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 www.microbe.tv/twiv.

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.

Mr. Paul Smallcombe
Records & Information Compliance Manager
Queen Mary University of London
Mile End Road
London E1 4NS

Dear Mr Smallcombe:

The PACE study of treatments for ME/CFS has been the source of much controversy since the first results were published in The Lancet in 2011. Patients have repeatedly raised objections to the study’s methodology and results. (Full title: “Comparison of adaptive pacing therapy, cognitive behaviour therapy, graded exercise therapy, and specialist medical care for chronic fatigue syndrome: a randomized trial.”)

Recently, journalist and public health expert David Tuller documented that the trial suffered from many serious flaws that raise concerns about the validity and accuracy of the reported results. We cited some of these flaws in an open letter to The Lancet that urged the journal to conduct a fully independent review of the trial. (Dr. Tuller did not sign the open letter, but he is joining us in requesting the trial data.)

These flaws include, but are not limited to: major mid-trial changes in the primary outcomes that were not accompanied by the necessary sensitivity analyses; thresholds for “recovery” on the primary outcomes that indicated worse health than the study’s own entry criteria; publication of positive testimonials about trial outcomes and promotion of the therapies being investigated in a newsletter for participants; rejection of the study’s objective outcomes as irrelevant after they failed to support the claims of recovery; and the failure to inform participants about investigators’ significant conflicts of interest, and in particular financial ties to the insurance industry, contrary to the trial protocol’s promise to adhere to the Declaration of Helsinki, which mandates such disclosures.

Although the open letter was sent to The Lancet in mid-November, editor Richard Horton has not yet responded to our request for an independent review. We are therefore requesting that Queen Mary University of London to provide some of the raw trial data, fully anonymized, under the provisions of the U.K.’s Freedom of Information law.

In particular, we would like the raw data for all four arms of the trial for the following measures: the two primary outcomes of physical function and fatigue (both bimodal and Likert-style scoring), and the multiple criteria for “recovery” as defined in the protocol published in 2007 in BMC Neurology, not as defined in the 2013 paper published in Psychological Medicine. The anonymized, individual-level data for “recovery” should be linked across the four criteria so it is possible to determine how many people achieved “recovery” according to the protocol definition.

We are aware that previous requests for PACE-related data have been rejected as “vexatious.” This includes a recent request from psychologist James Coyne, a well-regarded researcher, for data related to a subsequent study about economic aspects of the illness published in PLoS One—a decision that represents a violation of the PLoS policies on data-sharing.

Our request clearly serves the public interest, given the methodological issues outlined above, and we do not believe any exemptions apply. We can assure Queen Mary University of London that the request is not “vexatious,” as defined in the Freedom of Information law, nor is it meant to harass. Our motive is easy to explain: We are extremely concerned that the PACE studies have made claims of success and “recovery” that appear to go beyond the evidence produced in the trial. We are seeking the trial data based solely on our desire to get at the truth of the matter.

We appreciate your prompt attention to this request.


Ronald W. Davis, PhD
Professor of Biochemistry and Genetics
Stanford University

Bruce Levin, PhD
Professor of Biostatistics
Columbia University

Vincent R. Racaniello, PhD
Professor of Microbiology and Immunology
Columbia University

David Tuller, DrPH
Lecturer in Public Health and Journalism
University of California, Berkeley

TWiEVOTo a molecular biologist, the word ‘evolution’ evokes images of fossils, dusty rocks, and phylogenetic trees covering eons. The fields of molecular biology and evolutionary biology diverged during the twentieth century, but new experimental technologies have lead to a fusion of the two disciplines. The result is that evolutionary biologists have the unprecedented ability to evaluate how genetic change produces novel phenotypes that allow adaptation. It’s a great time to start a new podcast on evolution!

Molecular biology is an experimental approach that was born in 1953 with the discovery of the structure of DNA. Its goal is to understand how cells and organisms work at the level of biological molecules such as DNA, RNA, and proteins. Some of the experimental tools of molecular biology include recombinant DNA, nucleotide sequencing, mutagenesis, and DNA-mediated transformation. The experiments of molecular biology often involve simplified, or reductionist systems in which much of the complexity of nature is ignored. Variation in individuals, populations, and the environment are set aside. Data produced by the techniques of molecular biology can lead to decisive conclusions about cause and effect.

Evolutionary biology embraces variation, and in fact attempts to explain it. The basis for variation in organisms is usually inferred by associating phenotypes, sequences, and alleles. The problem with this approach is that alternative explanations are often plausible, and conclusions are rarely as decisive as those achieved with molecular biology. We can turn to Darwin’s finches as a good illustration of the difference between fields. Darwin hypothesized that variation in the beaks of finches was a consequence of diet, but how such variation occurred was unknown. It was not until 2004 that it was shown that beak shape and size could be controlled by two different genes.

The techniques of DNA sequencing, mutagenesis, and the ability to introduce altered DNA into cells and organisms have been the catalyst for the fusion of molecular biology and evolutionary biology into a new and far more powerful science, which Dean and Thornton call a ‘functional synthesis’. As a consequence, genotype can be definitively connected with phenotype, allowing resolution of fundamental questions in evolution that have been puzzles for many years.

Microbes are perfect subjects for study by evolutionary biologists, as they are readily manipulable and rapidly reproduce. However no organism is now very far from the eye of this new science. Subjects as diverse as insecticide resistance, coat color in mice, evolution of color vision, and much more are all amenable to scrutiny by the ‘functional synthesis’.

This Week in Evolution will cover all aspects of the functional synthesis, irrespective of organism. My co-host is Nels Elde, an evolutionary biologist at the University of Utah. Nels has appeared on This Week in Virology to discuss the evolution of virus-host conflict, and his lab’s story on the evolutionary battle for iron between mammalian transferrin and bacterial transferrin-binding protein was covered on This Week in Microbiology.

You can find This Week in Evolution at iTunes and at MicrobeTV.

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 www.microbe.tv/twiv.

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.

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 www.microbe.tv/twiv. Or you can watch the video below.

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.

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 www.microbe.tv/twiv.

On episode #364 of the science show This Week in Virology, Vincent, Rich, and Kathy speak with Ralph Baric and Vineet Menachery about their research on the potential of SARS-like bat coronaviruses  to infect human cells and cause disease in mice.

You can find TWiV #364 at www.microbe.tv/twiv.