GASTROENTEROLOGY 1993;104:955-963

The Discovery of the Hepatitis Viruses

ROBERT H. PURCELL
Heoatitis Viruses Section, Laboratow of Infectious Diseases. National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Beihesda, Maryland

he hepatitis viruses are an apt topic for an essay traduced in the United States as synonymous with in-
T commemorating the founding of the journal GAS- fective hepatitis. “Homologous serum jaundice” was
TROENTEROLOGY because the modern era in hepatitis introduced in a memorandum from the Ministry of
research began contemporaneously with the founding Health in Great Britain in 1943 to describe cases of
of the journal. hepatitis associated with injection of whole blood,
plasma, or serum. At about the same time in the
The “Silver Age” of Hepatitis United States and Great Britain, “serum hepatitis” was
Research used synonymously with homologous serum jaundice.
Viral hepatitis had a devastating effect on U.S. The terms “hepatitis A” for infectious hepatitis and
and foreign troops during World War II. Particularly “hepatitis B” for serum hepatitis were introduced by
troublesome were epidemics of hepatitis occurring MacCallum in 1947.
among recipients of vaccines, especially yellow fever These earlier volunteer studies in adults were con-
virus vaccine, which had been stabilized with pooled firmed and extended in volunteer studies in mentally
human serum. In addition, hepatitis following therapy retarded children at the Willowbrook State School in
with fresh frozen human plasma and following trans- New York during the latter half of the 1950s and the
fusion of whole blood was a serious emerging problem. first half of the 1960s by Saul Krugman of New York
This, coupled with the recognition of the existence of University and his colleagues. Both hepatitis viruses
community-acquired hepatitis unrelated to exposure were highly endemic in the school, and most children
to blood, provided the stimulus for a series of con- became infected naturally shortly after admission if
trolled studies in volunteers on the nature and trans- not entered into the studies. The studies at Willow-
missibility of presumed viral hepatitis. brook confirmed the existence of two distinct hepatitis
A major contribution of the volunteer studies, as viruses, distinguishable on epidemiological, clinical,
well as epidemiological studies of the time, was the and immunological grounds. They also showed an ap-
recognition of two types of viral hepatitis differing in parent lack of heterologous immunity between the two
primary route of infection and period of incubation. agents, and these data provided an explanation for sec-
These volunteer studies, principally among prisoners, ond cases of naturally occurring jaundice among resi-
yielded important information on some of the charac- dents of the Willowbrook State School (third cases
teristics of the two putative viruses, and methods for were never observed). Second cases of jaundice could
their inactivation and/or removal from blood prod- be duplicated in volunteers under experimental condi-
ucts are still in use today. Before that time, what we tions following inoculation with virus-containing
now know as viral hepatitis was known as “catarrhal serum. The two strains of hepatitis virus, obtained
jaundice.” In 1937, Findlay and MacCallum, in their from the serum of patient M.S. during his first and
first report from England on acute hepatitis among second bouts of hepatitis, were designated “MS-l” and
recipients of yellow fever vaccine, used the term “MS-2.” They were subsequently shown to be hepatitis
“common infective hepatic jaundice” as synonymous A virus (HAV) and hepatitis B virus (HBV), respec-
with catarrhal jaundice and in 1939 suggested the term
“infective hepatitis,” first suggested in 19 12 by Cock- 0 1993 by the American Gastroenterological Association
ayne. In 1943, the term “infectious hepatitis” was in- 0016~5085/93/$3.00
956 PURCELL
tively, and reagents from these experiments, carefully
collected and stored by Krugman, played an important
role in the certification (in some cases, refutation) of
new assays for the viruses and antibodies directed
against them. Important contributions of the Willow-
brook study included, in addition to delineation of hep-
atitis A and hepatitis B, demonstration of passive im-
munoprophylaxis against both diseases and the first
demonstration of active immunoprophylaxis against
hepatitis B. This last was a serendipitous observation:
Krugman had attempted to determine the heat stabil-
ity of HBV by diluting the virus-containing serum and
boiling it for 1 minute. The heated solution was subse-
quently inoculated into volunteers to assess residual
infectivity, and when hepatitis did not occur in the
volunteers, they were challenged with unheated serum
containing the virus to establish their susceptibility.
To his surprise, many of the recipients of the heated
serum were protected against challenge with live virus.
This observation was an important stimulus to the de-
velopment of hepatitis B vaccine.
However, the volunteer studies were not without
controversy. In one study, deaths occurred among
adult volunteers and led to the termination of most of
the other volunteer studies. Also, changes in social
values and mores led to conflicts over the ethics of the
studies in mentally retarded children and eventually
forced their termination, despite claims by Krugman
and others that the studies met all of the ethical stan-
dards in place at the time. It is easy to condemn these
studies from the perspective of 1993, but such retro-
spective applications of “political correctness” are dan-
gerous: undoubtedly many of our present acceptable
policies will be seen as unacceptable in the future when
viewed through a different prism.
Animal Models
Fortunately, the development of animal models
for the study of viral hepatitis paralleled the demise of
the volunteer studies. As early as 1961, viral hepatitis
among caretakers of nonhuman primates was recog-
nized. However, attempts to transmit viral hepatitis to
a variety of animal species, including nonhuman pri-
mates, yielded negative, equivocal, or nonreproduc-
able results, even when inoculation took place shortly
after capture to prevent inadvertent transmission in
captivity. It was not until 1967 that convincing evi-
dence of transmission of viral hepatitis to nonhuman
primates (marmoset monkeys) was published by the
late Friedrich Deinhardt and his colleagues at Presby-
terian-St. Luke’s Hospital in Chicago. The most im-
pressive data were obtained with an inoculum consist-
GASTROENTEROLOGY Vol. 104. No. 4
ing of acute-phase serum from a surgeon (G.B.) who
presumably acquired hepatitis from a patient. The GB
agent was subsequently characterized and believed to
be HAV. However, controversy over the marmoset
model arose when Wade Parks and Joseph Melnick of
the Baylor College of Medicine in Houston, attempt-
ing to confirm these findings, reported the detection of
hepatitis indistinguishable from GB hepatitis among
uninoculated marmosets and proposed that the GB
agent was, in fact, a latent virus of marmosets. In an
attempt to resolve this controversy, Deinhardt and his
colleagues inoculated marmosets with coded speci-
mens obtained from adult prisoner volunteers at the
Joliet prison in Joliet, Illinois, who had been experi-
mentally infected with Krugman’s MS-1 strain of
HAV by Joseph Boggs, of Northwestern University
Medical School in Chicago in studies sponsored by the
U.S. Army. Deinhardt’s group successfully broke the
code in this experiment, thereby establishing that mar-
mosets were, indeed, suitable for transmission studies
of HAV. Ironically, the GB agent was subsequently
shown to be serologically distinct from HAV. The na-
ture of the GB agent remains an enigma: it does not
appear to be any of the five recognized human hepati-
tis viruses.
Hepatitis B virus was first successfully transmitted
to chimpanzees in a collaboration between James
Maynard’s hepatitis laboratory in Phoenix (a labora-
tory of the Center for Disease Control) and my labora-
tory at the National Institutes of Health (NIH), Be-
thesda, Maryland, in the early 1970s. The inoculum
used was the MS-2 strain of HBV, supplied by Krug-
man. At about the same time, Lewellys Barker of the
Division of Blood and Blood Products of the Food and
Drug Administration, Bethesda, Maryland, and his col-
leagues were performing similar studies, and these two
reports established the chimpanzee as a suitable animal
model for the study of HBV. The transmission of
HAV to chimpanzees was reported in 1975 by Jules
Deinstag, who was a fellow in my laboratory, and by
Maynard’s group in separate studies.
Success in the transmission of HAV and HBV to
primates was followed by success in the transmission
of non-A, non-B (NANB) hepatitis virus to chimpan-
zees in 1978 by Harvey Alter (NIH), Edward Tabor
(Food and Drug Administration), Blaine Hollinger
(Baylor College of Medicine), and Alfred Prince (New
York Blood Center, New York, NY). Similarly, hepati-
tis D virus (HDV) was transmitted to chimpanzees in
1980 and hepatitis E virus (HEV) was transmitted to
cynomolgus monkeys in 1983 (see below). Eventually,
HAV and HEV were transmitted to several primate
April 1993 DISCOVERY OF HEPATITIS VIRUSES 957

species. However, only the chimpanzee was found to were actually the hepatitis B virions. The term “Aus-
be susceptible to all five human hepatitis viruses. We tralia antigen” was replaced with “hepatitis B surface
have prepared pools, of known infectivity, of each of antigen” (HBsAg) to denote its association with the
the hepatitis viruses as they have been discovered, and envelope of HBV.
these have been made available to the scientific com- Research on viral hepatitis began for me when I was
munity for challenge studies, characterization, etc. a fellow under Robert Chanock in the Laboratory of
Infectious Diseases at the NIH. We established a pro-
The “Golden Age” of Hepatitis spective study of transfusion-associated hepatitis, then
Research a serious problem among patients undergoing open-
If the period from 1943 to 1963 can be called heart surgery at the NIH, and this study was later
the Silver Age of hepatitis research, the period from merged with a similar one initiated by Paul Holland of
1964 to the present can be designated the Golden Age, the Clinical Center blood bank at the NIH and subse-
for it was in 1964 that Baruch Blumberg discovered quently expanded into the prospective study managed
the Australia antigen while studying genetic polymor- by Harvey Alter in the blood bank. This study has been
phisms of serum proteins at the NIH. Harvey Alter, a in almost continuous operation since 1964 and has
coauthor of the original paper and a fellow at the NIH been immensely productive. It was during the forma-
at the time, describes the discovery as follows: “The tive years of this program that I met and worked with
chronological events surrounding the Australia anti- John Walsh, another of the essayists in this series,
gen stand out as a monument to non-directed medical whose interest in gastroenterology was piqued by his
research and as a tribute to investigative perseverance. experience in the prospective transfusion-associated
This tale of serendipity began in the mid-1960’s when hepatitis study.
the Australia antigen was first reported by a geneticist By the late 196Os, a number of studies, including the
who had been seeking new inherited polymorphisms NIH study, had established that HBsAg-positive trans-
among serum proteins, by a blood banker looking for fused blood carried a high risk of transmitting hepatitis
non-cellular causes of febrile, non-hemolytic transfu- to the recipient. Despite the evidence, there was con-
sion reactions and by a technologist destined to be- siderable controversy over whether testing of blood
come a commercial airline pilot. A research interest in donors for HBsAg should be mandated. Nevertheless,
viral hepatitis was conspicuously absent in this investi- between late 1970 and mid- 1972, the American blood-
gative team. The significance of the Australia antigen, banking industry progressively expanded the testing in
found when the serum of an Australian aborigine response to public and legal pressure. However, it was
formed a precipitin line with the serum of a multiply- difficult to measure the impact of this move because a
transfused hemophiliac, was, at that time, unknown.“’ primary source of contaminated blood, commercial
Blumberg persistently pursued the antigen, both be- blood donors, was also removed from the donor popu-
fore and after he moved to the Institute for Cancer lation simultaneously. This was probably more impor-
Research in Fox Chase, Pennsylvania, and he and his tant than the screening for HBsAg, because it had al-
colleagues discovered (1) a low prevalence of Australia ready been shown in several prospective studies of
antigen in the United States but a high prevalence in transfusion-associated hepatitis that only one third to
individuals from developing countries, especially in two thirds of such disease was associated with HBV
Africa and Asia; (2) associations of Australia antigen infection. It was assumed that the remainder was
with patients with leukemia or Down’s syndrome; and caused by the hepatitis A virus, although the epidemi-
(3) an association with viral hepatitis, the common ology of HAV argued against it.
factor that eventually was shown to tie the other associ-
HAV
ations together. Kazuo Okochi (Tokyo University Hos-
pital) independently studied the antigen in the blood Buoyed by successes in the development of new
of transfusion recipients who developed hepatitis in sensitive assays for HBV, a number of research groups
Japan, and the work of Okochi and Alfred Prince, Da- launched a search for the hepatitis A virus. This led to
vid Gocke of Columbia University, and Paul Holland several false starts and premature reports of the isola-
of the NIH firmly established that the Australia anti- tion or detection of HAV or antibody to it, prompting
gen was associated specifically with hepatitis B. In Thomas Chalmers, then at the NIH, to comment upon
1970, David Dane and colleagues at the Middlesex our report of the successful visualization of HAV:
Hospital in London first described the 42-nm particles “Well, I see that the hepatitis A virus has been identi-
that came to be known as “Dane particles” and that fied . . . again.” The most compelling reason for
958 PURCELL GASTROENTEROLOGY Vol. 104, No. 4

searching for HAV in our Laboratory of Infectious new putative agent “non-A, non-B hepatitis virus” be-
Diseases was the discovery by another member of the cause we believed that there may have been more than
laboratory, Albert Kapikian of the Norwalk agent, the one such agent and we did not wish to repeat the mis-
most important cause of adult epidemic diarrhea. Ka- takes made previously in misinterpreting the epidemi-
pikian had learned the technique of immune electron- ology of HAV and HBV, and because we were confi-
microscopy during a 6-month sabbatical leave with dent that serological tests for NANB hepatitis virus
June Almeida of the Royal Postgraduate Medical would be rapidly forthcoming. So much for our pre-
School in London. The success of this technique in science! Alfred Prince, based on his own analysis of
visualizing the Norwalk agent, albeit after a very ex- transfusion-associated hepatitis cases and knowledge
tensive search, made the search for a virus with a simi- of our serological exclusion of HAV, also reported the
lar epidemiology seem worthwhile. Stephen Fein- existence of a third hepatitis virus and called it hepati-
stone, then a fellow in my group, learned the tis C virus (HCV), but it was not until the recent molec-
technique of immune electronmicroscopy (IEM) from ular identification of the virus (see below) that the
Kapikian, and the two of them spent many hours, name “hepatitis C virus” was resurrected. Jules Diens-
days, and eventually weeks searching for the virus in tag expanded the study of NANB transfusion-asso-
feces obtained from patients with viral hepatitis in the ciated hepatitis and, with James Mosley of the Uni-
South Pacific by Leon Rosen, a former member of the versity of Southern California in Los Angeles,
Laboratory. One time we thought we had found the documented NANB hepatitis in patients with spo-
virus in feces from patients with hepatitis on one of radic, community-acquired disease in the region, as
the islands, but this particle proved to be an artifact. had Victor Villarejos (Louisiana State University and
Eventually the virus was found by Feinstone in feces San Jose, Costa Rica) and Stephen Locarnini and Ian
supplied by Mark Conrad (Walter Reed Army Institute Gust (Fairfield Hospital, Melbourne, Australia) in
of Research, Washington, D.C.) from cases of experi- studies in Costa Rica and Australia, respectively.
mental hepatitis A in the prisoner volunteers who had
been inoculated with the MS-1 strain of HAV at the
HDV
Joliet Prison. In the mid 197Os, Mario Rizzetto, a young gas-
We learned subsequently that Philip Provost and troenterologist at the Ospedale Molinette in Turin,
Maurice Hilleman of the Merck Institute for Therapeu- Italy, discovered by immunofluorescence microscopy
tic Research in West Point, Pennsylvania, had made a “new” antigen in the nuclei of hepatocytes in liver
significant progress in the study of hepatitis A and biopsy specimens from Italian patients with chronic
were close to discovering the virus. Their work, with type B hepatitis. The antigen resembled hepatitis B
William Miller of the institute, eventually led to the core antigen (HBcAg) and indeed was originally
development of new serological techniques and the thought to be HBcAg until discrepant results obtained
isolation of HAV in cell culture, a key step to the with different fluorescein-labeled convalescent sera
development of vaccines. revealed it to be antigenically distinct from HBcAg but
related in some way to chronic infection with hepatitis
NANB Hepatitis Virus B virus. The antigen was never found in biopsy speci-
Immune electron microscopy, the method by mens from patients with HBsAg-negative hepatitis,
which HAV was visualized, also provided a serological and only a proportion of patients with HBsAg-positive
technique, albeit unwieldy and difficult, for detecting hepatitis had the antigen or antibody to it. The new
antibody to HAV (anti-HAV). One early application specificities were called delta antigen and anti-delta,
of this serological method was to examine sera from respectively. Rizzetto received a fellowship from the
patients with non-B transfusion-associated hepatitis NIH to study delta antigen and, over the subsequent 18
from the NIH posttransfusion study. Not surprisingly, months, in studies in our laboratory and in the George-
there was no relationship between the presence or ab- town University laboratory of John Gerin of Rock-
sence of anti-HAV and the occurrence of non-B trans- ville, Maryland, Rizzetto and his colleagues developed
fusion-associated hepatitis. Furthermore, none of the a sensitive radioimmunoassay for delta antigen and
patients who developed such disease developed anti- anti-delta. In addition, inoculation of chimpanzees
HAV. This study, performed by Feinstone and Kapi- with serum from Italian patients with chronic type B
kian with coded samples in collaboration with Harvey hepatitis and delta antigen resulted in the transmission
Alter and Paul Holland, provided the first direct evi- of delta antigen to these chimpanzees and, subse-
dence for a third human hepatitis virus. We called this quently, to other HBV-infected chimpanzees. These
April 1993 DISCOVERY OF HEPATITIS VIRUSES 959

studies revealed that the delta antigen was an internal young adults was the result of waning immunity fol-
component of a transmissible agent that was defective lowing previous exposure to HAV. However, when
and required coinfection with HBV for viral replica- serological tests for antibody to HAV became avail-
tion. Armed with sensitive and specific radioimmuno- able, it was clear that antibody persisted at high levels
assays for delta antigen and anti-delta and the knowl- for years in populations of developing countries. This
edge that the antigen was associated with a was consistent with other studies that pointed to long-
transmissible agent, Rizzetto mapped the seroepide- term, possibly lifelong immunity against reinfection
miology of the agent and identified the high-risk by HAV. Jules Dienstag, while a fellow in my labora-
groups with which it was associated. Molecular studies tory, was among those who questioned the etiology of
led to the identification of the genome of the delta the Delhi epidemic, based on his extensive studies of
antigen-associated agent. Surprisingly, it was very anti-HAV antibodies in different populations. He
small (1700 nucleotides), consisted of single-stranded tried unsuccessfully to locate clinical materials that
RNA, and was circular with a high degree of base pair- might have been saved from the Delhi epidemic to
ing. In these respects, it resembled the viroids and re- perform serological analyses. He was never successful,
lated agents of plants. Eventually the name was but after his departure from the laboratory, paired
changed to coincide with the nomenclature of the serum samples that had been stored from 1956 were
other hepatitis viruses: the delta-associated agent be- identified in the laboratory of Korshed Pavri, then Di-
came HDV. Thus, Rizzetto’s studies yielded a fourth rector of the National Institute of Virology, Pune, In-
human hepatitis virus, but a very strange one! HDV dia, who with others had studied the epidemic years
remains today the only recognized transmissible agent before. Eventually, after many months of red tape,
of its type in the entire animal kingdom. More interest- Pavri was able to release to us these samples and others
ing is the fact that it shares with transmissible viroids from more recent epidemics of hepatitis in India, and
and certain nontransmissible components of eucary- when they were serologically analyzed by Doris Wong,
otic cells the capacity of its RNA to function as an the results confirmed that the Delhi epidemic and
enzyme that effects autocatalytic cleavage and ligation. other water-borne epidemics in India were not caused
The discovery of HDV not only has provided a fasci- by HAV but presumably by a previously unrecognized
nating glimpse of the diversity of living things but also agent. Simultaneously with these studies, Mohammed
has pointed science in another direction: several Sultan Khuroo, another young gastroenterologist, was
groups are attempting to use the autocatalytic and studying a water-borne epidemic of hepatitis in the
transmissible properties of HDV to construct therapeu- mountainous Kashmir region of Northern India. The
tic agents capable of catalyzing the cleavage of other hepatitis in Kashmir had epidemiological characteris-
RNA molecules, such as the replicative intermediate tics similar to the Delhi epidemic, and when it was
form of the HBV genome. Rizzetto’s contributions in analyzed, this epidemic was also found to be serologi-
discovering HDV have been among the most signifi- tally unrelated to HAV infection. In 1983, Mikhail
cant and far reaching in the study of the basic science Balayan of the Institute of Poliomyelitis and Viral En-
of the hepatitis viruses. Like Blumberg before him cephalitides in Moscow reported on the self-ingestion
who made a chance discovery that might easily have of acute-phase stool suspensions from a water-borne
been discarded, Rizzetto pursued a paradox until he epidemic of non-A hepatitis in Central Asia. He de-
understood it. scribed the resultant clinical hepatitis, the recovery of
27-30-nm viruslike particles from his feces and devel-
HEV
opment of antibodies to these particles during conva-
In the winter of 1955-1956, a massive epidemic lescence. Furthermore, he transmitted the virus to
of water-borne hepatitis struck Delhi, India. The epi- cynomolgus monkeys, thereby establishing an animal
demic occurred when the receding waters of the Ya- model. Thus, the fifth recognized human hepatitis
muna River rechannelized and, for a period of time, virus was discovered and initially called epidemic, or
flowed in a retrograde fashion past the water-intake enterically transmitted NANB hepatitis virus and sub-
pipes of the central water treatment plant for Delhi, sequently designated HEV.
carrying raw sewage from a drainage ditch just down-
stream. More than 35,000 cases of jaundice occurred,
Molecular Virology
and the epidemic was intensively studied. One conclu- The molecular cloning of HCV by Michael
sion reached from the study of this epidemic was that Houghton at the Chiron Corporation in Emeryville,
it was caused by HAV and that the peak attack rate in California, and his colleagues was another first: the
960 PURCELL GASTROENTEROLOGY Vol. 104, No. 4

molecular characterization of a virus before it was ever from patients and experimentally infected chimpan-
visualized or isolated in cell culture. This milestone zees was a major source of information about this
was preceded by Houghton’s successful cloning of the virus. However, in 1978, a virus closely related to
genome of HDV. The cloning of HCV has resulted in HBV, the woodchuck hepatitis virus (WHV), was dis-
the development of screening assays that will all but covered in the woodchuck by Jesse Summers of the
eradicate transfusion-associated hepatitis. Similarly, Institute for Cancer Research in Fox Chase, Pennsyl-
the molecular cloning of HEV by Gregory Reyes and vania, in collaboration with Robert Synder of the Phil-
his colleagues at Gene Labs in Redwood City, Califor- adelphia Zoo, who had first noticed the high incidence
nia, has brought us much new knowledge about this of hepatocellular carcinoma (HCC) among captive
virus and has made possible the diagnostic and seroep- woodchucks at the zoo. In 1980, a similar virus was
idemiologic studies that will be important in under- identified in Beechey ground squirrels in northern Cal-
standing its epidemiology. The cloning of HCV and ifornia by Patricia Marion, William Robinson, and col-
HEV was achievable because of the development of leagues at the Stanford University School of Medicine
animal models for these viruses that permitted their in Stanford, California. In 1981, the first avian hepad-
biological amplification and characterization in vivo, navirus was recovered from ducks in the People’s Re-
and Daniel Bradley of the Centers for Disease Control public of China by Summers and his colleagues. Other
in Atlanta, Georgia, who played an important role in similar viruses were subsequently described in tree
the successes of Houghton and Reyes, was responsible squirrels in the United States and in herons in Ger-
for much of the new knowledge about these viruses. many.
The natural history of some of these nonhuman he-
Virus Variants padnaviruses proved to be remarkably similar to the
Variants of the hepatitis viruses have been rec- natural history of HBV in humans. Two of the viruses
ognized for some years but have recently taken on a have emerged as the most valuable for understanding
new cachet as the result of wide-spread interest in HBV infection: the duck hepatitis B virus, the only
HBV precore gene mutants, studied most extensively member of the group that will readily infect host hepa-
by Makoto Mayumi and his colleagues at the Jichi Med- tocytes in culture, has yielded much information about
ical School, Tochigi-Ken, Japan, and HBV S-gene mu- the replicative cycle of hepadnaviruses in studies by
tants, characterized by Howard Thomas and Arie Summers, as well as by many other groups; and the
Zuckerman (Royal Free Hospital, London) and Ales- biology of WHV infection and its relationship to he-
sandro Zanetti (Universita degli Studi diMilano, Mi- patocellular carcinogenesis in the natural host, the
lan, Italy), among others, and the possible association woodchuck, has also yielded new insights into the
of these variant viruses with fulminant hepatitis and pathogenesis of these diseases, as exemplified by the
escape from hepatitis B vaccination, respectively. The studies of Bud Tennant of Cornell University in collab-
recent finding of significant variation among strains of oration with John Gerin.
HCV has further stimulated interest in genetic hetero-
geneity of the hepatitis viruses. Finally, genetic vari- The Clinic and the Pathology
ants of HAV that are adapted to efficient growth in Laboratory
cell culture and that have lost their virulence are the
This is perhaps as good a place as any to ac-
bases of inactivated and live attenuated hepatitis A
knowledge the contributions of Dame Sheila Sherlock
vaccines. It is not surprising that genetic variation is a
and the late Hans Popper, among others, to our under-
fact of life of the hepatitis viruses, because viruses with
standing of the clinical and pathological aspects, re-
RNA genomes are known to be genetically heteroge-
spectively, of the hepatitis viruses. Over many decades,
neous, and all five of the recognized human hepatitis
they contributed to making hepatology a science, and
viruses have either an RNA genome (HAV, HCV,
their training of at least a generation of new scientists
HDV, and HEV) or a replication cycle that includes a
interested in viral hepatitis cannot be overemphasized.
replicative intermediate RNA form of the genome
Our professional and personal association with Hans
(HBV).
Popper began in the early 1970s and lasted until his
death. He contributed immensely to our understand-
HBV-like Viruses
ing of the hepatitis viruses and was a full participant in
Because HBV does not replicate in conven- the characterization of the pathology associated with
tional ways in cell cultures, a study of clinical materials each virus as it was discovered.
April 1993 DISCOVERY OF HEPATITIS VIRUSES 961

Diagnosis the development of serological markers for infection.

The association was identified in Asian populations by
Progress in understanding the hepatitis viruses
Kusuya Nishioka (Tokyo Metropolitan Institute of
has followed closely progress in the development of
Medical Science) and colleagues and was extended by
diagnostic tests for detecting infection and the host
others to include Africa and, in fact, most populations
immune response to it. This has been noted elsewhere
with a high incidence of HBV infection. The most
in this essay, but the significance of diagnostic and
convincing proof of an etiological association, how-
screening tests cannot be overemphasized. Thus, agar
ever, came from the elegant prospective study of male
gel diffusion was replaced in turn by counterelectro-
civil service workers in Taiwan by Palmer Beasley and
phoresis, complement fixation, passive hemagglutina-
his colleagues at the University of Washington Medi-
tion, and finally, radioimmunoassay (RIA) and en-
cal Research Unit in Taipei, Taiwan. This study identi-
zyme-linked immunosorbent assay (ELISA) for the
fied a relative risk of HBV carriers acquiring hepato-
detection of HBsAg and anti-HBs. To our surprise,
cellular carcinoma that was more than IOO-fold that of
anti-HBs detected by the earlier assays proved to be
noncarriers. Furthermore, Beasley and coworkers dis-
antibody stimulated by reexposure to the virus, and
covered that 40% of male HBV carriers would die of
primary anti-HBs responses were not detected until
causes related to their liver disease. Thus, HBV
RIA and ELISA became available. Similarly, IEM was
emerged as not only one of the world’s most important
replaced by immune adherence hemagglutination,
pathogens but also the first convincing human cancer
RIA, and ELISA for the detection of HBcAg and anti-
virus.
body to it and for the diagnosis of HAV infections.
However, despite the strong association between
Many of these assays were developed by some of the
HBV and HCC, many cases of HCC, particularly in the
same scientists mentioned in this essay. However, of
industrialized world, where HBV incidence is lower,
special note was the role played by commercial diag-
lacked markers of HBV replication, and the etiology of
nostic assays in unifying the serological results of scien-
these cases remained speculative and the basis of an
tists throughout the world. Scientists at Abbott Labora-
argument against the importance of HBV and HCC.
tories, among them Lacey Overby, Richard Decker,
With the cloning of HCV and the development of
and Isa Mushahwar, were responsible for the early de-
specific serological tests for diagnosing infection with
velopment of highly standardized assays for HBV,
this virus, it became apparent that many HCC cases
HAV, and HDV in turn that allowed scientists to
not associated with HBV infection had evidence of
compare their results directly with those of other scien-
HCV infection. This has now been established in a
tists. Thus, these commercial assays became the “gold
number of countries in Asia, Africa, and Europe, and
standard,” and coupled with the use of reference re-
the data for an etiologic association between HCV in-
agents from the Reference Reagents Branch, National
fection and HCC is convincing if not unequivocal.
Institutes of Allergy and Infectious Diseases, and inter-
This raises questions about the pathogenesis of HCC,
national standards distributed by the World Health Or-
because it has been assumed that the primary mecha-
ganization, the assays set the stage for rapid acceptance
nism of hepatocarcinogenesis in HBV infection is in-
of new data regardless of their origin. The recently
tegration of the viral genome into host chromosomal
developed commercial assays for detection of infec-
DNA with subsequent alterations in the function of
tions by HCV and HEV, originally developed by the
Chiron Corporation and Gene Labs, Inc., respectively, oncogenes and/or suppressor genes. However, there is
are now being widely used by the scientific community no known mechanism for a single-stranded RNA virus
in a similar manner. The development of polymerase such as HCV to integrate into the genome of the host,
chain reaction (PCR) by Kary Mullis while at the and indeed, such integration has not been found. Thus,
Cetus Corporation is having a similar impact on hepa- the alternative hypothesis that HCC results from
titis research, especially in regard to HCV and HEV, chronic damage to the liver, for whatever reason, has
which usually replicate to levels too low to be detect- gained credence. In fact, there may be multiple path-
able by other techniques. We can expect to see PCR ways from chronic viral infection of the liver to hepa-
and similar amplification technologies grow in impor- tocellular carcinoma.
tance over the next few years.
Therapy
HCC Therapy for chronic viral hepatitis has been the
The association between HBV infection and subject of intense interest during the past 50 years. A
HCC has generated much interest since shortly after number of therapies have been tried and discarded.
962 PURCELL GASTROENTEROLOGY Vol. 104, No. 4

The first successful use of an antiviral agent, leukocyte curred with the polio vaccines in the 1950s.
interferon, was reported in 1976 by Harry Greenberg Development of the vaccines was greatly helped by
and his colleagues in the laboratory of Thomas Meri- careful selection of candidate strains collected in Costa
gan at Stanford University. The same year, we reported Rica by Victor Villarejos and in Australia by Ian Gust.
the successful use of an interferon inducer in chimpan-
zees infected long-term with HBV. The restricted avail-
The Future
ability of interferon and the less than outstanding re- The Golden Age of viral hepatitis research has
sponse in many patients made this a therapy of limited not ended. The 1990s have already seen the licensing
usefulness until recombinant interferons became avail- in Europe of hepatitis A vaccine developed by
able and their efficacy for different types of hepatitis SmithKline Beecham, and the licensing in the United
measured in carefully controlled clinical studies by Jay States of this vaccine and a vaccine developed by
Hoofnagle and his colleagues at the NIH, as well as Merck, Sharp & Dohme cannot be far behind. Pros-
other groups. Now interferon is considered by many pects for the development of a hepatitis E vaccine dur-
the treatment of choice for selected cases of chronic ing this decade are also good. The development of a
hepatitis B, hepatitis C, and hepatitis D. Still, only a hepatitis C vaccine is less certain. The need for a hepa-
proportion of patients respond favorably, and there titis D vaccine is limited to carriers of HBV, because
remains a need for more universally applicable, less hepatitis B vaccine will also prevent hepatitis D in
expensive therapies for these chronic diseases. seronegative individuals. Thus, the prospects for con-
trol of viral hepatitis by immunoprophylaxis are gener-
Vaccines ally bright, if the vaccines actually reach the popula-
The study of the hepatitis viruses has been of tions most in need of them. Immunoprophylaxis of
more than academic interest. These agents are thought viral diseases continues to be among the most cost-ef-
to cause some 600,000 cases of clinical hepatitis in the fective of medical strategies, and this should carefully
United States per year and untold numbers in develop- be considered when contemplating strategies to con-
ing regions of the world. It has been estimated that trol the costs of health care. Nowhere is this positive
there are over 300 million cases of chronic hepatitis B cost benefit more evident than in the prevention of
worldwide and that almost half of these will die of chronic viral hepatitis and its sequelae of end-stage
liver-related diseases. Therefore, it is of some signifi- liver disease, cirrhosis, and liver cancer, the therapies
cance that considerable progress has been made in the for which are all exceedingly expensive and imperfect.
development of vaccines against these agents. The first Are there additional human hepatitis viruses to be
hepatitis vaccine against HBV was also a novel vaccine discovered? Up to 10% of transfusion-associated hepa-
prepared from viral antigens obtained by plasmapher- titis and up to 5% of community-acquired viral hepati-
esis of chronically infected patients. Its development tis have no identifiable etiology, even when the most
stemmed from the biophysical characterization of sophisticated diagnostic tests are applied. Similarly,
HBsAg by John Gerin, Hilleman’s group, and Blum- many cases of fulminant hepatitis cannot be ascribed
berg’s colleagues. This vaccine was licensed in 1981, to any of the five recognized viruses. Do these undiag-
following an efficacy trial under the direction of the nosed cases represent one or more additional hepatitis
late Wolf Szmuness with Cladd Stevens (New York viruses? Attempts to transmit an agent from such cases
Blood Center) in gay men in New York City that re- to primates have been either negative or not com-
mains a classic in its planning and execution. In part pletely convincing, and more effort needs to be ex-
because of the requirement for extensive safety tests pended in this area. However, the technology for iden-
for each lot of the plasma-derived product and the tifying infections with each of the five recognized
growing concern over acquired immunodeficiency viruses is now quite sophisticated, and the question of
syndrome, the plasma-derived vaccine was replaced in whether additional viruses exist will probably be an-
1986 with the world’s first recombinant vaccine. swered soon. Equally interesting, however, is the ques-
Similarly, hepatitis A vaccines have become a reality tion of whether other liver diseases of unknown etiol-
and should be licensed in the United States within a ogy may be caused by a transmissible agent. Among
year. One such vaccine is already licensed in Europe. those suggested are primary biliary cirrhosis, primary
Unlike hepatitis B vaccine, hepatitis A vaccine is the sclerosing cholangitis, giant cell hepatitis, and hepati-
product of classical vaccine development: the first vac- tis-associated aplastic anemia. All of these diseases
cines are inactivated whole virus preparations, possi- clearly have immunological components. However,
bly to be followed by live attenuated vaccines, as oc- the confirmation that autoimmune phenomena are as-
April 1993 DISCOVERY OF HEPATITIS VIRUSES 963

sociated with bona fide chronic hepatitis C and hepati- some who have made very significant contributions.
tis D renders less certain a sharp demarcation between Neither space nor time has allowed me to be su@i-
infectious and immunological etiologies. For that mat- ciently comprehensive in recognizing all of the impor-
ter, we still do not fully understand the role of immu- tant participants in this chronology. To those I failed
nology in the pathogenesis of any of the viral hepati- to mention, I apologize. This was not meant to be a
tides. Here, on the frontiers between infectious disease comprehensive review of the field but rather one trav-
and immunology, is where further skirmishes will be eler’s view of a convoluted journey through time and
fought. The weapons will be drawn from the high tech- space. Similarly, I have not included many references
nology of molecular biology. Regardless of the out- because I wanted this to be informal and I have used up
come, the winners will be the public who stand to gain more than my allotted space in recollections. Besides,
much from the rapid progress in hepatology that has as Garrison Keillor might quip, “Why should I assist
characterized the past 50 years and that undoubtedly in separating fact from fantasy?” Whether fact or fan-
will characterize the next half century. tasy, this has been a fantastic voyage, and I am glad to

Epilogue have been along for the ride.

In this essay, I have attempted to give you a References

feeling of the excitement and sense of community that 1. Alter, HJ. Hepatitis 6: a tribute to nondirected medical research.
existed among the principal players in hepatitis re- Semin Liver Dis 1981; 1:2-6.
search over the past several decades. This sense of a
common goal has resulted in extraordinary progress in Address requests for reprints to: Robert H. Purcell, M.D., Hepatitis
the understanding and control of viral hepatitis. Viruses Section, Laboratory of Infectious Diseases, National Instl-
I have also attempted to be fair in my recognition of tute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892.
the many scientists who have studied the hepatitis vi-
The author thanks Drs. Harvey Alter, Stephen Feinstone, and John
ruses and worked so long and diligently to unravel this Gerin, who have shared the ride, for careful reading of the manu-
complex puzzle. Undoubtedly, I have failed to mention script and for insightful comments and helpful suggestions.