TWiV 475: Everything is viral

In the first episode for 2018, the TWiV team reviews the amazing virology stories of 2017.

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Immune 3: Two epitopes, four serotypes, and a partridge in a pear tree

Cindy, Steph, and Vincent discuss recent problems with dengue virus vaccine, and a bi-specific monoclonal antibody against Zika virus.

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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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Zika virus blocks the neuron road

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.

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Zika virus has always been neurotropic

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.

TWiV 454: FGCU, Zika

Sharon Isern and Scott Michael return to TWiV for a Zika virus update, including their work on viral evolution and spread, and whether pre-existing immunity to dengue virus enhances pathogenesis.

 

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Does prior dengue virus infection exacerbate Zika virus disease?

Antibody dependent enhancementThe short answer to the question posed in the title of this blog is: we don’t know.

Why would we even consider that a prior dengue virus infection would increase the severity of a Zika virus infection? The first time you are infected with dengue virus, you are likely to have a mild disease involving fever and joint pain, from which you recover and develop immunity to the virus. However, there are four serotypes dengue virus, and infection with one serotype does not provide protection against infection with the other three. If you are later infected with a different dengue virus serotype, you may even experience more severe dengue disease involving hemorrhagic fever and shock syndrome.

The exacerbation of dengue virus disease has been documented in people. Upon infection with a different serotype, antibodies are produced against the previous dengue virus encountered. These antibodies bind the new dengue virus but cannot 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 (illustrated). The result is higher levels of virus replication and more severe disease. This phenomenon is called antibody-dependent enhancement, or ADE.

When Zika virus emerged in epidemic form, it was associated with microcephaly and Guillain-Barré syndrome, diseases that had not been previously known to be caused by infection with this virus. As Zika virus and dengue virus are closely related, because ADE was known to occur with dengue virus, and both viruses often co-circulated, it was proposed that antibodies to dengue virus might exacerbate Zika virus disease.

It has been clearly shown by several groups that antibodes to dengue virus can enhance Zika virus infection of cells in culture. Specifically, adding dengue virus antibodies to Zika virus allows it to infect cells that bear receptors for antibodies – called Fc receptors. Without Fc receptors, the Zika virus plus dengue antibodies cannot infect these cells. ADE in cultured cells has been reported by a number of groups; the first was discussed here when it appeared on bioRxiv.

The important question is whether antibodies to dengue virus enhance Zika virus disease in animals, and there the results are mixed. In one experiment, mice were injected with serum from people who had recovered from dengue virus infection, followed by challenge with Zika virus. These sera, which cause ADE of Zika virus in cultured cells, led to increased fever, viral loads, and death of mice.

These finding were not replicated in two independent studies conducted in rhesus macaques (paper one, paper two). In these experiments, the macaques were first infected with dengue virus, and shown to mount an antibody response to that virus. Over one year later the animals were infected with Zika virus (the long time interval was used because in humans dengue ADE is observed mainly with second infections 12 months or more after a primary infection). Both groups concluded that prior dengue virus immunity did not lead to more severe Zika virus disease.

Which animals are giving us the right answer, mice or monkeys? It should be noted that the mouse study utilized an immunodeficient strain lacking a key component of innate immunity. As the authors of paper one concluded, it’s probably not a good idea to use immune deficient mice to understand the pathogenesis of Zika virus infection of people.

When it comes to viral pathogenesis, we know that mice lie; but we also realize that monkeys exaggerate. Therefore we should be cautious in concluding from the studies on nonhuman primates that dengue virus antibodies do not enhance Zika virus pathogenesis.

The answer to the question of whether dengue antibodies cause Zika virus ADE will no doubt come from carefully designed epidemiological studies to determine if Zika virus pathogenesis differs depending on whether the host has been previously infected with dengue virus. Such studies have not yet been done*.

You might wonder about the significance of dengue virus antibodies enhancing infection of cells in culture with Zika virus. An answer is provided by the authors of paper one:

In vitro ADE assays using laboratory cell lines are notoriously promiscuoius and demonstrate no correlation with disease risk. For example, DENV-immune sera will enhance even the homotypic serotype responsible for a past infection in the serum is diluted to sub-neutralizing concentrations.

The conundrum of whether ADE is a contributor to Zika virus pathogeneis is an example of putting the cart before the horse. For dengue virus, we obtained clear evidence of ADE in people before experiments were done in animals. For Zika virus, we don’t have the epidemiological evidence in humans, and therefore interpreting the animals results are problematic.

*Update 8/12/17: A study has been published on Zika viremia and cytokine levels in patients previously infected with dengue virus. The authors find no evidence of ADE in patients with acute Zika virus infection who had previously been exposed to dengue virus. However the study might not have been sufficiently powered to detect ADE.

TWiV 432: Conjunction junction, what’s your function?

The TWiVites discuss Zika virus seroprevalence in wild monkeys, Zika virus mRNA vaccines, and a gamete fusion protein inherited from viruses.

You can find TWiV #432 at microbe.tv/twiv, or listen below.

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TWiV 430: The persistence of herpesvirus

The TWiX cabal discuss sexual transmission of Zika virus in mice, and how immune escape enables herpes simplex virus escape from latency.

You can find TWiV #430 at microbe.tv/twiv, or listen below.


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TWiV 429: Zika Experimental Science Team

Vincent meets with members of team ZEST at the University of Wisconsin Madison to discuss their macaque model for Zika virus pathogenesis.

You can find TWiV #429 at microbe.tv/twiv, or listen/watch right here.

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