| Avian influenza (AI) is a severe viral disease caused by influenza A virus (AIVs). H9N2 subtype avian influenza virus, was first isolated from turkey in North Americain 1966. Since then, it was out of control and soon became globally epidemic and caused a huge economic loss for the poultry industry. It is noteworthy that the H9N2 subtype avian influenza virus has been isolated from both poultries and mammalians, including humans and swines, and it also provides the internal genes to H5N1 avian influenza virus which transmitted to humans in Hongkong in 1997. Thus, it has been recognized as a potential threat causing new subtype avian influenza outbreak. We identified H9 viruses isolated from samples collected regularly in Yangzhou university veterinary hospital and live-poultry markets in Yangzhou from 2011 to 2014. We selected two strains of H9N2 subtype avian influenza virus in each year during 2011 to 2014 to investigate the antigenic and biological characteristicsof H9N2 AIVs.1. Isolation and identification of H9 subtype avian influenza virusWe isolated 37 H9 subtype avian influenza viruses from Veterinary Hospital of Yangzhou university and live-poultry markets from 2011 to 2014. two strains of H9 subtype avian influenza virus, which total were ten strains including Ck/GD/SS/94 and Ck/SH/F/98, were picked up in each year from 2011 to 2014, respectively, and the hyperimmune serum of which were made in SPF chicken. The EID50 and the HI cross-reactivity titer of ten H9N2 AIV strains were tested. The results showed that most of strains isolated in this study responsed well with the polyclonal antibody against Ck/GD/SS/94 and Ck/SH/F/98, indicating that no any antigenic variation were observed among isolated H9 viruses and Ck/GD/SS/94 or Ck/SH/F/98, except strains Ck/HB/11, Ck/JS/TM58/13 and Ck/JS/JT95/13, which HI titer against polyclonal antibody serum of Ck/GD/SS/94 was less than 8, and the HI titer of Ck/SD/C9QH/11 against polyclonal antibody serum of Ck/SH/F/98 was lower than 8 too, suggesting that the antigenic variation existed in Ck/HB/11, Ck/JS/TM58/13, Ck/JS/JT95/13 and Ck/GD/SS/94, and appeared between Ck/SD/C9QH/11 and Ck/SH/F/98.While the results of the HI cross-reactivity of H9N2 AIV strains showed that a lot of isolated strain displayed well response with the antibody serum of the viruse isolated in 2013-2014, and were less inhibited by the the serum of Ck/SD/C9QH/11 and Ck/JS/JT95/13 strains. Seven of viruses could replicate well in MDCK cells, while the replicated ability of Ck/JS/ZJ618/12, Ck/YZ640/12 and Ck/AH/WB/14 were weak, which mechanism are needed to further study.2. Sequencing and analysis of HA gene or whole genesThe HA gene of the 29 isolated strains and the whole genes of 8 isolated strains were sequenced and analyzed to study the genetic evolution of H9N2 subtype avian influenza virus in China. The results showed that the HA genes of the 29 isolates belonged to Beijing/94-like, most of the viruses isolated from Zhejiang, Shandong and Jiangsu provice belonged to the same branch. Hemagglutinin cleavage site of 335-338 amino acid sequence in 29 isolated strains were R-S-S-R, and were K-S-S-R in Ck/JS/YZ150/12, Ck/JS/YZ667/12, Ck/HLJ/DD/11, Ck/HB/11 and Ck/JS/JT12/11 strains, as the typical feature of low pathogenic avian influenza virus. There are 8 potential glycosylation sites in HA protein of most of 29 strains. Comparison to HA gene of Ck/SH/F/98 strain, there were T to I or V amino acids substitution in 220 site of isolated strains. The different amino acids appeared in 190 site ofof th HA gene in isolated strains and with Leu 234 amino acid residues, which is characteristics of Neu5Aca2-6Gal. The NA gene of 7 isolated strains belonged to the Beijing/94-like branch. But NA gene of recent isolates had 2 more potential glycosylation sites. NP, NS and PB1 genes of the isolated strains shared with Ck/SH/F/98-like branch; M genes of strains shared with Qa/HK/G1/97-like branch; PB2 and PA genes of the isolated strains belonged to G9/97-like branch. NP genes of CK/JS/TM58/13, CK/JS/JT95/13 and CK/JS/TM71/14 strains shared high homology with that of Ck/JS/SC537/13(H7N9), indicating that the newly isolated strain became the internal gene donor of H7N9 avian influenza virus, and esulted from the recombinant of G9-like, F/98-like and G1-like H9N2 avian influenza virus form chickens.3. Study on characteristics of pathogenicity and transmission of the isolated strainsTo study the pathogenicity and transmission of the isolated viruses, Ck/SD/C9QH/11, Ck/JS/JT12/11, Ck/JS/ZJ618/12, Ck/JS/YZ640/12, Ck/JS/TM58/13, Ck/JS/JT95/13, Ck/AH/WB/14, Ck/JS/TM71/14 were picked up for further research, and Ck/SH/F/98 was selected for control.120 3-week-old chickens were divided into 10 groups with 12 chickens in each group. One group was the blank control groupwithout infected any virus. In other each group,6 chickens were infected with the indicated virus as for challenged group,3 chickens lived the same cage with the challenged group, and 3 chickens put another cage with 50 cm from challenged group. After inoculation with viruses, the lung and spleen from chicken were taken at day 3 and 5 post-inoculation, half of which were processed to detect the replicated ability with SPF eggs, and another half were fixed in the 10% formalin to observe the pathological phenotype. Meanwhile, the trachea and cloacal swabs from each experiment group were collected at 3 and 5 days post-infection to test the transmission characteristics of the picked up viruses. The results showed that all selected strains possess the aerosol transmission ability, and have the stronger replicated ability in trachea than those in lung, while Ck/JS/ZJ618/12 replicated well in the trachea and lung of chickens. In consistence with replication characteristics, the trachea lesion caused by the virus isolated in 2013-2014 was more serious on day 3 than those on day 5 post-infection. In contrast, the lung pathological changes caused by the virus isolated in 2011-2012 were more serious on day 3 than those on day 5 post-infection. Conclusion, the aerosol transmission were observed in the viruses isolated in 2011-2014, and the stronger replicated ability and pathological lesion presented in chicken infected with the viruses isolated in 2013-2014, the detailed mechanism of which further need to be explored. |
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