed by the
unweighted pair group method using arithmetic averages on the previous
sets of pairwise distance. Significance of the grouping was assured by
bootstrap resampling (1,000 replicates). The following sequences from
the GenBank were included in the analysis (accession numbers given in
parentheses): subtype 1a (AF011751 and M62321); subtype 1b (AF165057
and D10934); subtype 1c (D14853); subtype 2a (AF177036 and D00944);
subtype 2b (D10077, D10649, D10988, and D45877); subtype 2c (AF041329,
L23457, L23458, and L38337); subtype 2i (X76411); subtype 3a (D17763
and D28917); subtype 3b (D10080, D11443, and D49374); subtype 4 (L23470, L29625, and Z29446). The partial HCV 5'NCR, core, and NS5B
sequences obtained from those samples which gave initially discrepant
typing results by the TRUGENE HCV 5'NC genotyping kit and
DEIA have been deposited in GenBank under accession numbers AF245282 to
AF245290 (HCV 5'NCR), AF233701 to AF23379 (HCV core), and AF233693 to AF233699 (HCV NS5B).
| |
RESULTS |
An HCV type and subtype could be determined for all 100 samples
with the TRUGENE HCV 5'NC genotyping kit, whereas one sample was not typeable by DEIA. The results obtained with the two tests were
in complete agreement in 91% of the cases. A different genotype assignment was achieved in two cases (1b versus 3a and 1b versus 4) and
divergent HCV 1 and 2 subtypes were determined for six isolates (Table
1).
For those samples that gave discrepant results with the TRUGENE
HCV 5'NC genotyping kit and DEIA, a phylogenetic analysis of
the 5'NCR sequences was performed to check for the reliability of HCV
subtype determinations by means of the GeneLibrarian database. Identical results were obtained in six cases. Samples S3, S7, and S8,
however, turned out to be HCV subtypes 1b and 2b in phylogenetic analysis, whereas typing by the GeneLibrarian database indicated the
presence of subtypes 1a and 2a/c, respectively (Fig.
1; Table 2). Direct sequencing and analysis of the
less-conserved HCV core and NS5B fragments revealed that the TRUGENE
HCV 5'NC genotyping kit provided the correct genotype
assessment (genotypes 3a and 4) for samples S5 and S6. Regarding the
discrepant subtyping results (samples S1 to S4, S7, and S8), DEIA was
correct in all six cases. Sample S9, which could not be typed by DEIA
and yielded subtype 2a/c by the TRUGENE HCV 5'NC genotyping
kit, belonged to the rare subtype 2i (Fig. 1; Table 2).
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FIG. 1.
Phylogenetic trees of samples S1 to S9 with discordant
HCV typing results by DEIA (GEN-ETI-K DEIA) and TRUGENE HCV
5'NC genotyping kit, derived from an analysis of partial 5'NCR (nt
96 to 278), HCV core (nt 461 to 676), and NS5B (nt 8353 to 8508)
sequences. As subtype prototypes, sequences from the GenBank were
included. Asterisks are used for discrimination of different isolates
from the same HCV subtype. The respective accession numbers are given
in the text.
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View this table:
[in this window]
[in a new window]
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TABLE 2.
Final HCV subtype assignment based on results of HCV core
and NS5B sequencing for samples that yielded discrepancies in
other assays
|
|
| |
DISCUSSION |
Nucleotide sequence analysis is the current "gold standard"
for identifying different HCV genotypes and subtypes but is generally regarded as not practical for routine clinical laboratory settings (8, 19). Therefore, a variety of surrogate HCV typing
procedures has been proposed during recent years, mainly based upon
amplification of the viral sequences by PCR, using either type-specific
primers (15), analysis of PCR products by hybridization with
genotype-specific probes (21, 25), or restriction fragment
length polymorphism (1, 22). The underlying assumption of
all these assays that the region (e.g., 5'NCR, core, or NS5B) analyzed
is representative of the whole HCV genome is generally supported by the
very consistent typing results which have been obtained so far using
assays based on sequence analysis of different regions of the HCV
genome (8, 19).
In this study, we have combined diagnostic HCV RNA detection with the
widely distributed Roche AMPLICOR HCV assay and direct sequencing of
the already available HCV 5'NCR PCR product by a newly developed
commercial system (26; TRUGENE HCV 5'NC
genotyping kit manual). Following this approach, HCV genotyping
including analysis of the sequence data was possible in about 5 h.
Genotyping by DEIA, besides requiring an additional nested PCR with
primers from the HCV core region, requires an overnight incubation with the biotinylated hybridization probes and, therefore, cannot be accomplished in less than 15 h. Comparison of the results obtained by the TRUGENE HCV 5'NC genotyping kit with those of the
already well-established DEIA (24) indicated an identical
HCV subtype assignment in 91% of all cases. Direct sequencing of the
183-bp 5'NCR fragment in our study, however, was not able to completely resolve all existing HCV genotypes and subtypes. This failure in three
cases was attributable to an inadequate subtype assignment by the
current version of the GeneLibrarian module, as shown by phylogenetic
analysis of the obtained 5'NCR sequences. Possibilities to improve the
overall performance of the TRUGENE system might, therefore, comprise
the inclusion of more HCV 5'NCR sequences in the GeneLibrarian
database, the modification of the general algorithm used for sequence
analysis, and the search for modes of HCV subtype assignment other than
by a simple alignment of sequences. The remaining discrepancies
recorded in this study between TRUGENE HCV 5'NC genotyping
kit and DEIA could not be resolved by a more-sophisticated sequence
analysis system and are the result of the high sequence conservation of
HCV 5'NCR (13). For instance, subtypes 1a and 1b in the
5'NCR fragment analyzed by TRUGENE HCV 5'NC genotyping
kit differ in only 1 nt in position 99, and subtype 2b only differs
from 2a by 2 nt at positions 124 and 164. A reliable discrimination of
subtypes 2a and 2c isolates is also not possible based upon sequence
analysis of the 5'NCR alone (20). Consequently, failures in
correct assignment of all HCV subtypes similar to those observed in
this study had already been demonstrated in several comparative
evaluations of HCV genotyping procedures based solely on the analysis
of the 5'NCR (3, 9-12, 14, 18, 21, 23), indicating that
less-conserved parts of the HCV genome, such as the core (used by DEIA)
or NS5B, are more suited for the identification of all existing HCV subtypes.
These inherent disadvantages of the 5'NCR for HCV typing are, however,
not crucial for clinical purposes. From the point of view of a
clinician, currently it is not necessary to know precisely the subtype
of the HCV strain present but to achieve a reliable discrimination
between HCV genotypes 1 and non-1 types. According to the most-recent
therapeutic recommendations, highly viremic patients chronically
infected with HCV genotype 1 isolates should be treated with
alpha-interferon and ribavirin for at least 1 year, whereas for
infections with non-1 types an initial course of 6 months is sufficient
(6). Since with the TRUGENE HCV 5'NC genotyping
kit no misclassifications between HCV genotypes 1 and non-1 types
occurred, the typing results obtained with this system are, in general,
reliable for clinical practice.
Taken together, the newly developed TRUGENE HCV 5'NC
genotyping kit and the MicroGene Clipper sequencing platform turned out to be a convenient analytical system that provides clinically valid HCV
typing results in about 5 h. The assay might be an attractive option for HCV genotyping in laboratories that already use the Roche
AMPLICOR HCV test for diagnostic RT-PCR.
| |
ACKNOWLEDGMENTS |
We are grateful to T. Teckentrupp for skillful technical assistance.
This study was supported in part by a grant to the German National
Reference Centre for Hepatitis C.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Virology, National Reference Centre for Hepatitis C, University of
Essen, Hufelandstr. 55, D-45122 Essen, Germany. Phone: 49 201 7233561. Fax: 49 201 7235929. E-mail: stefan.ross{at}uni-essen.de.
| |
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Journal of Clinical Microbiology, October 2000, p. 3581-3584, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
