More recently, Yoshihiro Kawaoka of the University of Wisconsin–Madison reported similar experiments with 1918 flu genes in mice, with similar results. But when he tested the HA and NA genes separately, he found that only the 1918 HA produced the intensive immune response, suggesting that for reasons as yet unclear, this protein may have played a key role in the 1918 strain's virulence.
These ongoing experiments are providing a window to the past, helping scientists understand the unusual characteristics of the 1918 pandemic. Similarly, these techniques will be used to study what types of changes to the current H5N1 avian influenza strain might give that extremely lethal virus the potential to become pandemic in humans. An equally compelling question is how such virulent strains emerge in the first place, so our group has also been analyzing the 1918 virus's genes for clues about where it might have originated.
Seeking the Source
The best approach to analyzing the relationships among influenza viruses is phylogenetics, whereby hypothetical family trees are constructed using viral gene sequences and knowledge of how often genes typically mutate. Because the genome of an influenza virus consists of eight discrete RNA segments that can move independently by reassortment, these evolutionary studies must be performed separately for each gene segment.
We have completed analyses of five of the 1918 virus's eight RNA segments, and so far our comparisons of the 1918 flu genes with those of numerous human, swine and avian influenza viruses always place the 1918 virus within the human and swine families, outside the avian virus group. The 1918 viral genes do have some avian features, however, so it is probable that the virus originally emerged from an avian reservoir sometime before 1918. Clearly by 1918, though, the virus had acquired enough adaptations to mammals to function as a human pandemic virus. The question is, where?
When we analyzed the 1918 hemagglutinin gene, we found that the sequence has many more differences from avian sequences than do the 1957 H2 and 1968 H3 subtypes. Thus, we concluded, either the 1918 HA gene spent some length of time in an intermediate host where it accumulated many changes from the original avian sequence, or the gene came directly from an avian virus, but one that was markedly different from known avian H1 sequences.
To investigate the latter possibility that avian H1 genes might have changed substantially in the eight decades since the 1918 pandemic, we collaborated with scientists from the Smithsonian Institution's Museum of Natural History and Ohio State University. After examining many preserved birds from the era, our group isolated an avian subtype H1 influenza strain from a Brant goose collected in 1917 and stored in ethanol in the Smithsonian's bird collections. As it turned out, the 1917 avian H1 sequence was closely related to modern avian North American H1 strains, suggesting that avian H1 sequences have changed little over the past 80 years. Extensive sequencing of additional wild bird H1 strains may yet identify a strain more similar to the 1918 HA, but it may be that no avian H1 will be found resembling the 1918 strain because, in fact, the HA did not reassort directly from a bird strain.