A collection of polioviruses

PoliovirusesIn midsummer 1986, five years after starting my poliovirus laboratory at Columbia University, I received a letter from Frederick L. Schaffer, a virologist at the University of California, Berkeley, asking if I would like to have his collection of poliovirus stocks. He was retiring and the samples needed a home, otherwise they would be destroyed. Of course I jumped at the opportunity to have a bit of virology history.

Three boxes full of dry ice arrived in the laboratory in August 1986. In them were sixty-six containers of different polioviruses that Dr. Schaffer had collected over the years. All three poliovirus serotypes were represented, both wild type and vaccine strains. The tubes were marked with dates ranging from 1948 to 1965. Most had come into Dr. Schaffer’s hands from well known poliovirologists, including Jonas Salk, Albert Sabin, Igor Tamm, Renato Dulbecco, and Charles Armstrong.

All of the samples were in glass containers, either tubes with a screw cap or a rubber stopper held in place with tape (pictured). A few were in glass bottles of the type that were used to grow cells. The collection held not a single plastic tube: these were the days before plastics entered the virology world. All were identified by hand-written labels on white cloth tape. Some of the labels in the photo read: Polio 1 Brunhilde (1963), Polio 2 MEF1, Polio 2 P712, HeLa P3, 10-21-62. Nearly all the samples were virus-containing cell culture supernatants.

Lansing poliovirusPerhaps the most amazing sample was a specimen labeled ‘Lansing poliomyelitis virus, 9/27/50, passage 379, C. Armstrong, NIH’. Within the glass vial was a intact mouse brain still attached to the spinal cord (there were no cell phones in 1986, hence no photo). The Lansing type 2 strain of poliovirus had been adapted to grow in mice by Charles Armstrong, and this was apparently the 379th intracerebral mouse-to-mouse passage! It was especially exciting for me to receive this sample, because my laboratory had been studying the ability of the Lansing strain of poliovirus to infect mice. I believe that the note accompanying the tube is in Dr. Armstrong’s writing.

I have since transferred most of the samples to plastic tubes which are stored in a -70C freezer. There is a trove of information to be obtained by studying these samples, but there are few poliovirologists left who are interested. Once poliomyelitis is eradicated – perhaps within the next 10 years – these samples, and similar ones throughout the world, will have to be destroyed.

Renato Dulbecco, 1914-2012

wee plaques 1952For the second time in a week I note the passing of an important virologist. Renato Dulbecco, together with David Baltimore and Howard Temin, received the 1975 Nobel Prize in Physiology or Medicine for discoveries about how tumor viruses interact with the genetic material of the cell. Dulbecco also devised my favorite virological method, the plaque assay, for determining the virus titer, the number of animal viruses in a sample.

Since the early 1920s bacteriologists had used the plaque assay to quantify the number of infectious bacteriophages (viruses that infect bacteria). Dulbecco noted in 1952 that “research on the growth characteristics and genetic properties of animal viruses has stood greatly in need of improved quantitative techniques, such as those used in the related field of bacteriophage studies.” One limiting factor was the development of suitable animal cell cultures that could be used to determine viral titer. By the 1950s the techniques for reliably producing and propagating human cell cultures were developed, and in 1951 the first immortal human cell line, HeLa, was isolated. Dulbecco took advantage of these advances and showed in 1952 that western equine encephalitis virus formed plaques on monolayers of chicken embryo fibroblasts (figure). Dulbecco also made the important observation that one virus particle is sufficient to produce one plaque. He drew this conclusion from his observation of a linear dependence of the number of plaques on virus concentration. This seminal advance made possible the application of genetic techniques to the study of animal viruses.

Dulbecco’s work on tumor viruses was focused on polyomaviruses – small DNA-containing viruses such as murine polyomavirus and SV40. He found that cells from the natural host of the virus – mice for polyomavirus and monkeys for SV40 – were killed as the viruses replicated and produced new viral progeny. However, these viruses did not replicate in or kill cells from other animals. For example, when hamster cells were infected with murine polyomavirus, no viral replication took place, the cells survived, and a few rare cell were transformed  – their growth properties in culture were altered and they induced tumors when injected into hamsters. Dulbecco later found that the polyomaviral DNA is a circular, double-stranded molecule; and that in non-permissive cells (in which the virus does not replicate) the viral DNA became integrated into the host cell chromosome. He also suspected that a viral protein called T (for tumor) antigen was a key to cell transformation.

Today we understand why polyomaviruses transform cells in which they do not replicate: infection does not kill these cells, and the rare transformed cells contain only viral DNA encoding T antigen. This protein is needed for viral replication in permissive cells because it drives cell proliferation, activating cellular DNA replication systems that are required for producing more viral DNA. In a non-permissive cell, T antigen drives the cell to divide endlessly, immortalizing it and allowing the accumulation of mutations in the cell genome that make the cells tumorigenic.

While the details of how DNA tumor viruses transform cells were being elucidated, other investigators were attempting to understand how another class of viruses – with RNA genomes – had similar effects on cells. In 1951 a young scientist named Howard Temin joined Dulbecco’s laboratory to study how Rous sarcoma virus (RSV) caused tumors. This virus had been discovered by Peyton Rous in 1911, but would only cause tumors in chickens, limiting progress. In Dulbecco’s laboratory, Temin found that RSV induced transformation of cultured chicken embryo fibroblasts – the same types of cells that were being used to develop the plaque assay for animal viruses. Temin took this transformation assay to his own laboratory, where he reasoned that a DNA copy of the RSV viral genome must be integrated into the chromosome of transformed cells. This led him to discover the enzyme reverse transcriptase in RSV particles, which produces a DNA copy of the viral RNA.

By embracing a new technology for the study of animal viruses – cell culture – Dulbecco set the study of both DNA and RNA tumor viruses on a path that would lead to understanding viral transformation, an achievement recognized by the 1975 Nobel Prize.

Dulbecco, R. (1952). Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus Proceedings of the National Academy of Sciences, 38 (8), 747-752 DOI: 10.1073/pnas.38.8.747

Detecting viruses: the plaque assay

One of the most important procedures in virology is measuring the virus titer – the concentration of viruses in a sample. A widely used approach for determining the quantity of infectious virus is the plaque assay. This technique was first developed to calculate the titers of bacteriophage stocks. Renato Dulbecco modified this procedure in 1952 for use in animal virology, and it has since been used for reliable determination of the titers of many different viruses.

polio-plaquesTo perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots are inoculated onto susceptible cell monolayers. After an incubation period, to allow virus to attach to cells, the monolayers are covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. When the plates are incubated, the original infected cells release viral progeny. The spread of the new viruses is restricted to neighboring cells by the gel. Consequently, each infectious particle produces a circular zone of infected cells called a plaque. Eventually the plaque becomes large enough to be visible to the naked eye. Dyes that stain living cells are often used to enhance the contrast between the living cells and the plaques. Only viruses that cause visible damage of cells can be assayed in this way. An example of plaques formed by poliovirus on a monolayer of HeLa cells is shown at left. In this image, the cells have been stained with crystal violet, and the plaques are readily visible where the cells have been destroyed by viral infection.

The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter. To determine the virus titer, the plaques are counted. To minimize error, only plates containing between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is used. Statistical principles dictate that when 100 plaques are counted, the sample titer will vary by plus or minus 10%. Each dilution is plated in duplicate to enhance accuracy. In the example shown below, there are 17 plaques on the plate made from the 10-6 dilution. The titer of the virus stock is therefore 1.7 x 108 PFU/ml.

plaque-assay

Next we’ll consider how the plaque assay can be used to prepare clonal virus stocks, a step that is essential for studying viral genetics.

Dulbecco, R., & Vogt, M. (1953). Some problems of animal virology as studied by the plaque technique. Cold Spring Harbor Symp. Quant. Biol., 18, 273-279