The neuraminidase of influenza virus

influenza virusThe 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.

TWiV 223: EEEV and the serpent

On episode #223 of the science show This Week in Virology, Vincent, Alan, and Kathy discuss new influenza virus NA inhibitors, detection of EEEV antibody and RNA in snakes, and replication of the coronavirus EMC in human airway epithelial cells.

You can find TWiV #223 at

Frederick Hayden on influenza antivirals

Frederick Hayden, Professor of Medicine and Pathology, University of Virginia School of Medicine, U.K., has focused on the use of antiviral agents to prevent and treat respiratory viral infections. His interests range from the use of in vitro assays to study viral susceptibility and antiviral mechanisms of action, to clinical trials utilizing experimentally induced and naturally occurring infections. Work from his laboratory includes the demonstration that intranasal administration of interferons can prevent transmission of rhinovirus colds, studies of transmission of drug-resistant influenza A viruses in families, and the antiviral activity and clinical use of influenza neuraminidase inhibitors. His laboratory currently focuses on the application of nucleic acid hybridization to study rhinovirus pathogenesis, elucidating the phenotypic and genotypic basis of antiviral drug resistance in rhinovirus and influenza viruses, and clinical testing candidate antiviral agents for influenza and rhinovirus infections.

I discussed the use of antiviral drugs to treat influenza with Dr. Hayden during ICAAC Boston 2010, as part of TWiV 99. View the video below, or at YouTube.

TWiV 99: ICAAC Boston 2010

Host: Vincent Racaniello

Vincent tours the 50th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in Boston, speaking with exhibitors and visitors, including Professors Derek Smith, Michael Schmidt, Frederick Hayden, and Myra McClure.

Many thanks to Chris Condayan and Ray Ortega of the American Society for Microbiology for recording and editing this episode.

Click the arrow above to play, or right-click to download TWiV #99 (45 MB .mp3, 62 minutes)

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Virology lecture #21: Antivirals

Download: .wmv (349 MB) | .mp4 (90 MB)

Visit the virology W3310 home page for a complete list of course resources.

Tamiflu-resistant pandemic influenza H1N1 virus selected by prophylaxis

viral_loadsThe emergence of oseltamivir (Tamiflu)-resistant 2009 H1N1 influenza virus in a Canadian family illustrates the basic concept that viral loads depend on the dose of antiviral drug.

Neuraminidase inhibitors like Tamiflu and Relenza are used to treat severe illness caused by the 2009 pandemic influenza A (H1N1) virus. The antiviral drugs may also be used to prevent infection in high-risk persons, a use called postexposure prophylaxis. For Tamiflu, that means taking 75 mg a day, compared with the same dose twice a day for treating a confirmed infection. Unfortunately, using sub-optimal levels of an antiviral drug is a recipe for disaster.

In this case, a boy with asthma developed confirmed H1N1 influenza and was given Tamiflu twice a day. Tamiflu was also prescribed once a day for all members of his household; this was presumably done to protect the boy’s father who has chronic obstructive pulmonary disease. Eight days after initiation of prophylaxis the father developed laboratory confirmed influenza. Sequence analysis revealed that the NA gene of the virus isolated from the boy and his father differed by just one amino acid change, H275Y, known to confer resistance to Tamiflu.

This experience illustrates the important fact that antiviral therapy has the potential to promote or prevent the emergence of drug resistant viruses. When viral replication is blocked, no drug resistant mutants can emerge. If an antiviral drug is given after the viral population has expanded, or if the amount of drug is not sufficient to block viral replication, genomes that contain mutations will replicate. If the number of viral genomes is small, the infection may be cleared by the immune response.

This concept is illustrated in the figure, in which median viral load is graphed versus time. Low doses of antiviral drug are not sufficient to block viral replication; there may be a transient drop in viral load but then replication resumes. At an intermediate dose of drug, viral load is initially reduced, but since replication is not completely blocked, resistant viruses emerge. At an optimal dose of drug all viral replication is blocked, viral loads drop dramatically, and no drug resistant mutants emerge.

In the Canadian family it’s likely that the sub-inhibitory (prophylactic) amount of Tamiflu taken by the father allowed some viral replication and hence the emergence of resistant mutants. The authors of the study correctly conclude:

These observations support the need for limiting the indications for postexposure prophylaxis. It also seems reasonable to rapidly convert prophylactic (once daily) regimens to therapeutic (twice daily) regimens as soon as influenza-like symptoms develop in a patient receiving prophylactic treatment. Monitoring for the H275Y mutation during outbreaks of 2009 H1N1 virus is important in order to rapidly identify transmission events that could lead to large-scale dissemination of an oseltamivir-resistant 2009 H1N1 virus, similar to what occurred with recent H1N1 virus seasonal strains.

Baz M, Abed Y, Papenburg J, Bouhy X, Hamelin ME, & Boivin G (2009). Emergence of Oseltamivir-Resistant Pandemic H1N1 Virus during Prophylaxis. The New England journal of medicine PMID: 19907034

TWiV 44: No hysteria

twiv_aa_2001Hosts: Vincent Racaniello, Dick Despommier, Alan Dove, and Jennifer Drahos

In episode #44 of the podcast “This Week in Virology”, Vincent, Dick, Alan, and Jennifer Drahos consider Marburg virus in Egyptian fruit bats, bacterial citrus pathogen found in shipping facility, canine parvovirus in Michigan, Relenza-resistant influenza virus, new HIV from gorillas, and public engagement on H1N1 immunization program.

Click the arrow above to play, or right-click to download TWiV #44 (54 MB .mp3, 78 minutes)

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Links for this episode:
Isolation of Marburg virus from Egyptian fruit bats
Inspectors find bacterial citrus pathogen in California
Parvovirus killing hundreds of dogs in Michigan
Relenza-resistant H1N1 identified in Australia (press and journal article)
New HIV from gorilla
CDC wants public comment on H1N1 vaccination
Original antigenic sin (article 1 and article 2)
Dr. Stanley Plotkin on Meet the Scientist (thanks Peter!)
audioBoo (iPhone app – thanks Jim!)
Audio clips (first and second) from the podcast No Agenda (thanks peripatetic apoplectic!)

Weekly Science Picks
Jennifer Piled Higher and Deeper (PhD Comics)
Vincent Giant Microbes (thanks Stephen!)
Dick Virology in the 21st Century
Alan Annals of the Former World by John McPhee

Send your virology questions and comments (email or mp3 file) to or leave voicemail at Skype: twivpodcast

TWiV 40: Tamiflu in the water

twiv-200Hosts: Vincent Racaniello, Dick Despommier, and Alan Dove

On episode #40 of the podcast “This Week in Virology”, Vincent, Dick, and Alan consider Reston ebolavirus in swine, historical perspective of H1N1 influenza virus emergence and circulation, Tamiflu-resistant H1N1, Tamiflu in Japanese river waters, transmission of H1N1 virus in ferrets, and pneumonia and respiratory failure from H1N1 in Mexico.

Click the arrow above to play, or right-click to download TWiV #40 (49 MB .mp3, 70 minutes)

Subscribe to TWiV in iTunes, by the RSS feed, or by email

Links for this episode:
Reston ebolavirus in Philippine swine
Historical perspective on H1N1 virus
Salk’s 1947 article on flu vaccine failure
Persistent legacy of 1918 H1N1 virus
Tamiflu resistant H1N1 virus (AP article)
Tamiflu in Japanese river waters
H1N1 infection of ferrets (article one and two)
Pneumonia and respiratory failure from S-OIV in Mexico
DNA-based equine WNV vaccine (thanks Peter!)
Fundamentals of Molecular Virology by Nicholas Acheson

Weekly Science Picks
Alan Coming to Life by Christiane Nusslein-Volhard
Monsters Inside Me from Discovery Channel
Vincent Microbeworld

Send your virology questions and comments (email or mp3 file) to or leave voicemail at Skype: twivpodcast

Tamiflu in river water

N1-oseltamivirTamiflu (Oseltamivir) is one of the few antiviral drugs available for treatment of influenza. Use of the drug has increased substantially because of the emergence of the 2009 H1N1 pandemic strain, against which no vaccine is yet available. A recent study has shown that low levels of oseltamivir can be detected in the aquatic environment. This finding raises the possibility that aquatic birds which harbor influenza virus could be exposed to the antiviral, leading to selection of drug resistant viruses.

Oseltamivir is an inhibitor of the influenza neuraminidase (NA) glycoprotein. This enzyme removes sialic acids from the surface of the cell, so that newly formed virions can be released. Neuraminidase inhibitors such as Tamiflu and Relenza function by preventing cleavage of sialic acid from the cell surface. These inhibitors act by binding to the sialic-acid binding pocket of the NA protein.

Tamiflu is s prodrug, oseltamivir phosphate, which is converted to the active form, oseltamivir carboxylate (OC) in the liver. The active form of oseltamivir is excreted in the urine, and is not removed by sewage treatment plants. It has therefore been suggested that OC may present in the aquatic environment, and could expose natural reservoirs of influenza virus to low levels of the antiviral.

Because Japan is the largest consumer of Tamiflu, the levels of OC were determined in the Yodo River system in the Kyoto and Osaka prefectures. This river was selected because it is distant from the sea and located in a densely populated area. Surface water was collected before (June 2007) and during (December 2007 and February 2008) the flu season. No OC was detected in water samples from June 2007. At the onset of the flu season, December 2007, the antiviral was found at levels between 2 and 7 nanograms per liter (ng/L). At the peak of the flu season, in February, levels increased to 12 – 58 ng/L. Levels of OC were higher in water samples taken near sewage treatment plants, compared with those obtained farther away. The amounts detected are close to the concentration of drug that causes 50% inhibition of virus replication (the IC50) in cell cultures, reported to be between 80–230 ng/L.

The authors suggest that dabbling ducks, a natural reservoir of influenza virus, could ingest OC. As influenza in dabbling ducks is a gastrointestinal infection, the virus would encounter oseltamivir in the gut, which could promote selection of viruses resistant to the drug.

It is not known whether OC in aquatic environments leads to influenza virus resistance to Tamiflu. Clearly additional studies must be done to determine whether the antiviral drug can be found in other waters around the world. Influenza viruses should be isolated from aquatic birds living in OC contaminated environments to monitor resistance to Tamiflu.

Söderström, H., Järhult, J., Olsen, B., Lindberg, R., Tanaka, H., & Fick, J. (2009). Detection of the Antiviral Drug Oseltamivir in Aquatic Environments PLoS ONE, 4 (6) DOI: 10.1371/journal.pone.0006064

Assembly of influenza virus

Our discussion of influenza virus replication has so far brought us to the stage of viral RNA synthesis. Last time we discussed the formation of viral RNAs, an event which takes place in the cell nucleus. Now we’ll consider how these RNAs participate in the assembly of new infectious viral particles, as illustrated in the following figure.


For simplicity, the nucleus is not shown. But remember that the viral RNAs have to be exported from the nucleus to the cytoplasm, where viral assembly occurs. First, the viral mRNAs are translated to produce all the proteins needed to synthesize a new virus particle. The mRNAs encoding the HA and NA glycoproteins are translated by ribosomes that are bound the the endoplasmic reticulum – the membranous organelle that assists in transporting certain proteins to the plasma membrane. As the HA and NA proteins are produced, they are inserted into the membrane of the endoplasmic reticulum as shown. These proteins are then transported to the cell surface via small vesicles that eventually fuse with the plasma membrane. As a result, the HA and NA are inserted in the correct direction in the lipid membrane of the cell. The M2 protein is sent to this location in a similar way.

The (-) strand viral RNAs that will be packaged into new virus particles are produced in the cell nucleus, then exported to the cytoplasm. These RNAs are joined with the viral proteins PA, PB1, PB2, and NP. Viral proteins other than HA, NA, and M2 are produced by translation on free ribosomes, as shown for M1. The latter protein binds to the membrane where HA, NA, and M2 have been inserted. The assembly consisting of viral RNAs and viral proteins – called a ribonucleoprotein complex or RNP –  travels to the site of assembly. The virion then forms by a process called budding, during which the membrane bulges from the cell and is eventually pinched off to form a free particle.

As new virions are produced by budding, they would immediately bind to sialic acid receptors on the cell surface, were it not for the action of the viral NA glycoprotein. This enzyme removes sialic acids from the surface of the cell, so that newly formed virions can be released. This requirement explains how the neuraminidase inhibitors Tamiflu and Relenza function: they prevent cleavage of sialic acid from the cell surface. In the presence of these inhibitors, virions bud from the cell surface, but they remain firmly attached. Therefore Tamiflu and Relenza block infection by preventing the spread of newly synthesized virus particles to other cells.