| Avian influenza is an infectious disease caused by influenza A virus, which normally lead to serious disease and mortality in domestic poultry industry. Since outbreak of H5N1 highly pathogenicity avian influenza (HPAI) at HongKong in 1997, it has been spreading in more than 60 countries of the world, resulted in lots of poultries and birds dead or killed, causing huge economic losses. In public health, by February 22, 2008, it had caused 366 people infected, of whom 232 people were died, and the mortality rate was as high as 60%. At the same time, the phenomenon of AIV spanning interspecies barriers had been reported unceasingly. It was reported that tigers, lions, cats, American panthers, Martens, dogs and other mammals could be infected by AIV. Because the range of host infected by AIV is expanding further, people worried more about: lion, tiger, dog and other mammals may become the new hosts of AIV, and AIV may be mutated in these animals thus leading to the capability of virus horizontal transmitted among mammals and infected people gradually strengthen, and obtained the capability to trigger a pandemic of influenza at last. Therefore, it is important to carry out the epidemiological investigation of AIV, and prevent and control the occurrence of this possible, potential threaten. We found H5N1 AIV can infect fox and cause death in 2007, and isolated 4 strains from the dead foxes. This is the first time to find that the H5N1 AIV can infect canine fox naturally and lead to its invasion and death in China. In order to find out the information of the H5N1 AIV infected fox, specific RT-PCR method of H5N1 AIV was used to detect lung tissue samples of farmed fox which showed pneumonia, high fever and other symptoms collected in Liaoning Province, 2007. MDCK cells were used to isolate 4 AIV from positive samples of RT-PCR. Identified by morphological and subtypes that they are H5N1 AIV. To determine the pathogenicity of these fox source of H5N1 AIV to cells and animals, the isolated viruses were used in cells and animals infected experiment. Fox source of H5N1 AIV were inoculated to Vero, F81, BHK-21, and other passage of renal cells lines came from monkeys, cats and hamsters. The results showed that fox source of H5N1 AIV can infect all of these passage of renal cell line; fox source of H5N1 AIV were inoculated MDCK cells, SPF chick embryo and mice, and the results showed that: TCID50 was between 10-6.8～10-7.4/50μl, EID50 was between 10-6.9～10-7.5/50μl, MLD50 was between 10-6.5～10-7.1/50μl.On the basis of virus isolation, in order to study the gene sources, genetic characteristics, evolution status and the molecular basis of infecting fox of the fox source of H5N1 AIV, of HA, NA gene were used with the virus isolated. Results of nucleotide sequencing analysis showed that the HA, NA gene of virus had highly homologous with the H5N1 AIV isolated from 1996 to 2007, and the homology was between 96% and 100%, which showed that the genes from fox source of the H5N1 AIV came from H5N1 AIV. Multiple sequence alignment of DNA Star program was used to analyze genetic characteristics of fox source of the H5N1 AIV and found that the basic split site in the HA protein of the virus isolated was composed with five basic amino acids (RRRKR), which fit the molecular characteristics of the H5N1 HPAIV. The amino acids of receptor binding sites are 222 Q and 224G, which showed that the fox source of H5N1 AIV can only combine with SAα2, 3 Gа1 receptor. On the basis of genetic evolution analysis, we carry out the animal infection empirical study. To identify the pathogenicity of the virus to the fox further, this fox source of H5N1 AIV (A/fox/LN/04/2007) was used in a fox artificial infection test. All the fox infected showed depressed spirit, lower appetite, fervescence, dyspneic respiration, and other symptoms; five foxes out of six in infected group were killed. Autopsy found that lung surface of infected fox had a large madder red consol focus; inoculated virus can be isolated from the lung, trachea, faucial tonsil and kidney tissue of infected fox, among which the highest content of virus is in lung, TCID50 is 10-3.9～10-5.8/50μl; with the pathohistologic light microscope observation, we can see diffuse alveolar damage, more macrophages infiltration in alveolar space and small amounts of protein-like serofluid exudation; with the immunohistochemistry, the positive antigen staining particles of the virus can be seen in bronchial epithelial cells and endochylema of minority alveolar macrophages.
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