Antibodies bound to poliovirusFor the first time since April of 1955, recipients of poliovirus vaccine will no longer receive all three serotypes. This past Sunday the World Health Organization orchestrated a synchronized switch from trivalent to bivalent oral poliovirus vaccine (OPV) in 150 countries.

The reason for the switch is clear: type 2 poliovirus was declared eradicated last year, and the only remaining cases are cause by vaccine-derived type 2 polioviruses. After oral administration of poliovirus vaccine, the virus replicates in the intestine, conferring immunity to subsequent infection. In all recipients of the vaccine the viruses lose the mutations that make them safe for humans. Consequently a small number of recipients, and their contacts, contract poliomyelitis from the vaccine.

To prevent further cases of poliomyelitis caused by circulating vaccine-derived polioviruses, WHO planned a synchronized, global switch from trivalent OPV to bivalent OPV on 17 April 2016. By July of 2016 all remaining stocks of the Sabin type 2 poliovirus strains, which are used to produce OPV, will also be destroyed.

My concern with this strategy is that type 2 vaccine-derived polioviruses continue to circulate. Whether they will continue to do so long enough to cause an outbreak of paralytic disease in the cohort of new infants that do not receive type 2 vaccine is a mattern of conjecture. In case there is an outbreak, monovalent type 2 oral poliovirus vaccine is being stockpiled by WHO. Of course, re-introduction of this vaccine will be accompanied by more circulating vaccine-derived poliovirus in the environment, and vaccine-associated disease, the very event WHO is trying to end with the trivalent to bivalent switch.

Type 3 poliovirus has not been isolated since 2012. Only type 1 poliovirus still causes outbreaks in two countries: Pakistan and Afghanistan. The inability to vaccinate in those countries, due to conflict, is delaying eradication. The recent killing of seven police officers who were protecting polio vaccinators by the Pakistani Taliban is an example of this difficulty.

Developing a great vaccine is not the only requirement for preventing infectious disease: you also have to be able to deploy it.

Image: Antibodies bound to poliovirus by Jason Roberts.

A major new feature of the fourth edition of Principles of Virology is the inclusion of 26 video interviews with leading scientists who have made significant contributions to the field of virology. These in-depth interviews provide the background and thinking that went into the discoveries or observations connected to the concepts being taught in this text. Students will discover the personal stories and twists of fate that led the scientists to work with viruses and make their seminal discoveries.

For the chapter on Infections of Populations, Vincent spoke with Thomas London, MD, of the Fox Chase Cancer Center, about his career and his work on hepatitis B virus.

TWiV 385: Failure

TWiVStuart Firestein, Columbia University neuroscientist and author of the book Ignorance, returns to TWiV for a chat about his latest work, Failure. This book is all about how experiments that don’t work, or provide the wrong conclusions, are essential for the progress of science.

You can find TWiV #385 at microbe.tv/twiv, or listen below.

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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 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.

TWiVMass die-offs of tilapia by a novel orthomyxo-like virus, Ian Lipkin’s editorial on the movie Vaxxed, and new vaccines to prevent dengue virus infections, including a human challenge model, are topics of episode #384 of the science show This Week in Virology. With guests Ian Lipkin and Nischay Mishra from the Center for Infection and Immunity.

You can find TWiV #384 at microbe.tv/twiv, or listen below.

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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.

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 microbe.tv/twiv or listen below.

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Syn3.0If the DNA sequence of a cell is like the operating system of a computer, then the smallest cellular OS has just been written. Called Syn3.0, it encodes everything needed to make a viable, autonomously replicating cell.

Mycoplasma is a genus of bacteria that are the smallest known free-living organisms. They also have the smallest genomes of any autonomously replicating cell: the DNA of Mycoplasma genitalium is just over one million base pairs in length and encodes 525 genes.

The entire 1,078,809 bp genome of Mycoplasma mycoses was synthesized in 2010 and transplanted into cells of another species, where it replaced the resident genome. In another nod to computer science, the authors refer to ‘installing’ the new genome into a cell, much like a new OS is installed on a hard drive.

This genome engineering tour de force was then followed by the synthesis of a reduced Mycoplasma genome. By combing the literature and carrying out extensive mutagenesis, genes were identified that were nonessential for growth in a rich culture medium. From the design of the new genome, to its installation into a new cell, took only 3 weeks.

The result, Syn3.0, has 438 protein coding genes and 35 RNA genes. Its 531,000 base genome is the smallest of any autonomously replicating cell found in nature. The doubling time of the cell is 180 minutes (compared with 16 hours for M. genitalium). The cells are smaller than the parent organism and are polymorphic in apperance (illustrated; image credit).

What is encoded by this minimal cellular OS?

Most of the genes (41%) are involved in expression of the genome: transcription, regulation, RNA metabolism, translation, protein folding, RNA, ribosome biogenesis, rRNA modification, and tRNA modification.

Seven percent of the synthetic genome is involved in preservation of genome information: DNA replication, DNA repair, DNA toplogy, DNA metabolism, chromosome segregation, and cell division.

Genes involved in cell membrane synthesis constitute 18% of the genome, and genes involved in cytosol metabolism, 17%.

Perhaps the greatest surprise is that 17% of the Syn3.0 genes have no known functions. Some of these genes are also present in other organisms and must have important roles. Their study should be stimulated by the creation of Syn3.0.

I would be very excited to see this technology applied to the study of viral genomes. For most small viral genomes it has already been determined that all of the genes are needed for replication in cell culture. For example, the genome of poliovirus, a 7,500 nucleotide RNA molecule, encodes about a dozen proteins. None of these protein coding sequences can be removed without destroying the ability of the virus to replicate.

However, viruses with larger genomes carry some genes that are dispensable for replication in cell culture. For example, the DNA genomes of adenoviruses, herpesviruses, and poxviruses encode proteins that can be deleted without affecting replication in cell culture. Many of these genes encode antagonists of the immune response, and have a role only during infection of an animal with an immune system.

Undoubtedly the most interesting application of the technology used to produce Syn3.0 would come from analysis of the genomes of giant viruses such as Mimivirus, Pandoravirus, and Pithovirus. The genomes of these viruses range from 600,000  to over 2.4 million base pairs in length. They encode mostly proteins of unknown function, as well as molecules not seen in other viruses, such as components of the protein synthesis apparatus. I hope that we will soon see the synthesis of reduced genomes of these giant viruses to identify the minimal gene set needed for production of infectious viruses in a host cell.

Put another way, what is the smallest operating system needed to run a giant virus?

TWiVOn episode #382 of the science show This Week in Virology, Nels Elde and Ed Chuong join the TWiV team to talk about their observation that regulation of the human interferon response depends on regulatory sequences that were co-opted millions of years ago from endogenous retroviruses.

You can find TWiV #382 at microbe.tv/twiv, or listen below.

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