Basic Science

Macedonian Journal of Medical Sciences. 2012 Mar 15; 5(1):30-39.

http://dx.doi.org/10.3889/MJMS.1857-5773.2011.0208
Basic Science
OPEN  ACCESS

HCV-Core Region: Its Significance in HCV-Genotyping and Type

Dependent Genomic Expression

Mohammad Ahmad Ansari, Raghavendra Lingaiah, Mohammad Irshad

All India Institute of Medical Sciences, Department of Laboratory MedicineClinical Biochemistry, Division Room No. 11, 2nd Floor, OPD
Block, Delhi, New Delhi 110029, India

Abstract
Citation: Ansari MA, Lingaiah R, Irshad M. HCV- Present study reports the standardization and use of polymerase chain reaction (PCR) followed by RFLP
Core Region: Its Significance in HCV-Genotyping
(PCR-RFLP) implicating HCV-core as a target region and its comparison with PCR-RFLP based on the
and Type Dependent Genomic Expression. Maced
J Med Sci. 2012 Mar 15; 5(1):30-39. http:// use of 5'NCR for HCV genotyping / sub-typing. The new assay produced 399bp PCR product from core
dx.doi.org/10.3889/MJMS.1957-5773.2011.0208. region and used a different set of restriction enzymes (MboI, AccI and BstNI) to digest these products
Key words: HCV; core; genotypes; expression; for HCV sub-typing. This assay allows a quick determination of HCV types / sub-types. HCV was typed
RFLP. / sub-typed using these two assays in patients with liver and renal diseases. Analysis of results
Correspondence: Prof. M. Irshad. Clinical demonstrated the two assay system to be comparable with some difference in few cases. Whereas
Biochemistry Division, Department of Laboratory genotype-3 was a common finding in liver disease groups, genotype-1 was more frequent in chronic
Medicine, A.I.I.M.S., New Delhi-110029, India. E-
mail: drirshad54@yahoo.com
renal failure cases. The PCR product from core based assays was also subjected to sequence analysis
for genotypes/sub-types and the findings were slightly different. This was further analysed by phylogenetic
Received: 19-Jul-2011; Revised: 24-Oct-2011;
Accepted: 22-Nov-2011; Online first: 07-Dec-2011 analysis which supported our findings.
Copyright: © 2011 Ansari MA. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Competing Interests: The authors have declared
that no competing interests exist.

Introduction [3]. HCV infects approximately 200 million people globally

Hepatitis C virus (HCV), a major etiological
agent of post-transfusion non-A non-B hepatitis was first
characterized by Choo et al in 1989 [1]. It is an enveloped
virus and belongs to genus Hepacivirus of the family
Flaviviridae [2]. HCV genome consists of single-stranded,
positive sense RNA of 9.6-kb length and a single open
reading frame (ORF) of 9033 to 9099 nucleotide flanked
by two non-coding regions (NCR) i.e. 5’ NCR and 3’
NCR. The ORF codes a long polyprotein of approximately
3000 amino acids and produces three structural proteins
(core, envelope E1 and E2) and seven nonstructural
proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B)
[4], has 12-13 million carriers in India [5]. Approximately
80% of HCV infected patients develop chronic hepatitis
and 20% of them develop cirrhosis [6]. The reported
prevalence of HCV in India is 15% to 25% in chronic liver
disease (CLD) patients [7, 8]. HCV infection is an
important cause of morbidity and mortality among patients
with end stage renal disease (ESRD), particularly among
renal transplant recipients also.
HCV-RNA has a high degree of heterogeneity
over the entire genome. It varies 30-35% and 20-25%
within types, and subtypes respectively [9]. Six major
genotypes and more than 120 subtypes of HCV have

30 http://www.mjms.ukim.edu.mk

been characterized [10]. These HCV genotypes have
distinct geographic distribution worldwide. Genotypes 1,
2 and 3 are widely distributed while other genotypes are
much more restricted to certain geographical areas. In
India, genotype 3 is reported to be the most prevalent,
followed by genotype 1 [11, 12]. These genotypes of
HCV have important epidemiological implications. The
duration and response to interferon therapy in HCV
infected patients depends and varies according to HCV
genotypes [13, 14].
Real-time PCR and nucleotide sequencing are
the most reliable methods for HCV genotyping and often
used as gold standard method [15]. Sequencing is a
complex and time consuming step. Other genotyping
methods include restriction fragment length
polymorphism (RFLP) [16, 17], type-specific polymerase
chain reaction (PCR) [18], hybridization based line probe
assay (LiPA) [19] and serotyping using type-specific
peptides of the HCV polyprotein [20]. Although these
methods are sensitive, they can identify only fewer
subtypes and cannot differentiate subtypes accurately
in most of the cases.
350 bases long fragment of 5’ non-coding region
(5’ NCR), is the most conserved part (~95%) of HCV
genomes and contain genotype specific motifs [21].
However, it has been analyzed that this region alone
cannot accurately distinguish genotype 1 and 6 and
subtypes of genotype 1 [22, 23]. The nearby core region
is also well conserved and has 81 to 88% nucleotide
sequence similarity [21]. Recent reports indicate that
sequence information of core region is sufficient to
differentiate all genotypes and several subtypes [24].
Thus, present study is an attempt to develop a
method for HCV typing based on a newly designed RT-
PCR, followed by restriction fragment length
polymorphism (RFLP) analysis using HCV-core as the
target region. This newly developed assay, simultaneous
with existing assay based on PCR-RFLP of 5’ NCR
region, was used in patients with liver failure and renal
diseases for HCV-genotyping and a comparison was
made. The procedure allows a quick identification of
nearly all major types and subtypes of HCV, and is more
accurate and easier to perform than similar methods so
far described. Present report also describes the relation
of HCV-core expression as indicated by the presence of
HCV-core protein in serum, with viral types / sub-types
in different disease groups.
Maced J Med Sci. 2012 Mar 15; 5(1):30-39.
Ansari et al. HCV-core in genotyping
Material and Methods
Patients and blood samples
A total number of 63 patients comprising 14
patients with chronic liver diseases (CLD), 4 patients
with cirrhosis, 12 patients with hepatocellular carcinoma
(HCC) and 17 patients with chronic renal failure (CRF)
before dialysis were included in this study plan. 16 of
these were excluded for genotyping as these could not
be found positive for HCV-RNA on conventional PCR
due to low viral load. The above case number is based
on the statistical analysis using SPSS Statistics 16.0. All
these patients were HCV positive as detected and
confirmed by Real Time PCR.
The venous blood (6-10 mL) was drawn and
transferred in plain tubes without anticoagulant from
HCV positive patients diagnosed with chronic liver
diseases (CLD), cirrhosis of liver, Hepatocellular
carcinoma (HCC) and chronic renal failure (CRF). The
patients were in adult age group and represented both
the sexes. They attended either Out Patient Department
or admitted to the Gastroenterology Unit/Haemodialysis
Unit of All India Institute of Medical Sciences, New Delhi.
Blood samples were collected after getting consent from
the patients. These patients were evaluated clinically
and biochemically and their sera were tested for various
hepatitis markers (HCV-antibody and HCV-core antigen)
and test parameters including liver function tests and
hemogram. The diagnosis of different types of diseases
was based on accepted clinical, biochemical and
histological criteria as outlined elsewhere [25]. The
study protocol was approved by the Institutional Ethical
Committee.
HCV- RNA extraction and cDNA
synthesis
HCV-RNA was extracted from 200 μL serum
sample using High Pure Viral Nucleic Acid Kit (total
nucleic acid isolation Kit-Roche Applied Science,
Germany) following manufacturer’s instructions. RNA
was eluted into 50 μL Elution buffer, aliquoted and stored
at -70°C. The extracted RNA was denatured at 65°C for
10 min before the reverse transcription step. cDNA was
synthesized by cDNA synthesis kit (Transcriptor High
Fidelity cDNA Synthesis Kit-Roche, Germany) at 42°C
for 30 minutes using 60ìM of random hexamer primer.
HCV- RNA detection on real time PCR
Patient sera were screened for HCV-RNA on
real time PCR using primers and probe of a highly
31
Basic Science
conserved region of HCV genome. HCV RNA was
detected using 5' NCR region specific primers (5'-CGG
GTG TAC TCA CCG GTT CCG-3' and 5'-AGC GTC TAG
CCA TGG CGT-3') and fluorescent labeled probe (MCY
CCC CCT YCC GGG AGA GCAT_ _DB). All positive
and negative controls were tested in parallel with test
samples throughout the entire procedures, starting with
RNA extraction.
Amplification of 5’ NCR on conventional
PCR
cDNA of HCV-RNA positive samples were
subjected to conventional PCR to amplify approximately
240 bp fragment of 5' NCR region according to following
steps : initial denaturation at 95°C for 7 min followed by
30 cycles of denaturation at 95oC for 15 sec, annealing
at 54oC for 30 sec, and extension at 72oC for 45 sec with
final extension at 72oC for 5 min. The second round of
PCR was performed with internal primers under same
thermal conditions as used for first round amplification
except annealing at 50oC for 30 sec. Three μL of the PCR
product was electrophoresed on 2% agarose gel
containing ethidium bromide and was visualized under
UV transilluminator.
HCV genotyping by RFLP method
PCR products obtained by above method were
subjected to HCV genotyping using RFLP method. These
amplicons were first purified using QIAquick Gel
Extraction Kit (Qiagen, Germany) and then digested
using two sets of restriction enzymes : RsaI+HaeIII and
MvaI+HinfI (from New England Biolabs, MA, USA) at
37°C for over night, to differentiate all six genotypes of
HCV (Fig. 1A & B) according to McOmish et al, 1993 [26].
The subtypes 1a, 1b and 2a, 2b, 3a & 3b were
differentiated using enzymes BstUI (at 60°C for 3hrs)
and ScrF1 (at 37°C for 3hrs), respectively (Fig. 2A & B).
Band pattern was analyzed on either 4% high quality
agarose gel or 10% polyacrylamide gel.
Amplification of core region
Sequences with the sequence Id’s (FJ795713,
EU420986, D11443, D16808, AJ291279, EU420985,
FJ795704, AJ291257, D16761, D10077, AJ231496,
FJ159776, AJ231497, NC_009824, D00831, D00574,
D00944, Z29471, U10198, AB079076, AB079077,
AB008441, AB008447, D14853, AY651061, AY051292)
were analysed to design degenerate primers to pick all
the HCV-RNA positive cases. First round amplification
was performed with primers 5'-TAC TGC CTG ATA
32
GGG TGC TT- 3' and 5'-AA GAT AGA G/AAA A/GGA
GCA ACC- 3' at 95°C for 5 min (initial denaturation)
followed by 35 cycles of denaturation at 95°C for 45 sec,
annealing at 46°C for 30 sec and extension at 72°C for
60 sec with final extension at 72°C for 5 min. The nested
PCR was performed with second round internal primers
5'-TCC TAA ACC TCA AAG AAA AAC C- 3' and 5'-ATG
TAC/T CCC ATG AGG/A TCG- 3' under same conditions
as used for first round amplification. Three μL of PCR
product was run on 2% agarose gel and visualized under
U.V. transilluminator. The 399bp PCR product was
obtained. These amplicons were purified from gel and
then sequenced on automated sequencer from Ms
Ocimum Biosolutions Ltd, Hyderabad, AP, India.
Amplicon sequences of HCV-core region were compared
to an online database for the best possible match using
the BLAST (Basic Local Alignment Search Tool) program
of National Center for Biotechnology Information
(www.ncbi.nlm.nic.gov).
HCV Genotyping by RFLP of CORE
region
Digestion pattern of 399 bp core region was
analyzed with restriction enzymes MboI, AccI and BstNI.
Production of fragments 307 bp and 75 bp with MboI are
predictive of genotype 1b (G1b). Restricted fragments
210 bp, 115 bp & 75 bp are predictive of genotype 2a
(G2a), fragments 142 bp, 75 bp, 67 bp & 38 bp are
predictive of genotype 2b (G2b), fragments 180 bp, 77
bp, 75bp & 68 bp are predictive of genotype 3b (G3b)
and fragments 325 bp & 75 bp and 245 bp, 80 bp & 75
bp are predictive of genotype 1a, 3a & 5a (G1a, G3a &
G5a) and genotype 4a & 6b (G4a &6b) respectively.
Similarly, digestion pattern with AccI producing two
fragments of either 231 bp & 169 bp or 309 bp & 90 bp
size shows the presence of genotype 1a (G1a) and
genotype 3a (G3a), respectively. Restricted fragments
of 203 bp & 196 bp show the presence of genotype 4a
(G4a) while genotype 5a and genotype 6b do not have
any restriction site for enzyme AccI. Enzyme BstNI
produces the fragments of 214 bp & 186 bp and 214 bp,
165 bp & 20 bp for genotype 5a and genotype 6b,
respectively.
Nested PCR product was added into reaction
mixture containing 10 Units of each restriction enzyme
[AccI, MboI and BstNI (New England Biolabs, MA, USA)]
individually at 37°C for overnight. Digestion products
were electrophoresed on 2.5% agarose gel to determine
the genotypes. The RFLP pattern was then evaluated
under UV light.
http://www.mjms.ukim.edu.mk

Phylogenetic analysis
Amplicon sequences of HCV core region were
compared to an online database for the best possible
match using the BLAST (Basic Local Alignment Search
Tool) program of National Center for Biotechnology
Information (www.ncbi.nlm.nic.gov) [27] and CLUSTAL-
X version 2.0. The evolutionary history was inferred by
using the Maximum Likelihood method based on the
Kimura 2-parameter model [28]. The tree with the highest
log likelihood (-1730.3173) is shown. The percentage of
trees in which the associated taxa clustered together is
shown next to the branches. When the number of
common sites was < 100 or less than one fourth of the
total number of sites, the maximum parsimony method
was used; otherwise BIONJ method with MCL distance
matrix was used. The tree is drawn to scale, with branch
lengths measured in the number of substitutions per site.
The analysis involved 20 nucleotide sequences. Codon
positions included were 1st+2nd+3rd+Noncoding. All
positions with less than 95% site coverage were
eliminated. That is, fewer than 5% alignment gaps,
missing data, and ambiguous bases were allowed at any
position. There were a total of 299 positions in the final
dataset. Evolutionary analyses were conducted in
MEGA5 [29].
Subgenomic sequences of known HCV
genotypes were retrieved from NCBI database (http://
www.ncbi.nlm.nih.gov/nuccore/
157781216?report=fasta&from=340&to=9405). Only
those sequences, which have our target region of HCV
Table 1: Prevalence and distribution of hcv genotypes / sub-types in different category of liver and renal diseases.
Maced J Med Sci. 2012 Mar 15; 5(1):30-39.
Ansari et al. HCV-core in genotyping
core, were taken for Phylogenetic study.
HCV Core Ag Assay
Sera samples were assayed for HCV core protein
according to the manufacturer’s instructions using EIA
kit from Ortho Diagnostics, UK. One hundred µL of
samples and controls were mixed with 100 µL of a pre-
treatment buffer. For the ELISA reaction, 200 µL of pre-
treated samples and controls were incubated for 95
minutes at 37°C with continuous shaking in the antibody-
coated wells of a microtiter plate. The plates were
washed and incubated for 30 minutes at 37°C with 200
µL of conjugate, washed again, and incubated for 30
minutes at 37°C with 200 µL of substrate. The optical
densities (ODs) were read in a spectrophotometer at
490 nm using a 620 nm reference. The samples and
controls were tested in duplicate and the mean OD of
each duplicate testing was used. The samples that
exhibited more than 25% variation between the two ODs
were considered invalid and retested. As recommended
by the manufacturer, the lower detection cutoff was
established for each run and corresponded to the mean
OD of the 2 negative controls plus 0.040. A sample was
considered positive only when the mean OD was higher
than the cutoff OD of the corresponding run.
Diagnostic Criteria
Patients with CLD and cirrhosis of liver were
diagnosed by histopathological criteria laid down by
International Study Group on Chronic Hepatitis [30]. All
33
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these CLD patients had persistent elevation of

transaminases level (at least twice the upper limit of
normal range) for more than six months and histological A)
evidence of chronic hepatitis on liver biopsy at the
beginning of follow-up. The diagnosis of Hepatocellular
Carcinoma is based purely on histological criteria. The
CRF was diagnosed using criteria as detailed elsewhere
[31].

Results
Present investigation reports the development
and application of PCR-RFLP using HCV-core as the
target region and its comparison with PCR-RFLP based
on use of 5’ NCR for HCV genotyping in different disease
groups. This study also describes possible relation
between HCV-core expression and HCV-genotypes.
Analysis of results achieved after determining HCV
genotype and their isotypes by PCR-RFLP assay in
patients infected with HCV and belonging to different

B)

A)

Figure 2: A) Digestion pattern of 5' NCR with enzyme BstU I. L: Low MW

B)
Figure 1: A) Digestion pattern of 5' NCR with enzyme Rsa I+Hae III. L:
Low molecular weight DNA marker; G : Genotype. B) Digestion pattern
of 5' NCR with enzyme Mva I+Hinf I. L: Low molecular weight DNA
marker; G: Genotype.
DNA marker; G1a: Genotype 1a; G1b: Genotype 1b. B) Digestion
pattern of 5' NCR with enzyme ScrF I. L: Low MW DNA marker; G3a:
Genotype 3a; G3b: Genotype 3b.
category of liver diseases and chronic renal failure are
shown in Table 1. A total number of 14 patients with CLD
indicates the presence of G-1 in 2 cases (14.2%), G-3 in
7 cases (50%), G-4 in 1 case (7.1%) and mixed genotypes
in 4 (28.7%) cases, respectively while using 5‘NCR as
the target region of HCV-genome. Of four patients with
cirrhosis, G-1 and G-3 were detected in 2 cases (50%)
each. Twelve cases with HCC were analysed and found
with a similar pattern of G-1, G-3, G-4 and mixed
genotypes. In 17 patients with CRF, G-1 was detected in
13 (76.5%) cases and mixed genotypes in 4 (23.5%)
cases, respectively. The pattern of subtypes against
each genotype is shown in Table 1. In order to make an

34 http://www.mjms.ukim.edu.mk
 Ansari et al. HCV-core in genotyping

another simple and quick assay for HCV-genotyping.

Of-course, a large number of cases need to be analysed
A) before reaching a conclusion.
On comparing the genotype pattern in these
disease groups with those obtained by sequencing, we
could find the presence of genotypes that further
supported by sequencing (Table 2). Moreover, 5‘NCR
region could help in the detection of more number of
cases as compared to HCV-core. However, the results
of genotyping using core-region were better substantiated
by sequence analysis of core region as compared to
those using 5‘NCR.
B)
A comparison of genotypes (1,2,3 and mixed)
with HCV–core expression, as indicated by the presence
of HCV-core protein in these disease groups,
demonstrated G-3 to be associated with more prevalence
of HCV in liver disease groups. At the same time, G-1

Figure 3: A) Digestion pattern of core region with enzyme Mbo I. L: Low

molecular weight DNA marker; G3a: Genotype 3a; G1b: Genotype 1b;
G2a: Genotype 2a. B) Digestion pattern of core region with enzyme
Acc I. L: Low molecular weight DNA marker; G1a: Genotype 1a; G1b:
Genotype 1b; G3a: Genotype 3a.

attempt for determining HCV-genotypes and their

subtypes in these cases with more accuracy, we
amplified, 399bp region of HCV-core, confirmed for
genotype by phylogenetic analysis (Fig. 4) and then
digested it with separate sets of restriction enzymes
(AccI, MboI and BstNI) as outlined in methods section.
Using defined pattern of bands on gel for different
subtypes (Fig. 3A & 3B), we analysed results in each
case and found the results as given in Table 1. The
pattern of genotypes and subtypes achieved in these
cases was not much different from the one observed
with use of 5’ NCR by PCR-RFLP.
We could have two major findings by the use of
HCV-core as compared to 5‘NCR: the first, less number
of subtypes of same category were picked up with use
of core as compared to 5‘NCR and second, there was a
predominant presence of G-3 in 83.3% in HCC as
compared to its prevalence in mere 58.3% cases with
use of 5‘NCR. In all other cases, there was not a
significant difference of type detection with the use of Figure 4: Molecular Phylogenetic anaylsis by Maximum Likelihood
either of two sub-genomic regions (Table 1). When method (Ref. 28,29) : Phylogenetic tree analysis of 399 bp fragment of
same set of sera were comparatively analysed using core region from 11 HCV isolates obtained in the studied population.
The accession number of standard genotypes used in the construction
these two assays, the difference was not very significant of the tree : D00831_G1a, D00574_G1b, D00944_2a, D10077_2b,
(Table 2). These data indicate the new assay, to be D16761_G3a, D11443_G3b, D16808_4a, Z29471_5a, U10198_6a.

Maced J Med Sci. 2012 Mar 15; 5(1):30-39. 35

Basic Science
Table 2: Comparative status of HCV - subtypes using PCR - RFLP and sequence analysis.
A total number of 20 sera from different disease groups as specified above, were genotyped by PCR – RFLP using 5’ NCR and HCV-core regions. The amplicons of HCV-core were also
sequenced and sub-typed by known-sequence-comparison criteria.
types had more expression in CRF patients, when
severity of disease in CLD patients was assessed in
relation to genotypes (Table 3).
Discussion
Hepatitis C virus (HCV) infection is a major
public health problem of the world. HCV infection leads
to not only serious liver diseases in majority of patients
but at the same time, remains non-responsive to antiviral
therapy in high proportion of cases. Whereas very little
success has been made in producing an effective vaccine
against HCV [32], antiviral treatment is the only way of
treating HCV infection. Unfortunately, majority of patients
either do not respond to the antiviral treatment or develop
the infection once again after a gap of time. As a result,
its early diagnosis and effective treatment remains a
major goal to be still achieved.
The genome of HCV is highly variable. Till date
six genotypes and more than 120 isotypes have already
been characterized [10]. The molecular forms originate
from frequent variations in certain sub-genomic regions
of HCV genome. The HCV-genotypes and their subtypes
Table 3: Relation between genotypes and core expression in different disease groups.
Above table indicates the presence of HCV-core protein in relation to genotypes in different disease groups. No., Indicates the number of sera analysed; No.+ve , Indicates the number of sera
positive for given parameter; ND, Not done.
36
have important implications, particularly, when response
of patients to antiviral therapy and its outcome is totally
dependent on the presence of HCV-genotype [13, 14].
HCV infection is a serious problem of India also where a
high proportion of chronic liver disease are caused by
HCV [7, 8]. Present study was planned to address the
problem of HCV infection by developing a simple and
easy assay for determination of HCV-genotypes and
find out the status of different genotypes and their
subtypes in different disease groups in north India.
Simultaneously, the present study was also aimed to
investigate some possible relation of HCV-core
expression, as indicated by the presence of HCV-core
protein in blood, with HCV-subtypes so that the presence
of this protein could be used as a simple and alternate to
foresee the possible presence of certain HCV subtypes
in human blood.
Earlier, the determination of HCV-genotypes
was done by methods, such as automated reverse
hybridization [33] and semi automated sequencing [34,
35]. These methods need a previously amplified genomic
product as starting material. 5‘NCR, a highly conserved
region is conveniently used for genotyping by these two
methods. However, differentiation of subtypes is not
http://www.mjms.ukim.edu.mk

always possible using this region and quite often leads
to sub-typing error [23, 36-38]. Nucleotide sequencing
followed by phylogenetic analysis of regions like NS5B
and HCV-core has also been recommended for HCV-
genotyping [23]. However, this procedure is impractical
and time consuming. It also does not detect mixed
genotypes infection [39].
RT-PCR followed by RFLP has been often used
for HCV-genotyping and their subtypes. In all these
assays the 5‘NCR has been frequently used for
amplification followed by digestion by different sets of
restriction enzymes to detect different subtypes.
However, like in other assay systems it is not possible to
detect all subtypes by using 5‘NCR [12]. Attempts have
been made to include the conserved part of other sub-
genomic regions also both in PCR-RFLP as well as Real
Time PCR [40]. Present study was one more attempt to
develop assays for determining HCV sub-types using
HCV core region for amplification followed by RFLP with
different sets of restriction enzymes. This assay was
found to be simple, quick and an alternate assay for
HCV-genotyping.
When the results achieved by this newly
developed assay, were compared with those found with
PCR-RFLP based on the use of 5‘NCR, we could not find
a remarkable difference in all disease groups. However,
use of core produced no confusion in distinguishing
HCV genotypes in compared with the use of 5‘NCR. In
fact, HCV core is another alternative region for HCV
genotyping. It is supported also by sequencing of
amplicon. This method is single step method producing
fast results. The difference of percent change was more
attributed to less number of cases included in some
disease groups. In fact, these diseases patients are
referred to our tertiary-care hospitals for treatment and
so, in spite of high prevalence of HCV in this area, we do
not get frequent patients with all these disease groups.
The study is continued and will cover more number of
cases to reach a logistic conclusion in the course of time.
However, one finding which is important, is that G-3
genotypes and its subtypes are common in our patient
populations. Whereas genotypes 2 & 3 are reported to
respond well to antiviral treatment, Genotype 1 & 4 show
either very little response or no response at all. In view
of these findings, it is quite apparent that the patients
infected with HCV infection in this area of north India
come under the category of responding well to antiviral
treatment. Our findings are in support of few other
studies reported from this country [11, 12].
HCV-core region is supposed to play an active
Maced J Med Sci. 2012 Mar 15; 5(1):30-39.
Ansari et al. HCV-core in genotyping
role in viral replication and immunopathogenesis of
HCV-infection. Recently, several studies are available
[41] showing the diagnostic significance of HCV-core
protein in sera. Several studies have reported
concordance between HCV-RNA and HCV-core protein
in sera of HCV infected patients [42]. Taking this into
view, we realized to find out whether core-expression
shows genotype/subtype dependence. When we
detected HCV-core protein in all these HCV-infected
patients and analysed the results in relation to the type/
subtype of HCV, we found a very peculiar phenomenon.
In liver disease patients, HCV-core protein had a frequent
association with G3, whereas in CRF patients, it was
common in G1 positive cases. Although there appears
to be an unlikely impact of organ disease on expression
of a particular HCV-genotype, however, it cannot be
ignored and needs extensive experimentation and
discussion in context of other contributing factors
ascribing to gene expression.
In conclusion, present study reports
standardization of another PCR-RFLP assay using HCV-
core as the target region and a different set of restriction
enzymes to digest the PCR products. This assay was
compared with PCR-RFLP based on 5’NCR region and
shown concordance in results achieved by two assays.
Use of these assays demonstrated the predominance of
genotype-3 as the major genotype in Indian patients’
population. HCV-core expression appears to depend on
HCV-genotype and the disease condition, however, it
needs further confirmation by including more number of
cases in this study plan.
Acknowledgement
The authors thank and appreciate the financial
aid provided by ICMR, New Delhi, India to conduct this
study. Authors are also thankful to Mrs. Suman Rawat
for preparing this manuscript.
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