Using Comparative Genomics to Explore the Genetic Code of Influenza Sangeeta Venkatachalam

The influenza pandemic of “Spanish flu” killed million people. considered the most deadly pandemic in recorded human history. Why?? The virus antigens were extremely different to those encountered previously so people carried no immunity to this virus strain and were highly susceptible to illness and even death. What was it?? It was caused by the H1N1 type. It has been found to be very similar to the bird flu of today mainly H5N1 and H5N2.

Resurrecting the 1918 influenza virus Flu victim frozen in Alaska Permafrost since Fragments of RNA are retrieved from fragments of long tissue, converted into DNA and sequenced. The overlapping sequences are pieced together to give the full gnome sequence. A DNA version is synthesized in the lab. The DNA is injected into canine kidney cells, which produces tens of virus particles. The virus is isolated from the cells and used to infect mice. They all die within 6 days.

Influenza Virus structure The Influenza virus A is a globular particle about 100 nm in diameter covered by in a lipid bilayer. Studded in the lipid bilayer are two integral membrane proteins. 500 molecules of hemagglutinin ("H") 100 molecules of neuraminidase ("N"). 8 pieces of RNA. The HA gene. – 3 distinct hemagglutinins, H1, H2, and H3 are found in human infections The NA gene. – 2 different neuraminidases N1 and N2 have been found in human viruses The NP gene – encodes the nucleoprotein. Influenza A, B, and C viruses have different nucleoproteins. Neuraminidase Hemaglutinin

Hemagglutinin & Neuraminidase Hemagglutinin the main antigenic determinant on the virus. immune system primarily recognizes and responds to hemagglutinin by making antibodies and mounting an immune defense. Based on how well a person's immune system can recognize the hemagglutinin the severity of the flu can be decided. Neuraminidase a glycoprotein expressed on the viral surface. Its principal biological role is the cleavage of the terminal sialic acid residues that are receptors for the virus' hemagglutinin (HA) protein. It is clear that successful virus replication and spread depends on tightly coordinated interactions among the virus’ genes.

Antigenic shift and drift Antigenic drift – The changes in the HA antigen shape, may cause the antibodies not to match up, and thus allowing the newly mutated virus to infect the body's cells. This genetic mutation is called. linklink Antigenic shift – when the genetic change enables a flu strain to jump from one animal species to another, including humans. is called antigenic shift. linklink

Investigation into changes or mutations in the gene for influenza N antigen. What did we do ? Run a multiple sequence alignment using versions of H3N2 viral sub-strain. Run a multiple sequence alignment using H3N2, H1N1 (1918) and H5N1 influenza A viral sub-strains. Use the sequence alignment to construct a phylogenic tree.

Multiple Sequence alignment of H3N2 sequences The NA gene sequences used here come from influenza viruses isolated from infected humans. The virus strain is H3N2 (referring to the H and N antigens on the virus). Each sequence has a name e.g. A_HongKong_68_H3N2 This indicates it is an influenza A virus from Hong Kong isolated in 1968.

Findings Some nucleotides not conserved (shown in black) most of the time these led to the same amino acid. most of the changes take place in the third nucleotide position. – Because the third nucleotide is where the binding between the tRNA and the mRNA is the weakest and mistakes in translation are most likely to take place here. Also the there often are several codons for a single amino acid and that the first two letters in a codon usually are the important ones, but that the third letter is occasionally significant. The NA gene is highly conserved between viral strains, especially the active site. If the amino acid sequence is altered too significantly, then the protein shape changes and can no longer function to cut the virus particle from the host cell membrane. As a result, the virus can no longer spread to infect new host cells.

Multiple Sequence alignment of H3N2 and H1N1 sequences We did alignment of this particular H1N1 virus from 1918 and the others found recently. The results show that the H5N1 virus seen in Thailand in 2004 are very close to each other in the tree. This may mean that the H5N1 might be able to create a pandemic like the H1N1 of 1918

Conclusion Knowledge of the genome of the 1918 virus gained so far may provide clues to help us avoid or prepare for another pandemic. resemble bird flu genomes more closely than those of human strains. Using the techniques available through bioinformatics we can analyze the virus strain which emerge newly and find out how deadly it can be by finding the closeness to viruses which have caused pandemics before.

References R. J. Webby, R. G. Webster, Science 302, “Are We Ready for Pandemic Influenza?”, (2003). Tumpey, T.N. et al. Science 310, “Characterization of the Reconstructed 1918 Spanish Influenza Pandemic Virus”, (2005). Palese, P. & Compans, R. W. Inhibition of influenza virus replication in tissue culture by 2- deoxy- 2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action (1976) J. Gen. Virol. 33, A. H. Reid et al., Initial Genetic Characterization of the 1918 "Spanish" Influenza Virus Proc. Natl. Acad. Sci. U.S.A. 97, (2000) Reid, A. H., Fanning, T. G., Hultin, J. V. & Taubenberger, J. K. Origin and evolution of the 1918 "Spanish" influenza virus hemagglutinin gene (1999) Proc. Natl. Acad. Sci. USA 96, Using bioinformatics to explore the genetic code