Computational protocol: Molecular Characterization of Subtype H11N9 Avian Influenza Virus Isolated from Shorebirds in Brazil

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Protocol publication

[…] The study was approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of the University of São Paulo (CEUA-USP 1752/2009), by the Brazilian Society of Laboratory Animal Science (Permit Number: 105, page 74, book 2) and by the Sistema de Autorização e Informação em Biodiversidade of the Instituto Chico Mendes de Conservação da Biodiversidade, Ministério do Meio Ambiente (SISBIO 21211–1, 25895–1).Three AIV isolates from shorebirds in Northern Brazil were studied: A/ruddy turnstone/Ilha de Canelas/A008/2008(H11N9), A/ruddy turnstone/Ilha de Canelas/A017/2008(H11N9), and A/ruddy turnstone/Ilha de Canelas/A051/2008(H11N9). These isolates had been obtained from orotracheal and cloacal swabs of ruddy turnstones (Arenaria interpres) captured at Canelas Island (Ilha de Canelas; 0°47’7”S 46°43’23”W), within a region known as “Reentrâncias Paraenses”. These were the only AIV isolates obtained in a previous study [] in which orotracheal and cloacal swabs from 556 aquatic birds captured at coastal areas of the Brazilian Amazon were tested for AIV by real-time RT-PCR, and virus isolation in embryonated eggs was attempted for PCR-positive samples.Viral RNA was extracted from allantoic fluid with the MagMAX AI/NDV RNA extraction kit. All 8 RNA gene segments were reverse transcribed using SuperScript®III (Invitrogen, Life Technologies) following the recommended protocol. Following the reverse transcription all 8 gene segments were amplified using universal primers () and Phusion polymerase (Thermo Fisher Scientific Products). The samples were then prepared for sequencing using the Nextera preparation kit (Illumina, Nextera XT DNA Library Preparation Guide, document # 15031942 Rev. E). Briefly, the amplified influenza gene segments were tagmented into about 300–500 base pair fragments which ligates the required Illumina adaptors. The tagmented fragments are then barcoded and purified using AMPure magnetic beads (Beckman Coulter). The enriched samples were then pooled and installed on the MiSeq instrument for cluster generation and sequencing. The resultant sequences were analyzed using CLC Genomics Workbench (Qiagen). The sequences of the segments were deposited on GenBank (accession numbers: KF824501-KF824506, KT932362-KT932379).Influenza gene sequences were obtained from the Influenza Research Database website (IRD— as available in 23 Sep 2015. All complete sequences of H11 and N9 were obtained; for the remaining segments, search criteria were selected to obtain complete segment sequences for the following groups: (1) avian strains worldwide, (2) H11N9 strains worldwide, (3) all strains from South America, (4) all strains from Antarctica, (5) avian strains from South America. Lineages that were incomplete or contained ambiguous nucleotide codes were excluded from the analysis; lineages retrieved from bats were also excluded. Sequences were aligned using the MUSCLE algorithm [], and trimmed to the coding DNA sequences (CDS) using MEGA 6.06c []. Representative centroids were obtained using USEARCH 8.1.1756 [], with varying levels of sequence identity (92–99%) being used to select approximately 350, 100 and 50 most representative centroids for sequence groups 1, 2 and 3, respectively. These representative centroids, along with the sequences from groups 4 and 5 and those obtained in this study, were used to construct maximum-likelihood trees for each of the viral genes using the GTR+G+I model (selected using jModelTest 2 []) as implemented in MEGA 6.06c, with 500 bootstrap replications.MegaBLAST [] was used to select the 50 GenBank sequences with highest identity to the CDS-trimmed sequences obtained in this study; this was done for each gene segment, resulting in a list of 400 high-identity sequences. This list was then used to determine high-identity sequence density for each state or province, i.e. the percentage of these 400 high-identity sequences that was recorded at that state or province. The migratory flyways [, ] and natural distribution of ruddy turnstones [] were also represented. […]

Pipeline specifications

Software tools CLC Genomics Workbench, MUSCLE, MEGA, USEARCH, jModelTest, BLASTN
Databases IRD
Applications Phylogenetics, WGS analysis, Nucleotide sequence alignment
Organisms Viruses, Human poliovirus 1 Mahoney, Arenaria interpres, Anas platyrhynchos