By David Tuller, DrPH

Earlier today, I e-mailed the following letter to Sue Paterson, University of Bristol’s Director of Legal Services, to clarify whether or not I had been sent a cease and desist letter (to cease and desist what, exactly?). Professor Esther Crawley made this claim at her public talk last Friday. I have never received any such letter. I cc’d the office of the university’s vice-chancellor, Professor Hugh Brady.


Dear Ms. Paterson—

I attended Professor Esther Crawley’s interesting talk at the University of Exeter on Friday, November 17th. During the question period, I introduced myself and politely asked her why she had accused me of libel and then failed to respond to my e-mail requests that she document this serious allegation. I was immediately asked to leave the room, which I did promptly and without fuss.

Before I left, however, Professor Crawley told me this: “You have been so unbelievably defamatory and unprofessional that I had to get my university to send you a cease and desist letter.“ As with her false charge of libel, she provided no documentation or evidence that anything I wrote was inaccurate or in error—which is of course necessary to sustain an accusation of defamation. (In contrast, calling me “unprofessional” is obviously well within Professor Crawley’s rights, even if I disagree.)

More significantly, her statement that Bristol had sent me a cease and desist letter was certainly news to me. I have received no such letter from Bristol, either by e-mail or by post. In our previous e-mail exchange involving my complaint to Bristol about Professor Crawley’s public accusation of “libellous” blogging, you made no reference to a cease and desist letter. Nor did you mention having sent such a letter in our more recent e-mail exchange involving my freedom of information request related to Professor Crawley’s ethically challenged school absence study.

I am writing now because it is obviously important to clarify this matter and set the public record straight as soon as possible. Did Bristol send me a cease and desist letter, or not? If Bristol sent me a letter that I did not receive, please let me know exactly when it was sent, where it was sent, and the name of the person who signed for it upon receipt, if anyone did. And please e-mail a copy to me immediately, so I understand what it is that Bristol expects me to cease and desist from. If Bristol did not send me a cease and desist letter, I suggest you inform Professor Crawley of that fact.

In our brief exchange, Professor Crawley also mentioned the notion of consulting with “the police” in relation to my activities. The clear implication to those attending the event was that I have engaged in behavior that poses some sort of danger or threat to Professor Crawley. Any objective observer of the situation would recognize that this implication is not only utterly preposterous but, like her unwarranted libel and defamation accusations, potentially harmful to my professional reputation. Last time I checked, writing vigorous commentary, seeking explanations for unsupported libel charges, and appearing at a public lecture to ask a polite but tough question are not criminal activities in the U.K.—even if Professor Crawley might wish that they were.

As I suggested in a previous e-mail to you, someone from Bristol’s legal department should sit down with Professor Crawley and explain very, very clearly that accusing people–and in this case me–of being “libellous” and “defamatory” without providing an iota of evidence of error or inaccuracy is not acceptable. She has now done this in public on at least three separate occasions. She is certainly skating on thin legal ice.

I have repeatedly invited Professor Crawley to send me her full response to my criticisms, promising to post it on Virology Blog at whatever length she chooses. I have also repeatedly offered to correct any mistakes or errors that she can document. It is obviously her decision whether or not to take me up on this, but her refusal to do so suggests that she prefers to insult critics rather than engage in robust debate about the self-evident and glaring flaws in her work.

I have stated before that I have no plans to sue Professor Crawley. But I also have no plans to cease and desist from analyzing and commenting on her research and her public presentations as I see fit. I have every right, as a journalist and public health academic, to make my case as forcefully as I choose. Professor Crawley obviously has every right to find my approach unpleasant, distasteful and offensive. But she apparently has serious difficulty distinguishing between opinions she dislikes and statements that are libelous and defamatory. I trust Bristol’s legal department is not operating under similar misconceptions or delusions.

In summary, if Bristol has previously sent me a cease and desist letter, please forward me a copy immediately. If not, I would certainly like an explanation as to why Professor Crawley would make such a false statement at a public event.


David Tuller, DrPH
Senior Fellow in Public Health and Journalism
Center for Global Public Health
School of Public Health
University of California, Berkeley

By David Tuller, DrPH

At noon last Friday, at the University of Exeter’s Mood Disorders Centre, Professor Esther Crawley gave a talk called “What is new in paediatric CFS/ME research.” When I saw a notice about the event the day before, I felt it might be my one chance to ask her directly about her concerns regarding my work and her accusation that I was writing “libellous blogs.” (If she were American, she would presumably have accused me of writing “libelous–one L–blogs”).

I also hoped to gain insight into some other issues that have troubled me: why she still believes PACE was a “great, great” trial, why her prevalence studies use “chronic fatigue” as a proxy for “chronic fatigue syndrome,” why she is not concerned about bias given that she conducts non-blinded studies relying on subjective rather than objective primary outcomes, etc.

So many questions! 
I’d heard that Professor Crawley is a compelling presenter with a strong dose of what many characterize as charm. This, I found out, is true. Unfortunately, she compellingly and charmingly presented a series of misleading arguments. The talk did not include any discussion of the obvious: that the biopsychosocial approach she has championed is losing ground rapidly, both internationally and in the U.K. Instead, she claimed that 22 percent of adults “recover” with treatment–without citing the reanalysis of the PACE trial data that demolished that claim.

She stated that two percent of teenagers have what she calls “CFS/ME”—even though other experts, like Johns Hopkins pediatrician Peter Rowe, consider this estimate to be wildly inflated because it is based solely on reports of chronic fatigue, with no clinical examinations to rule out other causes and no evidence of post-exertional malaise. She referenced the 2007 NICE guidelines, which she helped develop, as support for the rehabilitative treatments she favors–without mentioning that NICE has rejected the advice to reaffirm those guidelines and is instead planning a “full update” to replace the current version.

Professor Crawley did not explain why her much-hyped MEGA project, sponsored by the CFS/ME Research Collaborative, failed in major bids for funding from both the Wellcome Trust and the Medical Research Council. Also unmentioned was the U.S. National Institutes of Health’s decision to grant $2 million to MEGA’s rival, the highly regarded U.K. ME/CFS Biobank. At the CMRC conference in September, before the announcement of that NIH grant, Professor Stephen Holgate, the CMRC chair, had spoken with hopeful enthusiasm about an upcoming meeting with the U.S. agency.

Poor Professor Holgate. He appears not to understand that Professor Crawley might have caused irreparable damage to her professional reputation with some of her recent behavior, such as accusing legitimate critics of being vexatious and libelous and appearing to advise other researchers on how to evade their obligations under the U.K.’s freedom of information laws. Perhaps Professor Holgate also does not realize how much the CMRC itself might have undermined its own credibility by its willingness to overlook and condone such antics.

And perhaps he and Professor Crawley are not aware that U.S. health officials interested in ME/CFS, not just patients and advocates, might sometimes read Virology Blog to keep current with developments in the field. The American public health establishment has now rejected the PACE trial; this summer, the Centers for Disease Control removed the recommendations for GET and CBT from its website. Scientists who passionately defend the “greatness” of PACE and refuse to acknowledge that it has been effectively debunked should therefore not be too surprised if U.S. agencies reject any funding requests. 
In her Friday talk, Professor Crawley failed to mention new research documenting significant physiological dysfunctions among patients, such as the recent Newcastle University study indicating defective energy production in the mitochondria. She talked mostly about her own research but didn’t explain why anyone should trust open-label trials with subjective outcomes, like FITNET-NHS, her online CBT trial for kids. When she discussed her recently published SMILE study of the cult-like Lightning Process, she didn’t mention that even Action For ME, her close ally among the patient advocacy groups, raised real concerns about the trial and its findings.

Professor Crawley repeated the tiresome meme about the “harassment” purportedly faced by researchers in the field, supporting her point with screen shots of some of the overwrought and anti-patient articles that have appeared in the U.K. press in recent years. She did not mention that the First-Tier Tribunal last year sharply dismissed claims that patients had engaged in a campaign of threats against the PACE investigators. On a positive note, I’m pleased to report that Professor Crawley has apparently retired the slide that accused me of writing “libellous blogs.” Perhaps Bristol’s lawyers have informed her that making such a reckless, unsupported charge in high-profile public venues, including her inaugural lecture, puts her on slippery legal ground.

Last spring, after I first learned about the libel accusation, I e-mailed Professor Crawley repeatedly, seeking an explanation. In my e-mails, I offered to post her full response to my criticisms, at whatever length she wanted, on Virology Blog. I also offered to correct any mistakes she could document–something I do routinely, as a responsible journalist, even when I am not being accused of libel. I stopped e-mailing her after it became clear that she had no intention of ever responding or explaining anything. As a courtesy, however, I made sure to keep her colleagues on the CMRC leadership committee informed by sending them my blog posts about her activities.

(Even at this late date, I am happy to re-extend to Professor Crawley my longstanding offer to post on Virology Blog her full response to my concerns, along with her documentation of any inaccuracies or errors that would justify her libel accusation.)

At her Friday event, Professor Crawley spoke for about 45 minutes. Then she stopped and waited for questions. I raised my hand and introduced myself. When she heard my name, she looked seriously glum.

“Hi, David,” she said.

“Hi, Esther,” I responded.

Then things happened quickly and in a bit of a blur, so my memory of events might not be 100 percent accurate. I started asking about the bogus 22 percent “recovery” figure. She interrupted and asked in a tone of some dismay if I’d come all this way just to see her talk. I told her that I was already in the U.K., and that I had friends in Exeter. Sensing that my time for asking questions might be cut off, I dropped the 22 percent issue and asked why she had accused me of libel and then failed to explain herself.

At that point, Professor Crawley indicated that she was going to stop the talk; I guess she doesn’t mind attacking critics from the podium but cannot tolerate their presence in her audience. She said that my work has been unbelievably “defamatory”–but, as in the past, she did not cite any errors or inaccuracies that would warrant such a label. She also declared, if I heard her correctly, that Bristol University had sent me a cease and desist letter on her behalf. That was certainly news to me.

In fact, at the Invest in ME conference in June, I had heard that Professor Crawley wanted Bristol to send such a letter. I wasn’t sure what exactly I was supposed to “cease and desist” from. Exercising my right, as a public health professional and journalist, to investigate and express my opinions about Professor Crawley’s research? In any event, I have never received a cease and desist letter from Bristol. (Maybe it got lost in the mail. The U.S Postal Service can be very inefficient. Note to Bristol University: E-mail is more reliable.)

After this quick exchange, the moderator of the event stepped in and suggested that we were engaged in a private dispute beyond the scope of the presentation. I noted that it was most certainly not a private dispute, since Professor Crawley had leveled her libel accusation in public. Next, the moderator asked me to leave. I stood up, grabbed my bag, and left without a fuss.

I assume there was a lively discussion afterwards. I also assume Professor Crawley will use this incident to once again portray those who disagree with her as belligerent and vexatious. To be clear: My question was by nature tough and presumably unpleasant for Professor Crawley, but I spoke in a calm and reasonable tone. Perhaps, in retrospect, I should have asked about the flaws in her own research or stuck with my 22 percent question. Whatever. In the end, I asked the question I really wanted to ask, so I felt I had attained my objective.
So what’s the upshot? I believe that Professor Crawley wants to bully those who raise concerns about her work into silence rather than engaging in robust debate about the very real methodological and ethical problems with her studies. She also refuses to acknowledge what many experts have now recognized: The GET/CBT treatment approach for ME/CFS, based on the deconditioning/fear-of-exercise hypothesis, is scientifically bankrupt and is crumbling under the weight of its own absurdity. This isn’t happening fast enough for me and the patient community, but the trajectory of developments is obvious.

At this juncture, it would be fruitless to make further efforts to engage with Professor Crawley, either via e-mail or by attending another one of her performances. She’s expressed her thoughts about my work, I’ve expressed my thoughts about hers, and we have no common ground. But I will, of course, continue to press my arguments and opine on her research and activities as I see fit. Stay tuned!

TWiV 468: Zika by the slice

Amy Rosenfeld joins the TWiV team to talk about her career and her work on Zika virus neurotropism using embryonic mouse organotypic brain slice cultures.

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Lymphocytes after dark

B cell

When you are infected with a microbe, pieces of the pathogen are picked up by sentinel dendritic cells and brought to local lymph nodes. There the sentinels present their gifts to lymphocytes – B (pictured; image credit) and T cells – who then decide if they are foreign, in which case an immune response begins. These lymphocytes circulate throughout the body not continuously, but in a circadian manner – a 24 hour cycle.

In mice, lymphocyte numbers peak in lymph nodes about one hour after lights are turned off. During the day, these cells leave the lymph nodes. However, the same pattern was observed when mice were kept entirely in the dark, showing that the change in lymphocyte numbers is due to an endogenous circadian clock, not a response to a change in lighting.

The homing of lymphocytes into lymph nodes depends on a number of different proteins, including adhesion molecules and attractants. The levels of these proteins also peaked at night. When the receptor for one attractant, CCR7, was genetically ablated, no oscillations of lymphocyte numbers in lymph nodes was observed.

Dendritic cells, the major antigen presenting cells, are also more numerous in lymph nodes during night hours. This observation makes sense given that dendritic cells present antigen to lymphocytes in the lymph node.

Circadian rhythms are under the control of clock genes – the discovery of which was recently recognized by the Nobel Prize in Physiology or Medicine. Deletion of the gene encoding one such clock gene in B or T cells of mice caused loss of the rhythmicity of lymphocytes in lymph nodes. In these mice, the levels of CCR7 mRNAs also lost their rhythmic character. These observations show that circadian clocks regulate lymphocyte migration.

Earth has a 24 hour day and life has adapted this cycle by turning genes on and off only when needed. It seems reasonable that lymphocyte trafficking into lymph nodes would peak during the hours when animals are moving about and potentially encountering pathogens. But not all animals are diurnal – active during the day. Mice are nocturnal, a behavior that probably helps to avoid predators. It makes perfect sense that lymphocyte trafficking in mice peaks at the onset of night.

It seems likely that in humans, a diurnal species, lymphocyte numbers peak in lymph nodes during the day. Consistent with this idea, it has been shown that in humans, vaccination in the morning produces higher antibody titers compared with vaccination in the afternoon. The take away message is clear: do not get your vaccines at night!

By David Tuller, DrPH

Something’s weird over at BMJ Best Practice, a resource for clinical decision-making and an arm of the BMJ Publishing Group. Two days ago, Steven Lubet and I posted a blog praising the new guide written by Dr. James Baraniuk and apparently reviewed by Peter White, along with two other experts.

First, I want to acknowledge that many patients disagreed with us about the merits of the guide. We assessed this document based on how much better it was than previous terrible U.K. guidelines, like those from NICE–not based on how far it was from perfection. It was our understanding as well that this document would continue to be reviewed and revised, presumably for the better.

Are there passages that still read like verbiage from an earlier biopsychosocial-oriented version and would best be edited out? Yes, definitely. Should we have cited these and therefore been more equivocal in our comments? Perhaps. But the bottom line for me remains this: The guide contains very clear statements that CBT and GET are not indicated for patients with PEM and other core symptoms of ME, and it presents the illness as a serious physiological disease and not a psychological disorder. And that’s a big, big step forward, whatever the shortcomings. (Given the history, I recognize that my perspective on the matter might be too optimistic. I hope not.)

As we noted in an update yesterday, the document we reviewed was dated July 31, 2017. Shortly after the blog was posted, we learned that a more recently updated version of the guide, dated November 13th, did not include Professor White’s name as a reviewer. We have so far been unable to find out why or when that happened. Now that he is retired, his former e-mail address from Queen Mary University of London is no longer operative, so reaching him directly is a challenge.

Moreover, thanks to sharp-eyed patients, some other anomalies were soon revealed. Besides the removal of Professor White’s name as a reviewer, the new version of the guide has at least one other major change. In a section at the end on “Evidence Scores” (p. 58 in the July version of the guide and p. 63 in the November version), the following sentence has been deleted from the later edition:

“Graded exercise therapy and overall improvement: there is poor-quality evidence that graded exercise therapy results in greater overall improvement in symptoms and functioning.

Evidence level C: Poor quality observational (cohort) studies or methodologically flawed randomized controlled trials (RCTs) of <200 participants.”

No additional text was included to replace this assessment of the effect of GET on overall improvement. Since the new version is several pages longer than the July version, I assume other sections have also been changed, but I have not had time to compare the documents at length. Unfortunately, given the track record of the U.K. medical publishing establishment, I assume other changes might also have served to water down the evidence against CBT and GET.

Finally, I was also sent a three-page BMJ Best Practice patient leaflet purportedly drawn from the larger guide. This leaflet is dated November 13th, like the later version of the guide. Unlike the guide itself, the leaflet appears to portray chronic fatigue syndrome as a condition largely characterized by extended “tiredness.”

The leaflet only mentions in passing any possible physiological underpinnings. It does not discuss the lack of legitimate evidence supporting cognitive behavior therapy and graded exercise for patients with the core symptoms of ME, or that the latter especially is contra-indicated because of the symptom of post-exertional malaise. It says nothing about the enormous problems created by use of the Oxford criteria and other loose case definitions. Like the PACE trial itself, it is a piece of crap.

So BMJ Best Practice, what’s going on here?

Jon and Teddy Yewdell join the TWiV team to talk about their careers, their research, and the problems with biomedical research.

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By Steven Lubet, JD, and David Tuller, DrPH

Update: Nov 13th

We wrote this post based on a version of the guide that appeared recently and had been updated on July 31, 2017. That version lists Peter White as among three peer-reviewers. As commenters pointed out soon after the post went up, however, Peter White’s name is no longer on the peer-review list in the currently posted version of the guide, which indicates that it was updated this month. We are trying to determine when and why this change occurred.


Something has changed. 
That’s the only explanation for the recent publication of a “Best Practice” guide for “chronic fatigue syndrome” (behind a paywall, unfortunately) from the BMJ Publishing Group. This thing is good. It’s very good, in fact. One bottom line at this stage for any treatment guide is the following: Would it lead a clinician to prescribe cognitive behavior therapy or graded exercise therapy for patients with ME, as opposed to those suffering from a vague fatiguing illness? The answer here is an unequivocal no.
The guide doesn’t refute the PACE trial by name. It doesn’t have to. With its strong emphasis on the many physiological dysfunctions that characterize the illness, the guide represents a refutation not only of PACE itself but of the deconditioning/fear-of-exercise hypothesis—the foundational myth of the CBT/GET cult. It is hard to imagine that any organization associated with the British medical and academic establishment would have published this document just two years ago, before patients’ concerns about the PACE trial mushroomed into an international scientific scandal. Someone over at BMJ obviously understands that the field has moved beyond the simplistic and unproven claims of the biopsychosocial field.
The author, Dr. James Baraniuk, is an immunologist at Georgetown University and an expert on ME/CFS as well as Gulf War Illness. (Just this month, in a study in the journal Scientific Reports, he and a colleague reported distinctive molecular patterns in the cerebrospinal fluid of chronic fatigue syndrome patients, Gulf War Illness patients, and healthy controls after a round of exercise.) The best practice guide’s peer reviewers were Rosamund Vallings (Howick Health and Medical Centre, New Zealand), Abhijit Chaudhuri (Queen’s Hospital, Essex, UK), and, surprisingly, PACE lead investigator Peter White. Recommending an article for publication does not necessarily indicate agreement with its conclusions, but Professor White still deserves some credit for apparently approving this document, since it essentially demolishes the claims he and his colleagues have made for decades. 
Let’s be clear: While it was The Lancet that published the first PACE results, The BMJ has also failed to distinguish itself in the debate. Just one example: When the first PACE results were published, The BMJ stated in a news report that 30 percent of the participants receiving CBT and GET had been “cured”—an obvious misinterpretation of the trial’s findings that BMJ editors have never bothered to correct.

In the best practice guide, Dr. Baraniuk repeatedly makes the following salient points: 
*”Post-exertional malaise” or “exertional exhaustion” is the distinguishing characteristic of the disease; as noted in the guide’s summary, this symptom is a possible result of “autoimmune and metabolomic dysfunction that reduces mitochondrial ATP production.” 
*Oxford criteria studies should not be used to determine treatment approaches to patients identified by more narrow case definitions, such as for ME, that require post-exertional malaise and other core symptoms.
*Cognitive behavior therapy and graded exercise therapy are not indicated for patients with narrowly defined illness, whether or not these treatments might be indicated for patients with idiopathic chronic fatigue or fatigue arising from psychological causes. 

Dr. Baraniuk’s report therefore rejects CBT and GET as effective therapies for appropriately diagnosed ME/CFS patients, as the following quotes indicate:

*[T]he prospect that CBT can change the illness beliefs of a patient, and that graded activity can reverse or cure CFS, is not supported by post-intervention outcome data.

*There is widespread concern among CFS physicians that mandated exercise programmes can cause significant patient deterioration because of the exercise-induced musculoskeletal pain, neurocognitive impairment, weakness, and prolonged bed rest patients may require to recover from them.

*[I]n routine medical practice CBT has not yielded clinically significant long-term benefits in CFS.

This report puts the National Institute for Health and Care Exellence (NICE) to shame. NICE develops clinical guidelines that are widely followed in the U.K, and in other countries as well. A NICE surveillance team had the opportunity to review the same recent literature available to Dr. Baraniuk and recommended in June that the agency should make no changes to its 2007 guidance—which of course highlights CBT and GET as effective treatments.

In September, after patient organizations expressed overwhelming opposition to this recommendation, NICE rejected it and announced that the 2007 guidance would instead undergo a complete overhaul. The new NICE committee that will be tasked with developing fresh guidelines should thank Dr. Baraniuk for providing them with a pretty good road map.

It would be fascinating to learn the back-story of this report. What did the BMJ Publishing Group expect when Dr. Baraniuk took on this assignment? What did Professor White really think during his peer-review of a document that inflicts such serious damage on his legacy? Do Professor White and his colleagues finally recognize that the larger scientific community has rejected their flawed trial and that their long reign over this domain of research is finally coming to an end?

It is too soon to tell whether Dr. Baraniuk’s report will turn out to be an inflection point in the British understanding and treatment of ME/CFS. Old prejudices (and paradigms) die hard, and the PACE-influenced U.K. medical and academic establishment is deeply entrenched. Nonetheless, it may soon become impossible for anyone to continue to argue that valid research can be based on the Oxford criteria or that “best practices” can embrace the discredited CBT/GET model.

Written with Amy Rosenfeld, Ph.D.

By infecting organotypic brain slice cultures from embryonic mice, we have shown that Zika virus has always been neurotropic. The same culture system provides information on how Zika virus infection of the developing brain might lead to microcephaly.

The small heads observed in microcephalic children reflect a physically smaller brain – specifically, the neocortex is thinner than in a normal brain. The neocortex, only found in mammals, is the largest part of the cerebral cortex of the brain. It is composed of six distinct layers of neurons, which are established during embryonic development (illustrated below). First, glial cells originating from progenitor cells in the ventricular zone extend their processes throughout the cortex and anchor at the pia, the outer surface of the brain. These long fibers provide a scaffold on which neurons, produced from the same progenitor cells, migrate outwards to establish the six layers of the cortex. Movies have been made that show the migration of neurons on glial fibers, and they are amazing.

embryonic brain development

The glial fibers are visible as parallel tracks in our embryonic brain slice cultures stained with an antibody to vimentin, a protein component of the fibers (image below, left panel). When embryonic brain slice cultures were infected with Zika virus, the structure of the glial tracks was altered. Instead of parallel tracks, the fibers assumed a twisted morphology that would not allow neurons to travel from the ventricular zone to the developing neocortex (image below, right panel). Disruption of glial fibers was observed after infection with Zika viruses isolated from 1947 to 2016.

Zika fiber disruption

Image credit: Rosenfeld AB et al.

To determine if Zika virus-mediated disruption of glial fibers could impair neuronal migration, we isolated brains from embryonic mice as described above, but before virus infection, a plasmid encoding green fluorescent protein was injected into the ventricle. An electrical current was then applied to the brain to encourage uptake of the plasmid in neuronal progenitors lining the ventricle. The brains were then sliced, placed in culture, and infected with Zika virus.

Four days later, in the uninfected brain slices, green fluorescent neurons could be seen in the ventricular zone, and some of these had already migrated through the developing cortical plate to the pial surface. In brain slices infected with Zika virus, the migration of green neurons to the cortical plate was impaired, and the cells remained in the area where the plasmid was injected. This observation indicates that the disruption of glial fibers caused by Zika virus infection has caused fewer neurons to reach the cortical plate.

We think it is likely that Zika virus disruption of glial fibers during embryonic development contributes to microcephaly: if neurons cannot migrate to the pial surface, the neocortex will be thinner. Zika virus infection also inhibits the proliferation of progenitor cells that line the ventricular surface, which is likely a contributing factor to microcephaly. Other embryonic brain cells are infected with Zika virus, and these could play a role in microcephaly. Furthermore, there are other effects of Zika virus infection on the developing brain, including calcifications, hypoplasia (reduced cell density), lissencephaly (smooth brain), ventriculomegaly (enlarged ventricle), and brainstem dysfunction.

We are particularly interested in identifying the viral protein that disrupts the glial fibers in embryonic mouse brains. Once that protein is identified, it might be possible to understand the mechanisms by which the glial fibers are disrupted. Such information would not only lead to a better understanding of how Zika virus causes microcephaly, but should also provide a better understanding of how the brain develops.

TWiV 466: The Capsid Club

From Indiana University, Vincent and Kathy speak with Tuli Mukhopadhyay, John Patton, and Adam Zlotnick about their careers and their work on alphaviruses, hepatitis B virus, and rotaviruses.

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Third trimester embryonic mouse brains

Written with Amy Rosenfeld, Ph.D.

Zika virus has been infecting humans since at least the 1950s (and probably earlier), but epidemics of infection have only been observed in the past ten years and congenital Zika syndrome in the last two. Two hypotheses emerged to explain this new pattern of disease: evolution of the virus, or random introduction into large, immunologically naive populations. Results from our laboratory show that one component of these disease patterns – neurotropism, the ability to infect cells of the nervous system – has always been a feature of Zika virus.

If evolution has selected for Zika viruses that cause epidemics and congenital neurological disease, there are many steps in the infection pathway that could be affected. Let’s focus on the ability of Zika virus infection during pregnancy to cause microcephaly. Mutations that affect multiple stages of infection might be responsible. These could include any or all of the following:

  • Mutations that increase viremia in the human host, increasing the likelihood that virus will be captured by a mosquito taking a blood meal.
  • Mutations that increase viral replication in the mosquito vector.
  • Mutations that increase the ability of the virus to cross the placenta.
  • Mutations that allow efficient replication in the fetus.
  • Mutations that promote virus entry of the nervous system (neuroinvasion).
  • Mutations that enhance replication in neural cells (neurotropism).

This list is by no means exhaustive. The point is that no small animal model is likely to capture all of these steps. For example, no mouse model of Zika virus infection has so far lead to the development of microcephalic offspring. Therefore testing whether any of the the mutations observed in different Zika virus isolates are responsible for new disease patterns is likely impossible.

We have chosen to look at the question of how Zika virus disease has changed by looking at a very specific part of the replication cycle: growth of the virus in fetal brain, specifically in organtypic brain slice cultures. Here’s how it works: we remove the developing embryos from pregnant mice during the first, second or third trimesters of development (see photo). The fetal brain is removed, sliced (slices are about 300 nm thick), are placed into culture medium. The slices live up to 8 days, during which time brain development continues. The Vallee laboratory here at Columbia has used a similar system utilizing rats to study the genetic basis of microcephaly.

Next, we infect the embryonic brain slices with different isolates of Zika virus from 1947 to 2016, from Africa, Asia, South America, and Puerto Rico. All of the isolates replicated in brain slice cultures from the first and second trimesters of development. These observations show that Zika virus has been neurotropic since at least 1947. Similar observations have been made with the 1947 isolate using human neurospheres, organoids, and fetal organotypic brain slice cultures.

The incidence of microcephaly is greatly reduced when mothers are infected during the third trimester of development. Consistent with this observation, we found that organotypic brain slice cultures from the third trimester of mouse development support the replication of only two of seven Zika virus isolates examined – the original 1947 isolate from Uganda, and 2016 isolate from Honduras. Furthermore, these viruses replicate in different cells of the third trimester embryonic brain compared with second trimester brain. We are interesting in identifying the changes in the virus responsible for these differences.

Our approach asks only whether different Zika virus isolates can infect brain cells when the virus is placed directly on these cells. We cannot make any conclusions about the ability of the virus to invade the brain from the blood (neuroinvasion), or any of the other steps in infection listed above.

Our experimental system also reveals how Zika virus infection of the developing brain might lead to microcephaly, a topic that we’ll explore next week.