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The genotypes of the H9N2 avian influenza viruses have changed since 2013 when almost all H9N2 viruses circulating in chickens in China were genotype 57 (G57) with the fittest lineage of each gene. To characterize the H9N2 variant viruses from 2011 to 2014, 28 H9N2 influenza viruses were isolated from live poultry markets in China from 2011–2014 and were analyzed by genetic and biological characterization. Our findings showed that 16 residues that changed antigenicity, two potential N-linked glycosylation sites, and one amino acid in the receptor binding site of the HA protein changed significantly from 2011–2014. Moreover, the HA and NA genes in the phylogenetic tree were mainly clustered into two independent branches, A and B, based on the year of isolation. H9N2 virus internal genes were related to those from the human-infected avian influenza viruses H5N1, H7N9, and H10N8. In particular, the NS gene in the phylogenetic tree revealed genetic divergence of the virus gene into three branches labeled A, B, and C, which were related to the H9N2, H10N8, and H7N9 viruses, respectively. Additionally, the isolates also showed varying levels of infection and airborne transmission. These results indicated that the H9N2 virus had undergone an adaptive evolution and variation from 2011–2014.

BackgroundVaccines constitute a unique selective pressure, different from natural selection, drives the evolution of influenza virus. In this study, A/Chicken/Shanghai/F/1998 (H9N2) was continually passaged in specific pathogen-free embryonated chicken eggs with or without selective pressures from antibodies induced by homologous maternal antibodies. Genetic mutations, antigenic drift, replication, and pathogenicity of the passaged virus were evaluated.ResultsAntigenic drift of the passaged viruses occurred in the 47th generation (vF47) under selective pressure on antibodies and in the 52nd generation (nF52) without selective pressure from antibodies. Seven mutations were observed in the vF47 virus, with three in PB2 and four in HA, whereas 12 mutations occurred in the nF52 virus, with three in PB2, two in PB1, four in HA, one in NP, one in NA, and one in NS. Remarkably, the sequences of the HA segment from vF47 were 100% homologous with those of the nF52 virus. Both the vF47 and nF52 viruses showed enhanced replication compared to the parental virus F/98, but higher levels of replication and pathogenicity were displayed by nF52 than by vF47. An inactive vaccine derived from the parental virus F/98 did not confer protection against challenges by either the vF47 or nF52 virus, but inactive vaccines derived from the vF47 or nF52 virus were able to provide protection against a challenge using F/98.ConclusionTaken together, the passage of H9N2 viruses with or without selective pressure of the antibodies induced by homologous maternal antibodies showed genetic variation, enhanced replication, and variant antigenicity. Selective pressure of the antibody does not seem to play a key role in antigenic drift in the egg model but may impact the genetic variation and replication ability of H9N2 viruses. These results improve understanding of the evolution of the H9N2 influenza virus and may aid in selecting appropriate vaccine seeds.

Cadherin-17 (CDH17) belongs to the cell adhesion cadherin family with a prominent role in tumorigenesis. It is highly expressed in human hepatocellular carcinoma (HCC) and is proposed to be a biomarker and therapeutic molecule for liver malignancy. The present study aims to identify the transcription factors which interact and regulate CDH17 promoter activity that might contribute to the up-regulation of CDH17 gene in human HCC. A 1-kb upstream sequence of CDH17 gene was cloned and the promoter activity was studied by luciferase reporter assay. By bioinformatics analysis, deletion and mutation assays, and chromatin immunoprecipitation studies, we identified hepatic nuclear factor 1α (HNF1α) and caudal-related homeobox 2 (CDX2) binding sites at the proximal promoter region which modulate the CDH17 promoter activities in two HCC cell lines (Hep3B and MHCC97L). A consistent down-regulation of CDH17 and the two transcriptional activators (HNF1α and CDX2) expression was found in the liver of mouse during development, as well as in human liver cancer cells with less metastatic potential. Suppression of HNF1α and CDX2 expression by small interfering RNA (siRNA) significantly down-regulated expressions of CDH17 and its downstream target cyclin D1 and the viability of HCC cells in vitro. In summary, we identified the minimal promoter region of CDH17 that is regulated by HNF1α and CDX2 transcriptional factors. The present findings enhance our understanding on the regulatory mechanisms of CDH17 oncogene in HCC, and may shed new insights into targeting CDH17 expression as potential therapeutic intervention for cancer treatment.

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