On episode #262 of the science show This Week in Virology, Vincent returns to the University of Wisconsin – Madison to speak with Ann Palmenberg about her career in virology.
You can find TWiV #262 at www.twiv.tv.
One of my goals as a science communicator is to be Earth’s virology professor. To do this I teach an undergraduate virology course at Columbia University and at iTunes University. This past summer I ported my undergraduate virology course to Coursera.org where I reached 26,000 students. My next virology course at Coursera, How viruses cause disease, begins on 9 January 2014.
How viruses cause disease explores the interplay between viruses and their host organisms. The course begins with an overview of how infection is established in a host, then moves to a virologist’s view of immune defenses. Next we consider how the replication strategy and the host response determine the outcome of infection, such that some are short and others are of long duration. The mechanisms by which virus infections transform cells in culture are explored, a process that may lead to tumor formation in animals. We then move to a discussion of how viral infections are controlled by vaccines and antiviral drugs. After an introduction to viral evolution, we discuss the principles learned from zoonotic infections, emerging infections, and humankind’s experiences with epidemic and pandemic viral infections. The course ends with an exploration of unusual infectious agents such as viroids, satellites, and prions, followed by a discussion of the causative agent of the most serious current worldwide epidemic, HIV-1.
To create the Coursera courses, I divide the lecture videos from my undergraduate offering into 10-20 minute segments. I add annotations to indicate parts of the illustrations that I highlight during each lecture. Questions are also inserted in the videos to ensure that students are learning the desired principles. Weekly quizzes, a final exam, and discussion forums round out the Coursera experience.
Because others might benefit from the shorter videos, I have also made them available at YouTube. These videos are annotated, but do not have the built-in questions which are only available on Coursera. I would be pleased to learn how to add questions to YouTube videos.
On episode #261 of the science show This Week in Virology, Vincent meets up with Chantal and Jean-Michel at the first International Symposium on Giant Virus Biology in Tegernsee, Germany, to discuss their work on Mimivirus, Megavirus, and Pandoravirus.
You can find TWiV #261 at www.twiv.tv.
On episode #260 of the science show This Week in Virology, Vincent visits the University of Wisconsin, Madison and speaks with Tom, Tony, and David about their work on virus discovery at the AIDS Vaccine Research Laboratory.
You can find TWiV #260 at www.twiv.tv.
The hemagglutinin (HA) and neuraminidase (NA) glycoproteins of the influenza virus particle serve distinct functions during infection. The HA binds sialic acid-containing cellular receptors and mediates fusion of the viral and cell membranes, while the NA removes sialic acids from glycoproteins. Apparently this division of labor is not absolute: influenza viruses have been identified with NA molecules that serve as receptor binding proteins.
An influenza virus was created that could not bind sialic acid by introducing multiple mutations into the HA gene. This mutant virus was not expected to be infectious, but nevertheless did propagate to moderate titers in cell culture. A single amino acid change was identified in the NA protein of this virus: G147R, which is just above the active site of the enzyme (illustrated; active site marked with green spheres). Passage of the virus in cell culture produced a virus that multiplied to higher titers; improved growth was caused by a K62E change in the HA stalk. The results of site-directed mutagenesis showed that the G147R change allowed the NA protein to serve the receptor binding function normally provided by HA. It is not clear how the HA change leads to improved growth of the G147 virus.
Although the G147R NA can serve as receptor binding protein, the HA is still required for fusion: abolishing this activity by mutation or by treatment with a fusion-blocking antibody did not allow virus growth.
The influenza NA protein is an enzyme (sialidase) that cleaves sialic acids from cellular and viral proteins. The G147R NA is active as a sialidase, and this activity can be blocked by the antiviral compound oseltamivir, which is an NA inhibitor. Treatment of G147R-containing virus with oseltamivir also blocked virus binding to cells. Virus-like particles that contain G147R NA but not HA can attach to sialic acid-containing red blood cells. This attachment can be reversed by oseltamivir. After binding to red blood cells, these virus-like particles slowly fall off, a consequence of NA cleaving sialic acid receptors. These observations indicate that the G147R NA binds to sialic acids at the active site of the enzyme, and cleaves the same receptor that it binds.
Treatment of cells with a bacterial sialidase that removes a broad range of sialic acids only partially inhibits G147R NA-mediated binding to cells. In contrast, growth of wild type influenza virus is completely blocked by this treatment. Therefore the receptor recognized by G147R NA is not the same as that bound by wild type virus.
Changing the influenza virus NA to a receptor binding protein is not simply a laboratory curiosity: the G147R NA change was found in 31 of 19,528 NA protein sequences in the Influenza Virus Resource. They occur in seasonal H1N1 viruses that circulated before 2009, in the 2009 swine-origin pandemic H1N1 virus, and in avian H5N1 viruses. The presence of this change in phylogenetic clusters of seasonal H1N1 and chicken H5N1 sequences suggests that they are also found in circulating viruses, and are not simply sequence errors or the product of passage in the laboratory.
These observations emphasize the remarkable flexibility of the influenza viral glycoproteins in their ability to switch receptor binding function from HA to NA. They might also have implications for vaccines, whose effectiveness are thought to depend largely on the induction of antibodies that block the function of HA protein. The work underscores the importance of serendipity in science: the HA receptor binding mutant virus was originally produced as a negative control for a different experiment.
On episode #259 of the science show This Week in Virology, Vincent and Rich join Jackie at the University of Texas, Austin to talk about her work on mouse mammary tumor virus.
You can find TWiV #259 at www.twiv.tv.
It is well known that aquatic birds are a major reservoir of influenza A viruses, and that pandemic human influenza virus strains of the past century derive viral genes from this pool. The recent discovery of two new influenza A viruses in bats suggests that this species may constitute another reservoir with even greater genetic diversity.
A new influenza virus had previously been isolated from little yellow-shouldered bats (Sturnira lilium) in Guatemala. Three of 316 rectal swabs were positive when tested by a pan-influenza polymerase chain reaction assay. Viral sequences were also detected in liver, intestine, lung, and kidney tissues, suggestive of viral replication and not passage of ingested material through the intestinal tract. Analysis of the viral genome sequence revealed that A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10) is significantly diverged from all known influenza viruses.
When the same PCR approach was used to screen 114 rectal swabs from 18 different species of bats captured in Peru, a single flat-faced fruit bat (Artibeus planirostris) was positive. Viral sequences were also detected in liver, intestine, and spleen tissues from the same bat. Comparison of the sequences of all 8 genome RNA segments with those of the H17N10 Guatemalan isolate revealed sufficient divergence to justify naming it a new HA and NA subtype, A/flat-faced bat/Peru/033/2010 (H18N11).
Comparison of the nucleotide sequences of bat influenza A viruses from Peru and Guatemala with other influenza viruses leads to two amazing conclusions. First, 7 of the 8 viral RNAs of the bat influenza A viruses group separately from the RNAs of all other known influenza viruses. Second, the RNA sequences encoding four proteins, PB2, PB1, PA and NA, display greater genetic diversity than in all non-bat influenza virus sequences combined. The implication is that New World bats harbor a diverse pool of influenza viruses.
The H17 and H18 HA RNA sequences are, in contrast, far more related to known influenza virus HA and NA sequences. The implication of this observation is clear: some time after the bat and non-bat influenza A viruses diverged, a reassortment event occurred that introduced the HA of a non-bat influenza A virus into the genome of a bat influenza A virus.
Serological studies have revealed widespread circulation of these two new influenza viruses in bats. Sera from 55 of 110 (50%) Peruvian bats representing 13 different species were positive for antibodies against the viral HA or NA proteins. Twenty-one of these samples were positive for antibodies against both viral glycoproteins, while 30 were positive only for anti-HA18 antibodies and 4 were positive for only anti-N11 antibodies. These observations suggest that some bats are infected with reassortant viruses carrying the H18 or N11 genes. A study of sera from 8 different species of Guatemalan bats revealed antibodies to the H17 HA protein in 86 of 228 sera (38%).
A number of human viruses, such as SARS-coronavirus and Nipah and Hendra viruses, are known to have originated in bats. Can bat influenza A viruses infect humans and serve as a source of future pandemic strains? The answer to this question is not known, but the two new bat viruses cannot infect human cell lines in culture. However, it is possible that acquisition of other (e.g. avian or swine) influenza virus genes by reassortment could produce a virus with bat influenza virus genes that is capable of infection humans. The pathogenic and pandemic potential of such viruses is unknown. A first step to answering this question would be to determine if human populations with contact with bats have antibodies to the two new bat influenza A viruses.
The cell receptor for all known influenza A viruses is the carbohydrate molecule known as sialic acid The cell receptor for the two new bat influenza A viruses is not known, but it is clearly not sialic acid, a conclusion reached by studying the crystal structures and binding properties of the H17 (paper one and two) and H18 HA (illustrated) molecules. Furthermore, the crystal structures of the N10 (paper one and two) and N11 proteins reveal that their substrate cannot be sialic acid (the function of the influenza A virus NA is to remove sialic acids from the cell surface, allowing newly synthesized virions to move away from the cell). For this reason the N10 and N11 proteins are called ‘NA-like’.
Bats also harbor many other kinds of viruses, including hepatitis B viruses, Marburg virus, hepaciviruses, pegiviruses, paramyxoviruses, coronaviruses, and many more. They also contain parasites – specifically, malaria parasites. For more information, listen to these podcast episodes:
Hedging our bats (TWiV 258)
More bats out of hell (TWiP 62)
Hepaciviruses and pegiviruses in bats and rodents (TWiV 231)
Bats out of hell (TWiV 183)
Going to bat for flu research (TWiV 173)
Matt’s bats (TWiV 65)
On episode #258 of the science show This Week in Virology, Matt joins the TWiV team to discuss the discovery of a SARS-like coronavirus in bats that can infect human cells, and what is going on with MERS-coronavirus.
You can find TWiV #258 at www.twiv.tv.
The influenza virus particle is made up of the viral RNA genome wrapped in a lipid membrane (illustrated). The membrane, or envelope, contains three different kinds of viral proteins. The hemagglutinin molecule (HA, blue) attaches to cell receptors and initiates the process of virus entry into cells. I have written about the HA and its function during infection (article one and two) but not about the neuraminidase (NA, red) or M2 (purple) proteins. Let’s first tackle NA.
An important function of the NA protein is to remove sialic acid from glycoproteins. Sialic acid is present on many cell surface proteins as well as on the viral glycoproteins; it is the cell receptor to which influenza virus attaches via the HA protein. The sialic acids on the HA and NA are removed as the proteins move to the cell surface through the secretory pathway. Newly released virus particles can still potentially aggregate by binding of an HA to sialic acid present on the cell surface. Years ago Peter Palese showed that influenza virus forms aggregates at the cell surface when the viral neuraminidase is inactivated. The NA is therefore an enzyme that is essential for release of progeny virus particles from the surface of an infected cell.
The NA protein also functions during entry of virus into the respiratory tract. The epithelial cells of the respiratory tract are bathed in mucus, a complex protective coating that contains many sialic acid-containing glycoproteins. When influenza virions enter the respiratory tract, they are trapped in mucus where they bind sialic acids. This interaction would prevent the viruses from binding to a susceptible cell were it not for the action of the NA protein which cleaves sialic acids from glycoproteins. When the virus particle encounters a cell, it binds the sialic acid-containing receptor and is rapidly taken into the cell before the NA protein can cleave the carbohydrate from the cell surface.
The essential nature of the NA for virus production has been exploited to develop new drugs designed to inhibit viral release. Both Tamiflu (Oseltamivir) and Relenza (Zanamivir) are structural mimics of sialic acid that bind tightly in the active site of the NA enzyme. When bound to drug, the NA cannot remove sialic acids from the cell surface, and consequently newly synthesized virus remains immobilized. The result is an inhibition of virus infection because virions cannot spread from one cell to another.
This article is part of Influenza 101, a series of posts about influenza virus biology and pathogenesis.
On episode #257 of the science show This Week in Virology, the TWiV team consider how the kinase mTOR modulates the antibody response to provide broad protection against influenza virus, and explore the problems with scientific research.
You can find TWiV #257 at www.twiv.tv.
