Introduction to the virology of human hepatitis

Journal of ViroIogical Methods,

@ Elsevier~orth-Homed

INTRODUCTION

2 (1980) 7 -15 Biomedical Press

TO THE VIROLOGY

OF HUMAN HEPATITIS

ARIE J. ZUCKERMAN De~rt~ent

ofiMedicai Microbior

and WHO ~o~~a~orat~~gCentre for Reference

on Viral hepatitis, London School of Hygiene and Tropical Medicine, London

Human

viral hepatitis

and Research

WCIE 7HT, U.K.

has emerged as a major public health problem

occurring

in all

parts of the world. The general term viral hepatitis refers to infections caused by at least three different viruses: hepatitis A (infectious or epidemic hepatitis), hepatitis B (serum hepatitis) and the more recently identified form of hepatitis, non-A, non-B hepatitis. Based on epidemiological, clinical and more recent cross-challenge transmission experiments in chimpanzees, evidence has been obtained that there are at least two antiger&ally different non-A, non-B hepatitis viruses. Acute viral hepatitis is a generalised or systemic infection with particular emphasis on inflammation of the liver. The clinical picture of the infection ranges in its presentation from inapparent or subclinical infection, slight malaise, mild gastrointestinal symptoms and the anicteric form of the disease, acute icteric illness, severe prolonged jaundice to acute fulminant hepatitis. The incidence of individual symptoms and signs varies, therefore, both in different outbreaks and in sporadic cases. In addition, hepatitis B and non-A, non-B hepatitis may progress to chronic liver disease, which may be severe, and there is substantial evidence of a cIose association between hepatitis B virus and primary liver cancer. Hepatitis A and hepatitis B can now be differentiated for antigens and antibodies associated with these infections. non-B hepatitis are under development.

by specific laboratory tests Laboratory tests for non-A,

HEPATITIS A VIRUS

Hepatitis A virus is spread by the faecal-oral route, most commonly by person to person contact, and infection occurs readily in conditions of poor sanitation and overcrowding. The virus has now been identified as a small unenveloped cubic particle measuring 25-28 nm in diameter, and its principal characteristics are listed in Table 1. The natural host is man and the infection can be transmitted experimentally to certain species of marmosets and to susceptible chimpanzees. The virus has recently been cultivated in primary monolayer, explant and continuous cell lines of primate origin.

8

TABLE

1

Biophysical

and biochemical

characteristics

Size

of hepatitis

A virus

25-28

Capsid

cubic

assembly

cytoplasm

Virion Capsid

nm

symmetry

unenveloped

Density

in CsCl

Sedimentation Nucleic

1.34 g/ml constant

in sucrose

160 S

acid single-stranded

type mol. wt. Polypeptides

RNA

1.9 x lo6 (mol. wt.)

VP1

33,000-36,000

VP2

26,000-31,000

VP3

22,000-26,000

VP4

9,000-

14,000

Stability temperature

4°C -20°C

stable for weeks-months to -70°C

5O”C, 60 min

partially

60”C,lOh

mostly stable

acid, pH 3 1

inactivated inactivated

inactivated

20%, 4”C, 24 h

formalin

stable

6O”C, 60 min 1 OO”C, 5 min ether,

stable for years

stable

: 4000, 37”C, 72 h

inactivated

UV irradiation

inactivated

chlorine,

inactivated

Although

1 mg/l, 30 min

only one serotype has been identified

in volunteers

infected experimentally

with hepatitis A, in patients from different outbreaks of hepatitis in different geographical regions, in sporadic cases of hepatitis and in naturally and experimentally infected chimpanzees, there is evidence now that some strain differences may occur and that the antigenic differences may be marked. The virus is present in faeces of infected persons up to 14 days before the onset of symptoms and disappears rapidly during the acute phase of illness. Prolonged virus excretion and a persistent carrier state have not been demonstrated. Specific laboratory tests for hepatitis A antigen and antibodies include immune electron microscopy, immune adherence haemagglutination, radioimmunoassay and enzyme immunoassay (ELBA). Hepatitis A antibody munoassay in the early usually persists for many body develops very early

is always demonstrable by radioimmunoassay or enzyme-imphase of the illness and titres increase rapidly. The antibody years and possibly for life, and indicates immunity. Since antiin the infection, serological diagnosis can be established only

9

by titrations

of serial samples of serum, or by the demonstration

of the IgM class, which is the simplest and most economical

of hepatitis A antibody

method

of establishing

the

diagnosis of recent infection. Control gienic

of the infection

measures

is difficult.

and the sanitary

Spread of hepatitis

disposal of excreta.

A is reduced by simple hy-

Normal human

immunoglobulin

containing hepatitis A antibody may prevent or attenuate a clinical illness, while not always preventing the infection. Now that successful cultivation of hepatitis A virus in tissue culture has been reported, vaccines against this common infection are under development. HEPATITISBVIRUS Infection with hepatitis B virus leads to the appearance in the plasma during the incubation period of a specific antigen, hepatitis B surface antigen (orig~~ly referred to as Australia antigen) some two to eight weeks before biochemical evidence of liver damage or the onset of jaundice. The antigen persists during the acute illness and is usually cleared from the circulation during convalescence. Next to appear in the circulation is DNA polymerase associated with the core or nucleocapsid of the virus and e antigen, again preceding serum am~otr~sferase elevations. Antibody to the core is found in the serum two to four weeks after the appearance of the surface antigen and it is usually detectable during the early acute phase of the illness persisting after recovery. The next antibody to appear in the circulation is directed against the e antigen. Antibody to the surface antigen component is the last to appear, late in convalescence. More recently, precipitating antibodies reactin g with specificities on the complete virus particle have been described. These antibodies may be relevant to the clearance of c~c~ating hepatitis B virions and the termination of acute infection, and their absence in all but one of the patients with chronic active hepatitis examined might explain why the infection persists in such patients. Cell-mediated immunity appears to be important in terminating hepatitis

B infection

and, under certain circumstances,

in promoting

liver damage and in

the genesis of auto~m~ity. A carrier state of hepatitis B virus, which may be life-long, becomes established in some patients. Such a carrier state may be associated with liver damage ranging from minor changes in the nucleus of the hepatocytes to chronic active hepatitis and cirrhosis. A number of factors have been identified which increase the risk of developing the carrier state. The carrier state is commoner in males, more likely to follow infection acquired in childhood and more likely to occur in patients with natural or acquired immune deficiencies. Seroepidemiological surveys reveal that there is a large reservoir of persistent carriers of hepatitis B virus in the world, estimated to number about 176 million. The carrier state is characterised serologically by persistence of hepatitis B surface antigen and absence of free surface antibody. Core antibody is present often in high titre and there are reports that core antibody of the IgM class remains detectable. In some carriers hepatitis B DNA polymerase activity remains elevated, not infrequently fluctuating in

10

titre, and e antigen persists, whereas in others hepatitis B DNA polymerase is not detectable and anti-e is found. Hepatitis B e antigen has been reported to be commoner in young than in adult carriers, while the prevalence of anti-e appears to increase with age. The surface antigen complete

virion.

represents

excess virus-coat

The core of the virus particle

material. contains

The 42 nm particle is the

DNA-dependent

DNA poly-

merase, closely associated with a DNA template. The molecular weight of the DNA is 1.8-2.3 X 106. The DNA structure has been character&d by gel electrophoresis and restriction enzyme cleavage and shown to be a double-stranded and circular structure, approx~ately 36,000 nucleotides in length, cont~~g 2100 nucleotides. The endogenous DNA polymerase gap. The entire DNA of hepatitis

a s~gle-str~ded reaction appears

gap of 600to repair the

B virus has been cloned in E. coli with expression of viral

antigenic proteins. Laboratory tests for hepatitis 3 surface antigen and surface antibody Many laboratory methods of varying sensitivity and specificity are now available for detecting the surface antigen and its antibody. The most sensitive methods for detection of surface antigen and antibody are radioimmunoassays, including solid-phase radioimmunoassay and radioimmunoprecipitation. These two types of radioimmunoassay differ primarily in the techniques used for separating bound from free radiolabelled reagents. More recently, enzyme-linked immunosorbent assay (ELISA or enzyme-immunoassay) has been adapted for detection of surface antigen and surface antibody. This technique has been shown to have a sensitivity similar to that of radioimmunoassay. Reactions obtained with the highly sensitive techniques must be confirmed as specific by means of neutr~~tion or blocking with unlabelled surface antibody or surface antigen as approp~ate . Laboratory tests for hepatitis B core antigen and antibody Hepatitis

B core antibody

is a useful and consistent

marker of infection

with hepatitis

B virus, and persisting high titres suggest continuing viral replication, although this antibody remains detectable long after the cessation of viral replication. Various techniques are available for demonstrating core antibody ranging from immunofluorescence to radio~unoas~y. Core antibody may be found in serum in the absence of other markers of hepatitis B virus, and the possibility that under some c~c~stances blood from such individuals may contain infective virus in the absence of excess production of surface antigen is under investigation. Reference has been made to the possible value of the assay of IgM core antibody. Free core antigen has not been detected in the circulation. Fluorescent antibody techniques and thin-section electron microscopy demonstrate clearly the core antigen in the nuclei of hepatocytes. Core antibody is readily shown in the serum by indirect immunofluorescence using as a substrate cryostat sections of

11

liver obtained core antigen

at autopsy particles.

from patients

This technique

with hepatocytes

detection of core antibody are being increasingly immunoassay is under development. Tests for detection

containing

is widely used. Radioimmune introduced

a large number procedures

of

for the

into practice and enzyme

ofhepati~s Be antigen and anti-e

The presence of e antigen in the serum appears to correlate with the replication of hepatitis virus in the host. There is a highly significant correlation between e antigen, DNA polymerase activity and circulating complete virus particles and therefore infectivity. Anti-e, on the other hand, signifies reduced or relatively low infectivity, but virus production has been reported in the presence of antibody to e antigen in some carriers, Several serological methods are now available for detection of e antigen and its antibody. In order of increasing sensitivity these include ~munodiffusion, rheophoresis, ~mune adherence haema~ut~ation, passive haemagglut~ation and, more recently, enzyme-~munoassay and solid-phase radio~munoassay. Hepatitis B virus DNA polymerase A close correlation has been demonstrated between the presence of a DNA polymerase with unique properties and complete hepatitis B virus particles. The detection of specific DNA polymerase activity is therefore a useful marker of virus replication. This enzyme activity is often found early in the course of infection when large numbers of virus particles are present; it correlates with e antigen, and it persists in some persistently infected individuais with continuing viral replication. This rather specialised assay, therefore, might complement other screening serological procedures for early diagnosis, particularly

in high-risk settings such as haemodialysis

DNA polymerase

reaction

units. Another

is that the assay may discriminate

fective virus particles. The polymerase

between

application

of the

complete

and de-

activity is also being used for the evaluation

of a

number of antiviral compounds for the treatment of persistent hepatitis B infection. Frequent fluctuations in the enzyme levels are observed in serial samples from persistently infected patients and such variations should be taken into account while monitoring the effect of treatment. Finally, because of the close association of the DNA polymerase with the core of the virus, the reaction has been employed for the preparation of radiolabelled core antigen for use as reagents for assay of core antibody. The patterns of serological markers of infection with hepatitis B virus are outlined in Table 2. Immunopathogenesis and treatment of chronic liver disease associated with hepatitis B Possible ~unopathogenic mech~s~ involved in the progression of acute hepatitis B to chronic liver disease have been reviewed recently. Briefly, after replication and

12

TABLE

2

Interpretation HBsAg

of serological

HBeAg

tests for hepatitis

anti-HBe

anti-HBc IgM

f + + + +

+ + + +

anti-HBs

Interpretation

1gC

+I-

+ + f +

- i

+I-

+

+

Convalescence

t

+

Recovery

f

-

Recovery

with loss of detectable

_

-

Infection

with

detectable

HBsAg

+ + + +

B

+

+

Incubation Acute

period

hepatitis

Persistent

carrier

Immunisation. antigen from

B

without infection

state

hepatitis Repeated

infection, with

anti-HBs

B virus without exposure

to

or recovery

loss of detectable

anti-HBc

assembly of the virus in the liver cell, the virus is released from the cell and viral-associated antigens appear on the surface of infected hepatocytes. T-lymphocytes recognising these new antigenic determinants destroy the infected hepatocytes. The T-cells also stimulate B-cells to produce antibody to liver specific protein, a normal constituent of the hepatocyte cell membrane. The resulting antibody-dependent cell-mediated K-cell reaction against normal liver membrane antigens contributes to necrosis of the hepatocytes. Virus released from the cells stimulates ponents and the antibody complexes with moved by the reticuloendothelial system. ceases as well as the ‘helper effect’ for the lipoprotein, which together with a normally

antibody

directed against the viral com-

the virus. The immune complex is then reWith removal of the virus, T-cell reaction production of autoantibody to liver-specific functioning suppressor T-cell system leads to

cessation of liver cell necrosis and recovery from acute hepatitis. In surface antigenpositive chronic active hepatitis, as a result of a quantitative or qualitative defect in the production of antibody to the virus, the virus is not cleared and in turn infects other hepatocytes, and both mechanisms of immunological liver cell damage continue. In patients who progress to surface antigen-negative chronic active hepatitis, the production of antibodies directed against the virus is adequate and the virus is cleared. In these patients the defect probably lies in suppressor T-cell function, which is unable to switch off the autoimmune reaction against liver-specific protein. A further membrane antigen, liver membrane antigen, seems to be involved. The disease-specific autoantibody, liver membrane antibody, can be demonstrated in hepatocyte membranes. Genetic factors may also be involved, as indicated by an association with histocompatibility antigens HLA-Al

13

and B8, the associated high titres of autoantibodies frequency

of serological abnormalities

in first-degree

and viral antibodies,

and the increased

relatives.

Reviews on many studies show a highly significant occurrence of markers of hepatitis B infection in patients with primary hepatocellular carcinoma. Hepatitis B is ubiquitous in areas of the world where macronodular cirrhosis and primary liver cancer are common. It is possible, therefore, that patients with hepatocellular carcinoma are unduly susceptible to hepatitis B infection and to the development of the persistent carrier state. The question has been asked whether the infectious agent is the driver or the passenger? It has been suggested that an important factor in the possible aetiological association between hepatitis B infection and liver cell carcinoma may well lie in an early age of exposure to infection and high prevalence of persistent hepatitis B carriers. Indeed, in geographical regions where the prevalence of macronodular cirrhosis and primary liver cancer is high, infection with hepatitis B virus and the carrier state occur most frequently in early life, before the defence immune mechanisms have fully developed, and as many as 20% or more of the apparently

healthy

population

may be carriers. It seems likely,

therefore, that persistent hepatitis B infection occurs before the onset of chronic liver damage. Another possibility is that persistent infection with hepatitis B virus leads to cirrhosis and that carcinoma then arises from regenerative nodules by mechanisms in which the virus is not involved. However, this sequence does not explain liver cancer associated with persistent hepatitis B infection in about 20-30% of patients in the absence of cirrhosis. More recent laboratory studies have shown the presence of viral DNA base sequences in host cells derived from patients with persistent hepatitis B infection and chronic active hepatitis, and also from patients with hepatocellular carcinoma. These findings are consistent with hepatitis B viral DNA being integrated into host chromosomal DNA molecules. In addition, several cell lines which produce hepatitis B surface antigen in culture have been derived from primary liver cell tumours. The actual mechanisms involved in the pathogenesis of hepatocellular carcinoma remain unknown. It is possible that liver cancer is the cumulative result of several cofactors including genetic, nutritional and hormonal factors, mycotoxins, chemical carcinogens and other environmental factors, and that hepatitis B virus acts either as a carcinogen or as a cocarcinogen in persistently infected hepatocytes. Antiviral therapy Interferon is still being evaluated for the treatment of chronic hepatitis B in several centres. A number of reports indicate that the administration of human leucocyte interferon both in man and in persistently infected chimpanzees has an inhibitory effect on replication of hepatitis B virus. Ribavirin (Virazole) is a synthetic nucleoside analogue of guanosine and it has a broadspectrum, though modest, antiviral activity. Several clinical studies with ribavirin appeared to yield encouraging

results in acute hepatitis B as well as in persistent

carriers. However,

14

no significant chimpanzee

changes were noted in more recent studies either in patients

or in several

carriers of hepatitis B virus.

Adenine arabinoside (ara-A, vidarabine) acts as an analogue of the deoxyribonucleoside of adenine, and it has been shown to have significant antiviral activity against several DNA viruses. Several small studies have so far been carried out in patients with chronic liver disease. In most patients there was an immediate loss of DNA polymerase followed by a rebound in many of the patients when treatment was stopped. A similar temporary effect was found in infected chimpanzees treated with adenine arabinoside. Further trials with this potent drug are underway. i%e j7avonoid, (+)-cyanidanol-3. The use of this drug for the treatment of acute viral hepatitis has shown what appear to be marginal and mainly subjective beneficial effects. PASSIVE

AND ACTIVE

IMMUNISATION

AGAINST

HEPATITIS

B

Passive immunisation The availability of laboratory tests for hepatitis B surface antibody has allowed the selection of plasma for the preparation of high titre hepatitis B immunoglobulin. In general terms, it seems that hepatitis B immunoglobulin may confer temporary passive immunity and the most useful applications are: post-exposure prophylaxis after a single acute exposure to hepatitis B virus, such as when blood containing surface antigen is accidentally inoculated, ingested orally or splashed onto mucous membranes; and pre-exposure prophylaxis in endemic settings, such as haemodialysis units, where transmission of hepatitis B virus is known to occur and where preventive hygienic measures cannot be implemented.

There is also some preliminary

evidence that the administration

of hepatitis

B immunoglobulin to neonates at a risk of acquiring hepatitis may prevent the infection if the immunoglobulin is given at birth or within 48 h and subsequently at intervals for some months. Active immunisation There is an urgent need for a hepatitis B vaccine for groups which are at risk of acquiring the infection. Since the virus has not been cultivated in tissue culture, conventional vaccines cannot be prepared. Attention has therefore been directed to the use of purified 20-25 nm hepatitis B surface antigen spherical particles as the immunogen, the separated polypeptides of these particles with antigenic activity, to the use of antigen particles or the separated polypeptides from hepatitis B surface antigen-producing cell line from a human hepatoma cell line, and to the potential use of hepatitis B proteins expressed in clones of E. coli.

15

NON-A,

NON-B HEPATITIS

There is evidence antigenically

that there are at least two new forms of viral hepatitis

unrelated

to hepatitis

type A or type B. The infections

which are

have been trans-

mitted experimentally to chimpanzees. Non-A, non-B hepatitis is the most common form of hepatitis occurring after blood transfusion and the administration of certain plasma derivatives in some areas of the world. This type of infection also appears to account for some lo-20% of all recognised cases of acute sporadic hepatitis in adults. Specific laboratory tests are under development and it is evident that progress is being made towards the identification of these important

hepatitis viruses of man.

REFERENCES

Gardner

P.S. and C.R. Howard

Sherlock, Vyas,

G.N.,

S.N. Cohen

Symposium, Zuckerman,

(eds.),

1978, J. Med. Virol.

S. (ed.), 1980, in: Clinics in Gastroenterology,

A.J.,

University

and

R. Schmid

of California

(Franklin

1978,

Viral

Hepatitis.

Press, Philadelphia)

London)

Proceedings

p. 1. of the Second

p. 1.

1978, Bull. W.H.O. 56, 1.

Zuckerman,A.J.,

1979, Br. Med. J. 2,84.

Zuckerman,

and C.R.

A.J.

(Eds.),

3,1.

Vol. 9 gY.B. Saunders,

New York) p. 1.

Howard,

1979,

Hepatitis

Viruses

of Man (Academic

Press,

London

and