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67

Hepatitis Viruses

Learning Objectives
After reading and studying this chapter, you should be able
to:
Classify various hepatitis viruses.
Tabulate differences between various hepatitis viruses.
Compare various features of hepatitis A virus (HAV)
and hepatitis B virus (HBV).
Describe the following: Morphology of hepatitis B
virus; antigenic structure of hepatitis B virus; modes of
transmission of hepatitis B virus; hepatits B carriers.

INTRODUCTION
Viral hepatitis is a systemic disease primarily involving
the liver. At least six viruses, A through E and a newly
discovered G, are considered hepatitis viruses. (The
designation type F had been proposed for a putative
virus believed to cause transfusion-associated hepatitis,
distinct from type A to E. But it proved to be a mutant
of type B virus and not a separate entity. Type F was
therefore, deleted from the list of hepatitis viruses).
Although the target organ for each of these viruses is
the liver, they differ greatly in their structure, mode of
replication, and mode of transmission and in the course
of the disease they cause. Hepatitis A virus (HAV) and
Hepatitis B virus (HBV) are the best known, but three
non-A, non B hepatitis (NANBH) viruses (C, G, and E)
have been described, as has hepatitis D virus (HDV),
the delta agent (Table 67.1). Other viruses are associated
with hepatitis that cannot be ascribed to known agents,
and the associated disease is designated non-A to E
hepatitis.

HEPATITIS A VIRUS (HAV) INFECTIOUS HEPATITIS

Hepatitis A virus (HAV) causes infectious hepatitis and
is spread by the fecal-oral route. It is a subacute disease
of global distribution, affecting mainly children and
young adults.

Properties of Hepatitis Viruses

Feinstone and coworkers in 1973, using immunoelectron
microscopy (IEM) demonstrated this virus in the feces
of experimentally infected human volunteers. Sensitive
serologic assays and polymerase chain reaction (PCR)

Describe laboratory diagnosis of

Discuss prophylaxis of hepatitis B

hepatitis B virus.
infections or hepa-

titis B vaccine.
Describe the following: Hepatitis C virus or Type C
hepatitis; Hepatitis D virus or Delta agent; Hepatitis E
virus; Hepatitis G virus

methods have made it possible to detect HAV in stools

and other samples and to measure specific antibody in
serum.Chimpanzees and marmosets can be infected
experimentally. HAV can be grown in some human and
simian cell cultures and is the only human hepatitis virus
which can be cultivated in vitro. It has also been cloned.

Morphology
HAV is a 27 nm nonenveloped RNA virus belonging to
the picornavirus family (Fig. 67.1). Although, it was first
provisionally classified as enterovirus 72, but it has been
placed into a new genus, Hepadnavirus, on the basis of
its unique genome. Only one serotype is known.

Resistance
HAV is stable to treatment with 20 percent ether, acid
(pH 1.0 for 2 hours), and heat (60C for 1 hour). The virus
is destroyed by autoclaving (121C for 20 minutes), by
boiling in water for 5 minutes, by dry heat (180C for 1
hour), by ultraviolet irradiation (1 minute at 1.1 watts),
by treatment with formalin (1:4000, or by treatment
with chlorine (10-15 ppm for 30 minutes). It survives
prolonged storage at a temperature of 40C or below.

Pathogenesis
HAV is ingested and probably enters the blood stream
through the oropharynx or the epithelial lining of the
intestines to reach its target, the parenchymal cells of the
liver. The virus can be localized by immunofluorescence
in hepatocytes and Kupffers cells. Virus is produced in
these cells and is released into the bile and from there
into the stool. Virus is shed in large quantity into the

Table 67.1: Comparative feature of hepatitis viruses

Feature

Hepatitis A

Hepatitis B

1. Virus structure

HAV, 27 nm RNA, HBV, 47 nm DNA

Picornavirus
(Hepadnavirus)
(Hepatovirus)

Hepatitis C

Hepatitis D

Hepatitis E

HCV, 30-60 nm
RNA, Flavivirus
(hepacivirus)

HDV, 35-37 nm
Defective RNA
Deltavirus

HEV,32-34 nm RNA
Herpesvirus

Parenteral
Vertical, Sexual

Parenteral

Parenteral

Fecal-Oral

3. Age Affected

Children

Any age

Adults

Any age

Young adults

4. Incubation
Period (days)

15-45

30-180

15-160

30-180

15-60

5. Onset

Acute

Insidious

Insidious

Insidious

Acute

6. Illness

Mild

Occasionally severe

Moderate

Occasionally severe

Mild, except in pregnancy

7. Carrier state

Nil

Common

Present

Nil (only with HBV)

Nil

8. Oncogenicity

Nil

Present specially
after neonatal
infection

Present

Nil

Nil

9. Prevalence

Worldwide

Worldwide

Probably
worldwide

Endemic areas
(Mediterranean, N,
Europe, Central and
N. America)

Only developing
countriest lndia,
Asia, Africa, Central, America)

10. L
 aboratory
diagnosis

Symptoms and
anti-HAV IgM

Symptoms and
serum levels of HBs
Ag, HBe Ag, and
anti-HBc IgM

Symptoms and
anti-HCV ELISA

Anti-HDV ELISA

11. Specific
prophylaxis

Ig and vaccine

Ig and vaccine

Nil

HBV vaccine

Nil

Fig. 67.1: The picornavirus structure of hepatitis A virus. The

icosahedral capsid is made up of four viral polypeptides (VP1
to VP4). Inside the capsid is a single-stranded, positive-sense
RNA (ssRNA) that has a genomic viral protein (VPg) on the
5 end

stool approximately 10 days before symptoms of

jaundice appear or antibody can be detected.
HAV replicates slowly in the liver without producing
apparent cytopathic effects. A brief viremia occurs
during the preicteric phase, but ceases with the onset of
jaundice. Chronic viremia does not occur.

Epidemiology
HAV transmission is by the fecal-oral route in contaminated water, in food, and by dirty hands. Shellfish, especially clams, oysters, and mussels, are important sources

Chapter 67 Hepatitis Viruses

2. M
 odes of infec- Fecal-oral
tion

of the virus. HAV outbreaks usually originate from a

common source (e.g. water supply, restaurant, daycare
center). Under crowded conditions and poor sanitation,
HAV infections occur at an early age.
The epidemiology of type A hepatitis resembles that
of poliomyelitis. In the developing countries, infection is
acquired in childhood and by the age of ten, 90 percent
of the population possess antibody to the virus and are
immune. In India, type A hepatitis is the most common
cause of acute hepatitis in children, but is much less
frequent in adults.
Natural infection with HAV is seen only in humans.
Though primates such as chimpanzees have been shown
to acquire the infection from humans and transmit it to
human contacts, there is no evidence of any extrahuman
source of the virus in nature.

Clinical Features
The incubation period is 2 to 6 weeks. Disease in children
is generally milder than that in adults and is usually
asymptomatic. The clinical disease consists of two
stages: the prodromal or preicteric and the icteric stages.
The onset may be acute or insidious, with fever, malaise,
anorexia, nausea, vomiting and liver tenderness. These
usually subside with the onset of jaundice. The patient
starts to feel better within the next week or so and the
jaundice disappears within a month. Recovery is slow,
over a period of 4 to 6 weeks.

601

Section 4 Virology

B. Immunization

Fig. 67.2: Typical course of hepatitis type A

Hepatitis A is nearly always self-limiting, but relapses

have been reported.The disease is milder in children, in
whom many infections may be an icteric.
Complications such as fulminant hepatitis, fortunately
rare, are seen mainly in older people. Unlike HBV,
immune complex-related symptoms (e.g. arthritis, rash)
rarely occur in people with HAV disease.

Laboratory Diagnosis
Etiological diagnosis of type A hepatitis may be made
by demonstration of the virus or its antibody.
A. Demonstration of the virus: The virus can be visualized by immune electron microscopy (IEM) in
fecal extracts during the late incubation period and
the pre icteric phase, but seldom later.
B. Demonstration of antibody: The best way to
demonstrate an acute HAV infection is by finding
anti-HAV immunoglobulin M (IgM), as measured
by an enzyme-linked immunosorbent assay (ELISA)
or radioimmunoassay. Antibody IgM anti-HAV
antibody appears during the late incubation period,
reaches peak levels in 2 to 3 weeks and disappears
after 3 to 4 months. IgG antibody appears at about
the same time, peaks in 3 to 4 months and persists
much longer, perhaps for life (Fig. 67.2). Demonstration of lgM antibody in serum indicates current
or recent infection, while IgG antibody denotes
recent or remote infection and immunity.
C. Virus isolation: The virus is grown in human
simian cell cultures. It is notroutinely performed
because efficient tissue culture systems for growing
the virus are not available.

Prophylaxis
A. General Measures

602

The spread of HAV is reduced by interrupting the fecaloral spread of the virus. This is accomplished by avoiding potentially contaminated water or food, especially
uncooked shellfish. Chlorine treatment of drinking
water is generally sufficient to kill the virus.

There is only one serotype of HAV and HAV infects

only humans, factors that help ensure the success of an
immunization program. Natural infection with HAV,
clinical or subclinical, leads to lifelong immunity. There
is no cross-immunity between HAV and any of the other
hepatitis viruses:
1. Passive protection: Specific passive prophylaxis
by pooled normal human immunoglobulin (16%
solution in a dose of 0.2-0.12 ml/kg body weight)
intramuscular (IM), before exposure or in early
incubation period, can prevent or attenuate clinical
illness, while not necessarily preventing infection
and virus excretion.
2. Hepatitis A vaccine:
i. Formalin inactivated, alum conjugated vaccine
A safe and effective formalin inactivated, alum
conjugaged vaccine containing HAV grown in
human diploid cell culture is available for use
in children and adults at high risk for infection,
especially travelers to endemic regions. A full
course consists of two intramuscular injections
of the vaccine. Protection begins 4 weeks after
injection and lasts for 10 to 20 years.
ii.
Live HAV vaccineA live HAV vaccine has been
developed in China.

Treatment
Treatment is symptomatic. No specific antiviral drug is
available.

HEPATITIS B VIRUS (HBV)SERUM HEPATITIS

Type B hepatitis is the most widespread and the most
important type of viral hepatitis. Hepatits B virus (HBV)
infects the liver and, to a lesser extent, the; kidneys and
pancreas of only humans and chimpanzees. HBV establishes chronic infections, especially in those infected as
infants. It is a major factor in the eventual development
of liver disease and hepatocellular carcinoma in those
individuals. As there is an effective vaccine against HBV,
hepatocellular carcinoma becomes the only human cancer which is vaccine preventable.

Classification
HBV is assigned to a separate family Hepadnaviridae
(Hepatitis DNA viruses) which consists of two genera:
i. Orthohepadnavirus: Containing HBV as well as the
woodchuck and ground squirrel hepatitis viruses.
HBV is Hepadnavirus type 1.
ii. Avihepadnavirus: Containing the Pekin duck and
grey heron hepatitis viruses.

Structure
HBV is a 42 nm DNA virus with an outer envelope and
an inner nucleocapsid core, 27 nm in diameter, enclosing the viral genome and a DNA polymerase (Fig. 67.3).
1. Which is circular double stranded DNA.

Australia Antigen
In 1965, Blumberg, studying human serum lipoprotein
allotypes, observed in the serum of an Australian
aborigine, a new antigen which gave a clearly defined
line of precipitation with sera from two hemophiliacs
who had received multiple blood transfusions. This was
named the Australia antigen. By 1968 the Australia
antigen was found to be associated with serum hepatitis.
It was subsequently shown to be the surface component
of HBV. Therefore, the name Australia antigen was
changed to hepatitis B surface antigen (HBsAg).

Types of Particles
Under the electron microscope, sera from type B hepatitis patients show three types of particles (Fig. 67.4A
toC).
i. Spherical particle: The predominant form is a small,
spherical particle of (22 nm diameter).
ii. Tubular particle: The second type of particle is filamentous or tubular (22 nm diameter) of varying
length. Both types of particle consist solely of surplus virion envelope. The particles carry the hepatitis B surface antigen (HBsAg).
iii. Dane particle: The third type of particle, is a double walled spherical structure, (42nm in diameter).
This particle is the complete hepatitis B virus. It was
first described by Dane in 1970 and so is known as
the Dane particle.The outer surface, or envelope,
contains HBsAg and surrounds a 27 nm inner nucleocapsid core that contains HBcAg.

Antigenic Structure
1. Hepatitis B surface antigen (HBsAg): The envelope
proteins expressed on the surface of the virion and
the surplus 22 nm diameter spherical and filamentous particles constitute the hepatitis B surface antigen (HBsAg). HBsAg consists of two major polypeptides, one of which is glycosylated.
2. Hepatitis B core antigen (HBcAg): The antigen
expressed on the core is called the hepatitis B core
antigen (HBcAg).

3. Hepatitis B e antigen (HBeAg): A third antigen

called the hepatitis B e antigen (HBeAg) is a soluble
nonparticulate nucleocapsid protein. The HBeAg
and HBcAg proteins share most of their protein
sequence.
4. Viral genes and antigens

Genome

Chapter 67 Hepatitis Viruses

Fig. 67.3: Hepatitis B virus structure

Fig. 67.4A to C: Different types of particles of HBV: A. Spherical 22 nm particle. B. Double shelled 42 nm particle (Dane
particle). C. Tubular 22 nm particle

The nucleocapsid encloses the viral genome consisting

of two linear strands of DNA held in a circular
configuration. One of the strands (the plus strand) is
incomplete, so that the DNA appears partially double
stranded and partially single stranded. Associated with
the plus strand is a viral DNA polymerase, which has
both DNA-dependent DNA polymerase and RNAdependent reverse transcriptase functions. Although
a DNA virus, it encodes a reverse transcriptase
and replicates through an RNA intermediate. This
polymerase can repair the gap in the plus strand and
render the genome fully double stranded (Fig. 67.3).
The genome has a compact structure with four
overlapping genes. These include structural proteins
of the virion surface and core, a small transcriptional
transactivator (X), and a large polymerase (P) protein
that includes DNA polymerase, reverse transcriptase,
and RNase H activities (Table 67.2, Fig. 67.5).

HBV Subtypes
The particles containing HBsAg are antigenically complex. It contains two different antigenic components
the common group reactive antigen a, and two pairs of
type specific antigens d-y and w-r, only one member of
each pair being present at a time. HBsAg can thus be
divided into four major antigenic subtypes: adw, adr,
ayw and ayr. The subtypes do not seem to be important
in immunity because of the dominant antigen is shared
by all. The subtypes breed true, and the index case and
contacts in an outbreaks have the same subtype. The
finding of identical subtypes would, of course, not confirm the possibility, but differing subtypes would rule
it out.

603

Table 67.2: Genes coding for antigens of HBV

Gene

Regions

Antigen

S
S
(Having three regions S, Pre-S1 S + Pre-S2
and Pre-S2)
S + Pre-S1 and S2

Major protein (S)

Surface
Middle protein (M) antigen (HBsAg)
Large protein (L)Present only in virion

C
C
(Having two regions C and Pre-C) C + Pre-C

Core antigen (HBcAg)

HBeAg

DNA polymerase

HBx Ag (Nonparticulate antigen which leads to enhanced replication of HBV)

Section 4 Virology

in that DNA is synthesized from an RNA template by

reverse transcription. To replicate hepadnavirus DNA,
a full-length RNA copy is enclosed in core protein in
the hepatocyte nucleus. This is copied to DNA by the
polymerase, the RNA is destroyed and the DNA copied
to form double-stranded DNA as the virion matures.
HBV DNA and protein have also been identified in
extrahepatic sites such as bone marrow, spleen, lymph
nodes and circulating lymphocytes, but apparently no
damage is produced in these locations. The significance
of this extrahepatic presence is not understood.

Cultivation

Fig. 67.5: HBV genes and gene products

They show a distinct geographical distribution.

Subtype adw is common in Europe, Australia and the
America; adr is prevalent in South and East India and
the Far East ayw is common from West Asia through the
Middle East, to Western and Northern India; ayr is very
rare (Table 67.3). A number of other surface antigenic
reactivities (a, x, f, t, j, n, g) have been reported, but not
adequately studied.

Replication
HBV replicates within hepatocytes. Replication of
viral nucleic acid starts within the hepatocyte nucleus
where viral DNA can be free, extrachromosomal, or
integrated at various sites within the host chromosomes.
However, integration is not essential for viral replication. Replication resembles that seen in retroviruses,
Table 67.3: Antigenic types of HBsAg

604

Antigenic types

Distribution

adw

Worldwide

adr

Asia

ayw

India, Africa, Russia

ayr

India, Africa, Russia

HBV does not grow in any conventional culture system.

However, limited production of the virus and its proteins can be obtained from several cell lines transfected
with HBV DNA. HBV proteins have been cloned in bacteria and yeast. The chimpanzee is susceptible to experimental infection and can be used as a laboratory model.

Stability
HBV is a relatively heat stable virus. It remains viable at
room temperature for long periods. Heating to 60C for
10 hours inactivates virus by a factor of 100-1000-fold. It
is susceptible to chemical agents. Exposure to hypochlorite (10,000 ppm available chlorine) or 2 percent glutaraldehyde for 10 min will inactivate virus 100000-fold ,
though HBsAg may not be destroyed by such treatment.
The stability of HBsAg does not always coincide with
that of the infectious agent. HBsAg is not destroyed by
ultraviolet irradiation of plasma or other blood products, and viral infectivity may also resist such treatment.

Clinical Syndromes
Acute Infection
The clinical presentation of HBV in children is less
severe than that in adults, and infection may even be
asymptomatic. Clinically apparent illness occurs in as
many as 25 percent of those infected with HBV.
1. Preicteric phase: HBV infection is characterized by a
long incubation period (about 1-6 months) and an
insidious onset. Symptoms during the prodromal
period may include fever, malaise, and anorexia,
followed by nausea, vomiting, abdominal discomfort, and chills.

Chronic Infection
A proportion of cases (1-10 percent) remain chronically
infected. They may be asymptomatic carriers or may
progress to recurrent or chronic liver disease or cirrhosis. A few of them may develop hepatocellular carcinoma after many decades (Fig. 67.6).

Pathogenesis
The pathogenesis of hepatitis appears to be immunemediated. HBV replicates in the hepatocytes, reflected in
the detection of viral DNA and HBcAg in the nucleus
and HBsAg in the cytoplasm and at the hepatocyte
membrane. During the incubation period, high levels
of virus are present before the host immune response
develops and controls the virus. During replication
HBcAg and HBeAg are also present at the cytoplasmic
membrane. These antigens induce both B and T cell
responses. Damage to the hepatocyte can result from
antibody-dependent, NK and cytotoxic T cell action.
Hepatocytes carry viral antigens and are subject
to antibody-dependent NK cell and cytotoxic T cell

attack. In the absence of adequate immune response,

HBV infection may not cause hepatitis, but may lead
to carrier state. Therefore infants and immunodeficient
persons are more likely to become asymptomatic
carriers following infection.

Epidemiology
HBV is worldwide in distribution. There is no seasonal distribution. The infection is usually sporadic,
though occasional outbreaks have occurred in hospitals,
orphanages and institutions for the mentally handicapped. Natural infection occurs only in humans. There
is no animal reservoir. The virus is maintained in the
large pool of carriers whose blood contains circulating
virus for long periods, in some even lifelong.
The prevalence of HBV infection varies widely in
different parts of the world. The high prevalence areas
(10-20 percent) are East and South-East Asia, the Pacific
Islands, and tropical Africa. The CIS (ex-USSR), the
Indian subcontinent, parts of Africa, eastern and southeastern Europe and parts of Latin America are areas
of medium prevalence (2-20 percent). The prevalence
is low < 1 percent) in the rest of Europe, Australia and
New Zealand, Canada, and the USA.
India falls in the intermediate group, with higher
carrier rates in the southern part of the country and
lower rates in the northern part.
The rich and the poor countries also differ in the age
and modes of infection. In the rich countries, infection
occurs mostly in adolescents and young adults through
contaminated syringes and needles typically among
drug addicts, and through sex, particularly by homosexual intercourse. In the poor countries, infection occurs
usually at younger ages, either perinatally from mother
to baby, or horizontally among children. Perinatal and
horizontal infection in infants and neonates generally leads to asymptomatic infection, with circulating
HBeAg and HBV DNA, without any rise in transaminase
levels. This is due to their inability to mount an immune
response against the virus. Such cases become chronic

Chapter 67 Hepatitis Viruses

2. Icteric phase: The classic icteric symptoms of liver

damage (e.g. jaundice, dark urine, pale stools)
follow soon thereafter. Recovery is indicated
by a decline in the fever and renewed appetite.
Fulminant hepatitis occurs in approximately 1
percent of icteric patients and may be fatal.
HBV infection can promote hypersensitivity
reactions that are due to immune complexes of
HBsAg and antibody. These may produce rash,
polyarthritis, fever, acute necrotizing vasculitis,
and glomerulonephritis.
3. Convalecent phse: About 90 to 95 percent of adults
with acute hepatitis B infection recover within 1 to 2
months of onset. Mortality is about 0.5 to 2 percent,
but may be more in post-transfusion cases. About
1 percent of patients, particularly those having simultaneous delta virus infection develop fatal fulminant hepatitis.

605
Fig.67. 6: Clinical outcomes of acute hepatitis B infection

carriers, with an enhanced risk of developing hepatocellular carcinoma in later life. It is estimated that there
are 350 million HBV carriers; of these, 75 percent were
infected at birth. The global death rate from hepatocellular carcinoma is estimated at 250 000 per annum.

Mode of Transmission
HBV is a blood-borne virus and there are three important modes of transmission:
1. Parenteral transmission
2. Perinatal transmission
3. Sexual transmission

Section 4 Virology

1. Parenteral Transmission
HBV is transmitted only in blood and other body fluids,
including cervical secretions, semen, and breast milk.
Many other therapeutic, diagnostic, prophylactic and
even nonmedical procedures are now the main modes
of infection.
HBV is very highly infectious far more that HIV.
Because the titers of virus are so high in body fluids (106108 per ml), invisibly small quantities0. 00001 ml or
even lesscan transmit the infection. It is, therefore, easy
to understand that minor abrasions or cuts can serve as
portals of entry. These include shared syringes, needles
and other sharp items or endoscopes, personal articles
such as razors, nail clippers or combs, and practices
such as acupuncture, tattooing, ritual circumcision, ear
or nose piercing, and field camps for surgery or disease
detection by blood testing where separate sterile articles
may not be available. Professionals using sharp articles like barbers, dentists and doctors may unwittingly
transmit the virus if great care is not taken.

2. Perinatal Transmission
Vertical transmission from mother to child is one of
the most important routes. HBV can be transmitted to
babies through contact with the mothers blood at birth
and in mothers milk. Babies born to chronic HBV-positive mothers are at highest risk for infection.

3. Sexual Transmission
Since HBV is present in semen and vaginal secretions,
therefore, it can be transmitted by sexual contact. The
risk of transmission by heterosexual and homosexual contact increases with the number of partners and
the duration of such relationships. HBV infection has
occurred after artificial insemination.

Hepatitis B Carriers
Carriers are of two types:
1. Super carriers: They have HBeAg, high titers of HBsAg and DNA polymerase in their blood. HBV may
also be demonstrable in their blood. Very minute
amount of serum or blood from such carriers can
transmit the infection. About a quarter of the carriers in India are. HBeAg-positive.
2. Simple carriers: These are more common types of
carriers who have low titer of HBsAg in blood, with
negative HBeAg, HBV and DNA polymerase. They
transmit the infection only when large volumes of
blood are transferred as in blood transfusion. Many
super carriers in time become simple carriers.

HBV Markers
The main antigens HBsAg, HBcAg, and HBeAg each
induce corresponding antibodies. With the exception
of HBcAg, all these antigens and antibodies, together
with the viral DNA polymerase, can be detected in the
blood at various times after infection and are referred
to as markers, because their presence or absence in an
individual patient marks the course of the disease and
also gives a good idea of the degree of infectivity for
others (Table 67.4). HBcAg is readily detectable only in
the hepatocyte nuclei.

Laboratory Diagnosis
Specific Diagnosis
Specific diagnosis of hepatitis B rests on serological
demonstration of the viral markers and can be carried
out by detection of HBsAg, anti-HBs, HBeAg, anti-

Table 67.4: Interpretation of serological markers in HBV infection

Clinical condition

Serological tests
HBsAg

HBeAg

Anti-HBS

Anti-HBe

Anti-HBc
IgM

Late incubation period or early hepatitis

606

IgG

Acute hepatitis

Late/chronic HBV infection

Simple carrier

Super carrier

Past infection

Immunity following vaccination

*When +, it indicates high infectivity while indicates low infectivity.

HBe, IgM anti-HBc, IgG anti-HBc and HBV DNA in the

serum. The sequence of appearance of viral markers in
the blood is shown in Fig. 67.7. These can be detected by
sensitive and specific tests like ELISA and RIA.

1. Detection of Viral Markers

i. HBsAg: HBsAg is the first marker to appear in
blood after infection, being detectable even before
elevation of transaminases and onset of clinical
illness. HBsAg is usually detectable 2-6 weeks in
advance of clinical and biochemical evidence of
hepatitis and persists throughout the clinical course
of the disease. In the typical case, it disappears
within about 2 months of the start of clinical
disease, but may sometimes last for 6 months and
even beyond, but typically disappears by the sixth
month after exposure.
ii. HBcAg: High levels of IgM-specific anti-HBc are
frequently detected at the onset of clinical illness.
Because this antibody is directed against the 27 nm
internal core component of HBV, its appearance in
the serum is indicative of viral replication. HBcAg
is not demonstrable in circulation because it is
enclosed within the HBsAg coat, but its antibody,
anti-HBc appears in serum a week or two after the
appearance of HBsAg. It is therefore the earliest
antibody marker to be seen in blood, long before
anti-HBe or anti-HBs. As anti-HBc remains lifelong,
it serves as a useful indicator of prior infection with
HBV; even after all the other viral markers become
undetectable. Initially, antiHBc is predominantly
IgM, but after about 6 months, it is mainly IgG.
Selective tests for IgM or IgG anti-HBc therefore
enable distinction between recent or remote infection respectively.
iii. HBeAg: HBeAg provides information about relative
infectivity. Its presence denotes high infectivity and
its absence, along with the presence of anti-HBe,
indicates low infectivity. As it is invariably present
during acute hepatitis, its testing is indicated only
in chronic infection and carriers.

2. Viral DNA Polymerase

DNA polymerase activity, HBV DNA, and HBeAg,
which are representative of the viremic stage of hepatitis
B, occur early in the incubation period, concurrently or
shortly after the first appearance of HBsAg.

3. Polymerase Chain Reaction (PCR)

Molecular methods such as DNA:DNA hybridization
and PCR, at present used for HBV DNA testing are
highly sensitive and quantitative. HBV DNA level in
serum reflects the degree of viral replication in the liver
and so helps to assess the progress of patients with
chronic hepatitis under antiviral chemotherapy.

Chapter 67 Hepatitis Viruses

Fig. 67.7: Hepatitis antigens, antibodies and DNA in a

patient recovering from acute HBV infection

HBeAg appears in blood concurrently with HBsAg,

or soon afterwards. Circulating HBeAg is an indicator
of active intrahepatic viral replication, and the presence
in blood of DNA polymerase, HBV DNA and virions,
reflecting high infectivity. Before HBsAg disappears,
HBeAg is replaced by anti-HBe, signaling the start of
resolution of the disease. The disappearance of HBeAg
coincides with the fall of transaminase levels in blood.
Anti-HBe levels often are no longer detectable after 6
months.
The most useful detection methods are ELISA for
HBV antigens and antibodies and PCR for viral DNA.

4. Biochemical Tests
In acute viral hepatitis caused by various hepatitis
viruses, levels of serum transaminases (aminotransferases) are increased 5- to 100-fold. Both alanine and
aminotransferase and aspartate aminotransferase, rise
together late in the incubation period. Peak level is
obtained about the time jaundice appears and reverts
to normal in next 2 months. Serum bilirubin levels may
rise up to 25-fold.

Prophylaxis
Measures for the control of HBV infection are the same
as those for HIV infection.
A. General prophylaxis: General prophylaxis consists
in avoiding risky practices like promiscuous sex,
injectable drug abuse and direct or indirect contact
with blood, semen or other body fluids of patients
and carriers. Healthcare staff must take the obvious personal precautions, such as keeping cuts and
abrasions covered and wearing gloves when injecting or operating upon actual and potential highrisk patients.
B. Immunization: Both passive and active methods of
immunization are available.
1.
Passive immunization: Hyperimmune hepatitis B
immune globulin (HBIG) prepared from human
volunteers with high titer anti-HBs, administered IM in a dose of 300-500 IU soon after exposure to infection constitutes passive immunization. It may not prevent infection, but protects
against illness and the carrier state.

607

Section 4 Virology

608

HBIG must be given as soon as possible after

an accident and preferably within 48 hours. A
second dose is given 4 weeks later to those who
do not respond to current vaccines. If the victim
has not been vaccinated, HBIG should be used
and a course of active immunization started,
injecting the two materials into different body
sites.
2.
Active immunization: Active immunization is
more effective.
i. Plasma-derived hepatitis B vaccine: A vaccine
for hepatitis B has been available since 1982.
The initial vaccine was prepared by purifying
HBsAg associated with the 22 nm particles
from healthy HBsAg-positive carriers and
treating the particles with virus-inactivating
agents (formalin, urea, heat). This was immunogenic, but became unacceptable because its
source was human plasma, limited in availability and not totally free from possible risk
of unknown pathogens.
ii. Recombinant yeast hepatitis B vaccine: The
currently preferred vaccine is genetically
engineered by cloning the S gene of HBV in
bakers yeast. It consists of nonglycosylated
HBsAg particles alone. This vaccine is safe,
antigenic, free from side effects and as immunogenic as plasma-derived vaccine. It is given
with alum adjuvant, IM into the deltoid or,
in infants into the anterolateral aspect of the
thigh. Three doses given at 0, 1 and 6 months
constitute the full course. Seroconversion
occurs in about 90 percent of the vaccinees.
A special vaccine containing all antigenic
components of HBsAg (Pre-S1, Pre-S2 and S)
has been developed, which gives greater seroconversion. Clinical protection is believed
to last much longer. Booster doses are needed
only for those at high risk.
iii.
Recombinant chinese hamster ovary (CHO) cell
hepatitis vaccine Expression system of CHO
cells has been successfully used and the product is commercially available. This is the first
vaccine using mammalian cell expression
system.
iv.
Synthetic peptide vaccines: As the name indica
tes, these are chemically synthesized polypeptide vaccines. These are safe and cheap. These
are still under experimental stage.
v. Hybrid virus vaccine: Potential live vaccines
using recombinant vaccinia virus have been
prepared for hepatitis B, influenza, rabies,
Epstein-Barr and human immunodeficiency
viruses. Recombinant vaccines can be generated by incorporating foreign genes (HBsAg
sequences in case of HBV) into vaccinia virus
DNA. Recombinant vaccinia virus expresses

proteins (HBsAg in case of HBV) encoded

by foreign gene. The advantages of vaccinia
virus recombinant vaccine include low cost,
long shelf-life and possible use of polyvalent
antigens.

Treatment
No specific antiviral treatment is available for acute HBV
infection. Hepatitis B immune globulin may be administered within a week of exposure and to newborn infants
of HBsAg-positive mothers. Interferon alpha, alone or in
combination, with other antiviral agents such as lamivudine and famcyclovir, has been beneficial in some cases
of chronic hepatitis. There is no effective treatment for
the carrier state, though spontaneous resolution takes
place in some of them.

HEPATITIS C VIRUS (HCV)

HCV resembles flaviviruses in structure and organization, and has been classified as a new genus Hepacivirus
in the family Flaviviridae. HCV is a 50-60 nm virus with
a linear single stranded RNA genome, enclosed within
a core and surrounded by an envelope, carrying glycoprotein spikes.
The virus shows considerable genetic and antigenic
diversity. Various viruses can be differentiated by RNA
sequence analysis into at least six major genotypes
(clades) and more than 70 subtypes. The genome of
HCV encodes 10 proteins, including 2 glycoproteins
(E1, E2). Some genotypes are seen worldwide, while
others are localized. Because of this diversity there is
little heterologous or even homologous postinfection
immunity in hepatitis C.
The virus has not been grown in culture, but has been
cloned in Escherichia coli.
Mode of infection: Infection is mainly by blood transfusion and other modes of contact with infected blood
or blood products. Injectable drug abusers, transplant
recipients and immunocompromised persons are at
high risk. Sexual transmission is probably less important. The virus can be transmitted from mother to infant,
though not as frequently as for HBV. In some countries,
HCV infection has been associated with folk medicine
practices.
Infections by HCV are extensive throughout the
world. HCV infection is seen only in humans. The
groups at risk are broadly similar to those listed for
hepatitis B but their relative proportions are different.

Clinical Features
The incubation period is long, 15-160 days, with a mean
of 50 days. HCV causes three types of disease:
1. Acute hepatitis with resolution of the infection and
recovery in 15 percent of cases;
2. Chronic persistent infection with possible
progression to disease much later in life for 70
percent.

 3. Severe rapid progression to cirrhosis in 15 percent

of patients. Many patients (20-50%) develop
cirrhosis and are at high risk for hepatocellular
carcinoma (5-25%) decades later. HCV promotes
the development of hepatocellular carcinoma after
30 years in up to 5 percent of chronically infected
patients.

Laboratory Diagnosis

Prophylaxis
Only general prophylaxis, such as screening of blood
and blood products prior to transfusion, is possible. No
specific active or passive immunizing agent is available.

Morphology
HDV is enclosed within the hepatitis B surface antigen,
HBsAg, and has no recognizable morphology of its own.
The HDV is a defective satellite virus requiring HBV
as helper virus. HDV is a spherical, 36 nm particle with
an outer coat composed of the hepatitis B surface antigen surrounding the circular single stranded RNA
genome. The HDV RNA genome is very small (approximately 1700 nucleotides), and unlike other viruses, the
singlestranded RNA is circular. Delta agent is thus an
incomplete virus, reminiscent of the Dependoviruses
(Fig. 67.8). It has been proposed to be classified in a new
genus Deltavirus, because of its special features.

Pathogenesis
Its mode of transmission is the same as for HBV. Similar
to HBV, the delta agent is spread in blood, semen, and
vaginal secretions. However, it can replicate and cause
disease only in people with active HBV infections.
Types of infection: Two types of infection are recognized:
Coinfection and superinfection.
1. Coinfection: In coinfection, delta and HBV are
transmitted together at the same time. Coinfection clinically presents as acute hepatitis B, ranging
from mild to fulminant disease.
2. Superinfection: In superinfection, delta infection
occurs in a person already harbouring HBV. More
rapid, severe progression occurs in HBV carriers
superinfected with HDV than in people co-infected
with HBV and the delta agent.
No association has been noted between HDV and
hepatocellular carcinoma. In simultaneous acute HBV
and HDV infections, IgM anti-HBc will be detectable,

Chapter 67 Hepatitis Viruses

A. Antibody detection: The diagnosis and detection

of HCV infection are based on ELISA recognition
of antibody. The antigens used are various structural and nonstructural proteins cloned in E. coli.
Antibodies are directed against core, envelope, and
NS3 and NS4 proteins and tend to be relatively
low in titer. There have been three successive generations of such antigens, introduced to improve
sensitivity and specificity of serological diagnosis.
Even the third generation ELISA currently in use,
employing NS-5 region protein and synthetic peptides becomes positive only months after the infection and shows nonspecific reactions. Confirmation
by immunoblot assay is therefore recommended. In
HCV infection, antibodies appear irregularly and
late, limiting their diagnostic utility.
B. HCV RNA identification: Identification of HCV
RNA in blood provides more sensitive and specific results within a few days of exposure to HCV.
Reverse transcriptasepolymerase chain reaction,
branched-chain DNA, and other genetic techniques
can detect HCV RNA in seronegative people and
have become key tools in the diagnosis of HCV
infection.

the most severe forms of hepatitis in HBsAg-positive

patients. It is a viral parasite, proving that even fleas
have fleas.

Treatment
Recombinant interferon-alpha, alone or with ribavarin
is the only known treatment for HCV.

HEPATITIS D VIRUS (HDV)

This curious little agent was first detected in people
undergoing exacerbations of chronic HBV infections. In
1977, Rizzetto and colleagues in Italy identified a new
viral antigen in the liver cell nuclei of patients infected
with hepatitis B virus. This has been shown to be due to
the hepatotropic virus Delta or Hepatitis D virus (HDV)
is unique in that it uses HBV and target cell proteins to
replicate and produce its one protein. Delta is a defective
RNA virus dependent on the helper function of HBV for
its replication and expression. It acquires an HBsAg coat
for transmission. Therefore, it has no independent existence and can survive and replicate only as long as HBV
infection persists in the host. It is often associated with

Fig. 67.8: The delta hepatitis virion

609

while in acute HDV infection superimposed on chronic

HBV infections, anti-HBc will be of IgG class.

Section 4 Virology

Laboratory Diagnosis
The only way to determine the presence of the agent is
by detecting the delta antigen or antibodies. ELISA and
radioimmunoassay procedures are available for doing
this. Anti-delta antibodies appear in serum and can
be identified by ELISA. The IgM antibody appears 2-3
weeks after infection and is soon replaced by the IgG
antibody in acute delta infection. However, in chronic
infection, the IgM antibody persists for years.
For the rapid identification of delta particles in circulation, RNA sequences have been cloned and DNA
probes have been developed. The woodchuck has been
found to be a suitable experimental model for the study
of HDV infection.

Treatment
There is no known specific treatment for HDV hepatitis.

Prophylaxis
Because the delta agent depends on HBV for replication
and is spread by the same routes, prevention of infection with HBV prevents HDV infection. Immunization
with HBV vaccine protects against subsequent deltavirus infection.

HEPATITIS E VIRUSV (HEV) (ENTERICALLY

TRANSMITTED NANB OR EPIDEMIC NANB HEPATITIS)
Hepatitis E Virus (HEV) has been provisionally classified in the genus Hepativirus under the family Caliciviridae. HEV is a spherical nonenveloped virus, 32-34
nm in diameter, with a single stranded RNA genome.
The surface of the virion shows indentation and spikes.
Comparison of virus strains from different areas indicates that only one serotype of the virus exists.
HEV (E-NANBH) (The E stands for Enteric or epidemic) is predominantly spread by the feco-oral route,
especially in contaminated water.

Clinical Features

610

The incubation period ranges from 2 to 9 weeks with

an average of six weeks. Most cases occur in the young
to middle aged adults (15-40 years old). The symptoms
and course of HEV disease are similar to those of HAV
disease; it causes only acute disease. However, the
symptoms for HEV may occur later than those of HAV
disease, and response to serum immunoglobulin G may
be poor. The mortality rate associated with HEV disease
is 1 to 2 percent, approximately 10 times that associated
with HAV disease. HEV infection is especially serious
in pregnant women (mortality rate of approximately
20%). HEV infection during pregnancy may cause
a high rate of abortion and intrauterine death and
increased perinatal mortality in babies born to women

with fulminant hepatitis. Secondary attack rate among

household contacts is very low in type E hepatitis, 2-3
percent as against 10-20 percent in HA V infection.

Epidemiology
HEV is predominantly spread by the fecal-oral route,
especially in contaminated water. Hepatitis E has been
shown to occur in epidemics, endemics and sporadic
forms almost exclusively in the less developed parts of
the world. A substantial proportion of cases of acute
viral hepatitis occurring in young to middle-aged adults
in Asia and the Indian subcontinent appear to be caused
by HEV.
The largest such epidemic occurred in Delhi during
the winter of 1955-56, following a breakdown in the
water supply and sewage systems of Delhi caused by
floods, affecting over 30,000 persons within six weeks,
which affected pregnant women particularly severely.
HAV was blamed at first, but 20 years later, the cause
was eventually identified as a new agent, HEV. A similar epidemic of hepatitis E occurred between December
1975 and January 1976 in Ahmedabad city, India, again
due to contaminated water supplies. In India, HEV is
responsible for the majority of epidemic and sporadic
hepatitis in adults.

Laboratory Diagnosis
Several antibody assays are being developed, mostly
based on ELISA methods.
1. Exclusion of hepatitis A and hepatitis BExclusion of hepatitis A by IgM serology and hepatitis B
by absence of HBsAg and IgM anti-HBc.
2. Immunoelectron microscopyImmunoelectron
microscopic examination of patient feces for aggregated calicivirus-like particles using monoclonal
antibodies.
3. ELISA testsfor IgM and IgG anti-HEV.
4. Western blot assayA Western blot assay for IgM
and/or IgG antiHEV.
5. Polymerase chain reaction (PCR)Polymerase
chain reaction (PCR) assay for the detection of HEV
RNA (as cDNA) in patient feces or in acute-phase
sera.

Prophylaxis
General Measures
These depend on the maintenance of a clean water supply, and generally resemble those used to control HAV.

Immunization
Vaccines based on recombinant antigens are under
development, and show some promise.

HEPATITIS G VIRUS
So far, the alphabetic designations of the various hepatitis viruses has been reasonably simple. Two flaviviruslike isolates were obtained in 1995 from Tamarin monkeys inoculated with blood from a young surgeon (GB)

Laboratory Diagnosis
1. HGV is identified by detection of the genome by reverse transcriptase polymerase chain reaction (RTPCR) or other RNA detection methods.
2. Recently, an immunoassay has been developed to
detect anti-HG env. Serum HGV RNA indicates
viremia, whereas anti-HG env is associated with
recovery.

KNOW MORE
INDICATIONS FOR VACCINATION
Vaccination is recommended for infants, children, and
especially people in high-risk groups. Vaccination is useful even after exposure for new-borns of HBsAg-positive
mothers and people accidentally exposed either percutaneously or permucosally to blood or secretions from
an HBsAg-positive person. Immunization of mothers
should decrease the incidence of transmission to babies
and older children, thus also reducing the number of
chronic HBV carriers. Prevention of chronic HBV will
reduce the incidence of PHC.

)) KEY POINTS
At least six viruses, A through G (A, B, C, D, E and
G) are hepatitis viruses.

All the hepatitis viruses are RNA viruses, except

the hepatitis B virus (HBV), which is a DNA virus
belonging to the family Hepadnaviridae.
Mode of transmission is enteric in hepatitis A and
hepatitis E viruses while it is parenteral and sexual in
both hepatitis B and hepatitis C viruses.
Hepatitis A virus (HAV) can be demonstrated in
the stool by immunoelectron microscopy (IEM).
ELISA is the method of choice for detection of IgM
and IgG antibodies in the serum.
Prevention of HAV infection depends on: (a) vaccines containing formalin-inactivated HAV. (b)
Prophylaxis with hepatitis A immunoglobulin to
contacts within 2 weeks of exposure.
Hepatitis B virus (HBV) is a double-walled spherical
structure and measures 42 nm in diameter (Dane
particle). The outer surface or envelope of virus
contains hepatitis B surface antigen (HBsAg). It
encloses an inner icosahedral 27 nm nucleocapsid
(core), which contains hepatitis B core antigen
(HBcAg). Inside the core is the genome, a circular
double stranded DNA and a DNA polymerase.
The virion envelope consists of: 1. Hepatitis B surface antigen (HBsAg); 2. Hepatitis B core antigen
(HBcAg); 3. Hepatitis Be antigen (HBeAg). Hepatitis B surface antigen (HBsAg) is also named as Australia antigen.
HBV is a blood borne virus and there are three
important modes of transmission: 1. Parenteral
transmission; 2. Perinatal transmission; 3. Sexual
transmission.
Laboratory diagnosis: Specific diagnosis of hepatitis B rests on serological demonsration of the viral
markers and can be carried out by detection of
HBsAg, anti-HBs, HBeAg, anti-HBe, IgM antiHBc, IgG anti-HBc and HBV DNA in the serum.
The sequence of appearance of viral markers in the
blood is important. These can be detected by sensitive and specific tests like ELISA and RIA. HBV
DNA is also an indicator of viral replication and
infectivity. Molecular methods such as DNA:DNA
hybridization and PCR are used for HBV DNA
testing are highly sensitive and quantitative.

Chapter 67 Hepatitis Viruses

with acute hepatitis. It was termed GB, the patients initials. A similar virus was isolated from another human
specimen the same year. These isolates were called GB
viruses A, B and C respectively.
In 1996, an isolate closely resembling GBV-C was
obtained from a patient with chronic hepatitis. This has
been called hepatitis G virus (HGV).
Hepatitis G virus resembles HCV in many ways.
HGV is a flavivirus, is transmitted in blood, and has a
predilection for chronic hepatitis disease. It has not been
grown, but its RNA genome has been cloned.
HGV RNA has been found in patients with acute,
chronic and fulminant hepatitis, hemophiliacs, patients
with multiple transfusions and hemodialysis, intravenous drug addicts and blood donors. HGV appears to
be a blood-borne virus resembling HCV. Its role in hepatitis is yet to be clarified.
The virus is present worldwide. Majority of the individuals with HGV infection have no detectable evidence of liver disease. There have been, however, cases
of acute, fulminant and chronic hepatitis where HGV
is presently the only explanation for their liver disease.
There is no evidence of a causal relationship between
HGV infection and hepato-cellular carcinoma. HGV
infection results frequently in chronic viremia. It often
subsides after several years and anti-HG env antibody
develops.

Prophylaxis
Measures for the control of HBV infection areGeneral
prophylaxis and immunization.General prophylaxis
consists in avoiding risky practices like promiscuous
sex, injectable drug abuse and direct or indirect contact
with blood, semen or other body fluids of patients and
carriers.
1. Passive immunization: Hyperimmune hepatitis
B immune globulin (HBIG) administered IM in a
dose of 300-500 IU soon after exposure to infection.
2. Active immunization: Active immunization is
more effective such as plasma-derived hepatitis
B vaccine, recombinant yeast hepatitis B vaccine

611

(Three doses given at 0, 1 and 6 months constitute

the full course), recombinant chinese hamster ovary
(CHO) cell hepatitis vaccine, synthetic peptide
vaccines and Hybrid virus vaccine.

Hepatitis C Virus

Section 4 Virology

HCV can cause acute HCV infection, chronic HCV

infection, and cirrhosis and other complications
induced by hepatitis.
Blood or blood products and also organs of infected
patients are the major sources of infection.
For diagnosis, antibody to HCV antigen can be
detected by ELISA. The HCV RNA can be amplified
by RT-PCR.

Hepatitis D Virus
The hepatitis D virus (HDV) is an unusual, singlestranded, circular RNA virus and is unique in being
an incomplete virus, that requires hepadnavirus
helper functions for propagation in hepatocytes.
Transmission of HDV occurs parenterally.

Hepatitis E Virus (HEV)

Hepatitis E virus is the primary cause of enterically
transmitted non-A, non-B hepatitis.
It usually causes an acute, self-limiting disease similar to HAV.
Specific diagnostic tests for infection due to HEV
include PCR to detect HEV RNA, and ELISA, which
detects both IgG and IgM anti-HEV antibodies.
General measures for preventing HEV infection are
by improved standards of sanitation and chlorinat.
A vaccine may soon be available.
Hepatitis G virus (HGV) is a flavivirus, is transmitted in blood, and has a predilection for chronic
hepatitis disease.

612

2. Classify hepatitis viruses. Discuss the laboratory

diagnosis of infections caused by hepatitis B virus.
3. Draw a neat labelled diagram of hepatitis B virus.
4. Write short notes on:
Hepatitis A virus (HAV).
Hepatitis B virus or Danes particle.
Hepatitis B surface antigen (HBsAg) or Australia
antigen.
Hepatitis B virus markers.
Hepatitis C virus or (HCV).
Hepatitis D virus or Delta agent.
Non-A, Non-B hepatits.
Hepatitis E virus.
Hepatitis G virus.
Prophylaxis of hepatitis B or hepatitis B vaccine.

FURTHER READING
Bradley OW, Krawczynski K, Kane MA. Hepatitis E. In: Belshe
RB, editor. Textbook of human virology, ed 2, St Louis 1991,
Mosby.
British Medical Bulletin 1990;Hepatitis 2:46.
Hadler SC, Fields HA. Hepatitis delta virus. In Belshe RB,
editor. Textbook of human virology, ed 2, St Louis, 1991,
Mosby.
Hagedorn CH, Rice CM. The hepatitis C viruses. Curr Top
Microbiol Immuno l 2000;242:1-380.
Hepatitis NIAIO fact sheet. Available at http://www.niaid.
nih.gov/publications/hepatitis.htm.
Hollinger FB, Emerson SU. Hepatitis A virus. In: Fields Virology. 4th ed. Knipe OM et al (editors). Lippincott Williams
and Wilkins, 2001.
Hollinger FB and TJ Liary 2002. Hepatitis B virus. In. Fields
Virology, Vol 2. 4th ed. Philadelphia: Williams and WIlkins.

IMPORTANT QUESTIONS

Reyes GR, Baroudy BM. Molecular biology of NANBH agents:

hepatitis C and hepatitis E viruses. Adv Virus Res 1991;
40:57-102.

1. Name the hepatitis viruses. Describe the morphology and antigenic structure of hepatitis B virus.

Maillard ME and JR Gollan. Suppressing Hepatitis B. New

Eng J Med 2003;348:848.