The news that H5N1 viruses isolated from an uncle and niece in Egypt who died in December has been found to carry a genetic change suggestive of resistance to the main antiviral drug oseltamivir (Tamiflu) headlined the H5N1 newswires yesterday. Specifically, WHO announced that genetic sequencing had found the N294S change in the isolates (explanation below the fold). It is not clear at the moment whether the change occurred during treatment of the pair with the drug or the virus carried the change when it infected them. It is also not clear what the clinical significance of the change is. We need to explain further.
Oseltamivir (which we will call by its trade name, Tamiflu) is a neuraminidase inhibitor. Neuraminidase (NA) is a protein that acts like an enzyme (it catalyzes a specific biochemical reaction) and it is present on the virus in the form of a glycoprotein, one of the two glycoproteins that stud the viral surface (for more on the basics of glycoproteins in lay language see our series that starts here). The other glycoprotein is hemagglutinin (HA). There are 9 immunological classes of NA and 16 of HA and the specific HA/NA combination of these types are what distinguish the various influenza A subtypes named for them (e.g., H3N2 or H5N1). These are also the main elements that our own immune systems recognize. It is the HA glycoprotein that becomes the basis for most of the influenza vaccines.
The HA protein plays an important role at two critical points in viral infection of a host cell. The first comes when it binds to a host cell receptor of the right kind. Currently we believe the main receptor is something called a sialylglycan (we have discussed details in the glycoprotein series linked above). Once docked to the receptor the virus is “taken into” the cell by being enveloped by the cell’s membrane in a process called endocytosis. Think of a closed bag of liquid you push a small stone into the side of, making an indented pocket with a sort of “neck” to the outside. Now pinch off the neck part so that the stone is now inside the bag surrounded by its own little piece of the bag wall. It still isn’t really inside the bag yet because of the surrounding coating of bag wall, but if it could get through that it would be bathed by the liquid in the bag and truly “inside.” That’s what the virus needs to do because it wants access to the cell’s genetic and protein-making machinery so it can make copies of itself, its only real function. HA takes part in this process, too, helping the virus “fuse” with the surrounding little bit of bag wall and exit from its internal bubble into the cell proper.
That’s HA. What about NA? Here’s what is generally agreed upon. After the virus makes copies of itself and the parts fully assembled again to be budded from the surface of the host cell, it has to be cut loose from the same sialylglycans it used to attach, on entry. That is what NA does. But the mysteries of NA have not all been unraveled and at least two other functions have been suggested for it. Matrosovich et al. have suggested NA helps destroy the attachment of the virus to the many “decoy” sialylglycans found in the upper respiratory tract contained in mucus and other components not part of the cell membrane. In addition, Ohuchi et al. have recently put forward another role for NA. Once attached, it has been suggested that the virus needs to move around the surface a bit to find the right spot for the endocytosis, i.e., not every spot is suitable for the virus to invaginate into the cell, the next step in infection after attachment by HA. Without NA the virus is held to tightly on one spot and can’t move around enough to find the “door” to get in. It’s in the building foyer but still needs to find the front door and go through it. One of the mysteries about NA function is why it is good to break the sialylglycan bond on exiting but why thisisn’t bad when entering. It is likely that the answer is that there is a balance between HA and NA. Too much NA activity is bad for entry and too little is bad. This might explain why viral infectiveness is not just a matter of whether the HA can find a receptor but also whether it is paired with the right NA. In any event, neuraminidase inhibitors like Tamiflu work by decreasing NA function, making it hard for them to exit after budding at the surface (this is accepted) and possibly making it hard for them to enter as well, by getting them stuck at the surface before they can get in (speculated). So the story is more complicated than first thought. No surprise.
Antiviral drugs like Tamiflu and Relenza (generic name zanamivir) work because they look like the sialylglycan the virus is trying to find for its initial attachment. They become like the decoy receptors in mucus. The drugs bind in a chemical recognition site, a sort of “pocket” on the NA glycoprotein. In order for the drug to bind, there must be a fairly specific set of attachment points in the pocket and this is determined by the sequence of amino acids that make up the NA protein. which in turn is determined by the genetic sequence in the viral RNA. So if that genetic material changes (a mutation) it can alter the attachment points the drug needs and produce an NA that no longer binds the drug. Of course that NA might also not work very well for its function, either, and there is some suspicion that the resistant mutant viruses might be less fit than the normal virus (called the “wildtype”). A resistant virus has been isolated from a patient prior to this from a young Vietnamese girl (who may have been infected while caring for her 21 year old brother). She recovered completely. She was found to have circulating viruses with varying genetic sequences, one highly resistant to Tamiflu (carrying the H274Y mutation), one with only slight resistance
(H294N) N294S and one highly sensitive (wildtype, the same as the brother’s virus). Thus the virus may have mutated while replicating within the patient. On the other hand, de Jong has reported two cases that ended fatally and also had H274Y.
What are these mutation designations? The wildtype condition is the first letter in H274Y and gives the one letter code of the amino acid that was there before the change. 274 is the position along the length of the NA protein. The second letter is the amino acide after the mutation. Here the amino acid histidine (whose one letter code is H) has been changed or mutated to the amino acid tyrosine (one letter code Y) at position 274. We don’t know all the mutations that might confer Tamiflu or Relenza resistance, but based on experience the Japanese have had with the H3N2 virus and some experiments with human volunteers infected with H1N1 viruses, the mutations R292K, N294S and H274Y seem to be important to drug binding in the pocket. Much of the previous predictions were based on three dimensional structures of N2 type neuraminidase, the 3D structure of N1 not having been worked out. Last September the 3D structure of N1 was published and work is now proceeding using that information to understand better what is going on with resistance to neuraminidase inhibitors and possibly design new drugs for the purpose.
How do we know a virus is resistant to Tamiflu? It is done by detecting one of those three mutations in the genetic sequence. Since we have very little information on the clinical importance of those markers of mutation (the jump from sequence to biology is a big one), it is not clear what the significance of the N294S mutation is (it is an asparagine, N, to serine, S, mutation at position 294).
All this is explanation for this statement:
“What the resistance tests look for are markers associated with antiviral resistance,” though finding the markers did not necessarily mean Tamiflu would not work, said Dr. Angus Nicoll, flu director at the European Centre for Disease Prevention and Control.
[Dr. Frederick] Hayden said the mutations found in Egypt were different from Tamiflu-resistant H5N1 viruses found in patients two years ago in Vietnam. The Vietnamese strains were definitely resistant to Tamiflu, whereas the Egyptian viruses have only proven they are not as susceptible to the drug, he said. (AP)
This sounds suspiciously like spin. But in fact it is based on what we know, and more importantly, what we don’t know. Another good reason for never spinning. When you are telling it straight you want people to believe you.