Overview of Hepatitis C Virus in Pakistani population
Sumiya abbasi,kahkshan jabeen,Bushra uzair*
*Corresponding author:
Bushra uzair
Islamic International University
H-10, Islamabad
Contact # 051 908 561 71
1
ABSTRACT.
Hepatitis C virus (HCV) is an important human pathogen that causes acute and chronic hepatitis,
cirrhosis and hepatocellular carcinoma worldwide. HCV is common among the Pakistani
population. Approximately 10 million people could be carrier in Pakistan. HCV prevalence was
moderate in the general population but very high in injecting drug users and multi-transfused
populations. HCV enters the cell with CD81 and LSIGN, D-SIGN, Claudin-6/9 receptors(SRBI), (OCLN), (GAG), (LDLR), (EGFR), ephrin receptorA2 (EphA2), and Niemann-Pick C1-like
L1 (NPC1L1 and its single stranded RNA genome directly serves as the template for translation.
Accordingly, E1/E2 encode the outer membrane while nonstructural protein 4B (NS4B) induces
the specific membrane alteration, designated as membranous web (MW), that harbors this
complex. HCV NS3 is a serine protease/RNA helicase that plays a pivotal role in catalyzing the
cleavage of the single polyprotein encoded by HCV after infection of hepatocytes. The HCV
genome shows remarkable sequence variability 1 of E2 protein encoding region worldwide
leading into 6 genotypes, numerous subtypes and HCV exists in each infected patient as quasispecies. The genotype may be linked to the severity of the disease and to the efficiency of the
combination treatment with interferon and ribavirin. The epidemiology, pattern of transmission,
different genotypes and clinical consequences of the disease has been studied worldwide, but
little is known about the genomic organization and epidemiology of HCV infection in Pakistan.
This review article covers key areas of HCV particularly its genomic organization, prevalence,
life cycle, treatment options, genotypes and various factors associated with HCV infection in the
Pakistani population.
Key Words: HCV; genomic organization; genotypes; life cycle; treatment options.
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INTRODUCTION
Hepatitis C is a life threatening infectious disease of the liver which is caused by the hepatitis C
virus (HCV). An estimated 170-200 million people worldwide are infected with HCV (Conca
and Tarantino, 2009). HCV seroprevalence in Pakistan reported range between 2·4% and 6·5%.
(Second highest in the world) (Luby, 1997, Khattak et al., 2000, Mujeeb et al., 2002). Egypt,
with an estimated seroprevalence rate, 22% is reported to be highest in the world (Egypt data
sheet 2005).10% of the Pakistani population is chronically infected with this viral pathogen
(Farhana et al., 2009; Idrees and Riazuddin, 2009; Akbar et al., 2009). Still there is no vaccine
developed against HCV which is often referred to as a silent killer (Suresh D Sharma, 2009).
The existence of hepatitis C (originally "non-A non-B hepatitis") was postulated in the 1970s and
proved conclusively in 1989 (Choo et al., 1989). The global epidemiology of viral hepatitis A
and hepatitis B is well established, but HCV data remains limited, particularly in Pakistan (Raja
and Janjua, 2008). In 2004 it was estimated by the WHO that the annual deaths due to liver
cancer caused by HCV and cirrhosis were 3,08,000 and 7,85,000 respectively (World Health
Organization 2004).HCV infection is well recognized and pose severe health problem
worldwide, especially in the developing countries including Pakistan. HCV prevalence in
Pakistani population is nearly 5%. Its prevalence in injection drug users (IDUs) and the multitransfused population is high, suggesting that the reuse of syringes is common among the
injecting drug users, and blood transfusions are not properly screened here in Pakistan (Waheed
et al., 2009).
HCV cannot be contracted from or transferred to any other animal. Chimpanzees can be infected
with the virus or the full length viral RNA made in the laboratory, but they do not develop the
disease. The infection is often asymptomatic in most of the infected individual, but once
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established, chronic infection can progress to scarring of the liver (fibrosis) and development of
advanced scarring (cirrhosis) which is generally apparent after many years (Thomson et al.,
2003). In some cases, those with cirrhosis will go on to the development of liver failure or other
complications of cirrhosis including cancer or life threatening esophageal varices and gastric
varices (Asselah et al., 2003). A relationship between insulin resistance and hepatitis C has been
observed in HCV infected patients. Indeed, insulin resistance is frequently seen in patients with
chronic hepatitis C than in healthy controls (Romero-Gomez, 2006). In a cross-sectional survey
it was reported that HCV-positive persons have an increased risk for type II diabetes mellitus,
more than 3-fold than persons without HCV infection but, no association was seen between HBV
infected patients and diabetes type II (Mehta et al., 2000). In another study Diabetes mellitus was
investigated and seen more often in cirrhotic patients. However, in a cohort of 45 non-cirrhotic
HCV patients the prevalence of type II diabetes mellitus was 33% higher than in the control
group (Knobler et al., 2000). Genotyping is important to guide treatment because some viral
genotypes respond better to therapy than others.
HCV PREVALENCE IN VARIOUS GROUPS
Ten different studies showed that the percent prevalence of HCV in the general adult population
was 4.95% ±0.53% (Luby et al., 1997, Tunveer et al., 2008). Four different reports showed a
high 57% ± 17.7% prevalence of HCV among the IDUs (Kuo et al., 2006, Platt et al., 2009).
Thalassemic and hemophilic patients require life-long blood transfusions, so it is necessary to
obtain screened blood from a reputable source, because the multitransfused population is more
prone to blood-borne pathogens. Arif et al reported that only 15.8% of parents of thalassemic
children knew the importance of blood screening. Hamid et al reported that recapping of
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syringes is the key factor for receiving needle stick injuries in health care workers and that
transmission of HCV by needle stick injury ranges from 2% to 10%.
Janjua et al reported that 68% of individuals received injections during the previous three months
in Digri and Mirpurkhas, two districts of Pakistan, out of which only 54% were from freshly
opened syringes.
It has been reported from the US that up to 20% of new HCV infections are due to sexual
activity (Alter, 1999). The main problems in Pakistan are illiteracy, lack of awareness about
sexually transmitted diseases and low use of condoms among the sex workers. Saleem et al
reported in 2005 that 17% of female sex workers, 3% of male sex workers and 4% of hijras
(transgender men) consistently used condoms during the previous month; 67% of female sex
workers were illiterate, 34% of female sex workers were suffering from sexually transmitted
infections.
HCV GENOTYPES AND THEIR DISTRIBUTION
HCV has been categorized into different genotypes whose members have at least 67%
nucleotides identity with each other (Jose et al., 2002). HCV has a highly variable sequence,
allowing description of 11 genotypes and above 100 subtypes with different geographical
distributions. Clinical outcomes as well as response to antiviral therapy are strongly influenced
by HCV genotype (Eric et al., 2010).In Pakistan the prevalent genotype is 3a followed by
genotype1a and 2a while other genotypes are quite rare (Idrees and Riazuddin, 2008). Genotype
3a and 1a are also the foremost genotypes in Indian population. (Narahari et al., 2009). HCV
subtypes1a, 1b, 2a and 2b are found commonly in Unitesd States (Nainan et al.,
2006).Genotyping is important to guide treatment because some viral genotypes respond better
to therapy than others. The genetic diversity of HCV is one reason due to which it has been
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difficult to develop an effective vaccine. In Taiwan the most common genotype is 1b (Lee et al.,
2010). Type 4 is found in Middle East countries (Ayesh et al., 2009). Genotype 6 is found in
Hong Kong, Thailand, south China and south-east Asian population (Akkarathamrongsin et al.,
2010). Genotype 1 and 4 has been reported to be the common genotypes in African countries
(Muasya et al., 2008). In Sri Lankan population genotypes 1b and 2b are the leading genotypes
(Senevirathna et al., 2008) while in Korean population the most prevalent genotype is 1b
followed by 2a (Shin, 2006). More than two of every three persons who are infected by HCV
may continue to have the virus in their blood, which can transmit the infection to others.
Those at increased risk of developing HCV include:

Healthcare workers who expose themselves with infected blood from a cut or bruise or
from a device or instrument that has been infected ("contaminated")

Persons who inject illicit drugs into their veins and skin, with shared needles and syringes

Anyone who gets a tattoo or has his or her skin pierced with an infected needle

Persons with hemophilia who require large amounts of blood and blood products after
bleeding

Patients with kidney disease who have periodic dialysis and often requires the patient to
have blood transfusions
About one-fourth of patients with HCV do not belong to any of these high-risk groups. Although
blood transfusion is now a much less common cause of HCV infection than in earlier years, but
cases still occur. Also, sexual transmission is possible, and may take place with either
heterosexual or homosexual behavior (Ask.com, 2010).
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WHAT ARE THE SYMPTOMS OF HCV INFECTION?
About 75% of people have no symptoms when they first acquire HCV infection. The remaining
25% may complain of fatigue, loss of appetite, muscle aches or fever. Yellowing of the skin or
eyes (jaundice) is rare at this early stage of infection. Over time, the liver with chronic infection
may begin to experience the effects of the persistent inflammation caused by the immune
reaction to the virus. Blood tests may show elevated levels of liver enzymes, a sign of liver
damage, which is often the first suggestion that the infection may be present (Zechini et al.,
2004). Patients may become easily fatigued or complain of nonspecific symptoms. As cirrhosis
develops, symptoms increase and may include: weakness, loss of appetite, weight loss, breast
enlargement in men, a rash on the palms, difficulty with the clotting of blood, and spider-like
blood vessels on the skin.
GENOME ORGANIZATION
HCV is a member of the Flaviviridae and is the leading cause of chronic hepatitis and liver
cirrhosis in humans in the third world countries (Tanaka et al., 1996). The genome of HCV is a
9,600 nucleotides long positive-strand RNA that is translated into a polyprotein of
approximately 3,010amino acids (Tanaka et al., 1996). The 5’-NCR is highly conserved among
different HCV isolates and contains IRES that is essential for cap-independent translation of the
viral RNA. The 3’-NCR is composed of a short variable region which is a poly(U/UC) tract with
an average length of 80 nucleotides, and an invariant of 98 nucleotide RNA elements which is
designated the X-tail (Kolykhalov et al., 1996). The conserved elements in the 3’-NCR,including
a minimal poly(U) tract of about 25 bases, are essential for replication in cell culture (Yi Male et
al., 2003) and in vivo (Kolykhalov et al., 2000). Translation of the HCV yields a polyprotein
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precursor that is co- and post-translationally processed by cellular and viral proteases into the
mature structural and non-structural proteins.
Figure: 1 (Genetic organization and polyprotein processing of HCV)
Once initiated, translation of the HCV genome produces a large polyprotein which undergoes
proteolytic processing by both host signal peptidases and viral proteases to yield 10 mature viral
structural (C, E1, and E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B)
proteins (Figure 1).
The structural proteins and the p7 polypeptide are processed by the endoplasmic reticulum (ER)
signal peptidase whereas the non-structural proteins are processed by two viral proteases, the
NS2–3 protease and theNS3–4A serine protease. The HCV core protein is a highly basic, RNA8
binding protein, which presumably forms the viral capsid. The HCV core protein is a 191aa
precursor of 23-kDa. In addition to its role in viral capsid formation; the core protein has been
suggested to directly interact with a number of cellular proteins and pathways which are
important in the viral lifecycle (McLauchlan, 2000).
The two envelope glycoproteins, E1 and E2, are essential components of the HCV virion
envelope and necessary for viral entry and fusion (Nielsen et al., 2004). E1 and E2 have
molecular weights of 33-35 and 70-72 kDa, respectively. These proteins are involved in entry of
virion in the cell through cell surface receptors. p7 is a small, 63 aa polypeptide, that is shown to
be an integral membrane protein (Carrere-Kremer et al., 2002). NS2 is a non-glycosylated
transmembrane protein of 21-23 kDa. It consists of two internal signal sequences at aa positions
839-883 and 928-960, which are responsible for ER membrane association (Yamaga andOu,
2002). NS2, together with the amino-terminal domain of the NS3 protein, theNS2-3 protease,
constitutes a zinc-dependent metalloprotease that cleaves the site between NS2 and NS3
(Hijikata et al.,1993). NS3 is a multi-functional viral protein of 70kD containing a serine
protease domain in its N terminal third and a helicase/NTPase domain in its C-terminal twothirds. NS4A,which is a small protein of 54 amino acid residues, is a cofactor of NS3 protease
activity. The NS3-NS4A protease is essential for the HCV lifecycle. It catalyzes HCV
polyprotein cleavage at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions.
NS4B is an integral membrane protein of 261 aa with an ER or ER-derive membrane localization
(Hugle et al., 2001; Lundin et al., 2003). One of the functions of NS4B is to serve as a
membrane anchor for the replication complex (Egger et al., 2002). NS5A is a 56-58 kDa
phosphorylated zinc-metalloprotein that probably plays an important role in virus replication and
regulation of cellular pathways. NS5B belongs to a class of membrane proteins termed tail-
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anchored proteins (Ivashkina et al., 2002). An alternative reading frame (ARF) was identified in
the HCV core coding region, as a result of a –2/+1 ribosomal frame shift in genotype 1a which
has the potential to encode a protein of up to 160 amino acids and designated ARFP (alternative
reading frame protein) or F (frameshift) protein (Branch et al., 2005).
THE HCV LIFECYCLE
HCV only infects humans and chimpanzees. Hepatocytes are the main target cells but infection
of B cells, dendritic cells and other cell types has also been reported. All hepatitis C viruses are
made up of an outer coat (envelope) and have enzymes and proteins that allow the virus to
replicate within the cells of the body particularly liver cells (hepatocytes). Enveloped virus
particles interact with specific surface receptors CD81 (a tetraspanin protein) is found on the
surface of many cell types, including hepatocytes scavenger receptor class B type I (SR-BI), the
LDL receptor (LDLR), and claudin-1 have been proposed as HCV receptors (Bartosch et al.,
2006) Fusion of the viral and cellular membranes, triggered by the low pH of the endocytic
compartment leads to the release of a single-stranded (ss), positive-sense RNA genome into the
cytoplasm of a newly infected cell. This genome serves multiple roles within the virus life cycle:
first, as a messenger RNA (mRNA) for translation of the viral proteins; second as a template for
RNA replication; and third, as a nascent genome packaged within new virus particles. As in all
positive-strand RNA viruses, HCV RNA replication occurs in association with altered
cytoplasmic membranes.
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Figure: 2 (Lifecycle of HCV. (a) Viral entry; (b) cytoplasmic release and uncoating; (c) IRESmediated translation and polyprotein processing; (d) RNA replication; (e) packaging and
assembly; (f) virion maturation and release)
Little is known about the process of HCV RNA synthesis within the replication complex
(Cocquerel et al., 2006).With reference to related viruses, RNA synthesis is likely to be
semiconservative and asymmetric where the positive-strand genome serves as a template to make
a negative-strand intermediate and this negative strand then serves as a template to produce
multiple nascent genomes. Nascent products from these reactions are protected from nuclease
digestion by protease-sensitive factor and a detergent(Yang G. 2004). It is interesting to note that
protease treatment of permeabilized cells destroyed most NS proteins without compromising
RdRP activity which suggests that only a small fraction of NS proteins is actively engaged in
RNA replication. The function of the ‘excess’ NS proteins and composition of the HCV replicase
remain intriguing areas for future study.
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TREATMENT OPTIONS FOR HCV
When HCV was discovered in 1989, drugs targeting the viral proteins were expected to reach
clinics rapidly (Choo et al., 1989). But more than two decades on, patients and physicians are
still waiting. The current standard treatment for HCV infection involves weekly injections of
pegylated interferon-α, with oral ribavirin twice-daily. These drugs, both of which are general
inhibitors of viral infection, often have serious side effects, including depression and flu-like
symptoms. Still completion of the year-long course of treatment does not always cure the patient,
with success depending on characteristics of the virus, such as strain (the viral genotype), as well
as host attributes, such as genetic variations. Generally, patients infected with HCV genotypes 2
or 3 more readily achieve a sustained viral response (SVR) than those infected with HCV
genotype 1 (Jamall et al., 2008). A sustained virological response is achieved in 61% of the HCV
patients getting IFN therapy. Several studies have shown an association between HCV genotype
and both the responsiveness to interferon treatment and the extent of clinical progression of
chronic HCV infection (Yoshioka et al., 1992). Studies have shown that there is a strong
correlation of HCV genotypes with the grade and extent of steatosis, impaired glucose tolerance,
body mass index (BMI), atherogenic lipid profile and insulin resistance (Szanto et al., 2006) and
such genotype specific correlations are of high interest and clinical significance. It is noteworthy
that the interferon alpha therapy for HCV is associated with many side effects such as emotional
disturbances, cognitive impairment, chronic fatigue and psychotic states (Małyszczak et al.,
2006).
Viral enzymes are the prime targets for drug development, traditionally. Several HCV enzymes
are required for the virus’s replication, including two proteases (NS2-3 and NS3-4A), a helicase
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(NS3) and a polymerase (NS5B). Of these, NS3-4A and NS5B are the most attentive drug
targets, showing encouraging results in clinical trials (De Francesco et al., 2005, Kwong et al.,
2008). But the rapid and error-prone replication of HCV makes the emergence of drug resistance
a significant issue. To combat resistance, cocktails of antiviral drugs with diverse mechanisms of
action will probably be required, and researchers have therefore started looking beyond the viral
enzymes.
The antiviral drugs development has been hampered due to presence of several genotypes of
HCV and hundreds of quaspecies distributors. At NCVI-NUST scientists have successfully
obtained the consensus regions that can be targeted as peptide vaccine. The HCV pseudo type
particles expressing HCV envelope proteins have been successfully generated that offer a great
promise to be tested as vaccine specific for each genotype. Attempts are underway to develop
nonhuman primate model for HCV infection where various antiviral (anti-protease, anti-helicase,
anti-replicase) compounds will be tested.
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