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John Bertrand FIBMS from the London Region Virology Discussion Group looks at the brief history of virology.
Viruses as human pathogens can be identified on many pages of human history. At least one body found in an Egyptian tomb in the nineteen-thirties seems to have the lesions of smallpox. The famous photograph of an Egyptian frieze shows a man with clear signs of paralytic poliomyelitis. Yellow fever and other exotic viruses severely affected European explorers in many parts of the world. In more recent years, an number of people of varying fame (Franklin D. Roosevelt, Michael Flanders, Ian Dury) were afflicted by poliovirus, but managed to continue with their lives. Yet more recently, deaths from HIV and hepatitis B have deprived the world of some important talents, particularly afflicting the entertainment industry. Though the causes of viral disease in humans may have changed, these fascinating entities continue to show that they are endlessly resourceful in causing grief to all occupants of the planet, including plants, fish, birds and other mammals.
The history of virology as a scientific entity is quite short. The word, from the Greek meaning 'poison', has been in common use in the English language for many centuries. The work of the early bacteriologists showed that invisible agents were capable of causing infectious disease, but the technology to recognise these agents did not exist. The most important indicator of the infectious nature of these mysterious agents was shown by the work of Edward Jenner, and his courageous use of the material from cowpox to as a vaccine to prevent smallpox. An even more courageous demonstration was the use by Louis Pasteur of killed rabies as a vaccine to prevent the disease in a child bitten by a rabid animal. More recent attempts to control infectious disease have been rather more controlled, but were not, and are still not, without unexpected hazards.
Until after the Second World War, most virology was performed in laboratory animals or in fertile eggs. Animals could be used for the isolation of various viruses such as rabies and lymphocytic choriomeningitis; unfortunately, these diseases were relatively rare in Humans. Eggs were used to isolate influenza and herpes simplex, though their most important use was in the diagnosis of smallpox and its differentiation from varicella/zoster. There is no doubt that the technique was of enormous importance in managing this severe infection both in the UK and in the world. The use of eggs remains important in some aspects of work with ortho- and paramyxoviruses, being capable of producing virus viral antigens and vaccine virus to high titre. The eradication of smallpox (of which more below) and the advent firstly of cell culture and secondly of sensitive and specific antigen detection methods saw the end of the use of eggs in most diagnostic laboratories.
It is notable that many of the pioneers of virology in what one might call the modern era are still alive, and some are still working. John Enders (see below) died in the mid-1990's, as did Jonas Salk and Albert Sabin. David Dane, who first showed electron microscopic evidence of hepatitis B virus, died in 1998. Joseph L. Melnick, a co-worker of Enders, is still at work. The fifties and sixties must have been the most exciting and opportune period for a budding virologist to make his name. Perhaps the downside of the huge amount of work going on was a complete lack of control over viral nomenclature. Many viruses were named after diseases (measles, mumps, polio, influenza); many more were named after people (Epstein Barr). Yet others were named after places such as Coxsackie and Norwalk. The name ECHO (Enteric Cytopathic Human Orphan) is perhaps the most bizarre, being applied to those enteroviruses isolated from patients with no overt disease. Modern nomenclature is designed to provide some basic information about the virus, though the names cited above have become sufficiently well recognised to be retained by familiarity.
Arguably the most important development in virology was the demonstration by John F. Enders that poliovirus could be grown in monolayer cell cultures of non-neural tissue. Cell culture had been developing since 1900, with the most serious limiting factor being contamination. Use of stringent aseptic technique by people such as Alexis Carrell enabled scientists to show that cells could be grown and passaged in culture for long periods. The work of Hanks, Earle and Gey on balanced salt solutions, followed by Eagle and Morton, Morgan and Parker developing defined media for growing cells, provided that the media were supplemented with appropriate serum, usually calf, foetal calf or horse. The use of such undefined but successful basics for culture such as lactalbumen hydrolysate began to disappear. So also did occasional visits to the abattoir for calf blood, and the testing of the collected serum for its ability to support the cells in the department.
Enders discovery lead to the identification of a large number of hitherto unknown viruses, as well as the detection of a variety of agents suspected of being present. Polio itself was found to comprise three serotypes, and a variety of close relatives were demonstrated in faecal, throat and CSF, including Coxsackie A and B, and a number of viruses of similar behaviour patterns but no identifiable disease. These are the Enteric Cytopathic Human Orphan (ECHO) viruses, many of which are now proven to cause a variety of human illnesses.
A complete new family of adenoviruses was detected in the throats of people with adenoids; more significantly, these viruses were also found in samples from outbreaks of respiratory disease and conjunctivitis. In addition, it was noted that some of the viruses previously called ECHO were in fact reovirus, and at least one was found to be a rhinovirus. Through the fifties and sixties, over 100 more rhinoviruses were identified. As a result of the work on rhinoviruses, yet another new family was found, coronavirus, yet another cause of mild upper respiratory tract disease.
This large explosion of new discoveries led to some improvements in the health of the nation. The isolation of poliovirus enabled the development of killed vaccine by Jonas Salk. Use of this vaccine resulted in a rapid fall in the number of cases of poliomyelitis. Live attenuated vaccines for polio resulted from an improved knowledge of the biology and biochemistry of the viruses. Albert Sabin's vaccine is still in use in much of the world today, promoted particularly by the ease of administration (oral) and the relatively small dose required. In the UK, new cases of poliomyelitis are rare, and almost always imported. The large number of new viruses being discovered saw the appearance of virology laboratories, mysterious and slow-moving departments using fertile eggs and cell culture, and apparently taking days and sometimes weeks to come up with a diagnosis. The diagnosis itself often had little or no beneficial effects for the patient, apart from controlling further investigations, and providing some epidemiological information. The first diagnostic tests to assist directly in patient management followed the isolation of rubella virus in 1962.
It was well established that 'German measles' when acquired in the first trimester of pregnancy severely damaged the foetus. The study conducted by Norman Gregg, without the benefit of laboratory support, is a major milestone in virology. Isolation of the virus in 1962 (some 17 years after Gregg's study) was followed rapidly by the introduction of diagnostic tests. At first, these were cumbersome and time-consuming neutralisation tests in cell culture, but complement fixation and haemagglutination inhibition soon became available. Later, the first tests for a virus specific IgM were developed, using serum fractionation methods. It was now possible to investigate all cases of rubella-like illness in pregnancy, and all those in contact with rubella-like illness. Those who had experienced infection in the past could be reassured, while those who had developed rubella in early pregnancy could be advised of the risk to the foetus, and offered termination. Major screening programmes to identify women at risk of rubella in pregnancy were started.
Isolation of this virus also lead to the development of a vaccine, at first offered to pre-pubertal females, but now given to all children in the first 5 years of life. A combination of laboratory tests and the use of vaccine has seen a significant reduction in the number of cases of intra-uterine infection. Currently, the combination of measles and mumps vaccine with rubella has contributed to the increasing rarity of these childhood illnesses and, in the case of measles, their unpleasant and occasionally life-threatening sequelae.
Use of measles vaccination alone, prior to the introduction of the combined measles, mumps and rubella, had already significantly reduced the prevalence of disease. Better still, a combination of universal vaccination and stringent management of outbreaks was leading to the eventual eradication of the virus disease seen at the time as the most serious and frightening, smallpox. However, until the early 1970's, the diagnostic function of the laboratory was of limited interest and value to the wider population. This was all about to change, with the discovery of hepatitis B.
Hepatitis due to infection was clearly recognised. Krugman had shown using 'volunteers' that there were two types of disease. One was of short incubation and spread by the faecal-oral route; the other was of longer incubation and spread by the inoculation of infected blood. In 1968, Blumberg identified an antigen in an aboriginal Australian that became known as Australia antigen. It soon became clear that this antigen was present in many people who had received blood transfusions, and in some people with no obvious history of hepatitis. Some of these people could be shown to be persistent carriers of the antigen. At first, the antigen seemed mysterious in that there appeared to be no nucleic acid in it. We are all now aware that the antigen is a large excess of the surface protein of a hepadnavirus, first visualised in electron micrographs by David Dane.
Soon after the discovery of the virus, an outbreak of hepatitis in a renal unit resulting in a number of deaths occurred. The virus immediately assumed demonic status, with laboratories handling specimens in strict containment, and trying to control procedures carried out on those suspected of having the infection. The similarity with this era and the period surrounding our improving understanding of AIDS is striking. In addition, there were two laboratory releases of smallpox virus, causing three deaths from virus infection and leading to one suicide. The effect on laboratories was profound and beneficial, leading to major improvements in good laboratory practice and safety. Pathogens were classified according to risk, and Sir James Howie produced his definitive report on which most current safety procedures are based. At the time, however, the need to impose safe working practices was regarded as severely restrictive, slowing down the work, and cluttering laboratories with safety cabinets.
Thus, hepatitis B (particularly when combined with smallpox) reached a wide audience, and affected a far wider health care audience, including renal and other physicians, surgeons, all laboratory workers and porters and other ancillary staff. As time passed, however, the true risk and the true nature of this virus became more apparent. The risk to patients could be controlled by screening of blood and blood products (It will be no surprise today to note that haemophiliacs were at serious risk of persistent infection acquired from blood products - plus ca change!). In the UK it became apparent that there were risk groups such as homosexual men, promiscuous heterosexuals and intravenous drug abusers with prevalence levels far higher than background. In addition, the prevalence in populations from south of the Mediterranean Sea showed an ever-increasing rate of infection, reaching 5% in sub-Saharan Africa, and rising to 10% in China. Thus it could be shown that this was a highly successful human virus, spread most frequently from mother to child at birth.
The importance of this virus in transforming the approach of virologists to diagnosis cannot be underestimated. The period from discovery to development of a vaccine was under 15 years, made more remarkable by the fact that the virus has never been grown in high titres in vitro. Laboratory tests of high sensitivity, specificity and reproducibility were developed, enabling demonstration of virus, antibodies, infectivity markers, pre- and post-vaccination screens and methods of monitoring therapy. Use of antigen detection assays and increasingly sophisticated automation became a viable option, enabling the processing of large numbers of samples. To move briefly ahead in time, the work with hepatitis B promoted development in screening tests for rubella antibodies, hepatitis A and C markers, HIV tests, and syphilis screening, together with rapid progress in designing machines to deal with these assays.
To return to an approximation of chronological history, hepatitis B dominated virus laboratory and other laboratory work for most of the seventies. The discovery of gastoenteritis viruses (rotaviruses, enteric adenoviruses, Norwalk viruses, astroviruses, etc) in the late 1970's was important, as it showed that a variety of viruses could cause this disease, some of them food-borne. Some of them (small smooth round viruses, enteric coronaviruses) remain rather mysterious. Their discovery was overshadowed completely by the appearance in 1976 of Acquired Immunodeficiency Syndrome.
This disease was initially recognised by the appearance of Pneumocystis carinii pneumonia in people with no known underlying immunodeficiency. This pneumonia had been seen in transplant patients, and in those with haematological neoplasias. It was noted that most of the increasing number of patients were homosexual men or intravenous drug abusers, though there were a few haemophiliacs and heterosexual victims as well. An increasing variety of opportunistic diseases was seen, including Kaposi'' sarcoma, cytomegalovirus, EBV induced tumours, Toxoplasma gondii, parvovirus, the many gastroenteritis viruses, and unusual species of mycobacteria. Many patients died of these opportunistic infections.
In the early 1980's a retrovirus was found which is accepted by most as being the cause of the disease, though the precise mechanisms of pathogenesis are still not clear. The virus, called LAV in Europe, and HTLVIII in the USA, is now universally called Human Immunodeficiency virus (HIV) As with hepatitis B, the principal victims in the West are homosexual men, intravenous drug abusers and haemophiliacs, with some heterosexual transmission and increasingly rare cases of spread through donated blood. The real epidemic, however, was and remains in Africa and the developing world, with clear epidemiological evidence of heterosexual spread, and a high mortality in young adults.
This virus engendered considerable concern amongst laboratory workers, as it was assumed that the virus could be caught by a variety of routes. There was a period of reluctance to carry out laboratory tests on HIV positive or suspect patients because of the perceived risk, though this is happily in the past. Testing for antibodies became a political issue, as having a test, often regardless of result, affected an individuals ability to acquire personal loans, mortgages or life insurance. Informed consent from the patient remains a requirement before tests can be requested from the laboratory. Laboratories acquired increasingly sophisticated methods of confirming positive results on HIV and other blood borne viruses, as an erroneous result could be disastrous for the patient and relatives and partners. A screening programme for ever widening population groups developed. Advertisements on the risks of unprotected sexual activity proliferated on television, in the press and on advertising hoardings. Ill informed and inaccurate articles appeared in the popular press, and may have served to slow spread of virus by fear. The identification of this new disease, and the discovery of the completely new virus causing it, finally put virology in the front line of pathology specialities.
The discovery of hepatitis C virus caused less public drama, but was rather more scientifically rewarding. The use of molecular techniques to clone RNA from a laboratory infected chimpanzee, and to translate them into proteins, was laborious and time consuming. The testing of the various proteins against panels of sera was equally laborious but successful, and enabled the development of increasingly sensitive antibody assays, without the necessity of isolating a highly fastidious virus in vitro, a result that is still to occur. Indeed, it was only recently that an acceptable electron micrograph of hepatitis C virus was obtained. The identification of a variety of structural and non-structural proteins has enabled a range of diagnostic tests to be introduced. In common with all the other major new discoveries outlined here, more work was required of virology departments.
I have listed landmark discoveries that have had the most significant effect on the diagnostic service. Other new viruses identified in the last 30 years have had a lesser public profile, and some known viruses have assumed ever greater importance. Epstein Barr virus was the first virus to be associated with a human cancer, as well as being the cause of the majority of infectious mononucleosis. Cytomegalovirus is a significant infection in utero, in new-born children, and in the increasing population of immunocompromised patients. The identification of parvovirus B19 and human herpes virus type 6 resolved the aetiology of two childhood rashes, fifth disease and roseola infantum. The significance of some serotypes of human wart virus was brought sharply into focus by its clear association with cervical carcinoma. Screening for such viruses thus assumes a high degree of importance. There are many more viruses that could be cited here, all of which have added to the scope of the diagnostic service.
A number of events related to common, rare and new viruses stand out, if only for the lessons that could be learned. The eradication of smallpox was followed fairy closely by the accidental release of the virus from laboratories in London and Birmingham, leading to the deaths of three people due to virus infection, and one by his own hand. These events lead directly to the development of the current safety legislation, including the Howie report and the categorisation of pathogens according to risk.
A case of Lassa fever in a London teaching hospital made considerable headlines, but had a much happier outcome. Though there were many contacts with the index case, there were no secondary cases. The problems associated with rarely seen and highly dangerous viruses were highlighted. Without such incidents, it is possible that awareness of the potential hazard of the H5N1 influenza virus that appeared in Hong Kong in 1997 could have been less.
Since the discovery of hepatitis B there has been considerable awareness of the risk to staff from patients carrying the virus. There is a substantial list of patients with clear documentary evidence of acquisition while performing their normal duties. All staff exposed to patients or their body fluids should now be vaccinated, and infection from patients should be rare. A number of incidents in recent years have highlighted the risk to patients from staff carrying out invasive procedure, particularly surgeons. A major incident in a London teaching hospital saw nearly 300 patients put at risk, with 19 being infected. One of these remains a carrier of the virus. A second case sadly involved an individual who lied about his status, and subsequently received a prison sentence from the courts. Outbreaks take considerable time to investigate, but have a positive influence on how to proceed with staff undertaking invasive procedures, though it is more difficult to cope with people willing and able to lie about their status.
The huge growth in the amount of work carried out in virology, and the increasing number of departments performing microbial serology tests, has lead inevitably to major changes in technology and philosophy in departments. Technology in the 1960's was based around cell culture and animal work, with antibody tests reliant on these systems for the production of antigen and antisera. The viruses most commonly sought were polio and other enteroviruses,. rubella, and the other common viral diseases of childhood. Influenza epidemics occurred regularly, and herpes simplex and zoster viruses could be grown. It is a popular misconception that genital herpes appeared in the sexual freedom of the sixties; it has been around for centuries, and diagnostic tests have been carried out since cell culture became available. There was no rubella screening, no hepatitis testing, no HIV, no gastroenteritis or parvoviruses.
By the mid 1990's the change in investigations had changed dramatically. Poliomyelitis diagnosis was a rare event, and common childhood diseases such as mumps, measles and rubella had been greatly reduced by vaccination. There have been problems with rubella, as the virus continued to spread in the community, causing disease in males who did not receive vaccination at school, but the risk and occurrence of congenital rubella became increasingly small. Diagnosis and screening now concentrated on the blood borne viruses, chlamydia (not viruses, but found in virus labs by tradition) and rapid diagnosis of acute disease. The diagnosis of herpes simplex remains the same as it was in the sixties. Influenza and other respiratory viruses are now regularly diagnosed by rapid methods such as immunofluorescence and solid phase EIA, with many departments choosing not to isolate virus in cell culture. Serological diagnosis using paired sera is a relatively rare event; IgM tests for a variety of agents provide a timely result not available if one had to wait for a convalescent sample (which in serious disease might not become available). The most striking and important changes in the technology were the automation of EIA methods for antigen and antibody detection, and the development of molecular methods for the detection of exquisitely small amounts of viral nucleic acid.
Automation came to chemistry and haematology laboratories many years ago, and has become increasingly sophisticated. Developments were driven by increased numbers of tests and the increase in the number of analytes that could be detected. The need for diagnostic tests for hepatitis B did not in itself drive this development in virology, but the addition of hepatitis A, C, rubella screening, chlamydia detection, cytomegalovirus antibodies and Toxoplasma gondii antibodies made such developments imperative if the burgeoning workload was to be delivered to clinicians and their patients. Thus, many virology laboratories developed a section that resembled chemistry departments, with increasingly large random access analysers manipulating the samples and performing the assays. Automation and its rapidly diversifying function will have a profound and radical affect on the way pathology develops in the future.
Molecular biology methods such as polymerase chain reaction (PCR) will affect different areas of the speciality. These tests have enabled the detection of a number of viruses that will not grow readily (if at all) in vitro, but can be diagnosed presumptively by the presence of antibody. These include hepatitis C, papilloma viruses and HIV. The system will also rapidly demonstrate the presence of viruses in central nervous system disease, when early diagnosis is critical to facilitate treatment. Viruses that can be easily isolated from their normal location can be difficult to detect in the CNS. At the time of writing, the technique requires separate laboratory space for each phase of the test, in order to avoid contamination. Automation will resolve this problem in the near future, and molecular biology will become increasingly available for mass screening purposes.
The change in technology has been accompanied by an essential change in culture within virology departments. Cell culture based methods have an essential indolence about them. Good cell culture takes time and skill, and searching for cytopathic changes in the cultures requires patience both to search for the effect, and to wait anything up to thirty days for it to appear. It was commonly heard outside the laboratory that the department produced results only after the patient had recovered or died. While this was clearly not true all the time, the fact of waiting ten days for a viral specific change to appear did not lead to the rapid turn-round of results. In addition, waiting for convalescent serum before being able to start making a diagnosis was not designed to encourage users in the belief that virology could be timely. However, there is no doubt that a capability of making swift diagnoses of smallpox, polio and rubella were available.
Today, the necessity of making a swift diagnosis of smallpox has hopefully been removed forever, and poliomyelitis is now excessively rare. Virus laboratories, having excited and alarmed the real world with their discoveries, are now forced to join it, and produce results within a sensible timeframe, with same-day release of fully audited and controlled data being the minimum target. It is unusual to find a laboratory that is not capable of delivering a result on a respiratory sample within three hours of receipt. Given a good sample, the time could be much less. Blood tests results can be delivered on the day the specimen is received, though data on reactive samples may take a little longer as they must be confirmed. Even cell culture procedures have been accelerated, with early antigen tests on samples suspected of containing cytomegalovirus or adenovirus.
Perhaps the most important cultural change in virology has been the huge increase in the number of samples. In 1967, when I first stared work in the virology department, a total of seven staff (including the consultant and the person who did the washing-up, tested in the region of 3500 samples a year. By the mid-1970's specimen numbers had risen to over 30000, though staff numbers did not quite double. At the end of the current year, 16 MLSO staff had processed just over 100,000 samples, performing nearly 180,000 tests. Over 50,000 blood samples were tested, with most results going out within 24 hours. Over 7000 specimens were tested for acute virus infections with over half being reported on the day of receipt. There can be no doubt that virology now resides in the real world of ever increasing demand, and that future developments will only serve to increase the demand.
Article written: 2004