TWiVVincent speaks with Stan Lemon about his career in virology, from early work on Epstein Barr virus, through making essential discoveries about hepatitis A virus, hepatitis C virus, and rhinoviruses, on episode #389 of the science show This Week in Virology.

You can find TWiV #389 at microbe.tv/twiv, or listen below.

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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.

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.

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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Virology lecturesHave you always wanted to better understand viruses, but did not know where to start? I have the solution for you – my undergraduate virology course. The 2016 version has just ended, and all the lectures are available as videos, either on YouTube or here at virology blog (where you can also find lecture slides and study questions).

It will take some time for you to watch all the videos – each is about 70 minutes long – but the effort will be worth it. In the end you will know more virology than most of the world. With new viruses emerging annually, don’t you want to understand how they work? Go ahead, dive in.

Lecture 1: What is a virus?
Lecture 2: The infectious cycle
Lecture 3: Genomes and genetics
Lecture 4: Structure
Lecture 5: Attachment and entry
Lecture 6: RNA directed RNA synthesis
Lecture 7: Transcription and RNA processing
Lecture 8: DNA replication
Lecture 9: Reverse transcription and integration
Lecture 10: Translation
Lecture 11: Assembly
Lecture 12: Infection basics
Lecture 13: Intrinsic and innate defenses
Lecture 14: Adaptive immunity
Lecture 15: Mechanisms of pathogenesis
Lecture 16: Acute infections
Lecture 17: Persistent infections
Lecture 18: Transformation and oncogenesis
Lecture 19: Vaccines
Lecture 20: Antivirals
Lecture 21: Evolution
Lecture 22: Emerging viruses
Lecture 23: Unusual infectious agents
Lecture 24: HIV and AIDS
Lecture 25: Viral gene therapy

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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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.

 

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.

TWiVDid you know that the evolution of ancient retroviruses, millions of years ago, can be traced by studying their genomes in the chromosomes of contemporary animals? Ted Diehl and Welkin Johnson join the TWiV team to tell us how they did it with mammals. All without a single wet experiment! They also join in the discussion about virus dispersal by hand dryers.

You can find TWiV #386 at microbe.tv/twiv, or listen below.

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