TWiV 282: Tamiflu and tenure too

On episode #282 of the science show This Week in Virology, the TWiV team reviews a meta-analysis of clinical trial reports on using Tamiflu for influenza, and suggestions on how to rescue US biomedical research from its systemic flaws.

You can find TWiV #282 at

Cutting through mucus with the influenza virus neuraminidase

influenza virusNeuraminidase is one of three different viral proteins embedded in the lipid membrane of influenza virus (NA is blue in the illustration at left). This enzyme has a clear and proven role in virus release from cells. NA is also believed to be important during virus entry, by degrading the mucus barrier of the respiratory tract and allowing virus to reach cells. This role is supported by the finding that treatment of mucus-covered human airway epithelial cells with the NA inhibitor Tamiflu substantially suppresses the initiation of infection.  Further evidence comes from the recent finding that influenza virus binds to sialic acids in mucus and that NA cleaves these sugars to allow infection.

The mucus layer of the respiratory tract has a defensive role because it contains soluble glycoproteins that are rich in sialic acids, which are cell receptors for some viruses. When influenza virions enter the respiratory tract, they are thought to be trapped in mucus where they bind sialic acids, preventing infection of the underlying cells. The role of NA in penetration of the mucus layer was studied in frozen sections of human tracheal and bronchial tissues, in which the mucus layer is preserved. Influenza A virions bind to this mucus layer; the interaction is blocked when the tissues are first treated with a bacterial neuramindase, which removes sialic acids from glycoproteins. These observations indicate that the mucus layer of human airway epithelial cells contain sialic acid-containing decoys that bind influenza A viruses.

Human salivary mucins, which approximate the mucus of the human respiratory tract, can protect cultured cells from influenza virus infection. The protective effect can be achieved by simply adding the mucins to cells. The inhibitory effect is dependent on the sialic acid content of the mucins: fewer cells are infected when higher concentrations are used. In contrast, porcine salivary mucins do not substantially reduce influenza virus infection. The type of sialic acids and the virus strain also determine the extent of protection.

To determine the role of the viral NA in mucin-mediated inhibition, Tamiflu was mixed with virus before infection of mucus-coated cells. The presence of Tamiflu increased the inhibition of infection caused by mucins, indicating that the sialic acid-cleaving (sialidase) activity of NA is needed to overcome inhibition by human salivary mucins. When influenza virions are incubated with human salivary mucins linked to beads, sialic acids are cleaved from mucins, and this enzymatic activity is inhibited by Tamiflu. Human salivary mucins inhibit NA by binding to the active site of the enzyme.

These studies establish a clear role for the influenza viral NA in bypassing the defenses of the mucosal barrier. An important message is that not all strains of influenza virus are equally inhibited by mucus; presumably this property is one of many that determines viral virulence. The balance between how tightly the viral HA binds to sialic acids, and how well the NA cleaves them, is probably one predictor of how well we our protected by our mucus.


Secondary changes allow spread of oseltamivir resistant influenza virus

The influenza virus neuraminidase (NA) protein is required for virus release from the cell, a property exploited by the antiviral drugs oseltamivir (Tamiflu) and zanamavir (Relenza). During clinical testing of oseltamivir in 2001, some individuals shed drug-resistant viruses with an amino acid change from histidine to tyrosine (H274Y) in NA. Such viruses are not inhibited by oseltamivir because the amino acid change leads to  decreased binding of the drug. But these viruses replicated less well in cell culture, and had reduced infectivity in ferrets. It was concluded that oseltamivir resistant influenza virus mutants would not spread in the population. Why was this conclusion wrong?

During the 2008-09 flu season oseltamivir resistant influenza H1N1 viruses with the H274Y change began to spread, and within a year they were found in most seasonal isolates. It was hypothesized that these viruses contained other amino acid changes that masked the deleterious effect of H274Y. The H274Y mutation does not affect the catalytic activity of the NA: the ability to cleave sialic acid from glycoproteins. However it does lead to a decease in the amount of NA protein that is transported to the surface of infected cells.

Computational methods were used to identify amino acids in NA that could potentially compensate for the effect of H274Y. A single amino acid change at position 194 of NA, when present with H274Y, restored NA on the cell surface to normal levels.

Did a similar amino acid change in seasonal H1N1 strains allow the spread of oseltamivir resistant viruses with H274Y? Introduction of this amino acid change into the seasonal H1N1 strains A/Texas/91 and A/New Caledonia/99 causes a decrease in surface NA. However the same change has a lesser effect on surface NA in cells infected with A/Solomon Islands/2006. Two amino acid changes were identified in the NA protein of recent oseltamivir-resistant seasonal H1N1 viruses that restore surface levels of NA in the presence of H274Y: V234M and R222Q.

It seems likely that the amino acid changes V234M and R222Q emerged first in the NA of seasonal H1N1 viruses. Why these changes appeared is unknown, but they could be a consequence of random drift, antigenic selection, or a need to balance HA and NA activities. Once these changes were in place, oseltamivir resistant viruses with the H274Y could be selected, and because they had no defect in fitness, they spread globally.

The conclusion is that H274Y in NA attenuates the fitness of influenza virus by reducing the amount of NA on the cell surface. Spread of such viruses in the population is impossible without secondary amino acid changes that restore adequate levels of surface NA. H274Y probably causes a defect in NA folding or transport that is balanced by the secondary mutations.

These findings are another example of how drug resistance frequently comes with a cost to protein stability or folding, and prevents evolution unless compensated by secondary mutations.

There have been scattered isolations of oseltamivir-resistant, pandemic 2009 H1N1 influenza virus with the H274Y change. Will these viruses spread globally, or are they less fit, evolutionary dead ends? Introduction of the H274Y change into the NA of 2009 pandemic H1N1 virus leads to a large decrease in surface NA. Unless the 2009 swine-origin viruses already produce excess NA, viruses with the H274Y change are not likely to spread without secondary mutations that rescue NA surface expression.

Bloom JD, Gong LI, & Baltimore D (2010). Permissive secondary mutations enable the evolution of influenza oseltamivir resistance. Science (New York, N.Y.), 328 (5983), 1272-5 PMID: 20522774

Reinfection with 2009 influenza H1N1

immune-memoryIn healthy individuals, the first encounter with a virus leads to a primary antibody response. When an infection occurs with the same or a similar virus, a rapid antibody response occurs that is called the secondary antibody response. Antibodies are critical for preventing many viral infections, including influenza. But reinfection may occur if we encounter the same virus before the primary response is complete.

Recently three cases of confirmed infection with 2009 influenza H1N1 were reported in Chile. The first patient had laboratory confirmed infection; treatment with oseltamivir resolved symptoms after 48 hours. Twenty days later the patient developed a second bout of laboratory confirmed influenza which was treated with amantadine. The second patient acquired laboratory confirmed influenza in hospital, was treated with oseltamivir and recovered. Two weeks later, while still in hospital, the patient had a new episode of laboratory confirmed influenza infection. Treatment with oseltamivir again resolved the infection. The third patient also acquired laboratory infection in hospital, was successfully treated with oseltamivir, and was discharged. He was readmitted 18 days later with confirmed pandemic H1N1 2009, and again successfully treated with oseltamivir.

These individuals were likely resusceptible to reinfection with the same strain of influenza virus due to a confluence of unusual events. First, all three were reinfected within three weeks, before their primary adaptive response had sufficiently matured. Another contributing factor was the high level of circulation of the pandemic strain. This issue was compounded for patients two and three who probably acquired both infections while in the hospital (called nosocomial transmission).

Could reinfection also occur after immunization with influenza vaccine? Yes, if the immunized individual encounters the virus before the primary antibody response matures, which occurs in 3-4 weeks. This is more likely to occur during pandemic influenza when circulation of the virus is more extensive than in non-pandemic years.

Perez CM, Ferres M, & Labarca JA (2010). Pandemic (H1N1) 2009 Reinfection, Chile. Emerging infectious diseases, 16 (1), 156-7 PMID: 20031070

Influenza neuraminidase inhibitors work

tamifluIn the wake of a British Medical Journal article which concludes that Tamiflu has at best a modest effect, many readers have asked if influenza neuraminidase inhibitors function at all. If you’d like a good critique of this study, I suggest reading Paul Revere’s analysis at Effect Measure. For our part, we’ll examine some of the virological evidence for the effectiveness of Tamiflu.

One of the first human studies on the effectiveness of Tamiflu was published about ten years ago. The human subjects (117 healthy adult volunteers, 18-40 years of age, with hemagglutination-inhibition antibody titers 1:8 or lower) were infected intranasally with a seasonal H1N1 strain of influenza virus. Some subjects were given Tamiflu or placebo 26 hours before infection, while others were given the treatment 28 hours after inoculation. Frequency of infection, viral shedding in nasal washes, and clinical symptoms (such as temperature, nasal stuffiness, runny nose, sore throat, sneezing, cough, breathing difficulty, muscle aches, fatigue, headache, earache/pressure, feverishness, hoarseness, chest discomfort, overall discomfort) were then determined.

Administration of Tamiflu 28 hours after inoculation reduced viral shedding and clinical symptoms, as shown in the following two graphs. Note that virus titers peaked in treatment and placebo groups at roughly 36 hours after inoculation. Viral titers declined more rapidly in individuals given Tamiflu, and no virus was detected by 60 hours after infection.



When Tamiflu was given before infection (prophylaxis), 8 of 21 individuals became infected (38%), while 8 of 12 individuals given placebo were infected (67%). None of the patients given Tamiflu shed virus in nasal secretions, or reported respiratory illness. Virus was shed by 50% of individuals in the placebo group, and a third had clinical symptoms of infection.


The authors concluded that “In these trials, prophylaxis and early treatment with oral oseltamivir were both associated with significant antiviral and clinical effects in experimental human influenza.” In other words, if you know you have influenza, Tamiflu works reasonably well.

The results of these studies justified the larger scale clinical trials which were subsequently done to provide the data needed for approval of Tamiflu in humans.

Hayden FG, Treanor JJ, Fritz RS, Lobo M, Betts RF, Miller M, Kinnersley N, Mills RG, Ward P, & Straus SE (1999). Use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment. JAMA : the journal of the American Medical Association, 282 (13), 1240-6 PMID: 10517426

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

Tamiflu resistance of influenza H1N1 strains

124790416_5dc7e07b8f_mYesterday the New York Times ran an article on the resistance to Tamiflu of current influenza H1N1 strains circulating in the US. I wrote a post about this issue on 22 December 2008, so I’m happy to see the Times following my lead. But there is an issue with the Times article that I’d like to address here. According to the article:

The single mutation that creates Tamiflu resistance appears to be spontaneous, and not a reaction to overuse of the drug.

Drug-resistant viruses are not ‘reactions’ to overuse of the drug. The drug selects, from the diverse viral population in an individual, those viruses that can multiply in its presence. Usually the drug-resistant mutants are already in the host, and outpace other drug-sensitive viruses. Is that what the writer means by ‘spontaneous’? Not in this case. What apparently happened is that the mutation that causes drug-resistance, a change from histidine to tyrosine at position 274 of the viral NA protein, emerged in parts of the world were little Tamiflu is used. There was some other reason why this change was selected for in those populations. The article implies that the his->tyr change accompanied a second amino acid change at position 193 of the HA protein which improved the ability of the virus to infect people. This change did not affect resistance to Tamiflu, but apparently it only persisted when the change at 274 was also present. It so happened that the 274 change also conferred resistance to Tamiflu. Thus, when this virus arrived in parts of the world where Tamiflu is used, the resistance was noted. None of this is made particularly clear from the article.

I also have an issue with the author describing the amino acid changes in the ‘N’ and ‘H’ genes. The correct nomenclature is NA and HA. The author might have been mislead by the strain designation which uses only ‘H’, e.g. H1N1. It’s a small point but I believe that the devil is in the details.

What about the two other anti-influenza drugs? And the other strain currently circulating, H3N2?

Most of the flu in the US now is caused by H1N1 strains. So although the H3N2 strains are sensitive to Tamiflu, it’s not much help.

The Tamiflu-resistant H1N1 strains are sensitive to another drug, Relenza (zanamivir). But that drug must be inhaled and is not appropriate for everyone. However, these H1N1 strains are sensitive to Rimantadine, so its use is a good alternative. Most H3N2 strains are resistant to Rimantadine, which is why it has not been used much in recent years.

Nevertheless, our anti-influenza drug arsenal is much too small. It’s worth recalling the following information from Principles of Virology (ASM Press):

With about 1016 human immunodeficiency virus (HIV) genomes on the planet today, it is highly probable that somewhere there exist HIV genomes that are resistant to every one of the antiviral drugs that we have now or are likely to have in the future.

AIDS is no longer a death sentence because we have a deep arsenal of antiviral drugs that can control the infection. Patients are treated with a combination of three anti-HIV-1 drugs at a time. When resistance inevitably emerges, the patient is switched to another combination of three. The high levels of HIV-1 replication in many hosts, coupled with the large numbers of viral mutants that are produced, ensure that resistance will emerge.

Influenza virus shares similar features as HIV-1: high replication rates in many hosts, and the generation of large numbers of viral mutants. Therefore any antiviral strategy that employs only three drugs  is bound to fail. The difference with influenza, of course, is that an excellent vaccine is available, and should be used whenever possible. The antiviral compounds should only be used in the face of an outbreak when immunization has not been sufficiently comprehensive. However, I suspect that the use of Tamiflu and Relenza is far more prevalent than desired. How many people rush for a prescription at the first signs of a respiratory infection? And how many of those have already been immunized? This was not the intended use for these antiviral compounds.

If we want to seriously use antiviral to treat influenza (which I don’t think is a good idea except in certain cases), we need to have a far deeper arsenal of antiviral drugs.

Oseltamivir resistance in current H1N1 influenza virus strains

397723615_3493f379cb_mThe CDC has issued a health advisory which indicates that a high number of currently circulating H1N1 influenza viruses are resistant to the antiviral drug oseltamivir. Fifty H1N1 isolates obtained in the U.S. since October 2008 were examined for drug sensitivity; 98% of these isolates are resistant to oseltamivir. Influenza H3N2 isolates, however, are still susceptible to the drug.  The H1N1 isolates remain sensitive to zanamivir and amantadine. As a result of this finding, the CDC suggests that for treatment of influenza, “zanamivir or a combination of oseltamivir and rimantadine are more appropriate options than oseltamivir alone.” 

Oseltamivir and zanamivir are recently developed influenza antiviral drugs that inhibit the viral neuraminidase. This enzyme is required for infection and spread, and consequently its inhibition reduces viral production and disease. Both inhibitors were designed to mimic the natural ligand of neuraminidase, sialic acid. In theory, the more an inhibitor resembles the natural compound, the less likely the target can change to avoid drug binding while maintaining viable function. The results with oseltamivir do not support this theory. During the 2007-08 influenza season, 10.9% of H1N1 viruses tested in the U.S. were resistant to the drug. Although it is not known what percentage of the 2008-09 isolates will be resistant to oseltamivir, it is clear that resistance to this drug is rising.

It is not surprising to observe increasing resistance to oseltamivir, given the high mutation rates of RNA viruses. What is somewhat surprising is the absence of resistance to zanamivir. This difference may be in part due to structural differences between the two compounds – perhaps it is more difficult for the neuraminidase to be resistant to zanamivir and still have enzymatic function. The ways that the drugs are administered may also play a role – oseltamivir is taken orally, a route that is more widely accepted, while zanamivir is given by inhaler. Consequently, it is probably used less than oseltamivir.

One consequence is probably certain: the CDC recommendation that the two neuraminidase inhibitors be used in combination this year is likely to increase resistance to both neuraminidase inhibitors. This will be a difficult problem because there is widespread resistance among H3N2 strains against the only other licensed anti-influenza virus drug, the amantadines.