In 1977, an outbreak of seasonal flu struck China and Russia. A epidemic of seasonal flu was, of course, no surprise. But the viral strain responsible was something of a shock. The strain was H1N1 influenza A, which had not been seen in the wild for two decades. Where does a flu virus hide for 20 years, and how does it re-emerge? Evolutionary analysis provides clues.
The oldest known sample of H1N1 flu came from an Inuit woman who died at Brevig Mission, Alaska, during the great pandemic of 1918. That year flu sickened about one in five people world wide, and—in conjunction with bacterial pneumonia—killed at least 50 million. My grandmother was nine when the 1918 flu came to Omaha. She told me that by the time it was over, families were leaving their dead by the curb to be picked up by the coroner’s wagons.
For the rest of her long life, my grandmother remembered an argument between her parents, Rudi and Claudine. Claudine is on the right in the photo above, posing beside her sister Henrietta. Henrietta and her children had fallen ill. Rudi begged Claudine to stay at home, lest she catch the flu herself. Claudine, however, felt there was no choice. She packed a bag, walked across town, and nursed her kin back to health. Rudi’s concern notwithstanding, Claudine never got sick. I’m not sure there was a virus anywhere with the fortitude to take her on.
After wreaking havoc in 1918, H1N1 flu continued to circulate in humans until 1957. That year it was replaced by H2N2 flu. The letters H and N stand for hemagglutinin and neuraminidase, the two main proteins found on the surface of influenza virus particles. The numbers designate sets of surface proteins characterized by the ability of host antibodies to bind them. The H2N2 strain was descended from the same lineage as the 1918 H1N1 flu, but it had picked up new genes for hemagglutinin and neuraminidase—and for a component of viral RNA polymerase—from a flu strain circulating in birds.
When flu researchers realized in 1977 that H1N1 flu had reemerged after its long absence, they immediately compared it to earlier strains they had stored in their freezers. Despite the limitations of the technologies available at the time, it was clear that the 1977 H1N1 flu was closely related, though not identical, to an H1N1 strain collected in 1950. Perhaps during its hiatus from infecting humans, this lineage had been circulating in some unknown animal reservoir in which it evolves only slowly.
Recent studies using modern techniques suggest a different explanation. Joel Wertheim, for example, used sequence data to reconstruct an evolutionary tree for hemagglutinin genes of H1N1 flu strains collected from 1918 onward.
The figure above shows the same evolutionary relationships twice, with the branch lengths scaled to different metrics. In Tree A, branch lengths are proportional to calendar time. In Tree B, branch lengths are proportional to sequence divergence. In both trees, the section of the tree that includes the re-emergent 1977 strain and its descendants is boxed in blue. Note the difference in the length of the branch—marked by the arrow—that connects the post–1977 H1N1 flu lineage to the rest of the tree. The short length of this branch in Tree B indicates that during its absence from the human population the lineage that reappeared in 1977 evolved little, if at all.
Wertheim next prepared molecular clock plots for a variety of genes from various flu viruses. Students can explore the molecular clock in SimBio’s HIV Clock lab. Wertheim’s data included sequences from H1N1 isolates collected before and after 1977, H2N2 isolates, and H3N2 isolates. Each symbol on one of his plots represents an isolate. Position on the vertical axis shows the distance on a sequence-based evolutionary tree from the root to the isolate’s tip. Position on the horizontal axis shows the year the isolate was collected.
The plot above shows Wertheim’s analysis of the influenza nucleoprotein gene. This gene, which encodes a protein with many functions, is thought to be the viral component most responsible for host specificity. Post–1977 H1N1 strains are indicated by red O’s. All other strains, including pre–1977 H1N1 isolates, are indicated by blue X’s. Wertheim’s plots for seven other flu genes look similar.
As has been known for decades, flu sequences evolve in a highly clocklike fashion. Sequences change at a steady pace over time. Symbols on a molecular clock plot fall on lines with the rate of evolution indicated by the slope.
Note that the slope of the line on which the red O’s fall is the same as the slope for the blue X’s. Since it reappeared in 1977, H1N1 flu has evolved at the same rate as other strains. But the line for the post–1977 H1N1 is shifted to the right. Wertheim calculated the difference in the X intercept for post–1977 strains versus the X intercept for all other strains on each of his plots. The average difference was 27 years. Between 1950, when its closest relative was collected, and its own reappearance in the wild, post–1977 H1N1’s molecular clock stood still.
Some authors have suggested that the H1N1 flu spent twenty seven years frozen in ice in a lake. In principle this is possible, but there is no evidence for such a natural frozen reservoir. Others have suggested that the virus’s genome spent a quarter century integrated into the genome of a host cell. Other viruses can do this, but there is no evidence that flu can.
There is, however, another place where we already know flu viruses exist in suspended animation: laboratory freezers. The consensus opinion among flu researchers is that H1N1 was reintroduced into the wild by accident—most likely during a vaccine trial. Viruses thought to be dead or disabled turned out to be viable after all.
I’m a staunch supporter of vaccination. But examples like 1977 H1N1 flu demonstrate that vaccine research must be handled with care.
Joel Wertheim’s analysis, including the evolutionary trees and clock plot shown here, appears in Wertheim, J. O. 2010. The re-emergence of H1N1 influenza virus in 1977: a cautionary tale for estimating divergence times using biologically unrealistic sampling dates. PLoS ONE 5: e11184.
A concise evolutionary history of H1N1 influenza A appears in the introduction of Nelson, M. I., C. Viboud, et al. 2008. Multiple reassortment events in the evolutionary history of H1N1 influenza A virus since 1918. PLoS Pathogens 4: e1000012.
Completion of a sequence for the 1918 flu, derived mostly from the Brevig Mission isolate, is reported in Taubenberger, J. K., A. H. Reid, et al. 2005. Characterization of the 1918 influenza virus polymerase genes. Nature 437: 889–893.
An estimate of the number of people who died during the 1918 flu pandemic appears in Johnson and Mueller 2002 Johnson, N. P. A. S., and J. Mueller. 2002. Updating the accounts: Global mortality of the 1918–1920 “Spanish” influenza pandemic. Bulletin of the History of Medicine 76: 105–115.
Evidence of a connection between mortality during the 1918 flu and bacterial pnuemonia is assessed in Morens, D. M., J. K. Taubenberger, and A. S. Fauci. 2008. Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. Journal of Infectious Diseases 198: 962–970.
The origin of the 1957 H2N2 flu is discussed in Parrish, C. R., and Y. Kawaoka. 2005. The origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza A viruses. Annual Review of Microbiology 59: 553–586.
One contemporary analysis of the H1N1 flu that re-emerged appears in Scholtissek, C., V. von Hoyningen, and R. Rott. 1978. Genetic relatedness between the new 1977 epidemic strains (H1N1) of influenza and human influenza strains isolated between 1947 and 1957 (H1N1). Virology 89: 613–617.
Another contemporary analysis appears in Nakajima, K., U. Desselberger, and P. Palese. 1978. Recent human influenza A (H1N1) viruses are closely related genetically to strains isolated in 1950. Nature 274: 334–339.
Early documentation of clocklike evolution in flu lineages was reported in Fitch, W. M., J. M. Leiter, et al. 1991. Positive Darwinian evolution in human influenza A viruses. Proceedings of the National Academy of Sciences USA 88: 4270–4274.
Refutation of the pruported evidence that influenza experiences natural evolutionary stasis while frozen in lake ice appears in Worobey, M. 2008. Phylogenetic evidence against evolutionary stasis and natural abiotic reservoirs of influenza A virus. Journal of Virology 82: 3769–3774.
The consensus opinion about the most likely source of the re-emergent 1977 H1N1 flu is noted in Krasnitz, M., A. J. Levine, and R. Rabadan. 2008. Anomalies in the influenza virus genome database: new biology or laboratory errors? Journal of Virology 82: 8947–8950.
Just a few of the many reasons I support vaccination can be found in Moore, Z. S., J. F. Seward, and J. M. Lane. 2006. Smallpox. Lancet 367: 425–435.