Jon and Teddy Yewdell join the TWiV team to talk about their careers, their research, and the problems with biomedical research.

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By Steven Lubet, JD, and David Tuller, DrPH

Update: Nov 13th

We wrote this post based on a version of the guide that appeared recently and had been updated on July 31, 2017. That version lists Peter White as among three peer-reviewers. As commenters pointed out soon after the post went up, however, Peter White’s name is no longer on the peer-review list in the currently posted version of the guide, which indicates that it was updated this month. We are trying to determine when and why this change occurred.

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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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TWiV 466: The Capsid Club

From Indiana University, Vincent and Kathy speak with Tuli Mukhopadhyay, John Patton, and Adam Zlotnick about their careers and their work on alphaviruses, hepatitis B virus, and rotaviruses.

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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.

Theodora Hatziioannou joins the TWiV team to discuss a macaque model for AIDS, and how a cell protein that blocks HIV-1 infection interacts with double-stranded RNA.

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Marco VignuzziWhat would happen to an RNA virus if its genome were placed in a bad neighborhood? The answer is that fitness plummets.

RNA virus populations are not composed of a single defined nucleic acid sequence, but are dynamic distributions of many nonidentical but related members. In the past I have referred to these populations as quasispecies but that is no longer the preferred term: mutant swarms or heterogeneous virus populations should be used instead.

The term for all possible combinations of a viral genome sequence is sequence space; for a 10,000 nucleotide genome this would be theoretically 410,000 different genomes – a huge number, more than the atoms in the universe. Any RNA virus population occupies only a fraction of this sequence space, in part because many mutations are deleterious. Studies have shown that viral genomes occupy specific parts of sequence space, called neighborhoods, and movement to different neighborhoods is important for viability. If the viral genome is placed in a bad neighborhood – one that is detrimental for virus fitness – the ability to explore sequence space is restricted.

An example of the effect of changing viral sequence space is shown by a study in which hundreds of synonymous mutations (they did not change the amino acid sequence) were introduced in the capsid region of poliovirus (link to paper). Such rewiring, which placed the virus in a different sequence space, reduced viral fitness and attenuated pathogenicity in a mouse model. In other words, the viral genome was placed in a bad neighborhood, from where it could not move to other neighborhoods needed for optimal replication and pathogenesis. While the genome rewiring did not affect the protein sequence, it might have had deleterious effects on RNA structures or codon or dinucleotide frequency. For example, introduction of codon pairs that are under-represented in the human genome can produce less fit viruses.

A recent study avoids these potential issues by introducing changes in the viral genome that do not affect protein coding, RNA structures or codon or dinucleotide frequency, yet place the viral genome in a different sequence space (link to paper). All 117 serine/leucine codons in the capsid region of Coxsackievirus B3 were changed so that a single nucleotide mutation would lead to a stop codon, terminating protein synthesis and virus replication (this virus is called 1-to-Stop). The serine codons were changed to UUA or UUG; one mutation changes these to the terminators UAA, UGA, or UAG. Another virus was made in which two mutations were needed to produce a stop codon (NoStop virus).

1-to-Stop viruses replicated normally, but when mutagenized, they had significantly lower fitness than wild type or NoStop viruses. Extensive passage of the virus in cells, which would be expected to cause accumulation of mutations, had the same effect on fitness. When a high fidelity RNA polymerase was introduced into 1-to-Stop virus, it replicated like wild type virus. Similar results were obtained with an influenza virus when one of its 8 genome segments was rewired to produce 1-to-Stop and NoStop counterparts.

The 1-to-Stop Coxsackieviruses were attenuated in a mouse model of infection. Furthermore, mice infected with 1-to-Stop virus were protected against replication and disease after challenge with wild type virus. These observations suggest that rewiring viral RNA genomes could be used to design vaccines.

These findings show that recoding a viral genome places it an different sequence space than wild type virus, in which single mutations can lead to inactivation of viral replication. This new neighborhood is unfavorable (‘bad’) because the virus cannot readily move to other neighborhoods to accommodate the effects of mutation.

For more discussion of viral sequence space and rewiring viral genomes, listen to the podcast This Week in Evolution #24: our guest is Marco Vignuzzi (pictured), senior author on the second paper discussed here.

By David Tuller, DrPH

Last week I sent an e-mail with some questions to Sir Andrew Dillon, the chief executive of the National Institute for Health and Care Excellence (NICE). In particular, the questions involved the status of the ten-year-old guidance for CFS/ME, CG53, and of references to the illness elsewhere within the NICE system.

A few days ago, I received Sir Andrew’s response. He indicated that the current guidance remains in effect while NICE undergoes the process of developing the updated version. Whether and how much the NICE recommendations actually change in the updated version is apparently up to the new guideline committee.

Here’s what Sir Andrew wrote:

“The current guideline remains in force until it is replaced by the next version. The decision to update was based on the potential for the evidence published since the guideline was introduced, together with the views put forward by stakeholders to change the current recommendations. Whether that potential is realised is a matter for the new guideline committee to decide, as they apply our guideline development methods and undertake consultation with stakeholders. Other NICE guidance which refers to our CFS/ME recommendations will reviewed [sic] and amended if necessary once the new guideline has been published.”

This was the response I expected, more or less, but it was still disappointing. NICE was remiss in the first place to accept Oxford-criteria studies, including the PACE trial, as legitimate science, given their obvious flaws. The agency has since outlined the reasons why the current guidance is not fit for purpose and needs a full update. Now it has an opportunity—and a moral obligation, really–to take a more proactive stance and alert patients to the concerns raised about these potentially harmful treatments.

If these concerns are truly being taken seriously by NICE, how can the agency stand fully behind CG53 in the interim? Can it really be ethical for NHS clinics and doctors to continue to prescribe CBT and GET based on the current guidance without mentioning that these treatments are up for review and have already been dis-endorsed by the U.S. Centers for Disease Control? Who will be liable for any harms arising from the recommendations going forward, now that NICE has acknowledged the problems?

Whether NICE recognizes it or not, the current guidance has already acquired something of a lame-duck status, especially given widespread international rejection of CBT and GET. Any further argument from the CBT/GET ideological brigades that their approach remains the accepted standard of care can now be readily refuted. And that’s bad news for the public and private disability insurance agencies that have long relied on the CBT/GET paradigm to reject legitimate claims for ME/CFS-related benefits.

At Tufts University Dental School in Boston, Vincent speaks with Katya Heldwein and Sean Whelan about their careers and their work on herpesvirus structure and replication of vesicular stomatitis virus.

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Origin of virusesWe have previously discussed the idea that viruses originated from selfish genetic elements such as plasmids and transposons when these nucleic acids acquired structural proteins (see A plasmid on the road to becoming a virus). I want to explore in more detail the idea that the structural proteins of  viruses likely originated from cell proteins (link to paper).

Three ideas have emerged to explain the origin of viruses: 1. viruses evolved first on Earth, before cells, and when cells evolved, the viruses became their genetic parasites; 2. viruses are cells that lost many genes and became intracellular parasites; 3. viruses are collections of genes that escaped from cells. Missing from these hypothesis is how nucleic acids became virus particles – that is, how they acquired structural proteins. It seems likely that viral structural proteins originated from cellular genes.

An analysis of the sequence an structure of major virion proteins has identified likely ancestors in cellular proteins. Following are some examples to illustrate this conclusion.

A very common motif among viral capsid proteins is called the single jelly roll, made up of eight beta strands in two four-stranded sheets. Many cell proteins have jelly role motifs, and some form 60-subunit virus-like particles in cells. The extra sequences at the N-termini of viral jelly roll capsid proteins, involved in recognizing the viral genome, likely evolved after the capture of these proteins from cells.

The core proteins of alphaviruses (think Semliki Forest virus) has structural similarity with chymotrypsin-like serine proteases. The viral core protein retains protease activity, needed for cleavage from a protein precursor.

Retroviral structural proteins also appear to have originated from cell proteins, with clear homologies with matrix, capsid, and nucleocapsid proteins. The matrix Z proteins of arenaviruses are related to cellular RING domain proteins, and the matrix proteins of some negative strand RNA viruses are related to cellular cyclophilin. There are many more examples, providing support for the hypothesis that viruses evolved on multiple instances by recruiting different cell proteins.

Given this information on the origin of viral capsid proteins, we can modify the three hypotheses for the origin of viruses into one. Self-replicating, virus like nucleic acids emerged in the pre-cellular world and from the emerged the first cells. The replicating nucleic acids entered the cells, where they replicated and became genetic parasites. At some point these genetic elements acquired structural proteins from the cells and became bona fide virus particles. As cells evolved, new viruses emerged from them.

It is important to point out that the genes do not always flow from cells to viruses. We know that viral proteins can be returned to cells, where they serve useful functions. One example is syncytin, a retroviral protein used for the construction of the mammalian placenta.