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.


Zika virus in Brazilian non-human primates

Callithrix jacchusZika virus RNA has been detected in New World monkeys from the Northeast region of Brazil. This finding suggests that primates may serve as a reservoir host for the virus, as occurs in Africa.

The results of numerous serological surveys have shown that different Old World monkeys in Africa and Asia, including Rhesus macaques, Grivets, Redtail monkeys, and others, have antibodies that react with Zika virus. In these areas Zika virus is probably transmitted among monkeys in what is called a sylvatic cycle. Periodic outbreaks (epizootics) of Zika virus infections in nonhuman primates have been documented.

Where monkey reservoirs of Zika virus are present, humans may be infected with virus transmitted from a monkey. When non-human primates are absent, as on Yap Island, where an outbreak occurred in 2007, mosquitoes transmit the virus from human to human.

The Zika virus outbreak in Brazil has been thought to have been mainly transmitted between humans by mosquitoes. However, the results of this new study suggests that nonhuman primates could also be involved. The authors used polymerase chain reaction (PCR) to detect Zika virus RNA in sera or oral swabs from 15 marmosets and 9 capuchin monkeys in Ceará State where the virus is currently circulating. Four marmosets and three capuchins tested positive for Zika virus in this test.

Nucleotide sequence analysis of the PCR products from one marmoset and one capuchin monkey showed 100% identity with the strain of Zika virus that is circulating in Brazil.

The sampled animals were obtained from distant regions of the State. The marmosets were all free-ranging but had contact with humans, while 8 capuchins were pets and one was kept in a screening center for wild animals.

If these findings are confirmed and extended to other parts of Brazil, they would suggest that Zika virus might be spreading through non-human primates in that country. If so, they could serve as a reservoir for infection of humans via mosquito vectors.

An interesting question is when Zika virus entered monkeys in Brazil. It has been suggested that the virus entered Brazil in 2013 or 2014, and might have spread first in monkeys, first in humans, or both at the same time. I also wonder whether monkey to human transmission leads to a different disease than when virus circulates among humans.

Zika virus comics: Zanzare

Dr. Susan Nasif is a virologist and part of the team at Cimaza Comics that produces science-themed comics. In their latest creation, Zanzare, we are plunged head-first into the global mystery of Zika virus. We meet the mosquitoes (in Italian: zanzare) implicated in its spread; but the insects plead their innocence, saying it’s all a misunderstanding. They lay their case before the gods and demons of Zika’s victims, and ask for divine help. Will the mosquitoes be vindicated? Or will it all turn out that the zanzare are to blame after all?

Not even the authors know where Zanzare is heading. The comics follow weekly developments in the Zika investigation as it unfolds. The story is told through the lens of world mythology, but the virology presented comes straight from reputable journals. Thrilling and funny, Zanzare is a visionary mixture of ancient legend and up-to-the-minute fact.

The video below is an excerpt from this series, which is not yet released in book form. Their previous creation, Adventures of the Regatjes, is available here.

Zika virus, like all other viruses, is mutating

Zika virusNot long after the appearance of an outbreak of viral disease, first scientists, and then newswriters, blame it all on mutation of the virus. It happened during the Ebolavirus outbreak in West Africa, and now it’s happening with Zika virus.

The latest example is by parasitologist Peter Hotez, who writes in the New York Times:

There are many theories for Zika’s rapid rise, but the most plausible is that the virus mutated from an African to a pandemic strain a decade or more ago and then spread east across the Pacific from Micronesia and French Polynesia, until it struck Brazil.

After its discovery in 1947 in Uganda, Zika virus caused few human infections until the 2007 outbreak on Yap Island. The virus responsible for this and subsequent outbreaks in Pacific Islands is distinct from the African genotype, but there is no experimental evidence to suggest that sequence differences in the Asian genotype were responsible for the spread of the virus. For this reason I disagree with Dr. Hotez’ conclusion that mutation of the virus is the ‘most plausible’ explanation for its global spread. It is just as likely that the virus was in the right place at the right time to spark an outbreak in the Pacific.

We will never have experimental evidence that emergence of the Asian genotype allowed pandemic spread of Zika virus, because we cannot test the effect of individual mutations on spread of the virus in humans. Consider this experiment: infect a room of humans (and mosquitoes) with either the African or Asian genotype of Zika virus, then measure virus replication and transmission. If there is a difference between the two viruses, engineer specific mutations into the virus, reinfect another batch of humans, and continue until the responsible mutations are identified. Obviously we cannot do such an experiment! We could instead use animal models, but these have limitations in extrapolating results to humans. For this reason we have never identified any specific mutation that allows an animal virus to replicate more efficiently in humans.

The same experimental limitations do not apply to animals. An example is Chikungunya virus, spread by Aedes ageyptii mosquitoes. Before 2004, outbreaks of infection were largely confined to developing countries in Africa and Asia. The virus subsequently spread globally, due to a single amino acid change in the envelope glycoprotein which allows efficient replication in Aedes albopictus, a mosquito with a greater range than A. ageyptii. It was possible to prove this point by assessing the effects of changing this single amino acid on virus replication in mosquitoes. The same experiment cannot be done in humans.

There is no evidence that the Asian genotype of Zika virus is any more competent to replicate in mosquitoes than the African strain. Results of a study of replication of Asian genotypes of Zika virus revealed that Aedes aegypti and Aedes albopictus are not very good vectors for transmitting ZIKV. The authors smartly suggest that “other factors such as the large naïve population for ZIKV and the high densities of human-biting mosquitoes contribute to the rapid spread of ZIKV during the current outbreak.” In other words, don’t blame the Zika virus genome for the expanded range of the virus.

The Zika virus that has been spreading in Brazil, and which has been associated with microcephaly, shares a common ancestor with the Asian genotype. In a recent study of the genomes of 7 Brazilian isolates, there was no evidence that specific mutations are associated with microcephaly. Those authors conclude (also smartly):

Factors other than viral genetic differences may be important for the proposed pathogenesis of ZIKV; hypothesized factors include co-infection with Chikungunya virus, previous infection with Dengue virus, or differences in human genetic predisposition to disease.

It’s easy to blame mutations in the viral genome for novel patterns of transmission or pathogenesis. Viral mutations arise during every replication cycle, due to errors made by viral enzymes as they copy nucleic acids. RNA viruses are the masters of mutation, because, unlike the polymerases of DNA viruses, RNA polymerases cannot correct any errors that arise. As viruses spread globally through different human populations, it is not surprising that different genotypes are selected. These may reflect adaptation to various selective pressures, including different humans, vectors, climate, or geography. There is no reason to assume that such changes influence virulence, disease patterns, or transmission in humans. Whether they do so can never be tested in humans.

Blaming the viral genome is nothing new. At the onset of the 2014 Ebolavirus outbreak in West Africa there were many claims that the unprecedented size of the outbreak was a consequence of mutations in the viral genome. Genomic analysis of isolates early in the epidemic suggested that the large number of infections was leading to rates of mutation not previously observed. This work lead to dubious claims of  “Ebolavirus mutating rapidly as it spreads” and Ebolavirus is mutating (Time Magazine). Richard Preston, in the New Yorker article Ebola Wars quoted scientist Lisa Hensley:

In the lab in Liberia, Lisa Hensley and her colleagues had noticed something eerie in some of the blood samples they were testing. In those samples, Ebola particles were growing to a concentration much greater than had been seen in samples of human blood from previous outbreaks. Some blood samples seemed to be supercharged with Ebola. This, too, would benefit the virus, by enhancing its odds of reaching the next victim. “Is it getting better at replicating as it goes from person to person?” Hensley said.

And let’s not forget the absurd speculation, fueled by these data, that Ebolavirus would go airborne.

Within a year all this nonsense was proven wrong. Ebolavirus had not sustained mutations any faster than in previous outbreaks. Furthermore, the observed mtuations  did not change the virus into a more dangerous strain.

Go back to any viral outbreak – MERS-coronavirus, SARS-coronavirus, influenza virus, HIV-1 – and you will find the same story line. Mutation of the virus is leading to more virulence, transmission, spread. But in no case has cause and effect been proven.

Let’s stop blaming viral mutation rates for altered patterns of virus spread and pathogenesis. More likely determinants include susceptibility of human populations, immune status, vector availability, and globalization, to name just a few. Not as spectacular as ‘THE VIRUS IS MUTATING!’, but nearer to the truth.

Structure of Zika virus

Zika virus reconstructionSix months after Zika virus became a household word, we now know the three-dimensional structure of the virus particle. And it looks like very much like other flaviviruses, such as West Nile and dengue viruses.

In the old days, solving a virus structure was a big deal. A virus is, after all, a very large assembly of many proteins. To solve the structure of a virus – which will tell us the location of the amino acid chains in three dimensional space – was a technical tour de force. It was necessary to purify large amounts of virus particles, and then find the conditions to produce crystals, a hit and miss affair. If you were lucky to grow virus crystals – which could take a year or more – you then crossed your fingers to see if they diffracted in an X-ray beam. When X-rays are aimed at a crystal, the beams bounce off atoms in the crystals, and their reflections provide information on where the atoms are located. Finally you could collect the diffraction data, do a lot of math on a computer, and determine the three dimensional structure.

The first virus structure to be solved by X-ray crystallography was of a plant virus, tomato bushy stunt virus in 1976, followed by poliovirus and rhinovirus in 1985. Many X-ray structures of viruses have been solved, with resolutions less than 2 Angstroms that allow us to see not only the amino acid chain, but all the atoms in the side chains.

The Zika virus structure was not solved by X-ray crystallography. It was done by cryo-electron microscopy (cryo-EM) and image reconstruction. It’s easier and faster than X-ray crystallography, and can achieve comparable resolutions.

It is not necessary to produce crystals to determine structures by cryo-EM. Instead, samples of purified viruses are rapidly frozen and photographed with an electron microscope at very low temperatures. This procedure preserves native structure, and allows visualization of the contrast inherent in the virus particle. Photographs of thousands of virus particles – each in a slightly different orientation – are taken and processing computationally to create the final three-dimensional image.

The cryo-EM structure of Zika virus tells us how the virus particle is put together. It looks very much like other flaviviruses, which consist of a membrane surrounding the capsid, which in turn carries the viral RNA genome. Inserted into the membrane are 180 copies of the viral proteins E and M. Although inserted in a fluid lipid bilayer, they are arranged with a symmetry that reflects their contacts with the underlying icosahedral capsid. In the illustration, which I produced from the freely available cryo-EM data, you can clearly see five copies of the E glycoprotein (red) at one five-fold axis of symmetry.

One structural difference between Zika virus and other flaviviruses is a loop of amino acids exposed on the surface of the particle. This sequence of the E glycoprotein, and a sugar molecule attached to it, might be involved in regulating Zika virus tropism and pathogenesis. The ability of West Nile virus to enter the central nervous system of mice has been linked to glycosylation at a similar position, while cell receptors are thought to attach to sugars on the dengue virus capsid.

The authors of the Zika virus cryp-EM structure have produced an animation which illustrates aspects of the structure (below). Watch my lecture on virus structure for more information how viruses are put together.

Updated 7 April 2016 to provide an explanation of how the sugar attached to the E glycoprotein of Zika virus might regulate tropism and pathogenesis.

TWiV 383: A zillion Zika papers and a Brazilian

TWiVEsper Kallas and the Merry TWiXters analyze the latest data on Zika virus and microcephaly in Brazil, and discuss publications on a mouse model for disease, infection of a fetus, mosquito vector competence, and the cryo-EM structure of the virus particle. All on episode #383 of the science show This Week in Virology.

Audio and full show notes for TWiV #383 at or listen below.

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TWiV 381: Add viruses and Zimmer

TWiVOn episode #381 of the science show This Week in Virology, Carl Zimmer joins the TWiV team to talk about his career in science writing, the real meaning of copy-paste, science publishing, the value of Twitter, preprint servers, his thoughts on science outreach, and much more.

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TWiV 380: Viruses visible in le microscope photonique

TWiVOn episode #380 of the science show This Week in Virology, the TWiVeroos deliver the weekly Zika Report, then talk about a cryoEM structure of a plant virus that reveals how the RNA genome is packaged in the capsid, and MIMIVIRE, a CRISPR-like defense system in giant eukaryotic viruses.

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Zika virus infection of the nervous system

FlavivirusEvidence is mounting that Zika virus is neurotropic (able to infect cells of the nervous system) and neurovirulent (causes disease of the nervous system) in humans.

The most recent evidence comes from a case report of an 81 year old French man who developed meninogoencephalitis 10 days after returning from a 4 week cruise to New Caledonia, Vanuatu, Solomon Islands, and New Zealand (meningoencephalitis is infection of the meninges – the membranes that cover the brain – and the brain). His symptoms included fever, coma, paralysis, and a transient rash. A PCR test revealed Zika virus genomes in the cerebrospinal fluid, and infectious virus was recovered after applying the CSF to Vero cells in culture.

A second case report concerns a 15 year old girl in Guadeloupe who developed left hemiparesis (weakness of one side of the body), left arm pain, frontal headache, and acute lower back pain. After admission she developed dysuria (difficulty urinating) that required catheterization. PCR revealed the presence of Zika virus genomes in her serum, urine, and cerebrospinal fluid; other bacterial and viral infections were ruled out.

Until very recently Zika virus was believed to cause a benign infection comprising rash, fever, joint pain, red eyes, and headache. There is now strong evidence that the virus can cause congential birth defects, and growing evidence that the virus is neurotropic and neurovirulent. Previously the entire Zika virus genome was recovered from brain tissue of an aborted fetus.

Zika virus is classified in the family Flaviviridae, and other members are known to be neurotropic, including West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus. West Nile virus infection may lead to acute flaccid paralysis, meningitis, encephalitis, and ocular manifestations. Examination of additional cases of Zika virus infection will be needed to document the full spectrum of illness caused by this virus.

Update: Neurotropism of Zika virus is also indicated by the findings that the virus infects human cortical neural progenitors.

TWiV 379: A mouse divided

TWiVOn episode #379 of the science show This Week in Virology, Scott Tibbetts joins the TWiVirate to describe his work on the role of a herpesviral nocoding RNA in establishment of peripheral latency, and then we visit two last minute additions to the Zika virus literature.

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