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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Show notes at microbe.tv/twiv

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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Viral RNA is not infectious virus!

Zika RNA and virusA study of sexual transmission of Zika virus among mice (link to paper) demonstrates beautifully that viral nucleic acid detected by polymerase chain reaction (PCR) is not the same as infectious virus.

Male mice were infected with Zika virus and then mated with female mice. Efficient sexual transmission of the virus from males to females was observed. This observation in itself is very interesting but is not the focus of  my comments.

To understand the dynamics of sexual transmission, the authors measured Zika virus shedding in seminal fluid – by both PCR, to detect viral RNA, and by plaque assay, to detect infectious virus. The results are surprising (see figure – drawn in my hotel room).

Zika virus RNA persisted in semen for up to 60 days – far longer than did infectious virus, which could not be detected after about three weeks.

Many laboratories choose to assay the presence of viral genomes by PCR. This is an acceptable technique as long as the limitations are understood – it detects nucleic acids, not infectious virus.

Despite the presence of Zika virus RNA in seminal fluid for at least 60 days after infection, these mice are not likely to transmit virus after a few weeks. There is a lower limit of detection of the plaque assay – approximately 10 plaque forming units/ml – whether that would be sufficient to transmit infection is a good question.

Why Zika viral RNA and not infectious virus would persist for so long is an important and unanswered question that should definitely be studied.

Recently many papers have been published which demonstrate that Zika virus and Ebolavirus can persist in a variety of human fluids for extended periods of time. These results have been interpreted with alarm, both by scientists and by science writers. However, in most cases the assays were done by PCR, not by plaque assay, and therefore we do not know if infectious virus is present. Viral RNA would not constitute a threat to transmission, while infectious virus would.

The lesson from this study is very clear – in novel experimental or epidemiological  studies it is important to prove that any viral nucleic acid detected by PCR is actually infectious virus. Failing to do so clouds the conclusions of the study.

There are few excuses for failing to measure viral infectivity by plaque assays. Please don’t tell me it’s too much work – that’s a poor excuse on which to base selection of an assay. Even if your virus doesn’t form plaques there are alternatives for measuring infectious virus.

If you are wondering how a plaque assay is done, check out my short video below.

TWiV 426: I’m Axl, and I’ll be your cervid today

The sages of TWiV explain how chronic wasting disease of cervids could be caused by spontaneous misfolding of prion protein, and the role of the membrane protein Axl in Zika virus entry into cells.

You can find TWiV #426 at microbe.tv/twiv, or listen below.

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TWiV 422: Watching the icosahedron drop

The TWiVestigators wrap up 2016 with a discussion of the year’s ten compelling virology stories.

You can find TWiV #422 at microbe.tv/twiv, or listen below.

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Zika virus in Nicaragua with Eva Harris

I spoke with Eva Harris of the University of California, Berkeley, on the state of Zika virus in Nicaragua.

Zika in the Guys

In this episode of Virus Watch, we explore the finding that Zika virus infects the testis of mice, causing damage to the organ, reduced sperm production, and less fertility. The important question: does the same happen in humans?