Last February, Virology Blog posted an open letter to The Lancet and its editor, Dr. Richard Horton, describing the indefensible flaws of the PACE trial of treatments for ME/CFS, the disease otherwise known as chronic fatigue syndrome (link to letter). Forty-two well-regarded scientists, academics and clinicians put their names to the letter, which declared flatly that the flaws in PACE “have no place in published research.” The letter called for a completely independent re-analysis of the PACE trial data, since the authors have refused to publish the results they outlined in their original protocol. The letter was also sent directly to Dr. Horton.

The open letter was based on the extensive investigative report written by David Tuller, the academic coordinator of UC Berkeley’s joint program in journalism and public health, which Virology Blog posted last October (link to report). This report outlines such egregious failings as outcome thresholds that overlapped with entry criteria, mid-trial promotion of the therapies under investigation, failure to provide the original results as outlined in the protocol, failure to adhere to a specific promise in the protocol to inform participants about the investigators’ conflicts of interest, and other serious lapses.

Virology Blog first posted the open letter in November, with six signatories (link to letter). At that time, Dr. Horton’s office responded that he would reply after returning from “traveling.” Three months later, we still had not heard back from Dr. Horton–perhaps he was still “traveling”–so we decided to republish it with many more people signed on.

The day the second open letter was posted, Dr Horton e-mailed me and solicited a letter from the group. (He did not explain where he had been “traveling” for the previous three months.) Here’s what he wrote: “Many thanks for your email. In the interests of transparency, I would like to invite you to submit a letter for publication–please keep your letter to around 500 words. We will then invite the authors of the study to reply to your very serious allegations.”

Dr. Horton’s e-mail clearly indicated that the letter would be published, with the PACE authors’ response to the charges raised; there was no equivocation or possibility of misinterpretation. In good faith, we submitted a letter for publication the following month, with 43 signatories this time, through The Lancet’s online editorial system (see the end of this article for a list of those who signed the letter). After several months with no response, we learned only recently by checking the online editorial system that The Lancet had flatly rejected the letter, with no explanation. No one contacted me to explain the decision or why we were asked to spend time creating a letter that The Lancet clearly had no intention of publishing.

I wrote back to Dr. Horton, pointing out that his behavior was highly unprofessional and requesting an explanation for the rejection. I also asked him if he was in the habit of soliciting letters from busy scientists and researchers that his journal had no actual interest in publishing. I further asked if the journal planned to reconsider this rejection, in light of the recent First-Tier Tribunal decision, which demolished the PACE authors’ bogus reasons for refusing to provide data for independent analysis.

Dr. Horton did not himself apologize or even deign to respond. Instead, Audrey Ceschia, the Lancet’s correspondence editor, replied, explaining that the Lancet editorial staff decided, after discussing the matter with the PACE authors, that the letter did not add anything substantially new to the discussion. She assured us that if we submitted another letter focused on the First-Tier Tribunal decision, it would be “seriously” considered. I’m not sure why she or Dr. Horton think that any such assurance from The Lancet is credible at this point.

The reasons given for the rejection are clearly specious. The letter for publication reflected the matters addressed in the open letter that prompted Dr. Horton’s invitation in the first place, and closely adhered to his directive  to outline our “serious allegations”. If outlining these allegations was not considered publication-worthy by The Lancet, it is incomprehensible to us why Dr. Horton solicited the letter in the first place. Perhaps it was just an effort to hold off further criticism for a period of months while we awaited publication of the letter, unaware of the journal’s intention to reject it. It is certainly surprising that The Lancet appears to have given the PACE authors some power to determine what letters appear in the journal itself.

Dr. Tuller’s investigation, based on the groundbreaking analyses conducted by many savvy patients and advocates since The Lancet published the first PACE results in 2011, has effectively demolished the credibility of the findings. So has a follow-up analysis by Dr. Rebecca Goldin, a math professor at George Mason University and director of Stats.org, a think tank co-sponsored by the American Statistical Association. In short, the PACE study is a sham, with meaningless results. In this case, the emperor truly has no clothes. Dr. Horton and his editorial team at The Lancet are stark naked.

Yet the PACE study remains in the literature. Its recommendation of treatments that are potentially harmful to patients–specifically, graded exercise therapy and cognitive behavior therapy, both designed specifically to increase patients’ activity levels–remains highly influential.

Of particular concern, the PACE findings have laid the groundwork for the MAGENTA study, a so-called “PACE for kids” that will be testing graded exercise therapy in children and adolescents. A feasibility study, sponsored by Royal United Hospitals Bath NHS Foundation Trust, is currently recruiting participants. It is, of course, completely unacceptable that any study should justify itself based on the uninterpretable findings of the PACE trial. The MAGENTA trial should be halted until the PACE authors have done what the First-Tier Tribunal ordered them to do–release their raw data and allow others to analyze it according to the outcomes specified in the PACE trial protocol.

Today, because of the urgency of the issue, we are posting on PubMed Commons the letter that The Lancet rejected. That way readers can judge for themselves whether it adds anything to the current debate.

Please note that the opinions in this blog post are mine only, not those of any of the other signers of the Lancet letter, listed below

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

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

Jonathan C.W. Edwards, MD
Emeritus Professor of Medicine
University College London
London, England, United Kingdom

Leonard A. Jason, PhD
Professor of Psychology
DePaul University
Chicago, Illinois

Bruce Levin, PhD
Professor of Biostatistics
Columbia University
New York, New York

Arthur L. Reingold, MD
Professor of Epidemiology
University of California, Berkeley
Berkeley, California

******

Dharam V. Ablashi, DVM, MS, Dip Bact
Scientific Director – HHV-6 Foundation
Former Senior Investigator
National Cancer Institute, NIH
Bethesda, Maryland

James N. Baraniuk, MD
Professor, Department of Medicine
Georgetown University
Washington, D.C.

Lisa F. Barcellos, PhD, MPH
Professor of Epidemiology
School of Public Health
California Institute for Quantitative Biosciences
University of California
Berkeley, California

Lucinda Bateman MD PC
MECFS and Fibromyalgia clinician
Salt Lake City, Utah

Alison C. Bested MD FRCPC
Clinical Associate Professor of Hematology
University of British Columbia
Vancouver, British Columbia, Canada

John Chia, MD
Clinician/Researcher
EV Med Research
Lomita, California

Lily Chu, MD, MSHS
Independent Researcher
San Francisco, California

Derek Enlander, MD, MRCS, LRCP
Attending Physician
Mount Sinai Medical Center, New York
ME CFS Center, Mount Sinai School of Medicine
New York, New York

Mary Ann Fletcher, PhD
Schemel Professor of Neuroimmune Medicine
College of Osteopathic Medicine
Nova Southeastern University
Professor Emeritus, University of Miami School of Medicine
Fort Lauderdale, Florida

Kenneth Friedman, PhD
Associate Professor of Pharmacology and Physiology (retired)
New Jersey Medical School
University of Medicine and Dentistry of NJ
Newark, New Jersey

Robert F. Garry, PhD
Professor of Microbiology and Immunology
Tulane University School of Medicine
New Orleans, Louisiana

Rebecca Goldin, PhD
Professor of Mathematics
George Mason University
Fairfax, Virginia

David L. Kaufman, MD,
Medical Director
Open Medicine Institute
Mountain View, California

Susan Levine, MD
Clinician, Private Practice
Visiting Fellow, Cornell University
New York, New York

Alan R. Light, PhD
Professor, Department of Anesthesiology
Department of Neurobiology and Anatomy
University of Utah
Salt Lake City, Utah

Patrick E. McKnight, PhD
Professor of Psychology
George Mason University
Fairfax, Virginia

Zaher Nahle, PhD, MPA
Vice President for Research and Scientific Programs
Solve ME/CFS Initiative
Los Angeles, California

James M. Oleske, MD, MPH
Francois-Xavier Bagnoud Professor of Pediatrics
Senator of RBHS Research Centers, Bureaus, and Institutes
Director, Division of Pediatrics Allergy, Immunology & Infectious Diseases
Department of Pediatrics
Rutgers – New Jersey Medical School
Newark, New Jersey

Richard N. Podell, M.D., MPH
Clinical Professor
Rutgers Robert Wood Johnson Medical School
New Brunswick, New Jersey

William Satariano, PhD
Professor of Epidemiology and Community Health
University of California, Berkeley
Berkeley, California

Paul T Seed MSc CStat CSci
Senior Lecturer in Medical Statistics
King’s College London, Division of Women’s Health
St Thomas’ Hospital
London, England, United Kingdom

Charles Shepherd, MB BS
Honorary Medical Adviser to the ME Association
London, England, United Kingdom

Christopher R. Snell, PhD
Scientific Director
WorkWell Foundation
Ripon, California

Nigel Speight, MA, MC, BChir, FRCP, FRCPCH, DCH
Pediatrician
Durham, England, United Kingdom

Philip B. Stark, PhD
Professor of Statistics
University of California, Berkeley
Berkeley, California

Eleanor Stein, MD FRCP(C)
Assistant Clinical Professor
University of Calgary
Calgary, Alberta, Canada

John Swartzberg, MD
Clinical Professor Emeritus
School of Public Health
University of California, Berkeley
Berkeley, California

Ronald G. Tompkins, MD, ScD
Summer M Redstone Professor of Surgery
Harvard University
Boston, Massachusetts

Rosemary Underhill, MB BS.
Physician, Independent Researcher
Palm Coast, Florida

Dr Rosamund Vallings MNZM, MB BS
General Practitioner
Auckland, New Zealand

Michael VanElzakker, PhD
Research Fellow, Psychiatric Neuroscience Division
Harvard Medical School and Massachusetts General Hospital
Boston, Massachusetts

Mark Vink, MD
Family Physician
Soerabaja Research Center
Amsterdam, The Netherlands

Prof Dr FC Visser
Cardiologist
Stichting CardioZorg
Hoofddorp, The Netherlands

William Weir, FRCP
Infectious Disease Consultant
London, England, United Kingdom

John Whiting, MD
Specialist Physician
Private Practice
Brisbane, Australia

Marcie Zinn, PhD
Research Consultant in Experimental Neuropsychology, qEEG/LORETA, Medical/Psychological Statistics
NeuroCognitive Research Institute, Chicago
Center for Community Research
DePaul University
Chicago, Illinois

Mark Zinn, MM
Research consultant in experimental electrophysiology
Center for Community Research
DePaul University
Chicago, Illinois

TWiV 404: Not found

From the twiVivants, follow up on FluMist and Zoster vaccines, Zika virus update, and isolation of a multicomponent animal virus from mosquitoes.

You can find TWiV #404 at microbe.tv/twiv, or listen below.

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dose-response-plaque-assayThere are many examples of viruses with segmented genomes – like influenza viruses – but these genomes segments are packaged in  one virus particle. Sometimes the genome segments are separately packaged in virus particles. Such multicomponent viruses are commonly found to infect plants and fungi, but only recently have examples of such viruses that infect animals been discovered (paper link).
Sequence analysis of viruses isolated from Culex mosquitoes in Central and South American Countries revealed six new viruses with segmented RNA genomes, which was confirmed by gel electrophoresis of RNA extracted from virus particles.
Some of the virus isolates appear to lack the fifth RNA segment, and the results of RNA transfection experiments indicated that this RNA is not needed for viral infectivity.
RNA viruses with segmented genomes are common, but in this case, the surprise came when it was found that the dose-response curve of infection for these viruses was not linear. In other words, one virus particle was not sufficient to infect a cell (illustrated). In this case, between 3 and 4 particles were needed to establish an infection. These findings indicate that the viral RNA segments are separately packaged, and must enter a cell together to initiation infection.
These novel viruses, called Guaico Culex virus (GCXV) are distantly related to flaviviruses, a family of non-segmented, + strand RNA viruses. They are part of a clade of RNA viruses with segmented genomes called the Jingmenvirus, which includes a novel tick-borne virus isolated in China (previously discussed on this blog), and a variant isolated from a red colobus monkey in Uganda. These viruses are also likely to have genomes that are separately packaged.
An interesting question is to identify the selection that lead to the emergence of  multicomponent viruses that require multiple particles to initiate an infection. Perhaps transmission of these types of viruses by insect vectors facilitates the introduction of multiple virus particles into a cell. How such viral genomes emerged and persisted remains a mystery that might be solved by the analysis of other viruses with similar genome architectures.

The TWiV team takes on an experimental plant-based poliovirus vaccine, contradictory findings on the efficacy of Flumist, waning protection conferred by Zostavax, and a new adjuvanted subunit zoster vaccine.

You can find TWiV #403 at microbe.tv/twiv, or listen below.

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poliovirusAlthough the use of the live, attenuated (Sabin) poliovirus vaccines has been instrumental in nearly eradicating the virus from the planet, the rare reversion to virulence of these strains has lead to the World Health Organization to recommend their replacement with inactivated poliovirus vaccine (IPV). Unfortunately IPV is also not without shortcomings, including high cost, failure to induce intestinal immunity, and the need to keep the vaccine at low temperatures. An experimental poliovirus vaccine produced in plants could overcome these problems.

A new vaccine candidate was made by producing the poliovirus capsid protein VP1 in the chloroplast of tobacco plants (nuclear-directed antigen synthesis is often inefficient). VP1 was fused to the cholera toxin B (CTB) subunit which allows good transmucosal delivery of the protein. Leaves were freeze dried, ground to a powder, mixed with saline and fed to mice after subcutaneous inoculation with IPV. The results show that boosting with the plant-derived VP1-CTB protein lead to higher antibody neutralizing titers (against all three poliovirus serotypes) both in the blood and in fecal extracts, compared with mice inoculated with IPV alone.

The VP1-CTP protein within lyophilized plant cells was stable for 8 months at ambient temperatures. If immunogenicity is maintained under these conditions, it would eliminate the need for a cold chain to maintain vaccine potency, an important achievement.

The authors propose that plant-produced VP1-CTP protein could substitute for IPV once the use of OPV is discontinued. Whether this suggestion is true depends on confirmation, by clinical trial, of these findings in humans. Furthermore, oral administration of VP1-CTP plant cells alone produces no serum neutralizing antibodies, and whether VP1-CTP boosts immunity in OPV recipients remains to be determined. Because VP1-CTP does not provide protection in children who have never received IPV or OPV, it cannot be used if poliovirus circulation continues indefinitely in the face of a growing cohort that has not been immunized with IPV or OPV. Nevertheless the technology has promise for the development of other vaccines that are inexpensive and do not need low temperature storage.

Polio returns to Nigeria, Zika virus spreads in Miami, and virus infection of plants attracts bumblebees for pollination, from the virus gentlepeople at TWiV.

You can find TWiV #400 at microbe.tv/twiv, or listen below.

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Non-fatal attraction

bee attractionVolatile organic compounds that are emitted by plants – which play important roles in attracting insects – can be altered by virus infection. Infection by cucumber mosaic virus (CMV) increases the emissions that attract aphids, but the virus-altered plant juices are not so tasty, so the insect quickly leaves to spread virus to other plants (listen to TWiV #70 for a full discussion of this finding). Plant pollinators can also respond to plant virus-induced volatile compounds (paper link)

Cucumber mosaic virus is a (+) strand RNA virus that is a pathogen of tomato plants. The virus is spread by aphids, and bumblebees increase fruit yield by buzz-pollination (tomato plants are self-pollinating but bumblebees cause increased seed production).

To study the attraction of bumblebees to tomato plants, the authors encased single tomato plants in a tower topped with a screen and a small cup of 30% sucrose (shown in photo: credit). This arrangement allowed volatile compounds to waft through the screen above the plant.

Bumblebees clearly preferred to visit the CMV-infected over the uninfected plants. They also shunned plants infected with a mutant of CMV that cannot antagonize the RNA interference system of the plant – a major defense against viruses. The results suggest that CMV somehow alters plant produced volatile compounds to attract bumblebee pollinators, and that production of these compounds is regulated by microRNAs.

These findings – that CMV infected plants attract bees via the production of volatiles under the control of microRNAs – were confirmed using Arabidopsis, a model plant which can be genetically manipulated.

Chemical analysis of the volatiles produced by infected and uninfected tomato plants revealed that virus infection reduced the levels of two terpenoids, 2-carene and beta-phellandrene. These compounds are known to repel bumblebees, leading to the suggestion that their reduction may explain why bees prefer infected plants.

Is attraction of bumblebees by viral infection random, or does it have a purpose? CMV infection of tomato plants decreases seed production, an effect that is reversed when bumblebees are attracted to the plants and pollinate them.

The results show that CMV infection of tomato plants causes the emission of volatile compounds that attract pollinating bumblebees, which negate the inhibition of seed production by infection.

Bumblebees do not transmit CMV, so how did this non-fatal attraction come about? One can imagine that at one time CMV infection of plants did not attract bumblebees, and that infection severely depressed plant production. Then a random viral mutant arose that could induce the proper volatiles to attract bumblebees. This change provided a selective advantage because plant seed production was restored by bumblebee pollination. This is called a ‘payback’ hypothesis: the virus has a place to replicate, and the plant lives on due to virus-induced volatile compounds.

I have a bit of trouble with this payback idea, because at the base of it are human-like intentions. Why do we assume that what drives evolution are forces that make sense to us? The plant-centric view is that it was not a CMV mutant, but a plant mutant that arose, which could make the proper bumblebee attractants upon CMV infection. The selective force to maintain such a plant is very clear and involves no anthropocentric ‘payback’.

Whatever the origin of this non-fatal attraction, it merits further study, not only for developing better pollination methods, but for understanding the viral-host symbiosis.

Zika Virus in the USA

On this episode of Virus Watch we cover three Zika virus stories: the first human trial of a Zika virus vaccine, the first local transmission of infection in the United States, and whether the virus is a threat to participants in the 2016 Summer Olympic and Paralympic Games.

Zika virus spreads in the USA, a Zika virus DNA vaccine goes into phase I trials, and how mosquito bites enhance virus replication and disease, from the friendly TWiFolk Vincent, Dickson, Alan, and Kathy.

You can find TWiV #401 at microbe.tv/twiv, or listen below.

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Biting mosquitoMosquito saliva, which is injected into the host as a mosquito probes for a blood vessel, contains a collection of chemicals which include anticoagulants to prevent blood clotting, vasodilators to keep blood vessels wide, and anesthetics to prevent us from sensing the mosquito. Saliva also contains components that enhance viral replication, dissemination, and pathogenesis by inducing an inflammatory response that inadvertently promotes infection by providing new cell targets for infection (paper link).

To separate the bite from virus inoculation, mice were first exposed to Aedes aegyptii mosquitoes, and then infected at the bite site with two different mosquito transmitted viruses, Semliki Forest virus or Bunyamwera virus. Mosquito bites caused more virus replication at the inoculation site, greater dissemination of virus, and more lethality compared with control mice that received only virus.

How does mosquito saliva enhance virus replication and dissemination? Part of the story is that as the mosquito probes for a blood vessel, it causes damage that leads to vascular leakage and accumulation of fluid (edema) which inhibits movement of virus to draining lymph nodes.

But delaying dissemination of virus alone does not promote infection and disease. Mosquito bites cause an infiltration of neutrophils (a type of white blood cell) into the bite site. The edema at the bite site is enhanced by neutrophils, because depleting these cells from mice greatly reduced edema. This depletion also returned viremia to levels observed in unbitten control mice, and restored dissemination of virus to draining lymph nodes. Neutrophils are not susceptible to infection with Semliki Forest virus, and therefore cannot explain the increase in virus replication at the bite site.

Enhanced virus replication in the skin occurs because the neutrophils elaborate chemokines that attract macrophages, which can be infected by Semliki Forest virus and Bunyamwera virus. One of the chemokines produced by neutrophils that is a macrophage attractant – CCL2 – binds a receptor on macrophages. Mice lacking the gene encoding the CCL2 receptor are protected from bite enhancement of Semliki Forest virus enhancement.

When a mosquito bites a host, it delivers saliva along with a virus. The saliva induces an inflammatory response and attracts neutrophils into the bite site. The resulting edema holds virus at the bite site until chemokines produced by neutrophils attract macrophages, which are then infected. The virus produced disseminates widely, reaching secondary tissues and causing disease.

It seems likely that the ability to replicate in macrophages that are recruited to the bite site is a property that was selected during evolution of mosquito-transmitted viruses. By replicating in macrophages, the amount of virus in the blood is increased, as well as the likelihood that the virus will be picked up by another mosquito and transmitted to a new host – a powerful selection mechanism. The down side – increased disease in the mammalian host – is an accidental side effect.

Think about that the next time you are scratching that raised bump on your skin caused by a mosquito bite.