TWiV 393: Lovers and livers

Possible sexual transmission of Zika virus, and a cell protein that allows hepatitis C virus replication in cell culture by enhancing vitamin E mediated protection against lipid peroxidation, are the subjects discussed by the TWiVerati on this week’s episode of the science show This Week in Virology.

You can find TWiV #393 at microbe.tv/twiv, or listen below.

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Virus Watch: Counting Viruses

In this episode of Virus Watch, I show how to do my favorite assay in all of virology – the plaque assay.

TWiV 373: The distinguished virology career of Julius S. Youngner

On episode #373 of the science show This Week in Virology, Vincent speaks with Julius about his long career in virology, including his crucial work as part of the team at the University of Pittsburgh that developed the Salk inactivated poliovirus vaccine.

You can find TWiV #373 at microbe.tv/twiv. Or you can watch the video below.



The Wall of Polio, version 3.0

Wall of Polio 3.0Back in 2013 I built a Wall of Polio in my laboratory – a large stack of six-well cell culture plates that have been used to measure the concentration of polioviruses in various samples by plaque assay. It became a focal point of the lab at which many guests came to have their photographs taken. Sadly, the Wall fell twice. Now a new Wall – version 3.0 – has been completed.

The new Wall of Polio is in my office at Columbia University Medical Center, where it will not annoy the Fire Inspector (the former Wall partially blocked an aisle). Furthemore, the new Wall is glued together, so it will not come apart. Its construction is documented in the photographs below. The Wall of Polio 3.0 is built with 1,464 six-well plates of HeLa cells that were used to determine the titer of poliovirus. We have also already had a number of visitors to Wall 3.0.

Because the Wall is impressive, it attracts attention, which can then be used to explain the plaque assay and determining virus titer. Therefore it is simply another tool that I used to teach the world about virology.

When you visit, expect that I will ask to photograph you before the Wall. Only a few have refused.

 

TWiV 312: She sells B cells

On episode #312 of the science show This Week in Virology, the TWiVbolans discuss the finding that human noroviruses, major causes of gastroenteritis, can for the first time be propagated in B cell cultures, with the help of enteric bacteria.

You can find TWiV #312 at www.microbe.tv/twiv.

A saga of HeLa cells

HeLa spinnerWe have been using HeLa cells in my laboratory since 1982, when I arrived at Columbia University Medical Center fresh from postdoctoral work with David Baltimore at MIT. I brought with me a line of HeLa cells and used them for 30 years for our research on viruses. Here is a story of how we lost the cells and then found them ten months later.

As everyone knows, the continuous HeLa cell line was derived from a cervical tumor taken from Henrietta Lacks in 1951 (if you don’t know the story, you should read Rebecca Skloot’s The Immortal Life of Henrietta Lacks, or my shorter summary). When I arrived at the Baltimore lab in 1979, they were using cells derived from the S3 clone of HeLa cells that had been produced by Philip Marcus in the 1950s. I write ‘derived from’ because someone at MIT had further  cloned the S3 line and selected one that was particularly susceptible and permissive to poliovirus infection. This was the cell line that I took with me to Columbia in 1982.

Because we use so many HeLa cells each week, we grow them in spinner cultures (pictured). The cells are suspended in a glass bottle in nutrient medium and continuously stirred by a magnetic bar. The spinner bottle is placed on top of a stir plate, which contains a motor that drives a rotating magnet that in turn spins the bar in the bottle. When we need to produce monolayers of cells for experiments, we remove cells from suspension and plate them on plastic dishes. The HeLa S3 clone that we use grows very well in suspension and also forms excellent monolayers on plastic dishes.

Over the years we used the HeLa S3 subclone to conduct experiments with poliovirus, echoviruses, Coxsackieviruses, enteroviruses, rhinoviruses, and encephalomyocarditis virus. The cells could be infected with all these viruses, develop cytopathic effects, and form plaques, allowing titration of virus titers. They have been an essential part of my laboratory. The Wall of Polio is just one example of how important these cells have been for our work.

In December 2012, the spinner went down. The drive belt that turns the magnet in the spinner platform broke overnight; the cells settled out and died. Normally we would simply go to our stock of cells frozen in liquid nitrogen, thaw them out, and be up and working again within a week. Unfortunately, our liquid nitrogen tank had run dry one week in the summer of 2012, and all the cells had died. We tried recovering some of the HeLa cells that were frozen, but what grew out were not the same as our S3 subclone.

In the course of the next 9 months we tried HeLa cells from many different sources – laboratories here at Columbia, the American Type Culture Collection, and our colleagues elsewhere. None of the HeLa cells performed like our S3 subclone. Some HeLa lines did not grow well in spinner; others did, but formed poor monolayers. Still others did not support replication or plaque formation when infected with viruses we work on now – poliovirus, rhinoviruses, and encephalomyocarditis virus. I located former members of the Baltimore laboratory, hoping that they had taken the special HeLa cells with them, but I came up empty handed.

A few weeks ago I received an email from a former student who had heard my pleas for HeLa cell help on This Week in Virology. She remembered bringing some of our HeLa cells to her postdoctoral laboratory in Canada, and freezing them down when she left. I contacted the laboratory and found to my delight that our HeLa cells were indeed frozen there; a kind member of the laboratory grew up a stock of the cells, froze them, and shipped them off to us. I received them a week ago and put them into culture. They were the HeLa cells that I used to know: I recognized their morphology immediately. They grew beautifully as monolayers, and just today I set up a spinner culture. We are all looking forward to using the cells in our virology experiments.

Meanwhile, I have a large stock of belts for the magnetic spinner plate (I had to buy seventeen of them, to meet the $50 minimum order); I have placed 6 vials of the cells in our liquid nitrogen tank; and I plan to freeze additional vials in my colleagues’ freezers in case ours goes down again.

There are several lessons to be learned from this saga. First, because virologists are completely dependent on cells, they must take care that they have a reliable stock. This means having someone in the lab checking the level of liquid nitrogen every day, and ordering a new tank when the level is low. Second, it’s important to keep stocks of cells frozen elsewhere. We were very lucky to find them in Canada. Third, HeLa cell lines are very different. Finally, HeLa cells are special. I don’t know of any other cell line that grows well in spinner, makes beautiful monolayers, and allows us to work with so many different viruses. Thank you, Henrietta Lacks.

TWiV 217: I just flu in and my arms are shot

On episode #217 of the science show This Week in Virology, Vincent, Alan, Rich, and Dickson review influenza vaccines.

You can find TWiV #217 at www.microbe.tv/twiv.

A $707 million investment in cell-based influenza vaccine

novartis-holly-springs-usThe United States has provided a large sum of money to pharmaceutical giant Novartis to produce influenza vaccines grown in cell culture. According to CIDRAP:

Novartis’s US efforts have been spurred by two major federal contracts. In 2006 the firm won a $220 million HHS contract to develop cell-based flu vaccines, and in January 2009 HHS awarded a $487 million contract to help build the Holly Springs facility.

The facility consists of six connected buildings with 430,000 square feet of manufacturing, laboratory, and office space on a 167-acre site, Novartis reported in a news release. The company has about 400 employees there now and plans to expand to 500 by the end of 2012. Power said the facility is expected to be ready for full-scale commercial production in 2013.

Is this a good investment of US science dollars? Consider the following points:

  • A cell-based vaccine is expected to shorten by a few weeks the time required to produce a vaccine for a new influenza virus strain
  • No cell-based influenza vaccine is currently licensed for use in the US
  • Cell-based influenza vaccines use the same technology developed 50 years ago for egg-grown vaccine, with the exception that the virus is propagated in a different host
  • Inactivated influenza vaccine is not very good – it is 59% effective in individuals 18-65 years of age

We need a completely different influenza virus vaccine, not a retread of the existing process. Promising new approaches include virus-like particle vaccines produced in plants that can be made cheaply, rapidly, and appear to be immunogenic and safe in humans. Influenza vaccines that can protect against all viral strains now seem possible, and these vaccines will probably not be based on growing influenza viruses in eggs or cell culture.

I suspect that contemporary influenza vaccines produced in eggs or cell culture will be replaced with new vaccines within the next decade. With all this in mind, does it make sense to invest $707 million in a vaccine that has such a short life span? Even more money is at stake – according to CIDRAP, “Novartis is one of five companies that received sizable HHS contracts in 2006 to develop cell-based vaccines.” The new Novartis plant will not be able to supply all the influenza vaccine required by the US (and having a sole supplier is not a good idea). Are there enough individuals with severe egg allergies to justify this investment? Or is this a project that seemed like a good idea in 2006 but is now an anachronism?