Isolation, Identifaction And The NA Gene Sequence Analysis Of The Isolated SIV

Swine influenza is a highly infectious respiratory disease caused by Swine Influenza Virus infecting swine of different stages,sexes and breeds.At present, it can be discovered in many nations and causing immense economic losses in the swine industry.Therefore,the research on the epidemiology survey and the biological properties of SIV will be useful for the prevention,it also can help to control the human influenza in China.In the studying,samples were collected by nasal mucus,tracheal mucus, the infected lung from pigs with respiratory system problems in Guangxi,and detected by RT-PCR.Then the positive samples were inoculated 11-day-old chicken-embryos.3 strains were isolated to be swine influenza viruses preliminarily.And the results showed that GXHZ strain is belonged to H1N2 subtype,GXLZ,GXXY strain are belonged to H3N2 subtype by HI and RT-PCR.The EID₅₀of GXHZ,GXLZ,GXXY isolates and the challenge of GXHZ isolates were studied.The observations demonstrated that 11-day-old chicken-embryos did not die after being inoculated with the viruses via allantoic route,and 50%Egg Infective Doses(EID₅₀)of the 3 strains were 10^-5.3 EID₅₀/0.2ml,10^-4.7EID₅₀/0.2ml and 10^-5.5EID₅₀/0.2ml respectively.After the there were no visible clinical symptom and only isolated SIV in the fourth,fifth days from the infected pig repeat.The nucleotide sequences of NA gene of 3 isolates(named GXHZ,GXLZ, GXXY)were determined.The cDNA of neuraminidase(NA)gene of 3 isolates were reverse-transcribed and amplified by PCR respectively.PCR products were cloned and sequenced,and their amino acid sequences were deduced,compared with the other strains.The results suggested that the equences of NA genes of the 3 isolates were composed of 1410bp,and coding for 469 amino acids.The homology of nucleotide sequence and amino acid sequence among 3 isolated strains was high and more than 99.8%and 99.4 respectively.Compared NA gene of 3 strains with the H1N2,H3N2 subtype,the homology of nucleotide sequence and amino acid sequence was high,the homology of nucleotide sequence was 82.9～98.1%,97.0～99.7,the homology of amino acid sequence was 83.2～98.5%,96.8～99.4 respectively.And compared with the decade strains of EF556202(A/swine/Guangxi/17/05(H1N2)),EF566204(A/swine/Hainan/1/05 (H1N2)),EF556200(A/swine/Guangxi/13/06(H1N2)),F251412(A/Swine/Iowa/ 533/99(H3N2)),the homology of nucleotide sequence and amino acid sequence was 97.4～99.7%,97.9～99.4%respectively.But compared with D00715(A/sw/ Hong Kong/3/76(H3N2)),D00716(A/sw/Hong Kong/4/76(H3N2)),AB101674 (A/Hong Kong/1/68(H3N2)),CY009302(A/Swine/Colorado/1/77(H3N2)),the homology of nucleotide sequence and amino acid sequence only was 87.7～91.3%,87.4～91.7%respectively.Phylogenetic analysis of NA gene sequences indicated that the 3 isolates were belonged to N2 subtype,and they had a immediate relationship with H1N2 subtype isolatess of China,United States, and have high homology with EF556202(A/swine/Guangxi/17/05(H1N2)), EF566204(A/swine/Hainan/1/05(H1N2)),EF556200(A/swine/Guangxi/13/06 (H1N2)).Considering with the time,We also found that there were immediate genetic relationship between the 3 isolates and the H3N2 strains isolated after 1996,they all belong to the same subgroup.But they were different with the classical strain.