The Chicken or the Egg?
Possibly the next most important development in virology 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.
The Official Discovery of Influenza Virus
Also 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.
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.
Putting a Spin on it: 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 , invented and developed first by in Sweden as a purely analytical tool, and later by and in the USA as an . The ultracentrifuge revolutionised first, the physical analysis of proteins in solution, and second, the , by centrifugation at high speeds.
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 centrifugation at high speeds.
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 bacteria and eukaryotes came to be named: these are known as 70S (prokaryote) and 80S ribosomes, respectively, based on their different sedimentation rates.
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. 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 the infectious agent causing mosaic disease in tobacco – tobacco mosaic virus, or 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, 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.
Seeing is Believing: the Electron Microscope
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. This 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.
Click here for Part 3: Animal Cell Culture
and here for Part 1: Introduction
Copyright February 2012 by EP Rybicki and Russell Kightley, unless otherwise specified.