Thursday, 28 June 2012

Much a-flu about nothing revisited (post 2 of 2)

Welcome back to those who read yesterday’s post on the Kawaoka influenza paper. For those of you who have started reading this and haven’t read the other blog post, I’d suggest going back as we are halfway through a topic.

At the same time as Kawaoka’s team were working on their hybrid influenza virus in the USA another team lead by Ron Fouchier in the Netherlands were working on a similar project. The aim was again to look for mutations that would allow H5N1 to transmit effectively in a mammalian host. The big difference between the two studies is that Fouchier’s lab used a full blown H5N1 virus; as opposed to a hybrid. Fouchier’s paper was finally published last Friday in Science. Similarly to Kawaoka’s study, Fouchier began with an H5N1 virus isolated from a patient (in Indonesia). From this starting point the team added two mutations to the HA protein which are known to alter the specificity to α2,6 receptors (which, you may remember, is the first hurdle to allow mammalian spread). The mutations they added were Q222L and G224S. Some of the more astute readers may notice that this is our second encounter with position 224 of HA as the mutation N224K was seen in the Kawaoka paper. The fact that the position 224 of the virus from patients can either be a G or an N indicates how variable this virus can be. A third mutation was added in a protein called PB2 which forms part of the polymerase complex (a group of proteins needed to produce copies of the influenza genetic material). The mutation was E627K and allows the virus to replicate more effectively at the lower temperatures seen in the mammalian upper respiratory tract as opposed to the bird intestine (influenza is a gut infection in birds). The addition of these three mutations to the H5N1 virus did not allow for aerosol transmission between ferrets, even though the virus could bind to α2,6 receptors.
Cartoon of the influenza virus structure

Having failed to produce an H5N1 virus capable of aerosol transmission between ferrets with the three specific mutations, the team moved on to use the age old technique of passaging in order to force the virus’ evolution. Fouchier’s team took their triple mutant virus and inoculated a ferret intranasally (injected it to the ferret’s nose). After 4 days they would take virus from this ferret and do the same into a new ferret. After 10 ferrets had been infected in this manner the team produced viruses that were capable of aerosol transmission. Passaging is a beautiful example of how evolution works as only the viruses that have good replication are selected to grow in the new ferret, so replicative ability is selected for. From passage 7 to 10 the team induced sneezing in the ferrets to collect viruses that are best adapted for aerosol spread as well as replication. Thus this passaging process drives the evolution of viruses capable of a high level of replication in the upper respiratory tract and of aerosol transmission.

A coughing ferret
The team have therefore achieved their goal of making H5N1 viruses that are capable of aerosol spread, but that isn’t the end of the story. Fouchier’s team moved on to look at the additional mutations that had occurred to the triple mutant virus to allow aerosol transmission. They found that all the viruses capable of aerosol transmission had at least 9 mutations, including the initial 3. Interestingly, there were 5 mutations which were seen in all of the viruses capable of airborne spread; these being the 3 that were there initially along with T156A and H103Y of HA. The mutation of T156A is interesting as it comes very close to the N158D seen by Kawaoka and indeed causes the same effect of blocking a sugar binding to the protein. The H103Y mutation may play an important role in the stability of the HA protein similarly to the T318I of Kawaoka’s study.

Many press reports regarding the publication of the Fouchier study have claimed that only five mutations are needed for bird flu to become pandemic. I’d like to point out that that slightly misses the point. Five mutations are seen in all the viruses that became airborne in the study; however that does not mean these five mutations are sufficient for spread. It is likely that other mutations are also needed, hence the fact that the aerosol viruses all had at least nine mutations. What we can say is that between 5 and 9 mutations are needed as a minimum for aerosol spread of H5N1 in ferrets (I stress in ferrets as it comes back to the old point that ferrets are not humans). One other point that was sometimes missed in the press reporting of the paper was that none of the ferrets infected with the aerosol virus died, so similarly to Kawaoka’s study, there appears to be a loss of virulence when the virus becomes mammalian adapted.

Fouchier’s paper was published alongside a second influenza paper from Derek Smith’s lab at Cambridge (UK). This paper was looking for the presence of the mutations discovered by Kawaoka and Fouchier in the wild (both were co-authors on the paper). It was found that many H5N1 viruses are 3 and in some cases 2 mutations away from having Kawaoka’s 4 mutations and are 4 away from having the mutations cited by Fouchier. Again this became somewhat sensationalised in the press with headlines such as ‘bird flu is only two mutations away from pandemic.’ This drives me mad! If we turn those findings around they read somewhat differently; wild viruses have only 1 and in some cases 2 of the 4 mutations found by Kawaoka and only 1 of 5 mutations found by Fouchier. If we also add on the fact that these mutations are not necessarily sufficient for spread (remember Fouchier’s viruses had 5 core mutations, but all had at least 9) and the fact that they allow spread in ferrets, not necessarily humans, then it is starting to look less sensational. I’m not denying that bird flu has the potential to fairly easily mutate and become transmissible between humans, I’m just disappointed by the fear-mongering in some of the reporting.

Bird flu does have the potential to become a human pandemic, and pandemics are never good. I mentioned in my previous post that the current reported case fatality of bird flu is close to 60%. This number comes from the fact that there are very few confirmed cases of bird flu and of those that are known, 60% resulted in deaths. The thing is; flu is often a mild disease that people get better from in a couple of weeks so chances are many people won’t bother going to hospital and won’t be recorded as having bird flu unless it is serious, skewing the data (if it is serious enough for hospital then there is already an increased risk of death). Even if H5N1 is a highly virulent virus the Fouchier and Kawaoka studies both indicate that it loses virulence when it becomes airborne, so the idea that 60% of people who get it will die may be a long way off. Let’s not forget however, Spanish Flu of 1918 killed around 10-20% of those it infected, totalling 50-100 million deaths, so even if not at 60% bird flu is still a threat.

What needs to be taken from all this is that we now know the types of mutations needed for H5N1 to become mammalian transmissible. The two studies have shown that there are multiple routes for the virus to become airborne in the sense that they found different mutations; yet these mutations are seen to have similar characteristics. Changes are needed in the receptor binding site to allow α2,6 specificity. Mutations are needed to stabilise the protein to compensate for the apparent loss of stability seen when specificity is altered. It is likely that mutations are also needed in proteins other than just HA, for instance in PB2 (as in Fouchier’s paper). In order to effectively use this information it is essential to increase our understanding of the HA protein as this will allow even better surveillance for potentially risky mutations in the wild. While we continue to survey it is also essential that we work towards an H5N1 or universal flu vaccine and develop stockpiles of anti-viral drugs. That way, if a pandemic does start we are prepared and ready to respond as rapidly as possible. This was one of the major failings of the Swine Flu outbreak of 2009 in which people were caught off guard; hopefully we have learnt our lessons from that.

This blog was a bit more technical than some of my others so I hope I’ve managed to keep everyone interested. It is likely that this is not the last we will hear of bird flu so having a good understanding will help to avoid the fear-mongering which is so common in the press and allow you to assess the risk of a potential pandemic for yourself.

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