By recreating a key surface protein from the 1918 pandemic flu virus and testing its effects in mice, researchers have shown that the protein might have been an important reason for the virus´s extraordinary ability to kill.

An international team of researchers recreated hemagglutinin (HA) from the 1918 "Spanish flu" virus and spliced it into recent flu viruses adapted to humans and mice, according to a recent report in Nature. Mice that were exposed to the engineered viruses suffered severe lung infections that resembled those seen in people who died in the 1918 pandemic, according to the report.

"Replacing only one gene is sufficient to make the virus more pathogenic," said senior author Yoshihiro Kawaoka, a virologist at the University of Wisconsin–Madison (UWM) and the University of Tokyo, in a news release from UWM.

The 1918 flu pandemic killed tens of millions of people around the world. In recent years, researchers have extracted several of the virus´s genes from preserved tissue samples from pandemic victims and have sequenced them. HA and neuraminidase (NA), surface proteins that equip flu viruses to enter and leave human cells, are important targets of the body´s immune response; changes in their structure can make the virus more dangerous and enable it to infect new species.

What made the 1918 virus so lethal has been a mystery. The report by Kawaoka and colleagues says speculation has focused on living conditions at the end of World War I as well as on properties of the virus itself. The HA and NA of the virus contain no amino acid sequences known to be linked with high virulence, the report says. The researchers therefore sought to test the effects of the 1918 virus HA and NA on the pathogenicity of flu viruses in mice.

To do this, they took three recent influenza A strains (one of which is pathogenic in mice and two of which are not) and replaced their HA and NA genes either with the 1918 versions of both genes or with the 1918 HA gene and an NA gene from a recent strain. They then exposed groups of mice to the engineered viruses or the three natural viruses by intranasal inoculation.

All the viruses containing the 1918 HA gene, including those that in their natural form are harmless to mice, grew quickly in the mice´s lungs and eventually killed them, according to the report. Viruses containing both HA and NA from the 1918 virus were no more harmful than those containing the 1918 HA along with NA from a recent strain, which indicated that the 1918 version of NA didn´t contribute to viral pathogenicity.

In postmortem examinations, the researchers found signs that infection had spread much deeper into the lungs in mice exposed to viruses containing the 1918 HA gene than in the other mice.

"These highly virulent recombinant viruses expressing the 1918 viral HA could infect the entire lung and induce high levels of macrophage-derived chemokines and cytokines, which resulted in infiltration of inflammatory cells and severe haemorrhage, hallmarks of the illness produced during the original pandemic," the abstract of the report states. Further, the addition of the 1918 HA gene to a virus that is normally pathogenic in mice made the lung lesions "clearly more severe" than those in mice infected with the normal version of the virus.

"Severe lung infection was a hallmark of the illness produced by the original pandemic virus in humans, suggesting a possible, though not conclusive, association between the pandemic virus HA and its pathogenicity in humans, and indicating the need to examine the contribution of the HA to viral pathogenicity in other animal models," the report says.

Kawaoka´s group also looked at the ability of 1918 viral HA to recognize the cell-surface receptor molecule preferred by human-adapted flu viruses. Using a "competitive binding assay," the team found that the 1918 HA preferentially recognizes this receptor. Because the 1918 virus is believed to have originated in birds, this finding suggests that the virus must have circulated in humans long enough to develop a preference for the human type of receptor, the article says.

The researchers also report evidence that people who were alive in 1918 still have immunity to the 1918 virus. They examined the neutralizing activity of blood serum samples from people of various ages against viruses with the 1918 HA and NA genes and against more recent human flu viruses. Serum from those who survived the pandemic "showed markedly high activities" against the engineered virus, while samples from younger people had only limited activity against it.

If the 1918 virus reemerged today, "The only group with significant natural protection would be survivors of the 1918 pandemic, who still express high levels of antibodies against an antigen to which they were exposed over 80 years ago, a phenomenon referred to as original antigenic sin," the report says.

Despite their findings, Kawaoka and colleagues say that HA is probably not the sole cause of the virulence of the 1918 virus. "The virulence of influenza virus is probably a polygenic trait, in that ultimately we expect other gene products . . . to be implicated in the phenotype of the 1918 virus," they write.

Anthony Fauci, MD, director of the National Institute of Allergy and Infectious Diseases, recently concurred that there is more to be learned about what made the 1918 virus so destructive. "It is very clear that there was something about the virulence of this microbe that we don´t fully understand," he said in a Nov 15 press briefing on flu research.

Kobasa D, Takada A, Shinya K, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature 2004;431:703-7 [Abstract]