Hepatitis Viruses

Arie J. Zuckerman
General Concepts
Viral hepatitis has emerged as 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, both from acute infection and chronic sequelae which
include, in the case of hepatitis B, C and D, chronic active hepatitis and cirrhosis. Hepatocellular
carcinoma which is one of the ten most common cancers worldwide, is closely associated with
hepatitis B, and at least in some regions of the world with hepatitis C virus.
he hepatitis viruses include a range of unrelated and often highly unusual human pathogens.
Hepatitis A virus
Hepatitis ! virus "H!V#, classified as hepatovirus, is a small, unenveloped symmetrical $%!
virus which shares many of the characteristics of the picornavirus family, and is the cause of
infectious or epidemic hepatitis transmitted by the fecal&oral route.
Hepatitis B virus
Hepatitis B virus "HBV#, a member of the hepadnavirus group, double&stranded D%! viruses
which replicate, unusually, by reverse transcription. Hepatitis B virus is endemic in the human
population and hyperendemic in many parts of the world. ! number of variants of this virus have
been described. %atural hepadna virus infections also occur in other mammals including
woodchuc's, beechy ground squirrels and duc's.
Hepatitis C virus
Hepatitis C virus "HCV#, is an enveloped single&stranded $%! virus which appears to be
distantly related "possibly in its evolution# to flaviviruses, although hepatitis C is not transmitted
by arthropod vectors. (everal genotypes have been identified. )nfection with this more recently
identified virus is common in many countries. Hepatitis C virus is associated with chronic liver
disease and also with primary liver cancer in some countries.
Hepatitis D virus
Hepatitis D virus "HDV# is an unusual, single&stranded, circular $%! virus with a number of
similarities to certain plant viral satellites and viroids. his virus requires hepadna virus helper
functions for propagation in hepatocytes, and is an important cause of acute and severe chronic
liver damage in many regions of the world.
Hepatitis E virus
Hepatitis * virus "H*V#, the cause of enterically&transmitted non&!, non&B hepatitis, is another
non&enveloped, single&stranded $%! virus, which shares many biophysical and biochemical
features with caliciviruses. he most similar genome to H*V is found in a plant virus, beet
necrotic yellow vein virus, and there are similarities in the functional domains to rubella virus.
+inal ta,onomic classification is yet to be agreed upon.
Hepatitis * virus is an important cause of large epidemics of acute hepatitis in the subcontinent
of )ndia, Central and (outheast !sia, the -iddle *ast, parts of !frica and elsewhere. his virus is
responsible for high mortality "./&012#, during pregnancy particularly during the third trimester.
The GB hepatitis viruses
he 3B hepatitis viruses "3BV&!, 3BV&B and 3BV&C#. he 3B hepatitis viruses were cloned
recently and preliminary genomic characteri4ation shows that they are related to other positive&
stranded $%! viruses with local regions of sequence identity with various flaviviruses.
5hylogenetic analysis of genomic sequences showed that these viruses are not genotypes of the
hepatitis C virus.

Hepatitis A
6utbrea's of jaundice have been frequently described for many centuries and the term infectious
hepatitis was coined in .7.0 to describe the epidemic form of the disease. Hepatitis ! virus
"H!V# is spread by the fecal&oral route and continues to be endemic throughout the world and
hyperendemic in areas with poor standards of sanitation and hygiene. he seroprevalence of
antibodies to H!V has declined since 8orld 8ar )) in many countries, but large epidemics do
occur. +or e,ample, an outbrea' of hepatitis ! associated with the consumption of clams in
(hanghai in .799 resulted in almost :11,111 cases.
Hepatitis ! has an incubation period of about four wee's. he virus replicates in the liver.
$elatively large quantities of virus are shed in the feces during the incubation period before the
onset of clinical symptoms, and a brief viremia occurs. he severity of illness ranges from the
asymptomatic to anicteric or icteric hepatitis. he virus is non&cytopathic when grown in cell
culture. )ts pathogenicity in vivo, which involves necrosis of parenchymal cells and histiocytic
periportal inflammation, may be mediated by cellular immune responses. By the time of onset of
symptoms, e,cretion of virus in the feces has declined and may have ceased and anti&H!V )g-
increases in titer. !nti&H!V )g3 may be detected one to two wee's later and persists for years.
Distinctive properties
*lectron microscopic e,amination of concentrates of filtered fecal e,tracts from patients during
the early stages of infection reveals 0; nm icosahedral particles typical of the Picornaviridae
"+ig.;1&.#. H!V was classified in .79: in the genus *nterovirus "as enterovirus ;0# of the family
Picornaviridae, on the basis of its biophysical and biochemical characteristics, including stability
at low pH. !fter the entire nucleotide sequence of the viral genome was determined, comparison
with other picornavirus sequences revealed limited homology to the enteroviruses, and the virus
is now considered as a separate genus, hepatovirus.
FIGURE 70- Hepatitis A virus particles !oun" in !ecal e#tracts $% i&&unoelectron
&icroscop%' Both full and empty particles are present. he virus is 0; to 07 nm in diameter. "<
.0/,111.#
he H!V genome comprises about ;,/11 nucleotides "nt# of positive sense $%! which is
polyadenylated at the := end and has a polypeptide "V5g# attached to the /= end. ! single, large
open reading frame "6$+# occupies most of the genome and encodes a polyprotein with a
theoretical molecular mass of -r 0/0,111. he H!V polyprotein is processed to yield the
structural "located at the amino&terminal end# and non&structural viral polypeptides. -any of the
features of replication of the picornaviruses have been deduced from studies of prototype
enteroviruses and rhinoviruses, in particular poliovirus type ..
(atho)enesis
he clinical e,pression of infection with hepatitis ! virus varies considerably, ranging from
subclinical, anicteric, and mild illnesses in children to the full range of symptoms with jaundice
in adults. he ratio of anicteric to icteric illnesses varies widely, both in individual cases and in
outbrea's.
Hepatitis ! virus enters the body by ingestion and intestinal infection. he virus then spreads,
probably by the bloodstream, to the liver, a target organ. >arge numbers of virus particles are
detectable in feces during the incubation period, beginning as early as .1&.? days after e,posure
and continuing, in general, until pea' elevation of serum aminotransferases. Virus is also
detected in feces early in the acute phase of illness, but relatively infrequently after the onset of
clinical jaundice. )nterestingly, antibody to hepatitis ! virus that persists is also detectable late in
the incubation period, coinciding appro,imately with the onset of biochemical evidence of liver
damage. Hepatitis ! antigen has been locali4ed by immunofluorescence in the cytoplasm of
hepatocytes after e,perimental transmission to chimpan4ees. he antigen has not been found in
any tissue other than the liver following intravenous inoculation.
5athological changes produced by hepatitis ! appear e,clusively in the liver. (everal such
changes occur@ conspicuous focal activation of sinusoidal lining cellsA accumulations of
lymphocytes and more histiocytes in the parenchyma, often replacing hepatocytes lost by
cytolytic necrosis predominantly in the periportal areasA occasional coagulative necrosis in the
form of acidophilic bodiesA and focal degeneration.
Host De!enses
!ntibody to hepatitis ! virus develops late in the incubation period. (pecific hepatitis ! )g- is
found in the serum within . wee' from the onset of dar' urine, reaching ma,imum levels after .
wee' and declining slowly during the ne,t ?1&B1 days. (pecific )g3 antibody appears shortly
after )g- is detectable, reaching a ma,imum titer after B1&91 days. his antibody is protective
and persists for many years.
Epi"e&iolo)%
Viral hepatitis type ! "previously named infectious or epidemic hepatitis# occurs endemically in
all parts of the world, with frequent reports of minor and major outbrea's. he e,act incidence is
difficult to estimate because of the high proportion of subclinical infections and infections
without jaundice, differences in surveillance, and differing patterns of disease. he degree of
under reporting is believed to be very high.
he incubation period of hepatitis ! is :&/ wee's, with a mean of 09 days. (ubclinical and
anicteric cases are common and, although the disease has in general a low mortality rate, patients
may be incapacitated for many wee's. here is no evidence of progression to chronic liver
damage.
Hepatitis ! virus is spread by the fecal&oral route, person&to&person contactA and under conditions
of poor sanitation and overcrowding. Common source outbrea's are most frequently initiated by
fecal contamination of water and food, but waterborne transmission is not a major factor in
maintaining this infection in industriali4ed communities. 6n the other hand, many food&borne
outbrea's have been reported. his can be attributed to the shedding of large quantities of virus
in the feces during the incubation period of the illness in infected food handlersA the source of the
outbrea' often can be traced to uncoo'ed food or food that has been handled after coo'ing.
!lthough hepatitis ! remains endemic and common in the developed countries, the infection
occurs mainly in small clusters, often with only few identified cases.
)n .7;:, immune electron microscopy led to the identification of virus particles in e,tracts of
feces "+ig. ;1&0# during the early acute phase of illness, providing the long&awaited lead to
further studies of this infection. !vailability of viral antigen permitted, in turn, identification of
specific antibody, development of serologic tests for hepatitis !, and determination of
susceptibility to infection in human and nonhuman primates. Human hepatitis ! has been
transmitted to certain species of non&human primates shown to be free of homologous antibody,
thereby providing a model for e,perimental infection and, initially, also a source of reagents.
FIGURE 70-* I&&une a))re)ate o! hepatitis A virus !ollo+in) the a""ition o! convalescent
seru& to a !ecal e#tract "urin) the acute phase o! the illness , -00.000 /!ro& a series $%
Anthea Thornton an" A 0 1uc2er&an3'
he availability of specific serologic tests for hepatitis ! made possible the study of the
incidence and distribution of hepatitis ! in various countries. hese studies have shown that
infections with hepatitis ! virus are widespread and endemic in all parts of the world, chronic
e,cretion of hepatitis ! virus does not occur, the infection is rarely transmitted by blood
transfusion, and no evidence of progression to chronic liver disease has been found.
Dia)nosis
Various serologic tests are available for hepatitis !, including immune electron microscopy,
complement&fi,ation, immune adherence hemagglutination, radioimmunoassay, and en4yme
immunoassay. )mmune adherence hemagglutination, which had been widely used, is moderately
specific and sensitive. (everal methods of radioimmunoassay have been describedA of these, a
solid&phase type of assay is particularly convenient, very sensitive, and specific. Very sensitive
en4yme immunoassay techniques are used widely.
6nly one serotype of hepatitis ! virus has been identified in volunteers infected e,perimentally
with the -(&. strain of hepatitis !, in patients from different outbrea's of hepatitis in different
geographic regions, and in random cases of hepatitis !.
)solation of virus in tissue culture requires prolonged adaptation and it is, therefore, not suitable
for diagnosis.
Control an" (revention o! Hepatitis A
)n areas of high prevalence, most children are infected early in life and such infections are
generally asymptomatic. )nfections acquired later in life are of increasing clinical severity. >ess
than .12 of cases of acute hepatitis ! in children up to the age of si, are icteric, but this
increases to ?1&/12 in the B&.? age group and to ;1&912 in adults.
6f ../,//. cases of hepatitis ! in the C(! between .79: and .79;, only 72 of the cases, but
more than ;12 of the fatalities, were in those aged over ?7. )t is important, therefore, to protect
those at ris' because of personal contact with infected individuals or because of travel to a highly
endemic area. 6ther groups at ris' of hepatitis ! infection include staff and residents of
institutions for the mentally handicapped, day care centers for children, se,ually active male
homose,uals, intravenous drug abusers, sewage wor'ers, certain groups of health care wor'ers
such as medical students on elective studies in countries where hepatitis ! is common, military
personnel, and certain low socio&economic groups in defined community settings. 5atients with
chronic liver disease, especially if visiting an endemic area, should be immuni4ed against
hepatitis !. )n some developing countries, the incidence of clinical hepatitis ! is increasing as
improvements in socio&economic conditions result in infection later in life, and strategies for
immuni4ation are yet to be developed and agreed.
5assive protection may be obtained by the administration of pooled normal human
immunoglobulin, containing at least .11 )CDml of anti&H!V given intramuscularly at a dose of 0
)CD'g body weight. 5ost&e,posure prophyla,is, if given early enough, may prevent or attenuate a
clinical illness.
)nactivated hepatitis ! vaccines have been developed over the past decade and are now licensed
in many countries. he virus grows poorly in cell culture but yields have been improved by
adaptation and are sufficient to permit gradient purification. his virus is inactivated with
formaldehyde and the antigen adsorbed to aluminum hydro,ide and given intramuscularly. he
preparations are safe and highly immunogenic in man and have been shown to induce a
protective immune response in susceptible non&human primates and during e,tensive clinical
trials in man.
!ttenuated strains of H!V have been developed and may be useful potentially as vaccines. his
approach is attractive because live vaccines are cheaper to produce and tend to mimic the
antibody response induced by natural infection. !s with vaccine strains of polioviruses,
attenuation may be associated with mutations in the /= non&coding region of the genome which
affect secondary structure. here is also evidence that mutations in the region of the genome
encoding the non&structural polypeptides may be important for adaptation to cell culture and
attenuation. However, mar'ers of attenuation of H!V have not been identified. *,cretion in
feces occurs and reversion to virulence may also be a problem. 6n the other hand, there is also
concern that Eover&attenuatedE viruses may not be sufficiently immunogenic.
Hepatitis E
$etrospective testing of serum samples from patients involved in various epidemics of hepatitis
associated with contamination of water supplies with human feces indicated that an agent other
than H!V "or hepatitis B# was involved. *pidemics of enterically transmitted non&!, non&B
hepatitis in the )ndian subcontinent were first reported in .791, but outbrea's involving tens of
thousands of cases have also been documented in the C(($, (outheast !sia, %orthern !frica,
-e,ico and previously in )ndia. he average incubation period is slightly longer than for
hepatitis !, with a mean of si, wee's. he highest attac' rates are found in young adults, and
high mortality rates of up to 012 have been reported in women during pregnancy.
Virus&li'e particles have been detected in the feces of infected individuals by immune electron
microscopy using convalescent serum. However, such studies have often proved inconclusive,
and a large proportion of the e,creted virus may be degraded during passage through the gut. he
particles have a mean diameter of :0&:? nm. Cross reaction studies between sera and virus in
feces associated with a variety of epidemics in several different countries suggests that a single
serotype of virus is involved.
(tudies on hepatitis * virus "H*V# have progressed following transmission to susceptible non&
human primates. H*V was first transmitted to cynomolgus macaques and a number of other
species of mon'eys, including chimpan4ees, also have been infected. !ttempts to amplify the
virus by replication in cell culture have been unsuccessful.
Hepatitis * virus was cloned in .77. and the entire ;./ 'b sequence is 'nown. he organi4ation
of the genome is distinct from the Picornaviridae and the non&structural and structural
polypeptides are encoded respectively at the /= and := ends. H*V resembles the caliciviruses in
the si4e and organi4ation of its genome, as well as the si4e and morphology of the virion.
(equencing of the H*V genome has allowed the development of a number of specific diagnostic
tests. +or e,ample, H*V $%! was detected, using the polymerase chain reaction "5C$#, in fecal
samples obtained during a recent epidemic in Fanpur "%orth )ndia#. !n en4yme,
immunoabsorbent assay, which detects both )g3 and )g- anti&H*V, has been developed using a
recombinant H*V&glutathione&(&transferase fusion protein and used to detect antibodies in
sporadic cases of enterically&transmitted non&!, non&B hepatitis in children in *gypt.
5reliminary but significant progress has been made towards the development of hepatitis *
vaccine, using the trp*&C0 fusion protein. )n limited e,periments, : doses of the fusion protein,
which represents the carbo,yl two&thirds of the putative capsid protein, prevented the
development of biochemical evidence of hepatitis after challenge with wild&type virus.
Hepatitis B
Hepatitis B virus was originally recogni4ed as the agent responsible for Eserum hepatitisE, the
most common form of parenterally transmitted viral hepatitis, and an important cause of acute
and chronic infection of the liver. he incubation period of hepatitis B is variable with a range of
. to B months. he clinical features of acute infection resemble those of the other viral
hepatitides. !cute hepatitis B is frequently anicteric and asymptomatic, although a severe illness
with jaundice can occur and occasionally acute liver failure may develop.
Distinctive properties
he virus persists in / to .12 of immunocompetent adults, and in as many as 712 of infants
infected perinatally. 5ersistent carriage of hepatitis B, defined by the presence of hepatitis B
surface antigen "HBs!g# in the serum for more than si, months, have been estimated to affect
about :/1 million people worldwide. he pathology is mediated by the responses of the cellular
immune response of the host to the infected hepatocytes. >ong term continuing virus replication
may lead to progression to cirrhosis and hepatocellular carcinoma.
)n the first phase of chronicity, virus replication continues in the liver, and replicative
intermediates of the viral genome may be detected in D%! e,tracted from liver biopsies.
-ar'ers of virus replication in serum include HBV D%!, the (. proteins "HBs!g# and a soluble
antigen, hepatitis B e antigen "HBe!g# which is secreted by infected hepatocytes. )n those
infected at a very young age, this phase may persist for life but, more usually, virus levels decline
over time. *ventually, in most individuals, there is immune clearance of infected hepatocytes
associated with seroconversion from HBe!g to anti&HBe.
During the period of replication, the viral genome may integrate into the chromosomal D%! of
some hepatocytes and these cells may persist and e,pand clonally. $arely does seroconversion to
anti&HBs follow clearance of virus replication but, more frequently, HBs!g persists during a
second phase of chronicity as a result of the e,pression of integrated viral D%!.
4tructure o! the Virus
he hepatitis B virion is a ?0&nm particle comprising an electron&dense core "nucleocapsid# 0;
nm in diameter surrounded by an outer envelope of the surface protein "HBs!g# embedded in
membranous lipid derived from the host cell "+ig. ;1&:#. he surface antigen is produced in
e,cess by the infected hepatocytes and is secreted in the form of 00&nm particles and tubular
structures of the same diameter "initially referred to as !ustralia antigen#.
FIGURE 70-5 Electron &icro)raph o! seru& containin) hepatitis B virus a!ter ne)ative
stainin)' he three morphologic forms are shown intermingled in this photograph@ small
pleomorphic spherical particles 01 to 00 nm in diameterA tublar formsA ?0nm double&shelled
virus. "<?11.11#
he 00 nm particles are composed of the major surface protein in both non&glycosylated "p 0?#
and glycosylated "gp 0;# form in appro,imately equimolar amounts, together with a minority
component of the so&called middle proteins "gp :: and gp :B# which contain the pre&(0 domain,
a glycosylated // amino acid %&terminal e,tension. he surface of the virion has a similar
composition but also contains the large surface proteins "p :7 and gp ?0#, which include both the
pre&(. and pre&(0 regions. hese large surface proteins are not found in the 00 nm spherical
particles "but may be present in the tubular forms in highly viremic individuals# and their
detection in serum correlates with viremia. he domain which binds to the specific HBV receptor
on the hepatocyte is believed to reside within the pre&(. region.
he nucleocapsid of the virion consists of the viral genome surrounded by the core antigen
"HBc!g#. he genome, which is appro,imately :.0 'ilobases in length, has an unusual structure
and is composed of two linear strands of D%! held in a circular configuration by base&pairing at
the /= ends. 6ne of the strands is incomplete and the := end is associated with a D%! polymerase
molecule which is able to complete that strand when supplied with deo,ynucleoside
triphosphates.
6r)ani7ation o! the HBV Geno&e
he genomes of more than a do4en isolates of hepatitis B virus have been cloned and the
complete nucleotide sequences determined. !nalysis of the coding potential of the genome
reveals four open reading frames "6$+s# which are conserved between all of these isolates.
he first 6$+ encodes the various forms of the surface protein and contains three in&frame
methionine codons which are used for initiation of translation. ! second promoter is located
upstream of the pre&(. initiation codon. his directs the synthesis of a 0.? 'b m$%! which is
co&terminal with the other surface messages and is translated to yield the large "pre&(.# surface
proteins.
he core open reading frame also has two in&phase initiation codons. he EprecoreE region is
highly conserved, has the properties of a signal sequence and is responsible for the secretion of
HBe!g.
he third 6$+, which is the largest and overlaps the other three, encodes the viral polymerase.
his protein appears to be another translation product of the :./ 'b $%!, and is synthesi4ed
apparently following internal initiation of the ribosome.
he amino terminal domain is believed to be the protein primer for minus strand synthesis. here
is then a spacer region followed by the "$%! and D%!&dependent# D%! polymerase.
he fourth 6$+ was designated E,E because the function of its small gene product was not
'nown. However, E,E has now been demonstrated to be a transcriptional transactivator "+ig.;1&
?#.
FIGURE 70-- 4tructure an" )eno&ic or)ani7ation o! hepatitis B virus'
Host De!enses
!ntibody and cell&mediated immune responses to various types of antigens are induced during
the infection. However, these do not always seem to be protective and, in some instances, may
cause autoimmune phenomena that contribute to disease pathogenesis. he immune response to
infection with hepatitis B virus is directed toward at least three antigens@ hepatitis B surface
antigen, the core antigen, and the e antigen. he view that hepatitis B e,erts its damaging effect
on hepatocytes by direct cytopathic changes is inconsistent with the persistence of large
quantities of surface antigen in liver cells of many apparently healthy persons who are carriers.
!dditional evidence suggests that the pathogenesis of liver damage in the course of hepatitis B
infection is related to the immune response by the host.
he surface antigen appears in the sera of most patients during the incubation period, 0&9 wee's
before biochemical evidence of liver damage or onset of jaundice. he antigen persists during the
acute illness and usually clears from the circulation during convalescence. %e,t to appear in the
circulation is the virus&associated D%! polymerase activity, which correlates in time with
damage to liver cells as indicated by elevated serum transaminases. he polymerase activity
persists for days or wee's in acute cases and for months or years in some persistent carriers.
!ntibody to the core antigen is found in the serum 0&.1 wee's after the surface antigen appears,
and it is frequently detectable for many years after recovery. he titer of core antibody appears to
correlate with the amount and duration of virus replication. +inally, antibody to the surface
antigen component appears.
During the incubation period and during the acute phase of the illness, surface antigen&antibody
comple,es may be found in the sera of some patients. )mmune comple,es have been found by
electron microscopy in the sera of all patients with fulminant hepatitis, but are seen only
infrequently in nonfulminant infection. )mmune comple,es also are important in the
pathogenesis of other disease syndromes characteri4ed by severe damage of blood vessels "for
e,ample, polyarteritis nodosa, some forms of chronic glomerulo&nephritits, and infantile papular
acrodermatitis#.
)mmune comple,es have been identified in variable proportions of patients with virtually all the
recogni4ed chronic sequelae of acute hepatitis. Deposits of such immune comple,es have also
been demonstrated in the cytoplasm and plasma membrane of hepatocytes and on or in the
nucleiA why only a small proportion of patients with circulating comple,es develop vasculitis or
polyarteritis is, however, not clear. 5erhaps comple,es are critical pathogenic factors only if they
are of a particular si4e and of a certain antigen&to&antibody ratio.
Cellular immune responses are 'nown to be particularly important in determining the clinical
features and course of viral infections. he occurrence of cell&mediated immunity to hepatitis B
antigens has been demonstrated in most patients during the acute phase of hepatitis B and in a
significant proportion of patients with surface&antigen&positive chronic active hepatitis, but not in
asymptomatic persistent hepatitis B carriers. hese observations suggest that cell&mediated
immunity may be important in terminating the infection and, under certain circumstances, in
promoting immune&mediated liver damage and in the genesis of autoimmunity. !lso, evidence
suggests that progressive liver damage may result from an autoimmune reaction directed against
hepatocyte membrane antigens, initiated in many cases by infection with hepatitis B virus.
!lthough e,ogenous interferon may be effective in treating some patients with chronic hepatitis,
as yet endogenous interferon production has not been detected during the natural infection. -ore
studies to define the role of interferon are needed.
Epi"e&iolo)%
!lthough various body fluids "blood, saliva, menstrual and vaginal discharges, serous e,udates,
seminal fluid, and breast mil'# have been implicated in the spread of infection, infectivity
appears to be especially related to blood. he epidemiologic propensities of this infection are,
therefore, wide. hey include infection by inadequately sterili4ed syringes and instruments,
transmission by unscreened blood transfusion and blood products, by close contact, and by
se,ual contact. !ntenatal "rarely# and perinatal "frequently# transmission of hepatitis B infection
from mother to child may ta'e placeA in some parts of the world "(outheast !sia and Gapan#
able ;1&., perinatal transmission is very common.
Dia)nosis
Direct demonstration of virus in serum samples is feasible by visuali4ing the virus particles by
electron microscopy, by detecting virus&associated D%! polymerase, and by assay of viral D%!.
!ll of these direct techniques are impractical under general diagnostic laboratory conditions, and
specific diagnoses must therefore rely on serologic tests "able ;1&0#.
Hepatitis B surface antigen first appears during the late stages of the incubation period and is
easily detectable by radioimmunoassay or en4yme immunoassay. he antigen persists during the
acute phase of the disease and sharply decreases when antibody to the surface antigen becomes
detectable. !ntibody of the )g- class to the core antigen is found in the serum after the onset of
the clinical symptoms and slowly declines after recovery. )ts persistence at high titer suggests
continuation of the infection. Core antibody of the )g3 class persists for many years and provides
evidence of past infection.
(rotection o! hepatitis B
he discovery of variation in the epitopes presented on the surface of the virions and subviral
particles identified several subtypes of HBV which differ in their geographical distribution. !ll
isolates of the virus share a common epitope, a, which is a domain of the major surface protein
which is believed to protrude as a double loop from the surface of the particle. wo other pairs of
mutually e,clusive antigenic determinants, d or y and w or r, are also present on the major
surface protein. hese variations have been correlated with single nucleotide changes in the
surface 6$+ which lead to variation in single amino acids in the protein. +our principal subtypes
of HBV are recogni4ed@ adw, adr, ayw and ayr. (ubtype adw predominates in northern *urope,
the !mericas and !ustralasia and also is found in !frica and !sia. (ubtype ayw is found in the
-editerranean region, eastern *urope, northern and western !frica, the near *ast and the )ndian
subcontinent. )n the +ar *ast, adr predominates. But the rarer ayr occasionally may be found in
Gapan and 5apua %ew 3uinea.
he major response of recipients of hepatitis B vaccine is to the common a epitope with
consequent protection against all subtypes of the virus. +irst generation vaccines were prepared
from 00 nm HBs!g particles purified from plasma donations from chronic carriers. hese
preparations are safe and immunogenic but have been superseded in some countries by
recombinant vaccines produced by the e,pression of HBs!g in yeast cells. he e,pression
plasmid contains only the := portion of the HBV surface 6$+ and only the major surface protein,
without pre&( epitopes, is produced. Vaccines containing pre&(0 and pre&(., as well as the major
surface proteins e,pressed by recombinant D%! technology, are undergoing clinical trials.
)n many areas of the world with a high prevalence of HBs!g carriage, such as China and
(outheast !sia, the predominant route of transmission is perinatal. !lthough HBV does not
usually cross the placenta, the infants of viremic mothers have a very high ris' of infection at the
time of birth. !dministration of a course of vaccine with the first dose immediately after birth is
effective in preventing transmission from an HBe!g&positive mother in appro,imately ;12 of
cases, and this protective efficacy rate may be increased to greater than 712 if the vaccine is
accompanied by the simultaneous administration of hepatitis B immune globulin "HB)3#.
)mmuni4ation against hepatitis B is now recogni4ed as a high priority in preventive medicine in
all countries and strategies for immuni4ation are being revised and universal vaccination of
infants and adolescents is under e,amination as a possible strategy to control the transmission of
this infection. !bout :1 countries including the Cnited (tates now offer hepatitis B vaccine to all
children.
However, immuni4ation against hepatitis B is at present recommended in a number of countries
with a low prevalence of hepatitis B only to groups which are at an increased ris' of acquiring
this infection. hese 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 deficiency and
patients with malignant diseases. Viral hepatitis is an occupational ha4ard among health care
personnel and the staff of institutions for the mentally retarded, and those in some semi&closed
institutions. High rates of infection with hepatitis B occur in intravenous drug abusers, se,ually
active male homose,uals and prostitutes. )ndividuals wor'ing in high endemic areas are,
however, at an increased ris' of infections and should be immuni4ed. Houng infants, children and
susceptible persons "including travellers# living in certain tropical and sub&tropical areas where
present socio&economic conditions are poor and the prevalence of hepatitis B is high, should also
be immuni4ed.
Hepatitis B Anti$o"% Escape 8utants
5roduction of antibodies to the group antigenic determinant a mediates cross&protection against
all sub&types, as has been demonstrated by challenge with a second subtype of the virus
following recovery from an initial e,perimental infection. he epitope a is located in the region
of amino acids .0?&.?9 of the major surface protein, and appears to have a double&loop
conformation. ! monoclonal antibody which recogni4es a region within this a epitope is capable
of neutrali4ing the infectivity of hepatitis B virus for chimpan4ees, and competitive inhibition
assays using the same monoclonal antibody demonstrate that equivalent antibodies are present in
the sera of subjects immuni4ed with either plasma&derived or recombinant hepatitis B vaccine.
During a study of the immunogenicity and efficacy of hepatitis B vaccines in )taly, a number of
individuals who had apparently mounted a successful immune response and become anti&surface
antibody "anti&HBs#&positive, later became infected with HBV.
hese cases were characteri4ed by the co&e,istence of non&comple,ed anti&HBs and HBs!g, and
in :0 of ?? vaccinated subjects there were other mar'ers of hepatitis B infection. +urthermore,
analysis of the antigen using monoclonal antibodies suggested that the a epitope was either
absent or mas'ed by antibody. (ubsequent sequence analysis of the virus from one of these cases
revealed a mutation in the nucleotide sequence encoding the a epitope, the consequence of which
was a substitution of arginine for glycine at amino acid position .?/.
here is now considerable evidence for a wide geographical distribution of the point mutation in
hepatitis B virus from guanosine to adenosine at position /9;, resulting in an amino acid
substitution at position .?/ from glycine to arginine in the highly antigenic group determinant a
of the surface antigen. his stable mutation has been found in viral isolates from children several
years later and it has been described in )taly, (ingapore, Gapan, and Brunei, and from liver
transplant recipients with hepatitis B in the C(, 3ermany, and the CF who had been treated with
specific hepatitis B immunoglobulin or humani4ed hepatitis B monoclonal antibody.
he region in which this mutation occurs is an important virus epitope to which vaccine&induced
neutrali4ing antibody binds, as discussed above, and the mutant virus is not neutrali4ed by
antibody to this specificity. )t can replicate as a competent virus, implying that the amino acid
substitution does not alter the attachment of the virus to the liver cell.
Variants of HBV with altered antigenicity of the envelope protein show that HBV is not as
antigenically singular as previously believed and that humoral escape mutation can occur in vivo.
here are two causes for concern@ failure to detect HBs!g may lead to transmission through
donated blood or organs, and HBV may infect individuals who are anti&HBs positive after
immuni4ation. Variation in the second loop of the a determinant seems especially important.
-utants, variants, altered genotypes, and unusual strains are now being sought in many
laboratories.
HBV (recore &utants
he nucleotide sequence of the genome of a strain of HBV cloned from the serum of a naturally
infected chimpan4ee has been reported. ! surprising feature was a point mutation in the
penultimate codon of the precore region which changed the tryptophan codon "33# to an amber
termination codon "!3#. he nucleotide sequence of the HBV precore region from a number of
anti&HBe&positive 3ree' patients was investigated by direct sequencing 5C$&amplified HBV
D%! from serum. !n identical mutation of the penultimate codon of the precore region to a
termination codon was found in seven of eight anti&HBe positive patients who were positive for
HBV D%! in serum by hybridi4ation. )n most cases there was an additional mutation in the
proceeding codon. (imilar variants were found by amplification of HBV D%! from serum of
anti&HBe positive patients in )taly and 3reece. hese variants are not confined to the
-editerranean region. he same nonsense mutation "without a second mutation in the adjacent
codon# has been observed in patients from Gapan and elsewhere, along with rarer e,amples of
defective precore regions caused by frameshifts or loss of the initiation codon for the precore
region.
)n many cases, precore variants have been described in patients with severe chronic liver disease
and who may have failed to respond to therapy with interferon. his observation raises the
question of whether they are more pathogenic than the wild&type virus.
HBV an" Hepatocellular Carcino&a
8hen tests for HBs!g became widely available, regions of the world where the chronic carrier
state is common were found to be coincident with those where there is a high prevalence of
primary liver cancer. +urthermore, in these areas, patients with tumor almost invariably are
seropositive for HBs!g. ! prospective study in aiwan revealed that .9? cases of hepatocellular
carcinoma occurred in :,?/? carriers of HBs!g at the start of the study, but only .1 such tumors
arose in the .7,0/: control males who were HBs!g negative.
(outhern hybridi4ation of tumor D%! yields evidence of chromosomal integration of viral
sequences in at least 912 of HCCs from HBs!g carriers. here is no similarity in the pattern of
integration between different tumors, and variation is seen both in the integration site"s# and in
the number of copies or partial copies of the viral genome. (equence analysis of the integrants
reveals direct repeats in the viral genome often lie close to the virusDcell junctions, suggesting
that sequences around the ends of the viral genome may be involved in recombination with host
D%!. )ntegration seems to involve microdeletion of host sequences and rearrangements and
deletions of part of the viral genome also may occur. 8hen an intact surface gene is present, the
tumor cells may produce and secrete HBs!g in the form of 00 nm particles. 5roduction of
HBc!g by tumors is rare, however, and the core 6$+ is often incomplete and modifications such
as methylation may also modulate its e,pression. Cytoto,ic cells targeted against core gene
products on the hepatocyte surface seem to be the major mechanism of clearance of infected
cells from the liver, and cells with integrated viral D%! which are capable of e,pressing these
proteins also may be lysed. hus, there may be immune selection of cells with integrated viral
D%! which are incapable of e,pressing HBc!g.
he mechanisms of oncogenesis by HBV remain obscure. HBV may act non&specifically by
stimulating active regeneration and cirrhosis which may be associated with long&term chronicity.
However, HBV&associated tumors occasionally arise in the absence of cirrhosis, and such
hypotheses do not e,plain the frequent finding of integrated viral D%! in tumors. )n rare
instances, the viral genome has been found to be integrated into cellular genes such as cyclin !
and a retinoic acid receptor. ranslocations and other chromosomal rearrangements also have
been observed. !lthough insertional mutagenesis of HBV remains an attractive hypothesis to
e,plain its oncogenicity, there is insufficient supportive evidence.
>i'e many other cancers, development of hepatocellular carcinoma is li'ely to be a multifactorial
process. he clonal e,pansion of cells with integrated viral D%! seems to be an early stage in
this process and such clones may accumulate in the liver throughout the period of active virus
replication. )n areas where the prevalence of primary liver cancer is high, virus infection usually
occurs at an early age and virus replication may be prolonged, although the pea' incidence of
tumor is many years after the initial infection.
Hepatitis D
Delta hepatitis was first recogni4ed following detection of a novel protein, delta antigen "HD!g#,
by immunofluorescent staining in the nuclei of hepatocytes from patients with chronic active
hepatitis B.
Hepatitis delta virus "HDV# is now 'nown to require a helper function of HBV for its
transmission. HDV is coated with HBs!g which is needed for release from the host hepatocyte
and for entry in the ne,t round of infection.
wo forms of delta hepatitis infection are 'nown. )n 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. )n the second, an individual
chronically infected with HBV becomes superinfected with HDV. his may cause a second
episode of clinical hepatitis and accelerate the course of the chronic liver disease, or cause overt
disease in asymptomatic HBs!g carriers. HDV itself seems to be cytopathic and HD!g may be
directly cytoto,ic.
Delta hepatitis is common in some areas of the world with a high prevalence of HBV infection,
particularly the -editerranean region, parts of *astern *urope, the -iddle *ast, !frica and (outh
!merica. )t has been estimated that /2 of HBs!g carriers worldwide "appro,imately ./ million
people# are infected with HDV. )n areas of low prevalence of HBV, those at ris' of hepatitis B,
particularly intravenous drug abusers, are also at ris' of HDV infection.
Distinctive (roperties o! HDV
he HDV particle is appro,imately :B nm in diameter and composed of an $%! genome
associated with HD!g, surrounded by an envelope of HBs!g. he HDV genome is a closed
circular $%! molecule of .B;7 nucleotides and resembles those of the satellite viroids and
virusoids of plants and similarly seems to be replicated by the host $%! polymerase )) with
autocatalytic cleavage and circulari4ation of the progeny genomes via trans&esterification
reactions "ribosome activity#. Consensus sequences of viroids which are believed to be involved
in these processes also are conserved in HDV. Cnli'e the plant viroids, HDV codes for a protein,
HD!g.
his is encoded in an open reading frame in the antigenomic $%! but four other open reading
frames which are also present in the genome do not appear to be utili4ed. he antigen, which
contains a nuclear locali4ation signal, was originally detected in the nuclei of infected
hepatocytes and may be detected in serum only after stripping off the outer envelope of the virus
with detergent.
Hepatitis C
ransmission studies in chimpan4ees established that the main agent of parenterally acquired
non&!, non&B hepatitis was li'ely to be an enveloped virus some :1 to B1 nm in diameter. hese
studies made available a pool of plasma which contained a relatively high titer of the agent. )n
order to clone the genome, the virus was pelleted from the plasma. Because it was not 'nown
whether the genome was D%! or $%!, a denaturation step was included prior to the synthesis of
complementary D%! so that either D%! or $%! could serve as a template. he resultant cD%!
was then inserted into the bacteriophage e,pression vector lambda gt .. and the libraries
screened using serum from a patient with chronic non&!, non&B hepatitis. his approach led to
the detection of a clone "designated /&.&.# which was found to bind to antibodies present in the
sera of several individuals infected with non&!, non&B hepatitis. his clone was used as a probe
to detect a larger, overlapping clone in the same library. )t was possible to demonstrate that these
sequences hybridi4ed to a positive&sense $%! molecule of around .1,111 nt which was present
in the livers of infected chimpan4ees but not in uninfected controls. %o homologous sequences
could be detected in the chimpan4ee or human genomes. By employing a Ewal'ingE technique, it
was possible to use newly detected overlapping clones as hybridi4ation probes, in turn, to detect
further virus&specific clones in the library. hus, clones covering the entire viral genome were
assembled and the complete nucleotide sequence determined.
Dia)nosis o! HCV In!ection
(uccessful cloning of portions of the viral genome permitted the development of new diagnostic
tests for infection by the virus. (ince the original antigen was detected by antibodies in the serum
of an infected patient it was an obvious candidate for the basis of an *>)(! to detect anti&HCV
antibodies. ! larger clone, C.11, was assembled from a number of overlapping clones and
e,pressed in yeast as a fusion protein using human supero,ide dismutase sequences to facilitate
e,pression, and this fusion protein formed the basis of first generation tests for HCV infection.
he /&.&. antigen comprises amino acid sequences from the non&structural, %(?, region of the
genome and C.11 contains both %(: and %(? sequences.
)t is now 'nown that antibodies to C.11 are detected relatively late following an acute infection.
+urthermore, the first generation *>)(!s were associated with a high rate of false positive
reactions 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.
(econd generation tests include antigens from the nucleocapsid and further non&structural
regions of the genome. he former "C00# is particularly useful and antibodies to the HCV core
protein seem to appear relatively early in infection. hese second generation tests confirm that
HCV is the major cause of parenterally transmitted non&!, non&B hepatitis. $outine testing of
blood donations is now in place in many countries and prevalence rates vary from 1.0&1./2 in
northern *urope to ..0&../2 in southern *urope and Gapan. -ost of those with antibody have a
history of parenteral ris' such as a history of transfusion or administration of blood products or
of intravenous drug abuse. here is little evidence for se,ual or perinatal transmission of HCV
and it is not clear what are the natural routes of transmission.
he availability of the nucleotide sequence of HCV made possible the use of the polymerase
chain reaction "5C$# as a direct test for the genome of the virus. here is considerable variation
in nucleotide sequences among different isolates of HCV, and the /= non&coding region, which
seems to be highly conserved, is the preferred target for the 5C$.
Current data suggest that about 912 of infections with HCV progress to chronicity. Histological
e,amination of liver biopsies from asymptomatic HCV&carriers "blood donors# reveals that none
has normal histology and that up to ;12 have chronic active hepatitis andDor cirrhosis. 8hether
the virus is cytopathic or whether there is an immunopathological element remains unclear. HCV
infection is also associated with progression to primary liver cancer. +or e,ample, in Gapan,
where the incidence of hepatocellular carcinoma has been increasing despite a decrease in the
prevalence of HBs!g, HCV is now considered the major ris' factor.
here is no D%! intermediate in the replication of the HCV genome or integration of viral
nucleic acid and viral pathology may contribute to oncogenesis through cirrhosis and
regeneration of liver cells. HCV rarely seems to cause fulminant hepatitis.
Distinctive (roperties o! HCV
he genome of HCV resembles those of the pestiviruses and flaviviruses in that it comprises
around .1,111 nt of positive sense $%!, lac's a := poly! tract and has a similar gene
organi4ation. )t has been proposed that HCV should be the prototype of a third genus in the
family +laviviridae.
!ll of these genomes contain a single large open reading frame which is translated to yield a
polyprotein "of around :111 amino acids in the case of HCV# from which the viral proteins are
derived by post&translational cleavage and other modifications. he amino acid sequence of the
nucleocapsid protein seems to be highly conserved among different isolates of HCV. he ne,t
domain in the polyprotein also has a signal sequence at its carbo,yl&terminus and may be
processed in a similar fashion. he product is a glycoprotein which is probably found in the viral
envelope and is variably termed *.D( or gp:/. he third domain may be cleaved by a protease
within the viral polyprotein to yield what is probably a second surface glycoprotein, *0D%(. or
gp;1. hese glycoproteins have not been visuali4ed in vivo and the molecular si4es are estimated
from sequence data and e,pression studies in vitro. 6ther post&translational modifications,
including further proteolytic cleavages, are possible. hese proteins are the focus of considerable
interest because of their potential use in tests for the direct detection of viral proteins and for
HCV vaccines. %ucleotide sequencing studies reveal that both domains contain hypervariable
regions. )t is possible that this divergence has been driven by antibody pressure and that these
regions specify important immunogenic epitopes.
he non&structural region of the HCV genome is divided into regions %(0 to %(/. )n the
flaviviruses, %(: has two functional domains, a protease which is involved in cleavage of the
non&structural region of the polyprotein and a helicase which is presumably involved in $%!
replication. -otifs within this region of the HCV genome have homology to the appropriate
consensus sequences, suggesting similar functions. %(/ seems to be the replicase and contains
the gly&asp&asp motif common to viral $%!&dependent $%! polymerases "+ig. ;1&/#.
FIGURE 70-9 Hepatitis C Viral Geno&e'
Hepatitis C virus consists of a family of highly related but nevertheless distinct genotypes,
numbering at present B genotypes and various subtypes with differing geographical distribution,
and with a comple, nomenclature. he C, %(: and %(? domains are the most highly conserved
regions of the genome, and therefore these proteins are the most suitable for use as capture
antigens for broadly reactive tests for antibodies to HCV. he sequence differences observed
between HCV groups suggest that virus&host interactions may be different, which could result in
differences in pathogenicity and in response to antiviral therapy. )t is important, therefore, to
develop group& and virus&specific tests. he degree of divergence apparent within the viral
envelope proteins implies the absence of a broad cross&neutrali4ing antibody response to
infection by viruses of different groups.
)n addition to the sequence diversity observed between HCV groups, there is considerable
sequence heterogeneity among almost all HCV isolates in the %&terminal region of *0D%(.,
implying that this region may be under strong immune selection. )ndeed, sequence changes
within this region may occur during the evolution of disease in individual patients and may play
an important role in progression to chronicity.
Vaccine Develop&ent
5roblems in vaccine development include the sequence diversity between viral groups and the
substantial sequence heterogeneity among isolates in the %&terminal region of *0D%(..
%eutrali4ing antibodies have not been identified so far. he virus has not been cultivated in vitro
"cf. Hellow fever flavivirus, which has been cultured and from which vaccines have been
prepared#. %evertheless, approaches to vaccine development could be based on techniques used
for the development of vaccines against the Flaviviruses and Pestiviruses.
The GB Hepatitis Viruses
!bout :1 years ago, a series of transmission studies of human viral hepatitis were initiated in
small (outh !merican tamarins or marmosets, which were chosen because of their very limited
contact with man, implying that they were unli'ely to have been infected with human viruses. !
serum which was obtained on the third day of jaundice from a young surgeon "3B# with
jaundice&induced hepatitis in each of four inoculated marmosets and was passaged serially in
these animals. hese important observations remained controversial until the application recently
of modern molecular virological techniques. 5reliminary results indicate the identification of two
independent viruses, 3BV&! and 3BV&B, in the infectious plasma of tamarins inoculated with
3B.
3BV&! does not replicate in the liver of tamarins, whereas 3BV&B causes hepatitis. Cross&
challenge e,periments showed that infection with the original infectious tamarin inoculum
conferred protection from reinfection with 3BV&B but not 3BV&!. ! third virus, 3BV&C, was
isolated subsequently from a human specimen which was immunoreactive with a 3BV&B
protein. 3BV&C $%! was found in several patients with clinical hepatitis, and shown to have
substantial sequence identity to 3BV&!.
! series of studies including phylogenetic analysis of genomic sequences showed that 3BV&!,
B, and C are not genotypes of hepatitis C virus, and that 3BV&! and 3BV&C are closely related.
3BV&!DC and 3BV&B and the hepatitis C viruses are members of distinct viral groups. he
organi4ation of the genes of the 3BV&!, B, and C genomes shows that they are related to other
positive&strand $%! viruses with local regions of sequence identity with various flaviviruses.
he three 3B viruses and HCV share only limited overall amino acid sequence identity.
Diagnostic reagents were prepared with recombinant antigens, and limited testing was carried
out in groups of patients, blood donors and other selected individuals@ patients with non&!, B, C,
D, * hepatitis, multitransfused patients, intravenous drug addicts and other populations with a
high incidence of viral hepatitis. 5reliminary studies indicated the presence of antibody to each
of the 3B viruses in :2 to as many as .?2. he development and availability of specific
diagnostic reagents will establish the epidemiology of these newly identified viruses, their
pathogenic significance in man and their clinical and public health importance.
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