Genome sequencing and phylogenetic analysis

Viral RNA was extracted from the allantoic fluid using TRIZOL Reagent (Invitrogen Carlsbad, CA, USA) and reverse transcribed using the primer 5′-AGCRAAAGCAGG-3′. The PCR products of eight fragments of the H7N1 virus were sequenced with a set of specific sequencing primers. The sequence data were compiled with the SEQMAN program (DNASTAR, Madison, WI). All reference sequences used in this study were obtained from the National Center for Biotechnology Information (NCBI) GenBank database and the Global Initiative on Sharing All Influenza Data (See Supplementary Table 1). DNASTAR's MegAlign was used to perform the sequence homology and key amino acid site analyses. MEGA 6.06 software was used to perform multiple sequence alignments with the Clustal W algorithm, and a phylogenetic tree was generated with the neighbor-joining method and bootstrap test (1000 replicates) based on the sequences for the open reading frames. Potential glycosylation sites were analyzed with the NetNGlyc 1.0 online software (www.cbs.dtu.dk/services/NetNGlyc/). GenBank accession numbers are {"type":"entrez-nucleotide","attrs":{"text":"JQ973643","term_id":"387965632","term_text":"JQ973643"}}JQ973643-{"type":"entrez-nucleotide","attrs":{"text":"JQ973650","term_id":"387965649","term_text":"JQ973650"}}JQ973650 for BP/HuN/414/10 and {"type":"entrez-nucleotide","attrs":{"text":"KU663402","term_id":"998455631","term_text":"KU663402"}}KU663402-{"type":"entrez-nucleotide","attrs":{"text":"KU663409","term_id":"998455648","term_text":"KU663409"}}KU663409 for CP/XH/420/10.