The Discovery of the Hepatitis D Virus: Three Princes of Serendip and the Recognition of Autoantibodies to Liver‐Kidney Microsomes

Abbreviations

Ab

antibody

Ag

antigen

AIH

autoimmune hepatitis

AMA

anti‐mitochondrial antibody

ANA

antinuclear antibody

anti‐HBc

antibody to hepatitis B core antigen

CF

complement fixation

CFT

complement fixation test

CH

chronic hepatitis

CHB

chronic hepatitis B

EM

electron microscopy

FITC

fluorescein isothiocyanate

HBcAg

hepatitis B core antigen

HBeAg

HBV e antigen

HBnAg

hepatitis B virus nuclear antigen

HBV

hepatitis B virus

HCV

hepatitis C virus

HDV

hepatitis D virus

LKM

liver‐kidney microsomal

LM

liver microsomal antibody

LP

liver‐pancreas antigen antibody

NIH

National Institutes of Health

SLA

soluble liver antigen antibody

WHV

woodchuck hepatitis virus

Mario Rizzetto

An Auspicious Beginning

During my university years in Padua, where I followed in the footsteps of William Harvey (Fig. 1), I was fascinated by the emerging discipline of hepatology. In the 1950s, two discoveries opened the way to understanding the new and evolving entity of chronic hepatitis (CH). First was the observation by the Italian clinicians Fernando de Ritis, Mario Coltorti, and Giuseppe Giusti ¹ that serum transaminases (or aminotransferases, for the pedants among you) that once were the sole domain of the cardiologists ² were elevated in patients with hepatitis. Ironically, cardiologists now find that serum alkaline phosphatase and gamma‐glutamyl transferase are useful in assessing coronary artery disease. ³ Second was the almost coincident honing of the technique of liver biopsy by Dr. Giorgio Menghini ⁴ (Fig. 2). The allure of CH was that it seemed to be the missing link between cirrhosis (which had been known since antiquity) and acute hepatitis.

Fig 1.

William Harvey, circa 1650. Unknown artist. Printed with permission from the Royal College of Physicians, London UK.

Fig 2.

Giorgio Menghini (1916‐1983). Reproduced from Hepatology. ¹² Copyright 2003, American Association for the Study of Liver Diseases. Photograph courtesy of Professor Stefano Fiorucci and provided by Menghini's daughter Chiara.

Recognition of the Liver‐Kidney Microsomal Autoantibody

A first clue to the origin of CH came in the 1960s from the association of some cases with a battery of serum autoantibodies to normal human antigens, such as the antinuclear antibody (ANA), anti‐smooth muscle antibody, and others; and the diagnosis came to be known as autoimmune hepatitis (AIH) (see the essay by Albert Czaja, ⁵ “Autoimmune Hepatitis: Surviving Crises of Doubt and Elimination,” elsewhere in this series). Thus, these antibody‐positive CH cases were autoimmune hepatitides.

I was of course much interested and, luckily, I was awarded a 1‐year grant by the University of Turin to work as a research fellow in the Department of Immunology of the Middlesex Hospital in London, which was demolished in 2008 (Fig. 3), but in those days was a Mecca of Autoimmunity. My supervisor was the late Dr. Deborah Doniach (Fig. 4), a singularly creative, innovative, and brilliant investigator, who was often referred to as The Queen of British Immunology and who was also a highly cultured international personality.

Fig 3.

The Middlesex Hospital, by W.A. Donald. Provided by kind permission of the Trustees of University College London Hospitals NHS Foundation Trust.

Fig 4.

Deborah Doniach (1912‐2004). Photographed by Erica Leung for Middlesex Hospital, Mortimer Street, London, UK. Provided by kind permission of the Trustees of University College London Hospitals NHS Foundation Trust.

At Middlesex I learned the laboratory techniques of autoimmunity and participated in routine diagnostics using tissue autoantibodies. In the mid‐1960s, a young Geoff Walker (John Geoffrey Walker died on May 23, 2018), working with Deborah and fellow immunologist Ivan Roitt, and the formidable Dame of British Hepatology, Sheila Sherlock at the Royal Free Hospital, discovered the anti‐mitochondrial antibody (AMA) as a diagnostic marker of primary biliary cirrhosis, ⁶ now known as primary biliary cholangitis. The antibody target in the liver and kidney tubules was determined in tissue by characteristic immunofluorescent staining. By the time I arrived at the laboratory in January 1972, Dr. Doniach had observed that some sera stained liver and kidney with an immunofluorescence pattern resembling, but somewhat different from, that given by her original AMA. She considered this to be due to a second antibody against a different mitochondrial antigen. ⁷ She called the original antigen located in the inner mitochondrial membrane “mito‐1” and assumed that there was another antigen in the outer mitochondrial membrane, which she called “mito‐2.” ⁷

She charged me with the task of confirming that there were indeed two distinct mitochondrial antigens eliciting different fluorescent patterns. Using differential centrifugation of homogenates of rat livers, I prepared the mitochondrial and microsomal fractions that precipitated at 7000g for 10 minutes and 105,000g for 90 minutes, respectively. The mitochondria were then disrupted with sonication to expose the outer membranes and challenged with the mito‐2 antibody, using a standard complement fixation test (CFT); the microsomal fraction served as the negative control. But a problem arose from the very first experiments because the microsomal control fixed complement better than the mitochondrial fraction did every time I repeated the 5‐hour procedure. When I reported the findings to Deborah, she thought that I was “messy” and told me to be more careful. I went on with several new rounds of experiments, but to no avail. The microsomal fraction was always fixing better than the mitochondria. My relationship with Deborah then became tense; she was reluctant to accept my results and kept thinking that I was messy. I was discouraged and wondered whether I should accept defeat and return to Turin, when serendipity came to my rescue.

The inimitably English term “serendipity,” coined as a neologism by Hugh Walpole in 1754, ⁸ was inspired by the Persian fairy story of Three Princes of Serendip (Perso‐Arabic for Ceylon, now Sri Lanka), which originated, appropriately enough, from Peregrinaggio di tre giovani figliuoli del re di Serendippo, published in 1557 in Venice (which is located serendipitously just 39 km from Padua, where my career started some four centuries later), by Michele Tramazzino, who allegedly learned it from the Italian translator Christophero Armeno via his adaption of Book One of Amir Khusrau’s early 14th century Hasht‐Bihisht. In short, it is a tale of accidents and perspicacity, where serendipity is the art of discovering new things by learning from encountering unexpected observations. ⁸ Limited space does not permit here a full relating of the princes’ remarkable and successful adventures (Fig. 5), in which they displayed their powers of observation and their innate intelligence and wisdom by correctly interpreting, using Sherlock Holmes–style inductive reasoning, the meaning of unplanned yet fortunate discoveries ⁸ —the very essence of serendipity. ⁹

Fig 5.

The Three Princes of Serendipity. A. Murghal painting by Balchand, as illustrated on the book cover of R.K. Merton and E. Barber, The Travels and Adventures of Serendipity: A Study in Sociological Semantics and the Sociology of Science. Printed with permission from Victoria and Albert Museum, London, UK. © Victoria and Albert Museum, London.

What happened next in the laboratory was an unfortunate accident that was fortuitous, actually serendipitous, for me. The rotor of the ultracentrifuge broke down, possibly because of my overuse in the previous months. I was therefore no longer able to fractionate rat liver. However, given the fact that the concentrations of mitochondria and microsomes should have been different throughout the kidney, I resorted to an immunohistochemical technique in an attempt to demonstrate that mito‐1 and mito‐2 were not colocalized in the kidney, that is, that the two antigens were located in different subcellular structures as suggested by the results of my seemingly “messy” subcellular fractionation experiments. This alternative approach was made possible because rat kidney is small enough to be cut into single slices containing the whole longitudinal section of the organ.

The new experiment worked. Immunolocalization of the cognate antibody to the putative mito‐2 antigen was different from that for mito‐1 (Fig. 6), showing that mito‐2 was not in the mitochondria. ¹⁰ Deborah was visibly moved and immediately appreciated the implication of the result; it was not the antibody to mito‐2 but the newly named liver‐kidney microsomal (LKM) antibody (anti‐LKM) ¹¹ that was later to be recognized as the serological marker of a new type of CH, soon to be called AIH type 2 (Table 1).

Fig 6.

(A) Staining of cryostat sections of whole rat kidney with the putative anti‐M2 human antiserum conjugated with peroxidase. This low‐power photomicrograph image shows signals predominantly in the inner cortex, which contains mainly P3 portions of proximal tubules that are rich in microsomal (i.e., endoplasmic reticulum) areas. (B) Low‐power photomicrographs of the staining of mitochondrial areas of cryostat sections of rat kidney with anti‐M1‐human antiserum conjugated with peroxidase. Here the antibodies react with the entire cortex and medulla, giving maximum staining on the ascending loops of Henle and distal tubules. Reproduced with permission from Clinical and Experimental Immunology. ¹⁰ Copyright 1973, British Society for Immunology.

Table 1.

Classification of CH According to Etiology ¹¹

Hepatitis Type	HBsAg	HBV DNA	HDV Antibody (HDV RNA)	HCV Antibody (HCV RNA)	Autoantibodies
B	+	+/−	−	−	–
D	+	−	+	−	10%‐20% anti‐LKM‐3
C	−	−	−	+	2%‐10% anti‐LKM‐1
Autoimmune
Type 1	−	−	−	−	ANA
Type 2	−	−	−	−	LKM‐1
Type 3	−	−	−	−	SLA/LP
Drug induced	−	−	−	−	Some: ANA, LKM, LM
Cryptogenic	−	−	−	−	−

Modified with permission from Hepatology. ¹¹ Copyright 1994, American Association for the Study of Liver Diseases.

The Discovery of the Delta Antigen

Back in Turin at the end of 1973, I took a permanent position in the Division of Gastroenterology of the Ospedale Mauriziano, headed by Prof. Giorgio Verme, one of the first Italian hepatologists and our national expert in the histological diagnosis of CH; he had published his experience in one of the first Atlas of Liver Pathology publications in the world. Giorgio was for me a real maestro, in the classical meaning of the word, not only a Master of Medicine, but also a Master of Life by reason of his charisma, personality, and style. He gave his enthusiastic support for me to establish an immunofluorescence laboratory for the diagnosis of AIH. After running the laboratory for a few months, however, I realized that AIH was much less frequent in Italy than in Britain, and were I to dedicate all my energy to this topic, I would have remained idle for long intervals.

An alternative was at hand. The discovery of the hepatitis B virus (HBV) in the late 1960s (see Reuben’s ¹² review “The Thin Red Line” and also the essay by Robert Gish, ¹³ “We Are All Africans: A Highly Personal Migratory View of the History of Hepatitis B,” elsewhere in this series) lifted the vale over the landscape of CH of viral origin. At that time, no molecular assay was available to determine the replicative activity of HBV. As a surrogate for HBV replication, we started to test for hepatitis B core antigen (HBcAg) in the liver cells using immunofluorescence. Based on experience acquired in London, I prepared a fluorescein isothiocyanate (FITC)‐labeled anti‐core reagent from the IgG fraction of an HBsAg‐positive serum and used it as standard anti‐HBc (antibody to HBcAg) on unfixed cryostat sections of liver biopsies from patients with chronic hepatitis B (CHB). When the HBcAg was present in the liver, the nuclei displayed a granular fluorescence.

In a study in the mid‐1970s, we found that in patients with CHB, the intrahepatic core antigen invariably bound the homologous antibody in the form of immune complexes. ¹⁴ To detect the immune complexes, we devised a simple test. Unfixed cryostat sections of liver biopsies were covered with fresh normal human serum (containing complement) and were then incubated with FITC‐conjugated antisera to the C1q and C3 component of complement (Fig. 7). CF‐fixing biopsies displayed a nuclear fluorescence similar to the reference FITC anti‐HBc. This simple test conserved the precious standard FITC anti‐HBc. We first performed CFTs in all HBsAg⁺ biopsies and then confirmed the presence of the core antigen with the reference FITC‐conjugated anti‐HBc.

Fig 7.

Principles of the CFT in liver biopsy tissue to display intrahepatic immune complexes.

We had hardly started when we found that some HBsAg⁺ biopsies fixed complement but did not react with the reference FITC‐conjugated anti‐HBc. Was this an artefact? However, no CF reactivity was seen in dozens of HBsAg⁻ liver biopsies. As with the paradigm of the HBcAg/anti‐HBc system (antigen in liver/homologous antibody in serum), we conjugated with FITC a patient’s serum that displayed the unexpected CF, and with it challenged his/her own liver biopsy. The result was stunning: the antiserum reacted with an unknown antigen in many liver nuclei, producing strong green fluorescence (Fig. 8). Intrigued, we tested all CFT⁺ biopsies with the FITC‐conjugated anti‐HBc, in parallel with the new FITC‐conjugated antiserum. Most of the CFT⁺ biopsies reacted with either one or the other antiserum but rarely with both. Simultaneous challenge with FITC anti‐HBc and a rhodaminated antiserum against the unknown antigen confirmed that the two antigens were different, as shown by the separate green and red fluorescent staining in a liver containing both antigens (Fig. 9).

Fig 8.

Green immunofluorescence in the nuclei of human liver containing the delta antigen. Unpublished figure from the author’s laboratory.

Fig 9.

HBcAg in green fluorescence and delta antigen in red fluorescence in a human liver containing both antigens. Unpublished figure from the author’s laboratory.

We presumed that the new antigen might be a component of the core particle of HBV. However, Dr. Maria Grazia Canese, the electron microscopist collaborating with us, could not spot core particles, whereas a peroxidase‐labeled antiserum against the new antigen applied to sections prepared for electron microscopy (EM) deposited on clumps of amorphous nuclear material (Fig. 10). ¹⁵ The only plausible explanation for this puzzling appearance was that the new antigen was a hitherto undescribed component of HBV. Therefore, we named it the HBV nuclear antigen (HBnAg) and submitted our findings to a major medical journal for publication.

Fig 10.

Clumps of amorphous material in ultrathin sections of liver nuclei containing the delta antigen, stained with peroxidase‐conjugated anti‐delta. Unpublished figure from the author’s laboratory.

The paper was promptly rejected, starting an odyssey that went through four further submission/rejection cycles. The recurring reason for spurning our advances was that the data were too good to be true and, therefore, unacceptable. My disappointment led me to ask Deborah Doniach for help, as she was on the editorial board of the British journal Gut. Therefore, I sent her the manuscript and I asked for the Board to give it consideration, as publication seemed assured. Except that Dr. Christian Trepo, in Lyon, had localized the HBV e antigen (HBeAg) to the hepatocyte nuclei. Were HBnAg and HBeAg one and the same? Therefore, on a mission to Lyon and with both of our reagents in hand, Christian confirmed that his e and my n antigens were different. At last, the paper was acceptable for publication…once yet another problem was solved. It turned out that in the 1970s, the letter n had already been used for a variant of HBsAg. And so we renamed n as delta ¹⁶ because, as Christian reminded us, e‐alfa, e‐beta, and e‐gamma antigens had already been named, according to the Greek alphabet. At last the paper was ready for publication in Gut. ¹⁶

From the Delta Antigen to the Hepatitis D Virus

The report of a new HBV antigen was received with skepticism and even some sarcasm; some transalpine authorities joked that it had to be an antigen specific to Italians, perhaps as a result of eating spaghetti.

Given the widespread incredulity and its unique nature, the delta antigen would most likely have died out, alongside the several other HBV subtypes of the times. However, the US National Institutes of Health (NIH) became interested, and I had the good fortune to be given a grant from the United States to investigate the nature of delta in that prestigious institution, whither I went in 1978. I had the privilege and pleasure to work under the supervision of Drs. John Gerin and Robert Purcell, two outstanding individuals and world‐renowned virologists. The collaboration that I initiated between the NIH and the Gastroenterology Unit in Turin prospered most productively for almost two decades.

The first goal for the delta project was to devise a suitable serological assay to detect antibodies to the delta antigen (anti‐delta) in serum. Thanks to the help and expertise of Dr. James Shih, who became my bosom buddy throughout my time at the NIH, we were able to develop a specific and sensitive radioimmunoassay for anti‐delta, using as substrate the homogenate of a delta‐positive human liver. ¹⁷

To determine the nature of the delta antigen, we planned critical transmission experiments using the NIH colony of chimpanzees, which were the only animals susceptible to HBV infection, and thus to delta, too. In a first experiment, ¹⁸ an HBV‐naive chimpanzee was inoculated with the serum of an Italian HBsAg carrier who had delta in liver. After an interval consistent with the incubation period of acute hepatitis B, HBsAg appeared and increased in the blood. Once serum HBsAg rose, delta appeared in about 50% of the hepatocytes at its zenith and remained detectable for a few weeks, but disappearing before serum HBsAg was cleared. The animal developed a typical hepatitis B illness.

This first experiment confirmed that delta was a transmissible agent, seemingly a component of HBV. To assess whether and how a preexisting HBV infection would influence the subsequent superinfection with delta, we inoculated a healthy chronic HBsAg chimpanzee carrier with the same Italian HBsAg delta⁺ serum. ¹⁹ The outcome caught us off guard; in this primate of the Pan genus, delta⁺ liver cells were observed in the first liver biopsy taken 1 week after inoculation. In subsequent biopsies, the antigen spread to as many as 85% of hepatocytes at the peak. In parallel with the intrahepatic spreading of delta, blood HBsAg rapidly diminished, whereas severe hepatitis occurred in the previously healthy animal.

This experiment indicated that delta was different from HBV and suggested that it was a nonautonomous viral agent requiring biological help from HBV. Our best guess was that it might represent a defective particle from HBV, and one that could replicate only in the presence of the mature “parent” virus. The search started for the new infectious agent, but my attempts to find the delta antigen were frustrating. Once again, serendipity came to the rescue. One morning at coffee, Jim Shih suggested adding a drop of Nonidet P‐40 detergent to the reagent wells to clarify the mixture and perhaps allow for better detection by the radioactivity counter. This did the trick. In detergent‐treated chimpanzee serum taken at the height of acute delta hepatitis, the signal was overwhelming compared with preinoculation samples. ¹⁹ Clearly the delta antigen circulated in a protected particulate form, from which it was released by the detergent.

To answer the question concerning the physical form of delta, an antigen‐positive serum was pelleted by ultracentrifugation and examined under the EM by Dr. Canese, who had meanwhile joined me at the NIH. Instead of finding the rigid geometrical forms of a viral structure, she repeatedly found instead only a layer of fluffy soft particles that did not at all resemble classical viruses (Fig. 11). ¹⁹ Somewhat disappointed, Maria Grazia concluded that the antigen was probably enclosed in these nondescript particles and decided to work on them; the reward was immediate. Treating the pellet with guinea pig anti‐HBs serum, she found that the particles were coated by the HBsAg; thus, they were indeed of viral origin. With a viral candidate in hand, the next step, of course, was to look for a viral nucleic acid inside the particles. Surprisingly, the very first electrophoretic strip showed massive amounts of a nucleic acid that migrated faster and was distinctly smaller than the DNA of HBV (Fig. 12). Dr. Bill Hoyer, the experienced HBV molecular biologist of the laboratory, ended up treating us all to lunch when he lost his wager that the delta particle nucleic acid was obviously derived from the HBV genome. A few days later, Bill was most excited by the result of selective digestion of the delta nucleic acid with DNase and RNase that showed that the nucleic acid was digested by RNase, and thus it was RNA unrelated to the DNA genome of HBV (Fig. 12). ¹⁹

Fig 11.

EM of a 36‐nm delta particle, negatively stained with 1% phosphotungstic acid, the HDV virion, seen in delta‐positive serum. Unpublished figure from the author’s laboratory.

Fig 12.

The nucleic acid of the delta particle is distinctly smaller than woodchuck HBV. (Left) Temporal association of a low‐molecular‐weight nucleic acid with serum delta antigen activity, as shown by ethidium bromide staining after agarose electrophoresis. Lanes 1 to 4 show results from chimpanzee weekly serum samples; lane 5 contains WHV DNA (3.7‐kb pairs). (Right) Delta antigen‐associated nucleic acid after digestion by ribonuclease A (lane 1), the undigested control (lane 2), and after desoxyribonuclease I digestion (lane 3); lane 4 contains nucleic acid molecular weight markers. Reproduced from Proceedings of National Academy of Sciences of the United States of America. ¹⁹ Copyright 1980; National Academy of Sciences.

Thus, in 1980, was the delta virus officially born, after a somewhat rollercoaster gestation. ¹⁹ The rapid acceptance by the international scientific community and the demonstration of its worldwide distribution led early on to a change in denomination from Greek to Latin, as delta was replaced by D, as in HDV. We were careful not to usurp the letter C that was reserved for the imminent discovery of the agent of non‐A, non‐B hepatitis, which followed 9 years later.

Conclusions

Progress over the next 40 years was astonishing, and scarcely envisaged by me and the members of our laboratory in those early days in Turin (Fig. 13). HDV is now known to be an important human liver pathogen with unique biological properties unknown in animals (but see later for delta‐like relatives) and unlike conventional viruses. ²⁰ With a circular genome of only approximately 1700 nucleotides, it is the smallest human virus, more akin to viroids of plants than to the known animal viruses. Within the genome, a stretch of less than 100 nucleotides has the property of a ribozyme; it retains the genetic information but also acts as a catalyst cleaving its own RNA molecule as an essential step in the life cycle of the virus. HDV RNA is replicated with a rolling‐circle mechanism typical of plant viroids in a process that involves host RNA polymerase‐guided direct viral RNA‐to‐RNA synthesis, wherein the viral RNA genome is deceptively recognized as an endogenous host DNA.

Fig 13.

In the laboratory of the Division of Gastroenterology, 1983. From left: Dr. Patrizia Farci, Dr. Angelo Andriulli, Dr. Antonina Smedile, Dr. Osvaldo Crivelli, Dr. Ferruccio Bonino, Dr. Mario Rizzetto, and Mrs. Carla Lavarini. Author's personal photograph.

HDV causes hepatitis D, a disease that in its chronic form affects HBsAg carriers worldwide and runs a severe course to cirrhosis within 5 to 10 years in approximately 70% of cases, a rate that is three times higher than in HBV‐monoinfected individuals. ²¹ Prevention and cure of the disease remain formidable. Whereas HBV vaccines protect normal individuals, no anti‐HDV vaccine has yet been developed to protect susceptible carriers of HBsAg.

To date, the evolution of HDV has been thought to lie with humans, in an obligatory coevolution with HBV. However, delta‐like viruses recently have been found throughout the biological tree, from invertebrates to fish, birds, and reptiles. ²¹ , ²² , ²³ , ²⁴ More intriguing still, the HDV ribonucleoprotein has been successfully enveloped in the glycoprotein coat of vesiculoviruses, arenaviruses, and flaviviruses (such as hepatitis C virus [HCV], dengue virus, and West Nile virus), and it can be transmitted through the coat of viruses unrelated to HBV; coated in the E1/E2 glycoprotein of HCV, HDV has propagated its infection in the livers of humanized mice. ²⁵

The finding that the evolutionary pathway of HDV is divergent from humans and HBV opens up a wider scenario in delta research. Other invertebrate and vertebrate delta virus‐like agents are likely to be discovered, and their role and significance throughout evolution will be challenging. More provocative yet is the demonstration of HDV transmission in vivo through the capsid of other viruses. Studies are now in order to determine whether similarly disguised delta RNAs may occur in human infectious diseases of heretofore unknown cause.

Series Editor’s Postscript

When I invite seasoned investigators worldwide to contribute expert perspectives on their chosen fields to the current series on the History of Hepatology, I stipulate certain format parameters for this unusual genre. Such guidelines stipulate that the essays must be based, above all, on a solid core of scientific and historical facts. But to make the articles appealing and different from the usual run‐of‐the mill review and book chapter, we expect a personal view of the evolution of knowledge of the topic, with anecdotes, humor, and facts that may be unknown to even the informed student of hepatology, iconoclasm where justified, and a bevy of illustrations. In this pursuit, Mario Rizzetto has triumphed in spades. He describes the odyssey of his lifetime quest for what was then an unknown and, as it turned out, hepatitis virus like no other. He is self‐effacingly honest about the failures and barriers along the way. He modestly attributes much of his success to serendipity and gives credit that is due to a wide array of international scientists, starting with Deborah Doniach, his admired and esteemed early mentor (at the now‐razed‐to‐the‐ground Middlesex Hospital, London, UK), and later his cherished maestro, Prof. Giorgio Verme, his Chief of the Division of Gastroenterology in Turin. His experience in London resonates with me, as I too worked at the Middlesex Hospital and indeed shared a clinic once a week with the exceptional Deborah Doniach, whom I had the privilege, many decades later, to interview about the discovery of that same AMA (see Reuben ²⁶ ) that was so seminal in the formative years of his scientific career.