Upendra Thapa Shrestha

© U. T. Shrestha

Virus classification

Group IV
Group:
((+) ssRNA)

Order: Unassigned

Family: Flaviviridae

Genus: Hepacivirus

Species: Hepatitis C virus

 © U. T. Shrestha

Introduction
 Hepatitis C virus (HCV) is a small (55–65 nm in size),
enveloped, positive-sense single-stranded RNA virus of
the family Flaviviridae.
 It is the most important cause of parenteral non-A, non-
B hepatitis (NANBH) worldwide.
 Hepatitis C virus is the cause of hepatitis C and some
cancers such as liver cancer (Hepatocellular carcinoma,
abbreviated HCC) and lymphomas in humans
 Most patients infected with HCV have chronic liver
disease, which progresses to cirrhosis and hepatocellular
carcinoma.
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 Prior to identification of the virus, it was known as

NANBH to differentiate it from viral causes of
nonalcoholic hepatitis.
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Hepatitis C virus
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Morphology
 Hepatitis C virus is the only member of the genus
Hepacivirus in the family Flaviviridae of RNA-
containing virus.
 HCV appears to be closely related to hepatitis D, dengue
and yellow fever virus.
 It resembles flavivirus in structure and organization, and
hence has been classified as a new genus Hepacivirus in
the family Flaviviridae.
 Hepatitis C virus shows following morphological
features:
 © U. T. Shrestha

 It is a spherical, enveloped, 9.4 kb, single-

stranded RNA virus with a diameter of 55 nm.
 The genome encode 10 structural and regulatory
proteins.
 Structural proteins include the core and two
envelope proteins, namely, E1 and E2.
 These two envelope proteins undergo variation
during infection due to hypervariable regions
within their genes.
 The viruses are ether sensitive and acid sensitive
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HCV genome
 Monopartite, linear, ssRNA(+) genome of 9.4 kb.
 The genome 3’ terminus is not polyadenylated but forms a
loop structure.
 There is an internal ribosome entry site at the 5’ end that
mediates translation initiation.

GENE EXPRESSION
 The virion RNA is infectious and serves as both the
genome and the viral messenger RNA.
 The whole genome is translated in a polyprotein, which is
processed co- and post-translationally by host and viral
proteases.
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NTR = non-translated region LDL = low-density lipoprotein

Map of the hepatitis C virus (HCV) genome, depicting the 5 untranslated region
(5UTR), capsid core, envelope genes E1 and E2, viroporin p7, membrane-
anchored cysteine protease NS2, serine protease-helicase NS3, NS3 protease
co-factor NS4A, membrane remodeling protein NS4B, phosphoprotein NS5A,
RNA-dependent RNA polymerase NS5B, and the 3UTR.
 © U. T. Shrestha

Proteins encoded by the HCV genome

 HCV is formed by an enveloped particle harbouring a
plus-strand RNA of 9.4 kb.
 The genome carries a long open reading frame (ORF)
encoding a polyprotein precursor of 3010 amino
acids.
 Translation of the HCV ORF is directed via a 340
nucleotide long 5' non-translated region (NTR)
functioning as an internal ribosome entry site; it
permits the direct binding of ribosomes in close
proximity to the start codon of the ORF.
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 The HCV polyprotein is cleaved co- and post-

translationally by cellular and viral proteases into ten
different products, with the structural proteins (core (C),
envelops E1 and E2) and the nonstructural replicative
proteins.
Genomic Variation
 Hepatitis C virus shows a considerable degree of
genomic variations.
 There are six major genotypes (genotypes 1–6) and
numerous subtypes which differ in their worldwide
distribution.
 Molecular differences between these genotypes are
relatively large with as little as 55% genetic sequence
homology and more than 80 subtypes are described.
 This genetic variability is the main stumbling block
against the effort to develop an anti-HCV vaccine.
Comparison of Hepatitis A, B and C virus
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HCV Replication
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HCV Replication
Step I: Initial binding and internalization
 Probably involve glycosaminoglycans (GAGs) and
low-density lipoprotein receptor (LDL-R), which
may interact with viral envelope proteins or with virion-
associated lipoproteins.
 Entry depends directly on binding of E2 with the
tetraspanin CD81, as well as interactions with scavenger
receptor class B member 1 (SRB1) and further interactions
with the tight junction proteins claudin 1 (CLDN1) and
occludin (OCLN) and finally enters cells by receptor-
mediated endocytosis;
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Internalization
 Attachment of the viral envelope protein E to host
receptors mediates internalization into the host cell by
clathrin-mediated endocytosis.
 The viral genome is released from late endosomes
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Step II
 Fusion of virus membrane with host endosomal membrane
occurs and RNA; the viral RNA genome is released into the
cytoplasm.
 Being a positive sense RNA, viral RNA act as mRNA and
is therefore directly translated.
 Translation of HCV RNA is not cap dependent like other
cellular RNAs in which cap bind to ribosomal machinery
for translation.
 Translation of HCV RNA is initiated by binding the 5/-
IRES to ribosome.
 Translation of HCV RNA occurs at rough endoplasmic
reticulum and produces single polyprotein.
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Step III
 The single polyprotein formed is then cleaved by co and
post-translationally by cellular and viral proteases, to
produce structural and non structural proteins.
 With initial cleavages among the structural proteins
mediated by signalase and signal peptide
peptidase followed by cleavage of the NS2–NS3
junction by NS2-NS3 cysteine protease;
 The remaining junctions are cleaved by the NS3-NS4A
serine protease.
 NS4B recruits and rearranges endoplasmic reticulum
(ER) membranes
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Step IV
 Viral proteins, in conjunction with host cell factors,
induce the formation of a membranous compartment
(designated the membranous web (MW); the
principal site of viral replication) composed of single,
double- and multi-membraned vesicles as well as lipid
droplets (LDs)
 Hepatitis C virus like other single stranded viruses of
positive polarity induces alteration in membrane and these
changes in the membrane is termed as membraneous
web (MW)
 NS5B RNA-dependent RNA polymerase replicates the
genome by the synthesis of negative strand RNA.
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 This negative strand RNA serves as a template for the

synthesis of positive strand RNA.
 Replication and post-translational processing appear to take
place in a membranous web made of the non-structural
proteins and host cell proteins called "replication
complex", located in close contact with perinuclear
membranes
 Host miRNA mir-122 plays a essential role in initiating
replication.
 miR-122 is highly expressed in the liver, where it has been
implicated as a regulator of fatty-acid metabolism
 Reduced miR-122 levels are associated with hepatocellular
carcinoma. miR-122 also plays an important positive role in
the regulation of hepatitis C virus replication
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Step V
 RNA replication occurred the membranous web depend on
miR-122 and cyclophilin B, as well as conserved structural
elements at the 5 and 3 ends of the genome.
 Core protein associates with lipid droplets (LDs) in the
lipoprotein assembly pathway.
 Assembly of HCV particles initiates in close proximity to
the ER and lipid droplets, where core protein and viral
RNA accumulate and is facilitated by the viral ionic
channel p7
 The viral envelope is acquired by budding through the
ER membrane and is transported to the Golgi apparatus.
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Step VI
 HCV particles are released by exocytosis linked to
NS5A and other members of the replication complex
by interaction with NS2.
 Viroporin p7 is necessary for production of stable
viral particles coated with E1 and E2, which fold in a
cooperative manner and are glycosylated in a manner
consistent with ER but not Golgi processing.
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Pathogenesis and Immunity

 The ability of HCV to remain cell associated and
prevent host cell death is the main determinant of viral
pathogenicity, which causes persistent infection in the
liver.
 Presence of closely related but heterogeneous population
of virus genome is one of the important factors
responsible for persistence of HCV infection in the liver.
 Pathogenesis of hepatitis C virus infections include;
 Hepatocytes and possibly B lymphocytes are the
natural targets of HCV.
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 At least 50% of hepatocytes may be infected with HCV

in patients with chronic hepatitis.
 In most infected people, viremia persists and is associated
with a variable degree of hepatic inflammation and
fibrosis.
 Chronic hepatitis is characterized by lymphocyte
infiltration either within the portal tract or in the liver
lobule and portal and periportal fibrosis.
 Portal inflammation, interphase hepatitis, and lobular
necrosis are the main histopathological features of
chronic hepatitis caused by HCV.
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 Interface hepatitis (piecemeal necrosis) is a process of

inflammation and erosion of the hepatic parenchyma
at its junction with portal tracts or fibrous septa.
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Host Immunity
 Immunity to HCV may not be lifelong, and
serum antibodies to HCV are usually
protective.
 Cell-mediated immunity, mainly cytotoxic T
lymphocytes, contributes primarily to liver
inflammation and ultimately to tissue necrosis.
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Clinical Syndromes
 The incubation period of hepatitis C varies from 15 to
60 days with an average period of approximately 8
weeks.

 Hepatitis C virus can cause:

 (a) acute HCV infection,
 (b) chronic HCV infection, and
 (c) cirrhosis and other complications induced by
hepatitis.
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a. Acute HCV Infection

 Most patients with acute HCV infections are
asymptomatic and do not develop any jaundice.
 The symptoms of acute HCV infection tend to be mild
and may appear similar to those of HBV infections.
 In symptomatic cases, jaundice occurs in less than
25% of acutely infected patients,
 Whereas hepatomegaly is seen in one-third of cases.
 But most of the cases (80%) are asymptomatic and do
not develop any jaundice
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b. Chronic HCV Infection

 Hepatitis C virus is a major cause of chronic hepatitis
worldwide.
 Most patients with chronic hepatitis are asymptomatic
and may have nonspecific symptoms, such as fatigue or
malaise in the absence of hepatic synthesis
dysfunction.
 Chronic HCV infection is associated with varying
degrees of chronic inflammation and steatosis.
 Lymphocytic infiltrates are typically found in periportal
regions of the liver, though these do not correlate
strongly with liver disease progression.
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c. Cirrhosis and other complications induced

by Hepatitis C
 Hepatitis C virus is now a leading cause of hepatitis and
cirrhosis.
 An estimated 20% of patients with chronic hepatitis
progress to cirrhosis.
 This process may take an average of 20 years after
initial infection worldwide.
 Patients with this condition have a secondary risk of liver
failure, portal hyper tension, and other complications.
 Hepatocellular carcinoma is one of the most important
complications in 1–5% of patients with underlying
cirrhosis.
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 This condition usually develops after 30 years in patients

who are chronically infected and have cirrhosis.
 The increased total number of deaths due to HCV-
related complications, such as cirrhosis and hepatocellular
carcinoma, has been reported from many countries.
Progression of hepatitis C virus (HCV) infection:
Most HCV infections are persistent and stable, but a minority will progress
to cirrhosis within 20 to 30 years. Of those with cirrhosis, most are slowly
progressive, but 7% per year will develop either hepatocellular carcinoma
(HCC) or decompensated liver disease
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Reservoir, source and transmission of infection

 Hepatitis C is exclusively a human disease.
 Patients who are infected with the virus are the important
reservoir of infection.
 Blood or blood products and also organs of infected patients
are the major sources of infection.
 Hepatitis C can be transmitted by following methods:

Blood transfusion:
 Blood transfusion is the most important route of transmission of
HCV.
 The current risk of transfusion-derived HCV is estimated to be
one case in every 100,000 units transfused.
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Parenteral transmission:
 HCV is transmitted parenterally
 (a) through transfusion of infected blood or blood products,
 (b) transplantation of organs from infected donors, and
 (c) also by sharing of contaminated needles among intravenous
drug users.
 The use of intravenous drugs is most important risk factor
responsible for around 50% of both acute and chronic
infections.

Sexual transmission:
 Sexual transmission is believed to be responsible for
approximately 20% of cases of hepatitis C.
 The presence of coexisting sexually transmitted disease, such as
HIV, appears to increase the risk of transmission.
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Perinatal transmission:
 Perinatal transmission is possible and is observed in
fewer than 5% of children born to HCV-infected
mothers.
 The risk of perinatal transmission of HCV is higher in
children born to mothers who are coinfected with HCV
and HIV.

Other methods of transmission:

 Hemodialysis, tattooing, body piercing, and acupuncture
with unsterile equipments are other, but less frequent,
means of transmission of HCV.
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 Needle stick injury among healthcare workers who are

exposed to infected blood accounts for nearly 4% of
new infections.

 The possibility of acquiring HCV after needle stick

injury involving an infected patient appears to range
from 0% to 7%.
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Laboratory Diagnosis
 Laboratory diagnosis is most important to establish the
specific diagnosis of hepatitis caused by HCV.

Serodiagnosis
 Hepatitis C infection can be confirmed by employing
serological tests to detect antibodies to HCV.
 Antibodies are directed against core envelope and NS3
and NS4 proteins and tend to be relatively low in titer.
 Acute HCV antibodies are usually demonstrated in acute
infections 6–8 weeks after initial infection.
 Then antibodies that are produced persist throughout life
in chronic infection.
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 ELISAs, including second- and third-generation ELISAs, are

useful for screening of serum for anti-HCV antibodies.
 These assays are highly specific but cannot differentiate
acute infection from chronic infection.
 The ELISA which employs antibodies against core proteins
and nonstructural proteins 3, 4, and 5 is the most recent
third-generation enzyme immunoassay (EIA).
 The test can detect antibodies in the serum 8 weeks after the
onset of infection.
 Recombinant immunoblot assay using recombinant HCV
antigen is a highly specific test to detect HCV infection.
 This test, which is more specific than the ELISA, has been
used to confirm positive ELISA results.
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 The first enzyme immunoassay (EIA) for the detection of anti-

HCV antibodies, developed by using a recombinant HCV C100-3
peptide, had relatively poor specificity and sensitivity.
Seroconversion in patients with acute HCV infection is often not
detected until 3 months or longer after infection.
 The second-generation (G2) assay, introduced in 1991,
incorporated recombinant ant(NS3 and NS4) together with an
antigen from the core regionigens from nonstructural regions of
HCV.
 The G2 EIA was superior to the G1 EIA in both sensitivity and
specificity, and its use in blood banks has dramatically reduced the
incidence of post-transfusion hepatitis.
 The G3 EIA, which added an NS5 epitope, has increased the
reliability of the test and increase detection of anti-HCV earlier in
the course of infection.
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Molecular Diagnosis
 RT-PCR and branched DNA (bDNA) assays for signal
amplification are being used to detect HCV RNA in the
serum.
 HCV RNA testing is the most specific test for HCV
infection and useful in diagnosing acute HCV infections
before antibodies are developed.
 This is also helpful to
 (a) assess the HCV genotype,
 (b) to confirm false-positive cases, such as autoimmune
hepatitis, and
 (c) to predict the response to interferon therapy.
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 Hepatitis C virus genotyping is a recent method, which

is frequently helpful for predicting the likelihood of
response and duration of treatment.
 The genotyping is performed either by direct sequence
analysis or restriction fragment length polymorphism.
 Liver biopsy is the most accurate method to evaluate the
extent of HCV-related liver disease.
 It is usually recommended for all patients before the start
of antiviral therapy.
 However, it is generally not used to diagnose HCV.
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According to WHO……
Geographical distribution
 Hepatitis C is found worldwide.
 The most affected regions are Africa and Central and
East Asia.
 Depending on the country, hepatitis C infection can be
concentrated in certain populations (for example,
among people who inject drugs) and/or in general
populations.
 There are multiple strains (or genotypes) of the HCV
virus and their distribution varies by region.
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Transmission
 The hepatitis C virus is a blood borne virus.
 It is most commonly transmitted through:
 Injecting drug use through the sharing of injection
equipment;
 The reuse or inadequate sterilization of medical
equipment, especially syringes and needles in healthcare
settings; and
 The transfusion of unscreened blood and blood
products.
 HCV can also be transmitted sexually and can be
passed from an infected mother to her baby; however
these modes of transmission are much less common.
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 Hepatitis C is not spread through breast milk, food,

water or by casual contact such as hugging, kissing
and sharing food or drinks with an infected person.
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Screening and diagnosis

 Due to the fact that acute HCV infection is usually
asymptomatic, few people are diagnosed during the
acute phase.
 In those people who go on to develop chronic HCV
infection, the infection is also often undiagnosed because
the infection remains asymptomatic until decades after
infection when symptoms develop secondary to serious
liver damage.
 HCV infection is diagnosed in 2 steps:
 Screening for anti-HCV antibodies with a serological test
identifies people who have been infected with the virus.
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 If the test is positive for anti-HCV antibodies, a nucleic

acid test for HCV ribonucleic acid (RNA) is needed to
confirm chronic infection because about 15–45% of
people infected with HCV spontaneously clear the
infection by a strong immune response without the need
for treatment.
 Although no longer infected, they will still test positive
for anti-HCV antibodies.
 After a person has been diagnosed with chronic hepatitis
C infection, they should have an assessment of the
degree of liver damage (fibrosis and cirrhosis). This
can be done by liver biopsy or through a variety of non-
invasive tests.
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 In addition, these people should have a laboratory test

to identify the genotype of the hepatitis C strain.
 There are 6 genotypes of the HCV and they respond
differently to treatment.
 Furthermore, it is possible for a person to be infected
with more than 1 genotype.
 The degree of liver damage and virus genotype are
used to guide treatment decisions and management of
the disease.
Treatment
A combination therapy of pegylated
interferon and antiviral agent ribavirin
is the current option of treatment for
patients with chronic HCV infections.
 Other therapeutic options include the use of
protease inhibitors, ribozymes, and viral
vaccines.
Prevention and Control
 No vaccine against HCV is available.
 Immunoglobulin is not useful in preventing
transmission and, in fact, administration of
immunoglobulin has been associated with
HCV.
 Transmission of HCV can be prevented by
screening and preventing donation of
blood, organs, or semen from HCV-
positive donors.