The Discovery of Hepatitis Viruses: Agents and Disease

Abstract

Discovery of five hepatitis viruses A to E has followed distinctive definable phases. Human experiments at Willowbrook identified two forms of hepatitis namely infectious hepatitis and serum hepatitis. The discovery of Australia antigen in 1965 led to rapid scientific developments in viral hepatitis. SH antigen was detected in sera of patients with serum hepatitis and soon SH antigen and Australia antigen were found to be identical and selectively associated with serum hepatitis. In 1970, 42-nm Dane particles were detected in Australia antigen positive sera and linked to the virus of serum hepatitis. Subsequently, a new antigen-antibody system (e-antigen/antibody) was detected in such patients and associated with infectivity. Then, DNA polymerase was found in concentrated pellets containing Australia antigen. Hepatitis B virus (HBV) DNA cloning and sequencing of HBV followed these developments. In 1973, 27 nm hepatitis A virus (HAV)–like particles were visualized in stool samples obtained during acute phase of illness after inoculation of MS-1 strain in volunteers. Cloning and sequencing of HAV followed. In 1977, a new antigen-antibody system (δ antigen-antibody system) was identified by chance associated with HBV. Based on animal transmission studies, δ agent was found to be another virus called hepatitis D virus that is defective, requires the helper functions of HBV and interferes with HBV replication. The search for hepatitis C virus started when non-A, non-B hepatitis was recognised in multiply transfused patients with subsequent successful animal transmission. HCV was identified by a novel immunoscreening approach involving screening of cDNA libraries from infectious sera. The story of hepatitis E is historically linked to discovery of waterborne epidemic non-A, non-B hepatitis from Kashmir, India. Virus-like-particles of the agent were identified in stool samples of a human volunteer after a self-experimentation. HEV cDNA was detected in bile-enriched infectious samples and full-length HEV RNA genome was subsequently cloned and sequenced.

“The path traversed in the discovery of hepatitis viruses has been a fabulous journey and I was blessed to be onboard for a wonderful trip”

(Mohammad S. Khuroo).

Hepatitis viruses are major threats to human health. Five unrelated viruses, named after five English alphabets A to E, are all hepatotropic and known to cause syndrome of viral hepatitis. Hepatitis viruses are cause of significant global morbidity and deaths.¹ In 2005 alone, hepatitis A virus (HAV) was estimated to have infected 119 million individuals with 31 million icteric cases and 34,000 deaths,² whereas, hepatitis E virus (HEV) infected 20 million individuals with 3.4 million icteric cases and 70,000 deaths and 3000 stillbirths.³ Epidemics of HAV (foodborne) and HEV (waterborne) affect hundreds and thousands of populations and result in substantial morbidity, mortality and breakdown in trade and tourist activities.⁴ Hepatitis B virus (HBV) has infected more than 2 billion people globally, with 240 million chronic carriers of the virus and causes between 500,000 and 700,000 deaths annually,⁵ whereas, hepatitis C virus (HCV) has infected 150 million people and causes more than 350,000 deaths annually.⁶ Hepatitis D virus (HDV), dubbed as vanishing disease, has infected 15–20 million people.⁷

Early studies

Hippocrates (460–375BC) is credited to have given the first description of epidemic jaundice, which stays accurate even as of today.⁸ He recommended a special diet and ‘milekraton’ (a mixture of water and honey) and suggested the important concept of immunisation as a means to prevent hepatitis. He described the dramatic clinical syndrome of fulminant hepatitis as follows: “The patient soon raves, becomes angry, talks nonsense and barks like a dog, his nails become red and he loses his eyesight. Most patients die within a span of eleven days, few of them survive”.

Early knowledge on hepatitis viruses was gained on observations of epidemics of viral hepatitis in World War II⁹ and a series of controlled experiments on human volunteers. The first description of an epidemic of ‘serum hepatitis’ spread through vaccination using human lymph was given by Lurman.¹⁰ An outbreak of jaundice occurred in a Bremen factory employing 1200 to 1500 persons. Between October 1883 and April 1884 at least 191 employees fell ill. The number of cases in the rest of city was not unusual. The author investigated possible epidemiological factors, including water supply, domicile and alcohol. He concluded that by far the most likely cause of the epidemic was revaccination against small pox which was given to 1280 employees and their relatives on a single day in August 1983. Those vaccinated on other days were not affected. Dr. Lurman therefore firmly attributed the epidemic to the infection spread during the vaccination. In 1943, Beeson¹¹ reported on seven patients who had developed hepatitis one to four months after receiving transfusions of blood or plasma. He stressed the importance of surveillance of hepatitis following use of blood, blood products and vaccination. Epidemic jaundice hit military campaign at Dardanelles, Turkey.¹² Disease ran a benign course without any mortality. Cameron¹³ published data on another epidemic of jaundice in Palestine during world war II and based on volunteer studies established minimum incubation period disease of 32 days. Human transmission studies were extended by others^14,15 and established the routes of transmission of the disease. Dible et al.¹⁶ described the histopathological findings in needle liver biopsies of 56 patients with acute hepatitis, which led to abandonment of long-held belief of catarrhal jaundice. Gutzeit¹⁷ used liver biopsies to document progression of hepatitis to cirrhosis. He estimated that over 10 million people were affected by epidemic hepatitis in world war II. An epidemic of viral hepatitis, causing around 50,000 cases, was reported in United States Army servicemen in March 1942 during world war II, caused by the use of hepatitis-contaminated yellow fever vaccine. Seeff et al¹⁸ elegantly demonstrated that this epidemic was due to hepatitis B and that these veterans have high prevalence of antibodies to HBV (anti-HBs) even as of today.

Historically, discovering agents and diseases of hepatitis has seen distinct phases and has followed a remarkable uniform pattern which started with description of disease for each hepatitis virus. This led to the identification of antigen-antibody system and/or visualization of virus-like particles (VLPs) of the agent causing the disease. The cloning and sequencing of the viral genome with or without prior identification of cDNA followed, completing the discovery of the particular hepatitis virus (Table 1).

Table 1.

Landmarks Events in the Discovery of Five Hepatitis Viruses A to E. Discovery of Each Hepatitis Virus Followed a Distinctive Definable Event, Which Included Description of the Disease Caused by Each Hepatitis Virus, Followed by Identification of Antigen-antibody System And/Visualization of Virus-like particles. Cloning and Sequencing With or Without Prior Identification of cDNA Clones Followed to Establish the Viral Aetiology of the Agent. Author Along with the Institution and Relevant Reference is Included at the Bottom.

Hepatitis	Disease	VLP/Antigen	Virus (Cloning and Sequencing)
A	Krugman1 et al 1967 (infectious hepatitis)	Feinstone2 et al 1973 (VLP by IEM)	Ticehurst3 et al 1983
B	Krugman1 et al 1967 (Serum hepatitis)	Blumberg4 et al 1967 (Australia antigen on immunodiffusion) Dane5 et al 1970 (VLP-Dane particle on EM) Almeida6 et al 1971 (S & C antigens) Magnius7 et al 1972 (e antigen)	Burrell8 et al 1979
C	Alter9 et al 1975 (Non-A, non-B hepatitis)	Tabor10 et al 1978 (Transmission studies in Chimps)	Choo11 et al 1989 (identification of cDNA clone) Choo12 et al 1991
D	Rizzetto13 et al 1977 (delta antigen in liver biopsies)	Rizzetto14 et al 1980 (Delta Virus)	Wang15 et al 1986
E	Khuroo16 et al 1980 (Epidemic Non-A, Non-B Hepatitis)	Balayan17 et al 1983 (VLP on IEM)	Reyes18 et al 1990 (identification of cDNA clone) Tam19 et al 1991

1 = Willowbrook NY (JAMA 1967; 200:59), 2=NIH Bethesda MD (Science 1973; 182:1026), 3=NIH Bethesda MD (Proc Natl Acad Sci USA 1983; 80:5885), 4=Institute Cancer Research Philadelphia (JAMA 1967; 191:541), 5=Middlesex Hospital London (Lancet 1970; i:695), 6=Royal Postgraduate Medical School London (Lancet 1971; ii:1225), 7 = Stockholm Sweden (Acta Pathologica et Microbiologica Scandinavia 1972; Section B, 80:335), 8=Edinburgh UK (Nature 1979; 279:43), 9=NIH Bethesda MD (Lancet 1975; ii:838), 10=FDA Bethesda MD (Lancet 1978; i:463), 11 = Chiron CA USA (Science. 1989; 244(4902):359), 12 = Chiron CA USA (Proc Natl Acad Sci USA 1991; 88:2451), 13=Turin Italy (Gut 1977; 18:997), 14 = NIH Bethesda MD (Proc Natl Acad Sci USA 1980; 77:6124)., 15=Chiron CA USA (Nature 1986; 323:508), 16 = Kashmir India (Am J Med 1980; 68: 818), 17=Moscow USSR (Intervirology 1983; 20: 23), 18=Genelabs CA USA (Science 1990; 247:1335), 19=Genelabs CA USA (Virology 1991; 185(1):120).

Willowbrook experiments

Human experiments on viral hepatitis were most rewarding when Krugman et al.¹⁹ from New York University School of Medicine, NY, USA reported on his experiments carried out on children with intellectual disability located in the Willowbrook neighbourhood on Staten Island in New York City (Table 2). They admitted new entrants to the School in a special isolation facility and infected these with blood samples from patients of the School who had contracted hepatitis. The observations based on seven trials pointed to existence of two hepatitis viruses, the inoculums of which were named as MS-1 and MS-2. MS-1 inoculum belonged to a patient (Mir) during the first attack of hepatitis and inoculum MS-2 belonged to the same patient six months later during second attack of hepatitis. The two forms of hepatitis differed in clinical, epidemiological and immunological characteristics. Disease associated with MS-1 inoculum (infectious hepatitis) had short incubation period (30–38 days), brief transaminitis, abnormal thymol turbidity and high degree of infectivity. Disease associated with MS-2 inoculum (serum hepatitis) had longer incubation period (41–108 days), prolonged transaminitis (35–200 days), normal thymol turbidity and low infectivity. The hepatitis caused by MS-1 showed immunity to rechallenge with same inoculum but not with MS-2 inoculum. The studies by Krugman et al.¹⁹ were conclusive which could not be repeated today.

Table 2.

Human Experiments on Viral Hepatitis in Children at Willowbrook. Results of Seven Trials Which Pointed to Existence of Two Hepatitis Viruses are Defined. The Clinical, Epidemiological and Immunological Characteristics of MS-1- and MS-2–Related Diseases are Shown.

Trials	Date	Inoculum	Route	Subjects	Results
First Trial [F T]	Sept 23, 1964	WSP-5	Oral	13 NAS [11 EI & 2 C]	H=10 of 11 EI (6 had IH). AH = 1 of 2 C IP = 30–36 d in 6, 51–58 d in 3, 125 d in1
Second Trial [S T]	March 26, 1965	WSP-5	IM	13 FT [10 EI & 3 C]	H = 6 out of 10 EI (4 out of 6 had SA:- Sch, Mas, Wac, Mir]. IP = 38–102 d
Third Trial [T T]	Nov 22, 1965	MS-1	IM	14 NAS [8 EI & 6 C]	H = 7 of the 8 EI (1 had IH); IP = 31–53 d H = 6 of 6 C (I had IH); IP = 46–85 d
Fourth Trial [Fi T]	August 24, 1965	MS-2	IM	14 NAS [9 EI & 5 C]	H = 7 of 9 EI (1 had IH); IP = 41–69 d AH = 2 of 5 C; IP = 155–231 d
Fifth Trial [Ft T]	Feb 17, 1966	MS-2 (Ham) + μ-globulin	Oral	9 NAS [6 EI & 3 C]	AH = 5 of 6 EI; IP = 64–209 d All 3 C had no hepatitis
Sixth Trial [Si T]	May 27, 1966	MS-2 (Ham & Pel)	Oral	6 NAS	AH = 5 of 6 EI; IP = 88–108 d
Seventh Trial [St T]	March 28, 1966	MS-1	IM	16 [8 T T & 8 Fi T]	H = 0 of 8 T T (Immunity vs. MS-1) H = 8 of 8 Fi T (no immunity vs. MS-2))

	MS-1 Hepatitis	MS-2 Hepatitis
	Infectious hepatitis	Serum hepatitis
Incubation Period	15–50 days	43–180 days
Transmission	Oral + Parenteral	Oral + Parenteral (IP longer with oral) (Si T vs. Fi T)
Contact spread	High (T T)	Low
Transaminitis	Sharp and short lasting	Gradual and prolonged
Immunity	No cross immunity between MS-1 & MS-2. Homologous immunity following MS-1 & MS-2 infections.
Clinical profile	Indistinguishable
IH/AH	10/19	6/21
Bilirubin levels	1–3 mg/dl	1–3 mg/dl
Duration icterus	1–10 d (5d)	1–10 d (5d)
Weight gain	0.9–3.6 kg (25 weeks)	1.8–2.7 kg (30 weeks)
		Krugman et al JAMA 1967;200:365–373.

WSP-5= Willowbrook Serum Pool from 27 patients collected 3–7 days before jaundice; NAS= Newly Admitted Subjects; EI = Experimentally Infected; C=Controls; IP= Incubation Period; SA= Second Attack; H= Hepatitis; IH= Icteric Hepatitis; AH = Anicteric Hepatitis; MS-1 = Sample of subject Mir immediately prior to First Attack; MS-2 = Sample of subject Mir immediately before the Second Attack.

The volunteer studies of Krugman et al.¹⁹ were not without controversy.²⁰ These experiments were qualified as unethical by the World community because of several reasons. First the participants were infected deliberately which caused disease in the incumbent. Second the participants were mentally disabled children, and finally the study was conducted in an institution with unsanitary environment.²¹ There have been major changes in social values and ethics of the studies carried out in children with intellectual disability. This has led to eventual termination of such studies. Krugman stated that all children admitted to the facility were getting infected with viral hepatitis invariably. He was only infecting such children under controlled conditions and close supervision. He argued that his experiments met all ethical standards in place at the time.²² Krugman found defenders in high places. Franz Inglefinger,²³ who later became the editor of the New England Journal of Medicine said: “By being allowed to participate in a carefully supervised study and by receiving the most expert attention available for a disease of basically unknown nature, the patients themselves benefited … How much better to have a patient with hepatitis accidentally or deliberately acquired under the guidance of a Krugman than under the care of a (rights-minded) zealot”. Robert Purcell²⁴ from Laboratory of Infectious Diseases, NIH, Bethesda, MD USA on his experiences with discovery of hepatitis viruses wrote “It is easy to condemn these studies from the current perspective, but such retrospective applications of “political correctness” are dangerous: undoubtedly many of our present acceptable policies will be seen as unacceptable in the future when viewed through a different prism”.

Australia antigen

The breakthrough discovery of Australia antigen by Blumberg et al.²⁵ in 1965 led to rapid scientific developments in viral hepatitis. Blumberg discovered the Australia antigen while he was studying genetic polymorphisms of serum proteins during his tenure at NIH, Bethesda, MD. He postulated that multiply transfused people would develop antibodies against ‘pleomorphic’ serum lipoproteins and show up as antilipoprotein isoprecipitins in agar gel double diffusion experiments. In 1963, they tested haemophiliac sera, which were multiply transfused, for presence of isoprecipitins against 24 sera from general population including foreigners. Two haemophiliac sera showed a clearly defined precipitin line with one serum belonging to an Australian aborigine but with none of the others. Subsequent experiments showed that this protein system differed from that detected with the antilipoprotein antisera. The antigen thus identified in the Australian aborigine was named as Australia antigen. They tested sera from 107 multiply transfused patients for Australia antigen isoprecipitins (antibodies) and found it in 11 samples, with highest occurrence (8/28; 28.6%) in those with hemophilia. Then, frequency of Australia antigen was determined in 1704 sera from normal populations and 659 patients. The Australia antigen was absent in native American (0/700) but found in foreigners (38/1004; 3.78%) and highly prevalent in sera from patients with leukaemia (8/70; 11.4%). Authors postulated that those with Australia antigen may be susceptible to develop leukaemia or it may be a manifestation of the disease or related to a virus which may cause leukaemia and could help in diagnosis of early acute leukaemia. Harvey Alter²⁶ a coauthor of the original article and a fellow at the NIH at the time, describes the discovery of Australia antigen as follows: “The chronological events surrounding the Australia antigen stand out as a monument to non-directed medical research and as a tribute to investigative perseverance. This tale of serendipity began in the mid-1960s when the Australia antigen was first reported by a geneticist who had been seeking new inherited polymorphisms among serum proteins, by a blood banker looking for non-cellular causes of febrile, non-haemolytic transfusion reactions and by a technologist destined to become a commercial airline pilot. A research interest in viral hepatitis was conspicuously absent in this investigative team. The significance of the Australia antigen, found when the serum of an Australian aborigine formed a precipitin line with the serum of a multiply transfused haemophiliac, was, at that time, unknown”.

Australia antigen as a marker for serum hepatitis

Subsequently, Blumberg moved to Institute of Cancer Research at Philadelphia and showed that patients with Down's syndrome (25/84; 29.8%), Leukaemia (16/171; 9.0%) and hepatitis (5/48; 10%) had high prevalence of Australia antigen in their sera.²⁷ He proposed several hypotheses for this association but was far from the truth. It was left to Alfred Prince^28,29 at the New York Blood Centre, NY USA to spill the beans. He tested serum (serum S) from a haemophiliac who had received >10, 000 units of blood and in a double diffusion technique, detected an antigen (SH) in the serum collected during the incubation period of a multiply (16 units) transfused patient (Case MU-5), who had been operated for bleeding gastric ulcer and had developed long incubation serum hepatitis. Then, he found that SH antigen in 7 of the 9 cases of serum hepatitis and 3 of 2856 sera collected from the general population. He postulated that the SH antigen is located on the virus particle, and the virus causes some or all cases of serum hepatitis. The SH antigen and Australia antigen were soon found to be identical. Giles et al³⁰ reported on the selective association of SH/Australia antigen with MS-2 hepatitis and not with MS-1 hepatitis. Thus discovery of hepatitis B has been compared with “Vax Pack Hero game-Beat the Germs”³¹ and has been summarized as follows: “As a geneticist, Blumberg developed the idea that people from different backgrounds had varying susceptibilities to different infections and thus concluded that disease susceptibility is genetic. While working on this hypothesis, Blumberg discovered Australia antigen. Shortly thereafter Prince (a virologist) figured out that Australia antigen was part of the hepatitis B virus. With Blumberg and Prince's work as a foundation, a blood-derived vaccine was developed by Maurice Hilleman. Blumberg, who won the Nobel prize for discovering something without ever knowing what it actually did, mentions these two incredible men only in an aside. Many in scientific community believe that Nobel Prize should have included name of Alfred Prince”.

Dane particle

Electron microscopic examination of the sera with Australia antigen revealed presence of 2 types of particles namely 22-nm spheres and long filaments of the same width.³² As both these particles were found to be free of nucleic acids, these could not qualify to represent the virus of serum hepatitis. In 1970, Dane et al³³ from Middlesex hospital, London studied 16 Australia antigen positive sera by immune electron microscopy (IEM) and visualized 22-nm sphere particles in all samples and long filaments in few. However, 3 sera showed presence of 42-nm particles, with an inner nucleocapsid and outer coat akin to sphere/filament material. The authors postulated that the 42-nm particle is the virus of serum hepatitis and spheres/filaments are noninfectious surplus virus-coat material. Then, Almeida et al.³⁴ from Royal Postgraduate Medical School, London treated the 42-nm particles from patients with serum hepatitis with detergents and were successful in separating the outer coat and 27-nm inner particle of the virus. Convalescent sera from serum hepatitis reacted with inner component (not with the outer) to yield aggregates. Thus, core antigen/core antibody antigen-antibody system was defined. Magnius and Espmark³⁵ from Statens Bakteriologiska Laboratorium, Stockholm detected a new antigen complex (e-antigen) in Australia antigen–positive sera by immunodiffusion tests. The e-antigen was found in 18 of 23 Australia antigen–positive patients on haemodialysis, 6 of 43 Australia antigen–positive hepatitis and none of 17 Australia antigen–positive blood donors. Thirteen of the aforementioned sera contained antibodies against the new antigen (anti-e). The authors postulated that this new antigen system is associated with infectivity. Hirschman and colleagues³⁶ from Mount Sinai Hospital, New York, NY USA found DNA polymerase activity in concentrated pellets containing Australia antigen. These observations were confirmed by Robinson and Greenman.³⁷ Robinson et al.³⁸ from Stanford University School of Medicine, Stanford, California USA extracted the radioactive DNA band from sera containing Dane particles and on electron microscopy found double-stranded DNA molecule of approximately 0.78 um in length. They postulated that the DNA served as primer template for the DNA polymerase reaction carried out by Dane particle cores.

HBV DNA cloning sequencing

By this time, it was clear that 42-nm Dane particles had serological and biochemical properties of the HBV. Dane particles had outer envelope containing the hepatitis B surface antigen (HbsAg) and an inner core (27 nm) bearing a second unrelated antigen, the hepatitis B core antigen (HBcAg). Within the core antigen was a double-stranded circular DNA molecule, with DNA genome of around 2 x 10⁶ Da and an endogenous DNA-dependant DNA polymerase activity. Hepatitis B e antigen (HBeAg) was associated with the Dane particle and could be detected free in plasma of some infected individuals. However, progress on understanding the biology of the virus was limited as there was no tissue culture available for further studies on the virus. To circumvent this, Burrell et al.³⁹ from University of Edinburgh, UK isolated HBV DNA from Dane particles of HBsAg-/HBeAg-positive donors and digested with restriction endonucleases and expressed portion of the digest in to Escherichia coli plasmid and cloned. This made production of sufficient quantities of HBV DNA for structural and sequence analysis. Cells carrying the hybrid plasmid synthesised antigenic material that reacted specifically with antisera to hepatitis B antigens. Then, Sninsky et al.⁴⁰ from Stanford University, School of Medicine, Stanford, California, USA and Charnay et al.⁴¹ from Institute Pasteur, Paris, France cloned HBV DNA and performed endonuclease mapping of HBV DNA genome and showed cleavage sites of the genome for certain restriction endonucleases. Valenzuela et al.⁴² from University of California, San Francisco, California, USA cloned HBV DNA from HBV Dane particles and examined a full-length clone by restriction endonucleases. They reported on the nucleotide sequence of an 892-base pair fragment of the HBV DNA encoding for HbsAg, a 226 amino acid protein. Galibert et al. ⁸⁵ determined complete nucleotide sequence of 3182 nucleotides of HBV genome (subtype ayw). Eight open reading frames, able to code several proteins were located. HBV DNA was confirmed to be circular and encoded the genes for HbsAg, HBcAg, the putative endogenous DNA polymerase and an unexpected X gene. Availability of unlimited amounts of HBV DNA, HBsAg and HBcAg was used for diagnostics and vaccine development. This led to explosion of clinical, epidemiological, fundamental and experimental research on HBV.

Hepatitis A virus

Krugman et al.¹⁹ had conclusively described the clinical syndrome of viral hepatitis caused by HAV (MS-1 type). Human and animal experiments characterized the physiochemical properties of the etiologic agent.^43,44 The virus was found to be resistant to ether and heat and could be transmitted through faeces and blood collected during incubation period. Several groups of investigators reported on the isolation or detection of HAV, which all proved to be premature and false.^45,46 Finally, Feinstone et al.⁴⁷ from NIH, Bethesda, MD, USA successfully visualized 27-nm VLP on IEM in two of the four stools specimens, obtained during acute phase of illness after inoculation with MS-1 strain in prisoner volunteers at the Joliet Prison. The stool samples were incubated with the convalescent patient serum before examination. All six experimentally infected individuals had detectable antibodies to hepatitis A antigen on IEM in samples taken after and not in those collected before infection. Six other individuals from epidemics of hepatitis A had significant increase in graded antibody titres in paired samples. Two samples from naturally acquired hepatitis B and two others from experimental nonbacterial gastroenteritis did not have antibodies to hepatitis A antigen on IEM. The findings suggested that it is the etiologic agent for hepatitis A. The virus was transmitted to marmosets and chimpanzees, and hepatitis A antigen was demonstrated in liver biopsies on immunofluorescence.⁴⁸ The virus was propagated in cell cultures, an important step in development of vaccine.^49,50 Ticehurst et al.⁵¹ from NIH, Bethesda, MD, USA cloned and sequenced the genome of virus. A double-stranded cDNA was prepared from HAV RNA and inserted in to E. coli Pbr322 plasmid. Restriction endonuclease digestion and cross-hybridization of fragments included 99% of the viral genome. Sequence analysis revealed 414 bases open reading frame starting from the 3’ end of the genome, followed by two stop codons (closely spaced), a noncoding region of 60 bases and a tract of poly (A). This paved the way for production of recombinant vaccine.

δ antigen-antibody system

In 1977, Rizzetto et al.⁵² from Ospedale Mauriziano Umberto 1, Turin, Italy identified by chance a new antigen-antibody system (δ antigen-antibody system) associated with HBV. The δ antigen was localized by direct immunofluorescence only in the liver cell nuclei, and the δ antibody was found in sera of patients with chronic HBsAg carriers and was associated with severity liver disease. The δ antigen-antibody system was immunologically distinct from core, surface and e antigen-antibody systems.

Hepatitis D virus

The new antigen was at first thought to be a marker of the HBV. However, Mario Rizzetto collaborated with the National Institute of Health, Bethesda, MD, USA and Georgetown University School of Medicine and Dentistry, Washington, USA and unfolded an unexpected and amazing chapter in virology. Based on transmission studies in Chimpanzee, delta antigen was found not to be a component of HBV but a separate defective virus requiring HBV for its infection.^53,54 Inoculation of sera positive for HBsAg, as well as δ antigen (coinfection) resulted in hepatitis in susceptible recipient animals with resultant markers of hepatitis B, as well as δ agent. None of the animals with pre-existing antibodies to HBsAg (immune to HBV) inoculated with such sera developed hepatitis, markers for HBV infection and/or δ agent. However, inoculation of such sera in chimpanzees with pre-exiting circulating HBsAg (superinfection) resulted in earlier onset of synthesis of δ infection markers with extent and duration of disease greater than those previously unexposed to HBV. In addition, such animals showed suppression of pre-existing HBV gene products (HBsAg and HBcAg). Based on these experiments, δ agent appeared to be a transmissible agent, either an HBV variant or another agent that is defective, requires the helper functions of HBV and interferes with HBV replication. The agent was named as HDV to conform to the nomenclature of hepatitis viruses. In infectious sera, HDV particles were 36 nm in diameter and could be differentiated from 42-nm Dane particles and 22-nm spherical particles derived from HBV. The HDV consisted of an outer coat made of HBsAg and inner coat consisting of HDV antigen and an δ-associated RNA. HDV RNA has been cloned and sequenced by Wang et al⁵⁵ and Kos et al.⁵⁶ Biochemical and electron microscopic examination revealed that virus contained a circular and single-stranded RNA genome. The HDR RNA was polyadenylated and cDNA synthesized and cloned and sequenced. The nucleotide sequence of the cloned cDNA suggested that the template RNA is a circular molecule of 1678 nucleotides.

Non-A, non-B hepatitis

The search for hepatitis C virus started when Alter et al.⁵⁷ from NIH, Bethesda, MD, USA studied clinical and serological analysis of transfusion-associated hepatitis. Eight (66.6%) of the 12 transfusion-associated hepatitis cases which occurred in 108 prospectively followed multiply transfused open-heart surgery patients were found serologically unrelated to the HBV, HAV, the cytomegalovirus and the Epstein-Barr virus. Eight non-A, non-B hepatitis (NANBH) cases when compared with 4 hepatitis B cases had shorter incubation period (9.4 vs. 14.5 wk), lower peak aminotransferase (470 vs. 857 IU/dL) & serum bilirubin (1.1 vs. 7.8 mg/dL). One patient in each group progressed to chronic hepatitis. The authors postulated the existence of one or more previously unrecognised hepatitis virus(es). Two groups of investigators simultaneously transmitted the virus to chimpanzees. Alter et al.⁵⁸ from NIH, Bethesda, MD, USA transmitted NANBH to five chimpanzees by intravenous inoculation of serum or plasma from 4 patients with acute or chronic NANBH and a donor implicated in two cases of transfusion-associated hepatitis. All chimpanzees developed biochemical and histological evidence of hepatitis at a mean incubation period of 13.4 weeks. The authors suggested that NANBH is transmitted by an agent which can persist and remain infectious for long periods. Tabor et al.⁵⁹ from FDA, Bethesda, MD, USA transmitted NANBH to four colony-borne chimpanzees by intravenous inoculation of serum from 3 patients with clinical and serological evidence of chronic NANBH. All four chimpanzees showed elevated liver enzymes 2–4 weeks after inoculation and had evidence of hepatitis on liver biopsies. Shimizu et al.⁶⁰ from NIH, Bethesda, MD described two types of ultrastructural changes, exclusive of each other, in hepatocytes of chimpanzees with acute or chronic NANBH. These included cytoplasmic Tubule Forming Agents in one group of chimpanzees and nuclear aggregates of 22–27 nm particles in another group. The authors suggested the existence of two transmissible agents in NANBH, represented by cytoplasmic Tubule Forming Agents and nuclear aggregates of 22–27 nm particles. Several groups defined the physicochemical properties of the Tubule Forming Agent.^61,62 Concurrently, the epidemiology of NANBH after transfusion, its frequent acquisition within the community unlinked with blood transfusion and propensity of the infection to cause chronic hepatitis and cirrhosis was demonstrated.^63,64 Despite this progress and application of many molecular techniques, it proved elusive to definitely identify the NANBH agents well over a decade of intensive research by many laboratories throughout the world.⁶⁵ Numerous claims of identifying the virus were made which all proved to be wrong when tested against a famous infectious sera panel at NIH, Bethesda, MD.⁶⁶

Hepatitis C virus

Finally, a breakthrough was made when investigators at Chiron Lab initiated a novel blind immunoscreening approach involving the large-scale screening of bacterial proteomic cDNA libraries derived from NANB-infectious chimpanzee material using sera from NANBH patient as presumptive source of viral antibodies.^67,68 Six positive clones were identified. Five were derived from either added MS-2 bacteriophage RNA or host genes that encoding proteins apparently eliciting an autoimmune response. One small clone 5-1-1 of 150 base pairs failed to hybridize genomic DNA extracted from uninfected individuals and chimpanzee sera. In contrast, the clone hybridized to a positive, single-stranded RNA of 10,000 nucleotides in length from infected chimpanzees only. When expressed in bacteria or yeast, RNA encoded an antigen that bound to antibodies found only in parenterally transmitted NANB infected chimpanzees and not in those infected with HAV or HBV infected animals. Seroconversion of antibodies to 5-1-1 was detected in NANB infected animals and human and such antibodies were present in majority of patients with chronic NANBH and not in those with HAV or HBV infection. All these findings gave a convincing evidence that the etiologic agent for transfusion-associated NANBH was found. With availability of large amounts of recombinant HCV antigen, the first EIA test for NANBH-specific antibodies was produced.⁶⁹ To testify the discovery of hepatitis C virus at Chiron Lab, Harvey J. Alter⁶⁶ in his Master Perspective wrote: “Nineteen previous claims of NANB discovery had been laid to rest by this panel … My low level of expectation had dampened my sense of expectation. When I broke the code, I was surprised and excited to find that the Chiron assay had correctly identified every sample from chronically infected patients and found no reactivity in negative controls”. Finally, the complete sequence analysis of HCV genome allowed the genetic organization to be elucidated.^70,71

Epidemic NANBH

The story of hepatitis E is historically linked to discovery of Epidemic Non-A, Non-B Hepatitis (ENANBH) from the Gulmarg-Kashmir epidemic of November 1978.⁷² This massive unprecedented large-scale common source waterborne epidemic of hepatitis posed many intriguing problems. These included hard weather conditions, primitive healthcare structure, time factor to investigate a short-lasting massive epidemic, immediate need for a large survey team, availability of funding and personal health risk and thus needed a systematic approach and a strong conviction to explore the unknown. To face these challenges, Mohammad Khuroo⁷² working at the Government Medical College, Srinagar, Kashmir, India opted to live in the area, strengthen the health services, offer care at doorsteps and planned an ingenious study protocol to identify each and every next case of hepatitis over extended period. Four door-to-door surveys, carried out at 4–6 weeks intervals, were conducted in 200 affected villages over a population of 600,000 (Figure 1). Epidemic curve was highly compressed, lasted for 9 weeks with no secondary cases, with a weekly occurrence of up to 4000 icteric cases, amounting to total over 20,000 icteric cases with 600 deaths. The disease selectively affected young adults 15–45 years age. There was increased incidence and severity of the disease in pregnant women with substantial perinatal mortality.⁷³ Patients presented with acute hepatitic syndrome with significant cholestatic features in 20 percent of those affected. Around one-third of the population had features of anicteric hepatitis. A subset of patients had distinctive morphological changes in needle biopsies of liver. Disease was self-limiting with no evidence of chronic hepatitis or cirrhosis.⁷⁴ All patients had prior immunity to hepatitis A (IgG anti-HAV). Acute phase sera lacked acute markers of HAV & HBV infection (Non-A, non-B). Based on the aforementioned context, the existence of a new disease called ENANBH caused by another human hepatitis virus, hitherto not known and distinct from post-transfusion NANBH was announced.⁷²

Figure 1.

Gulmarg-Kashmir Epidemic non-A, non-B hepatitis 1978–1979. Ingenious door-to-door survey was carried out to identify the next case of hepatitis over extended period from Sept 4, 1978 to Nov 30, 1978. Four door-to-door surveys were carried out at 4 to 6-week intervals.

Sceptics

These data that a new form of hepatitis virus was the likely cause of this epidemic were presented at the annual meeting of Indian Society of Gastroenterology held at Pune, India on Oct 12, 1979 and at the ‘First International Conference of viral hepatitis’ held at New York on March 30, 1981.⁷⁵ All this led to a buzz and started a series of enquiries, support and collaborations. However, the existence of epidemic NANBH as a new disease entity did not go well with the sceptics, who were convinced that this was classical example of waterborne hepatitis A and believed that ‘hepatitis A’ testing carried out was faulty. This scepticism could be sensed in the words of (Late) Prof. Okuda in a letter dated Oct 15, 1979, “Dear Dr. Khuroo, …Regarding the outbreak of acute hepatitis in your area, I discussed it with Prof. Ishida, Dr. Alter and Dr. Mayumi. They are suspect that this was water-borne epidemic of hepatitis A. If you are certain that it was not, Dr. Mayumi is willing to test your sera…. Sincerely yours. Kunio Okuda”. To counter the ‘A vs. non-A controversy’, thousands of sera were shipped to many reputed laboratories of the world. Uniformly, reports from all such laboratories showed that all sera lacked acute phase markers of hepatitis A and thus the sera should be classified as ‘non-A’. This put the ‘A vs. non-A’ controversy to rest and established the existence of ‘ENANBH’ as a new disease.

A mis-step in hepatitis research

The reason for the ‘A vs. non-A controversy’ was related to studies carried out on Delhi epidemic 1955–1956, under the auspices of ‘Indian Council of Medical Research’ under overall charge of Dr. R. Viswanathan.⁷⁶ The epidemic underwent extensive animal transmission, cultivation and virus isolation studies and all turned out to be negative.⁷⁷ Of significance, Late Dr Melnick, the American epidemiologist/virologist, known for his work on polio virus and polio vaccine, was involved in virus isolation studies at National Institute of Virology Pune, India.⁷⁸ Based on these studies, the investigators concluded that Delhi epidemic was a classic example of epidemic hepatitis A and peak attack rate in young adults was due to waning immunity after previous exposure to HAV.²⁴ This mis-step in biomedical research on epidemic hepatitis delayed recognition of hepatitis E by two and a half decades. It was only after the existence of epidemic NANBH from Gulmarg Kashmir epidemic 1978 was announced, sera stored at National Institute of virology at Pune from 1955 to 1956 Delhi epidemic were retrieved and tested for acute markers of hepatitis A and found to be negative.⁷⁹ Thus, Delhi epidemic was found to be NANBH retrospectively 25 years after its occurrence and was not the first documented hepatitis E epidemic in history.

The lost opportunity

To look for the new agent, Mohammad Khuroo collaborated with Robert H. Purcell at NIH. Kashmir samples reached his lab in excellent conditions for animal transmission studies. However, transmission studies were delayed owing to shortage of Chimpanzees. Regarding this, Robert H. Purcell wrote on April 28th 1981: “Dear Mohammad, I have been enthusiastic about the collaborative study to test samples obtained from outbreaks of hepatitis in northern India. Our plan will be to inoculate primates with acute phase samples and serologically characterize any transmissible agent. Your clinical samples have arrived here in excellent condition. Because of shortage of chimpanzees, we have not been able to inoculate your samples to date but they will be inoculated as animals become available this month”.

Virus-like particles

Although the investigators at Kashmir were struggling to find the animals for transmission, (Late) Dr. Balayan from Russian Academy of Medical Sciences in Moscow, Russia showed extreme zest to explore the unknown. Through a self-experimentation, he identified VLP in his stool samples and this became the first stepping stone towards finding etiologic agent of ENANBH.⁸⁰ This event in history has been described by Dr. Balayan himself as follows: “In 1983, I was investigating an outbreak of non-A, non-B hepatitis among Soviet soldiers in Afghanistan. The outbreak had similar features as described in Kashmir epidemic 1979. I wanted to bring infected stool samples to my Moscow laboratory. I lacked refrigeration. So, I made a shake of yoghurt and stool from nine infected patients, drank it, went to Moscow and waited until a few weeks later when I developed severe attack of hepatitis. I then started collecting and analysing my own stool samples. In these I found virus-like-particles (VLP) on IEM, that produced liver injury in laboratory animals. I already had antibodies against hepatitis A virus (HAV) which did not protect me from the infection”.

Hepatitis E virus

The period from 1983 to 1991 of nine long years saw a major setback and a frustration for the investigators in the field of hepatitis E research. This was due to the fact that infectious samples (stool and sera) from patients and experimentally infected animals contained very few VLP, not enough for cloning and sequencing. This frustration ended, when Dr. Bradley at CDC, Atlanta Georgia, USA found large quantities of VLP, exceeding 1000/electron grid square, in the bile samples of experimentally infected macaques. Reyes et al (1990) partially cloned the virus from the infected bile of macaques.⁸¹ Virus enriched gallbladder bile from macaques was used as a source to construct cDNA libraries. One clone ET1.1 (1.3 KB) was found exogenous and hybridized a single-stranded polyadenylated RNA virus (7.6 kb) from infected liver of macaques. Soon, Tam et al⁸² from Genelabs group cloned and sequenced the entire 7.2 kb positive-strand RNA genome of the Burma strain of HEV. This completed the saga of discovery of HEV⁷⁵ and this event in history was recently described by Lemon et al⁸³ as “Thus, by 1992, a decade after discovery of the ET-NANB particle by Balayan et al in 1983⁸⁰ and a dozen years after Khuroo's description of the epidemic in Kashmir in 1980⁷², HEV was firmly established within the panoply of human hepatitis viruses”. Studies by Xiang J. Meng⁸⁴ working with Robert H. Purcell & Suzanne U. Emerson at the National Institute of Health identified swine strain of HEV, which led to the genetic and biologic diversity of HEV.

Conflicts of interest

The authors have none to declare.