All experiments were repeated 2-6 times with duplicate or triplicate samples for each condition unless indicated otherwise. A representative result of multiple independent experiments is present (n = 2-6) in each figure. Error bars shown in all figures represent standard deviation of the mean (n = 2-4). Dotted lines show detection limit except otherwise specified. Reporting standards: We followed the reporting standards for the Transcriptome Shotgun Assembly Sequence Database of NCBI, which are: Submitted sequences must be assembled from data experimentally determined by the submitter. Screened for vector contamination and any vector/linker sequence removed. This includes the removal of NextGen sequencing primers. Sequences cannot be less than 200 bp. Sequences should have no more than 10% n's or greater than 14 n's in a row. If the submission is a single-step, unannotated assembly and the output is a BAM file(s) these should be submitted as a TSA project to SRA. The Evolutionary Pattern of Glycosylation Sites in Influenza Virus (H5N1) Hemagglutinin and Neuraminidase Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of influenza viruses play crucial roles in transfaunation, membrane fusion and the release of progeny virions. To explore the distribution of N-glycosylation sites (glycosites) in these two glycoproteins, we collected and aligned the amino acid sequences of all the HA and NA subtypes. Two glycosites were located at HA0 cleavage sites and fusion peptides and were strikingly conserved in all HA subtypes, while the remaining glycosites were unique to their subtypes. Two to four conserved glycosites were found in the stalk domain of NA, but these are affected by the deletion of specific stalk domain sequences. Another highly conserved glycosite appeared at the top center of tetrameric global domain, while the others glycosites were distributed around the global domain. Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, and further focus on the H5N1 virus and conclude that the glycosites in H5N1 have become more complicated in HA and less influential in NA in the last five years. Influenza A viruses (IVs), which belong to the orthomyxoviridae family, consist of eight negative RNA strands. Hemagglutinin (HA) and neuraminidase (NA) are two glycoproteins that are encoded by the IV genome, expressed from segments 4 and 6, respectively. The selection due to various host immune systems and anti-flu drugs accelerate the mutation rates of viral proteins, especially for these two membrane proteins [1, 2] . There are 17 HA subtypes and 10 NA subtypes, designated H1-H17 and N1-N10, respectively. Over 118 combinations of IVs can be isolated from wild birds, which are also the natural reservoir of these viruses (except the H17N10 virus, which, until recently, was isolated only from bat) [3] [4] [5] . The species jumping ability of IVs can result in the infections of poultry and mammals, such as chicken, swine, equine or whale species, with different virulence levels [6] [7] [8] [9] . The H1N1, H2N2 and H3N2 viruses have been responsible for tens of millions deaths during the deadly history of human influenza epidemics. Furthermore, the H5N1, H7N7, H7N2, H7N3 and H9N2 viruses have been isolated from sporadic human infections and deaths [10] [11] [12] [13] . It is worth noting that the H5N1 virus is the most severe for human and avian species, with sudden onset and high mortality. The mortality rate in hundreds of patients who were hospitalized for H5N1 infections was roughly 59.05%, much higher than the mortality rates of the Spanish Flu or the 2009 influenza pandemic (H1N1) [11, 14, 15] .