Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

History of the Discovery of Hepatitis A Virus

Stephen M. Feinstone

Department of Biochemistry and Molecular Medicine, George Washington University School of Medicine, Washington, D.C. 20037 Correspondence: [email protected]

Disease outbreaks resembling hepatitis A have been known since antiquity. However, it was not until World War II when two forms of viral hepatitis were clearly differentiated. After the discovery of Australia antigen and its association with hepatitis B, similar method- ologies were used to find the hepatitis A virus. The virus was ultimately identified when investigators changed the focus of their search from serum to feces and applied appropriate technology.

iseases resembling hepatitis A, both in DIFFERENTIATION OF TWO FORMS OF Dindividuals and in outbreaks involving VIRAL HEPATITIS groups, were reported in China as early as 5000 years ago. Hippocrates noted a disease Viral hepatitis was a major problem for both the he called benign epidemic jaundice in “De Allies and the Axis during World War II. Early Morbis Internis” that certainly resembled hep- in the war, an outbreak of hepatitis related to atitis A. More accurate descriptions of hepatitis yellow fever vaccine, stabilized with human se- A began appearing in the 17th century often rum involving 49,233 clinically apparent cases associated with military campaigns. The first (Seeff et al. 1987), prompted a major hepatitis outbreak recorded in the United States was research effort. As the records on the vaccinees in 1812 in Norfolk, VA, and the disease was were very good, the incubation period was de- common among the Union troops during fined accurately as between 60 and 154 days.

www.perspectivesinmedicine.org the Civil War with more than 40,000 cases re- This outbreak as well as the more widespread ported. The association of hepatitis A with war problem of infectious hepatitis caused both the led to 19th-century terms such as “kriegsik- British and the Americans to initiate studies of terus” or “jaunisse des camps.” Hepatitis A viral hepatitis. It became clear that one form continued to afflict troops on both sides during of hepatitis spread quickly among troops and World War I and in the Second World War by 1943 hepatitis had become a real hindrance when there were estimates of 16 million cases of the war effort in North Africa and the of hepatitis among combatants and civilians Mediterranean. U.S. Army epidemiologic stud- (Sherlock 1984; Gust and Feinstone 1988; Fon- ies showed that epidemic or infectious hepatitis seca 2010). had a much shorter incubation period than se-

Editors: Stanley M. Lemon and Christopher Walker Additional Perspectives on Enteric Hepatitis Viruses available at www.perspectivesinmedicine.org Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740

1 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

S.M. Feinstone

rum hepatitis—18 to 25 days compared with a infections of some children and were controver- mean of 90 days for hepatitis following the yel- sial even during the era in which they were low fever vaccine (Havens 1968). Second, they conducted. They were justified by the investiga- found that some soldiers that had developed tors based on their belief that every child who hepatitis after immunization with the yellow entered the facility developed hepatitis soon fever vaccine could still develop a second bout after arrival. In the course of these studies, the of infectious hepatitis. They also noted that investigators identified a child with two distinct officers were more likely to develop infectious bouts of hepatitis from whom serum samples hepatitis than enlisted men and that the disease had been collected during the acute and recov- often followed outbreaks of diarrheal disease, ery stages of each bout. Additional studies suggesting a fecal–oral mechanism of spread. with these two samples, termed MS-1 for short These epidemiologic studies were accompa- incubation (infectious) hepatitis and MS-2 for nied by experimental infections of humans per- long incubation (serum) hepatitis, further de- formed by the Americans and British as well fined two distinct hepatitis viruses and, most as the Germans (Voegt 1942; Neefe et al. 1944, importantly, established a well-characterized, 1946; MacCallum et al. 1951). Transmission to known infectious inoculum for each (Krugman volunteers was not always successful. Preexisting et al. 1967). Krugman also determined the immunity in a high proportion of volunteers, as general period of infectivity for persons infected well as the inability to know whether any given with these viruses, showed that serum hepatitis inoculum was actually infectious, caused the often became chronic, and that standard results of these studies to be difficult to interpret. pooled human immune serum globulin was Nevertheless, these studies pointed to two protective against infectious hepatitis (Krugman distinct diseases, one with a short incubation and Giles 1970, 1972). This latter finding con- period transmitted by the fecal–oral route and firmed earlier studies done by the U.S. Army the other transmitted by serum with a relatively during World War II (Gellis et al. 1945). long incubation period. The investigators also Because of the continuing threat to military showed a lack of cross immunity between the operations posed by infectious hepatitis, the U.S. two types of infections and that some of the Army continued to sponsor research after the physical characteristics of the causative agents war under the aegis of the Armed Forces Epide- were distinct. These two forms of hepatitis miological Board. To obtain larger quantities became known generally as “infectious hepati- of the infectious hepatitis inoculum, the army tis” and “serum hepatitis.” It was not until the contracted for studies in which the Krugman early 1950s that the first description of these MS-1 inoculum was amplified by infecting www.perspectivesinmedicine.org diseases as “type A” and “type B” viral hepatitis volunteer inmates at the federal prison in Joliet, appeared in a report from an expert committee IL (Boggs et al. 1970; Melnick and Boggs 1972). of the World Health Organization (MacCallum Volunteers were inoculated with the MS-1 strain 1953). of hepatitis A, and the investigators collected Following the war, virology entered its gold- stools and serum samples throughout the course en era with the advent of tissue culture made of the illness, from the incubation period to possible by the development of defined media acute disease, and then during convalescence. and antibiotics (Robbins and Enders 1950). In addition, MS-1 infection was passed from Many viral agents were identified during this one volunteer to another. period but neither hepatitis agent was success- fully propagated in cell culture. Saul Krugman, EARLY ATTEMPTS TO ISOLATE AN Robert Ward, and Joan Giles conducted studies INFECTIOUS HEPATITIS A AGENT between 1956 and the early 1970s at the Willow- brook State School for intellectually handi- In early quests to isolate an infectious agent, capped children on Staten Island, New York. classic virologic methods, including inocula- These studies involved deliberate experimental tions of laboratory animals and embryonated

2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

History of the Discovery of Hepatitis A Virus

chicken eggs with hepatitis-related material, did Friedrich Deinhardt working at the Presbyteri- not yield positive results. With the development an-St. Lukes Hospital in Chicago, IL, reported of cell culture as a routine laboratory technolo- that a human hepatitis virus could be passaged gy, scientists could regularly propagate mamma- in marmoset monkeys (Saguinus sp., New lian cells in vitro and identify many viruses World nonhuman primates now generally known to be associated with human diseases considered to be tamarins) and that infected (Robbins and Enders 1950). animals developed evidence of liver disease Various cell culture methods were applied to (Deinhardt et al. 1967). One hepatitis-inducing the identification of the causative agent of hep- inoculum, GB, came from a surgeon who had atitis A (MacCallum 1972), but the most exten- developed hepatitis without a known exposure. sively reported study was performed by Wilton Deinhardt did extensive work to characterize the Rightsel and his colleagues at the Parke-Davis GB agent, but he could not specifically associate Company using a cell line termed Detroit-6, it with hepatitis A. Wade Parks and Joseph Mel- which was derived from human bone marrow nick, at Baylor College of Medicine in Houston, (Rightsel et al. 1956, 1961). These investigators TX, determined that the GB agent was likely a identified a cytopathic effect (CPE) in cells marmoset virus, but they also were unable to inoculated with various specimens thought to prove that these primates could be infected contain infectious virus. This CPE could be with the MS-1 virus (Parks and Melnick 1969; neutralized by convalescent serum from pa- Parks et al. 1969). The story of GB resurfaced tients with hepatitis A–like illnesses. Although in 1995 when two viruses, GBV-A and GBV-B, the neutralization studies were presumptive were identified by molecular cloning in animals evidence for specificity, further studies were in- infected with the eleventh animal passage of consistent for both the CPE and neutralization. the GB agent (Simons et al. 1995). Neither of The Detroit-6 cell culture studies were eventu- these agents infect humans. A related virus, ally abandoned at Parke-Davis, but Ruth Cole, a GBV-C, has been identified in humans, but member of the Parke-Davis team, returned to it is of very low or nonexistent pathogenicity. Melbourne, Australia, where she continued stud- This group of viruses are classified within the ies with Detroit-6 cells. Working at Fairfield Flaviviridae family, distantly related to classic Hospital, where almost every patient with hep- flaviviruses like dengue virus and to hepatitis atitis in the region was admitted, Cole and col- C virus (HCV) (Stapleton 2003). leagues performed a number of studies using various clinical inocula (Cole 1965; Ferris and SEROLOGIC STUDIES Cole 1966). Again, CPE was observed but sub- www.perspectivesinmedicine.org sequent independent studies, done with coded In 1965, while looking for serum protein poly- samples including multiple control samples, morphisms that would distinguish population failed to show that the observed CPE was spe- groups from around the world, Baruch Blum- cific for hepatitis A (Cross and Marmion 1966). berg at the Institute for Cancer Research in A group led by Joseph Melnick also studied Philadelphia, PA, and Harvey Alter and Sam infectious hepatitis in the Detroit-6 system using Visnich at the National Institutes of Health an MS-1 inoculum from the Joliet studies, and (NIH) in Bethesda, MD, observed a precipitin concluded that the observed CPE was caused band in an immunodiffusion gel reaction be- by a contaminant, most likely a parvovirus tween serum from a multiply transfused hemo- (Mirkovic et al. 1971). philiac (the potential source of antibody) and an Australian aboriginal man (Blumberg et al. 1965). They did not immediately understand the ANIMAL STUDIES nature of the antigen, but within a few years it Early studies suggested that the agent responsi- became clear that it was associated with hepatitis ble for hepatitis A was unable to infect any com- and then specifically with hepatitis B (Prince mon small laboratory animal. In 1967, however, 1968). However, the idea that one could use a

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 3 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

S.M. Feinstone

iac patients in a manner similar to the original studies that identified Australia antigen. In retrospect, although these investigators did un- derstand where the virus was most likely to be found, it made little sense to use hemophiliac sera as the source of antibody. Hemophiliacs were not at increased risk for infectious, type A hepatitis. They had access to convalescent sera from hundreds of patients admitted with clinical hepatitis A that were likely to contain specific antibody. Nevertheless, they did identify single precipitin bands in an agar gel diffusion assay with two of 37 sera, and four of six fecal specimens. They then used this technique to track the antigen in sucrose density gradients spun in an ultracentrifuge. The partially purified Figure 1. Milan antigen showing disc-like structures antigen was then used to raise antisera in rabbits. associated with serum lipoproteins (Taylor et al. After absorption, the rabbit sera also produced 1972). precipitin bands when reacted with stools from hepatitis patients. In a large study, a single precipitin band was identified in 90 of 220 pa- serologic approach to identify previously un- tients with acute hepatitis, compared with five of known and difficult-to-culture viral agents had 158 samples collected from patients with other been shown from these studies. diseases. The antigen was typically present in Similar antibody-based technologies were the first sample collected after hospitalization thus applied to search for a virus or a viral anti- and became undetectable an average of 18 days gen associated with hepatitis A with variable after admission (Ferris et al. 1970; Gust et al. results. The best characterized of these was the 1970, 1971). Milan antigen described by Salvatore Del Prete Immune EM (IEM) performed on clarified and colleagues (Del Prete et al. 1972a,b) at the fecal extracts revealed two different particles that University of Milan in Italy, who used an ap- appeared to be associated with small amounts of proach similar to Blumberg’s. Characterization antibody (Fig. 2). One of these particles was 15 of the antigen revealed it to have a density of to 25 nm in diameter, seemed to have a virus- www.perspectivesinmedicine.org only 1.06 gm/cm3 and electron microscopy (EM) studies revealed disk-like structures of varying size up to 50 nm, suggesting that Milan antigen was likely an abnormal serum lipopro- tein often associated with liver disease (Fig. 1) (Taylor et al. 1972). The serology-based studies generally fol- lowed the hepatitis B work, looking for an anti- gen in the serum of patients with acute hepatitis A. In contrast, Geoff Cross, Alan Ferris, and Ian Gust at the Fairfield Hospital in Melbourne un- derstood that feces were the best place to look for a virus responsible for hepatitis A, and they thus began a search for the virus in feces. Using acute Figure 2. Fecal antigen with both large particles of stool samples as a source of antigen, they reacted about 45 nm and small 20 nm particles. (Electron it with sera from multiply transfused hemophil- micrograph kindly supplied by Ian Gust.)

4 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

History of the Discovery of Hepatitis A Virus

like morphology, and appeared to be present as like the various morphologic forms associated both full and empty particles typical for nega- with HBV. tive-stained EM images of small nonenveloped These studies show the complexity of viruses. The investigators thought these parti- searching for a specific virus or viral antigen in cles could resemble 22 nm subviral HBsAg human stools by EM. Multiple particulate particles associated with hepatitis B. The sec- forms, many resembling viruses, are seen in ev- ond, less common particle was 40 to 45 nm in ery stool sample. Some of these particles may diameter and was considered to be similar to be breakdown products derived from food, the hepatitis B virion, otherwise known as the but some probably are actual viruses or bacteri- Dane particle (Dane et al. 1970). Antibody to ophages (Fig. 3). For this reason, any particle fecal antigen was thought to react with true seen in stool must be critically evaluated for spe- Dane particles but not with HBsAg, and anti- cificity before it can be associated with a specific body to Dane particle-enriched specimens infection. In the case of IEM studies, very specific seemed to aggregate the large particles observed antibody or well-characterized paired sera from in fecal antigen. Based on these results, Geoff patients with acute infections need to be used. Cross believed that there were two types of Because IEM is somewhat subjective, samples particles associated with hepatitis A, much should also be evaluated by a blinded observer. www.perspectivesinmedicine.org

Figure 3. Electron microscopic images of particles in human feces. (A) Small 22–25 nm particles without antibody commonly observed in stool mixed with immune serum globulin. We generally called these Clarke particles, as they are similar to 22 nm particles described in 1973 by Clarke and colleagues that were considered to be parvoviruses (Paver et al. 1973). The particles could be bacteriophages. (B) Honeycomb structure that we often saw in tools. (C) Particles observed in stools from a patient with hepatitis on Palau Island in Micronesia. This group of particles was observed in a partially purified specimen not reacted with antibody. (D) The Palau particles following addition of immune globulin and showing very heavy coating with antibody. (Photo from the author’s private collection.)

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 5 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

S.M. Feinstone

IDENTIFYING HEPATITIS A VIRUS: infected by HBV, become a carrier, or cleared A PERSONAL ACCOUNT the infection could be identified (Lander et al. 1971, 1972). This purified HBsAg also served as I came to the NIH in Bethesda, MD, in 1971 as a the basis for an experimental vaccine (Purcell Research Associate in the United States Public and Gerin 1975). Using these assays, Bob was Health Service (PHS). This was the height of the able to define much of the epidemiology of war in Vietnam and virtually every male doctor hepatitis B. The laboratory was very busy testing completing his internship was drafted. One samples coming in from all over the world. alternative to military service was to join the High stacks of microtiter plates were always on PHS and work at one of the agencies for which the benches of Doris Wong, Bob’s nearly career- that organization was responsible, including the long master technician, and Jose Valdesuso, NIH. This was obviously a very popular option who could do more work than can be imagined and PHS officers of that era became known as in the shortest possible time. the “yellow berets.” Acceptance to NIH was All this work was going on in a tiny “con- highly competitive and, for that reason, I never tainment” laboratory in the sub-basement of understood how I was selected. Applicants came Building 7 on the NIH campus because there to NIH for interviews in several laboratories was some fear of this virus among others work- and there was sort of a matching program for ing in the building. Bob had a tiny, sealed cubicle the available slots. I assume that I was selected inside the small laboratory where he used large because I had a long-standing interest in labo- amounts of radioactive iodine-125 to label the ratory research and had written two papers purified HBsAg that John Gerin produced in an from work I had done in medical school. I was off-campus laboratory. The sub-basement was a absolutely thrilled when I was selected by the very tight space, but it was also fun with Doris, Laboratory of Infectious Diseases (LID) headed Jose, Bob, and me. However, I felt like I was by Robert Chanock, one of the country’s leading beating my head on the wall working on cellular virologists. As the two other new associates were immunology, and when Bob asked me to take a pediatricians, they were assigned to projects crack at finding the virus responsible for hepa- dealing with infections of childhood, specifically titis A in stools, I was very grateful to get a new respiratory syncytial virus and viral diarrhea. I project. was training as an internist and was assigned to Albert Kapikian, who was head of the Epi- the viral hepatitis group headed by Robert Pur- demiology Section in LID, had just returned cell. As my previous research involved cellular from a sabbatical with June Almeida in the immunity to viral infections, I was originally United Kingdom. June had developed IEM as www.perspectivesinmedicine.org tasked to look at the importance of that arm of a technique for identifying viruses (Almeida and the immune system in hepatitis B infections. At Waterson 1969). Al was interested in discover- that time, T-cell function was barely understood ing novel, hard-to-grow viruses associated with and I was somewhat lost, heading down many acute respiratory infections. In fact, Al did dead ends and using inadequate technology. I identify the 692 strains of coronavirus in a har- floundered in that project for more than a year. vest from a tracheal organ culture that had been By this time, the hepatitis B virus (HBV) inoculated with washings from an adult with an envelope, HBsAg, had been identified, the acute upper respiratory illness (Kapikian et al. Dane particle was shown to represent the virion, 1973). Just next to Al’s laboratory was the and the genome was known to be DNA. Bob group working on viral diarrhea. Periodically Purcell and John Gerin had applied ultracentri- they would bring Al a sample that they thought fugation techniques to purify large quantities might contain a virus and eventually, using IEM, of HBsAg for use in high-sensitivity immuno- they identifi ed the Norwalk virus in stools from assays for both the antigen and the antibody. patients with acute diarrheal illness (Kapikian Using the radioimmunoprecipitation technique et al. 1972). These were beautiful studies, and (RIP), essentially every person who had been Bob Purcell immediately saw the potential for

6 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

History of the Discovery of Hepatitis A Virus

IEM in the search for the hepatitis A virus one of the patients studied in that outbreak (Fig. (HAV) that he knew should be in stool in 3C,D). The particle had a core and what ap- large quantities. At about this time, we also peared to be an envelope and they were not acquired new laboratory space where we had only aggregated by the immune globulin, they laminar flow hoods, a contained ultracentrifuge, were often so heavily covered with antibody that and other equipment that was not available in they looked like snowballs (Fig. 3D). In addition, the sub-basement laboratory. I believe Bob the size of the cores and therefore the entire would have started the hepatitis A work sooner particle varied tremendously. We spent months if we had a laboratory where we could safely studying this particle, and although we conclud- work with this highly contagious virus. As I ed that it was not the hepatitis A agent, we did was working on my immunology project and have enough data to associate it with the out- another research associate, Jon Gold, had re- break on Palau. We even had a final draft of a cently joined the laboratory and was working manuscript with all our data. I was rather inse- on hepatitis B polymerase, Bob asked both of cure about this particle, and we were not even us to share the hepatitis A project and keep sure that it was a virus with its very unusual and our other work going as well. After the first stool variable morphology. In addition, an enveloped extraction, Jon decided he was not interested in virus did not fit well with what was known then hepatitis A and I took it on full time. about the virus responsible for hepatitis A. Even The idea was to learn IEM from Al and work though we did not claim in the manuscript that with him to try to find the virus in various sam- the particle was the hepatitis A agent, I became ples that were available to us. We had one golden very worried that many investigators in the field set of samples from the volunteer studies that the would think that we really did believe it was the U.S. Army had sponsored at the Joliet Prison in virus. I spent some sleepless nights worrying Illinois (Boggs et al. 1970; Melnick and Boggs about this and determined to go back to the 1972). These volunteers had been infected with Joliet samples and go through them in a system- the MS-1 inoculum from the Krugman studies atic fashion. This of course meant making a at the Willowbrook State School. The object of lot of stool extracts—not the most desirable the Joliet studies was to produce large quantities task and one that was not at all popular with of infectious material that could be used by both the entire staff of Building 7. Working in lami- army and non-army investigators, and both nar flow hoods, the stool samples were extracted, stools and sera had been collected from the clarified, and filtered. Everything used for that volunteers. One sample, termed K30 (collected work was then carefully bagged and put in a from volunteer K on the 30th day after inocula- large autoclave to destroy the infectivity. The www.perspectivesinmedicine.org tion, while jaundiced), had been used to pass odors produced in that process pervaded the hepatitis to other volunteers and therefore was entire building, and everyone knew when it was known to contain infectious virus. Al and I used stool extraction day. standard immune globulin as a screening anti- Once we started this more systematic ap- body, and particles that appeared aggregated by proach to the Joliet stools, the virus appeared antibody were then checked for specificity using in one of the first samples that I tested, a sample paired sera from the volunteers that was avail- termed F33. Having spent about 1 year, and Al able in only small quantities. We looked at the many years, looking at stool filtrates, we knew K30 stool multiple times under different condi- the common structures observable by EM like tions, but never saw any particles that we could they were old friends. Among the many particles identify as specific for hepatitis A. in stool, we typically saw several virus-like par- As we did not find the virus in the Joliet ticles that might be bacteriophage, structures MS-1 stool, we started to examine stools from that were probably bacterial filaments, and hon- an outbreak of hepatitis on Palau Island in eycomb-like material for which I have no expla- Micronesia in 1967. We quickly identified large nation (Fig. 3A,B). So, when I first saw the quantities of an unusual particle in the stool of hepatitis A particle, it immediately popped out

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 7 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

S.M. Feinstone

skepticism and I might have had my enthusiasm quickly dampened. But I did run upstairs to tell Bob that I was nearly certain I had seen the HAV particle. When Bob saw it, I believe he was as excited as I was, and we immediately set out plans for proving it was indeed the virus respon- sible for hepatitis A. Al returned to the labora- tory soon after that and we went to work (Fig. 5). On a very personal note, the months that I spent with Al Kapikian in the basement of Building 7 on the NIH campus (recently razed), staring at the glowing phosphorescent screen of that antique Siemens 1-A EM (now on perma- Figure 4. Large immune aggregate of hepatitis A virus (HAV)reacted with immune globulin. Aggregates like nent display in the lobby of Building 50 on this is the way we originally observed the virus. (Photo the NIH campus) were some of the best in my from the author’s private collection.) career. Al taught me so much about science, of course. But we had wonderful conversations about our mutual love for baseball, of which as different. It almost glowed as a result of the Al had great historical knowledge. We talked antibody covering the particles (Fig. 4). On that about politics, as this was the Watergate era. Al day, Al was actually out because of illness and I taught me a lot about opera and I am to this day have always had regrets about that. Al had put in a regular operagoer. Al Kapikian was a wonder- a huge effort on this project, and he should have ful, kind, and generous man. I, like all his many been there on that day. However, with no evi- friends, miss him tremendously. dence other than the visualization of these Bob, Al, and I laid out a series of experi- antibody-covered particles, I was just a little re- ments to prove the specificity of the particle luctant to tell Bob before doing more experi- we had observed. We adopted two general ap- ments. Bob always maintained healthy scientific proaches to show the specificity of this particle. www.perspectivesinmedicine.org

Figure 5. Robert Purcell, Al Kapikian, and the author, seated at the Siemens 1A electron microscope that Al used to identify coronavirus 692, the Norwalk gastroenteritis virus, and HAV. Al loved that scope and never got used to a new one he was eventually forced to use. (Photo kindly supply by the National Institutes of Health.)

8 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

History of the Discovery of Hepatitis A Virus

One was to identify the particle in stool samples the samples after I had prepared the EM grids, so from other patients with acute hepatitis A. The that neither Al nor I knew what sample we were second was to show a specific antibody response looking at. We found the virus-like particles to the particle we had identified in the F33 stool. aggregated by antibody in two out of four stools As IEM is really a subjective process, Al had from acutely infected volunteers, but in none of tried to standardize it as much as possible. The their preinoculation stools. sensitivity of IEM is somewhere on the order of The second method we used to show these 106 particles/mL, and we understood that per- particles were specific for hepatitis A was to test haps not all stools, even from acutely ill patients, under code-paired sera for antibody directed to would have sufficient numbers of particles to the 27 nm particles. We quantified the antibody find the virus with a reasonable effort. There- on a 0 to 4+ scale (Fig. 6). A rating of “0” meant fore, Al’s system was to look at five good-quality there were no particles detected. The virus par- EM grid squares before we declared a sample ticles in the F33 stool were present at such a level negative. Of course, “negative” only meant that if they were not coated with antibody or that we did not find any particles in the five aggregated by antibody, we could not distin- examined squares. We also always did these guish the HAV particles from the other material evaluations under code. Every test was done in the stools. If the antibody were rated 4+ on the with an adequate number of controls to try to scale, it meant that the particles were so heavily keep bias out of the system. Doris Wong coded covered that the outlines of the particle were www.perspectivesinmedicine.org

Figure 6. Quantitation of antibody on hepatitis A virus (HAV) particles. (A) HAV particles purified and con- centrated from stool without antibody. (B) An immune aggregate of HAV with a low level of antibody rated 1+. (C) An immune aggregate of HAV particles with the antibody rated 2+. (D) An immune aggregate of HAV particles with antibody rated 3+. (Photo from the author’s private collection.)

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 9 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

S.M. Feinstone

obscured. Sera containing antibodies rated 4+ over the entire paper with her. I do not know if also tended to produce single particles or dou- she even sent it out for review, but it was very blets. The particles seen with lower-rated sera quickly accepted and published just 3 weeks later. were usually in aggregates, but when the anti- On publication, NIH called a press confer- body was rated about 1+, the aggregates tended ence and the finding was reported widely in the to be loose or appear to be almost falling apart. news media. We made the front page of the A serum antibody rating of 2+ to 3+ seemed to Washington Post early edition that comes out be near antigen/antibody equivalence, as we the evening before the publication date, but would see larger aggregates with those sera. we were knocked off the front page in the later First, we tested six serum pairs from individ- editions because of the resignation of Egil “Bud” uals who had been infected with the MS-1 strain. Krogh, who was head of the plumbers investi- All six of these went from no antibody in the gating leaks from the Nixon White House. preinoculation sample to easily detected anti- Appropriately, Krogh served prison time for body in the convalescent sample. Because these his role in the Watergate scandal. six serum pairs were from volunteers infected We worked so hard to get the paper out with MS-1 and our “antigen” was also MS-1, quickly, because we felt that there was some we considered the possibility that we were look- competition to find HAV. We knew the Austra- ing at something that had been passed with the lians had a similar approach to ours but had only MS-1 inoculum that was not the hepatitis A published on fecal antigen so far (Ferris et al. virus. Therefore, we tested serum pairs obtained 1970). If they were to change their experimental from patients with naturally acquired infection approach just slightly, they would find the vi- in two outbreaks of hepatitis A, one from rus. Stephen Locarnini (Fig. 7) was a graduate Massachusetts and one from American Samoa. student working with Alan Ferris at Monash All six of these patients showed either serocon- version or antibody increases on the 4-point scale. As controls, we also tested paired sera from two patients with acute hepatitis B and two patients with Norwalk-type gastroenteritis. None of those patients seroconverted or had increases in antibody levels. Therefore, we felt confident that these particles were, at a mini- mum, virus-like antigens associated with hepatitis A and were most likely the virus itself www.perspectivesinmedicine.org (Feinstone et al. 1973). We first saw the HAV particle on October 17, 1973. We published our results in Science on December 7. I believe it was nearly a speed record, but the rapid publication resulted from a lot of very intense work. We were getting new drafts of the paper out almost hourly and as soon as the new one was ready, Al, Bob, and I, as well as Dr. Chanock, would read each draft and edit Figure 7. A young Stephen Locarnini in the laboratory it immediately. Luckily, our laboratory had an at Monash University. Working with Alan Ferris and early word-processing machine so that the en- Ian Gust, Stephen was probably the second scientist to fi tire manuscript did not have to be retyped with identify hepatitis Avirus (HAV) particles and the rst fi to visualize the virus in feces from a patient with each draft. We hand carried the nal manuscript naturally acquired hepatitis A. As he had not seen to the American Association for the Advance- our paper before he first saw the virus, it can be said ment of Science (AAAS) offices in downtown that he identified it independently. (Photo kindly sup- Washington, met with the editor, and went plied by S. Locarnini.)

10 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

History of the Discovery of Hepatitis A Virus

University in Melbourne. Ferris had parted with so many particles that there was near antigen/ Geoff Cross and Stephen had decided to work antibody equivalence, resulting in most of the with Alan. Their December 7 issue of Science particles being sequestered in huge rafts that had not yet arrived by March of 1974 when were not uniformly distributed, and causing Stephen identified 27 nm virus-like particles us- me to miss them in looking at our standard ing EM to examine a precipitin band formed in a five grid squares. gel diffusion assay with an acute stool extract Because of the density that we found, we and hyperimmune rabbit sera raised against considered the possibility that HAV could be a acute phase stools. Stephen was quite excited, parvovirus, although it was a bit larger than until he walked into Alan’soffice and was given the diameters reported for most parvoviruses. the just-arrived Science issue with the words, Although we did not say it was a parvovirus or “These guys have discovered hepatitis A.” Obvi- a DNA virus, we did suggest it was a possibility. ously there was disappointment, but Stephen Later, when the group at Merck led by Philip quickly confirmed our results by finding similar Provost identified HAV in samples from an out- particles in naturally acquired, not experimen- break in Costa Rica and determined the density tal, hepatitis A. He also refuted a paper pub- to be ∼1.34 g/mL, they suggested that their virus lished in The Lancet by June Almeida, the moth- was an enterovirus and therefore different from er of IEM, who suggested that acute liver disease the virus that we described (Provost et al. 1975). could result in seroconversion to a multiplicity However, all of the morphologically identical of particle types present in feces, and who urged virus-like particles from these early studies caution in accepting that the particle we had were shown to be antigenically related, and the discovered was in fact HAV (Almeida et al. NIH particle, the Australian particle, and the 1974; Locarnini et al. 1974). Stephen went on Merck particle were all HAV. to complete his Ph.D. in a record 3 years, fol- IEM was a technique that could be used to lowed by a medical degree and a long, highly detect the virus and also to measure antibody to productive career in hepatitis research. it. There were so many things to do after the I was slated to leave Bob’s laboratory at the initial discovery, and as I was soon to leave the end of June 1974 to complete my clinical train- laboratory, I did not have time to develop a less ing. Therefore, I had very little time to continue labor-intensive antibody assay. I just continued the HAVstudies and I wanted to get a lot done as to use IEM to do as many things as I could in quickly as I could. We began a characterization that short time. As mentioned earlier, with the of the virus, which included determination of advent of highly sensitive assays for HBsAg and its buoyant density. Working in John Gerin’s anti-HBs, virtually all patients with hepatitis B www.perspectivesinmedicine.org laboratory, we performed cesium chloride or who had once been infected with hepatitis B density gradient ultracentrifugation studies us- could be identified. One of the most puzzling ing stool filtrate in which it was reasonably easy problems was the fact that <50% of patients to find particles by IEM. After ultracentrifuga- with transfusion-associated hepatitis could be tion and fractionation, I performed IEM on each shown to have hepatitis B. Some thought it fraction. I found a large number of particles was hepatitis A, but Bob Purcell recognized in the fraction corresponding to a density that the epidemiology of those posttransfusion of about 1.4 g/cm3 (Feinstone et al. 1974). cases, for example, the length of the incubation Although some forms of HAV do band at that period, the lack of secondary cases, and the like- density, it was quickly determined that the ma- lihood of chronicity, did not resemble hepatitis jor density for mature HAV is about 1.34 g/cm3 A at all. Therefore, one of the first things that (Provost et al. 1975; Lemon et al. 1985). My we did was to study patients with non-B post- guess is that after I found so many particles at transfusion hepatitis for hepatitis A using our 1.4 g/cm3, I was in a rush and simply did not specific, though cumbersome IEM assay. In col- spend adequate time on the other fractions. The laboration with Harvey Alter and Paul Holland fraction at 1.34 g/cm3 may also have contained at the NIH Blood Bank, where Bob Chanock and

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 11 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

S.M. Feinstone

Bob Purcell had established a very close collab- was a tremendous boost to viral discovery in oration on transfusion-associated hepatitis, we the 1950s and 1960s. IEM was a relatively new obtained the appropriate samples to evaluate. technology that was available to us in 1972. It We studied 22 open-heart surgery patients was of sufficient sensitivity, and the addition of who had received multiple blood transfusions antibody to standard EM added the specificity and who had developed hepatitis without any that was needed to prove that a virus-like particle serologic evidence for hepatitis B infection. We observed in stool filtrates from people with acute had pretransfusion and posthepatitis sera from hepatitis A was indeed HAV. Almost 20 years these patients and examined them under code later, HCV was identified with molecular for anti-HAV seroconversion or increases in an- techniques that were not available in the 1970s tibody levels. We could not find evidence that (Choo et al. 1989), and newer molecular tech- any of them had hepatitis A (Feinstone et al. nologies are of such sensitivity that virtually no 1975). Thus, the concept of a third form of viral virus can evade rapid detection today. However, hepatitis, non-A, non-B hepatitis, now known as the principles to assign specificity to any identi- hepatitis C was born. We presently have five, fied virus remain the same. Until specificity well-established types of viral hepatitis, includ- is proved, skepticism of any claim of etiology ing hepatitis D or Delta, and hepatitis E. is wise as sensitivity and specificity still have The identification of HAV began decades of an inverse relationship. research in Bob’s laboratory on hepatitis A that I was a research associate, basically a post- culminated in the development of cell culture– doc, at the time of the hepatitis Awork described adapted HAV and then vaccine development. here. Graduate students and postdocs are Ian Gust came to Bob’s laboratory on sabbatical the worker bees in most research laboratories, in 1978 and brought with him an Australian and when a significant or important result is isolate called HM175 that he had collected obtained, the postdoc typically gets some credit, from a family outbreak of hepatitis A (Gust but the finding is usually most associated with et al. 1985). This virus was highly infectious in the head of the laboratory who directed the re- marmosets and chimpanzees and was adapted search. For some reason, the discovery of hepa- to cell culture better than our other laboratory titis A is most often associated with my name strains (Daemer et al. 1981). It was the first strain and my career was launched on that one Science of HAV to be molecularly cloned and sequenced paper. Bob Purcell was instrumental in seeing (Ticehurst et al. 1983; Cohen et al. 1987), and that I received this credit and for that, I remain indeed it remains a standard HAV strain used in indebted. many laboratories today. Formalin-inactivated, www.perspectivesinmedicine.org cell culture–adapted HM175 virus was protec- tive against a heterologous virus challenge when REFERENCES used as an experimental vaccine by U.S. Army Almeida JD, Waterson AP. 1969. The morphology of virus– investigators in owl monkeys (Aotus trivirgatus), antibody interaction. Adv Virus Res 15: 307–338. and was also the antigen in an army vaccine that Almeida JD, Gay FW, Wreghitt TG. 1974. Pitfalls in the 2: – was the first to be tested in humans (Binn et al. study of hepatitis A. Lancet 748 751. Binn LN, Bancroft WH, Lemon SM, Marchwicki RH, LeDuc 1986; Sjogren et al. 1991). Eventually, it became JW, Trahan CJ, Staley EC, Keenan CM. 1986. Preparation the basis for the first licensed commercial of a prototype inactivated hepatitis A virus vaccine from hepatitis A vaccine (Innis et al. 1994). All of us infected cell cultures. J Infect Dis 153: 749–756. “ ” who worked on the hepatitis A project in Bob’s Blumberg BS, Alter HJ, Visnich S. 1965. A new antigen in leukemia sera. JAMA 191: 541–546. laboratory remain proud of the vaccine that has Boggs JD, Melnick JL, Conrad ME, Felsher BF. 1970. Viral significantly reduced the incidence of hepatitis hepatitis. Clinical and tissue culture studies. JAMA 214: A, wherever it has been broadly used. 1041–1046. The search for the cause of many diseases Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. 1989. Isolation of a cDNA clone derived with a suspected viral etiology has depended on from a blood-borne non-A, non-B viral hepatitis genome. the technology available at the time. Cell culture Science 244: 359–362.

12 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

History of the Discovery of Hepatitis A Virus

Cohen JI, Rosenblum B, Ticehurst JR, Daemer RJ, Feinstone Havens WP Jr. 1968. Viral hepatitis. In Internal medicine in SM, Purcell RH. 1987. Complete nucleotide sequence of world war II (volume III), Infectious diseases and general an attenuated hepatitis A virus: Comparison with wild- medicine (pp. 331–384). Office of the Surgeon General, type virus. Proc Natl Acad Sci 84: 2497–2501. Department of the Army, Washington, DC. Cole RA. 1965. Viral agents from patients with infective Innis BL, Snitbhan R, Kunasol P, Laorakpongse T, Poopata- hepatitis. Aust J Exp Biol Med Sci 43: 497–504. nakool W, Kozik CA, Suntayakorn S, Suknuntapong T, Cross GF, Marmion BP. 1966. Cell culture and infectious Safary A, Tang DB, et al. 1994. Protection against hepatitis hepatitis. Med J Aust 2: 1214–1217. A by an inactivated vaccine. JAMA 271: 1328–1334. Daemer RJ, Feinstone SM, Gust ID, Purcell RH. 1981. Prop- Kapikian AZ, Wyatt RG, Dolin R, Thornhill TS, Kalica AR, agation of human hepatitis A virus in African green Chanock RM. 1972. Visualization by immune electron monkey kidney cell culture: Primary isolation and serial microscopy of a 27-nm particle associated with acute passage. Infect Immun 32: 388–393. infectious nonbacterial gastroenteritis. J Virol 10: 1075– Dane DS, Cameron CH, Briggs M. 1970. Virus-like particles 1081. in serum of patients with Australia-antigen-associated Kapikian AZ, James HD, Kelly SJ, Vaughn AL. 1973. Detec- hepatitis. Lancet 1: 695–698. tion of coronavirus strain 692 by immune electron mi- Deinhardt F, Holmes AW, Capps RB, Popper H. 1967. croscopy. Infect Immun 7: 111–116. Studies on the transmission of human viral hepatitis to Krugman S, Giles JP. 1970. Viral hepatitis. New light on an marmoset monkeys. I: Transmission of disease, serial pas- old disease. JAMA 212: 1019–1029. 125: sages, and description of liver lesions. J Exp Med Krugman S, Giles JP. 1972. The natural history of viral hep- – 673 688. atitis. Can Med Assoc J 106: 442–446. Del Prete S, Constantino D, Doglia M, Fabiani MP, Borin R. Krugman S, Giles JP, Hammond J. 1967. Infectious hepatitis. 1972a. Epidemic hepatitis-associated antigen. Can Med 106: – Evidence for two distinctive clinical, epidemiological, and Assoc J 493 496. immunological types of infection. JAMA 200: 365–373. Del Prete S, Constantino D, Doglia M, Fabiani MP, Borin R, Lander JJ, Alter HJ, Purcell RH. 1971. Frequency of antibody Jacini A. 1972b. Epidemic hepatitis-associated antigen to hepatitis-associated antigen as measured by a new ra- (milan antigen). Study on normal population and spora- dioimmunoassay technique. J Immunol 106: 1166–1171. dic acute viral hepatitis. Am J Dis Child 123: 326–329. Lander HJ, Holland PV, Alter HJ, Chanock RM, Purcell RH. Feinstone SM, Kapikian AZ, Purceli RH. 1973. Hepatitis A: 1972. Antibody to hepatitis-associated antigen. Frequen- Detection by immune electron microscopy of a viruslike cy and pattern of response as detected by radioimmuno- antigen associated with acute illness. Science 182: 1026– precipitation. JAMA 220: 1079–1082. 1028. Lemon SM, Jansen RW, Newbold JE. 1985. Infectious Feinstone SM, Kapikian AZ, Gerin JL, Purcell RH. 1974. hepatitis A virus particles produced in cell culture consist Buoyant density of the hepatitis A virus-like particle in of three distinct types with different buoyant densities in cesium chloride. J Virol 13: 1412–1414. CsCl. J Virol 54: 78–85. Feinstone SM, Kapikian AZ, Purcell RH, Alter HJ, Holland Locarnini SA, Ferris AA, Stott AC, Gust ID. 1974. Letter: PV. 1975. Transfusion-associated hepatitis not due to 2: viral hepatitis type A or B. N Engl J Med 292: 767–770. Pitfalls in hepatitis A? Lancet 1007. 9: – Ferris AA, Cole RA. 1966. Detroit 6 cells and infective hep- MacCallum FO. 1953. Hepatitis. Br Med Bull 221 225. atitis. Med J Aust 2: 1213–1214. MacCallum FO. 1972. 1971 International Symposium on Viral Hepatitis. Historical perspectives. Can Med Assoc Ferris AA, Kaldor J, Gust ID, Cross G. 1970. Faecal antigen 106: – in viral hepatitis. Lancet 2: 243–244. J 423 426.

www.perspectivesinmedicine.org Fonseca JC. 2010. History of viral hepatitis. Rev Soc Bras Med MacCallum FO, McFarlan AM, Miles JA, Pollock MR, Wil- Trop 43: 322–330. son C. 1951. Infective hepatitis; studies in East Anglia during the period 1943–47. Spec Rep Ser Med Res Counc Gellis SS, Stokes J Jr, Brother GM, Hall WM, Gilmore HR, (G B) 273: 1–144. Beyer E, Morrissey RA. 1945. The use of human immune serum globulin (γ globulin) in infectious (epidemic) hep- Melnick JL, Boggs JD. 1972. Human volunteer and tissue 106: atitis in the Mediterranean theater of operations. I: Studies culture studies of viral hepatitis. Can Med Assoc J – on prophylaxis in two epidemics of infectious hepatitis. 461 467. JAMA 128: 1062–1063. Mirkovic RR, Adamova V, Boucher DW, Melnick JL. 1971. Gust ID, Feinstone SM. 1988. History. In Hepatitis A. CRC, Identification of the Kirk “hepatitis” virus as a member of Boca Raton, FL. the parvovirus (picodnavirus) group. Proc Soc Exp Biol 138: – Gust ID, Cross G, Kaldor J, Ferris AA. 1970. Virus-like par- Med 626 631. ticles in Australia-antigen-associated hepatitis. Lancet 1: Neefe JR, Stokes J, Reinhold JG, Lukens FD. 1944. Hepatitis 953. due to the injection of homologous blood products in 23: – Gust ID, Kaldor J, Cross GF, Waugh M, Ferris AA. 1971. human volunteers. J Clin Invest 836 855. Virus-like particles associated with a faecal antigen from Neefe JR, Gellis SS, Stokes J Jr. 1946. Homologous serum hepatitis patients and with Australia antigen. Aust J Exp hepatitis and infectious (epidemic) hepatitis; studies in Biol Med Sci 49: 1–9. volunteers bearing on immunological and other charac- 1: – Gust ID, Lehmann NI, Crowe S, McCrorie M, Locarnini SA, teristics of the etiological agents. Am J Med 3 22. Lucas CR. 1985. The origin of the HM175 strain of Parks WP, Melnick JL. 1969. Attempted isolation of hepatitis hepatitis A virus. J Infect Dis 151: 365–367. viruses in marmosets. J Infect Dis 120: 539–547.

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 13 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

S.M. Feinstone

Parks WP, Melnick JL, Voss WR, Singer DB, Rosenberg HS, JL Jr, Schiff ER, et al. 1987. A serologic follow-up of the Alcott J, Casazza AM. 1969. Characterization of marmo- 1942 epidemic of post-vaccination hepatitis in the United set hepatitis virus. J Infect Dis 120: 548–559. States Army. N Engl J Med 316: 965–970. Paver WK, Ashley CR, Caul EO, Clarke SK. 1973. A small Sherlock S. 1984. Landmark perspective: Landmarks in viral virus in human faeces. Lancet 1: 237–240. hepatitis. JAMA 252: 402–406. Prince AM. 1968. An antigen detected in the blood during Simons JN, Pilot-Matias TJ, Leary TP, Dawson GJ, Desai SM, the incubation period of serum hepatitis. Proc Natl Acad Schlauder GG, Muerhoff AS, Erker JC, Buijk SL, Chal- 60: – Sci 814 821. mers ML, et al. 1995. Identification of two flavivirus-like Provost PJ, Wolanski BS, Miller WJ, Ittensohn OL, McAleer genomes in the GB hepatitis agent. Proc Natl Acad Sci 92: WJ, Hilleman MR. 1975. Biophysical and biochemical 3401–3405. properties of CR326 human hepatitis A virus. Am J Med Sjogren MH, Hoke CH, Binn LN, Eckels KH, Dubois DR, Sci 270: 87–92. Lyde L, Tsuchida A, Oaks S Jr, Marchwicki R, Lednar W, Purcell RH, Gerin JL. 1975. Hepatitis B subunit vaccine: A fi et al. 1991. Immunogenicity of an inactivated hepatitis A preliminary report of safety and ef cacy tests in chimpan- vaccine. Ann Intern Med 114: 470–471. zees. Am J Med Sci 270: 395–399. Stapleton JT. 2003. GB virus type C/hepatitis G virus. Semin Rightsel WA, Keltsch RA, Tekushan FM, McLean IW Jr. 1956. Liver Dis 23: 137–148. Tissue-culture cultivation of cytopathogenic agents from patients with clinical hepatitis. Science 124: 226–228. Taylor PE, Almeida JD, Zuckerman AJ, Leach JM. 1972. Rightsel WA, Keltsch RA, Taylor AR, Boggs JD, McLean Relationship of Milan antigen to abnormal serum lipo- 123: – IW Jr, Capps RB, Weiss CF. 1961. Status report on tis- protein. Am J Dis Child 329 331. sue-culture cultivated hepatitis virus. I: Virology labora- Ticehurst JR, Racaniello VR, Baroudy BM, Baltimore D, tory studies. II: Clinical trials. JAMA 177: 671–682. Purcell RH, Feinstone SM. 1983. Molecular cloning and Robbins FC, Enders JF. 1950. Tissue culture techniques in characterization of hepatitis A virus cDNA. Proc Natl the study of animal viruses. Am J Med Sci 220: 316–338. Acad Sci 80: 5885–5889. Seeff LB, Beebe GW, Hoofnagle JH, Norman JE, Buskell- Voegt H. 1942. Zur aetiologie der hepatitis epidemica. Bales Z, Waggoner JG, Kaplowitz N, Koff RS, Petrini Muenchener Medizinische Wochenschrifte 89: 76–79. www.perspectivesinmedicine.org

14 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740 Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

History of the Discovery of Hepatitis A Virus

Stephen M. Feinstone

Cold Spring Harb Perspect Med published online April 30, 2018

Subject Collection Enteric Hepatitis Viruses

Hepatitis A Virus Genome Organization and Evolutionary Origins of Enteric Hepatitis Viruses Replication Strategy Anna-Lena Sander, Victor Max Corman, Alexander Kevin L. McKnight and Stanley M. Lemon N. Lukashev, et al. Adaptive Immune Responses in Hepatitis A Virus Enterically Transmitted Non-A, Non-B Hepatitis and Hepatitis E Virus Infections and the Discovery of Hepatitis E Virus Christopher M. Walker Stanley M. Lemon and Christopher M. Walker Small Animal Models of Hepatitis E Virus Infection Natural History, Clinical Manifestations, and Tian-Cheng Li and Takaji Wakita Pathogenesis of Hepatitis E Virus Genotype 1 and 2 Infections Rakesh Aggarwal and Amit Goel Acute and Persistent Hepatitis E Virus Genotype 3 Hepatitis A Virus and Hepatitis E Virus: Emerging and 4 Infection: Clinical Features, Pathogenesis, and Re-Emerging Enterically Transmitted and Treatment Hepatitis Viruses Nassim Kamar and Sven Pischke Stanley M. Lemon and Christopher M. Walker Epidemiology of Genotype 1 and 2 Hepatitis E Hepatitis A Virus Capsid Structure Virus Infections David I. Stuart, Jingshan Ren, Xiangxi Wang, et al. Kenrad E. Nelson, Alain B. Labrique and Brittany L. Kmush History of the Discovery of Hepatitis A Virus Comparative Pathology of Hepatitis A Virus and Stephen M. Feinstone Hepatitis E Virus Infection John M. Cullen and Stanley M. Lemon Epidemiology and Transmission of Hepatitis A Innate Immunity to Enteric Hepatitis Viruses Virus and Hepatitis E Virus Infections in the Zongdi Feng and Stanley M. Lemon United States Megan G. Hofmeister, Monique A. Foster and Eyasu H. Teshale Stem Cell−Derived Culture Models of Hepatitis E Nonhuman Primate Models of Hepatitis A Virus Virus Infection and Hepatitis E Virus Infections Viet Loan Dao Thi, Xianfang Wu and Charles M. Robert E. Lanford, Christopher M. Walker and Rice Stanley M. Lemon

For additional articles in this collection, see http://perspectivesinmedicine.cshlp.org/cgi/collection/

Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved