Viral Hepatitis

Viral hepatitis is a major public health problem throughout the world affecting several hundreds
of millions of people. Viral hepatitis is a cause of considerable morbidity and mortality in the
human population, from both acute infection and chronic sequelae which include, with hepatitis
B and hepatitis C infection, chronic active hepatitis, cirrhosis and primary liver cancer.
The hepatitis viruses include a range of unrelated and often unusual human pathogens:
 Hepatitis A virus (HAV): a small unenveloped symmetrical RNA virus, which shares many
of the characteristics of the picornavirus family. This virus has been classifi ed in the
hepatovirus genus, and is the cause of infectious or epidemic hepatitis transmitted by the
faecal–oral route.
 Hepatitis B virus (HBV): a member of the hepadnavirus group of double-stranded DNA
viruses which replicate by reverse transcription. Hepatitis B virus is endemic in the human
population and hyperendemic in many parts of the world.
 Hepatitis C virus (HCV): an enveloped single-stranded RNA virus which appears to be
distantly related (possibly in its evolution) to fl aviviruses, although hepatitis C is not
transmitted by arthropod vectors. Infection with this virus is common in many countries,
and it is associated with chronic liver disease and also with primary liver cancer.
 Hepatitis D virus (HDV): an unusual single-stranded circular RNA virus with a number of
similarities to certain plant viral satellites and viroids. This virus requires hepadnavirus
helper functions for propagation in hepatocytes, and is an important cause of acute and
severe chronic liver damage in some regions of the world.
 Hepatitis E virus (HEV): an enterically transmitted nonenveloped, single-stranded RNA
virus which shares many biophysical and biochemical features with caliciviruses. Hepatitis
E virus is an important cause of large epidemics of acute hepatitis in the subcontinent of
India, central and South-east Asia, the Middle East, parts of Africa and elsewhere; this
virus is responsible for high mortality during the third trimester of pregnancy.

Hepatitis A
Outbreaks of jaundice have been described for many centuries and the term ‘infectious
hepatitis’ was coined in 1912 to describe the epidemic form of the disease. HAV is spread by
the faecal–oral route and is endemic throughout the world and hyperendemic in areas with
poor standards of sanitation and hygiene. Commonsource outbreaks are initiated most
frequently by faecal contamination of food and water, and sporadic cases result from person-
to-person contact. The seroprevalence of antibodies to HAV has declined since World War II
in many industrialized countries. The exact incidence is diffi cult to estimate because of the
high proportion of subclinical infections and infections without jaundice, differences in
surveillance and differing patterns of disease. The extent of under-reporting is very high.
Hepatitis A is recognized as an important travel-related infection in travellers from low
prevalence areas to endemic countries.
The incubation period of hepatitis A is about 28 days. The virus replicates in the liver. Very
large amounts of virus are shed in the faeces during the incubation period, before the onset
of clinical symptoms and a brief period of viraemia occurs (Figure 39.1). The severity of illness
ranges from asymptomatic to anicteric or icteric hepatitis and rarely fulminant hepatitis. The
virus is noncytopathic when grown in cell culture. Its pathogenicity in vivo, which involves
necrosis of parenchymal cells and histiocytic periportal inflammation, may be mediated via the
cellular immune response. By the time of onset of symptoms, excretion of virus in the faeces
has declined and may have ceased and anti-HAV IgM, which is diagnostic of acute infection
and appears late during the incubation period, increases in titre. Anti-HAV IgG may be
detected 1–2 weeks later and persists for years. The virus does not persist and chronic
excretion of HAV does not occur. There is no evidence of progression to chronic liver disease.
Diagnostic tests are based mostly on enzyme-linked immunosorbent assays (ELISA).

Classification
Examination by electron microscopy of concentrates of fi ltere faecal extracts from patients
during the incubation period reveals 27 nm unenveloped spherical particles typical of the
Picornaviridae. The entire nucleotide sequence of the viral genome has been determined.
Comparison with other picornavirus sequences revealed limited homology to the
enteroviruses or, indeed, the rhinoviruses; however, the structure and genome organization
are typical of the Picornaviridae.

Figure 39.1 Electron micrograph showing the large number of hepatitis A virus particles in
faeces during the incubation period of the infection. (Reduced from 120 000. From a series
by Anthea Thornton and A. J. Zuckerman.)

HAV is now considered as a separate genus (Hepatovirus) within the Picornaviridae as are
the cardioviruses (of mice) and apthoviruses (foot and mouth disease viruses). There is one
human serotype of HAV. Seven genotypes have been identifi ed from wild-type strains, but all
have a highly conserved single immunodominant epitope which generates neutralizing
antibodies.

Organization of the HAV genome

The HAV genome comprises about 7500 nucleotides (nt) of positive sense RNA, which is
polyadenylated at the 3′ end and has a polypeptide (VPg) attached to the 5′ end. A single,
large open reading frame (ORF) occupies most of the genome and encodes a large
polyprotein.
The viral polyprotein is processed to yield the structural (located at the amino terminal end)
and non-structural viral polypeptides. Many of the features of replication of the picornaviruses
have been deduced from studies of prototype enteroviruses and rhinoviruses, in particular
poliovirus type 1. The virus replicates in the cytoplasm of hepatocytes.
HAV is stable at low pH and is resistant to degradation by environmental conditions.

Prevention and control of hepatitis A

Passive immunization
Control of hepatitis A infection is diffi cult. Since faecal shedding of the virus is at its highest
during the late incubation period and the prodromal phase of the illness, strict isolation of
cases is not a useful control measure. Spread of hepatitis A is reduced by simple hygienic
measures and the sanitary disposal of excreta.
Normal human immunoglobulin, containing at least 100 IU/ mL of anti-hepatitis A antibody,
given intramuscularly before exposure to the virus or early during the incubation period, will
prevent or attenuate a clinical illness. The dosage should be at least 2 IU anti-hepatitis A
antibody/kg body weight, but in special cases, such as pregnancy or in patients with liver
disease, that dosage may be doubled. Immunoglobulin does not always prevent infection and
excretion of HAV, and inapparent or subclinical hepatitis may develop. The effi cacy of passive
immunization is based on the presence of hepatitis A antibody in the immunoglobulin, but the
minimum titre of antibody required for protection has not yet been established. Immunoglobulin
is used most commonly for close personal contacts of patients with hepatitis A and for those
exposed to contaminated food. Immunoglobulin has also been used effectively for controlling
outbreaks in institutions such as homes for the mentally handicapped and in nursery schools.
Prophylaxis with immunoglobulin is recommended for persons without hepatitis A antibody
visiting highly endemic areas. After a period of 6 months the administration of immunoglobulin
for travellers should be repeated, unless it has been demonstrated that the recipient has
developed his or her own hepatitis A antibodies. Active immunization (see below) is strongly
recommended.

Active immunization: Hepatitis A vaccines

In areas of high prevalence most children have antibodies to HAV by the age of 3 years and
such infections are generally asymptomatic. Infections acquired later in life are of increasing
clinical severity. Less than 10% of cases of acute hepatitis A in children up to the age of 6
years are icteric but this increases to 40–50% in the 6–14 age group and to 70–80% in adults.
Of 115 551 cases of hepatitis A in the USA between 1983 and 1987, only 9% of the cases,
but more than 70% of the fatalities, were in those aged over 49. It is important, therefore, to
protect those at risk because of personal contact with infected individuals or because of travel
to highly endemic areas. Other groups at risk of hepatitis A infection include staff and residents
of institutions for the mentally handicapped, day care centres for children, sexually active male
homosexuals, intravenous narcotic drug abusers, sewage workers, healthcare workers,
military personnel and members of certain low socioeconomic groups in defi ned community
settings. Active immunization for travellers is strongly recommended. It is also recommended
that food handlers should be immunized. In some developing countries the incidence of clinical
hepatitis A is increasing as improvements in socioeconomic conditions result in infection later
in life and strategies for immunization are yet to be agreed. Immunization against hepatitis A
is also recommended for patients with chronic liver disease, and patients with chronic blood
clotting disorders.

Hepatitis A vaccines
The foundations for a hepatitis A vaccine were laid in 1975 by the demonstration that formalin-
inactivated virus extracted from the liver of experimentally infected marmosets induced
protective antibodies in susceptible marmosets on challenge with live virus. Subsequently
HAV was cultivated, after serial passage in marmosets, in a cloned line of fetal rhesus monkey
kidney cells (FRhK6), thereby opening the way to the production of hepatitis A vaccines. Later
it was demonstrated that prior adaptation in marmosets was not a prerequisite to growth of
the virus in cell cultures and various strains of virus have been isolated directly from clinical
material using several cell lines, including human diploid fi broblasts, and various techniques
have been employed to increase the yield of virus in cell culture. The vaccines are inactivated,
highly immunogenic and provide long-term protection against infection. Combined
preparations of killed hepatitis A vaccines with hepatitis B vaccine and other vaccines are
available or are under clinical trial. Combination vaccines are available: hepatitis A and B,
hepatitis A and typhoid, and other polyvalent vaccines are under development.

HEPATITIS E
Retrospective testing of serum samples from patients involved in various epidemics of
hepatitis associated with contamination of water supplies with human faeces led to the
conclusion that an agent other than hepatitis A or hepatitis B was involved. Epidemics of
enterically transmitted non-A, non-B hepatitis in the Indian subcontinent were fi rst reported in
1980, but outbreaks involving tens of thousands of cases have also been documented in the
former USSR, South-east Asia, northern Africa and Mexico. Infection has been reported in
returning travellers. The average incubation period is longer than that for hepatitis A, with a
mean of 6 weeks. The highest attack rates are found in young adults, and high mortality rates
(up to 20%) have been reported in women in the third trimester of pregnancy.
Virus-like particles measuring 28–34 nm in diameter have been detected in faecal
extracts of infected individuals by immune electron microscopy using convalescent serum.
However, such studies have often proved inconclusive because a large proportion of the
excreted virus may be degraded during passage through the gut. Cross-reaction studies
between sera and virus in faeces associated with a variety of epidemics and other viral isolates
in several different countries indicate that there are at least four major genotypes and
phylogenetic and sequence analyses defi ne at least nine different groups.
Studies on HEV have progressed following transmission to susceptible non-human primates.
Man is the natural host of HEV.2 A number of non-human primates such as chimpanzees,
cynomolgus monkeys, rhesus monkeys, pigtail monkeys, owl monkeys, tamarins and African
green monkeys are susceptible to natural (and experimental) infection with human strains of
HEV. Swine strains have been identifi ed and are able to infect humans. In endemic areas,
antibodies to HEV acquired naturally have been found in 42–67% of domestic farm animals:
cows, sheep and goats. In addition, there is evidence of widespread HEV or HEV like infection
in rodents in the USA, raising the possibility ofreservoirs of HEV infection in industralized
countries.
The problem of degradation of HEV in the gut was circumvented when the bile of infected
monkeys was found to be a rich source of virus. This material permitted the molecular cloning
of DNA complementary to the HEV (RNA) genome and the entire 7.5 kb sequence was
determined. The organization of the genome is distinct from the Picornaviridae and the non-
structural and structural polypeptides are encoded respectively at the 5′ and 3′ ends. HEV
resembles the caliciviruses in the size and organization of its genome as well as in the size
and morphology of the virion.

Laboratory tests
Sequencing of the HEV genome has resulted in the development of a number of specific
diagnostic tests. For example, HEV RNA was detected, using the polymerase chain reaction
(PCR), in faecal samples. An enzyme-linked immunosorbent assay (ELISA), which detects
both IgG and IgM anti-HEV, has been developed using a recombinant HEV-glutathione-S-
transferase fusion protein and used to detect antibodies in sporadic cases of infection in
children and adults and during a number of epidemics. Epidemics are usually associated with
warm weather and poor sanitation leading to faecal contamination of drinking water. Sporadic
cases occur where HEV is endemic and also in Western countries in individuals without a
history of travel to endemic countries.

Epidemiology
HEV is spread by the faecal-oral route. Consumption of drinking water contaminated with
faecal material has led to epidemics, and the ingestion of raw or uncooked shellfi sh has
caused sporadic infections and epidemics in endemic areas. The highest prevalence of
infection occurs in regions with low standards of sanitation and non-chlorinated drinking water.
The incubation period is 2–9 weeks, with an average of 6 weeks. Zoonotic spread of HEV
appears likely, particularly from swine and possibly rodents. For example, although hepatitis
E is not endemic in the USA and other developed countries, anti-HEV has been found in a
signifi cant proportion (up to 28% in some areas) of healthy persons in these countries.
Subclinical infection might be the explanation. Infection in town dwellers might be caused by
rodents. The prevalence of anti-HEV in blood donors (a highly selected sector of the
population) in Central Europe and North America is 1.4–2.5%, in South Africa 1.4%, Thailand
2.8% , Saudi Arabia 9.5% and 24% in Egypt.
The prevalence of anti-HEV in endemic regions is 3–26%, which is much lower than
expected, although HEV infections account for more than 50% of acute sporadic hepatitis in
some highly endemic areas.
Virus is excreted from the liver via the bile duct into the intestine and faeces. Viraemia and
shedding of HEV in the faeces reach a peak during the incubation period, and excretion in the
faeces continues for up to 14 days after the onset of jaundice. The quantity of virus in the
faeces is small, which is consistent with the low rate of secondary spread by person-to-person
contact. There is no evidence for sexual transmission or for transmission by transfusion.

Clinical features
The clinical spectrum of infection with hepatitis E is similar to infection caused by other
hepatitis viruses, and includes subclinical and anicteric infections, acute hepatitis with jaundice
and fulminant hepatitis. Cholestatic features are common. Hepatitis E does not progress to
chronic liver disease and there is no evidence of persistent infection. As with other forms of
viral hepatitis, hepatitis E is more likely to be asymptomatic, subclinical and anicteric in young
children.
Infection with hepatitis E is associated with a relatively high mortality of 1–4% of patients
admitted to hospital from the general population. Fulminant hepatitis in pregnancy may lead
to a mortality rate of 20% during the third trimester. Premature delivery and infant mortality of
up to 33% have been observed.

Control and prevention

Outbreaks are more common in countries with a hot climate, and have been reported from
many countries. Most outbreaks have occurred following heavy rain and fl ooding and
contamination of drinking water, contamination of well water, and untreated sewage gaining
access into city water treatment plants. Food-borne outbreaks have been associated with raw
or uncooked shell-fish. Therefore, the provision of safe (and chlorinated) drinking water and
safe disposal of sanitary waste are essential, including safeguarding the water supply from
animal waste from farm animals.
Smaller outbreaks and sporadic cases have been reported from many countries from
South-East Asia, Central Asia, the Middle East, Northern and Western Africa, Mexico and also
Italy and Spain. Sporadic cases have been reported from many other countries including
Taiwan, Japan, the USA, South America and many countries in Europe among returning
travellers and also among those who have not undertaken travel outside their own country. In
highly endemic areas, boiling is a good way of treating drinking water, and should be available
both for drinking and for brushing teeth. Bottled water or water in sealed cans of wellknown
brand names should be used for drinking. Raw or uncooked shellfi sh must be avoided, and
the other usual elementary food hygiene precautions are recommended.
These include not eating uncooked fruit or vegetables that are not peeled or prepared by
the consumer. Pregnant women travelling to countries where outbreaks have been reported
and countries where HEV is endemic should be counselled and the importance of the
precautions outlined above must be stressed.

HEPATITIS B
HBV was recognized originally as the cause of ‘serum hepatitis’, the most common form of
parenterally transmitted viral hepatitis, and an important cause of acute and chronic infection
of the liver in many countries. More than one-third of the world’s population has been infected
with HBV, and WHO estimates that it results in 1–2 million deaths every year. The incubation
period of hepatitis B is variable, with a range of between 1 and 6 months.
The clinical features of acute infection resemble those of the other viral hepatitides.
Frequently, acute hepatitis B is anicteric and asymptomatic, although a severe illness with
jaundice can occur and acute liver failure may develop. The virus persists in about 10% of
infected immunocompetent adults and in as many as 90% of infants infected perinatally,
depending on the ethnic group of the mother. About 350 million people worldwide are
persistent carriers of hepatitis B. Liver damage is mediated by the cellular immune response
of the host to the infected hepatocytes. Approximately 25% of all patients with chronic hepatitis
will progress to cirrhosis and about 20% of those with cirrhosis will develop hepatocellular
carcinoma. Hepatocellular carcinoma is one of the most common cancers worldwide.
During the fi rst phase of chronicity, virus replication continues in the liver and replicative
intermediates of the viral genome may be detected in DNA extracted from liver biopsies.
Markers of virus replication in serum include HBV DNA, the pre-S1 proteins (see below) and
a soluble antigen, hepatitis B e antigen (HBeAg), which is secreted by productively infected
hepatocytes. In those infected at a very young age this phase may persist for life but, more
usually, virus levels decline over time. Eventually in most individuals there is immune
clearance of infected hepatocytes associated with seroconversion from HBeAg to anti-HBe.
During the period of replication the viral genome may integrate into the chromosomal DNA of
some hepatocytes and these cells may persist and expand clonally. Rarely, seroconversion
to anti-HBs follows clearance of virus replication but, more frequently, the surface antigen
(HBsAg) persists during a second phase of chronicity as a result of the expression of
integrated viral DNA.

Structure of the virus

The hepatitis B virion is a 42 nm particle comprising an electrondense nucleocapsid or core,
27 nm in diameter, surrounded by an outer envelope of the surface protein (HBsAg) embedded
in membranous lipoprotein derived from the host cell (Figure 39.2). The surface antigen is
produced in excess by the infected hepatocytes and is secreted in the form of 22 nm particles
(initially referred to as Australia antigen) and tubular structures with the same diameter. The
22 nm particles are composed of the major surface protein in both non-glycosylated (p24) and
glycosylated (gp27) form in approximately equimolar amounts, together with a minority
component of the so-called middle proteins (gp33 and gp36) which contain the pre-S2 domain,
a glycosylated 55 amino acid Nterminal extension. The surface of the virion has a similar
composition but also contains the large surface proteins (p39 and gp42) which include both
the pre-S1 and pre-S2 regions. These large surface proteins are not found in the 22 nm
spherical particles (but may be present in the tubular forms in highly viraemic individuals) and
their detection in serum correlates with viraemia. The domain which binds to the specifi c HBV
receptor on the hepatocyte resides within the pre-S1 region.
The nucleocapsid of the virion consists of the viral genome surrounded by the core antigen
(HBcAg). The carboxy terminus of the core protein is arginine rich and this highly basic domain
is believed to interact with the genome. The genome, which is approximately 3.2 kb in length,
has an unusual structure and is composed of two linear strands of DNA held in a circular confi
guration by base pairing at the 5′ end. One of the strands is incomplete and the 3′ end is
associated with a DNA polymerase molecule which is able to complete that strand when
supplied with deoxynucleoside triphosphates. In the past, this endogenous DNA polymerase
reaction was used as a serological assay for the hepatitis B virion but this has now been
superseded by DNA-DNA hybridization and PCR. The 5′ ends of both strands of the genome
are modifi ed. The 5’ end of the complete strand is covalently linked to a protein and the 5′
end of the incomplete strand is an oligoribonucleotide. In both cases these moieties seem to
be primers for the synthesis of the respective strands during the genome replication. A motif
of 12 base pairs is repeated directly in the genome near to the 5′ ends of the two strands (DR1
and DR2, respectively) and these sequences play an important role in replication.

Mode of spread of hepatitis B virus

Specific laboratory tests for hepatitis B confirmed the importance of the parenteral routes of
transmission and blood-to-blood contact, and infectivity appears to be especially related to
blood. However, the infection is not spread exclusively by blood and blood products. There
are observations that under certain experimental circumstances the virus is infective by mouth
and the infection may be endemic in closed and semi-closed institutions and in institutions for
the mentally handicapped.

Mother-to-infant transmission
Transmission of hepatitis B from carrier mothers to their babies can occur during the perinatal
period and appears to be the single most important factor in determining the prevalence of the
infection in some regions, particularly in China and South-east Asia. The risk of infection in
the infant may reach 90% and appears to be related to ethnic groups. Infection of infants is
especially important because a large proportion of these infants will become carriers.
Infectivity is directly related to the presence of high titres of hepatitis B surface antigen and/or
hepatitis B e antigen in the mother’s circulation. When e antigen is present, as many as 95%
of newborn children are infected, usually in the perinatal period.
The prevalence of e antigen among surface antigen maternal carriers,and thus the infectivity
of mothers for their infants, varies markedly in different geographical areas and in different
ethnic groups.
The carrier state
The carrier state is defi ned on the basis of longitudinal studies as persistence of the hepatitis
B surface antigen in the circulation for more than 6 months.

Age distribution and the prevalence of infection

Two different patterns of age distribution of infection are recognized. In populations with a high
prevalence of hepatitis B virus, infection is usually acquired early in the life, and the highest
infection and carrier rates are seen among children and young adults, with lower prevalence
among older age groups. The e antigen has been found more commonly in young than in adult
carriers, while the prevalence of e antibody is higher in older age groups. These findings
suggest that young carriers could be the most infective.
In countries in which infection with hepatitis B virus is relatively uncommon, the highest
prevalence of hepatitis B surfaceantigen is found in the 20–40 age group. The highest rates
of infection are found among groups who have an increased risk of contact with blood or blood
products, as outlined above, including healthcare personnel, certain categories of patients,
intravenous drug abusers and male homosexuals who change partners frequently.

Prevention and control of hepatitis B

Passive immunization
Hepatitis B immunoglobulin (HBIG) is prepared from pooled plasma with high titre of hepatitis
B surface antibody (anti-HBs) and may confer temporary passive immunity under certain defi
ned conditions. The major indication for the administration of HBIG is a single acute exposure
to HBV, such as occurs when blood containing surface antigen is inoculated, ingested or
splashed on to mucous membranes and conjunctivae. The optimal dose has not been
established but doses in the range of 250–500 IU have been used effectively. It should be
administered as early as possible after exposure and preferably within 48 h, usually 3 mL
(containing 200 IU of anti-HBs/mL) in adults. It should not be administered 7 days following
exposure. It is generally recommended that two doses of HBIG should be given 30 days apart.
Results with the use of HBIG for prophylaxis in babies at risk of infection with HBV are
encouraging if the immunoglobulin is given as soon as possible after birth or within 12 h of
birth, and the chance of the baby developing the persistent carrier state is reduced by about
70%. More recent studies using combined passive and active immunization indicate an effi
cacy approaching 90%. The dose of HBIG recommended in the newborn is 1–2 mL (200 IU
of anti-HBs/mL).

Active immunization
Immunization against hepatitis B is required for groups which are at an increased risk of
acquiring this infection. These groups include individuals requiring repeated transfusions of
blood or blood products, prolonged in-patient treatment, patients who require frequent tissue
penetration or need repeated access to the circulation, patients with natural or acquired
immune defi ciency and patients with malignant diseases.

Site of injection for vaccination

Hepatitis B vaccination should be given intramuscularly in the upper arm or the anterolateral
aspect of the thigh and not in the buttock. There are over 100 reports of unexpectedly low
antibody seroconversion rates after hepatitis B vaccination using injection into the buttock.

Intradermal immunization
The high cost of hepatitis B vaccines is a serious economic obstacle to extensive immunization
against hepatitis B, which is needed in many countries in Africa and Asia. The possibility of
reducing the amount of antigen required for immunization by reducing the dose of vaccine or
by using the intradermal route has been
explored.
Immunization strategies and the kinetics of antibody production
Immunization strategies
Immunization against hepatitis B is recognized as a high priority in all countries, and strategies
for immunization have been implemented. Universal vaccination of infants and adolescents is
recommended, and more than 168 countries now offer hepatitis B vaccine to all children. A
few countries with a low prevalence of hepatitis B (such as the UK) recommend at present
immunization of only groups at an increased risk of acquiring this infection (see above).
There are three main approaches to developing new hepatitis B immunization strategies:
1. The introduction of universal antenatal screening to identify hepatitis B carrier mothers and
vaccination of their babies. It is important that any other strategies do not interfere with the
delivery of vaccine to this group. Immunization of this group will have the greatest impact
in reducing the number of new hepatitis B carriers. For children outside this group it is diffi
cult to estimate the lifetime risk of acquiring a hepatitis infection.
2. Vaccinate all infants.
3. Vaccinate all adolescents. This approach delivers vaccination at a time close to the time
when ‘risk behaviour’ would expose adolescents to infection. Vaccination could be
delivered as part of a wider package on health education in general, to include sex
education, the risk of AIDS, the dangers of drug abuse and smoking, and the benefi ts of
a healthy diet and lifestyle

The kinetics of anti-HBs response

The titre of vaccine-induced anti-HBs declines, often rapidly, during the months and years
following immunization. The highest anti-HBs titres are generally observed 1 month after
booster vaccination followed by rapid decline during the next 12 months and there after more
slowly.

HEPATITIS D
Delta hepatitis was fi rst recognized following the detection of a novel protein, delta antigen
(HDAg), by immunofl uorescent staining in the nuclei of hepatocytes from patients with chronic
active hepatitis B. Hepatitis delta virus (HDV) requires a helper function of HBV for its
replication. HDV is coated with HBsAg, which is needed for release from the host hepatocyte
and for entry in the next round of infection.
Two forms of delta hepatitis infection are known. In the first, a susceptible individual is co-
infected with HBV and HDV, often leading to a more severe form of acute hepatitis caused by
HBV. Vaccination against HBV also prevents co-infection. In the second, an individual
chronically infected with HBV becomes superinfected with HDV. This may accelerate the
course of the chronic liver disease and cause overt disease in asymptomatic HbsAg carriers.
HDV itself appears to be cytopathic and HDAg may be directly cytotoxic.
Delta hepatitis is common in some areas of the world with a high prevalence of hepatitis B
infection, particularly the Mediterranean region, parts of eastern Europe, the Middle East,
Africa and South America. It has been estimated that 5% of HbsAg carriers worldwide
(approximately 15 million people) are infected with HDV. In areas of low prevalence for
hepatitis B, those at risk of hepatitis B infection – particularly intravenous drug abusers are
also at risk of HDV infection.17

Structure and replication of HDV

The HDV particle is approximately 36 nm in diameter and is composed of an RNA genome
associated with HDAg, surrounded by an envelope of HBsAg. The virus reaches higher
concentrations in the circulation than HBV – up to 1012 particles per millilitre have been
recorded. The HDV genome is a closed circular RNA molecule of 1679 nucleotides with
extensive sequence complementarity that permits pairing of approximately 70% of the bases
to form an unbranched rod structure. The genome thus resembles those of the satellite viroids
and virusoids of plants, and similarly seems to be replicated by the host RNA polymerase II
with autocatalytic cleavage and circularization of the progeny genomes via trans-esterifi cation
reactions (ribozyme activity). Consensus sequences of viroids which are believed to be
involved in these processes are also conserved in the delta virus.
Unlike the plant viroids, however, HDV codes for a protein, HDAg. This antigen, which
contains a nuclear localization signal, was originally detected in the nuclei of infected
hepatocytes and may be detected in serum only after removing the outer envelope of the virus
with detergent.
Prevention and control of HDV are similar to those for hepatitis B. Immunization against
hepatitis B protects against HDV.The problem is protection against HDV superinfection of
established carriers of hepatitis B. Specific HDV immunization based on HDV antigens is
under development.

Laboratory diagnosis and epidemiology

Laboratory diagnosis of actue HDV infection is based on specifi c serological tests for anti-
HDV IgM or HDV RNA or HDAg in serum. Acute infection is usually self-limited and markers
of HDV infection often disappear within a few weeks.
Superinfection with HDV in chronic hepatitis B may lead to suppression of HBV markers
during the acute phase. Chronic infection with HDV (and HBV) is the usual outcome in
nonfulminant disease. Outbreaks of severe hepatitis with high mortality have been reported in
native Indians of the Amazon Basin and in areas of Central Africa.

Prevention and control

Prevention and control measures of HDV are similar to those for hepatitis B. Immunization
against hepatitis B protects against HDV. The diffi culty is protection against superinfection of
the many millions of established carriers of hepatitis B. Studies are in progress to develop
specifi c immunization in hepatitis B carriers against HDV based on HDAg.
Treatment with interferon at high doses for six months (or longer) results in biochemical and
virological improvement. However, many patients relapse when treatment is stopped.

HEPATITIS C
Before the identification of hepatitis C virus (HCV), transmission studies in chimpanzees
established that the main agent of parenterally acquired non-A, non-B hepatitis was likely to
be an enveloped virus some 30–60 nm in diameter. These experimental studies provided a
pool of plasma that contained a relatively high titre of the agent. In order to clone the genome,
the virus was pelleted from the plasma. Because it was not known whether the genome was
DNA or RNA, a denaturation step was included prior to the synthesis of cDNA so that either
DNA or RNA could serve as a template. The resultant cDNA was then inserted into the
bacteriophage expression vector l gt11 and the libraries screened using serum from a patient
with chronic non-A, non-B hepatitis.
This approach led to the detection of a clone (designated 5-1-1) which was found to bind
to antibodies present in the sera of several patients with non-A, non-B hepatitis. This clone
was used as a probe to detect a larger, overlapping clone in the same library. It was possible
to demonstrate that these sequences hybridized to a positive-sense RNA molecule of around
10 000 nt which was present in the livers of infected chimpanzees but not in uninfected
controls. By employing a ‘walking’ technique it was possible to use newly detected overlapping
clones as hybridization probes in turn to detect further virus-specifi c clones in the library. Thus
clones covering the entire viral genome were assembled and the complete nucleotide
sequence determined. The organization of the genome closely resembles those of the
pestiviruses and fl aviviruses.

Detection of HCV infection

Since the 5-1-1 antigen was detected originally by antibodies in the serum of an infected
patient it was an obvious antigen for the basis of an ELISA to detect anti-HCV antibodies. A
larger clone, C100, was assembled from a number of overlapping clones and expressed in
yeast as a fusion protein using human superoxide dismutase sequences to facilitate
expression. This fusion protein formed the basis of fi rst-generation tests for HCV infection.
The 5-1-1 antigen comprises amino acid sequences from the nonstructural, NS4, region of the
genome and C100 contains both NS3 and NS4 sequences. It is now known that antibodies to
C100 are detected relatively late following an acute infection. Furthermore, the fi rst generation
ELISAs were associated with a high rate of false positivity when applied to low-incidence
populations and there were further problems with some retrospective studies on stored sera.
Data based on this test alone should, therefore, be interpreted with caution.

Epidemiology and clinical features

Infection with HCV occurs throughout the world. Many of the seroprevalence data are
based on blood donors, who represent a selected population. The prevalence of antibodies to
HCV in blood donors varies from 0.02% to 1.25% in different countries. Higher rates have
been found in southern Italy, Spain, central Europe, Japan and parts of the Middle East, with
as many as 19% in Egyptian blood donors.
Most acute infections are asymptomatic: less than 30% of patients with acute infections
have non-specifi c symptoms and some develop mild jaundice. Fulminant hepatitis has been
described. Extrahepatic manifestations include mixed cryoglobulinaemia, membranous
proliferative glomerulonephritis and porphyria cutanea tarda.
Between 50% and 80% of patients do not clear the virus by 6 months and develop chronic
hepatitis. The majority have fl uctuating abnormal alanine transaminase levels, but some 30%
have normal levels.
Chronic hepatitis C infection leads to cirrhosis within two decades of the onset of infection
in at least 20% of patients. Chronic infection is also associated with an increased risk of
hepatocellular carcinoma, which occurs on a background of infl ammation and regeneration
related to chronic hepatitis over three or more decades. The risk of developing hepatocellular
carcinoma (HCC) is estimated at 1–5% after 20 years, but this varies considerably in different
areas of the world. It develops more commonly in men than in women.

MANAGEMENT OF ACUTE VIRAL HEPATITIS

There is no specifi c treatment. General measures include bed-rest and a generally
nutritious diet. Patients should be encouraged to exercise regularly if they feel well.
Consumption of alcohol should be avoided during the acute phase and continue to be modest
after convalescence.
Corticosteroids and non-steroidal antiinflammatory drugs are not indicated and should not
be used.

TREATMENT OF CHRONIC HEPATITIS B INFECTION

Specific treatment is now available following the demonstration that interferon-alfa inhibits
replication of HBV, and that prolonged treatment can lead to remission of the disease.
Antiviral therapy is aimed at patients with active disease and viral replication, preferably at
a stage before signs and symptoms of cirrhosis or signifi cant injury have occurred. Eradication
of the disease is possible in only a minority of patients. Permanent loss of HBV DNA and
HBeAg results in an improvement in necroinflammatory change(s), and reduced infectivity. It
is possible that the accompanying histological improvement reduces the risk of cirrhosis and
hepatocellular carcinoma.
Unfortunately, treatment of chronic hepatitis with interferon is effective in less than half of
those treated. It is relatively expensive, requires administration by injection and is not free of
side-effects. Nonetheless, recombinant interferon-alfa has been licensed for treatment of
chronic hepatitis B in the UK and several Europeancountries.
The interferons act by interaction with specifi c membrane receptors, thereby inducing a
number of enzymes and proteins, the best characterized of which are the 2′,5′-oligoadenylate
synthetases (2′,5′- A synthetases) and protein kinases. The expression of the class I major
histocompatibility antigen (MHC) genes is activated by all interferons, and those of class II by
interferon-gamma, to increase the expression of MHC at the cell surface, and thereby amplify
viral antigen recognition and display. Interferons also modify the cellular and humoral immune
response.
Three preparations of interferon-alfa are currently available, two of which are recombinant
preparations and one of which is prepared from a lymphoblastoid cell line. Approximately
40%–50% of patients respond. Highest response rates are usually seen in carriers with higher
baseline serum aminotransferase levels, lower levels of HBV DNA and without AIDS. Although
these factors provide some predictive information, none of these criteria is absolute, and
individual carriers, for example ethnic Chinese, with active disease or those patients with anti-
HIV antibodies but normal CD4 lymphocyte counts may respond, making the prediction of
treatment outcome somewhat diffi cult. The appropriate dose of interferon is not yet
established, but 5–10 mU three times weekly for 3–4 months is currently prescribed.
The subclinical exacerbation of the hepatitis frequently seen in responders suggests that
interferon acts by augmenting the immune response to HBV, perhaps triggered by the
inhibition of viral replication as well as the effects of interferon on cytotoxic T cells. Although
residual HBV DNA can be detected by PCR, the disease appears to be ameliorated.
Approximately 20% of patients who respond to treatment with clearance of HBeAg will also
clear HBsAg within a year of treatment, and up to 65% may later clear HBsAg after 6 years of
follow-up.
Pulsed corticosteroid treatment and interferon may also be of benefi t in patients without
elevated serum aminotransferases. This treatment regimen should be used with caution in
those patients with decompensated hepatitis B because of the risk of inducing severe hepatic
necrosis.
The major early side-effects of interferon include an infl uenzalike illness. Later side-effects
include malaise, muscle aches, headaches, poor appetite, weight loss, increased need for
sleep, irritability, anxiety and depression, hair loss, thrombocytopenia and leucopenia.
Unusual or severe side-effects include seizures, acute psychosis, bacterial infections,
autoimmune reactions, thyroid disease, proteinuria, cardiomyopathy, skin rashes and
interferon antibodies.
Pegylated interferon (PEG interferon) is used now.

Other antiviral drugs

A number of other agents have been used for the treatment of hepatitis B. These include
interferon-gama, aciclovir (acycloguanosine), 6-deoxyaciclovir, ganciclovir, foscarnet
(trisodium phosphonoformate), azido-3′ deoxythymidine triphosphate, 2′,3′-dideoxycytidine
and 2′,3′-dideoxyinosine, adenine arabinoside 5′ monophosphate (ara-AMP), phyllanthrus
amarus, interleukin 2, isoprinosine, thymosin, tumour necrosis factor, transfer factor, adenine
arabinoside 5′-monophosphate conjugated with lactosaminated albumin, interferon-gama plus
alfa, interferon-gama plus beta, and aciclovir plus interferon. Few of these agents are useful
clinically.
Lamivudine, a second-generation nucleoside analogue, inhibits both HBV DNA-dependent
and RNA-dependent DNA polymerase activity. This may cause suppression of HBV DNA
replication at four sites, and also has the indirect effect of restoring T cell hyporesponsiveness.
The decline in viral titre is rapid and dose related, and maximum inhibition is observed with
treatment with 100 mg by mouth once daily. While production of virus isinhibited rapidly,
production of viral protein which is dependent on the presence of the RNA pregenome is
unaffected by lamivudine. Reduction of viral protein concentrations depends on the
destruction of infected liver cells, and with immune control of HBV replication viral protein
production also declines.
Approximately 20% of patients clear HBeAg and HBV DNA within 1 year of starting
treatment with lamivudine. Long-term therapy may be required, and extended therapy is
feasible as, in contrast to interferon-alfa, lamivudine can be taken orally and isassociated with
a lower incidence of adverse events. Extended therapy with lamivudine has also been found
to produce significant improvements in liver histology and increasing levels of seroconversion.
In one cohort about 40% of patients seroconverted after 3 years. Seroconversion rates are
likely to be enhanced if patients with alanine aminotransferase (ALT) elevations are selected
(i.e. ALT levels >2x the upper limit of normal).
TREATMENT OF CHRONIC HEPATITIS C INFECTIONS
Treatment with pegylated interferon-alpha is indicated for patients with well-documented
chronic hepatitis C in whom other causes of chronic hepatitis have been excluded, and who
have at least a two-fold elevation of serum alanine aminotransferase. Interferon-alfa
ameliorates disease activity in approximately 50% of patients with hepatitis C after short
courses (6 months) of treatment. Liver biopsy histology provides useful information regarding
the extent of liver damage. Treatment should be started at a dose of 3x106 units, three times
weekly, and administered subcutaneously for 6 months. Treatment can be discontinued after
3 months if no response has occurred. However, approximately 50% of responders relapse
when treatment is stopped. Almost all of these relapses tend to re-respond to retreatment.
Ribavirin, a nucleoside analogue which is taken orally, has also been shown to inhibit HCV.
This drug may be a better choice for patients with cirrhosis, who respond poorly to interferon,
or it can be used in combination with pegylated interferon.