Virologists, start your poliovirus destruction!

I have worked on poliovirus for over thirty-six years, first as a posdoctoral fellow with David Baltimore in 1979, and then in my laboratory at Columbia University. The end of that research commences this year with the destruction of my stocks of polioviruses.

In 2015 there were 70 reported cases of poliomyelitis caused by wild type 1 poliovirus, and 26 cases of poliomyelitis caused by circulating vaccine derived polioviruses (cVDPV) types 1 and 2. The last case of type 2 poliovirus occurred in India in 1999, and the virus was declared eradicated in 2015. Consequently the World Health Organization has decided that all remaining stocks of wild type 2 poliovirus should be destroyed by the end of 2015.

My laboratory has worked extensively with type 2 polioviruses. Before we produced transgenic mice susceptible to poliovirus, we had studied the Lansing strain of type 2 poliovirus because it had the unusual ability to infect wild type mice (polioviruses normally only infect certain primates). We determined the nucleotide sequence of the viral genome, identified the capsid as a determinant of the ability of the virus to infect wild type mice, and showed that swapping an eight amino acid sequence of capsid protein VP1 from a type 1 strain with that from Lansing conferred the ability to infect non-transgenic mice. These findings indicate that the ability of the Lansing strain of poliovirus to infect mice is likely due to recognition by the viral capsid of a receptor in the mouse central nervous system. In the past year we took advantage of the ability to produce mouse neurons from stem cells to attempt to identify the murine cellular receptor for Lansing virus.

To prevent further cases of poliomyelitis caused by cVDPVs, WHO has decided that there will be a synchronized, global switch from trivalent OPV to bivalent OPV in April 2016. By July of 2016 all remaining stocks of the Sabin type 2 poliovirus strains, which are used to produce OPV, will also be destroyed.

No wild type 3 poliovirus has been detected since November 2012, and it is likely that this virus will be declared eradicated within the next several years. At that time we will have to destroy our stocks of type 3 poliovirus. That leaves wild poliovirus type 1, which circulates only in Pakistan and Afghanistan. Given the small number of cases of paralysis caused by this type, it is reasonable to believe that eradication will occur within the next five years. If this timeline is correct, it means that I will be destroying my last vials of poliovirus around 2020.

It is of course necessary to destroy stocks of wild and vaccine polioviruses to prevent reintroduction of the virus and the disease that it causes. The 1978 release of smallpox virus from a laboratory in the United Kingdom, which caused one death, lead to requests for reducing the number of laboratories that retained the virus. Today there are just two official repositories of smallpox virus in the United States and Russia.

It is rare for an investigator to be told to destroy stocks of the virus that is the subject of his or her research. Over the years we have published 81 papers on poliovirus replication, vaccines, and pathogenesis. While I realize that it is absolutely essential to stop working on this virus, I do so with a certain amount of sadness. What other emotion could I have for a virus on which I have expended so much thought and effort?

Image: Poliovirus by Jason Roberts

Correction: The synchronized switch in April 2016 is from trivalent to bivalent OPV, not OPV to IPV. Consequently I have removed comments related to an OPV-IPV switch.

TWiV 370: Ten out of 15

On episode #370 of the science show This Week in Virology, the TWiVomics review ten captivating virology stories from 2015.

You can find TWiV #370 at

TWiV 369: Camel runny noses and other JNK

On the latest episode of the science show This Week in Virology, a swarm of virologists discusses testing of a MERS coronavirus vaccine for camels, and how a neuronal stress pathway reactivates herpes simplex virus.

You can find TWiV #369 at

TWiV 368: Infected, you will be

On episode #368 of the science show This Week in Virology, a plaque of virologists explores the biology of Zika virus and recent outbreaks, and the contribution of a filamentous bacteriophage to the development of biofilms.

You can find TWiV #368 at

Viruses help form biofilms

Inoviridae virionBacteria frequently grow in communities called biofilms, which are aggregates of cells and polymers. An example of a biofilm is the dental plaque on your teeth. Biofilms are medically important as they can allow bacteria to persist in host tissues and on catheters, and confer increased resistance to antibiotics and dessication. Therefore understanding how biofilms form is crucial for controlling microbial infections. An advance in our understanding of biofilms formation is the observation that filamentous phages help them assemble, and contribute to their fundamental properties.

Pseudomonas aeruginosa is an important human pathogen which is a particular problem in patients with cystic fibrosis. The ability of this bacterium to form biofilms in the lung is linked to its ability to cause chronic infections. Pseudomonas aeruginosa biofilms contain large numbers of filamentous Pf bacteriophages (pictured; image credit). These viruses lyse cells and release DNA, which becomes one component of the biofilm matrix.

Mixing supernatants of P. aeruginosa cultures with hyaluronan, which is present in airways of cystic fibrosis patients, resulted in the formation of a biofilm – in the absence of bacteria. A major component of P. aeruginosa biofilms was found to be Pf bacteriophages. When purifed Pf bacteriophages were mixed with hyaluronan, biofilms formed. Similar biofilms also formed when the filamentous bacteriophage fd of E. coli was mixed with hyaluronan. Mixtures of Pf bacteriophages and various polymers (alginate, DNA, hyaluronan, polyethylene glycol) formed liquid crystals (matter in a state between a liquid and a solid crystal).

Pf phages were detected in sputum from patients with cystic fibrosis, but not in uninfected patients. Addition of Pf phage to sputum from patients infected with P. aeruginosa made the samples more birefringent, a property of liquid crystals. Compared with a strain of P. aeruginosa that does not produce Pf phage, colonies of virus-producing strains formed liquid crystals. These observations indicate that Pf phage help organize the bacteria into a biofilm matrix.

Some features of biofilms include their ability to adhere to surfaces, to protect bacteria from dessication, and to increase resistance to antibiotics. Addition of phage Pf increased biofilm adhesion and tolerance against dessication. Such addition also made the biofilm more resistant to aminoglycoside antibiotics, because these were sequestered in the biofilm. No phage-mediated increased resistance to ciprofloxacin was observed, probably because this antimicrobial does not interact with polyanions of the biofilm as do aminoglycosides.

These results show that presence of bacteriophage in a biofilm of P. aeruginosa helps organize the matrix while contributing to some of its fundamental properties. It seems likely that filamentous phages of other bacteria will play roles in biofilm formation, suggesting that targeting the phages in these matrices could be effectie strategies for treating biofilm infections.

TWiV 367: Two sides to a Coyne

On episode #367 of the science show This Week in Virology, two Coynes join the TWiV overlords to explain their three-dimensional cell culture model of polarized intestinal for studying enterovirus infection.

You can find TWiV #367 at

Exaptation: A cell enzyme becomes a viral capsid protein

Alphalipothrixvirus virionThe acquisition of a capsid is thought to be a key event in the evolution of viruses from the self-replicating genetic elements that existed during the pre-cellular stage on Earth. The origin of viral capsids has been obscure because their components are not similar to cellular proteins. The discovery that a viral capsid protein evolved from a CRISPR-associated nuclease provides insight into how viruses emerged.

Thermoproteus tenax virus 1 (TTV1) infects the hyperthemophilic archaeon Thermoproteus tenax, which grows at 86°C. The enveloped virus particles are flexible filaments 400 nm long and 40 nm in diameter (illustrated; image credit) built with four capsid proteins, TP1-TP4. The basic proteins TP1 and TP2 bind the 16 kb double-stranded DNA genome to form the nucleocapsid.

Thirty years after the discovery of TTV1, the capsid proteins remained ORFans – meaning that they had no sequence homology with viral or cellular proteins. Recently a more sensitive homology analysis revealed that TP1 is similar to Cas4, a nuclease that is a part of the prokaryotic CRISPR-Cas defense system.

Although TP1 clearly matches the Cas4 protein, it is not complete: codons at the carboxy-terminus are missing. A re-examination of the TTV1 genome sequence revealed a previously undetected open reading frame of 74 codons just downstream of the TP1 gene which are the missing C-terminal residues of the Cas4 nuclease. It is not known if this protein, called gp7, is produced in infected cells; it is not part of the virus particle.

Together the TP1 and gp7 proteins represent a full length Cas4 nuclease. TP1 is probably not catalytically active due to amino acid changes in the active site of the enzyme.

Why does TP1 lack the carboxy-terminal residues of Cas4? The amino terminus of the TP1 protein comprises a positively charged surface that might be involved in binding the viral DNA genome. The same surface in Cas4 is covered by the carboxy-terminal domain of the protein. This observation suggests that transformation of Cas4 from a nuclease into a viral capsid protein probably required removal of this shielding domain, so that the protein could bind the DNA genome.

How did a nuclease become a viral capsid protein? An ancestor of TTV1 might have encoded a Cas4-like protein with nuclease activity with a role in genome replication or repair. Mutations causing loss of nuclease activity might have been followed by truncation of the protein to expose the DNA binding domain, which then became a viral capsid protein. Support for this idea comes from the observation that a Cas4-like protein encoded in the genome of another archaeal virus, the rudivirus SIRV2, has nuclease activity.

Exaptation, a change in the function of a protein during evolution, is known to have taken place in the viral world. The case of Cas4 and TP1 shows that capsid components can evolve from proteins with a very different function.

TWiV 366: Doctorates down under

On episode #366 of the science show This Week in Virology, Vincent visits Melbourne, Australia, where he speaks with four PhD students about their research projects and what it’s like to get a doctorate down under.

You can find TWiV #366 at Or you can watch the video below.

Neurovirologist Richard T. Johnson, 84

Viral Infections of the Nervous SystemJohns Hopkins Neurovirologist Richard T. Johnson has died, and his obituary at Hub provides a good summary of his career. He had an important influence on my work early in my career.

The first edition of Dr. Johnson’s book, Viral Infections of the Nervous System, was published in 1982 – the year I began my laboratory at Columbia University. I was interested in studying poliovirus pathogenesis, so I immediately purchased the book (which I still have to this day – that’s a photo of it in the upper left). It served as an important resource and source of inspiration for many years, whether for writing grants, review articles, or thinking about viruses and their interaction with the central nervous system.

I always liked his sentence on page 5, under ‘Origins of Virology’: Virology as a discipline began in botany, not in medicine.

For me the most valuable chapter was ‘Pathogenesis of CNS Infections’ in which Johnson discussed how viruses move into and within that system. Many of the pages in that chapter are underlined and marked with comments (see photo below).

The blood-brain barrierYears later, when I was writing Principles of Virology, many of the concepts that I learned from Dr. Johnson made their way into that book. Dr. Johnson supplied me with an electron micrograph of the blood-brain barrier for use in our textbook.

A testimony to the value of this book for me is how many questions it raised, rather than answered. It truly influenced my thinking about poliovirus pathogenesis, for which I am very grateful to Dr. Johnson.

TWiV 365: Blood, feuds, and a foodborne disease

On episode #365 of the science show This Week in Virology, Vincent, Alan, and Kathy trace the feud over genome editing, a new virus discovered in human blood, and the origins of hepatitis A virus.

You can find TWiV #365 at