Protecting the lungs against influenza

Lungs Influenza viruses are a major public health threat. Each year, the typical seasonal flu epidemic affects millions of people with sometimes fatal outcomes, especially in high risk groups such as young children and elderly. The sporadic pandemic outbreaks can have even more disastrous consequences. In recent years it has become clear that the extreme pathogenicity of unusually “hot” strains of influenza such as the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is due to the “cytokine storm” that they unleash on infection.

The protein A20 is an important negative regulator of antiviral immune responses. A paper in PLoS Pathogens shows that the specific deletion of A20 in mouse bronchial epithelial cells improves the protection against influenza virus infections. This increased protection correlates with a dampened pulmonary cytotoxic T cell response and a strongly suppressed expression of the chemokine CCL2 during later stages of infection. This suggests that inhibiting A20 expression, for example by local administration of interfering RNAs, might be possible as a new therapeutic strategy to control severe disease caused by influenza A virus infection.

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection. (2016) PLoS Pathog 12(1): e1005410. doi:10.1371/journal.ppat.1005410
A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20AEC-KO mice during later stages of infection. When A20AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.

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Design and Evolution

Structures A half century of structural studies have shown the design principles controlling self-assembly of viral proteins into icosahedral virus capsids. A large repertoire of virus structures, mainly determined using X-ray crystallography and cryo-electron microscopy, are now conveniently available and carefully annotated in the VIPER database. These structures are not only important to the study of virus structure/function relationships, but they also help to decipher virus evolution.

Unlike cellular organisms, viruses defy conventional classification schemes based on bioinformatics analysis of macromolecular sequences. The huge selective pressure to keep viruses functional results in immense genomic variation that prevents a meaningful and informative taxonomy. Furthermore, icosahedral viruses are intrinsically limited in “structure space” by the structural constraints associated with assembling a coat protein into a 60-fold symmetric protein cage, the capsid, which can be achieved only in a limited and well-defined number of combinations.

The realization that capsid structure persists much longer than viral genomes or protein sequences led to the use of experimentally determined virus structures to define virus taxonomy and identify virus lineages. Amazingly, the tremendous variety and exhilarating complexity of icosahedral viruses existing in nature can be rationalized to four combinations by which one or a few major capsid proteins (MCPs) self-assemble to form an icosahedral lattice. These “modes” of assembly were used to define four viral lineages populating the virosphere:

  1. Picornoviridae, like the single-stranded foot-and-mouth disease virus in which the building block is an eight-stranded β-barrel MCP usually arranged parallel to the surface of the virus.
  2. Reoviruses, such as the double-stranded RNA Bluetongue virus that contains a mostly α-helical MCP arranged to form a core of 60 homodimers.
  3. Tailed bacteriophages like the Salmonella-phage P22, herpesviridae, and even certain archaeal viruses which build their capsid around the flexible HK97 MCP fold.
  4. PRD1-adenovirus type viruses in which a capsid lattice is made of vertical double β-barrels stacked against each other and orthogonal to the capsid that trimerize to form pseudo-hexameric capsomers. This fourth lineage, possibly the least well characterized, includes viruses that infect eukaryotes, bacteria, and archaeal and that often contain a lipid membrane underneath the icosahedral protein capsid.

A Greasy Aid to Capsid Assembly: Lessons from a Salty Virus. (2015) Structure, 23(10): 1777-1779.
Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single β-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single β-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly.

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Good Amyloid, Bad Amyloid – What’s the Difference?

Curli It’s quite remarkable that after so many years of studying prions we still don’t have a good understanding of their pathogenesis – how do they make normal cellular proteins turn evil? This interesting article suggests that it’s not the end stage amyloid deposits that we should be focussing on, but the toxicity (or otherwise) of the intermediate oligomers which generate the fibres and plaques.


Good Amyloid, Bad Amyloid—What’s the Difference? (2016) PLoS Biol 14(1): e1002362. doi:10.1371/journal.pbio.1002362
Amyloids have had some bad press. From Alzheimer to Parkinson, Huntington to Creutzfeldt-Jakob, amyloids are best known for their role in causing some very serious human diseases. The usual story is that the structure of a perfectly respectable, neatly folded (and usually soluble) protein is disrupted in some way, making it refold into a new “cross-beta” structure that predisposes multiple copies of that protein to tile together into a massive insoluble fibrous deposit. The trigger may be a mutation, an unusual cleavage pattern, a post-translational modification, or even (in the case of a class of amyloids called prions) infection by a ready-formed amyloid, but the end point tends to be the same – insoluble protein, cognitive problems, and eventual neurodegeneration.

Research has understandably been focused on the pathogenic amyloids that are involved in human neurological diseases, but several points have recently become clear. The first is that despite their suspicious presence at the scene of the crime, the insoluble deposits of amyloid may not themselves be the culprits and that instead it’s the smaller “oligomeric” assemblages or even the monomers of amyloidogenic protein (en route to fiber formation) that may cause the real damage. And the second is that not all amyloids are bad – some are biologically useful and have been selected for their beneficial function during evolutionary history.

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Sorry, it turns out you are human after all

Revised estimates for the number of human and bacteria cells in the body It’s often said that the bacteria and other microbes in our body outnumber our own cells by about ten to one. But that’s a myth that should be forgotten according to a new paper – the ratio between resident microbes and human cells is more likely to be one-to-one. The myth arises from a rough estimate made in 1972 which has persisted ever since.

Of course, this only applies to cells – the number of viruses in your body vastly outnumber all the cells.


Revised estimates for the number of human and bacteria cells in the body. (2016) bioRxiv
We critically revisit the ″common knowledge″ that bacteria outnumber human cells by a ratio of at least 10:1 in the human body. We found the total number of bacteria in the ″reference man″ to be 3.9·1013, with an uncertainty (SEM) of 25%, and a variation over the population (CV) of 52%. For human cells we identify the dominant role of the hematopoietic lineage to the total count of body cells (≈90%), and revise past estimates to reach a total of 3.0·1013 human cells in the 70 kg ″reference man″ with 2% uncertainty and 14% CV. Our analysis updates the widely-cited 10:1 ratio, showing that the number of bacteria in our bodies is actually of the same order as the number of human cells. Indeed, the numbers are similar enough that each defecation event may flip the ratio to favor human cells over bacteria.

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A host protein restricts bird flu in mammals

MxA Influenza A viruses circulate in various natural hosts, including mammals and birds. Transmission of influenza virus between mammals and birds occurs only rarely, owing to host restriction: an influenza A virus that is adapted to an avian host typically does not grow well in a mammalian host, and vice versa. When such restrictions are overcome and an avian virus transmits to humans, a pandemic can occur.

The Influenza PB2 protein is part of the influenza A polymerase enzyme complex, which copies the virus genome and is essential for replication. For many years, researchers have known that a specific domain of PB2 is involved in host restriction. A new paper in Nature (£) shows that a single bird gene, ANP32A, enables an avian-adapted PB2 protein to function efficiently in mammalian cells. Chicken and human ANP32A proteins are similar except for a stretch of 33 amino acids missing from the human protein. All avian ANP32A genes, except those of ostriches, encode these 33 amino acids, whereas all mammalian versions lack this region.

Further investigation of ANP32A’s role in virus replication may open the way to the development of new antiviral drugs.


Species difference in ANP32A underlies influenza A virus polymerase host restriction. (2016) Nature 529, 101–104 doi:10.1038/nature16474
Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals.

Virology: Host protein clips bird flu’s wings in mammals. (2016) Nature 529, 30–31 doi:10.1038/529030a

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New Polio Vaccine Strains for the Post-Eradication Era

Poliovirus When I started my scientific career by working on polio vaccines over 30 years ago, we were already thinking about newer, safer vaccines. Although polio eradication remains tantalizingly out of reach, we are close enough now to think in detail about what needs to be done after eradication. It won’t be as simple as with smallpox – polio vaccination will need to continue.


New Strains Intended for the Production of Inactivated Polio Vaccine at Low-Containment After Eradication. (2015) PLoS Pathog 11(12): e1005316. doi:10.1371/journal.ppat.1005316
New polio vaccines will be needed to safeguard global eradication: Sabin strains are known to evolve to fill the niche left by wild-strains so their long-term use is incompatible with eradication; most current inactivated vaccine is made from wild polioviruses so that production presents a significant biosecurity risk. We have developed new strains for Inactivated Polio Vaccine (IPV) production with negligible risk to the human population should they escape. Sabin’s live-attenuated vaccines are variants of wild strains selected by the use of unnatural cell substrates, hosts and growth conditions. Unsurprisingly these variants evolve back towards wild-type properties during replication in, and transmission between, their natural hosts. An understanding of the molecular basis of these pathways led us to design novel vaccine strains that are very highly attenuated and arguably cannot replicate in people and whose opportunities for reversion during replication in cell culture are severely restricted. At the same time, the strains can feasibly be produced on a large-scale and they are as immunogenic as current IPV. These attributes allow for safe vaccine production in the post-eradication world.

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Doomed? No, science can still save us


Recently there were stories in the media about a new superbug that might kill us all. This started with the discovery of bacteria in China which are resistant to the drugs used when all other treatments have failed. So is the Chinese superbug going to kill us all or are the media making a fuss over nothing? Well no, and no.

Bacteria resistant to antibiotics are not new. They’ve been around for millions of years – much longer than humans or medicine. This is because antibiotics are naturally occurring substances produced by bacteria in soil or water to kill off the competition and gain an advantage.

But the competition doesn’t want to be killed off, so for millions of years bacteria have been evolving ways to shrug off the effects of antibiotics. And they are damn good at it. Then in 1928 Sir Alexander Fleming discovered penicillin, and after a lot of effort during the Second World War it was developed into a useful drug. But even when he was accepting the Nobel Prize in 1945 Fleming warned: “It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally happened in the body.”

In an era of post-war optimism that wasn’t a message that anyone wanted to hear, so we chose not to.

In 1957 Harold Macmillan told us “you’ve never had it so good” – and in medical terms, he was right. We could treat nearly all bacterial infections, including deadly ones like tuberculosis, with a course of antibiotics. But complacency was setting in.

Back in the 1960s antibiotic-resistant bacteria were cropping up, but we stayed one step ahead of them by developing new antibiotics. This was a good business for drug companies –- while it lasted. Eventually, the pipeline ran dry. No new types of antibiotics have been discovered since the 1980s, and the bacteria have pulled level in the race.

Cue media stories of the post-antibiotic era in which simple infections are deadly, cancer therapy and transplants which wipe out the immune system are impossible, and childbirth is a dangerous lottery.

So are we doomed? Nope. The antibiotic era of medicine may be coming to an end, but we have the ability to go on beating the bugs. We could, if we wanted to, edit the human genome to make us resistant to AIDS and many other diseases – but there are reasons why we might choose not to do that. Rather than relying on nature for our antibiotics, we can use nanotechnology to manufacture new ones. We can outsmart some of the tricks bacteria use to make us sick by jamming their communication systems.

Given enough time and money to respond, science can save us. The alternative doesn’t bear thinking about.

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Oncolytic Virotherapy – Where Are We Going?

Oncolytic Viruses The last few years have seen an increased interest in immunotherapy in the treatment of malignant disease. In particular, there has been significant enthusiasm for oncolytic virotherapy, with a large amount of pre-clinical data showing promise in animal models in a wide range of tumour types. How do we move forward into the clinical setting and translate something which has such potential into meaningful clinical outcomes? This article reviews how the field of oncolytic virotherapy has developed so far and what the future may hold.


Evidence for Oncolytic Virotherapy: Where Have We Got to and Where Are We Going? (2015) Viruses 7(12), 6291-6312 doi: 10.3390/v7122938

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33 million healthy life years lost annually to foodborne diseases

The gut The global burden of foodborne diseases caused by contaminated food in 2010 has been estimated as 33 million Disability Adjusted Life Years. Of these years lost, 40% were in children under 5 years old. This is according to new estimates from a World Health Organisation (WHO) taskforce, which publishes its findings this week in a new PLOS Collection.

To measure the global and regional burden of foodborne disease, WHO established the Foodborne Disease Burden Epidemiology Reference Group (FERG), which now report their first estimates of the incidence, mortality, and disease burden due to 31 foodborne hazards in 2010. FERG found that the global burden of foodborne disease is comparable to HIV/AIDS, malaria or tuberculosis. Diarrheal disease agents, especially non-typhoidal Salmonella enterica, were responsible for the majority of deaths. Other major causes of foodborne disease deaths were Salmonella Typhi, Taenia solium (pork tapeworm) and hepatitis A virus. For those cases where illness, rather than death, is documented, the most frequent causes were diarrheal disease agents, particularly norovirus and Campylobacter spp. Among chemical agents evaluated by FERG, aflatoxin was found to cause the greatest burden.

The PLOS Collection also documents global variation in the impact of foodborne disease, with Africa being hardest hit, followed by sub-regions of South-East Asia and Eastern Mediterranean. Tthe burden of foodborne disease is borne particularly by children under five years old – although they represent only 9% of the global population – and people living in low-income regions of the world.

WHO Estimates of the Global Burden of Foodborne Disease. PLOS Collections, 2015

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