Computational protocol: Reassortant Highly Pathogenic Influenza A H5N2 Virus Containing Gene Segments Related to Eurasian H5N8 in British Columbia, Canada, 2014

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Protocol publication

[…] RNA was extracted from swab specimens or tissue homogenates using a MagMAX™-96 AI/ND Viral Extraction Kit (Ambion) and a MagMAX Express 96 particle processor. The 8 influenza A virus gene segments were amplified in a one-step RT-PCR using a universal primer set and a high-fidelity RT-PCR kit (Invitrogen; Superscript III One-Step RT-PCR System with Platinum TaqHigh Fidelity). RT-PCR amplicons were cloned into pCR4-Topo (Invitrogen) and used to transform OneShot TOP10 or Mach™-T competent E. coli. The resulting plasmids were sequenced using BigDye Terminator chemistry version 3.1 (Life Technologies) and an Applied Biosystems 3130xl Genetic Analyzer. A consensus sequence for each gene segment was arrived at based on the results of bidirectional sequencing of a minimum of 4 clones. Representative reference sequences with Eurasian and North American geographical associations were selected from NCBI and GISAID databases and aligned with the HA of the British Columbia H5N2 virus isolates using Clustal W. Phylogenetic analysis was conducted in MEGA6 using the maximum likelihood method based on the Tamura-Nei model. The initial trees for the heuristic search were obtained by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach. Five-hundred bootstrap replicates were used to determine the reliability of the inferred tree.All animal experiments were performed in strict accordance with the recommendations in the Canadian code of practice for the care and use of animals and was approved by the National Centre for Foreign Animal Disease (NCFAD) Animal Care Committee.The intravenous pathogenicity indices (IVPI) of the virus isolates were determined as described in the World Organization for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Briefly, a total of 10 specific pathogen free (SPF), 4- to 6-week-old White Leghorn chickens (Gallus gallus domesticus) were inoculated intravenously with 0.1 ml of each virus stock that had been diluted 1:10 in sterile PBS. The hemagglutination (HA) titer of the H5N2 broiler chicken isolate used in the IVPI was 1024 while the HA titer of the H5N2 turkey isolate was 32. Ten control birds were inoculated with 0.1 ml of PBS by the intravenous route. Birds were observed daily for a total of 10 days and given a clinical score at the end of each 24 hr period. The IVPI is the mean score per bird per observation period over the 10 day period. A maximum score of 3.00 means that all birds died by 24 hours post-inoculation. Viruses with IVPI > 1.2 are considered highly pathogenic. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Gallus gallus, Meleagris gallopavo