Studies On The Biological Characteristics Of AIV H9N2 Subtype From Ma Ya

Avian influenza, a highly infectious disease in the poultry, is caused by influenza virus type A, which belongs to the influenza virus genus, Orthomyxoviridae. As one of the member of the influenza viruses type A, the H9N2 subtype avian influenza virus is lowly pathogenic, rarely caused overt diseases in birds including ducks. However, it could cause worldwide pandemic in the poultry, and even infect the human. It is a remarkable fact that the waterfowls, especially the ducks, are the repository of the influenza viruses, helping the occurrence and spread of avain influenza. In 2007, a severe viral disease in a Ma Ya farm manifested clinical symptoms with heavy egg drop and large quantity low-quality eggs (e.g., soft-shelled eggs, shell eggs and abnormal eggs, etc). We collected samples, a H9N2 subtype avian influenza virus has been isolated and identified, designated as A/Duck/Fujian/FQ107/2007 (H9N2) (Dk/FQ107/07 for short). In order to comprehended and master the biological characteristics and the rules of homology and heredity evolution of H9N2 subtype avian influenza virus in ducks, the pathogenic, antigenic drifted and the rules of molecular genetic of the Dk/FQ107/07 strain were studied in the experiment, the theoretical and the experimental basis for prevention of H9N2 subtype avian influenza.The 50% embryo infective dose (EID₅₀), mean dead time (MDT) and intravenous pathogenicity index (IVPI) have been conducted in order to demonstrate the pathogenicity of Dk/FQ107/07 strain. The results of 10-4.32/0.1 mL for EID₅₀, 129.6 h for MDT, and 0.04 for IVPI, indicated that the H9N2 subtype avian influenza virus Dk/FQ107/07 was of low pathogenic. Allantoic fluid containing 10^5.02EID₅₀ 10^4.8_EID₅₀and 10^4.02EID₅₀ virus was inoculated into the 26-day-age chickens, 4-day-age Cherry valley ducks and 15-day-age BALB/c mice, respectively. At 21 days post-infection, the number of infected animals was calculated with signs and lesions indicative of infection observed. And the serum and tissues were collected for hemagglutination inhibition(HI) and detection by RT-PCR separately. After inculation, all of the animals were alive with good spirits, normal appetite and no obvious symptoms. The result of HI test showed that the chickens had a good immunogenic response to Dk/FQ107/07 strain at 7 days, 14 days and 21 days post-infection, while the Cherry valley ducks had less effect. The tissues of lung, liver, spleen, kidney and brain, collected from the infected BALB/c mice at 3-day-, 5-day-, 7-day- and 9-day-challenge, were detected by RT-PCR. The results showed that the only lungs collected on 3-days and 5-days after inoculation could be detected. The antibodies was first detected on 7-days after inoculation and stabilized on14-days and 21-days.The antigenic relativity between the Dk/FQ107/07 and two older isolates in Mainland China (A/Chicken/Guandong/FS/94 (H9N2) and A/Chicken/Beijing/1/94 (H9N2)) determined using cross hemagglutination inhibition. Beyond our expection, there were not obvious antigenic drifted with the variation ratio of 0.71.The complete genome of the Dk/FQ107/07 strain were amplified by RT-PCRs, and then cloned and sequenced. The results showed that HA gene was length 1 721 nt in size, containing a complete open read frame (ORF) which encoded a peptide of 560 aa. Amino acid sequence analysis showed that there were 7 potential glycosylation sites, 5 (at aa29, 83, 219, 299 and 306) of them located in the HA1 subunit, and the other 2 (at aa493 and 552) in the HA2 molecule. The cleavage site was of -PSKSSR↓GLF-, alongside a Gln at position of aa223 in HA protein, consisting with the characteristic of LPAIV. The HA gene had the highest similarity up to 98.9% with A/chicken/Shantou/5269/2005(H9N2), and had a close relationship with the first China's mammal animal-origin influenza isolate, named as A/Swine/HongKong/9/98(H9N2). All of them belongs to the H9N2/Y280 sublineage.The NA gene of Dk/FQ107/07 strain has a length of 1 428 nt, containing an ORF encoding 466 amino acids. Amino acid sequence analysis showed that there were 5 potential glycosylation sites (aa position of 10, 81, 160, 271 and 323). When compared with the first H9N2 subtype AIV strain in mainland, A/Chicken/Beijing/1/94(H9N2), the Dk/FQ107/07 strain losed T, E and I at 63, 64 and 65 sites, respectively, induces the 61site of glycosylation is lost. The NA gene had the highest similarity up to 99.5% with A/chicken/Shandong/7/96(H9N2), and belongs to the H9N2/Y280 sublineage.The NP gene was 1 565 nt in length, and ORF encoded 498 aa. The homology analysis of the NP gene revealed that Dk/FQ107/07 stain had the highest similarity up to 98.5% with A/chicken/Guangxi/55/2005(H9N2), and were in the same envolutionary branch with A/Duck/Fujian/1734/05(H5N1) and A/Duck/Fujian/9713/2005(H5N1).The M gene was 1 027 nt in length, encoding 348 aa containing a complete M1 and M2 protein. The NS fragment was 890 nt in size, and ORF encoded 337 aa, had a complete NS1 and NS2 protein. The phylogenetic analysis of M gene and NS gene revealed that they had the highest similarity up to 99.3% and 99.2% separately with A/chicken/Shandong/7/96(H9N2), and belongs to the H9N2/Y280 sublineage. The PB2 gene was 2 341 nt in length, and ORF encoded 759 aa. The phylogenetic analysis of PB2 gene revealed that they had the highest similarity up to 95.7% separately with A/Duck/Nanjing/2/97(H9N2). The PB1 gene a was 2 341 nt in length, and ORF encoded 757 aa. The phylogenetic analysis of PB1 gene revealed that they had the highest similarity up to 98.6% separately with A/Chicken/Guangdong/47/01(H9N2).all of them belongs to the H9N2/Y280 sublineage.The amplified fragment of PA gene was 2 233 nt, and encoded 716 aa. The phylogenetic analysis of PA gene revealed that Dk/FQ107/07 strain had the highest similarity up to 96.1% with A/chicken/China/Guangxi17/2000(H9N2), and were in the same envolutionary branch with A/Duck/Fujian/1734/05(H5N1) and A/Duck/Fujian/9713/2005(H5N1). On the other hand, analysis based on the PA gene revealed that Dk/FQ107/07 stain belongs to the H9N2/Y439 sublineage rather than to the H9N2/Y280 sublineage.According to Guan et al[11], A/Quail/HongKong/G1/97 (H9N2) strain had the highest nucleotide homology with the H5 subtype AIV, which directly infected and caused death of human occurred in Hong Kong in 1997, and had a role as the internal gene donor of H5 subtype AIV. The study, comparing the nucleotide homology between the Dk/FQ107/07 strain and A/Quail/HongKong/G1/97 (H9N2) strain, showed that only the M gene had the high similarity up to 96% with A/Quail/HongKong/G1/97 (H9N2) strain. However, other genes had relatively low similarity ranging from 23.9% to 94% with A/Quail/HongKong/G1/97 (H9N2) strain, indicating that the Dk/FQ107/07 strain had a far relation to A/Quail/HongKong/G1/97 (H9N2) strain.In short, the Dk/FQ107/07, a low pathogenitic H9N2 subtype Avian influenza virus strain, might have emerged through natural reassortment among avian influenza viruses of different sublineages.