Mutation(s) in the PB2 protein in the Netherlands

We frequently hear from the WHO and the CDC that the new H1N1 pandemic virus “has not mutated”. This is not true. The virus mutates frequently. When they make this inaccurate statement, it is not clear what they actually intended to say. Are they using the word “mutated” to mean reassortment? Or do they mean that no dangerous mutations have been observed? If the latter, this, also, is untrue.

Tonight, ProMed reports that a mutation has occurred in the Polymerase basic protein 2 (PB2) protein of the new H1N1 virus in the Netherlands [hat-tip, cactus at PFI_Forum]. To understand the significance of the mutation, some explanation of the PB2 protein might be useful. Influenza A has 8 genomic segments. These genomic segments code for at least 10 different proteins. The PB2 protein is coded for by the PB2 genomic segment. The PB2 protein is a polymerase. That is to say, it helps make more copies of RNA which is either used to make viral proteins or viral RNAs which get packaged inside viral proteins and serve as the genetic material for the new viruses. Thus, PB2 (and PB1 and PA), are essential for viral replication.

Each protein is made up of a series of amino acids. Each amino acid has a unique number. There is a “left” end and a “right” end to the proteins. By convention, the first amino acid is on the left end and the last amino acid is on the right. So, amino acid 1oo is 100 amino acids away from the first amino acid on the left end. There are 759 amino acids in the PB2 protein.

Strictly speaking, proteins don’t mutate, nucleic acids do. Nucleic acids code for proteins. That is, specific sequences of nucleic acids will produce specific proteins. A mutation is a change in a nucleic acid. This change may or may not change an amino acid in a protein.

A mutation in the nucleic acid of the PB2 gene of the new H1N1 has occurred. It resulted in a significant change in the PB2 protein.

From ProMed:

We would like to report 2 patients in The Netherlands, diagnosed with influenza pandemic A(H1N1) 2009 virus infection that had a mutation (E627K) in the basic polymerase 2 (PB2) protein. This mutation has previously been associated with increased efficiency of replication and possible virulence changes in other influenza A viruses.

The investigation identified a specific geographic region in the north of The Netherlands as the place where viruses with the same genetic background have circulated between mid July and mid August [2009]. No other cases carrying the PB2 mutation have been identified.

What this means is that at position (amino acid) 627 of the PB2 protein, there was a mutation in the viral genetic code that changed the amino acid from a glutamic acid (E) to a lysine (K). This is significant because flu A viruses that infect birds usually have a glutamic acid at position 627 while those that infect humans usually have a lysine at position 627. Although none of the people infected with this mutated form of the new H1N1 virus were seriously ill, the extended transmission of a virus that appears to be better adapted to humans is of concern.

From ProMed:

PB2 627K is consistently found in human influenza A viruses, but rarely in avian-derived viruses. The E627K mutation may result in enhanced virus replication efficiency in humans, possibly by adjustment to host body temperature or cellular cofactors, and has previously been shown to be associated with fatal cases of HPAI H5N1 and H7N7 virus infection in humans. Until now, A(H1N1)v viruses with Influenza pandemic (H1N1) 2009 (57): in PB2 have not been reported, and the clinical and epidemiological relevance of our finding remains unclear.

We do not know why people infected with this new, human-adapted form of H1N1 did not show severe symptoms. Possibly, it was because they were treated with Tamiflu. However, ferrets exposed to a similar mutation do not show increased severity of disease. Another possibility is that this mutation is necessary, but not sufficient, for increased virulence. Mutations can have different effects on different genetic backgrounds. What this means is that more than one mutation may be necessary to observe a change in behaviour of the virus. How likely are additional, significant mutations in H1N1 proteins?

One has already happened, again in The Netherlands.

A different mutation in the same protein had been observed in the Netherlands back in May [hat-tip, AccurateOne at PFI_Forum].

From ProMed

In the Netherlands, the 2nd laboratory confirmed human case of influenza A (H1N1) virus infection was reported on 7 May 2009. A 53-year-old woman returned on the 30 Apr 2009 from Cancun, Mexico. During the flight she developed an unproductive cough. Then, 2 days later on 2 May 2009, she had a temperature of 38.6C and a sore throat and consulted a general practitioner. Samples were submitted for diagnostic evaluation and both the patient and her husband were treated with oseltamivir.

The patient recovered completely and uneventfully, and samples collected 4 days later tested negative. The virus was analyzed for presence of antiviral resistance markers in the neuraminidase and for human adaptation markers in the PB2 protein by direct sequencing.

The sequence data suggested that the virus was susceptible to both oseltamivir and zanamivir. The amino acid 627 in PB2 (glutamic acid) was not human-host-adapted, similar to recent swine influenza A (H1N1) viruses. However, a glutamic acid to glycine amino acid substitution was detected at position 677 in PB2. This mutation was not observed in any of the A (H1N1) sequences submitted since 27 Apr 2009. Lam et al. (2008) postulated that this substitution could reflect adaptation to mammalian hosts of highly pathogenic avian influenza A (H5N1) viruses (1), as it was found to be under positive selection based on phylogenetics of Indonesian viruses. Based on the position of the mutation it might contribute to more efficient human-to-human transmission by enhanced replicative efficiency of the polymerase of the influenza A (H1N1) virus in humans.

Note, this first mutated form of the new H1N1 did not have the mutation observed in the most recent cases. Since it is not mentioned, the most recent mutated form probably did not have the mutation observed in first mutated virus. Thus, we have two different mutations in the PB2 protein, one at position 627 and one at position 677.

Could these two different mutations come together in the same virus? Yes, via a process called recombination. If a host is infected with both mutant strains of the new H1N1, these viruses could exchange parts of their PB2 genes to create a new virus with both mutations. Alternatively, the PB2 gene could change sequentially, first one mutation, then the other.

The CDC is looking for clusters of severe cases characterised by coughing blood. We are seeing clusters of fatal and severe cases. What is causing this? The answer is in the sequences.


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