Swine Influenza Virus Isolation, Identification And Serological Studies Of Swine Influenza

Swine influenza (SI) is caused by swine influenza virus (SIV), and it can induce an acute zymotic disease of respiratory tract. There were related reports about the outbreak of SI when the "Spanish" influenza was being in the USA, but until the late 1920s there was first SIV isolated from pigs.To gain insight into the epidemiology, genetic evolution of SIV and spread of SI in the central China including Henan, Hubei, Hunan, Guangdong, Fujian and Anhui, 32 viral samples were isolated from lungs of virus infected dead pigs. These virus isolates were popogated in chicken embryos. The positive cultures and the isolates of 2004 were further used for purification and biological researches. Subsequently, the genome (including NS, M, NA, NP, HA, PA, PB1, PB2 gene) of 8 SIV isolate strains were amplified by RT-PCR, cloned and the nucleotide sequences determined. Nucleotide sequences of the virus strains were compared and analyzed. Phylogenetic trees based on nucleotide sequences of the genome were constructed by MEAG (version 3.1) software, and their molecular epidemiology and genetic evolution analyzed. The HA protein of SWTJ/01/04 isolate strain was expressed. The purfied HA protein used to establish the indirect ELISA method and serological studies. The results were as follows:1. Results of etiology reseach: From 32 viral samples collected from lungs of virus infected dead pigs, one strain of H1N1, one strain of H3N8 and two strains of H5N1 subtype of SIV were isolated.2. Finity dilution cloning purification of SIV: eight SIV strains were obtained. The HA titers of the SIV strains were between 256 and 1024.3. Comparisons of biological characteristics of SIV: MDT of SIV isolates revealed that SWTJ/01/04, SWTS/01/04 and SWHB/05/06 were moderate pathogenic to chickens, with other five strains showing low pathogenicity to chickens. EID₅₀ of the SIV isolates showed no varied difference with the highest value of 10^-9.0/0.2mL from SWTS/01/04 and the others in the range of 10^-7.0-10^-8.5/0.2mL. TCID₅₀ of the SIV isolates varied greatly with no infectivity in the MDCK cell from SWTJ/01/04 and the others in the range of 10^-2.0-10^-8.5/0.1 mL. MLD₅₀ of SIV isolates showed many differences, which SWTJ/01/04, SWHN/01/06, SWCB/01/04, SWAH/01/06 and SWSX/01/04 did not cause the mouse death and other in the range of 10^-4.0-10^-6.0/0.1mL.4. Hemagglutinin thermostability and hemagglutination spectrtum tests: Large variations in hemegglutinin thermostability of 8 different regions of SIV isolates were detected, with SWTJ/01/04, SWHN/01/06, SWCB/01/04 and SWAH/01/06 being thermstabile and other 4 thermolabile. All SIV isolates were capable of agglutinating chicken, guinea pig and sheep erythrocytes. The diffenences in heamagglutination spectrum among SIV isolated mainly showed in the agglutination of pig, rabbit, mouse and cattle erythrocytes.5. RT-PCR, cloning and sequencing of the genome of SIV isolates: A universal primer using for RT and 8 specific primers using for the 8 genes were designed for amplification the genome of SIV isolates according to the descriptions by E. Hoffman. The cDNA of NS, M, NA, NP, HA, PA, PB1 and PB2 gene were successfully amplied by RT-PCR. Purification, cloning and sequencing of the amplified genes were done.6. Genetic evolution of SIV genome nucleotide sequence: The HA nucleotide sequence analysis indicated that HA gene formed 3 markedly different parts. H1 subtype of SIV isolates belonged to human influenza lineage. And the all 8 gene sequences of the lineage evolved from A/Brevig Mission/1/1918(H1N1) and showed the high homology to A/Taiwan/01/86(H1N1). H3 subtype of SIV isolates belonged to H3N8 subtype of horse influenza lineage. The highest nucleotide homology showed between 4 H5 subtype of SIV isolates and A/chicken/Hubei/327/2004(H5N1). The NA gene of SWTJ/01/04 and SWHN/01/06 showed the high homology to A/Yaiwan/01/86(H1N1). In this study H3N8 subtype of influenza virus isolated from pigs at first time. And the NS gene of SW/AH/01/06 divereged from A/Taiwan/01/86(H1N1) and the PB2 gene showed the highest homology to SWSX/01/04. The SWCB/01/04 isolate supplied NA gene to other N8 subtype isolates.7. Establishment of indirect ELISA detecting the SI: The purfied HA protein was used to antigen for establishing the method of detecting the antibodies of H1 subtype SIV. The protein concentration was 0.066 ug/well, and the serum diluted 40 to use. Comparing to the HI assay, the sensibility, specificity and coincidence were 90.9%,80.9% and 85.0%, respectively.8. Application in the serological epidemiology: Serological testing was performed on 2,029 pig serum samples of the central China including Hubei, Hunan, Anhui, Jiangxi, Henan and Shanxi province. Using the indirect ELISA, the average positive rate was 23.46%. And the rates above the average level in June, July and December were 25.35%, 24.92% and 51.42%, respectively. The highest antibody rate of region in the central China was Hunan proviee, with 24.24%. The Hubei and Henan provice were about 24.04% and 22.96%, respectively. In conclusion, this study conducted serological and aetiological surveys to study the ecology and epidemiology of SI in the central China. Results indicated that pig flocks in China were infected with H1, H3 and H5 subtype influenza virus. Comparsion of the genome of SIV isolates, this paper elucidated the genetic relationship and tendency of evolution according to the molecule level. And it determined the position of SIV isolates in the evolution of influenza virus.