Bioinformatics Workshop Summer 2006 Avian Influenza Bioinformatics Workshop Summer 2006

Avian Influenza The history of human influenza The Spanish flu pandemic of 1918 Emergence of ‘Bird Flu’ (avian influenza) Phylogenetic comparison of flu sequences Can bioinformatics prevent the next flu pandemic, or at least provide a warning? Problem based bioinformatics exercise

A Sense of Urgency

Bioinformatics Questions Can flu viruses be compared? Which flu genes are important? Where are those data organized? How do you compare viral strains? How do you interpret these results? (Yes; HA, NA, PB1; NCBI and flu databases; MSA sequence alignment and phylogenetics) (Compare alignments and addition / loss of aa residues with pathogenicity and epidemiology)

Influenza A Up Close and Personal SEM images of Influenza A virus – segmented genome is visible

Influenza A Genome Influenza A viral particle structure showing the two key genes, hemagglutinin HA and neuraminidase NA, used for attaching and release

Influenza A Genome

Influenza - An Emerging Disease Because all known influenza A subtypes exist in the aquatic bird reservoir, influenza is not an eradicable disease; prevention and control are the only realistic goals. If people, pigs, and aquatic birds are the principal variables associated with interspecies transfer of influenza virus and the emergence of new human pandemic strains, influenza surveillance in these species is indicated. Live-bird markets housing a wide variety of avian species together (chickens, ducks, geese, pigeon, turkeys, pheasants, guinea fowl), occasionally with pigs, for sale directly to the public provide outstanding conditions for genetic mixing and spreading of influenza viruses; therefore, these birds should be monitored for influenza viruses. Moreover, if pigs are the mixing vessel for influenza viruses, surveillance in this population may also provide an early warning system for humans http://www.cdc.gov/ncidod/eid/vol4no3/webster.htm

Ecology of Influenza A HxNy http://www.medicalecology.org/diseases/influenza/influenza.htm

Flu Attaching to Host Cells Flu binds to receptors in the trachea and also in the lungs Antigenic variable regions determine fit of virus and host cells Influenza will vary in structure of HA and NA – thus HxNy name

H5N1 Life Cycle H5N1 influenza Attachment Cytokine production Acute respiratory distress syndrome T cell activation Viral replication and release (host death) http://www2.niaid.nih.gov/Newsroom/FocusOn/Flu04/

Flu Virus - Reassortment Flu virions can aggregate on host cells, and through proximity, exchange RNA (reassortment) The greater the virion aggregation, the faster will be the process of flu adaptation through (RNA) reassortment

Antigenic Shift The genetic change that enables a flu strain to jump from one animal species to another, including humans, is called ‘antigenic shift’. It can happen in different ways, one of which is common on farms in Southeast Asia and China. http://www2.niaid.nih.gov/Newsroom/FocusOn/Flu04/

Background - Antigenic Shift Influenza A is a single stranded RNA virus containing 8 RNA gene segments, two of which code for the two antigenic surface proteins, hemagglutinin (HA) and neuraminidase (NA). These proteins are involved in entry (HA) and release (NA) from host cells during infection through the binding and cleaving of sialic acid on the host cell surface. It is likely that certain combinations of HA and NA are best suited for this interaction with sialic acid. [1] Antigenic shift is defined by a new subtype of HA and possibly of NA appearing in the population. Likely occurs when two viruses infect a single host, their gene segments are reassorted and viruses with a new combination of HA and NA proteins are created. Such antigenic shifts are believed to be the cause of pandemics, as the human population has no immunity to the new subtype. An example is the H2N2 pandemic of 1957. [1] Wagner, R, et al Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev Med Virol. 2002 May-Jun; 12 (3): 159-66.

Genetic Reassortment Leading to Pandemic Strains Antigenic shifts in Influenza A are caused by reassortment in pigs

Swine – Flu Mixing Vessels The trachea of pigs possess glycoproteins which can bind both bird / human flu strains Through RNA reassortment, influenza can obtain genes from both birds and humans Influenza then mutates to better adapt to the glycoproteins which are present in humans This ‘adapting period’ can take years, or less. Molecular Basis for the Generation in Pigs of Influenza A Viruses with Pandemic Potential JOURNAL OF VIROLOGY, Sept. 1998, p. 7367–7373

Note the insertion of the basic residues in HA1 / HA2 Highly Pathogenic H5N1 Note the insertion of the basic residues in HA1 / HA2 http://www.cdc.gov/ncidod/eid/vol4no3/webster.htm

Highly Pathogenic H5N1 Molecular changes associated with emergence of a highly pathogenic H5N2 influenza virus in chickens in Mexico. In 1994, a nonpathogenic H5N2 influenza virus in Mexican chickens was related to an H5N2 virus isolated from shorebirds (ruddy turnstones) in Delaware Bay, United States, in 1991. The 1994 H5N2 isolates from chickens replicated mainly in the respiratory tract, spread rapidly among chickens, and were not highly pathogenic. Over the next year the virus became highly pathogenic, and the hemagglutinin acquired an insert of two basic amino acids (Arg-Lys), possibly by classic recombination and a mutation of Glu to Lys at position 3 from the cleavage site of HA1/HA2. http://www.cdc.gov/ncidod/eid/vol4no3/webster.htm

Emergence of H5N1 Influenza in Hong Kong H5N1 enters domestic birds and then mammals http://www.cdc.gov/ncidod/eid/vol4no3/webster.htm

Emergence of H5N1 Influenza in Hong Kong The emergence of H5N1 influenza in Hong Kong. It is postulated that a nonpathogenic H5N1 influenza spread from migrating shorebirds to ducks by fecal contamination of water. The virus was transmitted to chickens and became established in live bird markets in Hong Kong. During transmission between different species, the virus became highly pathogenic for chickens and occasionally was transmitted to humans from chickens in the markets. Despite high pathogenicity for chickens (and humans), H5N1 were nonpathogenic for ducks and geese. http://www.cdc.gov/ncidod/eid/vol4no3/webster.htm

Flu Genetics of H5N1 in Asia Guan, Y. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 8950-8955

Bioinformatics Skills / Tasks Searching PubMed, PNAS, etc. Reading scientific articles (learn quickly) Finding and formatting sequences Uploading data to Biology Workbench Sequence alignment / phylogenetics Problem posing based on public flu data Suggest steps for monitoring / containing the spread of avian influenza into humans

Prerequisite Knowledge Using NCBI Thorough knowledge of tools / databases Sequence alignment MSA and phylogenetics for genes / proteins Biology Workbench Upload sequences and perform alignments Influenza – pathogenicity / epidemiology

HA Phylogenetics in Flu Reid, Ann H. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1651-1656

Hemagglutinin AA Changes Reid, Ann H. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1651-1656

Reid, Ann H. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1651-1656 Hemagglutinin CDS Reid, Ann H. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1651-1656

Neuraminidase Strains Compared 32 complete CDS from NA gene Mostly from H1N1 epidemics 1918, 1934, 1957, 1968, Compare avian / human / swine, and the location of the 1918 flu sequences Results suggest that the 1918 flu was a hybrid (reassortant) of avian / mammal Probably combined in swine in 1910 – 1915 Characterization of the 1918 Spanish Influenza NA

Neuraminidase Strains Compared

Following the Evolution of H5N1

Comparing H5N1 to Other Flu

Flu! The Influenza Sequence Database Read the latest Flu news Subscribe to a flu listserv for ‘flu alerts’ Compare flu sequences, save / archive to a ‘work file’ of ‘experiments’ and data Download alignments in Excel format View data in a ‘genome browser’ Research flu vaccine options for strains http://flu.lanl.gov/

Flu Sequence Dataset Explorer View flu data by taxonomy, segment, serotype, by year, host, and country http://www.ncbi.nlm.nih.gov/genomes/DatasetExplorer/FLU/fluPage.cgi Search, select, and align sequences Start with a small number of sequences Build a nonlinear map and tree Annotate with flu season date markers Use NCBI genomes to study flu as well! http://www.ncbi.nlm.nih.gov/genomes/taxg.cgi?tax=11320

1918 Flu: Bird-Human Hybrid The three-dimensional structure of a protein associated with the flu virus responsible for the devastating pandemic of 1918 was determined. The structure resembles that usually found in birds but with a slight alteration that makes it efficient at binding human cells. The Scripps team first determined the genetic sequence of the 1918 hemagglutinin protein, and then placed the code for that sequence in another type of virus to manufacture the protein. When they studied that protein's structure, they discovered the strong similarities to a type found in flu strains that infect birds (ScienceExpress 7/6/2005). Birds are natural hosts to all flu strains. Humans, by contrast, are usually infected only by those flu strains that have adapted to recognize lung cells. In 1997, however, doctors found the first cases of an avian flu virus, called H5N1, in humans, in Southeast Asia. If H5N1 enters swine (from birds) it can adapt to infect human cells. http://www.mos.org/cst/article/5755/1.html

Highly Pathogenic H5N1 Influenza Virus Infection in Migratory Birds 7/05

HA Binding – Glycan Arrays The binding and cleavage sites for HA are major sources of change in influenza A virus This is why vaccines have to be specific to a particular subtype of a virus currently circulating in a population. Sugar arrays can test for specificity of binding, a new method of predicting risk: Glycan Array Technology http://www.scripps.edu/newsandviews/e_20060116/flu.html

Methods (Modeling Current Subtypes) Model of a human population using NetLogo™ Script written using BioPerl to automatically BLAST all existing viruses against database of those known to infect humans (e.g. H1N1 vs H1N1, H3N2, H5N1, H1N1 and H9N2) Empirically determined values in order to maintain a level of infection 1 per ~150 people for the H1N1 and H3N2 strains [2] Well studied avian subtypes (e.g. H5N1) are then incorporated with viral attributes relative to H1N1 [2] CDC Weekly Report: Influenza, http://www.cdc.gov/flu/weekly/, 2005

Sequence Similarity Table

EU Urges Global Bird Flu Response “The H5N1 strain remained largely in South-East Asia until this summer, when Russia and Kazakhstan both reported outbreaks Scientists fear it may be carried by migrating birds to Europe and Africa but say it is hard to prove a direct link with bird migration” http://news.bbc.co.uk/1/hi/world/europe/4352340.stm

An Emerging Pandemic H5N1 has acquired AA changes to become highly pathogenic in both birds and humans. H5N1 has spread throughout Southeast Asia* It has not acquired the ability to rapidly infect humans, or affect human-human transmission. Birds are now adapting to H5N1, making the spread of the virus harder to monitor in Asia. Once H5N1 enters swine, it can both adapt to human cells, and ‘optimize’ its pathogenicity. * In early Winter 2006 H5N1 is now migrating across most of the globe

A Pandemic in Real Time How it Would Happen http://www.nature.com/nature/focus/avianflu/index.html Random humans are infected with avian subtypes of influenza (H5N1 or HxNy) Rapid mutation begins. Avian subtypes then spread, and reassort (inside humans) to create new subtypes (HxNy from H5N1). Viral attributes are based on the sequence similarity between the avian HA protein and the most similar HA protein capable of infecting humans to date – virulence and pathogenicity are ‘exchanged’. Without vaccine, the new virus spreads unimpeded

Visions of Past and Future

Summary Influenza A is a real bioinformatics story Think about the knowledge and skills you need to investigate bioinformatics of flu Ability to search literature of many types Ability to find, download, and format data Ability to choose and perform ‘experiments’ Start with recreating results in the papers Expand to using portal based flu data/tools

Key References Characterization of the 1918 Spanish Influenza NA Molecular Characterization of H5N1 virus Recent Avian Influenza Outbreaks: A Pandemic in the Making Emergence of avian H1N1 influenza viruses in pigs in China Molecular Basis for the Generation in Pigs of Influenza A with Pandemic Potential Scientists have uncovered the structure of the 1918 flu virus CDC – http://www.cdc.org/ WHO – http://www.who.org/ NCBI flu explorer – http://www.ncbi.nlm.nih.gov/ Flu! The Influenza sequence database http://flu.lanl.gov/