I spoke with virologist Ian Goodfellow, whose laboratory works on noroviruses, about why he went to Sierra Leone to establish an Ebolavirus diagnostic and sequencing laboratory. The obstacles he encountered were considerable, but the results were very useful. Recorded at the Emerging Infectious Diseases A to Z (EIDA2Z) conference hosted by the National Emerging Infectious Diseases Laboratories (NEIDL).
Prion diseases, also known as spongiform encephalopathies, are uniformly fatal, chronic degenerative neurological diseases caused by misfolding of a cellular protein, PrPC. Transmissible encephalopathies may be acquired by organ transplant, receiving contaminated blood, or the ingestion of contaminated food.
In the 1990s a new spongiform encephalopathy, variant Creutzfeld-Jakob disease or vCJD, began to appear in Great Britain. Variant Creutzfeld-Jakob disease is caused by prions acquired by the consumption of cattle with bovine spongiform encephalopathy, also a prion disease affectionately known as mad cow disease. To date 231 cases of vCJD have been reported, mainly in the UK and France.
Although the spread of BSE has been controlled by surveillance and feeding restrictions, it is estimated that millions of people were exposed to BSE prions. The concern is that some of these individuals might be infected but show no symptoms of disease. If they donate blood, they may transmit infection to others. It is known that several cases of vCJD have been transmitted from infected blood donors, so further transmission is a major concern. So far prion diseases have only been diagnosed after death, by detection of conformationally altered prion proteins in the brain.
Two sensitive and specific assays for vCJD prions have now been developed that show promise for non-invasive pre-symptomatic diagnosis of the disease. They are both based on a technology called protein misfolding cyclic amplification (PMCA, illustrated; image copyright ASM Press, 2015). A small amount of the normal human prion protein, PrPC (produced in transgenic mice) is mixed with plasma. The samples are incubated to allow formation of prion oligomers, followed by disruption by a pulse of sonication to disrupt the oligomers. The cycle is repeated multiple times, much like polymerase chain reaction (PCR) which is used to amplify small amounts of DNA. Prions are detected by western blot analysis after treatment with proteinase K. The misfolded, pathogenic prions, PrPSC , are not completely digested with this enzyme.
In one study, PMCA was used to analyze blood samples from 14 cases of vCJD and 153 controls, which included healthy individuals and those with other neurological diseases, including sporadic CJD (sCJD – not caused by ingestion of contaminated beef). All 14 samples from cases of vCJD were positive in the PMCA assay, but not any of the other samples.
In a second study, the PMCA assay was positive in samples from all 18 patients with vCJD. Of 134 control samples, just one was positive for vCDJ, from a patient with sCJD. Furthermore, the assay detected vCJD prions in archived blood samples from donors who gave blood before developing symptoms of the disease.
These findings suggest that the new assays can detect vCJD prions in the blood before the appearance of the neurological symptoms of spongiform encephalopathy. While additional samples must be analyzed to validate the results, they are nonetheless promising as a way to prevent spread of the disease via the blood supply. Unfortunately, if you are diagnosed with vCJD by one of these assays, that is the only positive outcome – there are as yet no treatments for any spongiform encephalopathy.
The TWiV academia discuss induction of diarrhea by the capsid protein of an astrovirus, and association of a fungal RNA virus with white-nose syndrome of North American bats.
You can find TWiV #423 at microbe.tv/twiv, or listen below.
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Virus infections initiate when virions bind to receptors on the cell surface. It is well known that cells can be made susceptible to infection by providing DNA encoding the virus receptor. For example, mice cannot be infected with poliovirus, but become susceptible if they are given the human poliovirus receptor gene. Now we have learned that providing the receptor protein is sufficient to make cells susceptible to infection (link to paper).
Bacteriophages determine the composition of microbial populations by killing some bacteria and sparing others. Bacteriophages are typically host specific, a property that is largely determined at the level of attachment to host cell receptors. How resistant and sensitive bacteria in mixed communities respond to phage infection has not been well studied.
Several phages (including SPP1, pictured) of the soil bacterium Bacillus subtilis first attach to poly-glycosylated teichoic acids (gTA), and then to the membrane protein YueB, leading to injection of DNA into the cell. Cells that lack the gene encoding either of these proteins are resistant to infection.
When a mixed culture of resistant and susceptible B. subtilis cells were infected with phage SPP1, both types of cells became infected and killed. Infection of resistant cells depended on the presence of susceptible cells, because no infection occurred in pure cultures of resistant cells.
Both infected and uninfected bacteria release small membrane vesicles that contain proteins, nucleic acids, and other molecules. Phage SPP1 can attach to membrane vesicles released by susceptible strains of B. subtilis, showing that they contain viral receptor proteins. Furthermore, phage SPP1 can infect resistant cells that have been incubated with membrane vesicles from a susceptible strain – in the absence of intact susceptible cells.
These results show that membrane vesicles released by susceptible bacteria contain viral receptors that can be inserted into the membrane of a resistant cell, allowing infection. Because phage infection can lead to transfer of host DNA from one cell to another, the results have implications for the movement of genes for antibiotic resistance or virulence. It’s possible that such genes may move into bacteria that have only ‘temporarily’ received virus receptors via membrane vesicle transfer.
These findings should also be considered when designing phage therapy for infectious diseases. The idea is to utilize phages that are host specific and can only destroy the disease-producing bacteria. It’s possible that the host range of such phages could be expanded by receptor protein transfer. As a consequence, unwanted genes might make their way into ‘resistant’ bacteria.
I wonder if membrane vesicle mediated transfer of receptors also occurs in eukaryotic cells. They shed membrane vesicles called exosomes, which contain protein and RNA that are delivered to other cells. If exosomes bear receptors for viruses, they might be able to deliver the receptors to cells that would not normally be infected. The types of cells infected by a virus would thereby be expanded, potentially affecting the outcome of viral disease.
Vincent Racaniello interviews Harmit Malik, PhD, Fred Hutchinson Cancer Research Center. Harmit and his laboratory are interested in a variety of problems that are characterized by evolutionary conflict.
This video is one of 26 video interviews with eminent virologists that are part of the supplemental material for Principles of Virology, 4th Edition, published by ASM Press. Other interviews in this series can be found at this link.
The TWiVnauts present another example of an infectious but replication incompetent vaccine, an insect specific arborvirus bearing chikungunya virus structural proteins.
You can find TWiV #421 at microbe.tv/twiv, or listen below.
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A new breed of vaccines is on the horizon: they replicate in one type of cell, allowing for their production, but will not replicate in humans. Two different examples have recently been described for influenza and chikungunya viruses.
The influenza virus vaccine is produced by introducing multiple amber (UAG) translation stop codons in multiple viral genes. Cloned DNA copies of the mutated viral RNAs are not infectious in normal cells. However, when introduced into specially engineered ‘suppressor’ cells that can insert an amino acid at each amber stop codon, infectious viruses can be produced. These viruses will only replicate in the suppressor cells, not in normal cells, because the stop codons lead to the production of short proteins which do not function properly.
When inoculated into mice, the stop-codon containing influenza viruses infect cells, and although they do not replicate, a strong and protective immune response is induced. Because the viral genomes contain multiple mutations, the viruses are far less likely than traditional infectious, attenuated vaccines to sustain mutations that allow them to replicate in normal cells. It’s a clever approach to designing an infectious, but replication-incompetent vaccine (for more discussion, listen to TWiV #420).
Another approach is exemplified by an experimental vaccine against chikungunya virus. The authors utilize Eilat virus, a virus that only replicates in insects. The genes encoding the structural proteins of Eilat virus were replaced with those of chikungunya virus. The recombinant virus replicates in insect cells, but not in mammalian cells. The virus enters the latter cells, and some viral proteins are produced, but genome replication does not take place.
When the Eilat-Chikungunya recombinant virus in inoculated into mice, there is no genome replication, but a strong and protective immune response is induced. The block to replication – viral RNA synthesis does not occur – is not overcome by multiple passages in mice. Like the stop-codon containing influenza viruses, the Eilat recombinant virus is a replication-incompetent vaccine.
These are two different approaches to making viruses that replicate in specific cells in culture – the suppressor cells for influenza virus, and insect cells for Eilat virus. When inoculated into non-suppressor cells (influenza virus) or non-insect cells (Eilat virus), a strong immune response is initiated. Neither virus should replicate in humans, but clinical trials have to be done to determine if they are immunogenic and protective.
The advantage of these vaccine candidates compared with inactivated vaccines is that they enter cells and produce some viral proteins, likely resulting in a stronger immune response. Compared with infectious, attenuated vaccines, they are far less likely to revert to virulence, and are easier to isolate.
These two potential vaccine technologies have been demonstrated with influenza and chikungunya viruses, but they can be used for other virus. The stop-codon approach is more universally applicable, because the mutations can be introduced into the genome of any virus. The Eilat virus approach can only be used with viruses whose structural proteins are compatible with the vector – probably only togaviruses and flaviviruses. A similar approach might be used with insect-specific viruses in other virus families.
Why do I call these vaccines ‘paradoxical’? Because they are infectious and non-infectious, depending on the host cell that is used.
Note: The illustration is from a t-shirt, and the single letter code of the protein spells out a message. However the title, ‘the gene stops here’, is wrong. It should be ‘the protein stops here. The 3’-untranslated region, which continues beyond the stop codon, is considered part of the gene.
The TWiV gurus describe how to use an orthogonal translation system to produce infectious but replication-incompetent influenza vaccines.
You can find TWiV #420 at microbe.tv/twiv, or listen below.
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In a recent editorial, the New York Times wrote about ‘the breakdown of a shared public reality built upon widely accepted facts’. As a scientist, I am appalled by the disdain for facts shown by many in this country, including the President-Elect. Unfortunately, science is not without its share of fake information.
The Times argues that at one time, nearly everyone had a unified source of news – the proverbial Walter Cronkite. Social media and the internet changed all that, allowing people to have their own sources of news, whether they be real or fake. The web developers in Macedonia who are paid $30,000 a month to spew out fake news are just part of the problem.
The goal of science is to discover how our world works. It’s about finding facts, not fake answers. Yet fake science has always been with us. Not long after Edward Jenner demonstrated vaccination against smallpox using pustules from milkmaids with cowpox, skeptics thought that this process would lead to the growth of cow-parts from the inoculated areas (see illustration). To this day anti-vaxers spew fake science which they claim shows that vaccines are not safe, do not work, or cause autism.
Fake science does not stop with anti-vaxers. There are people who deny climate change (including our President-Elect), despite easily accessible data showing that the trend is real. There are people who, bafflingly, claim that HIV does not cause AIDS, or that Zika virus does not cause birth defects, or that genetically modified plants will cause untold harm to people who consume them. The list of fake science goes on and on. The situation is appalling to any scientist who examines the data and finds clear proof that HIV does cause AIDS, and that Zika virus does cause birth defects.
There is also fake science perpetrated by scientists – those who publish fake data to advance their career. There are so many examples of such science fraud that there is a website to document the inevitable retractions – called RetractionWatch, of course. I find the existence of such a site lamentable.
That fake news can play such a large part in the operation of our society was something I only recognized recently. My initial reaction, as a scientist, was outrage that anyone would want to believe in, and adopt, lies. But this is a naive reaction, not only because bad behavior should always be expected of some humans, but because fake science has surrounded me for my entire career.
Nevertheless, I am a scientist who looks for the truth, and I simply cannot tolerate fabrication, whether in science or politics or in any field.
I don’t know how to solve the fake news and fake science problems. But the Times has a suggestion:
Without a Walter Cronkite to guide them, how can Americans find the path back to a culture of commonly accepted facts, the building blocks of democracy? A president and other politicians who care about the truth could certainly help them along. In the absence of leaders like that, media organizations that report fact without regard for partisanship, and citizens who think for themselves, will need to light the way.
I’m not sure that today’s profit-driven media organizations are the answer to the fake news problem. But I’ve always felt that scientists can help counter fake science. We all need to communicate in some way so that the public sees us as a single voice, advocating the huge role that science plays in our lives. That’s why here at virology blog, and over at MicrobeTV, you’ll always find real science.