Archive for the ‘biotechnology’ Category

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-)

www_sciencedirect_com_science__ob_PdfExcerptURL__imagekey_1-s2_0-S0140673601203833-main_pdf__piikey_S0140673601203833__cdi_271074__orig_article__zone_centerpane__fmt_abst__eid_1-s2_0-S0140673601203833__user_635696_md5_610868932f41b70483e225

 

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 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.

Ebola virus mutating, scientists say

29 January, 2015

Scientists at the Institut Pasteur in France who are tracking the Ebola outbreak in Guinea say the virus has mutated.

Source: www.bbc.com

I would be surprised it there weren’t evidence by now of adaptation to humans: never in any previous outbreak of EHD [Ebola haemorrhagic disease] has the person-person chain of transmission been sustained for so long, meaning never before has there been the opportunity for human-specific adaptations to become established.

The article points out that on consequence of mutation may be that the virus becomes less virulent, leading to a greater incidence of asymptomatic infection – of which there is already evidence from previous outbreaks, and which has been implicated in the lessening incidence of transmission because of increasing herd immunity.

However, this same property might lead to increased transmission to the non-exposed, because of a lack of signs that contacts with the infected person(s) should be avoided – and for a disease as lethal as EHD, even a reduced mortality rate still means you should avoid it at all costs.

The idea of developing a modified live measles virus vaccine as an Ebola virus vaccine vector, which is what the Institut Pasteur is apparently doing, seems to be a very good one.  Measles is still a major potential problem in that part of the world, necessitating regular infant immunisations, and coupling anti-measles with an anti-Ebola vaccine in those countries is probably very good use of both a proven vaccine and existing EPI infrastructure.

 

See on Scoop.itVirology News

More Surprises in the Development of an HIV Vaccine

14 November, 2014

More Surprises in the Development of an HIV Vaccine

In the current issue of Frontiers in Immunology, Jean-Marie Andrieu and collaborators, report results from non-human primate experiments designed to explore a new vaccine concept aimed at inducing tolerance to the simian immunodeficiency virus (SIV) (1). This approach, which is significantly different from other vaccine concepts tested to date, resulted in a surprisingly high level of protection. If the results are confirmed and extended to the human immunodeficiency virus (HIV), this approach may represent a game changing strategy, which should be welcomed by a field that has been marred by mostly disappointing results.

 

HIV Graphic from Russell Kightley Media

 

Source: journal.frontiersin.org

This is a commentary by two well-respected friends of mine on a very surprising result published by the Andrieu group recently, which seems to have been ignored by the mainstream HIV vaccine world.

This is not surprising, in that Andrieu is an outsider in this field – he is a cancer researcher – but is typical of the disappointing tendency in science to ignore contributions from outside the various "Golden Circles" that exist for various specialties.

Something that should elicit interest, though, is that this group has shown that a previously obscure 

"…population of non-cytolytic MHCIb/E-restricted CD8+ T regulatory cells [that] suppressed the activation of SIV positive CD4+ T-lymphocytes".

This is interesting because Louis Picker’s groups’ recent findings, announced at the recent HIVR4P conference in Cape Town, highlighted the involvement of MHC-E proteins in what amounted to a cure of SIV infection in macaques by a modified Rhesus cytomegalovirus (RhCMV) HIV vaccine vector (see here: http://www.iavireport.org/Blog/archive/2013/09/13/cmv-based-vaccine-can-clear-siv-infection-in-macaques.aspx). 

I tweeted at the time:

"Universal MHC-E-restricted CD8+ T cells – break all the rules for epitope recognition"

Could this be a link between the two mechanisms – both from way outside the orthodoxy, I will point out?

It will be interesting to see.

See on Scoop.itVirology News


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