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

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 

OPENACCESS 

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- 

Core Region: Its Significance in HCV-Genotyping 

and Type Dependent Genomic Expression. Maced 

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

dx.doi.org/10.3889/MJMS.1957-5773.2011.0208. 

Key words: HCV; core; genotypes; expression; 

RFLP. 

Correspondence: Prof. M. Irshad. Clinical 

Biochemistry Division, Department of Laboratory 

Medicine, A.I.I.M.S., New Delhi-110029, India. E- 

mail: drirshad54@yahoo.com 

Received: 19-Jul-2011; Revised: 24-Oct-2011; 

Accepted: 22-Nov-2011; Online first: 07-Dec-2011 

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. 

Present study reports the standardization and use of polymerase chain reaction (PCR) followed by RFLP 

(PCR-RFLP) implicating HCV-core as a target region and its comparison with PCR-RFLP based on the 

use of 5'NCR for HCV genotyping / sub-typing. The new assay produced 399bp PCR product from core 

region and used a different set of restriction enzymes (MboI, AccI and BstNI) to digest these products 

for HCV sub-typing. This assay allows a quick determination of HCV types / sub-types. HCV was typed 

/ sub-typed using these two assays in patients with liver and renal diseases. Analysis of results 

demonstrated the two assay system to be comparable with some difference in few cases. Whereas 

genotype-3 was a common finding in liver disease groups, genotype-1 was more frequent in chronic 

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 

analysis which supported our findings. 

Introduction 

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) 

30 

[3]. HCV infects approximately 200 million people globally 

[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 

http://www.mjms.ukim.edu.mk

Ansari et al. HCV-core in genotyping 

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. 

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 

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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 95 o C for 15 sec, annealing 

at 54 o C for 30 sec, and extension at 72 o C for 45 sec with 

final extension at 72 o C 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 50 o C 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 

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. 

32 



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 

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 pretreatment 

buffer. For the ELISA reaction, 200 µL of pretreated 

samples and controls were incubated for 95 

minutes at 37°C with continuous shaking in the antibodycoated 

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 

Table 1: Prevalence and distribution of hcv genotypes / sub-types in different category of liver and renal diseases. 


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Basic Science 

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 

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]. 

A) 

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) 

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. 

34 

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

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 



A) 

B) 

another simple and quick assay for HCV-genotyping. 

Of-course, a large number of cases need to be analysed 

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. 

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 

either of two sub-genomic regions (Table 1). When 

same set of sera were comparatively analysed using 

these two assays, the difference was not very significant 

(Table 2). These data indicate the new assay, to be 

Figure 4: Molecular Phylogenetic anaylsis by Maximum Likelihood 

method (Ref. 28,29) : Phylogenetic tree analysis of 399 bp fragment of 

core region from 11 HCV isolates obtained in the studied population. 

The accession number of standard genotypes used in the construction 

of the tree : D00831_G1a, D00574_G1b, D00944_2a, D10077_2b, 

D16761_G3a, D11443_G3b, D16808_4a, Z29471_5a, U10198_6a. 


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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 

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 

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 



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 HCVgenotyping 

[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 subgenomic 

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 


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 HCVcore 

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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