Computational protocol: Influenza A Viruses from Wild Birds in Guatemala Belong to the North American Lineage

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Protocol publication

[…] For viral isolation the swab supernatant of each specimen was filtered and BHI (brain heart infusion media) supplemented with antibiotics and antimycotics was added to a volume of 1.5 mL. A 200 µL volume of this mixture was then inoculated into the allantoic cavity of three, 9-day-old SPF embryonated chicken eggs per sample. Following incubation at 37°C for 72 hours, allantoic fluid was collected and tested for the presence of virus by HA assay and by Flu Detect (Synbiotics, San Diego, CA). After three blind passages in embryonated chicken eggs, samples without virus growth were considered negative for the presence of viable virus.Viral RNA was extracted from 200 µL of allantoic fluid with RNeasy Mini kit (Qiagen), according to manufacturer instructions. Extracted RNA was eluted in 40 µL of RNase-free water. After cDNA preparation, full-length PCR amplification of the influenza virus segments was performed followed by direct sequencing with the BigDye terminator kit (Applied Biosystems) on ABI 3100 Avant Genetic Analyzer (Applied Biosystems) or ABI 3500 Genetic Analyzer . Segments that could not be sequenced from PCR products were cloned into pCR 2.1 vector using a TA cloning kit (Invitrogen) and were sequenced using vector based M13 primers. At least two sequencing reactions were prepared for each gene. Partial and full-length sequences were acquired from overlapping partial sequences obtained with forward and reverse primers. Nucleotide sequences are assigned Genbank accession numbers CY067667 to CY067682 and CY096621 to CY096724. RNA extracts from original swab samples negative for virus isolation, were subjected to direct sequencing by the same method described above, in attempt to identify other virus subtypes.For each genome segment of each virus isolate, BLAST searches at the nucleotide level were initially performed to identify the most closely related viruses. Full-length genome segments from North American and selected Eurasian and South American viruses available at the Influenza Research database ( were then obtained to be included in the phylogenetic analysis. Sequences of each segment were initially aligned by Clustal V (Lasergene v.8.1.5., DNAStar, Madison, WI) and percent identities were calculated. Sequences from representative isolates were selected and aligned with ClustalW (Lasergene). Rooted phylogenetic trees were generated by Neighbor-Joining method with 1000 bootstrap replicates using PAUP 4.0b10 (Sinauer Associates, Inc., Sunderland, MA). […]

Pipeline specifications

Software tools Clustal W, PAUP*
Databases IRD
Application Phylogenetics
Organisms Anas platyrhynchos