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Molecular Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from Raccoon Dogs in China

Molecular Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from Raccoon Dogs in China,10.1371/journal.pone.0004682.t002,P

Molecular Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from Raccoon Dogs in China   (Citations: 9)
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Journal: PLOS One , vol. 4, no. 3, 2009
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    • ...ResultsComparison of 2009 H1N1 HA Sequences Isolated from China, Mexico, and the USAWe compared 23 H1N1 HA gene sequences isolated from humans in China with 11 from Mexico and 13 from the USA in 2009, and analyzed them by using DNASTAR software. The most common mutation sites and their frequencies in HA of H1N1 isolated in China are listed in table 1. HA protein had 3 RB sites (135–138 VTAA, 190–198 DQQSLYQNA, and 221–228 PKVRDQEG), 1 cleavage site (325–336, PSIQSRGLFGAI) and 8 glycosylation sites (13N, 14NST, 26NVTA, 90NGTC, 279NTTC, 290NTSL, 484NGTY, and 543NGSL). Amino acid substitutions of all Chinese strains were extremely rare in the 3 RB sites except GQ351314-China-Hongkong-2009-HA showed D?E at aa 225. Similarly, only 1 strain (GQ250161-Guangzhou-2009-HA) had a Q?L mutation at aa 328 observed in cleavage site. Sequences were conserved in all other strains, and no changes were found in any potential glycosylation sites (fig. 1).Phylogenetic tree analysis revealed that the HA genes from China, Mexico and the USA constituted the same branch. The evolutionary distances were small. No significant accumulation of geographical characteristics was observed (fig. 2).Molecular Evolution of the H1N1 HA Gene Isolated from Humans in China from 1947 to 200950 H1N1 HA gene sequences that were isolated from humans in China from 1947 to 2009 were downloaded from GenBank. Phylogenetic tree analysis revealed that the sequences clustered on the basis of their year of isolation. The sequences isolated in 2009 were only 70% similar to those isolated from 1947 to 2008 and constituted a separate branch (fig. 3).H1N1 HA mutations from 1947 to 2009 are listed in table 2. H1N1 HA has 8 glycosylation sites. Of these, only 2 sites (aa 279 and aa 290) underwent considerable changes; the other 6 glycosylation sites remained stable and conserved over the past ~60 years.During the past 60 years, the RB sites had a lot of changes. For example, the sequences of 135–138 cycle were VTAA, VTAS, VSAS, and VTAA in 1947, 1977–1996, 1999–2008, and 2009, respectively. But the most evident changes were seen in the 190–198 cycle from 1947 to 2009. Notably, there were some amino acid substitutions in the 221–228 cycle before 1999, but no mutations were observed thereafter.The cleavage site between HA1 and HA2 and the amino acid residues near it were directly associated with H1N1 pathogenicity. We observed that mutations occurred at only 2 sites, i.e. 326S?F and 331G?S in 1947 and 2006, respectively. The amino acids at these sites remained stable in other years. These data revealed that there were no highly pathogenic sequences (RXR/KRGLF). Thus, H1N1 viruses isolated in China have had low pathogenicity over the past ~60 years.Comparison of H1N1 HA Gene Sequences Isolated from Humans, Swine, and Birds in ChinaWe analyzed 32 H1N1 HA gene sequences (isolated from 6 human, 22 swine, and 4 avian) and compared their RB, glycosylation, and cleavage sites. The sequences in the first cycle (aa 135–138) in the RB sites were VTAA, VTAA/VSAS/TTVA/VTSA, and VTAA/VSSA/VTAS in humans, swine, and birds, respectively. The sequences in the second cycle (aa 190–198) were DQQSLYQNA, DQQSLYQNA/EQRAIYHTE/DQQTLYQNN/EQQTLYQNT/DQQSLYQNL and EQQSLYQNA/DQRAIYHTE/EQTRLYQNP in humans, swine, and birds, respectively. In the third cycle (aa 221–228) in the RB sites, only sequences at aa 225 and aa 227 changed. At aa 225, the sequences were D, G/E/N/D, and G in humans, swine, and birds, respectively; at aa 227, they were E, A/T/E, and A/E/S in humans, swine, and birds, respectively. At aa 226 and aa 228, the amino acids were Q and G, respectively, in all 3 species. The glycosylation site at aa 279 was absent in the HA of some of the swine and avian H1N1 isolates. The glycosylation site at aa 290 was absent only in the HA of swine H1N1 isolates; this site was present in human and avian H1N1 isolates. The other glycosylation and cleavage sites remained stable in the 3 species, with the exception of isolate EU874899 (fig. 4).Analysis of the phylogenetic tree of the H1N1 HA isolated from humans, swine, and birds indicated that the human strains aggregated together and differed completely from the swine and avian strains. There were 3 branches of swine strains, and the avian strains were closer to one of these branches (fig. 5).Prediction of Three-Dimensional Structure of H1N1 HA of Wild and Mutated StrainsThe most common mutation site of HA of H1N1 isolated in China in 2009 was aa 206 (S?T). Here, we compared the three-dimensional structure of a wild-type strain (GQ183617) and a mutant strain (GQ225365, with aa 206 S?T) isolated from humans. These 2 strains had the same structure in HA1 and HA2. This result indicated that this single mutation (aa 206 S?T) did not change the spiral structures and the pathogenicity of the virus. In contrast, when the mutation occurred in the cleavage sites, the 2 sequences (the strain with mutated cleavage sites, EU874899 and GQ225365 with no mutation in cleavage sites) had clearly differences in their structures. HA2 of the mutant had more a-helices (blue spiral structures) than the wild-type strain. The most evident differences were seen at and around the glycosylation site at aa 484. However, there were no differences near the 3 RB sites between the 2 strains (fig. 6).DiscussionThe H1N1 influenza genome has 8 gene segments, namely PB1, PB2, PA, HA, NP, NA, M, and NS, which encode at least ten kinds of protein antigens. American experts have speculated that the novel virus responsible for the 2009 influenza pandemic has 6 gene fragments reassorted from swine, avian, and human gene fragments [,...

    She-Lan Liuet al. Evolutionary Characterization of Human H1N1 Influenza Virus Hemaggluti...

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