Archive for the ‘Viruses’ Category

Viruses and Human Cancer: The Molecular Age

1 April, 2015

Hepatitis C virus

A suspicion that other viruses were involved in post-transfusion-(PTF) related hepatitis was first aired by Harvey J Alter and colleagues, after proof in 1972 that some PTF-related hepatitis cases had no HBV antigen associated with them.  By 1977 hepatitis A virus (HAV, infectious hepatitis) had been excluded as well, and the term “non-A, non-B” hepatitis (NANB) was coined.  By 1978, transmission studies using human serum injected into chimpanzees showed that

“Hepatitis was transmitted by serum derived from patients with chronic as well as acute hepatitis, strongly suggesting a chronic carrier state for the agent responsible for non-A, non-B hepatitis. Non-A, non-B hepatitis thus seems to be due to a transmissible agent which can persist and remain infectious for long periods”.

There was also evidence from Japan the same year that there might be a novel antigen – hepatitis C (HC) antigen – associated with NANB PTF hepatitis.  In 1979, it was suggested from ultrastructural studies in cells from infected chimpanzees that more than one NANB agent might exist; by 1980 Alter had concluded that that the NANB hepatitis agent(s) played a dominant role in the pathogenesis of PTF hepatitis. In 1987, in an interesting application of essentially the same technology used to characterise the first viruses, Alter’s group used polycarbonate membranes to filter the infectious agent, and showed it was  30-60 nm in diameter, and therefore highly unlikely to be a retrovirus, as had been suggested by some.

Also in 1987, Michael Houghton, Qui-Lim Choo, and George Kuo at Chiron Corporation collaborated with  DW Bradley at the CDC in using the much newer technology of constructing a random-primed cDNA clonal library from RNA extracted from human plasma in a lambda phage expression vector, and screening proteins expressed from the library against NANB hepatitis-infected patient serum.  They discovered one sequence that produced a protein fragment that bound antibodies, sequenced it, and used the sequence to “primer walk” through the entire genome by repeated cDNA generation, cloning and sequencing.  They published their finding in 1989 of

“…an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae”.

The agent was unique among viruses characterised until then, as no virus particle had yet been seen, let alone isolated. Alter and his team meanwhile tested for the presence of the virus in NANB patient samples, and in 1989 also published a paper – back-to-back with the previous – on

“An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis”.

The sequence of the putative agent allowed cloning and expression of a putative capsid protein in yeast, which allowed large-scale screening of patient samples and donated blood.  From their paper:

“These data indicate that HCV is a major cause of NANBH throughout the world”.

There was also already evidence that NANBH was associated with hepatocellular carcinoma (HCC) in Japan: M Sakamoto and colleagues showed that 90% of HBsAg-seronegative patients, who were also overwhelmingly HBV DNA-negative, showed evidence of chronic hepatitis in the non-cancerous liver, and 29% had a history of blood transfusion. This was followed as early as 1989 by evidence that 65% Italian HCC patients had antibodies to HCV, and again the same year by evidence from Spain that 75% of HCC patients had HCV antibodies, which

“…indicate[s] that HCV infection may have a role in the pathogenesis of hepatocellular carcinoma, even in patients with chronic liver disease apparently related to other agents such as alcohol”.

By 1990, K Kiyosawa and colleagues felt able to state that:

“These data suggest the slow, sequential progression from acute hepatitis C virus-related non-A, non-B hepatitis through chronic hepatitis and cirrhosis to hepatocellular carcinoma and support a causal association between hepatitis C virus and hepatocellular carcinoma”.

These findings rapidly led to the revelations that hepatitis C virus (HCV) was implicated in both an acute and relatively mild illness that lasts only a few weeks, and a chronic form that is usually more serious and can last lifelong.  Between 15–45% of infected persons spontaneously clear the virus within 6 months; however, the remaining 55–85% will develop chronic HCV infection.

Up to 150 million people globally are chronically infected with HCV.  Moreover – also from the WHO site – a significant number of these people will develop liver cirrhosis or liver cancer, and up to 500 000 people die worldwide every year from HCV-related liver disease.

There is as yet no vaccine against HCV infection, although trials are quite far advanced – and one candidate combination prime-boost strategy from Eleanor Barnes and coworkers seemed to show promise as of the end of 2014.  This consisted of

“…a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b” – which is using two well-characterised viruses as gene vectors to combat a third.

The authors make the point that the responses they achieved in human volunteers are similar to those seen in people who control natural infections.

Meanwhile, chemotherapy for chronic infections is both a realistic and well-established area: there are a number of treatments on the market already, and there have been significant recent developments which may make treatment even more effective.

HCV particles were finally characterised from cell culture-grown virus: both enveloped and non-enveloped pleomorphic spherical particles were found, of around 60 nm and 45 nm in diameter respectively.  This agrees well with the estimation by filtration of 30-60 nm previously determined in 1987.  The virus is classified as a ss(+)RNA genome flavivirus, similar to yellow fever virus, in the genus Hepacivirus, family Flaviviridae

Kaposi sarcoma herpesvirus

Moritz Kaposi in 1872 described what was originally called an “idiopathic multiple pigmented sarcoma of the skin”, which present as

“…disseminated blood- or bruise-coloured skin lesions (flat plaques or nodules) in the skin, usually on the lower extremities though sometimes on the hands and arms”.

What was subsequently called Kaposi Sarcoma, or KS, was at first thought to occur only among elderly men of Jewish, Arabic or Mediterranean origin; however by the 1950s it was realised it was quite common in sub-Saharan Africa, which led to the first suggestions that it might be caused by a virus.

In 1981-1982, however, the CDC received reports of KS occurring in otherwise healthy young homosexual men in California – often together with Pneumocystis carinii pneumonia, which was also previously very rare.  The disease was also much more aggressive, and spread beyond the skin into other tissues including bone, the mouth, gastrointestinal tract and lungs.

While there was at this time still no clue as to why this should be, the link to sexual acts as well as the previous observation that KS occasionally appeared in immune-suppressed organ transplant patients, led epidemiologists to discover the sexual transmission of immunodeficiency that led to the discovery of HIV and its causation of AIDS.

Valerie Beral and colleagues working at the CDC in the late 1980s used epidemiological data on KS in AIDS patients to build a compelling case for the tumour being caused by another sexually transmitted virus.  In a landmark paper in The Lancet, they announced in 1990, on the basis of painstaking and traditional-style investigation of 8 years’ worth of information from more than 90 000 people with AIDS collected in the US by the CDC since 1981, that:

“In the United States Kaposi’s sarcoma is at least 20000 times more common in persons with [AIDS] than in the general population and 300 times more common than in other immunosuppressed groups…Kaposi’s sarcoma was commoner among those who had acquired the human immunodeficiency virus (HIV) by sexual contact than parenterally, the percentage with Kaposi’s sarcoma ranging from 1% in men with haemophilia to 21% in homosexual or bisexual men. Women were more likely to have Kaposi’s sarcoma if their partners were bisexual men rather than intravenous drug users”.

The UK Cancer Research site on KS has an excellent account of the study as well as of its impact – one aspect of which was the proof in 1994 that indeed a virus was involved, using modern.  This was published by Y Chang and colleagues in Science, and detailed the use of the very modern technique of:

“Representational difference analysis … to isolate unique sequences present in more than 90 percent of Kaposi’s sarcoma (KS) tissues obtained from patients with acquired immunodeficiency syndrome (AIDS). These sequences were not present in tissue DNA from non-AIDS patients, but were present in 15 percent of non-KS tissue DNA samples from AIDS patients. The sequences are homologous to, but distinct from, capsid and tegument protein genes of the Gammaherpesvirinae, herpesvirus saimiri and Epstein-Barr virus. These KS-associated herpesvirus-like (KSHV) sequences appear to define a new human herpesvirus.”

This became human herpesvirus 8 (HHV-8), the newest of the seven viruses known to cause human cancers.

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Rinderpest and its eradication

30 March, 2015

Rinderpest virus infects cattle, buffalo and several species of antelope among other animals: it is a member of the genus Morbillivirus, family Paramyxoviridae, and is related to measles and mumps viruses in humans, distemper virus in dogs, and a variety of relatively newly-described viruses in marine mammals.  It also almost certainly gave rise to measles virus sometime around the 11th-12th centuries CE, as an originally zoonotic infection – sourced in domestic animals – took root in humans and began to be passed around (see here).

Wikipedia describes rinderpest virus as

“…an infectious viral disease of cattle, domestic buffalo, and some species of wildlife. The disease was characterized by fever, oral erosions, diarrhea, lymphoid necrosis, and high mortality.”  

And:

“The term Rinderpest is taken from German, and means cattle-plague.”

“Cattle plagues” have occurred throughout recorded history, often associated with the large-scale movement of animals accompanying military campaigns.  Europe was badly hit in the 1700s, with three epizootics in 1709–1720, 1742–1760, and 1768–1786, and a major British outbreak in 1865/66.

Of particular interest in South African folklore, an outbreak in the 1890s killed up to 90 percent of all cattle in southern and north-west Africa, and resulted in the deaths of many thousands of people who depended on them. It was devastating enough that people still remember it as a legendary time of hardship. Sir Arnold Theiler was instrumental in developing a vaccine that curbed the epidemic – by simultaneously injecting animals with blood from an infected animal and antiserum from a recovered animal. This protected animals for long enough to allow their immune systems to respond to the virus, but was rather risky, even though it was used for several decades.

In the 1920s J. T. Edwards in what is now the Indian Veterinary Research Institute serially passaged the virus in goats: after 600 passages it no longer caused disease, but elicited lifelong immunity. However, it could still cause disease in immunosuppressed cattle.

In 1924 the World Organisation for Animal Health (OIE) was formed, largely in response to rinderpest. This took on coordination of eradication efforts, which until then had been largely done on an individual country basis by means of vaccination.

This was followed by the Inter-African Bureau of Epizootic Diseases in 1950, with the aim of eliminating rinderpest from Africa. In 1962, Walter Plowright and R.D. Ferris used tissue culture to develop a live-attenuated vaccine grown in calf kidney cells.  Virus that had been passaged 90 times conferred immunity without disease even in immunosuppressed cattle, was stable, and did not spread between animals.  This vaccine was the one that allowed the prospect of eradicating the virus, and earned Plowright a World Food Prize in 1999.

Mass vaccination campaigns following outbreaks had, by 1972, eliminated rinderpest in all of Asia except for Lebanon and India. In the 1980s, a Sudan outbreak spread throughout Africa, killing millions of cattle, as well as much wildlife. The response was the initiation of the Pan-African Rinderpest Campaign in 1987, which made use of mass vaccination and surveillance to combat the disease. By the 1990s, all regions of Africa except for Sudan and Somalia were declared rinderpest-free.

By 1996, the complete nucleotide sequence of the virulent Kabete “O” strain of rinderpest had been obtained by Michael Baron and co-workers.  This could now be compared to that of the vaccine strain derived from it by Plowright and Ferris in 1962, that had been sequenced earlier, Despite the very different pathogenivities of the two viruses, there were only 87 base changes between them. It was interesting that  the Kabete strain – isolated in Kenya in 1910 – had been passaged by animal-to-animal transfer since then, and only 10 times since the derivation of the vaccine strain from it.  This provides a rare resource for determination of the determinants of pathogenicity.

The Food and Agriculture Organisation (FAO) has a Division of Animal Production and Health: their web site details a campaign known as the Global Rinderpest Eradication Programme (GREP), which has been going since 1994.  This had succeeded in reducing outbreaks to being small and infrequent by the late 1990s.  The last confirmed case of rinderpest was reported in Kenya in 2001. Final vaccinations were given in 2006; the last surveillance operations in 2009 failed to find any evidence of the disease.

By 14th October 2010, the BBC News site had this to say:

“The eradication of the virus has been described as the biggest achievement in veterinary history and one which will save the lives and livelihoods of millions of the poorest people in the world….”

If confirmed, rinderpest would become only the second viral disease – after smallpox – to have been eliminated by humans.”

A news item from the FAO site dated 25 May 2011, declared that:

“The national Delegates of Members of the World Organisation for Animal Health (OIE) declared today that rinderpest, one of the deadliest diseases of cattle and of several other animal species, is now eradicated from the surface of the earth.

At the organisation’s 79th annual General Session in Paris, France the national Delegates of OIE Members unanimously adopted Resolution 18/2011 which officially recognized, following thorough control by the OIE with the support of FAO, that all 198 countries and territories with rinderpest-susceptible animals in the world are free of the disease”.

When commenting on the significance of the achievement, John Anderson, the head of the FAO, described GREP’s announcement that rinderpest had been eradicated as:

“The biggest achievement of veterinary history“.

Like the smallpox eradication, even though much of the campaign happened in the era of modern virology, it was classical virological and disease control measures that were responsible for the success of the operation – with some assistance from molecular diagnostics towards the end.

This is only the second viral disease, ever, to have been eliminated.  And how was this possible?  Unlike smallpox, which has only humans as a natural and reservoir host (although it almost certainly also got into us from animals), rinderpest attacked a wider range of hosts.  However, it seemed mainly to have a reservoir in domesticated cattle, and it did not have an arthropod vector; moreover, the vaccine was cheap and effective.

This is momentous news: we may well have succeeded in ridding the planet of what has been a very significant disease of livestock and of wild animals, which has caused untold agricultural loss throughout recorded history, and which has resulted in enormous human hardship as well

We have also made a natural species go extinct – but it won’t be missed.  Like smallpox, it was completely sequenced some time ago, so we could theoretically recreate it if we ever needed to.

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Maize streak virus: the early history

30 March, 2015

The history of maize streak virus research is generally taken as starting in 1901, with the publication of the

The cover of the "Fuller Report"

The cover of the “Fuller Report”

by “Claude Fuller, Entomologist”. However, in the Report he does make reference to articles in the “Agricultural Journal” for August 3rd and 31st, 1900, and quotes personal sources as having noticed the disease of “mealie variegation” as early as the 1870s.  He comments that:

“…mealie growers…have been acquainted with variegated mealies…for at least 20 years…”, and “…Thomas Kirkman…has known the disorder for 30 years past…”.

His conclusions, although carefully arrived at, were very wrong. Fuller claimed the disease was due to soil deficiency or a “chemical enzyme” in soils, and could be combatted by intensive cultivation and “chemical manures”. However, his carefully-written account is still of great historical interest, and the observations are valuable as they are objective accounts of a skilled scientist.  The records of streaked grasses in particular are useful, as we still collect such samples to this day.  Fuller was later sadly a victim of one the first traffic accidents in what was then Lourenco Marques in Mozambique.

Streak symptoms in a maize leaf

The disease – now known as maize streak disease (MSD) – occurs only in Africa and adjacent Indian Ocean islands, where it is one of the worst occurring in maize.  The causal agent was discovered to be a virus by HH Storey in 1932, who termed it maize streak virus (MSV). The virus was found to be obligately transmitted by the leafhopper Cicadulina mbila, also by Storey, in 1928. In 1978, MSV was designated the type virus of the newly described group taxon Geminivirus.

Early studies indicated that there were several distinctly different African streak viruses adapted to different host ranges (Storey & McClean, 1930; McClean, 1947). These studies were based on the transmission of virus isolates between different host species and symptomatology.

In a subsequent study of streak virus transmission between maize, sugarcane, and Panicum maximum, the relatively new technique of immunodiffusion was employed, using antiserum to the maize isolate. From the results it was concluded that the maize, sugarcane, and Panicum isolates were strains of the same virus, MSV (Bock et al., 1974). The maize isolate was given as the type strain. The virus was only properly physically characterised in 1974, when the characteristic geminate or doubled particles were first seen by electron microscopy, and only found to be a single-stranded circular DNA virus in 1977 (Harrison et al., 1977).

Maize streak virus: photo from Robert G Milne in 1978

Maize streak virus: photo from Robert G Milne in 1978

The first isolates of MSV were sequenced in 1984 (Kenya, S Howell, 1984; Nigeria, P Mullineaux et al., 1984), and the virus was found to have a single component of single-stranded circular DNA (sscDNA), and to be about 2700 bases in size. The two isolates were about 98% identical in sequence. The second team took delight in noting that the first sequence was in fact of the complementary and not the virion strand.

A major advance in the field occurred in 1987, when Nigel Grimsley et al. showed that a tandem dimer clone of MSV-N in an Agrobacterium tumefaciens Ti plasmid-derived cloning vector, was infectious when the bacterium was injected into maize seedlings. Subsequently, Sondra Lazarowitz (1988) obtained the sequence of an infectious clone of a South African isolate (from Potchefstroom) – MSV-SA – and showed that it also shared about 98% identity with the first two sequences.

Since the early days other transmission tests and more sophisticated serological assays were performed on a wide range of streak isolates from different hosts and locales, and it was claimed that all forms of streak disease in the Gramineae in Africa were caused by strains of the same virus, MSV. This view changed as more and more viruses were characterised, however, and it became obvious that there were distinctly separate groupings of viruses that constituted different species: these were sugarcane streak viruses (SSV, see Hughes et al., 1993), the panicum streak viruses (PanSV, see Briddon et al., 1992), and the maize streak viruses. Together these viruses constituted an African streak virus group (see Hughes et al., 1992; Rybicki and Hughes, 1990), distinct from an Australasian striate mosaic virus group, and other more distantly related viruses (see here for the state of the art in 1997).  These studies together with a later one by Rybicki et al. in 1998 also pointed up the utility of the polymerase chain reaction (PCR) for amplification, detection and subsequent sequencing of DNA from diverse mastreviruses.

A more modern and comprehensive account can also be found here, in a recent review written for Molecular Plant Pathology.

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Human retroviruses and cancer

13 March, 2015

The very early discovery of avian viruses associated with cancer, and the subsequent failure for many years to isolate similar viruses from mammals, gave some researchers the idea that possibly birds were unique in this regard.  However, “RNA tumour viruses” or oncornaviruses, as they were known for a time, were first demonstrated to affect mammals when mouse mammary tumours were shown to be due to a virus by John Bittner in 1936, by transmission in milk. He also demonstrated vertical transmission, or inheritance of the virus. 

The nature of the agent was not known at the time, but by 1951 L Gross had shown that leukaemia could be passaged in mice using cell-free extracts.  In 1958 W Bernhard had proposed a classification of what were to become known as retroviruses on the basis of electron microscopy.  In 1964 a mouse sarcoma virus and a feline leukaemia virus had been isolated, and in 1969 bovine leukaemia was shown to be a viral disease.  1970 saw the description of reverse transcriptase from retroviruses, and in 1971 the first primate leukaemia virus – from gibbons – was described, and the first retrovirus (foamy virus) described from humans.  Bovine leukaemia virus was characterised as a retrovirus in 1976.

It is not surprising, therefore, that many labs tried to find cancer-causing disease agents in humans.  However, such effort had been put into finding oncornaviruses associated with human tumours, with such lack of success, that it led to people talking of “human rumour viruses” – a useful list of which can be seen here.  Nevertheless, by 1980 Robert Gallo’s group had succeeded in findingtype C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma”, which they called human T-cell leukaemia virus (HTLV).  The breakthrough was made possible by their prior discovery of “T cell growth factor”, now called interleukin 2 (IL-2), which meant human T cells could be successfully cultured for the first time.  A group of Japanese researchers described an “Adult T cell leukemia virus” (ATLV) in 1982: this proved to be the same as what became HTLV-1, given the description also in 1982 by Gallo’s group of another retrovirus associated with a T-cell variant of hairy cell leukaemia, which they dubbed HTLV-2. 

HTLV-1 is associated with the rare and genetically-linked adult T-cell leukaemia, found mainly in southern Japan, as well as with a demyelinating disease called “HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP)” and HTLV-associated uveitis and infective dermatitis.  The areas of highest prevalence are Japan, Africa, the Caribbean islands and South America.  HTLV-2 had a mainly Amerindian and African pygmy distribution, although it is now found worldwide, and causes a milder form of HAM/TSP, as well as arthritis, bronchitis, and pneumonia.  It is is also frequent among injecting drug users.  However, except for rare incidences of cutaneous lymphoma in people coinfected with HIV, and the fact of its origin in a hairy cell leukaemia, there is no good evidence that HTLV-2 causes lymphoproliferative disease.  The two viruses infect between 15 and 20 million people worldwide.  HTLV-1 infections can lead to an often rapidly fatal leukaemia.

By 2005 another two viruses had joined the family: HTLV-3 and HTLV-4 were described from samples from Cameroon that were presumably zoonoses – being associated with bushmeat hunters – and which are not associated with disease.  Interestingly, all the HTLVs have simian counterparts – indicating species cross-over at some point in their evolution.   Collectively they are known as the primate T-lymphotropic viruses (PTLVs) as they consitute an evolutionarily related group.  Another relative is bovine leukaemia virus.

The HTLV-1/STLV-1 and HTLV-2/STLV-2 relationships are relatively ancient, at more than 20 000 years since divergence.  However, their evolution differs markedly in that STLV-I occurs in Africa and Asia among at least 19 species of Old World primates, while STLV-2 has only been found in bonobos, or  Pan paniscus dwarf chimpanzees from DR Congo.  It is therefore quite possible that there are other HTLVs undiscovered in primates in Africa and elsewhere, that may yet emerge into the human population.

Human immunodeficiency virus type 1 (HIV-1) was for a time after its discovery in 1983 called HTLV-III by the Gallo group and lymphadenopathy virus (LAV) by the Montagnier group; however, evidence later obtained from sequencing and genome organisation showed by 1986 that it was in fact a lentivirus, related to viruses such as feline immunodeficiency virus (FIV) and the equine infectious anaemia virus discovered in 1904, and it was renamed.  Francoise Barre-Sinoussie and Luc Montagnier were awarded a half share in a 2008 Nobel Prize, commemorated here

HIV particle.  Russell Kightley Media

HIV particle. Russell Kightley Media

in Viroblogy.

HIV is indirectly implicated in cancer because it creates an environment through immunosuppression that allows the development of opportunistic tumours that would normally be controlled by the immune system: these include HPV-related cervical cancer, and Kaposi’s sarcoma caused by Human herpesvirus 8 (see later).  It is also possible that HIV may directly cause lymphoma development in AIDS patients by insertional activation of cellular oncogenes, although this appears to be rare.

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Epstein-Barr Virus and Hepatitis B Virus

13 March, 2015

Epstein-Barr Virus

While the early discoveries of Ellerman and Bang and Rous might have predicted that retroviruses would be found associated with human cancers, in fact it was a herpesvirus that was the first viral agent implicated in a human cancer.  This was what was called the Epstein-Barr virus – now Human herpesvirus 4 – that was discovered in 1964 by Michael Epstein, Bert Achong and Yvonne Barr in specimens from a  Burkitt lymphoma patient sent from Uganda by the surgeon Denis Burkitt.  The virus was later implicated in infectious mononucleosis or glandular fever, also known as the “kissing disease” or because it tends to get spread around by intimate contact between college students.

A human herpesvirus. Copyright Linda M Stannard

A human herpesvirus. Copyright Linda M Stannard

The virus is carried by up to 95% of adults worldwide, after mainly asymptomatic infections as children.  It is implicated in causing over 95% of nasopharyngeal carcinomas, nearly 50% of Hodgkin lymphoma, and about 10% of gastric carcinomas – for a total of nearly 200 000 cancers worldwide per year.  There is still no vaccine, although candidates are in clinical trial.

Hepatitis B virus

The next virus to be definitively linked to a human cancer was Hepatitis B virus (HBV), that had been discovered more or less accidentally during serological studies in the 1960s by Baruch Blumberg and colleagues.  However, a transmissible agent had been implicated in “serum hepatitis” as early as 1885, when A Lurman showed that contaminated lymph (serum) was to blame for an outbreak in a shipyard in Bremen after a smallpox prevention exercise.  Subsequently, reuse of hypodermic needles first introduced in 1909 was shown to be responsible for spreading the disease.

Blumberg’s “initial discoveries were based primarily on epidemiologic, clinical, and serological observations”; however, by 1968 the “Australia antigen” was seen to consist of 22 nm empty particles, now known to be composed of capsid protein or “surface antigen”, and by 1970 a 42 nm DNA-containing “Dane particle” was found which is now known to be the virion.  Blumberg had by 1972 patented a vaccine derived by purification of 22 nm particles composed of HBV surface antigen (HBsAg) from donor blood, a process pioneered by Maurice Hilleman.  By 1975 Blumberg and others had also implicated HBV in the causation of primary hepatic carcinoma, now known as hepatocellular carcinoma (HCC) and a serious complication of chronic infection with HBV, especially if acquired in early life.  The vaccine was licenced for use in 1982, meaning it was the first anti-cancer vaccine, and the first viral subunit vaccine.  Blumberg shared the 1976 Nobel Prize in Physiology or Medicine with D Carleton Gajdusek – who described the first prion-caused diseases – for “…their discoveries concerning new mechanisms for the origin and dissemination of infectious diseases”.

hbv particles

By 1979 the whole HBV genome had been cloned and sequenced, and molecular biology studies could start in earnest. A recombinant HBsAg produced in yeast was subsequently licenced in 1986, and has supplanted the earlier one.  It is being used in a many countries as part of the EPI (Extended Programme of Immunisation) bundle given to infants, as there is more risk of chronic infection the younger the person is that is infected.  Given that upwards of 2 billion people have been infected, and the over 300 million people that are chronically infected with HBV have a 15-25% risk of dying prematurely from HBV-related causes, there is the potential to make a very significant impact on liver disease.

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Papillomaviruses and human cancer

11 March, 2015

Human warts in all their forms – cutaneous, verrucous and genital growths and lesions – have been known since antiquity, and it was known since at least 1823 that at least some were infectious. Experiments done with human volunteers in the 1890s confirmed this, when it was shown that transplanting wart tissue resulted in typical disease.  As early as 1908, it was shown by a G Ciuffo that “verrucae volgare” – common warts – could be transmitted via a cell-free filtrate.  However, it was Richard E Shope who first showed that a papillomavirus was associated with animal tumours.  A useful review from 1931 on “Infectious oral papillomatosis of dogs” by DeMonbreun and the Ernest Goodpasture of egg culture fame covers the early history of the investigation of human disease as well as of animal papillomas very well, so we will not cover this further.

In light of later findings of the involvement of papillomaviruses, it was a prescient although premature observation by an Italian physician named Rigatoni-Stern in 1842 that cervical cancer appeared to be sexually transmitted, given that it occurred in married women, widows and prostitutes, but rarely in virgins and nuns.

Although papillomaviruses had been implicated as the first viruses known to cause a cancer in mammals as early as the 1930s, and the structurally very similar papovaviruses were similarly implicated in the late 1950s, it was only in 1972 that  Stefania Jabłońska proposed that a human papillomavirus (HPV; then called a papovavirus) was involved with the rare hereditary skin cancer called epidermodysplasia verruciformis.   

Meanwhile Harald zur Hausen had been investigating since 1974 the involvement of HPV in genital warts (condyloma accuminata) and squamous cell carcinomas, using DNA-based techniques such as hybridisation.  The rarely malignant condylomas had been shown to contain papillomavirus particles in some cases in 1968, with a better association in 1970; however, cross-hybridisation studies by zur Hausen’s group on DNA of these and common wart viruses showed no relationship despite their very similar morphologies. 

Virus particles from genital warts (6 &7) and a common skin wart (8).  Reproduced from Brit. J. vener. Dis., JD Oriel and JD Almeida, 46, 37-42, 1970 with permission from BMJ Publishing Group Ltd.

Virus particles from genital warts (6 &7) and a common skin wart (8). Reproduced from Brit. J. vener. Dis., JD Oriel and JD Almeida, 46, 37-42, 1970 with permission from BMJ Publishing Group Ltd.

Zur Hausen speculated on the role of HPVs in squamous cell carcinomas in 1977; Gérard Orth and Jabłońska and colleagues went on to define the “…Risk of Malignant Conversion Associated with the Type of Human Papillomavirus Involved in Epidermodysplasia Verruciformis” in 1979.

Because this was the new era of cloning and sequencing of DNA, the zur Hausen group and others went on to isolate and characterise a number of new HPVs associated with genital cancers and other lesions in the early 1980s.  In particular, they showed that HPV types 16 and 18 could be found both as free virus in cervical cell sample biopsies and integrated into the cell genomes of cell lines derived from cervical cancers.  A major finding in 1987 was that the legendary HeLa cell line – derived from a malignant cervical tumour from a Henrietta Lacks in 1951contains multiple copies of the HPV-18 genome.  The first HPV genome sequence (of type 1b) was obtained in 1982; the first genital type (6b, from condylomas) in 1983, and the first high-risk cancer virus (type 16) in 1985.

Later work involving large international surveys showed by 1995 that 99.7% of cervical cancers contained DNA from so-called “high risk” HPVs, leading to the conclusion that these were the necessary cause of cervical cancer, and that around 70% of these cancers were caused by HPVs 16 and 18.  Since then, HPVs have been found in more than 80% of anal cancers, 70% of vulval and 40% of vaginal cancers, around half of all penile cancers, and in roughly 20% of head and neck cancers.  If 16% of cancers are due to infection, and HPVs cause or are implicated in 30% of these, then they are a significant cause of cancers worldwide.

Harald zur Hausen was awarded a half share of the 2008 Nobel Prize in Physiology or Medicinefor his discovery of human papilloma viruses [sic] causing cervical cancer”.  I blogged on this at the time, here.

Work on vaccines against papillomaviruses (PVs) started early, after demonstrations presumably in the 1930s that domestic rabbits inoculated with the cottontail rabbit PV (CRPV) could become immune to reinoculation after recovery, and in 1962 that a “…formalin-treated suspension of bovine papilloma tissue” provided protection against challenge, but was not therapeutic.  However, progress was stymied by the fact that it proved impossible to culture any of the PVs, and challenge material had to be made from infected animal tissue, even though it had been shown that isolated viral DNA was infectious.

This changed after the advent of molecular cloning, when whole viral genomes could be prepared in bacteria.  Model systems for use in PV vaccine research by 1986 included cattle and bovine PVs, rabbits and CRPV and rabbit oral PV, and dogs and canine oral PV.  It had also been demonstrated that the L1 major structural protein of type 1 BPV produced in recombinant bacteria was protective against viral challenge in calves.  Jarrett and colleagues demonstrated, in 1991 and 1993 respectively, that they had achieved prophylactic and therapeutic immunisation against cutaneous (ie: skin; caused by BPV-2) and then mucosal (respiratory tract; BPV-4) bovine PVs, using E coli-produced proteins.  L1 and L2 proteins were protective against BPV-2, while L2 was protective against BPV-4 infection.  They suggested BPV-4 was a good model for HPV-16 given its mucosal tropism.

By the early 1990s several groups had demonstrated that it was possible to make PV virus-like particles (VLPs) by expression in eukaryotic systems such as yeast or animal cells of the L1 major virion protein either alone, or together with the minor protein L2.  In 1991 Ian Frazer’s group showed that expression of HPV-16 L1 and L2 together but not separately in animal cells via recombinant vaccinia virus, resulted in 40 nm particles resembling the virion being made.  In 1992 John Schiller’s lab showed VLP formation by L1 alone, with both BPV-1 and HPV-16 L1 genes expressed in insect cells via a baculovirus vector. In 1993 came the demonstration that expression of the plantar wart-causing HPV-1 L1 gene alone and L1 and L2 genes together in animal cells via vaccinia virus, as well as of the genital wart-causing HPV-11 L1 expressed in insect cells, resulted in VLP formation.  By 1995, it had been shown that immunisation of rabbits with CRPV L1-only or L1+L2 VLPs, and of dogs with canine oral PV L1 VLPs, protected completely against viral challenge.

hpv vlps

This groundwork made it possible for Merck and GlaxoSmithKline to develop and to push through to human trial and licensure, two independent VLP-based vaccines.  Merck’s vaccine – Gardasil – is quadrivalent, consisting of a mixture of VLPs made in recombinant yeasts from expression of L1 genes of HPV types 6 and 11, to protect against genital warts, and types 16 and 18, for cervical lesions and cancer.  GSK’s offering – Cervarix – is a bivalent HPV-16 and -18 vaccine only, consisting of VLPs made via recombinant baculoviruses in insect cell culture.  These are only the second anti-cancer vaccines on offer, and have gone on to blockbuster status within months of their release: Gardasil was licenced in June 2006, and Cervarix in October 2009.

Both appear to protect very well against infection with the types specified, but not to affect established infections.  Their long-term efficacy against cervical cancer is still to be established, although Gardasil has certainly lessened the incidence of genital warts in Australia post introduction in 2007.  There is now also a VLP-based vaccine for canine oral PV.

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The Discovery of Filoviruses

10 March, 2015

The discovery of filoviruses: Marburg and Ebola

Marburg virus

In 1967, the world was introduced to a new virusthirty-one people in Marburg and Frankfurt in Germany, and Belgrade in the then Yugoslavia, became infected in a linked outbreak with a novel haemorrhagic fever agent. Twenty-five of them were laboratory workers associated with research centres, and were directly infected via contact with infected vervet monkeys (Chlorocebus aethiops) imported to all three centres from Uganda.  Seven people died.   In what what was a remarkably short period of time for that era – given that this was pre-sequencing and cloning of nucleic acids, let alone viruses – it took less than three months for scientists from Marburg and Hamburg to isolate and characterise what was being called “green monkey virus” virus. The new agent was named Marburg virus (MARV), after the city with the greatest number of cases.

The first electron micrograph of the virus clearly exhibits the filamentous nature of the particles, complete with the now-famous “shepherd’s crook”.

The virus disappeared until 1975, when an Australian hitchhiker who had travelled through what is now Zimbabwe was hospitalised in Johannesburg, South Africa, with symptoms reminiscent of Marburg disease. He died, and his female companion and then a nurse also became infected with what was suspected to be yellow fever or Lassa viruses. In an example for later outbreaks, this led to rapid implementation of strict barrier nursing and isolation of the patients and their contacts, which resulted in quick containment of the outbreak – with recovery of the two secondary cases. MARV was later identified in all three patients.

Ebola virus

Ebola viruses burst from obscurity in 1976, with two spectacular outbreaks of severe haemorrhagic fever in people – both in Africa. In the better-known outbreak for which the viruses were later named, Ebola virus (EBOV) was first associated with an outbreak that eventually totalled 318 cases, starting in September 1976.  This was in the Bumba Zone of the Equateur Region in the north of what was then Zaire, and is now the Democratic Republic of the Congo (DRC).  The index case in the outbreak, as well as many of those subsequently infected, was treated in the Yambuku Mission Hospital. He was injected with chloroquine to treat his presumptive malaria: within a few days fever symptoms developed again; within a week, several others who had received injections around the same time also developed fevers which in several cases had haemorrhagic complications. 

Interestingly, women 15-29 years of age were most affected by the disease: this was strongly correlated with their attending antenatal clinics at the hospital, where they regularly received injections.

Apparently the hospital had only five old-style syringes and needles, and these were reused without proper sterilisation.  Nearly all cases in this outbreak either received injections at the hospital, or had close contact with those who had. 

Most people were infected within the first four weeks of the outbreak, after which the hospital was closed because 11 of 17 staff had died.  Another  269 people died, for a total estimated case-fatality rate of 88%.

The incubation period for needle- transmitted Ebola virus was 5 to 7 days and that for person to person transmitted disease was 6 to 12 days.

Interestingly, in post-epidemic serosurveys in DRC, antibody prevalence to the “Zaire Ebola virus” has been 3 to 7%: this indicates that subclinical infections with the disease agent may well be reasonably common.

The team that discovered the virus at the Antwerp Institute of Tropical Medicine in Belgium, did so after receiving blood samples in September 1976 from a sick Belgian nun with haemorrhagic symptoms who had been evacuated from Yambuku to Kinshasa in the DRC, for them to investigate a possible diagnosis of yellow fever.  Following her death, liver biopsy samples were also shipped to Antwerp – where the team had already ruled out yellow fever and Lassa fever.  Because of the severe nature of the disease, and its apparently novel agent, the World Health Organisation (WHO) arranged that samples be sent to other reference centres for haemorrhagic viruses, including the Centres for Disease Control (CDC) in Atlanta, USA.

The Belgian team were the first to image the virus derived from cell cultures on an electron microscope – when it was obvious that the only thing it resembled was Marburg virus. 

Image copyright CDC / Frederick A Murphy, 1976

Image copyright CDC / Frederick A Murphy, 1976

The CDC quickly confirmed that it was Marburg-like, with possibly the most famous virus image in the world, but that it was a distinct and new virus.

This meant it needed a name – and it was given one derived from the Ebola River that was supposed to be near the town of Yambuku.

Google map of the area where the first Ebola haemorrhagic fever outbreaks occurred

Google map of the area where the first Ebola haemorrhagic fever outbreaks occurred

Another, minor outbreak of the virus occurred in June 1997 in Tandala in north-western DRC: one young child died, and virus was recovered from her – and subsequent investigations showed that “two previous clinical infections with Ebola virus had occurred in 1972 and that about 7% of the residents had immunofluorescent antibodies to the virus”. This further reinforced the idea that subclinical infections were possible.

Sudan virus

In June 1976 – before the Yambuku epidemic in DRC –  an outbreak of a haemorrhagic fever began in the southern Sudanese town of Nzara.  The presumed index case was a storekeeper in a cotton factory, who was hospitalised on June 30th, and died within a week.

There were a total of 284 cases in this outbreak: there were 67 in Nzara, where it is presumed to have originated, and where infection spread from factory workers to their familes.  There were also 213 in Maridi, a few hours drive away – where, as in Yambuku, the outbreak was amplified by “nosocomial” or hospital-acquired transmission in a large hospital. In this case, transmission seems to have been associated with nursing of patients.  The incubation period in this outbreak was 7 – 14 days, with a case mortality rate of 53%.

Two viral isolates were made from sera from Maridi hospital patients in November 1976. Antibodies to the now-identified “Ebola virus” from DRC were detected in 42 of 48 patients clinically-diagnosed patients from Maridi – but in only 6 of 31 patients from Nzara.  However, it was subsequently shown that the Sudan and DRC Ebola viruses were different enough from one another to be separate viral species (see later), which undoubtedly affected the results.

Interestingly, 19% of the Maridi case contacts had antibodies to the virus – with very few of them with any history of illness.  This strongly indicates that the Sudan virus can cause mild or even subclinical infections.

An indication of the possible origin of the epidemic is the fact that 37% of the workers in the Nzara cotton factory appeared to have been infected, with 6 independently-acquired infections – and that this was concentrated in the cloth room, where there were numerous rats as well as thousands of insectivorous bats in the roof.  However, subsequent study of antibodies in the bats failed to detect evidence of infection, and no virus was isolated from bat tissue.

There was another outbreak of the same type of Ebola haemorrhagic fever in the area of Nzara in July – October 1979: this resulted in 34 cases, 22 of them fatal, with the index patient working at the cotton factory and all others being infected via the hospital he was admitted to.  It is interesting that antibodies to the Sudan virus were detected in 18% of adults not associated with the outbreak, leading the report’s authors to speculate that the virus was endemic in this region.

It was thought that the Sudan and DRC outbreaks were linked: the original WHO Bulletin report on the Sudan outbreak even speculates that extensive truck-borne commercial goods traffic between Bumba in DRC and Nzara in what is now South Sudan could have caused the DRC outbreak.  However, comparisons between the viruses isolated from the two epidemics later showed that they were distinct, both in terms of virulence, and antigenicity – meaning the Sudan virus got its own name.

Epidemics and outbreaks have resulted from person to person transmission, nosocomial or in-hospital spread, or laboratory infections. The mode of primary infection and the natural ecology of these viruses are unknown. Association with bats has been implicated directly in at least 2 episodes when individuals entered the same bat-filled cave in Eastern Kenya. Ebola infections in Sudan in 1976 and 1979 occurred in workers of a cotton factory containing thousands of bats in the roof. However, in all early instances, study of antibody in bats failed to detect evidence of infection, and no virus was isolated form bat tissue.

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Happy centenary, phages!

17 February, 2015

Here am I, writing a not-so-brief history of the the discovery of viruses, and I miss The Centenary of the Phage!  How did THAT happen?!

Seriously: it took an email from Virologica Sinica alerting me to their commemorative issue, to jolt me into a better state of historical awareness.

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I wrote elsewhere:

Eaters of Bacteria: The Phages

Two independent investigations led to the important discovery of viruses that infect bacteria: in 1915, Frederick Twort in the UK accidentally found a filterable agent that caused the bacteria he was growing to lyse, or burst open.  While he was not sure whether or not it was a virus, Félix d’Hérelle in Paris published in 1917 that he had discovered a virus that lysed a bacterial agent he was culturing that causeddysentery, or diarrhoea.  He named the virus “bacteriophage”, or eater of bacteria, derived from the Greek term “phagein”, meaning to eat.

The discovery of bacteriophages was a landmark in the history of virology, as it meant that for the first time it was relatively easy to work with viruses: many kinds of bacteria could be grown in solid or liquid culture quite easily, and the life cycle of the viruses could be studied in detail.”

"Twort" by Obituary Notices of Fellows of the Royal Society, Vol. 7, No. 20. (Nov., 1951), pp. 504-517.. Licensed under Public Domain via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:Twort.jpg#mediaviewer/File:Twort.jpg

“Twort” by Obituary Notices of Fellows of the Royal Society, Vol. 7, No. 20. (Nov., 1951), pp. 504-517.. Licensed under Public Domain via Wikimedia Commons – http://commons.wikimedia.org/wiki/File:Twort.jpg#mediaviewer/File:Twort.jpg

And so it has come to be: the study of phages helped to establish virology as a science, in the era before tissue culture and accurate assay of animal viruses; the birth of molecular biology was pretty much due to the famous Phage Group – and phages turn out to be possibly the most abundant form of life in the known galaxy.

Moreover, the wheel of phage therapy espoused by Félix d’Hérelle has turned full circle, with formerly-scorned Soviet-era institutes now suddenly courted by biotech companies: the Virologica Sinica issue has a an editorial review on the subject, and there is another review on the history of the Eliava Institute in Tbilisi, Georgia, complete with a picture of d’Hérelle there in the 1930s.

So, congratulations Frederick Twort, on the centenary of your discovery.  Your “ultramicroscopic viruses” have gone from strength to strength; your name is remembered – albeit shamefully late – and we really should think of how to put phages more into the public eye.

Figuratively and literally, possibly B-)

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PS: I discover to my delight that there is an entire site devoted to The Year of Phage, which has some amazing art as well as an entire book available for download.  Get yours NOW!

A Short History of the Discovery of Viruses – Part 4

11 February, 2015

RNA Genomes and Modern Virology

RNA as genetic material

While it had been known since Bawden and Pirie’s work in 1937 that TMV particles contained RNA, followed later by a number of other viruses, it must be remembered that DNA had only really been accepted as the genetic material of cells and viruses after the Hershey-Chase experiment in 1952 and the Watson-Crick demonstration of the nature of DNA in 1953.  Moreover, the way in which the information in DNA was used to make proteins was still very obscure in the 1950s, given that the proof that RNA was used as a template for the production of proteins was only provided in 1961 by Marshall Nirenberg.

It was hailed as a major development in molecular biology, therefore, when between 1955 and 1957, Heinz Fraenkel-Conrat, B Singer and Robley C Williams demonstrated that it was possible to reconstitute fully infectious TMV from separately-purified preparations of coat protein and RNA.  At the time it was assumed that neither of the two components was infectious on its own; however it was subsequently shown by Fraenkel-Conrat and Singer, and separately by A Gierer and G Schramm, that purified TMV RNA was in fact infectious – albeit several hundred times more weakly per unit mass than the native or reconstituted particles.

While this was revolutionary in itself, the clinching experiment was the proof that mixed reconstitution – or the reassembly of a RNA of one strain of TMV with the coat protein of another – followed by infection of plants resulted in particles made of protein specified by the RNA component rather than being determined by the protein donor.  This work possibly represents the birth of molecular virology as a sub-discipline within molecular biology, given that the molecular nature of viruses had so conclusively been shown – vindicating the prescient remark made by the virus pioneer Thomas Rivers in 1941, on the occasion of the presentation of a gold medal to Wendell Stanley, that:

“In fun, it has been said that we do not know whether to speak of the unit of this infectious agent [TMV] as an “organule” or a “molechism”” (p.7, CA Knight, Chemistry of Viruses 2nd Edn., 1975. Springer-Verlag, Wien)

Further important developments with TMV included the demonstration in 1958 by Gierer and KW Mundry that TMV mutants with altered genomes could be produced by treatment of virions with nitrous acid, which only alters nucleic acids, and the sequencing of the TMV coat protein in 1960 by two groups including Fraenkel-Conrat and Stanley and Knight in one, and Schramm in the other.

Between 1953 and 1954, an interesting class of new viruses was discovered in humans, birds, and later in other animals too.  These were dubbed “respiratory enteric orphans” based on where they were found, and the fact they were not associated with any disease – which gave rise to the name “reovirus”, and their description as a distinct group of viruses by

Human rotavirus, in the family Reoviridae.  Russell Kightley Media

Human rotavirus, in the family Reoviridae. Russell Kightley Media

Albert Sabin in 1959.  By 1962 the unique double-layered capsid morphology had been seen and the virions shown to contain RNA, and then in 1963 PJ Gomatos and I Tamm showed using physical and chemical techniques that the viruses as well as the similar wound tumour virus isolated from plants had a genome consisting of double-stranded (ds) RNA – a finding unprecedented in biology at the time.  Gomatos and W Stoeckenius  went on to show in 1964 – by electron microscopy – that the reovirus genome was also segmented – another unprecedented finding for viruses. In the 1963 paper the authors remark that “…all attempts to isolate the nucleic acid of reovirus in an infective form have failed” – which distinguished these viruses from the ssRNA viruses previously looked at – not surprisingly, given the requirement for a different replication method for dsRNA compared to viruses like TMV or poliovirus (see here).

Ribosomes translating protein from a messenger RNA molecule

Ribosomes translating protein from a messenger RNA molecule

A major highlight in molecular biology in 1961 was Marshall Nirenberg and Heinrich Matthaei’s 1961 demonstration of “…an assay system in which RNA serves as an activator of protein synthesis in E. coli extracts”, or the proof in an in vitro translation system that RNA was the “messenger” that conveyed genetic information into proteins.

In 1962, A Tsugita, Fraenkel-Conrat, Nirenberg and Matthaei used the still extremely novel in vitro translation system with purified TMV genomic RNA, and were able to show that:

“The addition of TMV-RNA to a cell-free amino acid incorporating system derived from E. coli caused up to 75-fold stimulation in protein synthesis (C14-incorporation). Part of the protein synthesized formed a specific precipitate with anti-TMV serum.”, indicating that TMV coat protein had been made.

This was the first demonstration of in vitro translation from any specific mRNA, and incidentally also direct proof that the single-stranded TMV genome was “messenger sense”.  They also concluded that their result showed that the newly-determined “genetic code” – the nucleotide triplets that code for individual amino acids – was universal, given that it was a tobacco virus RNA being translated by a bacterial system.

Later in 1962, D Nathans and colleagues used coliphage f2 RNA as template for translation in the same type of bacterial extract.  They showed that polypeptides corresponding to the coat as well as other proteins were made, showing that it was the input virion RNA that was responsible.

Modern virology

The proof that RNA was both the “messenger” that conveyed information from DNA to be made into protein, and was in fact a genetic material in its own right, made possible a revolution in virology that transformed it into the science we know today.  The new molecular biology together with well-established physical and biochemical techniques for molecular characterisation, coupled with the ability to reliably culture bacterial, plant and now animal viruses as well, enabled an explosion of discovery that continues to this day. 

A tour de force experiment in the modern molecular biological era was the in vitro synthesis of an infectious phage RNA genome by S Spiegelman and coworkers in 1965, using only purified Qbeta coliphage single-stranded virion RNA and the purified viral replicase.   They remarked:

“The successful synthesis of a biologically active nucleic acid with a purified enzyme is itself of obvious interest. However, the implication which is most pregnant with potential usefulness stems from the demonstration that the replicase is, in fact, generating identical copies of the viral RNA. For the first time, a system has been made available which permits the unambiguous analysis of the molecular basis underlying the replication of a self-propagating nucleic acid.”

In 1967 there followed the demonstration that the same could be done for a single-stranded (ss)-DNA virus: M Goulian and colleagues reported in that they had successfully made a completely synthetic and infectious PhiX174 coliphage genome, by means of a series of syntheses using purified virion ssDNA, E coli DNA polymerase and a “polynucleotide-joining enzyme”, or DNA ligase.  It is instructive that the authors offer this as evidence for the involvement of the same enzymes in E coli chromosomal replication, the mechanism for which which was still obscure at the time.  Their justification for their work:

“If enzymatic synthesis of infectious bacteriophage DNA were achieved, it would be made clear at once that relatively few, if any, mistakes had been made in replicating a DNA sequence of several thousand nucleotides.”

– was undoubtedly borne out, in yet another example in the growing number of cases of the use of viruses to demonstrate important facets of cellular biology.

Naked nucleic acids as infectious agents: viroids

A potato disease that had been known in the New York and New Jersey state areas in the US since the 1920s was the source of an exciting discovery by Theodor (Ted) Diener and WB Raymer, reported in Science in 1967.  The potato spindle tuber disease agent had proved recalcitrant over many years to being characterised or isolated; all that was known was that it could be transmitted mechanically using sap, or via grafting, and that no fungi, bacteria or viruses could be isolated from diseased material.  Diener and Raymer showed that:

“Infectious entities, extractable, with phosphate buffer, from tissue infected with potato spindle tuber virus and inciting symptoms on tomato that are typical of this virus, have properties incompatible with those of conventional virus particles. …[Their properties] suggest that the extractable infectious agent may be a double-stranded RNA.”

By 1971 Diener had determined that

“…the infectious RNA occurs in the form of several species with molecular weights ranging from 2.5 × 104 to 1.1 × 105 daltons. No evidence for the presence in uninoculated plants of a latent helper virus was found. Thus, potato spindle tuber “virus” RNA, which is too small to contain the genetic information necessary for self-replication, must rely for its replication mainly on biosynthetic systems already operative in the uninoculated plant.”

This was a revolutionary concept: an infectious, pathogenic entity in the form of a naked RNA that was too small to encode a replicase or any other protein.  He proposed the term “viroid” to designate this and similar agents, a term that persists up to today.  By 1979, they were known to be single-stranded circular RNA molecules with a high degree of sequence self-complementarity, which results in them appearing as “highly base-paired rods”.

Reverse transcription and tumour viruses

While it was apparent in the 1960s that there were single-and double-stranded DNA and RNA viruses, it was only in 1970 that two back-to-back papers in Nature, by Howard Temin and S Mituzami, and David Baltimore respectively, revealed a highly novel viral replication strategy.  They showed that “RNA tumour viruses” such as the agents found by Ellerman and Bang and Peyton Rous contained an enzyme activity named reverse transcriptase – a colloquial term for RNA-dependent DNA polymerase – in their virions, which converted the single-stranded RNA genomes into double-stranded DNA. Later this was shown to result in resulted in insertion of the DNA into the host cell genome, vindicating Howard Temin’s 1960 proposal that “…a RNA tumor virus can give rise to a DNA copy which is incorporated into the genetic material of the cell”.

When Francis Crick formulated his ”Central Dogma” in 1956, it was indisputable that genetic information flowed from DNA to progeny DNA, from DNA to RNA, and from messenger RNA to protein – while he only postulated no return information flow from protein, it was generally assumed that this was also true for RNA

In the words of David Baltimore, in his Nature article:

“Two independent groups of investigators have found evidence of an enzyme in virions of RNA tumour viruses which synthesizes DNA from an RNA template. This discovery, if upheld, will have important implications not only for carcinogenesis by RNA viruses but also for the general understanding of genetic transcription: apparently the classical process of information transfer from DNA to RNA can be inverted.”

This gives rise to a modified Central Dogma, where information flows from DNA to DNA, from DNA to RNA, from RNA to RNA, from RNA to DNA, and from RNA to protein.  It is interesting that RNA seems central to this flow – which, incidentally, strengthens the proposal that RNA is the original genetic material.

Baltimore and Temin both received a share of the Nobel Prize in Physiology or Medicine 1975 for their discovery of reverse transcriptase – and shared it with Renato Dulbecco, who was credited with clarifying the process of infection and of cellular transformation by DNA tumour viruses.  He used the double-stranded (ds) DNA polyomavirus SV40: this was originally isolated from monkeys, but shown to cause a variety of tumours in a number of experimental animals, hence the name “poly-oma”. 

He and colleagues showed that polyomavirus grew and could be assayed normally in certain cell cultures, but caused tumour-like transformation of cells in others in which it did not grow.  They showed that transformed cell chromosomes contained covalently integrated viral DNA termed a provirus, which was active in producing mRNA which made virus-specific proteins.  Thus, his work was the first to show how DNA viruses might cause cancer, and he and his colleagues deserved their award “…for their discoveries concerning the interaction between tumour viruses and the genetic material of the cell.

Viral genome cloning and sequencing: the new age

The techniques of recombinant DNA technology – or the artificial introduction of genetic material from one organism into the genome of another – were pioneered between 1971 and 1973 by Paul Berg, Herbert Boyer and Stanley Cohen.  In 1971 Berg performed an in vitro exercise in which a segment of the lambda phage genome was ligated into the purified DNA of SV40, which had been linearised using the then-new restriction endonuclease, EcoRI.  Cohen, Annie Chang, Boyer and Robert Helling took the technology further in 1973 by showing that:

“The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules.”

Cloning had arrived – made possible in part by use of viruses.  The fundamental nature of this advance of molecular biology was rewarded by a half share of the 1980 Nobel Prize in Chemistry to Paul Berg.

Nucleotide sequencing, or the determination of the order of bases in nucleic acids, started with laborious, difficult techniques such as the two-dimensional fractionation of enzyme digests of 32P-labelled for RNA described by Frederick Sanger and colleagues in 1965.  DNA sequencing followed in 1970: Ray Wu described the use of E coli DNA polymerase and radiolabelled nucleotides to sequence the single-stranded ends of phage lambda DNA. He and colleagues followed this with a more general method in 1973, using extension of synthetic oligonucleotide “primers” annealed to target DNA

Walter Gilbert and Allan Maxam published in February 1977 an immediately popular paper entitled “A new method for sequencing DNA”.  This became known as Maxam-Gilbert sequencing, or the chemical method, as it entailed sequencing by chemical degradation.  Also in 1977, however, Frederick Sanger and colleagues adapted the Wu technique to come up with the so-called Sanger method, or “DNA sequencing with chain-terminating inhibitors“: this soon became the industry standard for at least the next twenty years, because it was easier and cheaper than the chemical method.

Gilbert and Sanger were awarded a share of the Nobel Prize in Chemistry in 1980, for their contributions concerning the determination of base sequences in nucleic acids“.

MS2 phage sequencing

A highlight of Ed Rybicki’s introduction to the world of viruses was discovering during his Honours year in 1977, the paper in Nature in 1976 by Walter Fiers and his coworkers on completing the genome sequencing of the ssRNA E coli phage, MS2.  They had previously also been responsible for the first ever gene sequence, in 1972: this was of the coat protein gene from the same virus.  This was a landmark publication, because it completed the work of years by their group by sequencing the replicase gene, using the ribonuclease digestion and genome fragmentation and two-dimensional electrophoresis technique from Sanger.  Moreover, they proposed a secondary structure for the replicase gene based on intrasequence complementarity, and described it eloquently as follows:

“The secondary structure of the coat gene resembles a flower, and there are similar foldings in other parts of the molecule; the secondary structure of the whole viral RNA therefore constitutes a bouquet”.

Their achievement looks modest in retrospect, in this era of high-throughput sequencing – however, it is worth remembering that at this time in 1976,

“MS2 is the first living organism for which the entire primary chemical structure has been elucidated”. 

Depiction of the linear sequence of MS2 phage.  The maturation (M), coat (CP) and replicase (Rep) genes and proteins were known at the time of sequencing; the lysis gene that partially overlaps the Rep open reading frame was shown to be functional only in 1982

Depiction of the linear sequence of MS2 phage. The maturation (M), coat (CP) and replicase (Rep) genes and proteins were known at the time of sequencing; the lysis gene that partially overlaps the Rep open reading frame was shown to be functional only in 1982

While this comprised just 3569 nucleotides, encoding only three genes, this  is sufficient to constitute a self-replicating entity with an independent evolutionary history.

The immediate value of their work was that it provided a basis for understanding the biology of the interaction of the genome with the bacterial cell at the molecular level.  Moreover, the proposed secondary structures also helped explain how such a simple genome managed to temporally regulate its own expression – by means of long-distance interactions between different areas of the sequence.

PhiX174 phage sequencing

The next complete viral genome sequenced was that of the circular single-stranded DNA coliphage PhiX174, in 1977 by Sanger and his team in Cambridge, using the new sequencing technique invented by them.  The abstract of their paper reads:

“A DNA sequence for the genome of bacteriophage phi X174 of approximately 5,375 nucleotides has been determined using the rapid and simple ‘plus and minus’ method. The sequence identifies many of the features responsible for the production of the proteins of the nine known genes of the organism, including initiation and termination sites for the proteins and RNAs. Two pairs of genes are coded by the same region of DNA using different reading frames.“

This was the first complete genome sequenced for any DNA-containing organism, and a satisfying conclusion to many decades of work on the virus.  One of the most interesting features of the sequence was the fact that several of the 11 genes  are highly overlapping: that is, the same DNA sequence is used to encode completely different genes in different open reading frames.  This represented an economy of use of genetic information that was hitherto unknown. 

Ed Rybicki was also able to greatly impress his Honours external examiner – one DR Woods – by launching into a detailed account of the sequencing and the genetic implications, when asked “What did you find interesting in the literature this year?”

SV40 sequencing

The simian vacuolating virus 40, or SV40, was discovered in 1960 by Ben Sweet and Maurice Hilleman as a contaminant of live attenuated polio vaccines made between 1955 and 1961: this was as a result of use of vervet or African green monkey cells that were inadvertently infected with SV40 to grow up the polioviruses.  As a consequence, between 1955 and 1963 up to 90% of children and 60% of adults – 98 million people – in the USA were inadvertently inoculated with live SV40.  Given the demonstration by  Bernice  Eddy and others in 1962 that hamsters inoculated with simian cells infected with SV40 developed sarcomas and ependymomas, the class of viruses including SV40 and MPyV described earlier became known as “polyomaviruses”, and DNA tumour viruses.  However, and despite considerable concern over many years, SV40 has not been shown to cause or to definitively be associated with any human cancers.

Still, it had become an object of considerable interest as mentioned earlier in connection with Renato Dulbecco, and it was accordingly the next virus to be completely sequenced.  This was by Walter Fier’s group: they determined by Maxam-Gilbert sequencing that the circular dsDNA genome comprised 5224 base pairs, and had an interesting organisation.  In their words:

“Particular points of interest revealed by the complete sequence are the initiation of the early t and T antigens at the same position and the fact that the T antigen is coded by two non-contiguous regions of the genome; the T antigen mRNA is spliced in the coding region. In the late region the gene for the major protein VP1 overlaps those for proteins VP2 and VP3 over 122 nucleotides but is read in a different frame.”

Linear depiction of the circular SV40 genome and its protein coding capacity.  Regions of RNA spliced out of of transcribed genomic sequence, and the direction of transcription, are shown as red arrows.  Genes shown are those depicted in the current Genbank sequence entry.

Linear depiction of the circular SV40 genome and its protein coding capacity. Regions of RNA spliced out of of transcribed genomic sequence, and the direction of transcription, are shown as red arrows. Genes shown are those depicted in the current Genbank sequence entry.

This was the first time that RNA splicing had been demonstrated for an entire genome; indeed, it had only been discovered in 1977 when two separate groups of researchers showed that adenovirus-specific mRNAs made late in the replication cycle in cell cultures were mosaics, being comprised of sequences from noncontiguous or separated sites in the viral genome.  This was subsequently found to be a common feature in eukaryotic but not prokaryotic mRNAs.

The SV40 genome showed major gene overlaps, as for the PhiX174, again demonstrating the effectiveness with which viruses could pack protein coding capability into a small genome

Sequencing of a viroid

Also in 1978, Heinz Sänger’s group published the sequence and the predicted secondary structure of potato spindle tuber viroid.  This was the first RNA genome to be sequenced using the still relatively new method of generating complementary DNA (cDNA) from RNA by use of reverse transcriptase.

They stated in their Nature paper abstract that:

“PSTV is the first pathogen of a eukaryotic organism for which the complete molecular structure has been established”.

This was absolutely true, as the detailed 3D structure of SV40 was not known, even though the genome sequence was.

Sequencing of the first human polyomavirus

In October and December of 1979, two groups published the complete nucleotide sequences of two strains of the polyomavirus known as BKV: this had been first isolated from a renal transplant patient with those initials in 1971, and found to be present in about 80% of healthy blood donors.  It causes only mild infectionsfever and respiratory symptoms – on first infection, and then subsequently infects cells in the kidneys and urinary tract, where it can remain causing no symptoms for the lifetime of infected individuals.  It is associated with renal dysfunction in immunocompromised people, and “BK nephropathy” in transplant patients, when immunosuppressive drugs allow destructive viral multiplication within the donated organ.

The viral sequences differed by 190 nucleotides from one another, and the first-published MM strain sequence was shown to have a strikingly similar arrangement of putative genes to SV40, and to share 70% sequence homology and 73% predicted amino acid sequence homology with SV40.  It also shared 75% genome sequence homology with the JC polyomavirus, first isolated in 1971 from a patient with progressive multifocal leukoencephalopathy (PML).  It is found in between 70 and 90% of humans, but as with BKV, is only associated with disease in immunosuppressed or immunodeficient people.

The hepatitis B virus genome

The involvement of HBV in human disease has already been described, as has its potential to cause human cancers.  The complete genome sequence of an E coli genomic clone of the “subtype ayw” strain of HBV was reported by Francis Galibert and co-workers in 1979.  This consisted of 3182 nucleotides, arranged as what had previously been shown by physicochemical techniques on DNA isolated from virions to be a circular structure with a “-” strand with a short gap, and an incomplete “+” strand of varying length.  The reason for this was puzzling, and was attributed to virion assembly requirements, although the covalently closed circular form of this and related viruses (duck hepatitis), like genomes of polyoma- and papillomaviruses, had been isolated.  The reason for this would have to wait a few years, and would result in a new class of viruses being recognised that would reflect very deep evolutionary links between viruses and cellular elements.

The HBV genome. "HBV Genome" by T4taylor - Own work based on File:HBV_genome.png. Licensed under CC BY-SA 3.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:HBV_Genome.svg#/media/File:HBV_Genome.svg

The HBV genome.
“HBV Genome” by T4taylor – Own work based on File:HBV_genome.png. Licensed under CC BY-SA 3.0 via Wikimedia Commons – http://commons.wikimedia.org/wiki/File:HBV_Genome.svg#/media/File:HBV_Genome.svg

Cauliflower mosaic virus

The first of what are now known to be caulimoviruses – family Caulimoviridae – was described in 1933 as dahlia mosaic virus. Cauliflower mosaic virus (CaMV) was described in 1937, and shown to have particles containing a DNA genome in 1968.  This was visualised by electron microscopy by two groups in 1971 as relaxed open circles or also as linear forms – unlike the supercoiled DNAs of papilloma- or papovaviruses.  By 1977 it was known that the “nicked circular form” was infectious, and fragments of the genome had been cloned in E coli. Physical and biochemical characterisation of the genome in 1978 showed that it consisted of three discrete lengths of single-stranded DNA – alpha, beta and gamma, with alpha being the full genome-length – that annealed to one another to give a circular double-stranded form about 8 000 nucleotides in length.  By 1979 it was known that only the alpha strand was transcribed to give mRNA.

The complete sequence of the viral genome was published in 1980, and was predicted to encode six proteins: this has since been upped to eight, with two small ORFs (VII and VIII) being shown to produce proteins.  The genome is transcribed into only two mRNAs – named 35S and 19S,  on the basis of their sedimentation properties – and contains one discontinuity in the alpha or coding strand, and two in the non-coding sequence.  Cloned DNA was also shown to be infectious in 1980, even if excised as a linear molecule.

Diagram showing a depiction of the CaMV genome in red, with single-strand discontinuities shown as yellow triangles.  mRNA species are shown in blue: the 35S RNA is longer-than-genome-length, with a ~200 bp repeat at the 5’ and 3’ ends.  ORFs as presently known are shown in green.  Redrawn from Figure 8.1 of REF Mathews’ “Plant Virology”, 3rd edition.

Diagram showing a depiction of the CaMV genome in red, with single-strand discontinuities shown as yellow triangles. mRNA species are shown in blue: the 35S RNA is longer-than-genome-length, with a ~200 bp repeat at the 5’ and 3’ ends. ORFs as presently known are shown in green.
Redrawn from Figure 8.1 of REF Mathews’ “Plant Virology”, 3rd edition.

A new class of viruses

In 1971, David Baltimore of reverse transcriptase and future Nobel Prize fame published a review in which he described the “Expression of Animal Virus Genomes” as it was then understood.  This described a scheme for classifying viruses on the basis of the pathways taken to make messenger RNA – because, in his words,

“The specific mechanism for mRNA synthesis used by a given virus depends on the structure of the viral genetic material”,

which in turn meant that the different means used to arrive at the same end were useful in functionally grouping viruses together.

He proposed 6 classes of viral expression.  These were:

  • Class I: all dsDNA viruses
  • Class II: ssDNA viruses
  • Class III: dsRNA viruses
  • Class IV: ssRNA viruses whose mRNA sequences are subsets of the virion RNA sequence (ie: ss(+)RNA viruses)
  • Class V: ssRNA viruses with genomes which  are complementary in sequence to the mRNA sequences (ie: ss(-)RNA viruses)
  • Class VI: ssRNA viruses which have a DNA intermediate in their lifecycle

It is worth remembering that this was proposed only for animal viruses, and that it was not even known at the time whether retroviruses (Class VI) actually made a DNA genome in cells or what their mRNA looked like.  Additionally, remembering that this predated both cloning and sequencing of viral genomes or mRNAs, it was a triumph of biochemistry and molecular biology at the time that as much as was known.  For example, it was only discovered in 1968 that dsRNA-containing reovirus particles also contained an RNA polymerase activity.  Baltimore himself only discovered in 1970 that the ssRNA-containing vesicular stomatitis virus virions also contained an RNA polymerase.  He made the simple but bold and prescient proposition that:

“The discovery of virion [RNA-dependent RNA] polymerases has provided a rationale for an old puzzle in virology. Why is it that infectious nucleic acid can be extracted from the virions of some viruses but not others? Virion polymerases provide the clue to this puzzle; where they exist the nucleic acid is noninfectious, where the nucleic acid is infectious they could not exist or at least they could not serve an obligate role.”

This has held up surprisingly well, to the extent that the Baltimore Classification has been enshrined in the teaching of virology – by me, among others – for more than 40 years, and has been extended to cover all viruses, as the similarities in their route to mRNA synthesis became apparent, whether they infected bacteria, animals or plants.  In light of the discovery detailed below, it is interesting that HBV virions were discovered in 1975 to contain both circular dsDNA and a DNA polymerase activity.  The virion DNA pol could be used in vitro to convert the partially-dsDNA to a 3 kb completely dsDNA.

Baltimore also wisely made room for “…extending the class designations”, which meant that a very important new class of viruses could be neatly slotted in as Class VII, after their confirmation in 1983.  These were dsDNA viruses which have an RNA intermediate in the life cycle, or pararetroviruses as they came to be known – and the first two representatives were the very different duck hepatitis  B virus (DBV) and CaMV, which had been thought of up till then as classical dsDNA viruses

DBV was discovered in 1980, then investigated by Jesse Summers and William Mason, who determined in 1982 that:

“Subviral particles resembling the viral nucleocapsid cores were isolated from persistently infected liver and shown to have a DNA polymerase activity that utilizes an endogenous template and synthesizes both plus- and minus-strand viral DNA. Synthesis of the viral minus-strand DNA utilized an RNA template that was degraded as it was copied. Viral plus-strand synthesis occurred on the completed minus-strand DNA”.

On the basis of this evidence, they proposed a pathway for replication of HBV-like viruses by reverse transcription of a RNA intermediate

The next member of the reverse transcribing Class VII pararetroviruses was described in 1983, by Pierre Pfeiffer and Thomas Hohn.  Interestingly, like retroviruses and unlike the very different HBV model, their model for CaMV replication included a probable tRNA primer for RNA-dependent DNA synthesis initiation.  It is also interesting that CaMV virions did not contain a DNA polymerase activity, unlike HBVs or retroviruses, indicating significant differences in the mode of replication.  However, the demonstration in 1983 of amino acid sequence homology between the retroviral reverse transcriptase and putative polymerases of both HBV and CaMV, cemented the dawning realisation of a whole new class of viruses.

Ed Rybicki remembers vividly the excitement at the Seventh John Innes Symposium in 1986 on “Virus replication and genome interactions”, where evidence was presented for both HBV and CaMV – and strong public disbelief expressed as to whether HBV replicated by this route by none other than Peter Duesberg, who later went on to also doubt that HIV caused AIDS.

Infectious, cloned poliovirus RNA

In 1958, working from the example of Gierer and Scramm with TMV, Hattie Alexander and colleagues demonstrated that RNA extracted from concentrated, partially purified preparations of polioviruses types I and II, that was free of protein and DNA, was infectious in cultured HeLa cells and human amnion cell monolayers. Moreover, the RNA produced progeny virus characteristic of that used to produce the RNA.  The concept of RNA genomes was still new enough at the time to prompt their conclusion that:

“It would seem, therefore, that the virus RNA is the essential infectious agent”.

In the same year, the same was shown by Fred Brown for another distantly related picornavirus, foot and mouth disease virus.  By 1968, it was known – thanks to MF Jacobson and Baltimore and others – that several picornaviruses related to poliovirus appeared to have just one open reading frame (ORF) encoding a single polypeptide encoded in their genomic RNA, and did not make a smaller mRNA. 

In June of 1981, Naomi Kitamura and coworkers published the complete nucleotide sequence of poliovirus I.  They showed it was

“[an] RNA molecule [which] is 7,433 nucleotides long, polyadenylated at the 3′ terminus, and covalently linked to a small protein (VPg) at the 5′ terminus. An open reading frame of 2,207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO”.

In August 1981, Vincent Racaniello – working in Baltimore’s lab – reported a set of three cDNA clones in E coli spanning the whole of the poliovirus I genome, and also sequenced the genome

Later in 1981, Racaniello used the three clones to construct one contiguous cDNA clone in pBR322, which he successfully used to transfect vervet monkey (=African green monkey) kidney cell cultures, producing infectious wild-type virus with which he produced characteristic plaques in HeLa cells.  This was the first proof that an infectious cDNA construct could be made for an RNA virus – and it is interesting that the construct should not have worked, as there was no mammalian promoter to produce the viral RNA, and it had to rely on an adventitious or cryptic promoter in the plasmid sequence. In any case, it was an excellent example of success of the “just try it” school of experimentation. He has written an excellent account of this – “Thirty years of infectious enthusiasm” – in his popular blog.

Tobacco mosaic virus sequenced

In 1982, P Goelet and five coworkers published the complete nucleotide sequence of the Vulgare strain of tobacco mosaic virus.  They did this by using reverse transcriptase and synthetic oligonucleotide primers to generate a set of short, overlapping complementary DNA fragments covering the whole TMV genome, cloning these in bacteriophage M13, and using the Sanger dideoxy method to determine the sequences :…of more than 400 independently derived cDNA clones”.  Their complete sequence confirmed earlier partial sequences derived by direct RNA sequencing techniques, as well as confirming quite pronounced sequence variability within the isolate, especially at the 5’ end.

The length of the consensus sequence they produced has to be dug for in the paper, incidentally, as nowhere do the authors in fact simply state what it is – or in fact give a diagram of the genetic organisation! 

Depiction of the TMV genome and transcription and translation strategy

Depiction of the TMV genome and transcription and translation strategy

They report a 6 395 nucleotide sequence, with just three major ORFs.  However, the presence of an amber or leaky stop codon explained how two different N-coterminal proteins could be made by translation of genomic RNA, and separate ribosome binding sites for the two genomic 3’ ORFs plus the already proven existence of subgenomic 3’-coterminal mRNAs, explained how the other proteins could be made. Their was also found to be a unique hairpin loop-encoding sequence region for assembly initiation – nicely rounding out pioneering work by PJ Butler and colleagues and Genevieve Lebeurier and others – both published in January 1977, incidentally – on the physical mechanism of assembly, which had shown a single site for assembly nucleation.

The circle closes: TMV understood

The sequencing of TMV almost, but not quite, brought to a complete circle the journey of discovery that started with its description as a “contagium vivum fluidum” in 1898.  Closing the circle required a complete molecular understanding of the virus particle, which was achieved with the publication from Gerald Stubbs’ group of the 3.6 Angstrom resolution structure for TMV in 1986, and then the refined 2.9 A structure in 1989.  In their words,

The final model contains all of the non-hydrogen atoms of the RNA and the protein, 71 water molecules, and two calcium-binding sites.

This allowed the building of molecular models that helped explain the chemical and physical basis for virion self-assembly, as well as accounting for the positioning in particles of the whole RNA and the whole of the more than 2000 individual protein subunit sequences.

TMV atomic model

The structure also brought to a conclusion a journey that had started as early as 1936, after Stanley’s demonstration that TMV could be crystallised.  Gerald Stubbs has written an excellent account in the book “Tobacco Mosaic Virus: One Hundred Years of Contributions to Virology” of how JD Bernal and I Fankuchen used “liquid crystalline” gels of TMV provided by Bawden and Pirie, and RWG Wyckoff and RB Corey used crystals of TMV, to obtain X-ray diffraction patterns.  While Bernal and Fankuchen were not able to do more than obtain the radius of the virions, and the dimensions of the protein subunits as well as their repeat along the virion axis, they laid the foundations for others to work not only on helical viruses, but also on DNA and RNA helices.

Rosalind Franklin of DNA structure fame took on the study of TMV structure using the then-new method of isomorphous replacement to first, in 1956, locate the RNA within virions, and then in 1958 with Kenneth Holmes, to determine that the virions were indeed helical in structure – something first proposed by James Watson in 1954, although he did not see they were also hollow.  Indeed, Crick and Watson used her TMV structure results to bolster their proposal in 1956 that virions  of small viruses were built up according to simple rules of symmetry from identical protein subunits surrounding the nucleic acid.

Franklin died in 1958, which may have denied her a share of the Nobel Prize for the structure of DNA that went to Watson, Francis Crick and Maurice Wilkins in 1962.

Holmes and Stubbs continued the work on TMV, however, and published a 6.7 A structure in 1975: this showed that an 11 A structure seen by Bernal and Fankuchen in 1936 was in fact alpha helices within subunits.  They went on with Steven Warren in 1977 to obtain a 4 A structure which showed the structure of RNA and the RNA binding site of the protein.  Stubbs celebrated his group’s subsequent refinements of the structure with this statement in the centenary book on TMV:

“…in 1989, Bernal and Fankuchen’s remarkable patterns finally yielded the fulfillment of Franklin’s vision with the publication of the 2.9 A resolution structure…”

A modest statement for a landmark achievement: the finalising of the complete molecular understanding of the first virus discovered.  The circle was finally closed, 91 years after Beijerinck first showed how a filter could define a new class of organisms.

Click here for Part 1: Filters and Discovery

here for Part 2: The Ultracentrifuge, Eggs and Flu

here for Part 3: Phages, Cell Culture and Polio

Copyright Edward P Rybicki and Russell Kightley, February 2015, except where otherwise noted.

A Short History of the Discovery of Viruses – Part 3

29 January, 2015

Phages, Cell Culture and Polio

The Phage Group and the birth of molecular biology

Some of the more fundamental discoveries in modern biology were facilitated either by the study of viruses, or by use of viruses as tools for exploring host cell mechanisms.  The foundations for this work were laid by Felix d’Hérelle and others, working after 1917 with bacterial viruses in cultured bacteria.  Indeed, Macfarlane Burnet’s first important work was in 1929, showing by use of plaque counting that a single bacterial cell infected with a single phage produced 20 – 100 progeny some 20 minutes following infection.  The fact that phages adsorbed irreversibly to their hosts as part of the infection process was shown by AP Krueger and M Schlesinger in 1930 – 1931.  Schlesinger later showed between 1934 and 1936 that the bacteriophage he worked with consisted of approximately equal amounts of protein and DNA, the first proof that viruses might be nucleoprotein in nature.

However, it took until 1939 for the former physicist Max Delbrück, working with the biologist Emory Ellis at Caltech, to elucidate the growth cycle of a sewage-isolated Escherichia coli bacteriophage in a now-classic paper simply entitled “The Growth of Bacteriophage”.  This used the simple technique of counting plaques in a bacterial lawn in a Petri dish, following infection of a standard bacterial inoculum with a dilution series of a phage preparation.

Their principal finding was that viruses multiply inside cells in one step, and not by division and exponential growth like cells.. This was determined using the so-called “one-step growth curve”, which allowed the accurate determination of the titres of viruses released from bacteria that had been synchronously infected.  This allowed calculation of not only the time of multiplication of the virus, but also the “burst size” from individual bacteria, or the number of viruses produced in one round of multiplication.  This was a fundamental discovery, and allowed the rapid progression of the field of bacterial and phage genetics

Indeed, Wolfgang Joklik wrote in 1999:

Conceptualization of the one-step growth cycle completely changed virology. From then on, populations of host cells were infected with multiplicities greater than 1 infectious unit per cell, which meant that infection was synchronous and that virus replication was amenable to biochemical and, therefore, molecular analysis. This study represents the beginning of molecular virology, molecular biology, and molecular genetics.”

One important facet of this work was that it showed that infection could be caused by single phages: the power of the plaque assay meant that even dilutions of phage preparations that contained only a single particle could produce a detectable plaque.

The Phage Group was started in the 1940s after Delbrück and Salvador Luria – also famous for inventing the Luria broth used to this day to grow bacteria –  met at a conference.  They soon began to collaborate, and in 1943 published the famous Luria–Delbrück experiment or Fluctuation Test: this showed that resistance to phage infection in bacteria could arise spontaneously and without selection pressure.  This was fundamental to understanding bacterial evolution and the development of antibiotic resistance in particular.

Also in 1943, they added Alfred Hershey to the group.  An important early result of their joint work was the proof that co-infection of one bacterium with two different bacteriophages could lead to genetic recombination, or mixing of the phage genomes. 

Hershey and his assistant Martha Chase subsequently went on in 1952 to perform the legendary Hershey-Chase experiment in order to prove whether or not DNA was the genetic material of the phage: this purportedly used a new high speed Waring blender Hershey had purchased for his wife, but which never made it to her.  This was published as “Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage“, and essentially cemented the central role of DNA as the material of heredity.

The Hershey-Chase Experiment

Building on an observation by RM Herriott in 1951 that phage “ghosts”virus particles that had lost their DNA due to osmotic shock – could still attach to and lyse their target bacteria, they grew up preparations of the E coli bacteriophage T2 separately in the presence of the radioisotopes 35S and 32P, to label the protein and nucleic acid components of the phage respectively.  They confirmed the earlier observations by showing that “plasmolysed” phage ghosts retained nearly all of the 35S and the ability to bind to phage-susceptible bacteria and bind phage-specific antibodies, while the free DNA fraction retained nearly all of the 32P, which was DNAse-susceptible, unlike DNA in intact phages.  Their conclusion was that:

The ghosts represent protein coats that surround the DNA of the intact particles, react with antiserum, protect the DNA from DNase…, and carry the organ of attachment to bacteria”.

However, their most exciting result was achieved by investigating whether “…multiplication of virus is preceded by the alteration or removal of the protective coats of the particles”.  They did this by allowing adsorption of phages to bacteria in liquid suspension for different times, then shearing off adsorbed phage particles from the bacteria using the blender.  Pelleting the bacteria by centrifugation and assaying radioactivity allowed them to determine that over 75% of the 35S – incorporated into cysteine and methionine amino acids – remained in the liquid, or outside the bacteria, whereas over 75% of the 32P – incorporated into the phage DNA – was found inside the bacteria.  They concluded that:

“…the bulk of the phage sulfur remains at the cell surface during infection, and takes no part in the multiplication of intracellular phage. The bulk of the phage DNA, on the other hand, enters the cell soon after adsorption of phage to bacteria.”

Subsequent production of phage from the infected bacteria that contained next to no radioisotope-labelled protein, but did contain labelled DNA, showed that DNA was probably the genetic material, and that protein was not involved in phage heredity.

Aside from their ground-breaking discoveries, the main influence of the Phage Group was felt via their establishment of the yearly summer phage course at Cold Spring Harbor Laboratory. From 1945 through to the 1960s, Delbrück and colleagues taught the fundamentals of bacteriophage biology and experimentation to generations of biologists, which helped to instill a culture of rigorous mathematical and analytical techniques in attendees – many of whom went on to help establish the emerging field of molecular biology.

Indeed, not only did Delbrück, Luria and Hershey receive the 1969 Nobel Prize for Physiology or Medicine for their work on bacteriophages, but Luria’s first graduate student James Watson was also awarded the prize in 1962 for his work with Francis Crick on elucidating the structure of DNA.  It is a not particularly well known fact that Watson honed his analytical skills for 3-D reconstructions from X-ray data of DNA with data from TMV, which he helped to show had helical virions.

Animal cell culture

Possibly the most important development for the study of animal viruses since their discovery was the growing of poliovirus in cell culture: this was reported in 1949 by John Enders, Thomas Weller and Frederick Robbins from the USA, and was rewarded with a joint Nobel Prize to them in 1954.  They did this around the same time as David Bodian and Isabel Morgan identified three distinct types of poliovirus.

In the words of the Award Ceremony presentation speech,

“The use of cultures of human tissues has permitted attacks on many virus problems previously out of reach because of the lack of susceptible laboratory animals. Already at an early stage Enders, Weller and Robbins discovered agents representing a previously unknown group of viruses. Other scientists have systematically pursued this line and the answer to the question of the causes of a number of common-coldlike diseases now seems to be at hand. Weller has succeeded in cultivating the agents causing varicella and herpes zoster, Enders that of measles, viruses previously almost inaccessible for study. The method has also been successfully applied to several problems in the field of veterinary medicine.”

While both bacterial and plant viruses could be both grown and assayed in “culture” – bacterial cells for phages, and plants for viruses like TMV – it was very difficult to grow and work with animal viruses, and especially to assay them, or measure their concentration.  While the pock assay done on egg membranes for influenza virus was very useful, it was not applicable to many viruses.  Indeed, people working with animal and human viruses were envious of the advantages enjoyed by their colleagues working with bacteriophages and plant viruses, because their assay systems were far more generally useful, even if local lesion assays on leaves for plant virus were limited compared to the precision obtainable for bacteriophages using pure cultures of bacterial cells on Petri dishes.  Titration or assay of poliovirus, for example, required the injection of virus preparations into the brains of monkeys, or later, in the case of the Lansing or Type II poliovirus strain, into brains of mice.

The technological advances that led to the breakthrough were incremental, and in fact had occurred over a period of over sixty years: Wilhelm Roux is credited with creating the first “tissue culture” with animal cells, by maintaining extracts of chicken embryos in warmed saline in 1885.  Other early workers had used minced-up chick embryos as far back as the early 1900s; roller-tube cultures had been in use for some time for studying viruses; a number of human and other tissues had been used to culture viruses.  Part of the development was, however, the increased ease of making the necessary reagents, such as ultrafiltered bovine serum, and a greater understanding of the requirements of cells for successful growth in culture.  Another major enabling factor was the post-Second World War availability of antibiotics, which meant contaminating microorganisms could be killed in culture – which had been impossible previously.

Enders, Weller and Robbins started with a suspended cell culture of human embryo skin and muscle tissue – a technique first described in 1928 – with the idea of studying varicella zoster herpesvirus.  However, in a case of chance favouring the prepared mind(s), the proximity of these tissue cultures and the Lansing strain of poliovirus in the same lab led to them using this instead, as part of an effort to determine whether all polioviruses exclusively multiplied in human nervous tissue.

Their cultures were started by inoculation with a suspension of infected mouse brains, and re-inoculation of mice with tissue culture fluids demonstrated that the virus was multiplying.  Injection of fluid into monkey brains after three passages of tissue culture resulted in typical symptoms of paralysis.  Later, Types I and III poliovirus were also successfully cultured – and suspended cell cultures of intestine, liver, kidney, adrenals, brain, heart, spleen, lung and brain derived from human embryos were also found to support growth of various polioviruses.

Renato Dulbecco in 1952 adapted the technique to primary cultures of chicken embryo fibroblasts grown as monolayers in glass flasks.  Using  Western equine encephalitis virus and Newcastle disease virus of chickens, he showed for the first time that it was possible to produce plaques due to an animal virus infection, and that these could be used to accurately assay infectious virus titres.  He and Marguerite Vogt went on in 1953 to show the technique could be used to assay poliovirus – and went on to show that the principle of “one virus, one plaque” first established with phages, and later to plant viruses, could be extended to animal viruses too.

Adaptation of the culture technique to roller-tubes allowed higher yields of virus – and the possibility of direct observation of the effects of virus multiplication on large sheets of cells, rather than in clumps and pieces of tissue from suspension cultures.  These effects were termed “cytopathogenic” (now generally cytopathic) for the direct damage and morphological changes to cells that could be seen and measured, and roller-tubes made it far easier and quicker to do this by simple staining of cultures with various reagents such as haemotoxylin and eosin.

adeno haema

The technique of looking at cells for cytopathic effects (also abbreviated as CPE) quickly found application in assays of infectivity – and therefore of concentration – of poliovirus preparations.  It was also possible to do neutralisation assays with immune human sera.  There was also the observation that passaging the Lansing strain through cell suspensions reduced its virulence in mice, and similar passage of Type I poliovirus significantly reduced virulence in rhesus macaques.  These developments together were part of the advances that led to the development of live poliovirus vaccines soon afterwards.

The development of polio vaccines

Poliomyelitis – the disease caused by polioviruses – became increasingly common as population densities grew, to the point where in the the USA in 1952, there were 58 000 cases of the disease, compared to 20 000 normally – and up to 500 000 people (mainly children) died worldwide

A failed attempt at producing a vaccine in 1936 by a Maurice Brodie involved the use of ground-up infected monkey spinal cords to produce a formaldehyde-killed vaccine: Brodie tested the vaccine on three thousand children, none of whom developed immunity.  Hilary Koprowski in 1948 tested a live type II poliovirus attenuated by passage in rat brains, on himself and a colleague – with no ill effects, but no test of immunogenicity or efficacy.  In 1950 he went on to test the vaccine on 20 children in a home for the disabled, with positive results for immunogenicity.  It is claimed that Albert Sabin’s live attenuated virus (see below) was supplied by Koprowski; however, events overtook him and the other groups supplied the viruses that have been used to largely eradicate the disease, even though Koprowski went on to do huge clinical trials in Africa.

It took the development of cell culture techniques for poliovirus, the finding that there were three distinct types of the virus, as well as the proof in 1953 that immune globulins alone could protect against infection, to enable the successful development of vaccines still used today.  This is very well documented elsewhere; this account will summarise the most important features of the development.

Inactivated polio vaccines

The 1952-1953 polio epidemics in the US led to major public concern, and national efforts to develop vaccines.  Jonas Salk and his team at the University of Pittsburgh.  They produced virulent poliovirus types 1, 2 and 3 in culture in monkey kidney-derived Vero cells, and then used formaldehyde to inactivate the viruses to create an injectable vaccine.   After trials in animals proved that the “Inactivated Poliovirus Vaccine” or IPV was safely killed, it was trialled from 1954 in what was possibly the biggest medical experiment in history, involving 1.8 million children in the US.  By 1955 it was possible to announce that IPV was 60–70% effective against poliovirus type 1, and over 90% effective against types 2 and 3.  The vaccine was licenced in 1955, and immediately used in campaigns for vaccination of at-risk children.  

See heat map showing the number of cases of polio per 100 000 people across the USA.  Copyright Wall St Journal, 2015.

While the vaccine did not in fact prevent infection by the virus – which infects the gastrointestinal tract via the oral route – it prevented disease by means of eliciting a largely IgG-dependent circulating antibody response, which neutralised any virus entering the circulatory system and thus prevented viraemia and subsequent involvement of the nervous system.  This means that it is an excellent vaccine to use as an end-stage weapon in the fight against polio, as unlike the live attenuated vaccine, there is no “shedding” of live virus.

Attenuated live polio vaccines

While others (including Koprowski) were involved in attempting to develop attenuated live vaccines, it was Albert Sabin’s trivalent live attenuated vaccine that was eventually successful.  This was developed by repeated passage in animal and then cell culture, that resulted in the effective abolition of neurovirulence of all three poliovirus types – accompanied by a significant number of mutations in the viral genomes.  After a successful safety trial in institutionalised children in the US in 1954, Sabin worked closely with scientists and the authorities in the former USSR, and in particular Mikhail Chumakov, to first manufacture the vaccine, then to perform large-scale clinical trials between “…1955 and 1961, [when] the oral vaccine was tested on at least 100 million people in the USSR, parts of Eastern Europe, Singapore, Mexico, and the Netherlands”.  While the US was initially reluctant to use the vaccine, the Russian-made product was distributed worldwide, and rapidly usurped the dominance of IPV.  By 1963, however, the trivalent product was licenced in the US, and from 1962-1965 about 100 million doses were used.

Advantages of the “Oral Polio Vaccine” or OPV were that it could be given much more easily – as droplets, into the mouth – that it multiplied efficiently in the gut, meaning doses could be small, but not in nervous tissue so that it caused no disease, and that it elicited mucosal immunity that could prevent infection as well as disease.

Disadvantages of the live vaccine are that it requires a cold chain for transport, otherwise it loses infectivity; that it can be shed in stools by vaccinees, meaning that uncontrolled community spread is possible.  This can result in vaccine-associated paralytic poliomyelitis, either due to reversion of the vaccines to virulence by mutation, or more rarely because of immune deficiencies  in those exposed.  Because of this, and the possibility of persistence of vaccine strains in populations even in the absence of overt disease, the final stages of poliovirus eradication probably require use of IPV in areas where there is no longer any endemic wild-type poliovirus.

While there have been serious concerns about contamination of poliovirus vaccines with live SV40 virus – a known tumour-causing agent – the consensus opinion appears to be that there is no danger.

The rapid development of human virology

These observations also quickly found application with a wide variety of other human and animal viruses, which triggered an explosion in these fields that led to them rapidly overtaking plant and bacterial virology in terms of understanding how the viruses replicated, and developing assays and vaccines for them.  Indeed, the poliovirus work was rapidly followed in the same lab by the isolation of herpes zoster and herpes simplex viruses; the agent of measles was characterised by Thomas Peebles and Enders via tissue culture by 1954; adenoviruses were discovered in 1953 by Wallace Rowe and Robert Huebner and shown to be associated with acute respiratory disease soon afterwards, by Maurice Hilleman and others.

Click here for Part 1: Filters and Discovery

here for Part 2: The Ultracentrifuge, Eggs and Flu

and here for Part 4: RNA Genomes and Modern Virology

Copyright Edward P Rybicki and Russell Kightley, February 2015, except where otherwise noted.


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