| In recent years, avian influenza outbreak all over the world caused by a variety of different subtypes of avian influenza viruses have occurred continuously worldwide in poultry and wild birds. And through the natural reassortment and mutation, some viruses have obtained the capabilities of crossing the species barrier to infect mammals and human, which often result in mammals and human disease and even death. Therefore, avian influenza has become today’s one of the most serious public health problems, which has a direct impact or threat on animal and human health. Wild birds, as a natural reservoir of avian influenza virus, play an important role in virus persistence, variation, transmission and spread. Therefore, for the early warning prevention and control of avian influenza, it is of great significance to strengthen the active surveillance and research of wild bird influenza viruses, and fully understand their ecological distribution, epidemiological features, and genetic evolution rules.In this study, epidemiological surveillance of avian influenza viruses in wild birds in Anhui province in the spring and winter seasons of 2014-2015 was performed by virus isolation and identification and analysis of genetic evolution, for understanding the situation of the avian influenza virus infection in wild birds. The potential threat of some of these wild bird influenza virus strains to human and mammals were assessed by experimental infection in mice and cells. Finally, the differential expression of the cytokine storm factors on CEF and DEF induced by the representative strains of these viruses was preliminarily analyzed for predicting the possible outcome of the innate immune in chicken and duck.The main research contents and results are as follows:First:Epidemiological surveillance of AIVs in wild birds in Anhui province from 2014 to 2015The whole genomes of 38 strains of avian influenza virus were sequenced and analyzed. All of the 38 strains belong to the low pathogenic avian influenza virus determined by cleaved site of amino acids in HA genes. Although the HA genes of two H9N2 strains isolated in 2015 displayed characteristics of the low pathogenic avian influenza virus, the 627 site animo acid in PB2 protein is K, which is a signal of enhanced pathogenicity to mammals, indicating that the influenza viruses in wild birds evolve continuously, which with the trend towards enhancing the virulence and having the potential threats to the mammals and human. The results of comparative analysis showed that there existed complicated reassortment or rearrangement in the internal genes which could come from the viruses of different subtypes. Especially, some strains were reassortant viruses between North America and Eurasian, their N2 gene, PA and NS genes were derived from the North American branches. Wild birds play an important role in the spread and restructuring of avian influenza virus, due to they can carry the low pathogenic avian influenza viruses along with their long distant migration.Second:Pathogenic assessment of the representative AIV strainsTo understand the potential threat to mammals of these wild birds influenza virus, the Babl/c mice, as a mammalian model, were infected with the influenza virus. The result showed that most of the strains from wild birds had the capability to infect mice, and the infected mice experienced different degrees of weight loss but not associated with other clinical symptoms. Virus replication was only confined to the respiratory organs in mice (turbinate and lungs), and different levels of replication could be found in different subtypes or strains. The result in this study showed that avian influenza viruses originated from wild birds had no pathogenicity in mice, but they could directly infect mice without adaptive process in advance, and may have potential threat to mammals. In view of these wild bird influenza viruses having presented good replication in mice, two H6 subtype strains (one H6N1 and one H6N2) were chosen to perform an experimental infection in human alveolar epithelial cancer cells, which as target cells, for evaluating the replication capability of these viruses in the human respiratory tract and further inferring the threat to human beings. The result showed that both of these two strains could replicate well on A549 cells, which indicated that these H6 subtype wild bird viruses may have a certain hazardous risk to human.Third:Comparison and assessment of differential expression of cytokine storm factors in different cells induced by the representative AIVsChicken and duck are the two main reservoirs of influenza virus and also are the main livestock products of human consumption. However, the two hosts have obvious difference in their resistance to the influenza virus. The highly pathogenic avian influenza virus infection often leads to obvious symptoms with high mortality in chickens. Ducks infected with highly pathogenic influenza virus can sometimes present obvious clinical symptoms either, but the mortality rate is very low. and ducks infected with low pathogenic influenza virus do not have any clinical symptoms. So it is believed that duck has much higher tolerance to influenza viruses than chicken. In this study, chicken embryo fibroblast cells and duck embryo tibroblast cells, which were used as the target cells, were infected with four stains of H6 virus which could replicate well the in respiratory organs of mice. Then we compared and analyzed the expression levels of innate immune storm factors (inflammatory factor, anti-inflammatory factor TLR.7, antigen recognition receptors and antiviral factor) in these two cells and gave a tentative evaluation for the difference of body innate immune response in chicken and duck if infected by wild bird avian influenza viruses. The results showed that the innate immune response could be induced in both chicken and duck cells, but the response in chicken cells were stronger than in duck cells, especially high level expression of IFN-α, the antiviral factor, in chicken cells. Compared with in chicken cells, most of the innate immune factor expressions in duck cells were regulated negatively. These results coincide with the ideas that ducks often have the longer incubation period and the lower viral titer after AIV infection. |
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