Purging the PERVs

pigThere aren’t enough human organs to meet the needs for transplantation, so we have turned to pigs. Unfortunately pig cells contain porcine endogenous retroviruses, PERVS, which could infect the transplant recipient, leading to tumor formation. But why worry? Just use CRISPR to purge the PERVs.

The genomes of many species on Earth are littered with endogenous retroviruses. These are DNA copies of retroviral genomes from previous infections that are integrated into germ line DNA and passed from parent to offspring. About 8% of the human genome consists of ERVs. The pig genome is no different – it contains PERVs (an acronym made to play with). The genome of an immortalized pig cell line called PK15 contains 62 PERVs. Human cells become infected with porcine retroviruses when they are co-cultured with PK15 cells.

The presence of PERVS is an obvious problem for using pig organs for transplantation into humans – a process called xenotransplantation. The retroviruses produced by pig cells might infect human cells, leading to problems such as immunosuppression and tumor formation. No PERV has ever been shown to be transmitted to a human, but the possibility remains, especially with   the transplantation of increasing numbers of pig organs into humans.

The development of CRISPR/Cas9 gene editing technology made it possible to remove PERVs from pigs, potentially easing the fears of xenotransplantation. This technology was first used to remove all 62 copies of PERVS from the PK15 cell line. But having PERV-free pig cells doesn’t help humans in need of pig organs – for that you need pigs.

To make pigs without PERVs, CRISPR/Cas9 was used to remove the PERVs from primary (that is, not immortal) pig cells in culture. Next, the nuclei of these PERV-less cells was used to replace the nucleus of a pig egg cell. After implantation into a female, these cells gave rise to piglets lacking PERVs.

In theory such PERV-less piglets can be used to supply organs for human transplantation, eliminating the worrying about infecting humans with pig retroviruses. But first we have to make sure that the PERV-free pigs, and their organs, are healthy. The more we study ERVs, the more we learn that they supply important functions for the host. For example, the protein syncytin, needed to form the placenta, is a retroviral gene, and the regulatory sequences of interferon genes come from retroviruses. There are likely to be many more examples of essential functions provided by ERVs. It would not be a good idea to have transplanted pig organs fail because they lack an essential PERV!

TWiV 454: FGCU, Zika

Sharon Isern and Scott Michael return to TWiV for a Zika virus update, including their work on viral evolution and spread, and whether pre-existing immunity to dengue virus enhances pathogenesis.

 

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TWiV 453: Neurovirology with Diane Griffin

From the Vector-Borne Viruses Symposium in Hamilton, Montana, Dickson and Vincent speak with Diane Griffin about her career and her work on understanding viral infections of the central nervous system.

 

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TWiV 452: Kiss that frog

Lynda Coughlan joins the weekly virtual bus companions for a discussion of a host defense peptide from frogs that destroys influenza virus, and mouse models for acute and chronic hepacivirus infection.

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Kermit’s urumi

Hydrophylax bahuvistaraFrogs don’t get flu (as far as I know) but their skin contains a peptide that inhibits the replication of influenza virus (link to paper).

Frog skin contains host defense peptides (HDPs), part of the innate immune defenses of many species. They were first found in amphibians by Michael Zasloff, who, as part of his research, performed surgery on frogs and then returned them to an aquarium – which was not sterile. He wondered why the frogs always healed without signs of infection, which lead him to discover the antimicrobial peptides, called magainins, in frog skin. HDPs had been first discovered years earlier in the silk moth.

Amphibian HDPs are active against bacteria, fungi, viruses, and protozoa. To discover HDPs that inhibit influenza virus, 32 HDPs from skin secretions of the Indian frog Hydrophylax bahuvistara were screened by mixing them with virus followed by a plaque assay. One peptide was found to potently inhibit influenza virus replication without cell toxicity. It was called urumin, after the whip sword known as urumi.

Urumin inhibits infectivity of influenza H1N1 viruses far better than H3N2 viruses. The reason is that the peptide targets the viral H1 hemagglutinin, one of two glycoproteins in the viral envelope. Furthermore, the peptide appears to interact with the conserved stalk region of the HA glycoprotein, and not with the globular head.

Currently two different antiviral drugs, oseltamivir and relenza, are used to control influenza virus infection. Viruses resistant to these drugs were still inhibited by urumin, indicating that should urumin ever be licensed, it would be useful in the event that oseltamivir and relenza resistant viruses became more common.

Examination of urumin treated virus particles by electron microscopy revealed that they are disrupted by the peptide. How urumin breaks influenza virus particles is not known. However, the HDP nisin destroys bacteria by first binding to a bacterial membrane component, then moving into the membrane. After binding to HA, urumin might in a simlar way disrupt the membrane of influenza virus particles.

Urumin also reduced disease, death, and the amount of virus in the lung in mice intranasally infected with influenza virus.

These observations suggest that urumin is worthy of additional study as an influenza virus inhibitor. HDPs are attractive antimicrobial compounds because resistance to their mechanisms of action is lower than for other types of inhibitors. However, enthusiasm for urumin is dampened because, despite extensive study, no HDP has yet been approved by the US Food and Drug Administration for use in humans. The obstacles to therapeutic success of HDPs have not been identified.

TWiV 450: Ben tenOever and RNA out

Ben tenOever joins the TWiVoli to discuss the evolution of RNA interference and his lab’s finding that RNAse III nucleases, needed for the maturation of cellular RNAs, are an ancient antiviral RNA recognition platform in all domains of life.

 

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The Traditional Lecture is Not Dead. I Would Know – I’m A Professor

Virology 2017Wired Magazine recently published an article with a headline distinctly opposite of mine, which claims that the traditional lecture is dead. I disagree, and here is why.

The thesis of the article, by Rhett Allain, is that modern technologies have made the traditional lecture obsolete. The traditional lecture is one during which a teacher stands at the front of the room and ‘disseminates knowledge to students’. Allain claims – rightly so – that animated videos like The Mechanical Universe are far more engaging. He suggests that, to teach physics, just show the students episodes from this show. If they have questions, just pause the show.

He claims that showing these videos – or equivalents for other subjects – beats most lectures. Lectures in which teachers drone on and on.

Well guess what – I teach a virology course at Columbia University, and at the end of the year, most of the students say its one of the best, or the best class they have taken in their college years. I don’t show The Viral Universe or any other videos during my class. I talk to the students about my knowledge of viruses, gained from researching them for over 30 years.

Not every virology lecturer has my experience in the field. Many of them learn virology from a book. I agree that lectures given by those individuals are dead.

I do record each lecture and post them at YouTube, so that the students in the class, or anyone in the world for that matter, can watch them. It’s a new technology that Allain likes. I like allowing the students to time-shift their learning: some never come to class. But it’s still me making the videos and sharing my knowledge via a traditional lecture format.

Allain is also fond of the flipped classroom – assign a video for students to watch before class, and then use class time to discuss it. I love this idea. But I still think that for my introductory virology class, it’s better for me to talk to them. To walk around the room, without notes, look them in the eye, and muster all my passion and love for the field and send it their way. And don’t think that doesn’t matter – many of my students tell me that my passion for the subject is what makes them interested in viruses.

Research says that most students learn better by doing. I do pause a few times during each lecture to have students complete an online quiz – something Allain also likes. It gives me time to see if what I’m saying is sinking in, and to clarify complex material.

When I lecture, students come to me afterwards with questions, and some even walk with me to the subway, to talk about viruses. How can a video provide that experience?

Allain’s response might be that if anyone wants to teach a virology course, just play my lectures and discuss them in class. I’m all for that. But every spring semester, I’ll be in front of the class, talking about viruses, and making new videos. There is no substitute for a expert who is passionate about their subject. I realize that every physics course can’t be taught by Einstein, but he can teach at least one, and the students at his university will love it.

Not every passionate researcher will make a great lecturer, and for them,  videos and flipped classrooms are a great way to teach. But for those passionate researchers who can teach – why not put them in front of a class and inspire the next generation? I do it every year.

TWiV 447: Un-impacting an elephant

The glorious TWiVerati un-impact their email backlog, anwering questions about viruses, viruses, viruses, viruses, viruses, and more. You should listen – our fans ask great questions!

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TWiV 446: Old sins die hard

The TWiV hosts review an analysis of gender parity trends at virology conferences, and the origin and unusual pathogenesis of the 1918 pandemic H1N1 influenza virus.

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The first human virus discovered

PlaqueOn the wall of a Columbia University Medical Center building just across the street from my laboratory is a plaque commemorating two participants in the discovery of a mosquito vector for yellow fever virus.

The plaque reads:

Aristides Agramonte, Jesse William Lazear, Graduates of the Columbia University College of Physicians and Surgeons, class of 1892. Acting Assistant Surgeons, U.S. Army. Members of the USA Yellow Fever Commission with Drs. Walter Reed and James Carroll. Through devotion and self-sacrifice they helped to eradicate a pestilence of man.

Yellow fever, known in tropical countries since the 15th century, was responsible for devastating epidemics associated with high rates of mortality. The disease can be mild, with symptoms that include fever and nausea, but more severe cases are accompanied by major organ failure. The name of the illness is derived from yellowing of the skin (jaundice) caused by destruction of the liver. For most of its history, little was known about how yellow fever was spread, although it was clear that the disease was not transferred directly from person to person.

Cuban physician Carlos Juan Finlay proposed in 1880 that a bloodsucking insect, probably a mosquito, was involved in yellow fever transmission. The United States Army Yellow Fever Commission was formed in 1899 to study the disease, in part because of its high incidence among soldiers occupying Cuba. Also known as the Reed Commission, it comprised four infectious disease specialists: U.S. Army Colonel Walter Reed (who was the chair); Columbia graduates Lazear and Agramonte, and James Carroll. Lazear confirmed Finlay’s hypothesis in 1900 when he acquired yellow fever after being experimentally  bitten by mosquitos who had fed on sick patients. Days later, he died of the disease.

The results of the Reed Commission’s study proved conclusively that mosquitoes are the vectors for this disease. Aggressive mosquito control in Cuba led to a drastic decline in cases by 1902.

The nature of the yellow fever agent was established in 1901, when Reed and Carroll injected filtered serum from the blood of a yellow fever patient into three healthy individuals. Two of the volunteers developed yellow fever, causing Reed and Carroll to conclude that a “filterable agent,” which we now know as yellow fever virus, was the cause of the disease.

Sometimes you don’t have to wander far to find some virology history.

Update 6/16/17: The statement on the plaque that Agramonte and Lazear “helped to eradicate a pestilence of man” is of course incorrect, as yellow fever has never been eradicated. Recent large outbreaks of yellow fever in Brazil and Angola are examples of the continuing threat the virus poses, despite the availability of a vaccine since 1938.