Posts Tagged ‘rinderpest’

Moratorium on using live rinderpest virus lifted for approved research

30 July, 2013

See on Scoop.itVirology News

Benefits of future research should be carefully balanced against potential risks

Paris, 10 July 2013 – A moratorium on using live rinderpest virus for approved research has been lifted by the Food and Agriculture Organization of the United Nations and the World Organisation for Animal Health (OIE).

The moratorium followed the adoption of a Resolution in May 2011 by all OIE Member Countries that urged members to forbid the manipulation of rinderpest virus containing material unless approved by the Veterinary Authority and by FAO and OIE.

The two organizations have now put in place strict criteria and procedures to follow in order to obtain official approval for any research proposals using rinderpest virus and rinderpest virus-containing materials. One of the most crucial requirements is that the research should have significant potential to improve food security by reducing the risk of a reoccurrence of the disease. This procedure replaces an earlier complete ban on handling the virus.

Rinderpest was formally declared eradicated in 2011, but stocks of rinderpest virus continue to exist in laboratories. In June 2012, a moratorium on handling the virus was imposed after an FAO-OIE survey found that the virus continues to be held in more than 40 laboratories worldwide, in some cases under inadequate levels of biosecurity and biosafety.

When rinderpest was officially eradicated, FAO and OIE member countries committed themselves to forbid the manipulation of rinderpest virus-containing material unless approved by the national veterinary authority as well as by FAO and OIE.

Paramyxovirus EM courtesy of Linda Stannard

Thanks to Len Bracher for alerting me to this.

Ed Rybicki‘s insight:

This is an interesting sequel to the eradication of wild rinderpest virus, which I have covered in some detail here on ViroBlogy: see here ( and here (

The article covers an interesting prospect: that it may be possible to use attenuated, safe vaccines against the related peste des petits ruminants virus (PPRV) not only to protect against any resurgence of rinderpest, but also to eradicate this rather nasty virus.

Which is, apparently, spreading at rather an alarming rate, and is an obstacle to small ruminant production (

So maybe this is “Now for Number 4!” time.

See on

A Short History of the Discovery of Viruses – Part 2

7 February, 2012

The Ultracentrifuge, Eggs and Flu

The ultracentrifuge

A technical development that was to greatly advance the study of viruses was begun in 1923, but only reached fruition by the 1930s: this was the ultracentrifuge, invented and developed first by Theodor (“The”) Svedberg in Sweden as a purely analytical tool, and later by JW Beams and EG Pickels in the USA as an analytical and preparative tool.  The ultracentrifuge revolutionised first, the physical analysis of proteins in solution, and second, the purification of proteins, viruses and cell components, by allowing centrifugation at speeds high enough to allow pelleting of subcellular fractions.

Analytical centrifugation and calculation of molecular weights of particles gave some of the first firm evidence that certain proteins, and virus particles, were large, regular objects.  Indeed, it came to be taken as a given that one of the fundamental properties of a virus particle was its sedimentation coefficient, measured in svedbergs (a unit of 10-13 seconds, shown as S20,W).  This is also how ribosomes of pro- and eukaryotes came to be named: these are known as 70S (prokaryote) and 80S ribosomes, respectively, based on their different sedimentation rates.

The Official Discovery of Influenza Virus

In 1931, Robert Shope in the USA managed to recreate swine influenza by intranasal administration of filtered secretions from infected pigs.  Moreover, he showed that the classic severe disease required co-inoculation with a bacterium – Haemophilus influenza suis – originally thought to be the only agent.  He also pointed out the similarities between the swine disease and the Spanish Flu, where most patients died of secondary infections.  However, he also suggested that the virus survived seasonally in a cycle involving the pig, lungworms, and the earthworm, which is now known to be completely wrong.

This notwithstanding, he found that people who had survived infection during the 1918 pandemic had antibodies protecting them against the swine flu virus, while people born after 1920 did not, which showed that the 1918 human and swine flu viruses were very similar if not identical. This was a very relevant discovery for what happened much later, in the 2009 influenza pandemic, when the same virus apparently came back into the human population from pigs after circulating in them continuously since 1918.

Shope went on in 1932 to discover, with Peyton Rous, what was first called the Shope papillomavirus and later Cottontail rabbit papillomavirus: this causes benign cancers in the form of long hornlike growths on the head and face of the animal. This may explain the sightings in the US Southwest of the near-mythical “jackalope”.

Influenza viruses in pigs

Influenza viruses in pigs

Patrick Laidlaw and William Dunkin, working in the UK at the National Institute for Medical Research (NIMR), had by 1929 successfully characterised the agent of canine distemper – a relative of measles, mumps and distemper morbilliviruses – as a virus, proved it infected dogs and ferrets, and in 1931 got a vaccine into production that protected dogs.  This was made from chemically inactivated filtered tissue extract from infected animals.  Their work built on and completely eclipsed earlier findings, such as those of Henri Carré in France in 1905, who first claimed to have shown it was a filterable agent, and Vittorio Puntoni, who first made a vaccine in Italy from virus-infected brain tissue inactivated with formalin in 1923.

Influenza and Ferrets: the Early Days

Continuing from Laidlaw and Dunkin’s work in the same institute, Christopher Andrewes, Laidlaw and W Smith reported in 1933 that they had isolated a virus from humans infected with influenza from an epidemic then raging.  They had done this by infecting ferrets with filtered extracts from infected humans – after the fortuitous observation that ferrets could apparently catch influenza from infected investigators!  The “ferret model” was very valuable – see here for modern use of ferrets – as strains of influenza virus could be clinically distinguished from one another.

Eggs and Flu and Yellow Fever

Influenza virus and eggs: large-scale culture

Frank Macfarlane Burnet from Australia visited the NIMR in the early 1930s, and learned a number of techniques he used to great effect later on.  Principal among these was the technique of embryonated egg culture of viruses – which he took back to Melbourne, and applied to the infectious laryngotracheitis virus of chickens in 1936.  This is a herpesvirus, first cultivated by JR Beach in the USA in 1932: Burnet used it to demonstrate that it was possible to do “pock assays” on chorioallantoic membranes that were very similar to the plaque assays done for bacteriophages, with which he was also very familiar.  Also in 1936, Burnet started a series of experiments on culturing human influenza virus in eggs: he quickly showed that it was possible to do pock assays for influenza virus, and that

“It can probably be claimed that, excluding the bacteriophages, egg passage influenza virus can be titrated with greater accuracy than any other virus.”

Max Theiler and colleagues in the USA took advantage of the new method of egg culture to adapt the French strain of yellow fever virus (YFV) he had grown in mouse brains to being grown in chick embryos, and showed that he could attenuate the already weakened strain even further – but it remained “neurovirulent”, as it caused encephalitis or brain inflammation in monkeys.  He then adapted the first YFV characterised – the Asibi strain, from Ghana in 1927 – to being grown in minced chicken embryos lacking a spinal cord and brain, and showed in 1937 that after more than 89 passages, the virus was no longer “neurotrophic”, and did not cause encephalitis.   The new 17D strain of YFV was successfully tested in clinical trials in Brazil in 1938 under the auspices of the Rockefeller Foundation, which has supported YFV work since the 1920s.  The strain remains in use today, and is still made in eggs.

Virus purification and the physicochemical era

Given that the nature of viruses had prompted people to think of them as “chemical matter”, researchers had attempted from early days to isolate, purify and characterise the infectious agents.  An early achievement was the purification of a poxvirus in 1922 by FO MacCallum and EH Oppenheimer. 

Much early work was done with bacteriophages and plant viruses, as these were far easier to purify or extract at the concentrations required for analysis, than animal or especially human viruses. 

CG Vinson and AM Petre, working with the infectious agent causing mosaic disease in tobacco – tobacco mosaic virus, or TMV – showed in 1931 that they could precipitate the virus from suspension as if it were an enzyme, and that infectivity of the precipitated preparation was preserved.  Indeed, in their words:

“…it is probable that the virus which we have investigated reacted as a chemical substance”.

Viruses in Crystal

An important set of discoveries started in 1935, when Wendell Stanley in the USA published the first proof that TMV could be crystallised, at the time the most stringent way of purifying molecules.  He also reported that the “protein crystals” were contaminated with small amounts of phosphorus.  An important finding too, using physical techniques including ultracentrifugation and later, electron microscopy, was that the TMV “protein” had a very high molecular weight, and was in fact composed of large, regular particles.  This was a very significant discovery, as it indicated that some viruses at least really were very simple infectious agents indeed.

TMV particle: 95% protein, 5% RNA

However, his conclusion that TMV was composed only of protein was soon challenged, when Norman Pirie and Frederick Bawden working in the UK showed in 1937 that ribonucleic acid (RNA) – which consists of ribose sugar molecules linked by phosphate groups – could be isolated consistently from crystallised TMV as well as from a number of other plant viruses, which accounted for the phosphorus “contamination”.  This resulted in the realisation that TMV and other plant virus particles – now known to be virions – were in fact nucleoproteins, or protein associated with nucleic acid.

Stanley received a share of the Nobel Prize in Chemistry in 1946 for his work on TMV: it is instructive to read his acceptance speech from the time to realise what the state of the science that was becoming virology was at the time.  He wrote:

“Since the original discovery of this infectious, disease-producing agent, known as tobacco mosaic virus, well over three hundred different viruses capable of causing disease in man, animals and plants have been discovered. Among the virus-induced diseases of man are smallpox, yellow fever, dengue fever, poliomyelitis, certain types of encephalitis, measles, mumps, influenza, virus pneumonia and the common cold. Virus diseases of animals include hog cholera, cattle plague, foot-and-mouth disease of cattle, swamp fever of horses, equine encephalitis, rabies, fowl pox, Newcastle disease of chickens, fowl paralysis, and certain benign as well as malignant tumors of rabbits and mice. Plant virus diseases include tobacco mosaic, peach yellows, aster yellows, potato yellow dwarf, alfalfa mosaic, curly top of sugar beets, tomato spotted wilt, tomato bushy stunt, corn mosaic, cucumber mosaic, and sugar cane yellow stripe. Bacteriophages, which are agents capable of causing the lysis of bacteria, are now regarded as viruses”.

Two of the most interesting things about the article, however, are the electron micrographs of virus particles – Stanley had one of the first electron micrsoscopes available at the time -  and the table of sizes of viruses, proteins and cells that had been determined by then by techniques such as ultracentrifugation and filtration: TMV was known to be rodlike, 15 x 280 nm; vaccinia was 210 x 260 nm; poliomyelitis was 25 nm; phages like T2 were known to have a head-and-tail structure.

Seeing is Believing: the Electron Microscope

First Electron Microscope with Resolving Power Higher than that of a Light Microscope. Ernst Ruska, Berlin 1933 Wikipedia CC BY-SA 3.0,

First Electron Microscope with Resolving Power Higher than that of a Light Microscope. Ernst Ruska, Berlin 1933
Wikipedia CC BY-SA 3.0,

The development of the electron microscope, in Germany in the 1930s, represented a revolution in the investigation of virus structures: while virions of viruses like variola and vaccinia could just about be seen by light microscopy – and had been, as early as 1887 by John Buist and others – most viruses were far too small to be visualised in this way. 

While Ernst Ruska received a Nobel Prize in 1986 for developing the electron microscope, it was his brother Helmut who first imaged virus particles – using beams of electrons deflected off virus particles coated in heavy metal atoms.  From 1938 through the early 1940s, using his “supermicroscope”, he imaged virions of poxviruses, TMV, varicella-zoster herpesvirus, and bacteriophages, and showed that they were all particulate – that is, they consisted of regular and sometimes complex particles, and were often very different from one another.  He even proposed in 1943 a system of viral classification on the basis of their perceived structure.

While electron microscopy was also used medically to some extent thereafter – for example, in differentiating smallpox from chickenpox by imaging particles of variola virus and varicella-zoster virus, respectively, derived from patients’ vesicles – its use was limited by the expense and cumbersome nature of sample preparation. For example, the micrographs in Stanley’s 1946 paper were all done with samples “…prepared with gold by the shadow-casting technique”.

The use of the cumbersome technique of metal shadow-casting, and the highly inconvenient nature of electron microscopy as a routine tool all changed from 1959 onwards, when Sydney Brenner and Robert Horne published “A negative staining method for high resolution electron microscopy of viruses”.  This method involves the use of viruses in liquid samples deposited on carbon-coated metal grids, and then stained with heavy-metal salts such as phosphotungstic acid (PTA) or uranyl acetate.

This simple technique revolutionised the field of electron microscopy, and within just a few years much information was acquired about the architecture of virus particles. Not only were the overall shapes of particles revealed, but also the details of the symmetrical arrangement of their components. Some beautiful examples can be seen here, at the Cold Spring Harbor site.

Depiction of the effects of using a heavy metal salt solution to negatively stain particles on a carbon film. The stain (dark) pools around the particles (light).  Human rotavirus particles, stained from below (left) and by immersion (right).
Images copyright LM Stannard

Depiction of the effects of using a heavy metal salt solution to negatively stain particles on a carbon film. The stain (dark) pools around the particles (light). Human rotavirus particles, stained from below (left) and by immersion (right).
Images copyright LM Stannard

Click here for Part 1: Filters and Discovery

here for Part 3: Phages, Cell Culture and Polio

and here for Part 4: RNA Genomes and Modern Virology

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

A Short History of the Discovery of Viruses – Part 1

6 February, 2012

The following text may or may not appear in a book of some kind in the future.  However, I thought I may as well share it – both for general education purposes, as well as for comment.  Enjoy!

A Short History of the Discovery of Viruses

While people were aware of diseases of both humans and animals now known to be caused by viruses many hundreds of years ago, the concept of a virus as a distinct entity dates back only to the very late 1800s.  Although the term had been used for many years previously to describe disease agents, the word “virus” comes from a Latin word simply meaning “slimy fluid”.

Porcelain filters and the discovery of viruses

The invention that allowed viruses to be discovered at all was the Chamberland-Pasteur filter.  This was developed in 1884 in Paris by Charles Chamberland, who worked with Louis Pasteur.  It consisted of unglazed porcelain “candles”, with pore sizes of 0.1 – 1 micron (100 – 1000 nm), which could be used to completely remove all bacteria or other cells known at the time from a liquid suspension.  Though this simple invention essentially enabled the establishment of a whole new science – virology – the continued development of the discipline required a string of technical developments, which I will highlight as appropriate.

Pasteur Germ Proof Filter, c. 1890, Pasteur-Chamberland Filter Co., Dayton, Ohio – Museum of Science and Industry (Chicago)


As the first in what was to be an interesting succession of events, Adolf Eduard Mayer from Germany, publishing in 1886 on work done in Holland from 1879, showed that the “mosaic disease” of tobacco – or “mozaïkziekte”, as he named it in his paper – could be transmitted to other plants by rubbing a liquid extract, filtered through paper, from an infected plant onto the leaves of a healthy plant.  However, he came to the erroneous conclusion that it must be a bacterial disease.

The first use of porcelain filters to characterize what we now know to be a virus was reported by Dmitri Ivanovski in St Petersburg in Russia, in 1892.  He had used a filter candle on an infectious extract of tobacco plants with mosaic disease, and shown that it remained infectious: however, he concluded the agent was probably a toxin as it appeared to be soluble.

The Dutch scientist Martinus Beijerinck in 1898 reported similar experiments with bacteria-free filtered extracts, but made the conceptual leap and described the agent of mosaic disease of tobacco as a “contagium vivum fluidum”, or contagious living fluid, because he was convinced the infectious agent had a liquid nature. The extract was completely sterile, could be kept for years, but remained infectious.  The term virus was later used to describe such fluids, also called “filterable agents”, which were thought to contain no particles.  The virus causing mosaic disease is now known as Tobacco mosaic virus (TMV).  A paper commemorating Ivanovsky and Beijerinck’s work – “One Hundred Years of Virology” – was published in Journal of Virology 1992 to honour both pioneers.

The first animal viruses

The second virus discovered was what is now known as Foot and mouth disease virus (FMDV) of farm and other animals, in 1898 by the German scientists Friedrich Loeffler and Paul Frosch.  Again, their “sterile” filtered liquid proved infectious in calves, providing the first proof of viruses infecting animals – a fact commemorated by an article in 1998 in the Journal of General Virology.  Indeed, some believe that the true discoverers of viruses were these two scientists, as they concluded the infectious agent was a tiny particle, and was not a liquid agent.  The two went further by showing that it was possible to vaccinate cows and sheep against the disease using filtered vesicle extract that had been heated sufficiently to destroy its infectivity: this was possibly the first use of an inactivated virus as a prophylactic vaccine.

In 1898 G Sanarelli, working in Uruguay, described the smallpox virus relative and tumour-causing myxoma virus of rabbits as a virus – but on the basis of sterilisation by centrifugation rather than by filtration.

The first human virus: yellow fever

The first human virus described was the agent which causes yellow fever: this probably originated in Africa, but was spread along with its mosquito vector Aedes aegyptii to the Americas and neighbouring islands by the slave trade.  Indeed, the  declaration of independence from France by Haiti in 1804 was made possible in part by the devastating effect of the disease on the French army sent to quell a slave revolt there. The virus was discovered and reported in 1901 by the US Army physician Walter Reed, after pioneering work in Cuba by Carlos Finlay reported in 1881 hypothesising that mosquitoes transmitted the deadly disease

The agent became the subject of intense study because, in the Spanish-American war in Cuba in the 1890s, about 13 times as many soldiers died of yellow fever as died from wounds. The subsequent eradication of mosquitoes in Panama is probably what allowed the completion of the Panama Canal – stalled because of the death rate among workers from yellow fever and malaria.

Rinderpest and rabies

The paper describing rinderpest as a virus disease

A finding that was later to have great importance in veterinary virology was the discovery by Maurice Nicolle and Adil Mustafa (also known as Adil-Bey), in Turkey in 1902, that rinderpest or cattle plague was caused by a virus.  This had been for several centuries the worst animal disease known worldwide in terms of mortality: for example, an epizootic or animal epidemic in Africa in the 1890s that had started in what is now Ethiopia in 1887 from cattle imported from Asia, had spread throughout the continent by 1897, and killed 80-90% of the cattle and a large proportion of susceptible wild animals in southern Africa.  Many thousands of people died of starvation as a result.  The virus is, incidentally, only the second to have ever been eradicatednearly 100 years after its discovery.

Viruses and Vaccines

Sir Arnold Theiler, a Swiss-born veterinarian working in South Africa, had been appointed as state veterinarian for the Zuid-Afrikaansche Republiek prior to 1899, on the strength of his having produced a smallpox vaccine for miners in the Johannesburg area.  He then developed a crude vaccine against rinderpest by 1897, without knowledge of the nature of the agent: this consisted of blood from an infected animal, injected with serum from one that had recovered – something also shown to work with FMDV by Loeffler and Frosch.  This risky mixture worked well enough, however, to eradicate the disease in the region.  He went on to do the same thing successfully for African horsesickness virus and other disease agents, in an institute (Onderstepoort Veterinary Institute) that still works on the virus.

The description in Annales de l’Institut Pasteur by Remlinger and Riffat-Bay in 1903 of the agent of rabies as a “filterable virus was the culmination of many years of distinguished work in France on the virus, started by Louis Pasteur himself. While Remlinger credited Pasteur with having the notion in 1881 that rabies virus was an ultramicroscopic particle, the fact is that Pasteur and Emile Roux had also, in 1885, effectively made a vaccine against rabies by use of dried infected rabbit spinal cords, without any knowledge of what the agent was.

Title page of the original article in Annales de l’Institut Pasteur Volume 17 of 1903

It is interesting that the same volume of the Annales which reported the rabies agent also has a discussion on whether or not the smallpox agent variola virus and the vaccine against it, vaccinia virus, were differently-adapted variants of the same thing, or were different viruses.

More and more viruses

The viral nature of many disease agents started to be made evident around this time, as more researchers started investigating known diseases.  In 1904, E Baur in Germany described an infectious variegation of Abutilon that could only be transmitted by grafting, that was not associated with visible bacteria.  This is now known to be due to Abutilon mosaic virus, now known to be a single-stranded DNA geminivirus.

Abutilon mosaic

Incidentally, the earliest recorded description of a plant disease was probably in a poem in 752 CE by the Japanese Empress Koken, describing symptoms in eupatorium plants.  It was shown in 2003 that the striking yellow-vein symptoms were caused by a geminivirus infection.

Interestingly, also in 1906, A Zimmermann proposed – in a paper entitled “Die Krauselkrankheit des Maniok” – that the agent of mosaic disease of cassava that had first been described from German East Africa (now Tanzania) in 1894, was a filterable virus.  This was the second geminivirus discovered, although this was only proved in the 1970s.

Cassava affected by a recombinant African cassava mosaic virus in western Kenya, 1997

Cassava affected by a recombinant African cassava mosaic virus in western Kenya, 1997.  Insets, from left: healthy cassava, mild disease, severe disease

In 1906, Adelchi Negri – who had previously discovered the Negri bodies in cells infected with rabies virus – showed that vaccinia virus, the vaccine for the dreaded smallpox caused by variola virus, was filterable.  This was the final step in a long series of discoveries around smallpox, that started with Edward Jenner’s use of what was supposedly cowpox, but may have been horsepox virus to protect people from the disease in 1796.

An Egyptian stele thought to represent a polio victim (1403–1365 BC).  Note the characteristic withering of one leg.

An Egyptian stele thought to represent a polio victim (1403–1365 BC). Note the characteristic withering of one leg.

The disease now known as poliomyelitis was first clinically described in England in 1789, as “a debility of the lower extremities”.  However, it had been known since ancient times, and had even been depicted clearly in an Egyptian painting from over 3000 years ago.

An important development in human virology in 1908, therefore, was the finding by Karl Landsteiner and Erwin Popper in Germany that poliomyelitis or infantile paralysis in humans as it was known then, was caused by a virus: they proved this by injecting a cell-free extract of a suspension of spinal cord from a child who had died of the disease, into monkeys, and showing that they developed symptoms of the disease.

Viruses and cancer

In 1908, Oluf Bang and Vilhelm Ellerman in Denmark were the first to associate a virus with leukaemia: they successfully used a cell-free filtrate from chickens with avian leukosis to transmit the disease to healthy chickens.

The first solid tumour-causing virus, or virus associated with cancer, was found by Peyton Rous in the USA in 1911.  He showed that chicken sarcomas, or solid connective tissue tumours, could be transmitted by grafting, but also that a filterable or cell-free agent extracted from a sarcoma was infectious.  The virus was named for him as Rous sarcoma virus, and is now known to be a “retrovirus”,as is chicken leukaemia virus,in the same virus family as HIV.

Eaters of Bacteria: The Phages

"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 -

“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 –

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.  Although he showed that it could pass through porcelain filters, and could be transmitted to other colonies of the same bacteria, he was not sure whether or not it was a virus, and referred to it as “the bacteriolytic agent”.  It is interesting that he was actually attempting to grow vaccinia virus in culture, and that it was a contaminating staphylococcus that he noticed was being lysed by his infectious agent.


The original article published by Twort in The Lancet in 1915

Subsequently, Félix d’Hérelle in Paris published in 1917 that he had discovered a virus that lysed a bacterial agent he was culturing in liquid broth – a Shigella – that caused human dysentery, or diarrhoea.  He named the virus “bacteriophage”, or eater of bacteria, derived from the Greek term “phagein”, meaning to eat.  He showed a number of interesting properties of his shigella-specific bacteriophages, including that they could be adapted to other Shigella species or types by passaging them repeatedly, and that they protected rabbits against infection by lethal doses of bacteria

D’Hérelle’s main interest in his new discovery was in using them as a therapeutic agent for bacterial infections in humans: sadly, this idea did not take off in Europe or the Americas, largely due to the unreliability of the ill-understood phage preparations, although it was extensively exploited in the former USSR. Indeed, he mentored George Eliava who went on to found the Eliava Institute in Tbilisi, Georgia, which became a major centre for the use of bacteriophage cocktails against persistent bacterial infections in humans.  A review on phage therapy from the Institute was recently published to mark the centenary of Twort’s discovery in 1915.

D‘Hérelle’s main interest in his new discovery was in using them as a therapeutic agent for bacterial infections in humans: sadly, this idea did not take off in Europe or the Americas, although it was extensively exploited in the former USSR.

The 1896 paper from Annales de l’Institut Pasteur

Interestingly, and as reported in ViroBlogy previously, what could have been the first discovery of phages was probably described by Ernest Hankin, who had previously proved in India that cholera was caused by bacteria.  In 1896 in Annales de l’Institut Pasteur, he documented that river water downstream of cholera-infested towns on the Jumma river in India contained no viable Cholera vibrio – and that this was a reliable property of the water, and was probably responsible for limiting the spread of cholera

While he did not prove the presence of a “filterable agent”, he was recognised by d’Hérelle and others as having contributed to the discovery of bacteriophages.  In fact, d’Hérelle went to India in 1927, and put cholera phage preparations into wells in villages with cholera patients: apparently the death toll went down from 60% to 8%.

Influenza A viruses in waterbirds – Russell Kightley Media

The virus as human plague: the Spanish Flu

Possibly the worst human plague the world has ever seen swept across the planet between 1918 and 1922: this was known as the Spanish Flu, from where it was first properly reported, and it went on to kill more than 50 million people all over the world.  We now know it to have been H1N1 influenza type A: modern reconstruction of the virus from archived tissue samples and frozen bodies found in permafrost has shown it probably jumped directly into humans from birds.

Most medical authorities at the time thought the disease was caused by bacteria – however, MJ Dujarric de la Rivière, and Charles Nicolle – brother of Maurice – and Charles  Lebailly in France, separately proposed in 1918 that the causative agent was a virus, based on properties of infectious extracts from diseased patients.  Specifically, they found that the infectious agent derived from bronchial expectoration of an infected person was filterable, caused disease in monkeys via nasal administration and human volunteers via subcutaneous injection, and was not present in the blood of an infected monkey.  However, many scientists at the time still doubted that influenza was a viral disease – despite this contemporary comment in the British Medical Journal of 1918.

Conclusions from the Nicolle and Lebailly paper

Translation of this passage (courtesy of Mrs Francoise Williamson):


1⁰ The bronchial  expectoration of people suffering from flu, collected during the acute period, is virulent.
2⁰   The monkey (M. cynomolgus)  is sensitive to the virus  by sub-conjunctival and nasal inoculation.
3⁰   The flu agent is a filterable organism.  The inoculation  of the filtrate has indeed reproduced the illness in two of the people injected subcutaneously;  on the other hand when given intravenously it  appears to be ineffective. (two failures out of two tries).
4⁰ It is possible that the influenza virus does not occur in the patient’s blood.  The blood of a monkey with influenza, inoculated subcutaneously, did not infect man;  the negative blood result of subject 2 at D, is however, not convincing, the blood route seeming to be ineffective for  the flu virus transmission.”

Other agents of other diseases were found to be “filterable viruses” in the 1920s, including yellow fever virus by Adrian Stokes in 1927, in Ghana.  Indeed, the US bacteriologist and virologist Thomas Rivers in 1926 counted some sixty-five disease agents that had been identified as viruses.

Virus Assays: Counting the Viruses in the 1920s

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.  In fact, this later led to the birth of molecular biology, as described here

However, the beauty of working with phages was that they could be assayed – or counted in terms of infectious units – so easily, either by the plaque technique or by infections of liquid cultures.  This was not true of viruses of plants or of animals in the absence of similar culture techniques; these could only be assayed in a much more crude method using whole organisms.  One such method was by determining infection endpoints by serial dilution of inoculum, such as the now-famous ID50, or dose infecting 50% of the experimental subjects.

This changed in 1929 for plant viruses, with the demonstration by the plant virus pioneer FO Holmes that local lesions caused by infection of particular types of tobacco by TMV could be used as a means of assaying the infectivity of virus stocks.  This was then extended to other virus/host combinations, and allowed the rapid and quantitative assay of virus stocks – which, as it had done for phages, allowed the study of the properties of plant viruses, and led to their biological isolation and then purification.

TMV-induced local lesions in N. tabacum cv. glutinosa

TMV-induced local lesions in N. tabacum cv. glutinosa

Eggs and animals for virus culture

Chicken eggs for virus growth and assay

Possibly the next most important methodological development in virology after the discovery of phages was the proof that embryonated or fertilized hen’s eggs could be used to culture a variety of important animal and human viruses.  Ernest Goodpasture, working at Vanderbilt University in the USA, showed in 1931 that it was possible to grow fowlpox virus – a relative of smallpox – by inoculating the chorioallantoic membrane of eggs, and incubating them further. 


While tissue culture had in fact been practiced for some time – for example, as early as the 1900s, investigators had grown “vaccine virus” or the smallpox vaccine now called vaccinia virus in minced up chicken embryos suspended in chicken serum – this technique represented a far cheaper and much more “scalable” technique for growing pox- and other suitable viruses.

An important first in a chain of related discoveries was the one by Howard Andervost, at Harvard University in 1929, who showed that human herpes simplex virus could be cultured by injection into the brains of live mice.

This led to the demonstration in 1930 by the South African-born Max Theiler – son of Sir Arnold – also at Harvard, that yellow fever virus could be similarly cultured: this allowed much easier handling of the virus, which until then had to be injected into monkeys in order to multiply it in their livers.  In addition, it allowed the development of attenuated or weakened strains of virus, by him and in parallel by a French laboratory, by serial passage or repeated transmission of the virus between mice. He also incidentally caught yellow fever from one of his mice through a laboratory accident. Culturing in mouse brains also allowed the successful animal testing of vaccine candidates, and of protective antisera, for which Theiler was awarded the Nobel Prize in 1951.  

Until 2008, this was the first and only recognition of virus vaccine work by the Nobel Foundation.

A consequence of this work was the landmark in medical virology that was the development of human vaccines against yellow fever virus, by Wilbur Sawyer in the USA in 1931: this followed on Theiler’s mouse work in using brain-cultured virus plus human immune serum from recovered patients to immunize humans – very similar to Theiler Senior’s strategy with rinderpest, more than thirty years later.

here for Part 2: The Ultracentrifuge, Eggs and Flu

here for Part 3: Phages, Cell Culture and Polio

and here for Part 4: RNA Genomes and Modern Virology

Copyright Edward P Rybicki and Russell Kightley, February 2012, except where otherwise noted.

Deliberate extinction: now for Number 3

3 August, 2011

I have written previously about the Rinderpest virus eradication campaign – and now it appears as though the final nail has in fact been hammered into the coffin’s lid while I wasn’t looking.  I thank my son Steven for noticing!

It was officially announced on 26th June, in Rome – the headquarters of the Food and Agriculture Organisation (FAO) – that “…for only the second time in history, a disease has been wiped off the face of the earth”.

From a New York Times article:

The long but little-known campaign to conquer rinderpest is a tribute to the skill and bravery of “big animal” veterinarians, who fought the disease in remote and sometimes war-torn areas — across arid stretches of Africa bigger than Europe, in the Arabian desert and on the Mongolian steppes.

The victory is also proof that the conquest of smallpox was not just an unrepeatable fluke, a golden medical moment that will never be seen again. Since it was declared eradicated in 1980, several other diseases — like polio, Guinea worm, river blindness, elephantiasis, measles and iodine deficiency — have frustrated intensive, costly efforts to do the same to them. The eradication of rinderpest shows what can be done when field commanders combine scientific advances and new tactics.

… The modern eradication campaign began in 1945, when the Food and Agriculture Organization was founded. But it became feasible only as vaccines improved. An 1893 version made from the bile of convalescent animals was replaced by vaccines grown in goats and rabbits and finally in laboratory cell lines; a heat-stable version was developed in the 1980s.

The interesting thing about Rinderpest virus is that it is probably a consequence of human’s development of agriculture and especially the keeping of livestock, that got it into animals in the first place: it is closely enough related to measles virus that it probably only diverged from it some time around CE 1000.

So it’s not just us that get animal viruses – our pets and our livestock can get them from us, too.

But it’s now time to concentrate on the next two: polioviruses and measles.  Vaccinate, brothers and sisters, vaccinate!!

Rinderpest: gone, but not forgotten – yet.

5 November, 2010

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 MicrobiologyBytes).

Electron micrograph of a morbillivirus particle showing the membrane, matrix, and inner helical nucleocapsid. Image by LM Stannard

The ICTVdB generic description of morbilliviruses is as follows:

Virions consist of an envelope and a nucleocapsid. Virus capsid is enveloped. Virions are spherical to pleomorphic; filamentous and other forms are common. Virions measure (60-)150-250(-300) nm in diameter; 1000-10000 nm in length. Surface projections are distinctive spikes of haemagglutinin (H) and fusion (F) glycoproteins covering evenly the surface. Surface projections are 9-15 nm long; spaced 7-10 nm apart. Capsid/nucleocapsid is elongated with helical symmetry. The nucleocapsid is filamentous with a length of 600-800(-1000) nm and a width of 18 nm. Pitch of helix is 5.5 nm.

The Mr of the genome constitutes 0.5% of the virion by weight. The genome is not segmented and contains a single molecule of linear negative-sense, single-stranded RNA. Virions may also contain occasionally a positive sense single-stranded copy of the genome (thus, partial self-annealing of extracted RNA may occur). The complete genome is 15200-15900 nucleotides long.

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.”

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.

With very little fanfare, I might point out: as a practicing teaching virologist, I was totally unaware of it.  Anyway: they state that:

Rinderpest has been a dreaded cattle disease for millennia, causing massive losses to livestock and wildlife on three continents. This deadly cattle plague triggered several famines and caused the loss of draught animal power in agricultural communities in the 18th, 19th and 20th centuries.

…which is a little of an understatement: Wikipedia tells us that

“Cattle plagues recurred throughout history, often accompanying wars and military campaigns. They hit Europe especially hard in the 18th century, with three long pandemics which, although varying in intensity and duration from region to region, took place in the periods of 1709–1720, 1742–1760, and 1768–1786. There was a major outbreak covering the whole of Britain in 1865/66.”

“Later in history, an outbreak in the 1890s killed 80 to 90 percent of all cattle in Southern Africa, as well as in the Horn of Africa [and resulted in the deaths of many thousands of people who depended on them]. Sir Arnold Theiler was instrumental in developing a vaccine that curbed the epidemic. [my insert / emphasis] More recently, a rinderpest outbreak that raged across much of Africa in 1982–1984 cost at least an estimated US$500 million in stock losses”.

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“.

The 19th century southern African outbreak was devastating enough that people still remember it as a legendary time of hardship – and then there was the 1980s outbreak.  Another South African interest in rinderpest is that the legendary Sir Arnold Theiler had a hand in making a vaccine: he did this around the turn of the 20th century, 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 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.

But a memory may be all rinderpest is any more – as the GREP site says the following:

“The last known rinderpest outbreak in the world was reported in 2001 (Kenya). Based on the above-mentioned investigations, FAO is confident that all rinderpest virus lineages will prove to be extinct.”

This was also announced via the BBC on the 14th October, 2010.  They said:

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.

And the significant bit:

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

Let us reiterate that: 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.

From GREP:

Though the effort to eradicate rinderpest has encountered many obstacles over the past several decades, the disease remains undetected in the field since 2001. As of mid 2010, FAO is confident that the rinderpest virus has been eliminated from Europe, Asia, Middle East, Arabian Peninsula, and Africa. This has been a remarkable achievement for veterinary science, evidence of the commitment of numerous countries, and a victory for the international community.

Amen.  However – it’s not quite time to celebrate as the certification is only planned for 2011.

And now for mumps, and measles too.


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