On episode #353 of the science show This Week in Virology, the TWiVniacs discuss twenty-eight years of poliovirus shedding by an immunodeficient patient, and packaging of the innate cytoplasmic signaling molecule cyclic GMP-AMP in virus particles.

You can find TWiV #353 at www.twiv.tv.

Glass PoliovirusAn immunodeficient individual has been excreting poliovirus in his stool for 28 years. Such chronic excreters pose a threat to the poliovirus eradication program.

Since its inception in 1988 by the World Health Organization, the poliovirus eradication program has relied on the use of the infectious, attenuated vaccine strains produced by Albert Sabin. These viruses are taken orally, replicate in the intestine, and induce protective immunity. During replication in the gut, the Sabin strains lose the mutations that prevent them from causing paralysis. Nearly every individual who receives the Sabin vaccine strains excretes so-called vaccine-derived polioviruses (VDPVs) which are known to have caused outbreaks of poliomyelitis in under-immunized populations.

Immunocompromised individuals who produce very low levels of antibodies (a condition called agammaglobulinemia) are known to excrete VDPVs for very long periods of time – years, compared with months in healthy individuals. Seventy-three such cases have been described since 1962. These individuals receive the Sabin vaccine in the first year of life, before they are known to have an immunodeficiency, at which time they must receive antibodies to prevent them from acquiring fatal infections.

The most recently described immunocompromised patient was found to excrete poliovirus type 2 vaccine for 28 years (the time period is determined by combining the known rate of change in the poliovirus genome with sequence data on viruses obtained from the patient).  The VDPV is neurovirulent (causes paralysis in a mouse model), antigenically drifted, and excreted in the stool at high levels.

Because the polio eradication plan calls for cessation of vaccination at some future time, these immunocompromised poliovirus shedders pose a threat to future unimmunized individuals. The global number of such patients is unknown, and there is no available therapy to treat them – administration of antibodies does not clear the infection. The development of antivirals that could eliminate the chronic poliovirus infection is clearly needed (and ongoing). It will also be necessary to conduct environmental surveillance for the presence of VDPVs – they can be identified by properties that distinguish them from VDPVs produced by immunocompetent vaccine recipients.

While the WHO eradication plan now includes a shift to using inactivated (Salk) poliovaccine, this strategy would not impact the existing immunocompromised poliovirus shedders. Should a VDPV from these individuals cause an outbreak of polio in the post-vaccine era, it will be necessary to control the outbreak with Salk vaccine, or an infectious poliovirus vaccine that cannot revert to virulence during replication in the intestine. Polioviruses with a recoded genome are candidates for the latter type of vaccine.

Image credit: Jason Roberts


Shelves and mentors

1 September 2015

Palese laboratoryWhen I became Peter Palese’s first Ph.D. student in 1976, his laboratory at Mt. Sinai School of Medicine in New York City was in dire need of shelves. The laboratory benches (pictured) had no room for storing the many bottles of reagents that I was beginning to generate.

When I told Peter that his laboratory needed shelves, he told me to get them. I looked in the yellow pages (there was no internet at the time) and found a dealer in downtown Manhattan. When I told Peter, he responded, to my amazement, that he would drive me there to get them.

Within minutes we were in Peter’s car, driving far downtown to a small garage filled with shelf parts. The proprietor loaded some into Peter’s car and we returned to Mt. Sinai, where we carried the metal pieces up the elevator into the laboratory. There I bolted them together and made one set of shelving for the low lab bench, and a second set for a higher bench. The photo of Peter shows what the shelves looked like just a few years ago.Peter Palese

A few days ago Matt Evans, a professor in the same department as Peter, told me that the Palese lab was being renovated and that the shelves that I had built were being discarded. He knew that I had made the shelves because I talk about them when I give a seminar. Matt was kind enough to send photos of the shelves, and of the low lab bench on which they once rested. It’s amazing that the shelves lasted for 39 years!

There is a reason why I’m writing this story, and why I tell it whenever I give a seminar. I had selected Peter to be my Ph.D. mentor, which meant that I listened to him. When he told me to build the shelves, I built them. When I went to David Baltimore’s laboratory as a postdoctoral fellow, I listened to David. When either Peter or David asked me to do something, I did it. I selected them as mentors because they had more experience in virology than I had and I wanted to train with them. Therefore when they asked me to jump, I said, ‘how high’?

Palese shelvesI am not saying that mentors should have the ability to make their trainees do whatever they ask. Of course it is important to question; if you don’t understand why your mentor is asking you to do a specific experiment, by all means ask them! Being an effective trainee means listening and questioning. But remember that in the end they have more experience doing science than you have. If you don’t want to listen, why did you pick them in the first place?

I have been very lucky to have trained many fine virologists during my 33 years at Columbia University. Most have listened to my suggestions. The best have always asked questions, leading to many wonderful two-way interactions. But some chose to consistently ignore my suggestions for experiments or projects. I have seen similar conduct in other laboratories here and elsewhere. This behavior baffles me. To this day, if Peter or David asked me to do something, I would immediately comply. They will always be my mentors.

Palese shelvesUpdate: Gayathri, a Ph. D. student in the Palese lab, sent me a before and after photo of the shelves.

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On episode #352 of the science show This Week in Virology, Vincent meets up with Michele Banks in Washington, DC to discuss her career as a creator of science-themed art.

You can find TWiV #352 at www.twiv.tv.

TWiV 351: The dengue code

23 August 2015

On episode #351 of the science show This Week in Virology, the Masters of the ScienTWIVic Universe discuss a novel poxvirus isolate from an immunosuppressed patient, H1N1 and the gain-of-function debate, and attenuation of dengue virus by recoding the genome.

You can find TWiV #351 at www.twiv.tv.

The individuals who believe that certain types of gain-of-function experiments should not be done because they are too dangerous (including Lipsitch, Osterholm, Wain-Hobson,) cite the 1977 influenza virus H1N1 strain as an example of a laboratory accident that has led to a global epidemic. A new analysis shows that the reappearance of the 1997 H1N1 virus has little relevance to the gain-of-function debate.

Human influenza viruses of the H3N2 subtype were circulating in May of 1977 when H1N1 viruses were identified in China and then Russia. These viruses spread globally and continue to circulate to this day. The results of serological tests and genetic analysis indicated that these viruses were very similar to viruses of the same subtype which circulated in 1950 (I was in the Palese laboratory in 1977 when these finding emerged). Three hypotheses were suggested to explain the re-emergence of the H1N1 virus: a laboratory accident, deliberate release, or a vaccine trial.

Rozo and Gronvall have re-examined the available evidence for the origin of the 1977 H1N1 virus. While there is ample documentation of the extensive work done during the 1970s in the Soviet Union on biological weapons, there is no evidence that Biopreparat had attempted to weaponize influenza virus. The release of the 1977 H1N1 virus from a biological weapons program is therefore considered unlikely.

It is more likely that the 1977 H1N1 virus was released during testing of influenza virus vaccines. Many such trials were ongoing in the USSR and China during the 1960s-70s. C.M. Chu, a Chinese virologist, told Peter Palese that the H1N1 strain was in fact used in challenge studies of thousands of military recruits, an event which could have initiated the outbreak.

The hypothesis that the 1977 H1N1 virus accidentally escaped from a research laboratory is formally possible, but there are even less data to support this contention. Shortly after this virus emerged, WHO discounted the possibility of a laboratory accident, based on investigations of Soviet and Chinese laboratories. Furthermore, the H1N1 virus was isolated at nearly the same time in three distant areas of China, making release from a single laboratory unlikely.

It is of interest that with the onset of the gain-of-function debate, which began in 2011 with the adaptation of influenza H5N1 virus to aerosol transmission among ferrets, the ‘laboratory accident’ scenario for the emergence of the 1977 strain has been increasingly used as an example of why certain types of experiments are ‘too dangerous’ to be done (See graph, upper left). For example, Wain-Hobson says that ‘1977 H1N1 represented an accidental reintroduction of an old vaccine strain pre-1957, probably from a Russian research lab’. Furmanski writes that ‘The virus may have escaped from a lab attempting to prepare an attenuated H1N1 vaccine’. In the debate on gain-of-function experiments, the laboratory escape hypothesis is prominently featured in public presentations.

The use of an unproven hypothesis to support the view that some research is too dangerous to do is another example of how those opposed to gain-of-function research bend the truth to advance their position. I have previously explained how Lipsitch incorrectly represented the results of the H5N1 ferret transmission studies. We should not be surprised at this tactic. After all, Lipsitch originally called for a debate on the gain-of-function issue, then shortly thereafter declared that the moratorium should be permanent.

Rozo and Gronvall conclude that the use of the 1977 influenza epidemic as a cautionary tale is wrong, because it is more likely that it was the result of a vaccine trial and not a single laboratory accident:

While the events that led to the 1977 influenza epidemic cannot preclude a future consequential accident stemming from the laboratory, it remains likely that to this date, there has been no real-world example of a laboratory accident that has led to a global epidemic.


On episode #350 of the science show This Week in Virology, Vincent speaks with Katherine High about her career and her work on using viral gene therapy to treat inherited disorders.

This episode is drawn from one of twenty-six video interviews with leading scientists who have made significant contributions to the field of virology, part of the new edition of the textbook Principles of Virology.

You can find TWiV #350 at www.twiv.tv.

arenavirusI have a soft spot in my heart for Lassa virus: a non-fictional account of its discovery in Africa in 1969 inspired me to become a virologist. Hence papers on this virus always catch my attention, such as one describing its origin and evolution.

Lassa virus, a member of the Arenavirus family, is very different from Ebolavirus (a filovirus), but both are zoonotic pathogens that may cause hemorrhagic fever. It is responsible for tens of thousands of hospitalizations, and thousands of deaths each year, mainly in Sierra Leone, Guinea, Liberia, and Nigeria. Most human Lassa virus outbreaks are caused by multiple exposures to urine or feces from the multimammate mouse, Mastomys natalensis, which is the reservoir of the virus in nature. In contrast, outbreaks of Ebolavirus infection typically originate with a crossover from an animal reservoir, followed by human to human transmission. Despite being studied for nearly 50 years, until recently the nucleotide sequences of only 12 Lassa virus genomes had been determined.

To remedy this lack of Lassa virus genome information, the authors collected clinical samples from patients in Sierra Leone and Nigeria between 2008 and 2013. From these and other sources they determined the sequences of 183 Lassa virus genomes from humans, 11 viral genomes from M. natalensis, and two viral genomes from laboratory stocks. All the data are publicly available at NCBI. Analysis of the data lead to the following conclusions:

  • Lassa virus forms four clades, three in Nigeria and one in Sierra Leona/Liberia (members of a clade evolved from a common ancestor).
  • Most Lassa virus infections are a consequence of multiple, independent transmissions from the rodent reservoir.
  • Modern-day Lassa virus  strains probably originated at least 1,000 years ago in Nigeria, then spread to Sierra Leone as recently as 150 years ago. The lineage is most likely much older, but how much cannot be calculated from the data.
  • The genetic diversity of Lassa virus in individual hosts is an order of magnitude greater than the diversity of Ebolavirus. Furthermore, Lassa virus diversity in the rodent host is greater than in humans, likely a consequence of the longer, persistent infections that take place in the mouse.
  • The gene encoding the Lassa virus glycoprotein is subject to high selection in hosts, leading to variants that interfere with antibody binding.
  • Genetic variants that arise in one rodent are not transmitted to another.

Perhaps the most important result from this work is the establishment of laboratories in Sierra Leone and Nigeria that can safely collect and process samples from patients infected with Lassa virus, a BSL-4 pathogen.

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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.


On episode #349 of the science show This Week in Virology, Vincent, Alan and Rich explain how to make a functional ribosome with tethered subunits, and review the results of a phase III VSV-vectored Ebolavirus vaccine trial in Guinea.

You can find TWiV #349 at www.twiv.tv.