title: Phylodynamics of avian influenza clade 2.2.1 H5N1 viruses in Egypt.
abstract: BACKGROUND
  Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are widely distributed within poultry populations in Egypt and have caused multiple human infections. Linking the epidemiological and sequence data is important to understand the transmission, persistence and evolution of the virus. This work describes the phylogenetic dynamics of H5N1 based on molecular characterization of the hemagglutinin (HA) gene of isolates collected from February 2006 to May 2014.
  
  
  METHODS
  Full-length HA sequences of 368 H5N1 viruses were generated and were genetically analysed to study their genetic evolution. They were collected from different poultry species, production sectors, and geographic locations in Egypt. The Bayesian Markov Chain Monte Carlo (BMCMC) method was applied to estimate the evolutionary rates among different virus clusters; additionally, an analysis of selection pressures in the HA gene was performed using the Single Likelihood Ancestor Counting (SLAC) method.
  
  
  RESULTS
  The phylogenetic analysis of the H5 gene from 2006-14 indicated the presence of one virus introduction of the classic clade (2.2.1) from which two main subgroups were originated, the variant subgroup which was further subdivided into 2 sub-divisions (2.2.1.1 and 2.2.1.1a) and the endemic subgroup (2.2.1.2). The clade 2.2.1.2 showed a high evolution rate over a period of 6 years (6.9 × 10(-3) sub/site/year) in comparison to the 2.2.1.1a variant cluster (7.2 × 10(-3) over a period of 4 years). Those two clusters are under positive selection as they possess 5 distinct positively selected sites in the HA gene. The mutations at 120, 154, and 162 HA antigenic sites and the other two mutations (129∆, I151T) that occurred from 2009-14 were found to be stable in the 2.2.1.2 clade. Additionally, 13 groups of H5N1 HPAI viruses were identified based on their amino acid sequences at the cleavage site and "EKRRKKR" became the dominant pattern beginning in 2013.
  
  
  CONCLUSIONS
  Continuous evolution of H5N1 HPAI viruses in Egypt has been observed in all poultry farming and production systems in almost all regions of the country. The wide circulation of the 2.2.1.2 clade carrying triple mutations (120, 129∆, I151T) associated with increased binding affinity to human receptors is an alarming finding of public health importance.
authors.affiliation: Food and Agriculture Organization of the United Nations (FAO) - Emergency Center of Transboundary Animal Diseases (ECTAD), 11 Al Eslah El Zerai St, P.O. Box, 2223, Giza, Egypt. araby85@hotmail.com.|National Laboratory for Veterinary Quality Control on Poultry Production (NLQP), Animal Health Research Institute, P.O. Box, 264, Giza, Egypt. araby85@hotmail.com.
authors.forename: Abdelsatar
authors.initials: A
authors.lastname: Arafa
authors.affiliation: Food and Agriculture Organization of the United Nations (FAO) - Emergency Center of Transboundary Animal Diseases (ECTAD), 11 Al Eslah El Zerai St, P.O. Box, 2223, Giza, Egypt.
authors.forename: Ihab
authors.initials: I
authors.lastname: El-Masry
authors.affiliation: National Laboratory for Veterinary Quality Control on Poultry Production (NLQP), Animal Health Research Institute, P.O. Box, 264, Giza, Egypt.
authors.forename: Shereen
authors.initials: S
authors.lastname: Kholosy
authors.affiliation: National Laboratory for Veterinary Quality Control on Poultry Production (NLQP), Animal Health Research Institute, P.O. Box, 264, Giza, Egypt.
authors.forename: Mohammed K
authors.initials: MK
authors.lastname: Hassan
authors.affiliation: Food and Agriculture Organization of the United Nations (FAO), Viale delle Terme di Caracalla, 00153, Rome, Italy.
authors.forename: Gwenaelle
authors.initials: G
authors.lastname: Dauphin
authors.affiliation: Food and Agriculture Organization of the United Nations (FAO), Viale delle Terme di Caracalla, 00153, Rome, Italy.
authors.forename: Juan
authors.initials: J
authors.lastname: Lubroth
authors.affiliation: Food and Agriculture Organization of the United Nations (FAO) - Emergency Center of Transboundary Animal Diseases (ECTAD), 11 Al Eslah El Zerai St, P.O. Box, 2223, Giza, Egypt. Yilma.Makonnen@fao.org.
authors.forename: Yilma J
authors.initials: YJ
authors.lastname: Makonnen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001499:Bayes Theorem; D001665:Binding Sites; D001717:Birds; D016000:Cluster Analysis; D004534:Egypt / Q000453:epidemiology; D019143:Evolution, Molecular; D005838:Genotype; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification* / Q000235:genetics*; D005585:Influenza in Birds / Q000453:epidemiology* / Q000821:virology*; D009154:Mutation; D010802:Phylogeny; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011991:Receptors, Virus / Q000737:chemistry / Q000378:metabolism; D012641:Selection, Genetic
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011991:Receptors, Virus
keywords: Clade 2.2.1; Egypt; H5N1 HPAI; Phylodynamic; Poultry; Sequence and phylogenetic analysis; Virus evolution