Computational protocol: Eco-Virological Approach for Assessing the Role of Wild Birds in the Spread of Avian Influenza H5N1 along the Central Asian Flyway

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Protocol publication

[…] Phylogeographic mapping of HPAI H5N1 viruses that overlapped with the Central Asian Flyway was performed to qualitatively assess the trajectory of the virus compared to the northward migration of bar-headed geese and ruddy shelduck. We chose to include sequences from poultry and wild birds as transmission of the virus is thought to involve exchange between the two avian populations. Hemagglutinin (HA) nucleotide sequences for low and highly pathogenic H5N1 influenza were obtained from the Genbank database hosted by the National Institutes of Health ( and the Global Initiative on Sharing of All Influenza Data ( Metadata associated with each sequence provided the geographic location for the outbreak. Where possible we attempted to classify each site at the provincial or state level. We aligned H5N1 (HA) nucleotide data using the software package ClustalW-MPI . Sequence ends were trimmed to the reading frame. We removed sequences that broke the protein reading frame. The final alignment consisted of 3304 sequences and 1776 aligned positions. A phylogeny was obtained via tree search of 100 replicates using the maximum likelihood (ML) criterion implemented in RAxML under the GAMMAI model of nucleotide substitution . The tree was rooted to A/goose/Guangdong/1/96 and included sequences from January 1959 (A/chicken/Scotland/1959) to June 2010 (A/Hubei/1/2010). We used character optimization in Tree Analysis Using New Technology (or ‘TNT’) to identify clades of HPAI H5N1 that circulate in the Central Asian Flyway. Sequences within each clade were realigned and new proximal outgroups were chosen. Transmission events were calculated using a modified version of the method developed by Slatkin and Maddison . To find transmission events, different geographical regions were treated as character states of a single multistate character, and were mapped onto trees obtained from phylogenetic analyses using standard optimization methods. To assess directionality of strain evolution, the change command in TNT was used to count the minimum and maximum number of transmission events for pairs of geographical character states for all global parsimonious reconstructions of the multistate character. When the number of state changes for a geographical state pair was >0, this result was interpreted as a possible transmission route from one region to another. Patterns of viral evolution were not assessed by migration season owing to the small number of sequences available for wild birds in the Central Asian Flyway. We visualized these transmission routes and their polarity with ROUTEMAP, . […]

Pipeline specifications

Software tools Clustal W, RAxML, TNT
Application Phylogenetics
Organisms Anser indicus, Tadorna ferruginea, Tadorna tadorna, Human poliovirus 1 Mahoney
Diseases Influenza in Birds, Infection