TWiV 241: The ferret looks ill

On episode #241 of the science show This Week in Virology, Vincent, Alan, Rich and Kathy review how human placental trophoblasts confer viral resistance via exosome-mediated delivery of microRNAs, and isolation of the first human influenza virus in 1933.

You can find TWiV #241 at

TWiM 30: Unraveling melioidosis and insulin resistance

On episode #30 of the science show This Week in Microbiology, Vincent, Elio, and Michael review how a toxin from Burkholderia pseudomallei inhibits protein synthesis, and the role of the gut microbiome in modulating insulin resistance in mice lacking an innate immune sensor.

You can find TWiM #30 at

TWiM 16: ICAAC Live

This Week in Microbiology

Hosts: Vincent RacanielloMichael Schmidt, Arturo Casadevall, Stuart Levy, and David Livermore.

VincentMichael, Arturo, Stuart, and David converse about antimicrobial resistance and why most fungi do not cause disease, at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC).

Click the arrow above to play, or right-click to download TWiM 16 (65 MB, .mp3, 90 minutes).

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Send your microbiology questions and comments (email or mp3 file) to, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at and tag them with twim.

TWiM 6: Antibacterial therapy with bacteriophage: Reality or fiction?

bacteriophage modelHosts: Vincent Racaniello, Cliff Mintz, Michael Schmidt, and Elio Schaecter

On episode #6 of the podcast This Week in Microbiology, Vincent, Cliff, Michael and Elio review the use of bacteriophages to manage infections, and the presence of antibiotic resistance genes in bacteriophages from urban sewage and river water.

Click the arrow above to play, or right click to download TWiM #6 (57 MB .mp3, 82 minutes).

Subscribe to TWiM (free) on iTunesZune Marketplace, via RSS feed, by email or listen on your mobile device with the Microbeworld app.

Links for this episode:

The model of bacteriophage T4 shown in the photo is described here.

Send your microbiology questions and comments (email or mp3 file) to, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at and tag them with twim.

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

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.