Molecular Epidemiology Of Swine Influenza Virus In Some Regions Of China

Swine influenza is an acute respiratory disease caused by swine influenza virus (SIV). Swine influenza surveillance is of great significance for veterinary and public health.To facilitate swine influenza surveillance, RT-PCR assays were developed to detect SIV type and subtypes (H1, H3, H9, N1, and N2), using six pairs of primers based on conserved sequence of the M, HA and NA genes of SIV. Specificities of our RT-PCR assays were very good and sensitivities of detection were about 102-103EID50 SIV. These RT-PCR assays could be used in detecting SIV in clinical samples.To learn the information about prevalence of SI in China, a total of 600 samples were collected from 9 provinces (Heilongjiang, Henan, Shandong, Zhejiang, Anhui, Jiangxi, Beijing, Guangdong, and Guangxi) and 22 viruses were isolated, including one H1N1 virus, four H3N2 viruses, and 4 H9N2 viruses. To determine the pathogenicity of the nine viruses in mammals and horizontal transmission level in mammalian population, these viruses were inoculated into mice. The result shows that the nine viruses were lowly pathogenic for mice, didn't cause obvious clinical signs, and couldn't transmit in mice. All the nine viruses could be isolated from lungs of inoculated mice at 4 to 8 days post infection. Classical H1N1 SIV and avian origin H1N1 SIV have been reported widely in swine population worldwide. But human origin H1N1 SIV was reported occasionally. In 2006 we isolated a human origin H1N1 SIV from a pig in a farm of Guangdong province. We made full use of another two human origin H1N1 swine viruses isolated and sequenced by scientists of Huazhong Agricultural University of China to analyze their genetic evolution. All the eight gene segments of the three viruses are highly homologous to recent (2000) and early (1986) human H1N1 influenza viruses, respectively. Phylogenic analyses of eight genes and molecular analyses of HA and NA also show also that the three viruses might derive from recent or early human H1N1 influenza viruses. Existence of these influenza viruses, especially older viruses, in pigs is an invaluable characteristic in studying influenza virus epidemiology.Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts or"mixing vessels"for the generation of pandemic influenza viruses through reassortment or adaption to the mammalian host. In the study, for the first time, we summarize and report the coexistence of wholly human-like H3N2 viruses, double-reassortant H3N2 viruses and triple-reassortant H3N2 viruses in pigs in China, by analyzing the eight genes of swine influenza A (H3N2) viruses in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In 1980s, two triple-reassortant viruses were isolated in pigs, which have been reported. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP) and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts or"mixing vessels", and emphasizes the importance of reinforcing swine influenza virus surveillance in China.The H9N2 subtype influenza virus is a remarkable member of the influenza A viruses because it can infect not only chickens, ducks and pigs, but also humans. In March 2007, during the outbreak of highly pathogenic PRRS, 50 samples were collected from different pig farms in Guangxi province. RT-PCR assays were carried out to detect PRRSV and SIV. Interestingly, four PRRSVs and SIVs were isolated from the same lung samples. All the SIV isolates were closely related to each other in all eight gene segments, and so one of four SIV isolates was selected as the typical isolate and was designated as A/swine/Guangxi/7/07(H9N2). The eight gene segments of the four viruses are highly homologous to A/Pigeon/Nanchang/2-0461/00 or A/Wild Duck/Nanchang/2-0480/00. Phylogenetic analyses show that the four viruses are of avian origin and may be the descendants of A/Duck/Hong Kong/Y280/97-like viruses. To further understand the genetic evolution of avian origin H9N2 SIV, we selected another 24 viruses from GenBank. The result show that all avian origin H9N2 viruses isolated in China could be divided into 10 genotypes (A-J). The five viruses of genotype B and the five viruses of genotype C derived from A/Duck/Hong Kong/Y280/97 and A/Chicken/Shanghai/F/98, respectively. The rest are reassortant viruses. All these deepen the understanding of genetic evolution of avian origin H9N2 SIV and provide necessary data for SI control.To get overall information about the prevalence of SI in China, the serological studies of nine provinces were carried out by HI assay using H1, H3, H5 and H9 subtype influenza viruses as antigens. The positive rates of H1 subtype influenza was relatively low, with prevalence ranging from 0 to 20.8%, indicating that H1 subtype human influenza viruses might sporadically infect pigs in China. The positive rates of H3 subtype influenza was 29.6~97.1%, indicating human origin H3 SIV might exist in pigs widely. The positive of H5 and H9 subtype influenza were 1.1~13.2% and 6.5~31.6%, respectively. In addition, we established indirect ELISA method for detecting H3 subtype SIV antibody rapidly.Recently, H5N1 subtype SIV has been isolated from pigs in Fujian province. To further impulse research in molecular mechanism and genetic engineering vaccines for SI, we established a reverse genetics system for Fujian isolate, Sw/Fj/NP/01, which was the first strain of H5N1 swine influenza virus in world. Our work about rescuing virus is still ongoing.