New Technology Effectively Gauges Specificity of Influenza Strains, Including 1918 Spanish Flu

By Jason Socrates Bardi

A team of researchers led by scientists from The Scripps Research Institute has used a new technology called a glycan array to survey samples of coat proteins from various strains of human and avian viruses, including from the deadly 1918 influenza outbreak know as the Spanish Flu.

Led by Scripps Research Professors James Paulson and Ian Wilson the work is a demonstration of the utility of the glycan array, which is a grid of sugars that resemble those found on the outside of human cells. By looking at the specificity with which the influenza proteins bind to these sugars, it is possible to gauge how adapted a given strain is for entering human cells.

"This opens the door to the possibility of using the glycan array as a surveillance tool for monitoring individual strains of influenza in birds and humans," says Paulson, who is the director of the Consortium for Functional Glycomics at The Scripps Research Institute.

Significantly, in addition to surveying samples from the 1918 influenza virus, the team also looked at closely related recombinant forms of this protein, which represented mutants of the protein's binding site. By doing so, they were able to identify specific amino acid changes responsible for shifting the specificity of an influenza virus.

"It would appear that two mutations could change the specificity dramatically going from avian to human," says Wilson, who is a member of the Department of Molecular Biology and the Skaggs Institute for Chemical Biology at Scripps Research.

An Epidemic to End All Epidemics?

Near the end of World War I, as thousands of American troops were pouring off transports and marching to the western front, a deadly infection was on the march as well. Influenza was no stranger to humanity then, as it is no stranger today.

Influenza of various strains causes a common viral infection of the lungs that affects millions of people annually and contributes to tens of thousands of deaths per year. But there was something remarkable about the strain of influenza that marched on humanity in 1918. It caused hundreds of thousands of deaths in the United States and tens of millions worldwide, inflicting unusually high mortality rates that reached 70 percent in some communities. In fact, the Spanish Flu took more lives than World War I and became the largest and deadliest influenza outbreak in recorded history—unmatched in its magnitude even though two similar outbreaks occurred in 1957 and 1968.

The theory is that terrible influenza outbreaks like these occur when a virus in birds jumps directly into humans or reassorts and infects another species, such as the pig, and then jumps into humans. What makes this so scary is the possibility that another strain of influenza could emerge as deadly as the 1918 Spanish Flu outbreak.

This fear has created a great deal of interest in finding out what it was about the 1918 strain that made it so deadly.

A Crucial Structure

Research on the molecular biology of the virus that caused the 1918 outbreak was complicated by the fact that in 1918, the cause of the disease was not known. Viruses were not identified as the cause of influenza until the 1930s.

Because the virus degrades easily, lung tissue samples taken in 1918 are generally unreliable sources. However, scientists obtained fragments of RNA from biopsies from soldiers who died from influenza in 1918 that were preserved and maintained in the Armed Forces Institute of Pathology. Another sample was taken from an Inuit woman who had succumbed to the infection and had been buried in the Alaskan permafrost. A few years ago, Jeffery Taubenberger and his colleagues at the Armed Forces Institute of Pathology were able to piece together enough fragments to reconstruct the sequence of the gene that coded for the viral protein hemagglutinin.

Hemagglutinin is the main antigenic determinant on the virus—it is what the human immune system primarily recognizes and responds to by making antibodies and mounting an immune defense. How deadly an individual influenza infection is depends on how well one person's immune system recognizes the hemagglutinin. A large, glycosylated protein that forms from three identical 550 amino-acid chains, hemagglutinin is abundantly displayed on the surface of the influenza virus. It is also the receptor responsible for the virus's ability to infect cells of the host organism.

During an infection, the virus enters the airways and travels to the epithelial cells lining the lungs. There, the hemagglutinin on the surface of the virus binds to lung epithelial cell receptors containing sialic acid, which allows the virus to be internalized into the epithelial cell, through something known as the endosomal pathway, and this establishes an infection.

Last year, Wilson, Scripps Research Assistant Professor James Stevens, Ph.D., and their colleagues made enough of the protein to crystallize and went on to solve the structure using x-ray crystallography. Significantly, the researchers compared the structure of the 1918 influenza hemagglutinin to hemagglutinin proteins from other human, avian, and pig viruses.

One of only a handful of proteins made by the virus, the hemagglutinin protein structure that Stevens, Wilson, and their colleagues solved revealed details that were crucial to understanding the 1918 pandemic.

The structure has features primarily found in avian viruses, but with some human characteristics. This suggests why the virus may have been so deadly. Avian-to-human transmission is rare, and because of this the surface proteins of the 1918 virus were different from those found on other flu viruses. People's immune systems were unaccustomed to them and unable to fight off the Spanish Flu.

An avian origin of the virus also suggests an explanation for one of the most unusual features of the 1918 outbreak—that mortality was particularly high among young adults, the age group that is usually least impacted by the flu. Influenza is normally more deadly to the elderly and preadolescents within a population, but in 1918, there were a surprising number of deaths among 15 to 34 year olds. The avian nature of the structure suggests the older age group may have been partially protected by exposure to a similar virus in an earlier epidemic.

Sugar Arrays 

Wanting to see how easily a virus can adapt from one species to another through mutations to the hemagglutinin protein, Wilson and Stevens set out to compare the receptor specificity of the 1918 virus with that of modern avian viruses. They made recombinant forms of the 1918 virus hemagglutinin and several other "mutants" where they changed particular amino acids in its binding site. (These recombinant proteins were completely benign, since they were created using technologies that at no time required samples of the actual virus.)

Then, they began to collaborate with Paulson and Scripps Research investigator Ola Blixt, Ph.D. Blixt and Paulson,with several individuals in the Consortium for Functional Glycomics, had recently overseen the creation of a technology designed to study how proteins like hemagglutinin bind to sugars.

The technology, known as a functional glycan microarray, is a glass slide onto which are printed hundreds of different sugar or "glycan" chains, covering the major types of relevant sugars to which sugar-binding proteins bind.

The array offers scientists a cutting-edge general research tool that allows them to analyze the specificities of all types of glycan binding proteins (GBPs), and the new tool will make it easier and faster for scientists to determine how a diversity of human glycan binding proteins interact with carbohydrates in biological systems. This is significant because glycosylation, the attachment of carbohydrate chains to proteins, is a crucial part of biology, and some scientists estimate that half of all proteins encoded by the human genome have sugars attached to them at some point after they are made. Carbohydrates carry information and are responsible for important biological functions, playing a central role in intercellular communication, protein folding, and cell adhesion. Some viruses, like influenza, use sugars on the outside of human cells to gain entry into human cells.

Significantly, the glycan microarray was able to distinguish between specificities of influenza strains that differed only in terms of a single sialic acid linkage—one sugar molecule to which the influenza recognizes. Thus the microarray readily distinguished avian viruses that preferentially bind to a2-3 linked sialic acids on receptors of intestinal epithelial cells, from human viruses that are specific for the a2-6 linkage on epithelial cells of the lungs and upper respiratory tract.

The ability to so finely distinguish specificities is important, says Paulson, because scientists can now look at a whole pattern of hemagglutinins from different viral isolates, including animal reservoirs of emerging influenza strains, and compare them to one another.

The array is not commercially available but it is available to the scientific community through the consortium. For more information on the Consortium for Functional Glycomics, see the consortium's home page at: http://functionalglycomics.org.

The article, Glycoarray Analysis of the Hemagglutinins from Modern and Pandemic Influenza Viruses Reveals Different Receptor Specificities" by James Stevens, Ola Blixt, Laurel Glaser, Jeffery K. Taubenberger, Peter Palese, James C. Paulson, and Ian A. Wilson will be published in an upcoming issue of the Journal of Molecular Biology. The article is available online at: http://dx.doi.org/10.1016/j.jmb.2005.11.002.

This work was supported by funds from the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute of General Medical Sciences (NIGMS), and the Skaggs Foundation for Research.

Much of the work was carried out in conjunction with the Consortium for Functional Glycomics, of which Paulson serves as director and principal investigator. The consortium is funded by a $37 million "glue" grant awarded by the National Institute of General Medical Sciences, one of the National Institutes of Health, and has brought together some 230 independently funded researchers at 140 different institutions around the world, including several in San Diego.

Send comments to: mikaono[at]scripps.edu

Ribbon representation of the hemagglutinin HA0 trimer from the 1918 influenza virus. Image courtesy of James Stevens.