Influenza: conundrums from cadavers

For several years, Dr J. Taubenberger and his collaborators have been painstakingly reconstructing the genome of the notorious strain of influenza A which killed 20–50 million people worldwide in 1918. The investigators have now completed this task, reconstructed infectious virus containing all 8 RNA segments using reverse genetics and studied its replication and pathology in mice. They used ‘mix-and-match’ experiments with a contemporary H1N1 virus to define the phenotype of viruses containing one or more of the 1918 RNA segments and to identify genetic determinants of pathogenicity. The results are surprising and challenge some of the conventional theories proposed to explain the extreme pathogenicity seen in 1918. By analogy with other influenza viruses, several features of key proteins encoded by the 1918 strain would have suggested immediately some potential mechanisms for the enhanced pathogenicity. For example, the strains which caused pandemics in 1957 and 1968 were each reassortants between extant human viruses and novel avian strains. In 1957, the avian virus donated haemagglutinin, neuraminidase and PB1 genes to form the H2N2 pandemic virus while, in 1968, the avian donor contributed haemagglutinin and PB1 genes to form the H3N2 pandemic virus [1,2]. In contrast, the 1918 strain is not a reassortant [3,4] but appears to have transferred directly from avians to humans. However, while avian strains normally have a preference for sialic acid with an 2–3 linkage, the 1918 strain binds sialic acid in the mammalian 2–6 conformation. In order to activate the haemagglutinin into its infectious form, the H0 protein must be cleaved into H1 and H2 moieties. These then associate through disulphide bonds into a complex of trimers able both to bind sialic acid and then trigger fusion of the viral/cellular membranes. Highly pathogenic avian strains have a polybasic site which allows the H0 protein to be cleaved intracellularly by ubiquitous proteases to produce the infectious form of haemagglutinin. In contrast, human and avian strains of normal virulence have a single basic amino acid at the cleavage site, are cleaved extracellularly by proteases and require trypsin to be added exogenously to cell cultures in order to obtain efficient cell to cell infection in vitro. The 1918 haemagglutinin contains a single basic amino acid cleavage site and yet replicates in cell cultures without the aid of trypsin. To facilitate cleavage of haemagglutinin, some neuraminidase molecules bind plasminogen and present it to H0 [5]. However, the neuraminidase of the 1918 strain has not lost the N-linked glycosylation site required to achieve this, although it can transfer to other haemagglutinins the phenotype of non-requirement for trypsin [6]. One possibility is that the 1918 neuraminidase may bind another cellular enzyme able to act in an analogous way to plasminogen, but no such function has been identified to date. The non-structural protein NS1 is known to antagonise interferon by preventing activation of interferon regulatory factor 3 and NF B [7,8], so it would not have been surprising to find that the 1918 strain was particularly potent at inhibiting this part of the innate immune response. However, a virus containing the 1918 NS1 protein was attenuated in mice [9]. The most obvious difference between the 1918 strain sequences and those of normal influenza strains is that the three proteins (PB1, PB2, PA) which make up the polymerase have a series of mutations. Compared to consensus avian strains, the 1918 virus has seven changes in PB1, five in PB2, and seven in PA which, in combination, confer increased replicative ability on the virus [4]. These changes in polymerase explain the increased quantity of 1918 virus produced in cell cultures or in mice and, in combination with the 1918 haemagglutinin, are also directly responsible for lethality in chicks [6]. Thus, it is somewhat worrying to see that some of these putative adaptive changes have been observed in the single fatal human case of avian H7N7 from Holland in 2003 as well as in the strains of H5N1 avian influenza virus currently adapting progressively to infect humans in Asia [4]. E D I T O R I A L