Antiviral Research 131 (2016) 109e123

Contents lists available at ScienceDirect

Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

A historical perspective on the discovery and elucidation of the

hepatitis B virus
Timothy M. Block a, *, Harvey J. Alter b, W. Thomas London c, Mike Bray d
a
Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, PA 18902, USA
b
Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD 20892, USA
c
Fox Chase Cancer Center, Philadelphia, PA 19046, USA
d
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

a r t i c l e i n f o a b s t r a c t

Article history: The discovery in 1965 of the “Australia antigen,” subsequently identified as the hepatitis B virus surface
Received 8 February 2016 antigen (HBsAg), was such a watershed event in virology that it is often thought to mark the beginning of
Received in revised form hepatitis research, but it is more accurately seen as a critical breakthrough in a long effort to understand
15 April 2016
the pathogenesis of infectious hepatitis. A century earlier, Virchow provided an authoritative explanation
Accepted 18 April 2016
Available online 20 April 2016
of “catarrhal jaundice,” which did not consider an infectious etiology, but the transmission of jaundice by
human serum was clearly identified in two outbreaks in 1885, and the distinction between “infectious”
and “serum” hepatitis was recognized by the early 1920s. The inability to culture a virus or reproduce
either syndrome in laboratory animals led to numerous studies in human volunteers; by the end of
World War II, it was known that the diseases were caused by different filterable agents, and the terms
“hepatitis A” and “B” were introduced in 1947 (though some long-incubation cases then designated B
must in retrospect have been hepatitis C). The development of a number of liver function tests during the
1950s led to the recognition of anicteric infections and the existence of chronic carriers, but little more
could be done until an infectious agent had been identified. Once Blumberg and colleagues had found a
specific viral marker, the vast amount of accumulated epidemiologic and clinical data, together with
huge numbers of stored serum samples, enabled rapid progress in understanding hepatitis B, and
revealed the existence of a vast population of chronically infected people in Asia, Oceania and Africa. In
this article, we place the identification of the Australia antigen within the historical context of research
on viral hepatitis. Following a chronological review from 1865 to 1965, we summarize how the discovery
led to improved safety of blood transfusion, the development of a highly effective vaccine and the
eventual identification of the hepatitis C, D and E viruses. This article forms part of a symposium in
Antiviral Research on “An unfinished story: from the discovery of the Australia antigen to the develop-
ment of new curative therapies for chronic hepatitis B.”
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Fifty years ago, the fortuitous detection of an abundant protein
in the serum of an Australian aboriginal person provided the long-
sought key to the specific diagnosis of hepatitis B. Even though the
finding occurred in the course of studies completely unrelated to
viral hepatitis, the investigators pursued the mysterious protein
until it was finally identified as the hepatitis B virus (HBV) surface
antigen (HBsAg). The discovery of this viral marker solved with one
* Corresponding author.
E-mail address: tim.block@bblumberg.org (T.M. Block).
stroke a problem that had frustrated researchers for decades e the
inability to distinguish between forms of viral hepatitis on a basis
other than the epidemiological setting and the patient's clinical
history. The detection of a circulating viral antigen also detected
cases of hepatitis unaccompanied by jaundice and revealed the
existence of vast numbers of chronically infected people.
The discovery of the Australia antigen is often regarded as the
beginning of hepatitis B studies, but it is more accurately viewed as
a breakthrough in a long history of research (Table 1). Virchow
claimed in 1865 that “catarrhal jaundice” was not infectious in
origin, but the ability of minute quantities of human serum to
transmit an icterogenic agent was dramatically demonstrated in

http://dx.doi.org/10.1016/j.antiviral.2016.04.012
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110 T.M. Block et al. / Antiviral Research 131 (2016) 109e123

Table 1
Milestones in the history of hepatitis B. Early descriptions refer to epidemic or “catarrhal” jaundice, many cases of which may also have been caused by hepatitis A, C or E virus.
The term “serum hepatitis” became common in the early 20th century, and “hepatitis B” was introduced in 1947.

Year Observation/event Reference

1800e400 Descriptions of jaundice in Babylonian clay tablets and writings of Hippocrates. (Papaevangelou et al., 1983)
BCE
1600e1900s Outbreaks of jaundice common in urban populations and armies on campaign.
1865 Virchow explains etiology of “catarrhal jaundice” as duodenal edema obstructing the (Virchow, 1865)
ampulla of Vater.
1885 Two outbreaks of catarrhal jaundice in Germany following smallpox vaccination. (Lurman, 1885; Jehn, 1885)
1900e40s Outbreaks of jaundice associated with i.v. salvarsan inoculation and blood testing in (Stokes et al., 1920; Flaum et al., 1926)
diabetes clinics.
1930s Liver biopsies detect hepatic inflammation in patients with jaundice, casting doubt on (Roholm and Iversen, 1939)
“catarrhal” origin.
1930e1940s Outbreaks of jaundice linked to injection of measles and mumps convalescent plasma. (Propert, 1938; MacNalty, 1938; Beeson et al., 1944)
1930e40s First “liver function tests” introduced into clinical practice. (Maclagan, 1947)
1930s Yellow fever vaccine containing human serum causes cases of jaundice in Brazil and the UK. (Findlay and MacCallum, 1938; Soper and Smith, 1938; Fox
et al., 1942)
1942 Yellow fever vaccine containing human serum causes massive outbreak of hepatitis B in US (Sawyer et al., 1944; Turner et al., 1944)
Army.
1937e40s Recognition of chronic hepatitis in patients with previous catarrhal jaundice. (Polack, 1937; Kornberg, 1941)
1944 Serum and infectious hepatitis shown to be caused by distinct filterable agents in volunteer (MacCallum, 1945; MacCallum and Bradley, 1944; MacCallum,
studies. 1944)
1947 “Infectious hepatitis” renamed hepatitis A, “serum hepatitis” renamed hepatitis B. (Anonymous, 1947)
1950s Chronic viral carriers identified in human volunteer studies. (Neefe et al., 1954; Murray et al., 1954)
1965 “Australia antigen” detected in serum of an Australian aborigine and some American (Blumberg et al., 1965; Alter and Blumberg, 1966)
leukemia patients.
1967 Australia antigen associated with hepatitis B. (Sutnick et al., 1968; Blumberg et al., 1968; Blumberg et al.,
1969)
1972e73 Hepatitis B transmitted to chimpanzees. (Barker et al., 1973; Maynard et al., 1972)
1969e72 Chronic hepatitis B linked to the development of liver cancer. (Smith and Blumberg, 1969; Sutnick et al., 1972)
1972 First testing of donor blood for Australia antigen. (Alter et al., 1970)
1972 First vaccine to protect against hepatitis B. (Blumberg and Millman, 1972)
1973 Hepatitis A virus identified by immuno-EM in stool specimens. (Feinstone et al., 1973)
1977 Discovery of delta antigen leads to identification of HDV. (Rizzetto et al., 1977)
1978 Non-A, non-B hepatitis virus hypothesized to exist. (Alter et al., 1978)
1983 Hepatitis E identified. (Balayan et al., 1983)
1986 First clinical trial of interferon-a therapy of chronic hepatitis B. (Hoofnagle et al., 1986)
1989 Hepatitis C virus cloned from human serum. (Choo et al., 1989)
1998 First direct-acting antiviral (lamivudine) for chronic hepatitis B approved by FDA. (Lai et al., 1998)

two outbreaks 20 years later. Based on the route of exposure and
length of the incubation period, the distinction between short-
incubation or “infectious” and long-incubation or “serum” hepati-
tis was clearly recognized by the 1920s, but scientists were
consistently unable to cultivate an infectious agent or reproduce
either disease in laboratory animals. The occurrence of several
serum-transmitted outbreaks in the 1930s, and a massive epidemic
of jaundice in soldiers given the yellow fever vaccine in 1942, led to
numerous studies in human volunteers, and by the end of World
War II it was well established that infectious and serum hepatitis
were caused by different filterable agents. The terms “hepatitis A”
and “hepatitis B” were introduced in 1947. (From our current
perspective, it's clear that some long-incubation cases then desig-
nated hepatitis B must in fact have been hepatitis C). The devel-
opment of a variety of liver function tests during the next two
decades revealed the occurrence of anicteric hepatitis and the ex-
istence of many chronically infected individuals, but the causative
agents still could not be isolated.
By the time Blumberg and his colleagues noticed an unusual
band in a double-diffusion gel in 1965, a huge amount of clinical
and epidemiologic data had accumulated, together with thousands
of frozen serum samples, so that the introduction of a specific
diagnostic assay for hepatitis B made rapid progress possible. The
discovery of the Australia antigen had two major consequences: it
provided a way for physicians in the industrialized countries to
reliably diagnose asymptomatic carriers of hepatitis B, improving
the safety of blood transfusion, and it revealed the existence of
many millions of chronically infected people. The predominant
distribution of antigen positivity in Oceania, East Asia and Africa
that initially puzzled Blumberg and his colleagues was the first
glimpse of a vast human tragedy.
In this article, we place the discovery of the Australia antigen
within the 150-year history of research on viral hepatitis. We first
discuss early explanations of jaundice, then review a series of epi-
sodes that began in 1885, in which outbreaks of what we now
recognize as viral hepatitis followed smallpox vaccination, the
introduction of glass syringes and injectable medications, treat-
ment with pooled immune globulin, vaccination against yellow
fever and blood transfusion. After describing the path of research
by Blumberg and his co-workers that led to the fortuitous detection
of the Australia antigen, and recounting how their relentless pur-
suit of the strange protein led to its identification as the HBsAg, we
summarize the multiple consequences of the discovery, including
improved safety of the blood supply, recognition of a link between
chronic hepatitis and hepatocellular carcinoma, the creation of a
highly protective vaccine and the eventual discovery of the other
major blood-borne agent of chronic hepatitis, the hepatitis C virus,
and the other cause of infectious hepatitis, the hepatitis E virus.
2. Explaining infectious jaundice: from Hippocrates to
Virchow to the modern era
Viral hepatitis has numerous physical manifestations, but the
most obvious is the jaundice seen in many patients. This striking
physical sign is recorded in Babylonian clay tablets and mentioned
in the Old Testament, but the lengthy time interval, measured in
weeks, between exposure to the causative agent and the develop-
ment of illness prevented recognition of jaundice as an infectious
 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
disease until recent times. Although Hippocrates described a
number of cases and proposed various causes and treatments, and
used the terms ikteros and kirros in his descriptions, he was not
aware that the condition could be transmitted from person to
person, and attributed jaundice to a humoral imbalance
(Papavramidou et al., 2007).
Over the succeeding two millennia, outbreaks of illness
accompanied by jaundice were common among crowded urban
populations and in armies, both in barracks and in the field.
Numerous epidemics were described in Europe and the Mediter-
ranean region in the 18th and 19th centuries, and many thousands
of cases were recorded during the American Civil War. Impure
water was sometimes suspected as the source of the disease, but
the association with foul-smelling places also made airborne
spread by a “miasma” seem quite possible.
In 1865, the renowned pathologist Rudolf Virchow weighed in
on the cause of jaundice, but he also did not recognize it to be in-
fectious in nature. An ardent opponent of the germ theory of dis-
ease, he believed that all illnesses represented derangements of
cellular function that arose within the host. After hearing of an
autopsy of a jaundiced individual in whom the opening of the
common bile duct was swollen and blocked by mucus, he
announced that the condition resulted from inflammation of the
duodenal mucosa, leading to obstruction of the ampulla of Vater
(Virchow, 1865). Since it was a condition of the intestinal tract,
jaundice must result from gastronomic misadventures such as
over-eating, and recommended therapies were therefore directed
toward soothing the inflamed duodenum, such as by imbibing
magnesium salts. Thanks to Virchow's enormous prestige among
European and American physicians and the endorsement of his
theory by Sir William Osler, the concept of “catarrhal jaundice” was
generally accepted in medical practice well into the 20th century,
and the term was still being used by Harvard physicians in 1944
(Altschule and Gilligan, 1944).
Studies during World War I provided the first evidence that
jaundice did not actually originate from the gastrointestinal tract.
As in all wars, outbreaks were common in armies at the front, and
pathologists who examined soldiers killed while suffering from
“catarrhal jaundice” noted the presence of diffuse microscopic ab-
normalities in the liver, with severe parenchymal injury and round-
cell inflammatory infiltrates. Coincidentally, an infectious etiology
also became available, as the identification of the spirochete of
Weil's disease in 1916, and the recognition of its role in large, water-
borne outbreaks of jaundice, offered the possibility that many cases
were caused by this agent (Adler, 2015). However, the organism
was typically recovered from patients with a history of heavy
exposure to contaminated water, and it could not be implicated in
urban outbreaks in the USA or Europe.
Jaundice was not recognized to be a process originating within
the liver until the 1930s, when clinicians in Denmark began to
perform liver biopsies, using a hollow needle much like that
employed today. In a study of 38 patients, Roholm and Iversen
concluded that “ … catarrhal jaundice … actually is due to a diffuse
hepatitis that is characterized by inflammatory phenomena …
[and] degeneration of the parenchymatous cells” (Roholm and
Iversen, 1939). They also noted the development of connective
tissue in the liver of some patients, which they suggested might be
the beginning of cirrhosis, and concluded that “the theory about the
catarrhal pathogenesis is not tenable.”
3. The search for infectious agents
We are now familiar with viruses A, B, C, D and E as causes of
human hepatitis, but their identification was one of the most
difficult problems in the history of virology. The existence of
111
“filterable agents,” smaller than bacteria and unable to propagate in
culture medium, was discovered in the early 1890s, when the
Russian botanist Dmitri Ivanovskii showed that mosaic disease of
tobacco plants could be produced by treating leaves with liquid that
had passed through a fine-pored porcelain (Chamberland) filter
(Ivanovskii, 1892). Over subsequent decades, ultramicroscopic
agents (named “viruses” by 1900) were proven to be responsible for
canine distemper, rabies, poliomyelitis, influenza and other dis-
eases, as investigators reproduced the conditions in laboratory
animals by inoculating them with filtered material. The early 1930s
saw further advances, as it was found that many viruses could be
propagated in embryonated eggs and sequentially passaged in the
brains of newborn mice. The introduction of tissue culture in the
early 1950s was a further breakthrough, permitting the detection
and propagation of viruses in simple flask systems and the sharing
of strains among laboratories.
None of these advances benefited hepatitis research. Despite
repeated efforts over many decades, researchers were unable to
isolate a causative agent in cell culture or produce jaundice in an-
imals by inoculating them with specimens from jaundiced humans.
As described below, investigators succeeded by the late 1930s in
demonstrating that both infectious and serum hepatitis were
caused by filterable agents, but lacking animal models, they were
obliged to obtain such proof by inoculating human volunteers with
filtered material. For both ethical and practical reasons, this
markedly slowed the pace of research.
4. Serum hepatitis as a companion of medical progress,
1885e1945
Vaccination against smallpox, the glass syringe, injectable
antimicrobial drugs, passive immunization with convalescent
immunoglobulin, the yellow fever vaccine and blood transfusion
have all been of enormous benefit to humanity, but each of these
medical advances has left a trail of jaundice in its wake. In each
case, the fundamental problem was the inability of clinicians to
detect the presence of chronic hepatitis in apparently healthy, non-
jaundiced individuals, rendering their serum a source of infection,
whether injected in large quantities through transfusion, employed
as a “stabilizer” in vaccine production or transferred by a contam-
inated syringe.
4.1. Smallpox vaccination
The introduction of smallpox vaccination in the 1790s must
occasionally have resulted in the inadvertent transmission of
hepatitis, especially when arm-to-arm transfer of “lymph” was
performed, but such a complication was not recognized for almost a
century, until an outbreak of jaundice occurred following the mass
vaccination of shipyard workers in Bremen, Germany (Lurman,
1885). Based on their knowledge of jaundice in military settings,
clinicians initially blamed the epidemic on contaminated water, or
suspected “miasmatic” transmission from the local environment,
but since some groups of workers remained healthy, while others
showed a high incidence of jaundice, neither hypothesis could
explain the distribution of cases.
After eliminating various possibilities, the only shared factor
that corresponded to the observed distribution of illness in the
Bremen shipyard was a history of receiving smallpox vaccine when
the disease broke out earlier in the year. Of 1289 vaccinated
workers, 191 developed jaundice after an interval of several weeks
to 6 months, but the condition did not spread to their family
members or other close contacts. It was also noted that, once the
outbreak ended, it did not recur, and no cases of jaundice were
observed among new employees. Further investigation also
112 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
showed that jaundice only developed in workers who had been
inoculated with certain lots of vaccine, which differed in the source
of human serum used in their preparation. It was therefore
concluded that epidemic jaundice had been caused by an agent
present in human serum, but the problem was not pursued further.
Remarkably, a similar outbreak occurred elsewhere in Germany
in the same year, when inmates of an insane asylum in Merzig,
more than 500 km to the south of Bremen, were vaccinated against
smallpox (Jehn, 1885). Five different groups of patients were
immunized, using vaccine composed of glycerinized lymph from
different sources; more than 25% of one group of inmates became
jaundiced over the following 2e8 months, but few cases were seen
in the other groups. The occurrence of two large outbreaks of
jaundice among smallpox vaccinees in the same country the same
year suggests a common source of virus-containing serum, but
none was identified.
4.2. Jaundice following the injection of medications
From the beginning of the 20th century, the development of an
increasing number of antimicrobial medications and the syringes
needed to deliver them provided new opportunities for the inad-
vertent transmission of viral hepatitis. The introduction of
parenterally-administered salvarsan (arsphenamine) therapy of
syphilis in 1909 and the subsequent discovery of several more
injected medications for the treatment of venereal disease resulted
in outbreaks of jaundice whose etiology was not finally established
for more than three decades.
The first major report on the occurrence of “post-arsenical
jaundice” was published in 1920, when doctors at the Mayo Clinic
noted a sudden increase in the number of cases of jaundice among
patients treated for syphilis (Stokes et al., 1920). It could not be
attributed to drug toxicity, as it usually followed treatment by
several months, or to syphilis itself, as the patients' condition was
usually improving at the time jaundice appeared. The authors
considered a wide range of possible causes, including infectious
agents, and did not exclude a possible connection with the recent
massive influenza pandemic. A few years later, a report from
Sweden described an outbreak of jaundice among patients in a
diabetes clinic, in which lancets were repeatedly re-used to obtain
blood samples (Flaum et al., 1926). As with the 1885 outbreak in
Bremen, the clinicians concluded that jaundice most likely resulted
from the accidental transfer of an infectious agent that only pro-
duced visible disease several months after exposure.
Outbreaks of jaundice associated with arsphenamine
(salvarsan) treatment continued to occur into the 1940s. Some in-
vestigators, realizing that the illness in syphilitic patients resem-
bled catarrhal jaundice in otherwise healthy individuals, performed
liver biopsies, and observed similar microscopic features in the two
groups. Writing in 1944, Beattie and Marshall found it most likely
that “salvarsan jaundice” was unrelated to the medications them-
selves, but “the spread occurred through inoculation of an infective
agent” and that it was the same disease that occasionally occurred
after administration of human blood products (Beattie and
Marshall, 1944). Suspecting that infection resulted from the trans-
fer of minute amounts of serum on imperfectly sterilized syringes,
they decided to give each patient his own syringe, and no cases of
jaundice occurred thereafter.
A year later, an experimental study in volunteers provided
definitive proof of the transmission of an infectious agent, when
serum from a soldier who developed hepatitis during the 14th
week of arsenical therapy was inoculated into ten volunteers, and
seven became jaundiced 42e70 days later (MacCallum, 1945). At-
tempts to transmit the disease further by administering nasopha-
ryngeal washings from those cases to an additional 17 volunteers
were unsuccessful, confirming serum as the source of infection.
4.3. Transmission of hepatitis by prophylactic immune serum
The 1930s also saw a number of epidemics of hepatitis when
pooled human serum was administered to children to prevent
measles and mumps. In one instance, seven young residents of a
long-term institution were inoculated to protect them against
measles; all became jaundiced some 78e82 days later, and three
died (Propert, 1938). The fatal cases were noted to resemble a se-
vere form of catarrhal jaundice. In another outbreak, pooled mea-
sles convalescent serum collected from 26 donors was given as a
preventive to young children, resulting in 41 cases of jaundice with
eight deaths (MacNalty, 1938).
Prophylaxis with pooled immune serum came into widespread
use during World War II, in an effort to prevent the spread of res-
piratory viral infections among soldiers in Army camps. In one
outbreak observed in Britain, the administration of mumps
convalescent serum to 266 men resulted in jaundice in some 40% of
them (Beeson et al., 1944). The serum had been passed through a
Seitz filter to remove possible bacterial contaminants, indicating a
viral causation of the illness. No cases of jaundice were seen in
uninoculated soldiers or among the neighboring civilian popula-
tion. The occurrence of these cases resulting from the administra-
tion of human serum led to use of the term “homologous serum
hepatitis” (Anonymous, 1947).
4.4. Hepatitis B virus contamination of yellow fever vaccine
The introduction of vaccination against yellow fever in the early
1930s was also followed by outbreaks of hepatitis. The first British
vaccine was prepared by mixing live, mouse-brain-passaged virus
with human immune serum, to which normal serum was added
during manufacture of the final stocks. In 1938, the British physi-
cians Findlay and MacCallum reported that, during the previous
five years, 89 cases of jaundice had been observed among 3100
vaccinees (about 3% of recipients) (Findlay and MacCallum, 1938).
In most cases, jaundice developed 2e3 months after vaccination,
though some cases were delayed as long as seven months. The
authors noted that the illness was consistent with epidemic
catarrhal jaundice, but the long incubation period resembled that
observed after salvarsan treatment.
Yellow fever vaccination was also introduced in Brazil in the
mid-1930s, using a Rockefeller Foundation preparation of neuro-
tropic virus “stabilized” through the addition of human immune
serum, and cases of jaundice were soon observed (Soper and Smith,
1938). Suspecting that the vaccine strain of yellow fever virus might
be causing the jaundice, the researchers replaced the original virus
with the highly attenuated 17E strain, and also deleted human
immune serum from the preparation, but normal serum continued
to be employed as a “stabilizer” in the production of large vaccine
stocks. By early 1939, more than one million people had received
the new vaccine, with no instances of jaundice. Later that year,
however, cases began to appear; as only certain vaccine lots were
icterogenic, it appeared that one or a few serum donors were source
of the hepatitis agent (Fox et al., 1942). Once a completely serum-
free vaccine was introduced in late 1940, jaundice was no longer
observed.
Unfortunately, the lessons learned in Brazil were not applied in
the USA, even though the Rockefeller laboratories produced vaccine
for both countries, and in 1942 the US Army experienced a massive
hepatitis epidemic. Earlier that year, the Army medical staff had
decided that all soldiers who might be deployed to yellow fever-
endemic areas should be vaccinated; there was also concern that
the movement of troops might introduce yellow fever into Asia,
 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
which was still free of the disease. To support a massive increase in
vaccine production, the Rockefeller labs obtained serum from blood
donated by medical school volunteers; a subsequent investigation
found that several of the donors had a history of catarrhal jaundice,
and one of them was actually jaundiced at the time of blood
collection (Sawyer et al., 1944; Thomas et al., 2013). Serum from
these donors was linked to vaccine lots that were highly ictero-
genic; of the 330,000 men vaccinated with these lots, 1 in 7 became
ill. Fortunately, the Navy used different vaccine lots, and saw almost
no cases of jaundice.
By the end of 1942, more than 50,000 cases of acute hepatitis
had occurred, with some 100 deaths. At Camp Polk, for example,
5000 soldiers were vaccinated on one day, and just over 1000 of
them became jaundiced (Turner et al., 1944). The onset of jaundice
was seen 60e154 days after inoculation (a mean of 96 days),
consistent with serum hepatitis. The illness began insidiously,
without fever, in contrast to the abrupt onset of malaise and fever
typical of infectious hepatitis. In a few cases, fatal fulminant hep-
atitis developed during the fourth to sixth week of illness. Person-
to-person transmission at Camp Polk was limited to four wives of
vaccinated soldiers, consistent with the low infectivity of serum
hepatitis, but indicating the possibility of spread through intimate
contact (Freeman, 1946).
HBV was not actually proven to be the cause of the Army
outbreak until 1985, when a study of surviving veterans found that,
among 221 men who had become jaundiced after vaccination in
1942, 97% had antibodies to viral antigens, and one was HBsAg-
positive (Seeff et al., 1987). Of 171 men who did not become ill af-
ter receiving a vaccine from an icterogenic lot, 77% had circulating
antibodies to HBsAg and HBcAg, while only 13% of an age-matched
control group showed similar evidence of prior infection. Analysis
of the records of some 70,000 veterans found that men who had
received the contaminated vaccine had only a slightly increased
risk of hepatocellular carcinoma, suggesting that few became
chronically infected (Norman et al., 1993).
4.5. Wartime studies of transmission of hepatitis by normal
immune globulin and blood transfusion
In June 1943, Morgan and Williamson described nine cases of
jaundice among 56 patients who had been treated with large vol-
umes of pooled human serum for a variety of conditions, with onset
49e107 days after treatment (Morgan and Williamson, 1943). The
authors noted the resemblance of these cases to the jaundice that
occurred after yellow fever vaccination. In response to this report,
an editorial in the British Medical Journal advised against further use
of the product (Anonymous, 1946).
Outbreaks of jaundice resulting from the administration of
virus-containing serum to a large number of people over a short
period of time were relatively easy to recognize. In contrast, it was
much more difficult to detect individual cases of hepatitis that
followed blood transfusion, because incubation periods measured
in weeks to months prevented specific attribution, and the detailed
records needed to link a case of jaundice to earlier transfusion were
often lacking. Nevertheless, several researchers described
transfusion-associated jaundice during World War II. For example,
clinicians at Grady Hospital in Atlanta reported seven cases that
developed 1e4 months after transfusion of whole blood or plasma
(Beeson, 1943). Additional analysis found that, of 79 cases of “acute
catarrhal jaundice” or “toxic hepatitis” that had been seen at Grady,
six were preceded by blood transfusion. The authors suggested
that, given the link between transfusion and hepatitis, a sample of
each unit of blood be retained for analysis.
Another study reviewed all battle casualties admitted to a large
Army hospital, and identified 103 patients who developed hepatitis
113
after receiving blood transfusions (Grossman et al., 1945). To see if
the situation might be alleviated, the authors performed a trial in
which every other patient admitted after receiving a transfusion in
the field was given gamma-globulin. They observed a marked
reduction in cases of hepatitis.
5. Wartime research on viral hepatitis: volunteer studies
As noted in the US Army's official medical history
(US_Army_Medical_Services_in_World_War_II), hepatitis was not
considered to be a significant military problem at the beginning of
World War II. Although the armed forces of other countries had
suffered severely from infectious hepatitis or catarrhal jaundice
during World War I, especially under conditions of prolonged
trench warfare in Europe and in the Dardanelles campaign, few
cases had been seen among American troops. However, the situa-
tion changed dramatically when the massive outbreak of vaccine-
transmitted serum hepatitis in soldiers in 1942 was followed by
epidemics of infectious hepatitis in the North African, Italian and
Pacific theaters. By 1943, large-scale research programs on viral
hepatitis were being conducted under Army sponsorship in the
USA, and to a lesser extent in the UK. The American program was
directed by members of the Army's Hepatitis Study Group, and in
Britain, research was led by the Jaundice Committee of the Medical
Research Council.
When the work began, clinicians were aware of the existence of
two distinct syndromes, serum and infectious hepatitis, with
markedly different epidemiological patterns and incubation pe-
riods, but they knew little about their biological nature or means of
prevention. Wartime research therefore focused on characterizing
sources of infection, the course of illness, pathways and duration of
infectivity and whether the syndromes were caused by one or more
pathogens. Because no microbial agents had been isolated, and
neither disease could be reproduced in laboratory animals, all ex-
periments were conducted in human volunteers, in ways that
would not meet today's ethical standards for clinical research. In
the USA, these included inmates of state and federal prisons in New
Jersey, Connecticut and Michigan and conscientious objectors
working at various institutions in the northeastern states. In the US
Army's official medical history of World War II, Paul and Gardner
reviewed all published reports of American, British and German
investigators from 1944 to 1946, in which feces, serum, nasopha-
ryngeal washings or urine from patients was administered to vol-
unteers by various routes (Voegt, 1942). The principal findings from
these studies are summarized below.
5.1. Infectious hepatitis
Research on infectious hepatitis achieved significant advances
during the war. The disease was shown to be caused by a filterable
agent, which survived heating at 56 C for 30 min and was trans-
missible by feeding or inoculation of fecal extracts or blood of
symptomatic patients (MacCallum and Bradley, 1944; Havens,
1945). The virus resisted chlorination and persisted in frozen
samples for more than a year. In contrast to earlier theories of
“catarrhal jaundice”, researchers found that the disease did not
spread by the respiratory route (Findlay and Willcox, 1945). The
incubation period ranged from 20 to 30 days. Stool specimens were
highly infectious during the active illness, but there was little evi-
dence that patients incubating or recovering from the disease
transmitted infection (Havens, 1946a; Francis et al., 1946).
Volunteers who had recovered from infectious hepatitis were
immune to re-infection, and soldiers who had contracted the dis-
ease in the USA were resistant to viruses circulating in the Medi-
terranean area, suggesting relative global homogeneity of virus
114 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
strains (Neefe et al., 1946). However, soldiers who became jaun-
diced after yellow fever vaccination in 1942 were not protected
from infectious hepatitis when fighting in Italy in 1943, indicating
that the causative agents were not closely related. Normal human
immune globulin, prepared from pooled donated blood, was
effective for pre-exposure prophylaxis of infectious hepatitis, as
tested in the field during the Italian campaign, suggesting that most
adult blood donors had antibodies to the virus.
5.2. Serum hepatitis
Considerable progress was also made in elucidating the etiology
of serum hepatitis, principally by inoculating volunteers with ma-
terial from soldiers who became jaundiced after yellow fever
vaccination. Two years after the epidemic, Oliphant and co-workers
reported that it had been caused by a filterable agent, which
resisted heating to 56 C and survived freezing and desiccation
(Oliphant, 1944). It could be inactivated by exposure to UV light and
by prolonged heating. Pooled normal immune globulin was less
effective in preventing serum than infectious hepatitis, indicating
that fewer members of the general population had a history of
infection.
In contrast to infectious hepatitis, the agent of serum hepatitis
was not successfully transmitted to volunteers by feeding stool
extracts of patients, and there was no evidence of cross-immunity
between the two diseases. The incubation period ranged from 60
to 150 days, and the experimental inoculation of volunteers
revealed that virus was present in serum as long as 87 days before
the onset of jaundice (Neefe et al., 1944). However, in contrast to
later studies showing the development of a silent carrier state in
some patients, virus was rarely detected in the serum of conva-
lescent patients (Havens, 1946b). Seen in retrospect, this is
consistent with the ability of most adults to recover completely
from acute hepatitis B, and explains the apparent rarity of chronic
hepatitis in soldiers who received the yellow fever vaccine.
6. From “catarrhal jaundice” to hepatitis A and B
The authority of Virchow and Osler had so firmly established
catarrhal jaundice as a clinical entity that the term was still being
used to describe cases of hepatitis during World War II. In the USA,
it was not officially replaced by “infectious hepatitis” until 1943
(Anonymous, 1943). In the same year, the term “homologous serum
jaundice” was introduced by the British Ministry of Health to
denote jaundice that followed the inoculation of blood products
(MOH, 1943).
To reduce confusion, MacCallum proposed in 1947 that the
terms “epidemic jaundice”, “infectious hepatitis” and “acute
catarrhal jaundice” all be replaced by “hepatitis A”, while those
conditions which had been known as “serum hepatitis”, “long-in-
cubation hepatitis” and “post-transfusion hepatitis” be re-named
“hepatitis B” (Anonymous, 1947). His proposal was rapidly
accepted, and in 1952, an authoritative review identified the
Table 2
Clinical differentiation of hepatitis A and B in the mid-20th century. Modified from (Stokes, 1952).
Feature
Incubation period
Secondary cases
Source of infection
Age susceptibility
Oral administration of infective serum
Heterologous immunity
Protection by gamma-globulin
characteristics of hepatitis A and B, as shown in Table 2.
The most important characteristic of hepatitis B missing from
the table is its ability to persist as a subclinical process of chronic
hepatic infection. Only with the discovery of the Australia antigen,
and its detection in the serum of a significant percentage of chil-
dren and adults in Asia and Africa, was its global importance
identified.
No further change in the terminology of hepatitis was made for
more than two decades, until the recognition that hepatitis B virus
was not responsible for all cases of post-transfusion hepatitis led to
a new, rather awkward label “non-A, non-B” in the early 1970s,
eventually to be replaced by “hepatitis C” (see below).
7. The discovery of chronic hepatitis B
7.1. Liver function tests
Wartime studies in adult volunteers revealed a great deal about
serum or long-incubation period hepatitis (now renamed hepatitis
B), but because the investigators were almost entirely limited to
clinical observations, they had difficulty recognizing patients who
did not become jaundiced. Similarly, they remained unaware that a
small percentage of patients who appeared to recover from serum
hepatitis actually remained chronically infected. However, the
progressive introduction of a variety of liver function tests per-
formed on serum samples increasingly made it possible for doctors
and researchers to detect and monitor cases of anicteric hepatitis.
The earliest assays of hepatic dysfunction were based on the
liver's role in bile metabolism, and measured levels of bilirubin in
the serum and conjugated bilirubin in the urine. Tests such as
thymol turbidity and cephalin flocculation detected alterations in
serum albumin and globulins, but provided only a nonspecific in-
dex of liver disease (Maclagan, 1947). Over time, more sensitive
quantitative assays were developed, based on the liver's role in
removing foreign substances from the bloodstream. In the brom-
sulphthalein retention test, a bolus of dye was inoculated into the
bloodstream and its clearance was measured by testing sequential
serum samples. Beginning in the 1940s, assays were also developed
that assessed damage to hepatocytes resulting in the release of
intracellular enzymes into the serum. The first to be quantitatively
measured was alkaline phosphatase, but it was subsequently found
that two other enzymes, serum glutamic-oxaloacetic transaminase
(SGOT), later renamed aspartate aminotransferase (AST), and
serum glutamic-pyruvic transaminase (SGPT), later renamed
alanine aminotransferase (ALT), were more closely associated with
liver injury (Breen and Schenker, 1971).
7.2. The discovery of chronic hepatitis
In 1937, Polack published the first report of the use of liver
function tests to detect persistent hepatic inflammation in Danish
patients who had otherwise appeared to recover from an episode of
jaundice (Polack, 1937). Many cases were in children, who clearly
Hepatitis A Hepatitis B
20e40 days 60e120 days
Common Rare
Feces, contaminated water, blood Blood
Common in childhood, rare over 40 More common over 40
Disease No disease
None None
Yes No
 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
had no history of alcoholic liver disease; he identified eight patients
in whom abnormal tests had persisted for 3e8 years.
Four years later, similar observations were made at the Uni-
versity of Rochester, where Kornberg screened hospital records and
identified 16 individuals who had been treated at some time in the
past for “catarrhal jaundice” (Kornberg, 1941). For all of them, re-
covery from the acute illness had been “rapid and uneventful,” but
10 still displayed abnormal retention of injected bilirubin in an
excretion test, while showing few or no symptoms referable to liver
disease. He concluded that “… long-lasting impairment of liver
function … is a frequent residuum of catarrhal jaundice.”
At about the same time, pathologists reported the unexpected
finding of cirrhosis in several deceased individuals with no history
of alcoholic liver disease, but who had recovered many years earlier
from an episode of catarrhal jaundice (Ratnoff and Patek, 1942).
That report stimulated another retrospective study by physicians at
Harvard Medical School, who performed liver function tests on 36
persons who had been treated for catarrhal jaundice as long as 29
years previously (Altschule and Gilligan, 1944). They were sur-
prised to discover that 9 of them had plasma bilirubin levels higher
than anyone in a normal control group, even though none reported
symptoms of chronic illness.
7.3. Silent carriers and the threat to blood transfusion
The risk of viral hepatitis posed by products containing human
serum was unmistakably revealed when the inoculation of a large
group with vaccines or immune globulin was followed by an
outbreak of jaundice. In contrast, hepatitis following transfusion of
an individual unit of blood was much more difficult to detect,
mainly because of the lengthy interval between exposure and
illness, and only very careful record-keeping would make it
possible to link a jaundiced patient to a specific donor. Such studies
were impossible under wartime conditions, but were taken up by
research hospitals in the subsequent two decades.
In 1954e6, a series of papers in the JAMA focused on the prob-
lem of chronic hepatitis carriers. In the first article, Stokes and his
colleagues reviewed research to date, based on extensive screening
with LFTs, and estimated that “… probably 0.2e0.5% of the general
[US] population carry hepatitis B virus in their blood” (Stokes et al.,
1954). They tracked blood transfusions at the University of Penn-
sylvania and identified several donors with evidence of chronic
hepatitis for at least three years. A similar study at the University of
Pennsylvania identified eight donors without a history of jaundice
whose blood had caused post-transfusion hepatitis in 14 patients
(Neefe et al., 1954). To obtain “final proof” that the transfusions
were the cause of hepatitis, NIH investigators inoculated serum
from each of the 8 donors into adult volunteers at federal peni-
tentiaries, and found that 7 sera produced hepatitis in at least one
recipient (Murray et al., 1954).
Efforts to reduce the hazard of blood transfusion took three
approaches: attempts to inactivate virus in blood products,
screening of donated blood by LFTs and exclusion of risky donors. In
an example of the first strategy, NIH researchers tried to sterilize
serum from a known infectious pool by exposing it to ultraviolet
light (Murray et al., 1955). They performed serial ten-fold dilutions
of the treated material and inoculated 1 mL aliquots into volunteers
at federal prisons. UV treatment proved to be ineffective, as hepa-
titis occurred in the recipients of both irradiated and untreated
serum down to a 10 4 dilution.
A number of research groups assessed the potential of LFT
screening of donated blood to reduce the rate of post-transfusion
hepatitis. For example, clinicians at Memorial Sloan-Kettering
found that the incidence of hepatitis in transfusion recipients was
1.5% when the SGOT of the donor blood was less than 100, but 5.5%
115
for units with higher values (Bang et al., 1959). Elevated SGOT
values were much more common in blood from paid donors.
Related studies found that persons at risk of serum hepatitis, such
as heroin addicts, frequently had elevated SGOT and SGPT levels,
and liver biopsies often confirmed the presence of chronic
inflammation (Potter et al., 1960). However, LFT screening was
clearly not the solution to the problem of post-transfusion hepa-
titis, as Norris and colleagues observed that “… the rejection of all
donors with abnormal tests would seriously handicap blood
banking” (Norris et al., 1956). Another reviewer observed that,
because tests such as thymol turbidity and SGOT were nonspecific,
the safety of transfusion could not be assured, so that the admin-
istration of a single unit of blood was never justified (Alsever, 1959).
The third approach to improving blood safety was based on the
growing awareness that professional blood donors were much
more likely to be chronic carriers of hepatitis B virus than volun-
teers (Allen and Sayman, 1962). For example, a study of open-heart
surgery patients at UCLA concluded that excluding paid donors was
a more efficient way of preventing post-transfusion hepatitis than
screening blood for abnormal LFTs (Adashek and Adashek, 1963).
Similarly, a survey of nine Boston hospitals found that those which
used blood from paid donors had a higher rate of post-transfusion
hepatitis than those which relied on volunteers (Grady and
Chalmers, 1964).
8. Experiments at the Willowbrook State School, before
discovery of the Australia antigen
As described above, hepatitis researchers during World War II
and the following decade performed numerous experiments in
prisoners, soldiers, conscientious objectors and other adult volun-
teers. This practice would understandably be objectionable by to-
day's standards, but was acceptable under the ethical criteria of the
day, in part because no one realized that infection with hepatitis B
virus could result in life-long disease. Also approved at the time, but
unacceptable by today's standards, was a long-term study at the
Willowbrook State School on Staten Island, which involved the
experimental inoculation of hundreds of severely handicapped
children and adults with hepatitis virus. The project was initiated
by New York University pediatrician Saul Krugman and his col-
leagues in the early 1950s, in an effort to understand and poten-
tially reduce the remarkably high incidence of hepatitis that
plagued the institution.
Willowbrook provided a life-long home for severely handi-
capped children and adults from the southern New York area.
When first established in the early 1940s, its population was
limited to a few hundred residents, but after 1950 the number
rapidly expanded, and by 1957 the school housed more than 5000
children and adults. The severely over-crowded conditions, plus the
difficulty of maintaining basic hygiene, resulted in a high incidence
of viral hepatitis. Most infections occurred in the first six months
after entering the school; in 1955, more than 100 cases were
recognized in residents, and 23 in staff members (Ward et al., 1958;
Krugman and Ward, 1958).
Krugman and colleagues initially assumed that they were
dealing only with infectious hepatitis, which was preventable with
gamma-globulin. In a series of studies involving several thousand
residents, they observed a dose-dependent protective effect (Ward
et al., 1958). As such passive immunity would necessarily be tran-
sient, they then decided on an approach that might achieve lasting
immunity, by treating children with gamma-globulin, then feeding
them virus-containing material obtained from the stools of symp-
tomatic patients. To determine if this “passive-active” immuniza-
tion strategy provided solid protection, they would need to
challenge their subjects with virus, rather than wait for them to be
116 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
accidentally exposed.
The plan was reviewed and approved by New York state and
Armed Forces committees, and with the consent of the parents, the
study was carried out using newly admitted children, who were
housed in a special research building isolated from the rest of the
school. The investigators found that, although immunized children
were in fact resistant to subsequent challenge with the same virus,
some of them still came down with jaundice after they joined the
general school population, indicating that more than one type of
hepatitis virus was circulating (Krugman et al., 1967). Subsequent
research at the Willowbrook School therefore focused on identi-
fying viral strains, determining routes of infection and the period of
infectivity, assessment of cross-immunity and further testing of
gamma-globulin. The findings of these extensive studies are sum-
marized below; they essentially duplicated those already obtained
in experiments employing adult volunteers during and after World
War II.
9. Discovery of the Australia antigen
By the early 1960s, hepatitis A had been well characterized: it
was highly contagious; spread by the fecal-oral route, often pro-
duced large outbreaks, was often subclinical, was always transient
and resulted in protective immunity. Hepatitis B, in contrast, was
still poorly understood. Because the agent was usually transferred
in serum, epidemics were rare, and the most significant problem
was post-transfusion hepatitis, which was difficult to study. Liver
function tests, biopsies and experimental challenge of volunteers
had shown that some persons without a history of jaundice could
harbor the virus for years. In 1962, musing on this phenomenon,
MacFarlane Burnet noted that “perhaps 1% of apparently healthy
people have the virus in their blood,” and those silent carriers are
responsible for its maintenance in human populations (Burnet,
1962). He continued “… I think the greatest intellectual prize that
a virologist can hope for is that some day he should be the first to
explain the real natural history of serum hepatitis.”
That day was only a few years away, but the key discovery was to
be made, not by a virologist, but through a collaboration between a
geneticist, a blood bank specialist and others working outside the
field of viral hepatitis. The geneticist, Baruch (“Barry”) Blumberg,
would follow up on an initial clue with a rapidly expanding series of
studies that identified the causative agent of hepatitis B, produced
new diagnostic tests that improved the safety of blood transfusion,
revealed a massive global population of chronically infected people,
identified the relationship of chronic hepatitis B to hepatocellular
carcinoma and led to the first hepatitis vaccine. His efforts were
rewarded with a Nobel Prize.
9.1. The value of widely-directed research
Barry Blumberg was a very unusual man. Born in Brooklyn and
initially educated in a yeshiva school, he developed an early fasci-
nation with explorers, and Lewis and Clark were his childhood
heroes. Following Navy service in World War II, he began graduate
studies in mathematics, but perhaps because it offered greater
opportunities for travel, he soon switched to medicine. After
studying parasitology in his third year of medical school, he spent a
summer in Surinam, where he was struck by the diversity of the
local population, which included indigenous Indians, descendants
of slaves and immigrants from Europe and Asia, and by their
apparent variation in susceptibility to local infectious diseases
(Blumberg, 2002). His first research paper, published the year he
received his M.D., described differences in the course of lymphatic
filariasis among members of those population groups (Blumberg
et al., 1951).
Blumberg's interest in human diversity continued during his
residency at Bellevue and a fellowship in arthritis, but the events
that determined his future career occurred during subsequent
graduate studies in biochemistry at the University of Oxford. First, a
lecture by E. B. Ford on natural variation in butterflies introduced
him to the concept of polymorphism, defined as “the persistence of
… two forms of a species in the same habitat in such proportions
that the rarest of them cannot be maintained by recurrent muta-
tion” (Ford, 1957; Blumberg, 1964). Applied to human health, this
implies that common variants among human populations must
provide some sort of survival benefit, and studying them may
elucidate mechanisms of disease resistance.
An opportunity to directly study this question came through
Blumberg's collaboration with Tony Allison, an Oxford researcher
who had investigated the geographic distribution of the sickle-cell
trait in Africa and had shown that it enhanced resistance to fal-
ciparum malaria (Allison, 1954). In 1957, Blumberg and Allison
traveled to Nigeria, where they collected large numbers of blood
specimens from several distinct human groups and from a variety
of domestic animals. Later that year, Blumberg joined the staff of
the US National Institutes of Health, which enabled him to carry out
his own field study in Alaska, where he again collected blood
samples, both from diverse human populations and from a number
of animal species (Fig. 1). He and Allison used starch gel electro-
phoresis to identify differences in the concentration and mobility of
haptoglobins and other serum proteins in the samples they had
collected, resulting in a number of publications (Allison et al., 1958;
Blumberg et al., 1959, 1960).
The development that led to the discovery of the Australia an-
tigen came in 1961, when it occurred to Allison and Blumberg that
human blood proteins might differ sufficiently that transfusion
recipients could develop antibodies against proteins different from
their own (Allison and Blumberg, 1961). Individuals who had
received many transfusions would be most likely to possess such
antibodies, so the researchers obtained sera from chronically ill
patients and used them to probe their extensive collection of blood
Fig. 1. Baruch S. (Barry) Blumberg on a 1958 field trip to Alaska, where he collected
blood samples from an isolated group of Inuit people. Blumberg's collection of frozen
sera from populations around the world made possible the discovery of the Australia
antigen, and was invaluable in defining the vast extent of human infection by the
hepatitis B virus. From (Blumberg, 2002), with permission.
 T.M. Block et al. / Antiviral Research 131 (2016) 109e123 117

samples, employing the technique of agar gel immunodiffusion,

which had been introduced by the Swedish bacteriologist Orjan €
Ouchterlony in the late 1940s as a method of diagnosing diphtheria
(Ouchterlony, 1949). Patient serum was placed in the central well of
an agar plate and test samples in surrounding wells; as proteins
diffused through the gel, the formation of antigen-antibody com-
plexes produced visible bands. Their study soon paid off, when they
found that a person who had received more than 50 transfusions
possessed an antibody (termed a “precipitin”) that bound a lipo-
protein in some samples in their serum collection (Allison and
Blumberg, 1961). They noted that the patient had recently begun
having febrile transfusion reactions, and proposed that the pre-
cipitin was responsible.
The discovery that some complications of blood transfusion
might be the result of diversity in serum proteins brought Blum-
berg into contact with Harvey Alter, an NIH researcher just begin-
ning his career in blood banking. Alter was investigating sources of
transfusion complications other than immunity to cellular ele-
ments, and the method of agar gel diffusion to visualize antigen-
antibody binding appeared to be just what he needed. Screening
soon revealed that a dozen multiple-transfusion recipients at the
NIH Clinical Center had precipitins in their serum (Blumberg et al.,
1964). Such antibodies were most common in patients with thal-
assemia; the antigens they bound were identified as lipoproteins by
their uptake of a lipid-binding dye, Sudan black.
Had this observation been the only outcome of the collaboration
between Blumberg and Alter, the present review might form part of
a symposium on the history of blood banking. However, because
recipients of multiple blood transfusions are at risk of hepatitis B,
and many samples in Blumberg's freezers came from geographic
regions where chronic HBV infection is highly prevalent, it was
Fig. 2. Detection of the Australia antigen by double immunodiffusion in an agarose gel
inevitable that screening would eventually produce an unusual (the Ouchterlony technique), in the initial report by Blumberg, Alter and Visnich. The
band in an Ouchterlony plate. This occurred in 1964, when Alter upper well contains serum from a leukemia patient, the lower well serum from a
used precipitin-containing serum from two hemophilia patients to patient with hemophilia. From (Blumberg et al., 1965), with permission.
screen a panel of 24 samples from Blumberg's global collection, and
in one of them e a specimen from an Australian aboriginal person e
London, Alton Sutnick and other investigators. In an initial study,
he observed a precipitin line that did not take up Sudan black, but
they followed up on observations that people with Down's syn-
instead stained strongly red with azocarmine, indicating a high
drome frequently developed leukemia, and screened blood samples
protein content (Blumberg et al., 1965). As Alter recently noted, “We
from an large institution housing mentally handicapped in-
initially called this the ‘red antigen’ for its staining properties, but
dividuals; they found that nearly 30% of Down's patients were Au-
later debated whether to call it the Bethesda antigen for the place
positive (Sutnick et al., 1968; London et al., 1969). However, in a
where it was identified, or the Australia antigen for the person in
follow-up investigation focusing on persons with the same diag-
whom it was found” (Alter, 2014). The designation Australia antigen
nosis, but who lived in smaller institutions, they found only a 3%
(Au) was ultimately chosen, following the current nomenclature for
prevalence of Au, and when they screened newborns with Down's
hemoglobin variants.
syndrome and older patients living with their parents, none was
Alter was unable to detect the antigen in a collection of 150
antigen positive (Sutnick et al., 1968). This striking association be-
samples representing a healthy US population, but when he turned
tween antigenemia and the subject's social setting was the first
his attention to patients at the NIH, he found it in the serum of 11 of
indication that Au might be a trait acquired under the conditions of
107 individuals who had received 10 or more transfusions; 8 of
crowding and poor sanitation that characterized large institutions
them were hemophilia cases (Fig. 2). Additional testing yielded
for severely mentally handicapped people, and might therefore be
even more interesting results: although none of 700 normal
of infectious origin.
American blood donors was positive for Au, the antigen was pre-
sent in the serum of 8 of 70 leukemia patients (Alter and Blumberg,
1966). As there was considerable speculation at that time that 9.3. Linking Au to viral hepatitis
leukemia originated through viral infection, it seemed possible that
Au was a component of a “leukemia virus.” However, Blumberg's As the investigators continued to screen serum samples for Au,
focus on human polymorphism suggested an alternative explana- they found it in patients with a puzzling variety of conditions,
tion, that Au was a human protein associated with an inherent ranging from lepromatous leprosy to lymphocytic and granulocytic
predisposition to leukemia. leukemia, renal failure, thalassemia, hemophilia and other diseases
that required multiple transfusions. Testing of the large numbers of
9.2. Initial characterization of the Australia antigen sera collected by Blumberg on his global travels also revealed that
Au was common among the populations of Oceania and other
Further investigation of Au as a marker of disease susceptibility tropical regions.
followed Blumberg's move from NIH to the Institute for Cancer Had a series of chance observations not come to the rescue, it
Research in Philadelphia, where he joined forces with Thomas might have taken years for the researchers to discover a common
118 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
factor among these diverse groups. The first was made by one of
Blumberg's technologists, Barbara Walter, whose laboratory spe-
cialty was producing purified Au by sucrose gradient centrifugation
of serum samples. She had routinely served as the lab's Au-negative
control, but when she became ill and noticed that her urine had
turned darker than normal, she found that her own serum had
become Au-positive (but only on a single day) (Blumberg et al.,
1968). She recalled no accidental needle sticks or other obvious
exposure to Au-containing material. Second, an antigen-negative
adult with Down's syndrome in a cohort being followed by Sut-
nick and London became ill, with abnormal liver function tests, and
Au appeared in his serum (Sutnick et al., 1968). Further screening of
institutionalized Down's patients found that many were persis-
tently Au-positive; their sera also had elevated SGPT levels, indic-
ative of hepatitis, which was confirmed in some cases by liver
biopsy. Suspicion of an infectious etiology was further strength-
ened when an antigen-negative investigator who had immunized
rabbits with Au-positive serum himself developed a high titer of Au,
though without becoming ill.
In addition to these individual observations, the link between
Au and hepatitis was more firmly established when the antigen was
detected in 20% of 125 acute viral hepatitis patients in Philadelphia
and New York, but in none of 138 patients with other liver diseases
(London et al., 1969). It was much more common in patients with
posttransfusion hepatitis than with infectious hepatitis; in retro-
spect, detecting Au in patients with a clinical diagnosis of infectious
hepatitis simply represented the inherent inaccuracy of a diagnosis
based only on a patient's history. The antigen rapidly disappeared
from the serum of most convalescent hepatitis patients, but per-
sisted for years in some people with leukemia, thalassemia and
renal failure, and was also chronically present in many residents of
tropical countries. When Blumberg and colleagues made use of
liver function tests to screen these Au-positive individuals, they
found that many of them had elevated serum SGPT (ALT) levels,
indicating that they suffered from chronic hepatitis (Blumberg
et al., 1968).
Taking a similar approach to Blumberg and Alter, but publishing
somewhat later, Alfred Prince at the New York Blood Center
attempted to identify an agent of hepatitis B by testing sequential
serum samples from patients who had received multiple trans-
fusions. He also made use of the Ouchterlony double-diffusion
method, with test serum from an individual with hemophilia
who had received more than 10,000 units of blood products over
his lifetime. Prince identified a number of patients with post-
transfusion hepatitis, in whom a rise in serum SGPT (ALT) was
accompanied by the appearance of a precipitin band, which he
designated “SH” (Prince, 1968a). Using samples provided by Saul
Krugman, he demonstrated a similar antigen in blood samples from
children with long-incubation hepatitis at the Willowbrook State
School. Later that year, after carrying out parallel testing, Prince
concluded that “the Australia antigen and SH antigen are closely
related, and perhaps identical” (Prince, 1968b).
Additional evidence linking Au to hepatitis was reported by
Okochi and colleagues in Japan, who screened transfused blood and
found that patients who received antigen-positive units subse-
quently developed abnormal liver function tests and became
antigen-positive (Okochi and Murakami, 1968). A virus connection
was further supported when electron microscopy of sucrose
gradient-fractionated serum samples revealed 20-nm particles
resembling picornaviruses, which formed large aggregates when
anti-Au antibodies were added (Bayer et al., 1968). In a similar
study, particles concentrated by cesium chloride gradient were
seen to resemble small virions, but they contained little nucleic acid
(Millman et al., 1969). The authors therefore proposed that “the
bulk of material identified as Australia antigen may be an
incomplete virus or virus capsid,” as was subsequently found to be
the case. A year later, Dane and colleagues were able to demon-
strate by immune-EM that HBsAg existed as both circular and
tubular sub-viral particles and as complete enveloped virions,
which came to be known as “Dane particles” (Dane et al., 1970).
9.4. Further studies at the Willowbrook State School
In 1967, Krugman and his colleagues reviewed data obtained
during the previous decade and presented evidence for the exis-
tence among their school population of two types of infectious
hepatitis, which they labeled MS-1 and MS-2 (Krugman et al.,
1967). The MS-1 agent had a short incubation period, was readily
spread by the oral route and sometimes by injection, while MS-2
had a longer incubation period, was transmissible by injection
and occasionally through oral feeding. Gamma-globulin prophy-
laxis prevented MS-1 infection, but not MS-2. Once it became
possible to test for Australia antigen, the researchers began
screening the thousands of serum samples in their freezers, and
found that Au was absent from the serum of children who had been
inoculated with MS-1 serum, but present in MS-2 infections,
identifying it as hepatitis B (Giles et al., 1969; Krugman and Giles,
1970). Of greatest concern from our current ethical viewpoint,
some children and adults given MS-2 serum by intramuscular in-
jection or oral feeding became chronically infected, and may ulti-
mately have developed cirrhosis or hepatocellular carcinoma.
One of the few discoveries made at the Willowbrook School that
was directly beneficial to the experimental subjects occurred by
accident, as the investigators were assessing methods of inacti-
vating virus-containing material. It was already known that MS-1
and MS-2 sera kept at 56 C for 30 min remained infectious, so
the investigators decided to determine the effect of placing vials for
1 min in boiling water. Children fed the boiled MS-1 serum did not
develop hepatitis, but also were not resistant to a subsequent
challenge oral challenge with the virus (Krugman et al., 1970).
Inoculation of boiled MS-2 serum also did not produce hepatitis,
but in contrast to MS-1, the recipients became resistant to subse-
quent virus challenge (Krugman et al., 1971). These observations
suggested the possibility of a heat-inactivated hepatitis B vaccine.
In retrospect, the experimental infection of hundreds of
mentally handicapped children and adults at Willowbrook State
School produced little new knowledge. Much of the information
duplicated findings obtained in adult volunteers during World War
II; for example, the announcement in 1967 that two types of hep-
atitis virus circulated at the school came 20 years after MacCallum's
designation of viral hepatitis A and B. However, the vast collection
of well-characterized serum samples from Willowbrook patients
was later employed by researchers to test new diagnostic methods,
to characterize HBV antigens and antibody responses and for the
identification of hepatitis A virus.
9.5. Proving causation: satisfying Koch's postulates
Less than four years after the initial report of a “new” antigen in
leukemia serum, screening of samples from Blumberg's vast global
collection (which is today stored at the Hepatitis B Foundation's
Baruch S. Blumberg Institute) allowed him to state that “tens of
millions of asymptomatic people carry the antigen chronically,” and
that “it is closely associated with, or may itself be, a causative agent
of viral hepatitis” (Blumberg et al., 1969). However, proof of a causal
role of Au in hepatitis would not be straightforward. Robert Koch's
classic prescription for proving the microbial etiology of a disease
called for an agent to be isolated from a sick individual, propagated
in pure culture and inoculated into a healthy subject, in which it
must produce the same disease and from which the agent can in
 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
turn be isolated. This proved impossible for viral hepatitis:
although several reports had been published describing the isola-
tion of a virus in tissue culture or the production of diseases in a
laboratory animal, none of the claims had held up on further
testing.
Establishing a causal link between Au and hepatitis B would
therefore, as a minimum, require evidence that the antigen was
present in sites of hepatic injury, and that it increased in quantity
like a replicating agent over the course of illness. The first condition
was satisfied when Blumberg and colleagues found that
fluorescein-labeled anti-Au serum bound to granules in the nuclei
of hepatocytes in liver biopsies of hepatitis B patients (Millman
et al., 1969). EM studies of biopsied tissue subsequently revealed
aggregates of virus-like particles within hepatocyte nuclei, which
bound antibody-coupled ferritin (Huang et al., 1972).
Convincing evidence that Au was a component of a replicating
agent was obtained by Barker and colleagues, employing serum
samples kept frozen since the mid-1950s. In the earlier study
summarized above, Moore et al. inoculated volunteers with serial
dilutions of untreated or UV-irradiated serum from a chronically
antigenemic individual (Murray et al., 1955). The original untreated
serum produced clinical hepatitis in most recipients, but as it was
progressively diluted, illness became less common, and volunteers
who received plasma diluted 10 4 or more appeared to remain
healthy. However, when Barker's group tested samples from the
volunteers, they found that even those who had received as little as
a 10 7 dilution became antigenemic 40e120 days after inoculation,
indicating that the agent replicated, even if it did not cause illness
(Barker et al., 1970). The authors concluded that “HAA [hepatitis-
associated antigen] is a virus commonly associated with long-
incubation or serum hepatitis.”
In a follow-up study, Barker and colleagues obtained further
evidence of a causal role of Au in hepatitis B, by inoculating
chimpanzees with the same stock of pooled plasma from the 1955
study (Barker et al., 1973). The animals showed a range of responses
similar to those seen in humans: none became visibly ill, but two
showed biochemical and histologic abnormalities, with antigen
demonstrable in hepatocytes by immunofluorescence; two had
antigen in serum, followed by the development of specific antibody,
and a fifth showed only the late development of anti-HBs antibody
(which is now known to be equivalent to anti-Au antibody). The
findings were confirmed in a collaborative study by CDC in-
vestigators and Purcell's lab at NIH, employing Willowbrook MS-2
serum as the infective material, which found that neither of two
inoculated chimps became ill, but one became antigenemic 70 days
after challenge (Maynard et al., 1972; Purcell, 1993).
10. Applications of Au screening and further discoveries
10.1. Clearing the blood supply
Even before Au had been firmly linked to hepatitis B, Alter and
Holland considered the evidence sufficient to call for the screening
of all donated blood before transfusion (Alter et al., 1970). The
recognition that Au was especially common in paid donors also led
to the increasing adoption of an all-volunteer system by hospital
blood banks. In a few years, these two measures resulted in a
greater than 70% reduction in transfusion-associated hepatitis
(Alter et al., 1975). As described below, the failure of screening to
completely eliminate the problem led to the recognition of “non-A,
non-B” hepatitis and the eventual discovery of the hepatitis C virus.
10.2. The identification of hepatitis A virus
Wartime studies in adult volunteers, followed by experiments at
119
the Willowbrook State School, clearly established that the agent of
hepatitis A was shed in large quantities in feces during the late
incubation period and the course of illness. Once Albert Kapikian
and his colleagues at the NIH had pioneered the method of
immuno-electron microscopy to identify Norwalk virus in stool
specimens (Kapikian et al., 1972), it was relatively straightforward
for a new NIH investigator, Stephen Feinstone, to apply the same
technique to hepatitis A (Feinstone et al., 1973).
To make sure that the pathogen they identified was in fact the
agent they sought, the investigators used the well characterized
Willowbrook MS-1 virus to infect adult volunteers. Stools were
collected before and during the course of illness, and a saline sus-
pension was filtered, then mixed with antibodies from individuals
who had recovered from the same infection. Examination by elec-
tron microscopy revealed clumps of virus-like particles in the stools
of subjects during the course of illness, but not in those collected
before infection. No antigenic cross-reactivity was found between
the newly discovered agent and the hepatitis B virus.
10.3. From “non-A, non-B” hepatitis to hepatitis C
Within a few years after the discovery of the Australia antigen,
efforts by Alter and his colleagues had made screening for HBsAg
mandatory in US blood banks. It was hoped that rigorous testing,
combined with the exclusion of paid donors, would put an end to
post-transfusion hepatitis, but although a significant decrease was
seen, cases continued to occur. When the researchers examined the
situation in 1972, it seemed possible that screening might simply
have missed some hepatitis B-infected donors with low antigen
levels (Alter et al., 1972), but the introduction of the much more
sensitive radioimmunoassay soon made it clear that another
pathogen must be responsible.
By the 10th anniversary of the identification of the Australia
antigen, investigators had become convinced of the existence of an
additional agent of serum-transmitted hepatitis that also produced
a chronic carrier state. In New York City, Prince and colleagues
followed 204 open-heart surgery patients and identified 36 cases of
post-transfusion hepatitis that were negative for HBsAg by radio-
immunoassay and negative for cytomegalovirus (Prince et al., 1974).
At the NIH, investigators studied 22 patients with post-transfusion,
HBsAg-negative hepatitis, and were able to exclude the recently
identified hepatitis A virus by the lack of an antibody response
(Feinstone et al., 1975). In 1978, two groups proved the existence of
a novel infectious agent, by inoculating chimpanzees with serum
from blood donors who had been the source of non-A, non-B
hepatitis (Alter et al., 1978; Tabor et al., 1978). Although the animals
did not become ill, they displayed elevations in circulating ALT, AST
and bilirubin and typical inflammatory changes on liver biopsy.
Largely because the level of circulating virus in patients with
chronic hepatitis C is some thousand-fold lower than in hepatitis B,
making it impossible to visualize viral particles by immuno-EM, the
pathogen continued to evade researchers for more than a decade.
Only in 1989 did the virus finally yield to an assault employing all
available molecular tools, when RNA viral sequences were cloned
from the blood of a patient with chronic non-A, non-B hepatitis
(Choo et al., 1989). Within a few years, blood banks added screening
for HCV to that already in place for HBsAg, virtually eliminating
viral hepatitis as a hazard of blood transfusion.
10.4. Discovery of the delta agent
During the decade following the discovery of the Australia an-
tigen, hepatitis B was becoming well characterized, but in 1977 an
unexpected element appeared with a report from Mario Rizzetto's
lab in Torino, Italy. While studying the cellular distribution of HBV
120 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
antigens in liver biopsies, Rizzetto and colleagues noted that a FITC-
labeled anti-HBc antiserum produced positive staining both in bi-
opsies in which core particles were visible by EM and in some in
which no core particles could be found. They therefore reported the
existence of a novel HBV antigen distinct from HBs, c or e, which
they named “delta” (Rizzetto et al., 1977). The new antigen was only
present in the serum of patients with circulating HBsAg, and it was
associated with the presence of severe liver damage.
Subsequent studies in HBV-infected chimpanzees, aided by the
development of a highly sensitive radioimmunoassay, established
that the delta antigen was not a component of HBV, but was a
product of replication of a second infectious agent that requires
HBV for its own replication. The novel viroid-like defective virus,
designated hepatitis D virus (HDV), employs a form of HBsAg as its
own surface antigen (Rizzetto et al., 1980, 1981). Its 1.7 kb genome
makes it the smallest agent known to infect humans. The charac-
teristics of HDV, its replication in humans and efforts at antiviral
therapy are reviewed in detail by Alfaiate et al. (Alfaiate et al., 2015).
10.5. Discovery of hepatitis E virus
Just as HBV was found not to be the only cause of transfusion-
associated hepatitis, by the late 1970s it was recognized that HAV
was not the only cause of “infectious” hepatitis. An outbreak of
water-borne disease in Kashmir in 1978 resulted in more than
50,000 recognized cases and nearly 2000 deaths, but patients
showed no serologic evidence of HAV infection (Khuroo, 1980).
When a similar disease occurred among Soviet troops in
Afghanistan, Russian virologist Mikhail Balayan heroically investi-
gated its pathogenesis by swallowing filtered fecal extracts,
resulting in acute hepatitis and the identification of virus-like
particles in his own stool by immune-EM (Balayan et al., 1983).
The virus was then further characterized, including the production
of hepatitis in cynomolgus macaques, in collaborative studies by
scientists in Moscow and the US CDC (Bradley et al., 1988).
The hepatitis E virus (HEV) is a positive-sense, single-stranded
RNA virus, the sole member of the family Hepeviridae. Hepatitis E is
the most common form of acute hepatitis worldwide; the World
Health Organization estimates that some 20 million infections
occur each year, with 3 million symptomatic cases and 60,000
deaths. Genotypes 1 and 2 circulate among humans in southern
Asia and Africa, and may cause fulminant hepatitis in pregnant
women. A licensed vaccine is in use in China. In contrast, genotypes
3 and 4 are maintained in wild and domestic swine, principally in
Europe, and cause sporadic human infections through ingestion of
undercooked pork (Khuroo and Khuroo, 2016). Chronic hepatitis E
in immunocompromised people has been treated with pegylated
interferon and ribavirin (Debing and Neyts, 2014).
11. Further studies of hepatitis B
11.1. Association with liver cancer
By the late 1960s, hepatocellular carcinoma (HCC, hepatoma)
had been recognized to occur most commonly in hepatitis patients
with postnecrotic cirrhosis, suggesting that chronic Au-
antigenemia might be predictive of cancer. Blumberg's first
attempt to detect such a link was unsuccessful, as a survey of 65
hepatoma patients in Hong Kong, East Africa and the USA found a
similar prevalence of antigen as in other liver diseases (Smith and
Blumberg, 1969). However, within two years Sutnick, London and
Blumberg were able to cite five published papers reporting a sig-
nificant association between chronic hepatitis B and HCC, including
a 31% prevalence of serum Au among hepatoma patients in
Australia, 31% in Greece and 40% in Uganda (Sutnick et al., 1971).
Reviewing the high prevalence of Au HCC in Africa and Asia,
Sutnick and colleagues proposed in 1972 that cancer results from a
series of selections: of all people with acute hepatitis B, a fraction
become chronically infected; some of those develop chronic hep-
atitis, which in a smaller subset leads to postnecrotic cirrhosis, of
which a still smaller fraction develop cancer (Fig. 3) (Sutnick et al.,
1972). These speculations were eventually confirmed by long-term
prospective studies of large cohorts of HBsAg-positive and -nega-
tive individuals in Taiwan, which revealed that the former had a
200-fold greater risk of developing HCC (Beasley et al., 1981, 1983).
This association was also strongly supported by elegant epidemi-
ologic studies in South Africa (Kew et al., 1983).
11.2. Mechanisms of chronic hepatitis B and oncogenesis
There is now general agreement among researchers that HBV
replication rarely kills hepatocytes; instead, cellular injury and
necrosis result primarily from the host's immune assault against
cells expressing viral antigens, particularly HBcAg. The attack is
mediated by cytotoxic T cells and inflammatory cytokines, and the
degree of damage often correlates directly with the patient's im-
mune status (Chisari and Ferrari, 1995). The recognition that
chronic hepatitis B is largely immune-mediated, and that a
persistent necroinflammatory response determines the outcome of
infection, has helped to elucidate the pathogenesis of the disease.
In contrast, the molecular mechanisms by which HBV causes
hepatocellular carcinoma remain elusive. Viral DNA enters the
hepatocyte nucleus, but no specific oncogenes have been recog-
nized, and viral insertion into the human genome appears to be a
random event. Carcinogenesis may therefore simply be a side-
effect of prolonged viral infection and the opportunities it pro-
vides for chance mutations in the regenerating liver, especially in
patients with cirrhosis (Block et al., 2003). This concept would
explain the increased frequency of HCC that is also seen in persons
with other types of liver disease, including chronic hepatitis C,
alcoholic liver disease, non-alcoholic steatohepatitis and hemo-
chromatosis. More complex mechanisms, yet to be elucidated, are
probably involved as well.
11.3. HBV vaccines and the interruption of perinatal transmission
The ability to screen serum for Au revealed an immense global
burden of chronic hepatitis B, especially in Africa and Asia, making
the development of a preventive vaccine a priority. However the
traditional approach to vaccine development, by attenuating a virus
through sequential passage in tissue culture, was not possible in
this case, as HBV could not be induced to replicate in vitro. Blum-
berg and his colleagues therefore made use of the only available
source of antigen, and devised a vaccine based on purified, inacti-
vated HBsAg recovered from the serum of patients with chronic
hepatitis B (Blumberg and Millman, 1972).
Although the prototype vaccine generated solid immunity in
chimpanzees (Buynak et al., 1976; Purcell and Gerin, 1978) and was
protective in humans (Szmuness et al., 1981), its acceptance by the
Fig. 3. An early proposal by Sutnick and colleagues for the etiology of hepatocellular
carcinoma arising from chronic hepatitis B infection. From (Sutnick et al., 1972), with
permission.
 T.M. Block et al. / Antiviral Research 131 (2016) 109e123
public health community was limited by concerns about possible
contamination with other human pathogens, particularly HIV. The
subsequent development of a vaccine consisting entirely of yeast-
expressed gene products was therefore an important advance
(McAleer et al., 1983). All current HBV vaccines contain recombi-
nant microorganism-generated polypeptides.
Once large-scale vaccination was under way, the most dramatic
proof of its benefit came from Taiwan, where studies showed that
the risk that a child born to an HBsAg/HBeAg-positive mother
would become a chronic carrier diminished from 90% to less than
5% if the newborn received the HBV vaccine and hepatitis B gamma
globulin (Beasley et al., 1983; Chen et al., 1987). A 30-year retro-
spective study has documented the striking reduction in the inci-
dence of HCC in Taiwan resulting from vaccination (Chiang et al.,
2013).
11.4. The first wave of antiviral therapies
Because clinicians initially proposed that chronic hepatitis B
resulted from an impaired immune response, exogenous stimula-
tion with interferon-a was the first therapy attempted for the dis-
ease (Hoofnagle et al., 1986; Perrillo et al., 1990). Unfortunately,
interferon is less than ideal, as it requires parenteral administration,
adverse reactions are common and its efficacy is limited. Patients
with elevated serum ALT levels and low-to-moderate viremia were
the most responsive to IFN treatment, but only a minority were able
to clear the virus and achieve lasting clinical benefit (Korenman
et al., 1991). Nonetheless, the fact that some 5e8% of IFN re-
cipients eventually lost circulating HBsAg and developed anti-
HBsAg antibody provided a somewhat convincing demonstration
that chronic hepatitis B was “treatable,” and in some cases even
“curable” (Asselah et al., 2007).
The other approach to the management of chronic hepatitis B
has made use of antiviral drugs originally developed for the treat-
ment of HIV infection. The discovery that HBV, despite having a
DNA genome, replicates through an RNA intermediate, using a
virus-specified reverse transcriptase (RT), was one of the major
surprises in 20th century virology (Summers and Mason, 1982).
This unusual replication mechanism proved to be more than a
laboratory curiosity, as screening showed that several drugs
developed for the treatment of HIV infection were also active
against HBV (Gish et al., 2015a).
Lamivudine was the first RT inhibitor to be used for the man-
agement of hepatitis B. Its introduction proved to be a break-
through, making it possible to rapidly control HBV viral loads with
an oral drug. Although such therapy was not curative, it was still
highly beneficial, as long-term treatment was shown to halt pro-
gression to liver cirrhosis, and even reverse the process, which had
not previously been thought possible (Lai et al., 1998; Villeneuve
et al., 2000; Bonis et al., 2001; Chang et al., 2010). The HBV poly-
merase therefore became the leading target for the development of
therapeutics, and at least five orally active drugs are now available.
RT inhibitors are well tolerated and can achieve impressive re-
ductions of viremia in most patients, but they require continuous
use and can be foiled by the evolution of drug-resistant viral vari-
ants (Locarnini, 2005). Their benefit is also limited: even after 5e10
years of effective viral suppression, death from cancer or cirrhosis is
only reduced by 50e60% (Lok, 2011; Gordon et al., 2014).
12. The search for a cure
Because of the persistence of viral cccDNA in the nuclei of
infected hepatocytes, currently available antiviral therapy cannot
“cure” chronic hepatitis B, except in a very small proportion of
cases. Thus, although current medications provide a major
121
therapeutic benefit, the severe consequences of chronic hepatitis B
continue to affect vast numbers of the global population.
A new wave of antiviral strategies, aimed at eradicating HBV
from the liver and achieving a definitive cure, are the subject of
articles in the present symposium in Antiviral Research. These novel
approaches target a variety of steps in the viral life cycle. The
introductory paper reviews the range of antiviral approaches
currently under development (Block et al., 2015), and a second
describes the HBV replication cycle, the course of illness and cur-
rent therapies (Gish et al., 2015a). A third article examines the use
of murine models to study the pathogenesis of chronic hepatitis B
and for antiviral drug evaluation (Cheng et al., 2015); a fourth
provides a comprehensive review of co-infection with the hepatitis
delta virus (Alfaiate et al., 2015); and a fifth discusses the cell sur-
face HBV receptor and its potential as a target for antiviral therapy
(Yan et al., 2015).
The remaining articles in the symposium cover the following
experimental approaches to therapy:
 cccDNA as a potential antiviral target (Guo and Guo, 2015);
 new drugs providing more effective suppression of the HBV
reverse transcriptase (Clark and Hu, 2015)
 therapeutic vaccines to potentiate host immune response
(Zhang et al., 2015);
 the use of immunomodulators to enhance anti-HBV responses
(Isorce et al., 2015);
 strategies employing RNAi to suppress HBsAg production (Gish
et al., 2015b);
 pharmacological activation of innate antiviral responses (Chang
and Guo, 2015);
 the HBV core protein as a potential drug target (Zlotnick et al.,
2015);
 the possible use of CRISPR/Cas9 technology to eliminate cccDNA
(Kennedy et al., 2015);
 the viral ribonuclease H as a target for antiviral therapy (Tavis
and Lomonosova, 2015).
A final paper provides a computation chemistry perspective on
drug development (Morgnanesi et al., 2015).
Under various names, hepatitis B has been the subject of med-
ical investigation and laboratory research for 150 years, but prog-
ress in diagnosing the disease, identifying chronic carriers and
preventing further transmission was markedly accelerated by the
discovery of the Australia antigen 50 years ago. It is our hope that
current research efforts aimed at finding a definitive cure, in
combination with global infant vaccination, will lead to the even-
tual eradication of the disease.
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