1. Bioinformatics Workshop Summer 2006
Avian Influenza
Bioinformatics Workshop
Summer 2006

2. 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

3. A Sense of Urgency

4. 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)

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

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

7. Influenza A Genome

8. 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

9. Ecology of Influenza A HxNy

11. 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

12. H5N1 Life Cycle H5N1 influenza Attachment Cytokine production
Acute respiratory distress syndrome
T cell activation
Viral replication and release (host death)

13. 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

14. 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.

15. 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 May-Jun; 12 (3):

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

17. 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

18. Note the insertion of the basic residues in HA1 / HA2
Highly Pathogenic H5N1
Note the insertion of the basic residues in HA1 / HA2

19. 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 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.

20. Emergence of H5N1 Influenza in Hong Kong
H5N1 enters domestic birds and then mammals

21. 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.

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

23. 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

24. 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

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

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

28. 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,

30. 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

31. Neuraminidase Strains Compared

33. Following the Evolution of H5N1

34. Comparing H5N1 to Other Flu

36. 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

38. Flu Sequence Dataset Explorer
View flu data by taxonomy, segment, serotype, by year, host, and country
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!

39. 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.

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

41. 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

42. 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,

43. Sequence Similarity Table

44. 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”

45. 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

46. A Pandemic in Real Time How it Would Happen
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

47. Visions of Past and Future

48. 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

49. 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 –
WHO –
NCBI flu explorer –
Flu! The Influenza sequence database