No problem not being nice to Dickson in this episode, because he’s absent for a discussion of a new giant virus that replicates in the cytoplasm yet transiently accesses the nucleus to bootstrap infection.

You can find TWiV #440 at microbe.tv/twiv, or listen below.

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nucleusAmong the multitudes of eukaryotic viruses with DNA genomes, some replicate in the cell nucleus, while others avoid the nuclear bureaucracy and remain in the cytoplasm. But biology is not always so rigid: a new giant virus has been found that replicates in the cytoplasm, where it seems to recruit components of the nuclear transcription machinery (link to paper).

Noumeavirus was isolated from a pond near – where else? – Noumea airport in New Caledonia. The 200 nanometer icosahedral particles infect the amoeba Acanthamoeba castellani and have a double-stranded DNA genome of 376,207 base pairs encoding 452 proteins. Sequence comparisons revealed Noumeavirus to be a new member of the family Marseilleviridae, which  includes other previously discovered giant viruses.

Other members of the Marseilleviridae replicate in the cytoplasm of the host cell, so it was assumed that the related Noumeavirus would do the same. However an analysis of the proteins in purified virus particles revealed an absence of components of the transcriptional machinery – which is needed for ths synthesis of mRNA. RNA polymerase, for example, is readily detected in other cytoplasmic viruses such as Mimivirus and poxviruses.

If proteins involved in transcription are not present in the Noumeavirus particle, and the virus does not enter the nucleus, how are viral mRNAs produced?  It appears that early in infection, the required proteins are moved from the nucleus to sites of viral replication in the cytoplasm. When nuclear proteins were labled with green fluorescent protein, within one hour after infection they can be seen moving out of the nucleus into the cytoplasm to sites of viral replication. The nuclear integrity remains intact, as host DNA does not leave the organelle. This recruitment of nuclear proteins is transient:  after 2-4 hours proteins are no longer leaving the nucleus.

This series of events suggests that nuclear proteins needed to initiate viral mRNA synthesis are recruited from the nucleus to sites of viral replication in the cytoplasm. Once viral mRNAs are made, the viral transcriptional machinery can be assembled and the nuclear proteins are no longer needed. The authors call this ‘remote control of the host nucleus.’

Confirmation of this hypothesis will require the demonstration that nuclear proteins involved in viral mRNA synthesis are recruited to early sites of viral replication in the cytoplasm. It will also be essential to identify the mechanism by which these nuclear proteins are extracted. Perhaps one or more virion proteins, such as an abundant 150 amino acid protein of unknown function, is involved.

Other giant viruses, such as Mimivirus, package the viral transcriptional machinery in the virus particle and are independent of the cell nucleus. At the other extreme are viruses that undergo transcription and DNA synthesis entirely in the nucleus (e.g., herpesviruses). Perhaps Noumeavirus is a relic of an evolutionary transition between the two replication strategies.

Image credit

by David Tuller

During a recent talk at the annual conference of the British Renal Society, pediatrician and staunch PACE proponent Esther Crawley accused me of libeling her. I wasn’t at her presentation, but her slides were captured and tweeted. Dr. Crawley’s lecture recounted her heroic struggle against the dark forces of anti-science—presumably, those pesky ME/CFS advocates who challenge her work. One slide included a mention of “libellous blogs,” along with a screen shot of one of my Virology Blog posts. Hm.

This libelous Virology Blog post—“Trial By Error, Continued: The New FITNET Trial for Kids”–was about Dr. Crawley’s flawed research into ME/CFS and her proposed study of Internet-based cognitive behavior therapy for kids. The post explained how Dr. Crawley’s research conflates “chronic fatigue” and “chronic fatigue syndrome,” thus dramatically increasing the apparent prevalence of the illness. In the post, I also took aim at Dr. Crawley’s FITNET-NHS protocol and an earlier Dutch study of the same online intervention. I will not review the arguments here, but everything I wrote was based on facts.

In that post and elsewhere, I have expressed my strong opinion, as a public health academic and professional, that Dr. Crawley’s research is misleading. In pushing that perspective, I have used sharp and snarky rhetoric to ensure my voice was heard. Maybe I’ve even been obnoxious. But that just makes me sharp, snarky and obnoxious. It does not make me libelous. Something has to be untrue for it to be libelous, and Dr. Crawley has not identified any actual errors in my work.

It’s not surprising that Dr. Crawley would dislike my opinions and find them offensive. But my opinions are fair comment and based on my interpretations of the documented facts. There is an appropriate legal remedy for libel, and it is not to stand in front of a crowd of nephrologists and make baseless but serious accusations. Dr. Crawley should know better. And she should vet her slides with lawyers before she pulls a stunt like that again.

Having now informed Britain’s community of renal experts that I have engaged in libelous blogging—and that my colleague, Dr. Vincent Racaniello, has engaged in libelous blog-publishing–Dr. Crawley should explain herself. Several days ago, I asked her via e-mail to provide evidence for her accusation; in other words, to tell me what is inaccurate in my posts. I offered, of course, to correct any inaccuracies—something I do even when I’m not being accused of libel. I offered to run any statement she sent as part of my post, without editing or trimming it. Dr. Crawley did not respond to my e-mail.

In addition to her position as a professor of child health at the University of Bristol, Dr. Crawley is deputy chair of the CFS/ME Research Collaborative (CMRC). Her reckless accusation of libel raises questions about her judgment, and it certainly casts a shadow over any organization in which she plays a leadership role. I have e-mailed the other members of the CMRC board to express my dismay at Dr. Crawley’s accusation. I have also asked them to publicly disavow it.

To be sure, Dr. Crawley might feel under pressure at the moment, having recently suffered a humiliating public setback. She is deeply involved in the ME/CFS Epidemiology and Genomics Alliance, or MEGA, an ambitious proposed research project that grew out of a CMRC initiative. In March, MEGA announced that the Wellcome Trust had rejected its preliminary application for funding, meaning the group was not invited to submit a full proposal.

Wellcome is a major source of non-governmental funding for health and medical research, so this rejection of the MEGA application is a huge blow. Although the reasons for Wellcome’s rejection were not disclosed, we can speculate on some of the possibilities. Perhaps the application from Dr. Crawley and her colleagues was simply sub-par–poorly argued or inadequate for any number of reasons. It is possible the Wellcome reviewers were perplexed at the MEGA request, since the project’s goal of collecting samples from ME/CFS patients appears similar to what a respected and well-established organization, the U.K. ME/CFS Biobank, is already doing successfully. It would be reasonable for Wellcome to wonder whether funding a completely new parallel project for the same illness would be an effective use of their resources. I assume other grant-makers might have similar questions.

Another intriguing possibility is that the Wellcome reviewers have actually gotten wind of the growing international controversy over the PACE trial. Perhaps they recognize that the evidence base behind the CBT/GET approach is fast eroding, now that many experts outside the orbit of Dr. Crawley, Sir Simon Wessely, and the Science Media Centre have reviewed the study and assessed it harshly.  Given the changing attitudes, the Wellcome reviewers might even have wondered why Dr. Crawley still defends PACE so vigorously, as when she told an interviewer late last year that it was a “great, great” trial.

Whatever the reason for Wellcome’s rejection of the MEGA application, the bad news for PACE supporters keeps coming. In March, more than 100 scientists, clinicians and other experts–from Berkeley, Columbia, Stanford, Harvard, University College London, King’s College London, and elsewhere–signed an open letter to Psychological Medicine. (I helped organize the open letter, and also signed it.) The open letter, posted on Virology Blog, demanded retraction of the “recovery” findings published by Psychological Medicine in 2013. In outlining the study’s multiple missteps, the open letter bluntly declared that “such flaws are unacceptable in published research; they cannot be defended or explained away.”

Given Dr. Crawley’s recent endorsement of PACE’s greatness, does she consider all the signatories of that open letter to be libelous, or just me? It is likely that many if not all of those experts would agree with my opinion that Dr. Crawley’s research conflates “chronic fatigue” and “chronic fatigue syndrome” in a misleading manner. Would that qualify all of them as libelous?

Fortunately for the patient community, scientific and academic concern over the PACE enterprise continues to build. The Journal of Health Psychology (JHP) has just published a series of blistering commentaries—or perhaps libelous, depending on your perspective–about what is now being referred to as “PACE-gate.” The commentaries reflect the genuine surprise in the broader research community at PACE’s methodological lapses. No one, it seems, has ever before come across a clinical trial in which, as in PACE, participants could actually be “recovered” on key outcomes at baseline, before any treatment at all.

The JHP is based in the U.K., so publication of the commentaries represents a welcome departure from the stubborn, longstanding reluctance of the British academic and media establishments to seriously question the PACE investigators and their supporters, like Dr. Crawley, on scientific grounds. Hopefully medical journals and news organizations will soon start conducting their own independent investigations into this huge, publicly funded disaster. Maybe they will even ask why the entire U.K. medical establishment accepts as legitimate a clinical trial in which participants could be simultaneously defined as disabled enough for entry and yet “recovered” on key outcomes. (My own JHP commentary focuses on how the PACE investigators offer non-answers instead of answers, and then claim to have answered all of the questions.)

No matter how many times they try, PACE proponents are unable to provide credible and logical explanations for the irregularities of the research—at least, credible enough to make the questions disappear. Instead, they have accused critics of this and that malfeasance, all the while complaining about being persecuted themselves. I understand the urgency behind their increasingly strained and even laughable efforts to defend this indefensible body of research—these scientists are fighting for their reputations. But they are losing that fight because they are so clearly wrong on the science. In disseminating false accusations of libel, Dr. Crawley has merely embarrassed herself and exposed the desperation and intellectual weakness of the position she is seeking to defend.

Paul Bieniasz joins the TWiV team to talk about the co-option, millions of years ago, of an endogenous retrovirus envelope protein by hominid ancestors for host defense against viral infection.

 

You can find TWiV #439 at microbe.tv/twiv, or listen. below.

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AlistipesThe microbes that live on and in us provide a host of functions that are essential for our health. Changes in the composition of these microbial communities correlate with a variety of disease states. Results of a new study (link) reveal altered populations of intestinal bacteria and metabolic disturbances in ME/CFS patients.

The study subjects were 50 patients with ME/CFS from four sites across the US (meeting 1994 CDC Fukuda and 2003 Canadian consensus criteria) and 50 healthy controls. Some of the ME/CFS patients (21/50) reported a diagnosis of irritable bowel syndrome, absent in all the controls. Whether IBS leads to ME/CFS or is a consequence is unclear.

Genomic DNA was extracted from a fecal sample from each patient and subjected to high-throughput sequencing. Bacterial sequences were identified after computational subtraction of human genomic, mitochondrial, and ribosomal sequences.

The results show that bacterial taxa in ME/CFS patients with and without IBS were distinct. The most reliable markers of ME/CFS with IBS were increased abundance of Alistipes (pictured) and a decrease in Faecalibacterium genera of bacteria. In contrast, an increase in Bacteriodes and a decrease in Bacteroides vulgatus were associated with ME/CFS without IBS.

The bacterial genes identified in the sequence analysis were used to predict alterations in metabolic pathways. Some pathways are altered only in ME/CFS patients, while others are linked to IBS. Enrichment in the pathway of vitamin B6 biosynthesis appeared to be independent of IBS. This vitamin plays a role in many aspects of metabolism, neurotransmitter synthesis, histamine synthesis, hemoglobin synthesis and function, and more, and is a cofactor for many essential reactions.

The unsaturated fatty acid biosynthesis pathway was also found to be reduced in ME/CFS patients independent of IBS. A reduction of specific fatty acids has been linked to pro-inflammatory responses and immune activation in ME/CFS patients.

These and other metabolic findings in ME/CFS patients validate additional work on bacterial metabolic pathways and the metabolome – the set of small-molecule chemicals – of ME/CFS patients.

Others have previously shown increased levels of cytokines in the plasma and cerebrospinal fluid of ME/CFS patients who had been ill for a short period of time. No such association was found in the current study, perhaps because most subjects had been ill for extended periods of time.

The dysbiosis and bacterial metabolic disturbances identified in this study of ME/CFS patients are intriguing. The results suggest that abundance of certain bacterial taxa could be used as diagnostic markers for the disease. The more important question is whether these changes are a cause or a consequence of ME/CFS. Answering this question is relevant to the potential for using microbiome transplants, or metabolic therapeutic strategies to ameliorate the disease.

On the eve of the March for Science, the TWiV team gathers at ASM Headquarters in Washington, DC with guests Stefano and Susie to talk about the state of science communication.

You can find TWiV #438 at microbe.tv/twiv, or watch above/listen below.

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vrrThis Saturday I will be participating in the March for Science in Washginton, DC. It’s all about celebrating science and the role it plays in each of our lives. Frankly, I could have participated in a March for Science one, two, three, or any number of years ago, because these issues have always been important.

Over ten years ago, well into my science career, I recognized the need for scientists to come off the sidelines (to quote March for Science) and start telling the world what it is that we do. It’s why I wrote a virology textbook; started virology blog; produce five science podcasts; teach a virology course and record all the lectures for YouTube, and much more. These activities have been my March for Science.

Whenever I visit a university to give a science talk, I spend the last 15 minutes telling the audience (mainly scientists) why they need to tell the public what we do. I tell them to let the world know that our lives are long and prosperous because of science. I emphasize that every scientist needs to communicate, so that the public sees us all together championing the way science benefits the planet.

The March for Science, held in many different cities, will give the world a view of scientists together defending the roles that science plays in our lives. It’s a large scale view of what I have done myself over the years, and what I have encouraged other scientists to do.

It is unfortunate that the March for Science had to be triggered by an administration that shows disdain for science and facts. I bet we could have organized a similar march years ago. But the march is happening now, and whether or not we are branded as left or right doesn’t matter – science doesn’t care about your political party. Neil de Grasse Tyson put it perfectly: “The good thing about science is that it’s true whether or not you believe in it”.

More important than the March for Science is what happens afterwards. Does it build a “global movement to defend the vital role science plays in our health, safety, economies, and governments” as envisioned by its organizers, or is it back to business as usual for most scientists?

I don’t know the answer, but I do know that there are many scientists who do engage the public, and their work will continue. The work that me and my co-hosts do to bring science and scientists to everyone will go on, as it has before the March.

That’s why I will be wearing a This Week in Virology t-shirt to the March in Washington DC. It’s the way I’ve been communicating science, making a difference by reaching as many people as I can. That’s the spirit of the March for Science, which will go beyond one Saturday in April.

The TWiVsters reveal new giant viruses that argue against a fourth domain of life, and discovery of viruses in the oceanic basement.

You can find TWiV #437 at microbe.tv/twiv, or listen below.

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Viruses under the sea

CORKsViruses infect every living organism on the planet, but not every habitat has been explored for their presence. The igneous ocean crust had not yet been examined for viruses, but seek and ye shall find: there are plenty of viruses under the seas.

The oceanic basement is an enormous ecosystem that lies at the bottom of the seas, beneath a thick layer of sediment. It is composed of igneous rock through which percolates 20 million cubic kilometers of water. Previous study of cores from this region has revelealed the presence of prokaryotes, but no one had looked for viruses.

Facilitating the study of the oceanic basement are seafloor observatories that have been placed into existing boreholes. Two have been placed in 3.5 million year old rock in the northeastern Pacific Ocean. They penetrate hundreds of meters through sediment and into the basement (illustrated; image credit) and are fitted with plumbing that allows sampling of uncontaminated fluids from different depths in the basement rock.

Analysis of fluids recovered from these sites revealed both prokaryotes (8,000 per ml) and virus particles (90,000 per ml). Ribosomal RNA sequence analysis showed that bacteria dominated these communities, with some Archaea but virtually no eukaryotes.

Examination of the fluids by electron microscopy showed virus particles of different kinds: tailed and untailed icosahedral particles, untailed globular particles, and rod and spindle shaped. My favorite is the lemon shaped particle, for its form and implied taste.

To provide information on the viral genomes in the oceanic basement, sequences were determined from total cellular DNA (material retained on a 0.2 micron filter) extracted from the samples. Most viral sequences likely had archaeal hosts. Some prophage sequences were identified – viral genomes integrated into host DNA – which allowed more certain identification of the infected cell.

Most of the identified archaeal and bacterial virus sequences came from the families Myoviridae and Siphoviridae (think tailed, icosahedral viruses). One complete circular DNA genome identified is 55,906 nucleotides in length with 81 open reading frames. Twenty of these encode proteins with recognizable functions, such as capsid proteins, a primase and a DNA polymerase. No genes encoding tRNAs, such as those found in giant viruses, were identified.

Some sequences were similar to those of giant viruses like mimiviruses and phycodnaviruses. These viruses are known to only infect eukaryotes. Eukaryotic genomes were rare in the basement metagenome collections (1% of the community in one location).

I am not surprised that viruses have been found in ocean’s basement. Still, I’m amazed when I think about how far down they are, in warm water (65 degrees C) in 3.5 million year old rock.

To paraphrase Samuel E. Wright (Under The Sea):

The viruses are always greener
In somebody else’s lake
You dream about going up there
But that is a big mistake
Just look at the viruses around you
Right here under the ocean floor
Such wonderful viruses surround you
What more is you lookin’ for?

 

At Cornell University in Ithaca, New York, Vincent speaks with Susan, Colin, and Gary about the work of their laboratories on parvoviruses, influenza viruses, and coronaviruses that infect dogs, cats, horses and other mammals.

You can find TWiV #436 at microbe.tv/twiv, or listen below.

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