Statistical Phylogenetics

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Statistical Phylogenetics

• Avian Influenza Virus
• By Kyle Walsh, Lisa Booth, and Carley Matanin

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DNA Background

• Structure of DNA

Nucleotide Pairing Adenine (A)Thymine
(T) Guanine (G)Cytosine (S) Nucleotide
Groups PurinesA and G PyrimidinesT and C
www.ashingtonhigh.northumberland.sch.uk
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DNA Background

• Reading of DNA
• Nucleotides read in triplets
• Each triplet codes for an amino acid

www.genome.ou.edu
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DNA Background

• Mutations
• Substitutions One nucleotide is exchanged for
  another
• Ex. GACCTA ? GAGCTA
• Gaps Nucleotides are either added or deleted
  from the DNA
• Ex. GACCTA ?? GACTTCA

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Phylogenetics Background

• Study of evolutionary relatedness among various
  organisms based on their DNA
• Uses variations in DNA sequences to determine the
  path of evolution

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Phylogenetics Background

• Maximum Parsimony Tree
• Path of evolution which requires the fewest
  changes

http//research.amnh.org/siddall/methods/day3.htm
l
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Phylogenetics Background

• Maximum Likelihood tree
• Work backwards from a given tree topology to
  determine if it is the most likely tree topology
  based on the probabilities of specific DNA
  mutations.
• Bayesian Method
• Find the distribution of trees that would produce
  the observed data.

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Avian Influenza Background
www.channel4.com www.itn.co.uk www.kfyrtv.com
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Background Info

• Avian Influenza viruses occur naturally in wild
  birds
• wild birds carry influenza viruses in their
  intestines (but virus usually does not make wild
  birds sick)
• highly contagious among birds and can make
  domesticated birds (chickens, ducks, turkey)
  sick can be fatal

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Bird Flu in Humans?

• transmission from birds to humans is possible,
  but is not sustained
• virus obtained from direct or close contact with
  infected poultry or surfaces (secretions and/or
  excretions)
• can also be transmitted through an intermediate
  host (ex. Pig)

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Human Cases

• more than 200 human cases have been confirmed
  since 1997
• virus first seen in Hong Kong in 1997 on farms
  and in markets
• outbreaks began in South-East Asia in 2003 and
  have infected nine countries including Korea,
  Viet Nam, Japan, Thailand, Cambodia, Laos,
  Indonesia, China and Malaysia
• infecting humans at a 50 mortality rate

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Subtypes

• humans can be infected with influenza types A, B,
  and C viruses
• influenza A is classified into subtypes according
  to surface glycoproteins hemagglutinin (HA) and
  neuraminidase (NA)
• highly pathogenic influenza A subtype H5N1 HA is
  what our study focused on

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Low vs High Pathogenic Viruses

• viruses classified based on its capability of
  causing a disease
• Low Pathogenic Avian Influenza (LPAI) associated
  with mild disease in poultry such as ruffled
  feathers and drop in egg production
• High Pathogenic Avian Influenza (HPAI) leads to
  severe illness and mortality in poultry H5N1 is
  a highly pathogenic virus

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Possibility of a Pandemic

• Influenza A viruses composed of eight separate
  gene segments
• allows for creation of new strands of the virus
  via reassortment or adaptive mutation

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Possibility of a Pandemic

• Reassortment- exchange of genetic material
  between human and avian viruses during
  coinfection (infection of both human and avian
  viruses at the same time)
• new strands could increase transmissibility among
  humans and an influenza pandemic could occur

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Goals

• To determine the nature of H5N1 sub-lineages by
  assessing if the phylogeny of a series of these
  viruses is ordered based on geography, time, or
  host species.
• To determine if any of these factors are primary,
  and if so, if any act as secondary factors of the
  phylogeny of the H5N1 virus.
• To assess host specificity of the H5N1 virus.

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Methods

• Clustal X software was used to do a multiple
  sequence alignment. We aligned data from 35 H5N1
  HA genes and 3 H5N2 HA genes were aligned for an
  out group comparison. (Courtesy of NCBI Gen
  Bank)
• 38 sequences from Gen Bank were named according
  to location, ID , species, and year of
  collection.
• Example- hk55ck03 is from Honk Kong, case ID 55,
  from a chicken in 2003.

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Methods continued

• PHYLIP (the PHYLogeny Inference Package) was used
  to infer phylogenies from the data. Parsimony
  and Maximum Likelihood trees were produced using
  the PHYLIP software.
• The MrBayes program was used for Bayesian
  estimation oh phylogeny therefore producing trees
  conditioned on the observed sequences.
• MrBayes uses the Markov chain Monte Carlo (MCMC)
  technique to approximate the posterior
  probabilities of trees.
• Our study used 3.5 million generations to produce
  the Bayesian tree.
• Trees were visually interpreted to determine
  whether location, species, or time had the
  greatest impact.

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Fig. 1. Map of eastern Asia showing Fujian,
Guangdong, Guangxi, Guizhou, Hong Kong, Hunan,
and Yunnan where influenza surveillance was
conducted and the locations of Poyang and Qinghai
Lakes
Chen, H. et al. (2006) Proc. Natl. Acad. Sci. USA
103, 2845-2850
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Results
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1
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.65
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1
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.96
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.99
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Vietnam 2003-2005
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Thailand 2004-2005
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Hong Kong 1997-2003
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Conclusions

• The phylogeny of the H5N1 HA virus is primarily
  based on geography, the secondary factor being
  time.
• The H5N1 HA virus is mainly found in birds,
  however it has been known to infect humans, pigs,
  and large cats.

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Future Direction

• Study economic trade routes and migratory bird
  routes to determine their effect on the spread of
  H5N1 virus
• Study the virulence of H5N1 and see if it is
  reflected in the phylogeny
• Study how an LPAI mutates to an HPAI virus

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Future Direction

• Perform another study using DNA sequences from
  countries further west, such as Iraq, Jordan, etc
  and determine where the virus originated.
• Study June 2006 family of eight infected
  individuals to see where the virus originated and
  how it was transmitted.

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References

• http//www.cdc.gov/flu/avian/gen-info/facts.htm
• http//www.who.int/mediacentre/factsheets/avian_in
  fluenza/en/index.html
• http//www.who.int/csr/disease/avian_influenza/tim
  eline.pdf
• Yang, Z.Y., Duckers, H.J., Sullivan, N.J., et al.
  Identification Of The Ebola Virus Glycoprotein As
  The Main Viral Determinant Of Vascular Cell
  Cytotoxicity And Injury. Nature Medicine 886-8,
  2000.
• Chen, H., Smith, G.J.D., Li, K.S., et al.
  Establishment of multiple sublineages of H5N1
  influenza virus in Asia. PNAS 2845-2850, 2005.
• Smith, G.J.D., Naipospos, T.S.P., Nguyen, T.D.,
  et al. Evolution of H5N1 Influenza virus in avian
  and human hosts in Indonesia and Vietnam.
  Virology 258-268, 2006.

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• Thank you Jeff and Dr. Pearl!!!