The Zika Effect

Zika virusHaving worked on viruses for over 40 years, I know a fair number of people in the field, and I am amazed at how many of them have started to work on Zika virus. What exactly is attracting virologists to this emerging virus?

There are probably many reasons why Zika virus would be of interest to a research lab – what I call the Zika Effect – but here are what I think are the three main factors.

First, Zika virus has become medically important in the past year, as it has spread globally and is infecting many people each day. There are many unanswered questions about the virus, and for a scientist, there is nothing better than unanswered questions (except maybe getting money to answer the questions – see below). Because the virus is causing human disease, these questions have an immediacy – such as, does the virus cause birth defects; does the virus cross the placenta, and if so, how; how does the virus enter the central nervous system and cause disease, to name just a few. Because of the nature of Zika virus infection, the virus has attracted not only virologists, but neurobiologists, cell biologists, developmental biologists, and structural biologists. In short: scientists love answering questions, and when it comes to Zika virus, they are not in short supply.

Second, Zika virus is not dangerous to work with – a biosafety level 2 laboratory (BSL-2) is all that is needed. Most virologists carry out their work under BSL-2 containment, so if you are working on influenza virus, poliovirus, herpesvirus, and a host of other viruses, you are ready to work with Zika virus. This situation is in contrast to that which took place in 2015 with the ebolavirus outbreak in west Africa. Work on ebolavirus must be conducted under BSL-4 containment – which few virologists have access to (for a look inside a BSL-4 laboratory, check out the documentary Threading the NEIDL). Consequently far fewer laboratories began work on ebolaviruses after that outbreak.

The third reason for the Zika effect is the reward: the promise of a publication in a high profile scientific journal, a promotion, a new job, and new grant funding for the laboratory. Not the purest motivation, but a reality: in the United States, government funding of scientific research has been flat for so many years that any new opportunity is seized. Many laboratories are on the brink of extinction and reach out to any funding opportunity. Few will admit that funding or publication drives their interest in Zika virus, but there is no doubt that it is a major factor. If research money were plentiful, and if luxury journals were not so tightly linked to career success, there would likely be fewer entrants in the Zika race. And a race it is – at least in these early days, when low-hanging fruit is ripe for picking, papers roll out on a weekly basis and it is difficult to compete without a large research group.

The fact that so many laboratories are working on Zika virus is not only impressive but encouraging: it means that the scientific establishment is flexible and nimble. There is no doubt that the more minds engaged on a problem, the greater the chance that important questions will be answered. But working on Zika virus is not for the faint of heart – which I document on a weekly basis in Zika Diaries, a personal account of our foray into this seductive virus.

TWiV 390: Building a better mosquito trap

TWiVProject Premonition, a Microsoft Research project that uses drones to capture mosquitoes and analyze them for pathogens, preprint servers, and three mouse models for Zika virus induced birth defects are the topics of episode #390 of the science show This Week in Virology.

You can find TWiV #390 at microbe.tv/twiv, or listen below.

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Virus Watch: Zika virus and microcephaly

Three papers have been published showing that Zika virus can cross the placenta in mice, replicate in the fetus, and cause microcephaly. In this video from Virus Watch I summarize these data and their implications.

Science publishing has a Zika problem

Zika virusScience publishing has a problem. I agree with Nobel Laureate Randy Schekman, who wrote that prestigious science journals like Cell, Nature, and Science – which he calls ‘luxury journals’ – are damaging science.  The succession of articles on Zika virus nicely illustrates this problem.

The big three in science publishing – Science, Nature, and Cell – have published many papers on Zika virus since the beginning of 2016. Many of these have had a turnaround time of a week or two – the time between when the papers were submitted, and when they were published online. A rapid turnaround time is unusual, and not compatible with proper peer review of the work. Indeed, many of the papers have been clearly rushed into print, and lack proper controls and clear explanations of what has been done.

The recent publication in Cell Host & Microbe of a description of an infectious DNA clone of Zika virus is a perfect illustration of the problem with luxury journals. Infectious DNA clones of viral genomes are nothing new – the first were described the late 1970s and 1980s. They are important reagents, allowing manipulation of the viral genome to study replication and pathogenesis. But publishing a reagent has never been enough to get into a high profile journal.

As a postdoctoral fellow with David Baltimore in 1981, I was fortunate to publish the first report of an infectious DNA clone of an animal virus – poliovirus – in Science (At the time there were no luxury journals. Years later Nobel Laureate Paul Berg asked why we chose to publish in such a lowly journal). A few years later, I submitted a paper to the Journal of Virology describing the construction of an infectious DNA clone of a different serotype of poliovirus which had the unique ability to infect mice. The paper was rejected because, I was told, it didn’t contain any new results.

The first infectious DNA clone of a calicivirus – the family that includes noroviruses, agents of human gastroenteritis – was published in 1995 in Virology. The senior author told me the paper was rejected from the Journal of Virology because an infectious clone is ‘just a tool’.

The Journal of Virology is a solid journal that publishes many important articles in the field. But no one would mistake it as a luxury journal.

Some infectious DNA clones of viruses have been published in prominent journals – for example, Ebolavirus and influenza virus in Science (2000 and 2001). Zika virus is a flavivirus, and the first infectious DNA of a member of this virus family was for yellow fever virus, published 27 years ago in PNAS. Subsequently there have been many reports of infectious DNA clones of other flaviviruses, notably, West Nile virus, published in Virology in 2001. This virus, which entered the United States, gained quite a bit of attention in the press.

Technically, there is nothing novel about making an infectious DNA clone of Zika virus. It is an important reagent, just as infectious DNA clones are important for the study of all viruses. But the paper reports no experimental results using the Zika virus infectious DNA that advance the field. In my opinion, the infectious DNA clone of Zika virus should not have been published in a high profile journal.

Clearly the paper was published because Zika virus is hot and it will garner the journal a great deal of publicity, a consideration that should not determine whether an article should be published or not. It is the science that should drive publication – and the luxury journals have lost track of this fact.

Schekman points out that the reputations of luxury journals reputations as the “epitome of quality” is only “partly warranted”: they don’t always publish outstanding work, and they are not the only journals to publish great science. He feels that they “aggressively curate their brands, in ways more conducive to selling subscriptions than to stimulating the most important research”. They are driven by impact factor, which Schekman and others, including myself, think is wrong. Highly cited papers are not necessarily correct; they might be “eye-catching, provocative or wrong”. He says that editors accept papers that will ‘make waves’ and therefore influence, inappropriately, the direction of science. He favors open-access journals that are edited by scientists, and so do I.

In my view there are two main forces that have corrupted science publishing. The first is one that Schekman notes: that these journals are in the business of selling subscriptions. The Cell and Nature journals are owned by for-profit publishing companies. This situation is problematic because the drive for profit is not necessarily compatible with the need to publish high quality science. Editors know that controversial or prominent (e.g., Zika) papers will drive advertising revenue, but this should not even be a consideration when deciding what to publish. The publication of scientific data should not be a for profit enterprise. Unfortunately, Science magazine, which is published by the non-profit AAAS, seems to be driven by the same corrupting influences.

A second problem is that decisions at the luxury journals are typically not made by working scientists, but by full-time editors. A professional editor cannot possibly know the field as well as a working scientist, who spends his or her days in the trenches of science: designing experiments, interpreting data, guiding students and postdoctoral fellows, reviewing manuscripts, writing grants, going to meetings, and much more. The result is that the working scientist is fully immersed in science every day, all year, and is in the best position to know what work is significant, advances the field, and should be considered for publication.

These two factors control what kinds of papers are published. The luxury journals want high-impact papers that are of broad interest. But the problem is that it’s not always clear exactly where a paper fits in. Many of us have had the experience of submitting a paper to Cell, Science, or Nature, only to be told ‘it’s not of sufficient interest’. But the real reason is that the paper won’t sell advertising, or subscriptions; or perhaps the editor who made the decision simply doesn’t sufficiently understand the field.

A paper on an infectious Zika virus DNA clone will help Cell Host & Microbe get more advertising. A year ago, the journal would not even have reviewed the paper.

It’s no secret that publishing controls our scientific careers. Decisions about important things like hiring, promotion, tenure, and grant funding revolve around what you have published and where. I’ve been on many tenure or grant review committees, and it’s common to count the number of Cell, Nature, and Science publications as a metric of quality. The same occurs when examining job candidates for professorial positions.

In other words, the luxury journals are controlling the careers of scientists. Journals motivated by profit, run by professional editors who are not scientists, are deciding who is hired, promoted, tenured, and who gets grant money.

Unfortunately it is a system that scientists have created and nurtured until it has become an absurd and untenable situation, and it has to change. The PLoS journals and eLife are helping to do that, but what is also needed is to diminish the importance of the luxury journals to the careers of scientists. That is a much harder goal to achieve, as all my colleagues who are sending their Zika virus papers to luxury journals, will admit.

Zika virus crosses the placenta and causes microcephaly in mice

Zika virusI am convinced that Zika virus causes microcephaly in humans, but it would be valuable to have an animal model to study how the virus crosses the placenta and damages the fetus. As with many questions about Zika virus, answers are coming very rapidly, and three different groups have now provided substantial insight into this problem.

When a Samoan isolate of Zika virus was injected into the brain of embryonic day 13.5 mice, the virus replicated mainly in neural progenitor cells, but also in many other brain cells (link to paper). At 5 days after infection, brain size was markedly reduced compared with uninfected littermates. Infection of neural progenitor cells disrupted their normal differentiation program which leads to the production of mature neurons. Gene expression profiling of infected brains showed an increase in the production of cytokines, suggesting that these proteins might play a role in disease development. The expression of genes that have been previously associated with microcephaly was reduced. The authors conclude that “these effects are likely to account for microcephaly in human fetuses or newborn babies.”

In a different approach (link to paper), pregnant mice (not fetuses as in the previous study) were infected with a Brazilian Zika virus strain, and newborns were studied. Zika virus was detected in many tissues of newborns, especially in brain. Newborn mice displayed overall reduced growth, cortical malformations, and reduced cortical cell number and cortical layer thickness, all defects associated with microcephaly in humans. The infected mice also had ocular abnormalities similar to those that have been observed in babies with congenital Zika virus syndrome. Of note was the observation that the ability of Zika virus to cross the placenta was dependent on the strain of mouse used. I would not be surprised if the ability of viruses (or any microbe) to cross the human placenta is also determined in part by the genetics of the host.

The authors of this study suggest that human to human passage of Zika virus in humans for ~60 years led to a strain that can cross the placenta and infect the fetus. Consistent with their hypothesis, they show that replication of a Brazilian Zika virus strain in human brain organoids (three-dimensional models of the human brain produced from human pluripotent stem cells), greater morphological abnormalities and a reduction in size compared with effects cause by infection with an African strain of the virus. Furthermore, they find that a Brazilian Zika virus strain does not replicate in chimpanzee organoids, while the African strain does well. However, they do not address their hypothesis directly by determining in their mouse model whether an African strain of Zika virus can cross the placenta and infect the fetus.

A third report provides insight into how Zika virus might cross the placenta (paper link). This model consists of immunocompromised mice in which the type I interferon response has been ablated either genetically or by the administration of antibodies to the receptor for this cytokine. Pregnant mice are infected subcutaneously in the footpad with a French Polynesian Zika virus strain at embryonic days 6.5 and 7.5; embryos are then sacrificed 7 or 8 days later. No microcephaly was observed, but virus was detected in the head of the fetus. Replication was also detected in placenta and is accompanied by placental damage. Infected placentas are smaller, have damaged blood vessels, and display evidence of cell death and virus replication in various types of trophoblasts that make up placental tissues. In contrast, dengue virus did not replicate in the placenta or in the fetus.

These observations suggest that Zika virus can cross the placenta and move from the maternal blood into the fetus. The authors conclude that “The cellular and ultrastructural evidence of ZIKV infection in trophoblasts and fetal endothelium suggests that maternal viremia leads to compromise of the placental barrier by infecting fetal trophoblasts and entering the fetal circulation.”

Previously it was shown that term placentas are resistant to Zika virus infection due to the production of type III interferon (paper link). The authors of the mouse study described above suggest that Zika virus may cross the placenta early in pregnancy due to infection of extravillous trophoblasts. These cells are abundant early but not later in pregnancy.

These three reports provide conclusive evidence that Zika virus can cross the placenta and cause fetal defects in mice, and provide models to understand the biology and pathogenesis of infection.

I am astounded at the unprecedented, rapid and frequent publication of new research results on Zika virus biology and pathogenesis. This phenomenon reflects the willingness and ability of large and mature groups of investigators from diverse fields (virology, neurobiology, cell biology) to tackle a new problem and make rapid progress.

I predict that we will learn more about Zika virus in the next year than we have about any other microbe in a similar period of time.

Virus Watch: Building Zika virus

The results of recent structural studies have given us the ability to display the structure of Zika virus and of the viral E protein bound to antibody. In this video from Virus Watch I explain how the Zika virus particle is built, and how it interacts with an antibody that blocks infection, in beautiful three dimensional imagery.

TWiV 388: What could possibly go wrong?

TWiVPreprint servers, the structure of an antibody bound to Zika virus, blocking Zika virus replication in mosquitoes with Wolbachia, and killing carp in Australia with a herpesvirus are topics of episode #388 of the science show This Week in Virology, hosted by Vincent, Dickson, Alan, and Kathy.

You can find TWiV #388 at microbe.tv/twiv, or listen below.

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Virus Watch, Zika Edition

Readers of virology blog know my fondness for the long form. Many appreciate an in-depth discussion of virology in a blog post, video, or podcast, but this format is not for everyone. I know that I have been missing many individuals who would like to know more about viruses, but do not have the time or interest to spend an hour or more a week doing so. For those individuals, I have started Virus Watch.

Virus Watch is a weekly video series that explores the amazing world of viruses. They will be short (less than 10 minutes), with clear animation and focused on one or two stories. I released the first episode this week, which is about recent research on Zika virus. This virus will certainly be our focus for some time, but I also will explore other viruses in the series.

I am fascinated by viruses, which have I studied for my entire career, and I want you to be fascinated with them as well. You can find Virus Watch at my YouTube channel, or view the first video below.

TWiV 387: Quaxxed

TWiVOn episode #387 of the science show This Week in Virology, Nina Martin joins the TWiV team to talk about the movie Vaxxed, her bout with dengue fever, and the latest research on Zika virus.

You can find TWiV #387 at microbe.tv/twiv, or listen below.

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Antibodies to dengue virus enhance infection by Zika virus

Zika virus

Model of Zika virus particle. E glycoprotein dimer is expanded at left.

It has been speculated that the development of neurological disease and fetal abnormalities after Zika virus infection may be due to the presence of  antibodies against other flaviruses that enhance disease. In support of this hypothesis, it has been shown that antibodies to dengue virus enhance infection of cells by Zika virus.

There are four serotypes of dengue virus, and infection with one of the serotypes generally leads to a self-resolving disease. When a different serotype is encountered, antibodies to the first serotype bind virus but do not block infection. Dengue virus then enters and replicates in cells that it does not normally infect, such as macrophages. Entry occurs when Fc receptors on the cell surface bind antibody that is attached to virus particles. The result is higher levels of virus replication and more severe disease. This phenomenon is called antibody-dependent enhancement, or ADE.

Because dengue and Zika viruses are antigenically related, an important question is whether antibodies to dengue virus can enhance infection with Zika virus. To answer this question, the authors used two broadly neutralizing anti-dengue virus monoclonal antibodies that had been previously isolated from patients who recovered from infection. These antibodies recognize the viral E glycoprotein; specifially, a loop of the protein involved in fusion of the viral and cell membranes. The amino acid sequence of this fusion loop region is the same in dengue virus and in Zika virus.

The two anti-dengue virus mAbs bind the Zika virus E glycoprotein and also recognize Zika virus infected cells. However, when mixed with Zika virus, they do not neutralize infectivity in a cell line made from rhesus monkey kidneys. But when the antibodies were tested in Fc receptor-bearing cells, Zika virus infection was enhanced by over 100 fold. In the absence of dengue virus antibodies, levels of Zika virus RNA are very low.

Serum from four patients who had recovered from dengue virus infection was also examined for enhancement of Zika virus infection. All four sera contain antibodies that neutralized all four serotypes of dengue virus, but only two neutralized Zika virus infection. All four human sera enhanced Zika virus infection of Fc receptor-bearing cells. Enhancement of Zika virus infection could be blocked when Fc receptors were blocked with anti-Fc receptor antibodies before virus infection. A control serum from a patient in Canada that did not contain antibodies to dengue or Zika viruses did not enhance Zika virus infection.

These findings have one caveat: enhancement of Zika virus infection by antibodies against dengue virus was measured by PCR amplification of infected cell RNA, not by measuring the yield of infectious virus. The assumption is that increased intracellular viral RNA means more virus released from the cell, but this remains to be confirmed.

It will be important to confirm these findings in animal models of Zika virus infection, and in humans. If true, they have wide implications. If antibodies against dengue virus enhance Zika virus infection in humans, more severe disease might be observed in areas such as Brazil where both viruses co-circulate. It will be necessary to determine if Guillain-Barré syndrome, other neurological complications, and birth defects correlate with antibodies to dengue virus. Perhaps Fc receptors on the placenta and neural tissues allow entry of Zika virus only when bound to dengue virus antibody. It is also possible that antibodies to Zika virus might enhance dengue virus disease.

These observations do not bode well for Dengvaxia, a tetravalent dengue virus vaccine that has been recently licensed in Brazil, Mexico, and the Philippines. Might anti-dengue virus antibodies induced by this vaccine make Zika virus disease more severe? This outcome would be a tragedy, as many years of work has gone into making this vaccine to prevent severe disease caused by dengue virus infections. Second generation dengue virus vaccines such as TV003 are already moving through clinical trials.

It is essential to determine as soon as possible if antibodies induced by Dengvaxia and TV003 enhance Zika virus disease. If so, it will be necessary to assess whether deployment of this vaccine should proceed.

Dengvaxia consists of the yellow fever virus vaccine strain 17D in which the E and prM viral membrane proteins are substituted with those of dengue virus. In contrast, the attenuated TV003 vaccine has only the dengue virus genome. Would a vaccine consisting of TV003 plus an attenuated Zika virus vaccine solve potential problems of antibody dependent enhancement of disease?

Update 4/28/16: Over on Twitter someone asked, “any idea why we DON’T see severe disease in parts of Africa/Asia where dengue and Zika co-circulate”? Good question, too long an answer for Twitter. The easiest is that in humans, there is no antibody depencent enhancement of Zika virus infection by dengue antibodies. But if there is ADE, then there are a number of possible explanations. First, there have not been enough cases in Africa, nowhere near the numbers in the Pacific and South America.

There were certainly some cases of Guillain-Barré syndrome associated with some of the Pacific outbreaks – which I would consider more serious disease and could be potentiated by dengue antibodies.

I also think that Brazil is hyperendemic for dengue virus, with multiple serotypes circulating and people having multiple infections.

But the recent outbreaks are much larger than before, and dengue antibody induced complications of Zika virus infection might only be observed in larger outbreaks.

The authors of the paper discussed in this post suggest that the introduction of Zika virus into a completely naive population could also be a factor, as the age of exposure. Maybe a robust anti-Zika virus antibody response, in a non-naive population, can temper any effects of dengue mediated ADE.