| Swine influenza(SI), caused by swine influenza virus(SIV), is a contagious respiratory disease which often occurred in swinery and very difficult to eradicate. Influenza virus has a high host specificity, in natural conditions, avian influenza virus(AIV) and human influenza virus would not happen genetic recombination, while, research shows that which can be done through the intermediate host of swine to restructure new strain. In the surface of swine somatic cells have both a-2,3 galactosyl glycoside receptors and a-2,6 galactosyl glycoside receptors, among them the former has a strong hydrophilic with AIV while the later has stronger affinity with human influenza virus. For this, swine can infect human influenza virus,AIV and SIV, swine plays a role of mixer of influenza virus. Serologic tests of SI for monitor the distribution and epidemiology situation, separation and identification as well as phylogenetic analysis of SIV have important economic value and public health significance.Shandong located in the lower reaches of Yellow River, near Bohai and Yellow sea, across the sea of Korean peninsula and Japan islands. Except that, Shandong is a big province of raising pigs, SI occurred frequently, in addition, Shandong is the migration route of birds,which amplified the influenza gene pool. It is very important to bring out serological investigation for SI and phylogenetic analysis for SIV in Shandong province. In this study,swine blood samples, totally 1049 copies, were collected in various regions of Shandong province from 2013 to 2014. Samples were tested with the H1 subtype SIV ELISA antibody detection kit to understand the distribution and epidemiology situation in Shandong province.The study shows that SI highest occurred in spring and winter, especially in December and March. For the region, the positive rate in southern and southwest area was higher,respectively 56.8% and 55.3%, while in jiaodong region was lower, just 9.6%.In addition, swine nose swabs were also collected for the separation of SIV. Swine nose swabs samples after processing were inoculated in 10-day-age SPF chicken embryos, two strain virus in ZC and NJ region were separated respectively named as A/Swine/ZC/90/2014(H1N1)and A/Swine/NJ/03/2014(H1N1). Sequence published in NCBI were referred to design primers, through RT-PCR amplified eight gene sequences, after gel extraction connected with pMD-18 T vector, then transformed Trans5 a competent cell, selected positive plasmid for monoclonal sequence. After phylogenetic analysis, it shows that HA sequences of the two strains isolated both have seven glycosylation sites, five in HA1 and two in HA2; the cleavage sites are QSR/GL, without basic acid insertion, which can infer two strains are low pathogenic SIV. For eight sequences, HA, M, NA, NP of two strains have the highest homology with H1N1 subtype SIV, while, NS, PA, PB1, PB2 have the highest homology with H3N2 subtype SIV. The result inferred that strains isolated may be a recombination by H1N1 subtype and H3N2 subtype.A/Swine/ZC/90/2014(H1N1)was passaged in 10-day-age SPF chicken embryos, and the E20 virus were processed comparison analysis with the primary virus. The result shows that until E20, the virus does not appear larger variation. Only HA and NP protein appear three and two amino acid variation.In this study, A/Swine/ZC/90/2014(H1N1)was also used for preparation of high immune serum. HI and ELISA were adapted to monitor antibody titer, and the results show that the antibody would arrive to the peak in the sixth week, if collections were not in time, the titer would decline. IFA was used for verification of the high immune serum, and the fluorescence signal indicated the preparation of high immune serum has good specificity. |
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