Epidemiological Investigation Of SIVs In Southern China And Comparative Transcription Of Mouse Lungs Infected Wild-type And Mouse-adapted GDK6 Viruses

Swine Influenza Virus(SIV)is one of the major diseases that influence the large scale breeding farms.The simple infection of pigs with SIVs presents mild clinical signs in the field and experimental conditions,including fever,slight cough,sneezing,and nasal discharge,high morbidity and low mortality.However,SIV can impair the host immune system in a variety of ways,subsequently suppressing the immune response to other pathogens.Due to unique geographical and environmental factors and the large breeding scale of poultry and swine,southern China is considered an important reservoir of influenza virus.Multiple lineages of influenza viruses have emerged and become established in southern China,including Classical Swine Influenza Virus(CS),Eurasian Avian-like Swine Influenza virus(EA)swine-like H3N2 lineages,North American triple reassortant(NTR)and Human Seasonal Influenza virus(HS).After the pandemic,pdm/09-like virus was repeatedly transmitted to pigs in many countries and reassorted with other endemic viruses.To examine the trend in SIV,an active surveillance program has been conducted since 2012 in southern China.A total of 10188 sero-samples were collected from Guangdong,Jiangxi,Hunan,Fujian,Guangxi and Yunnan provinces from Sep.2012 to June 2016,and were estimated using Hemagglutination Inhibition test(HI).The resultes show that the positive rate of the antibody against to pdm/09 was decreased to 9.11%(95%CI: 8.06%-10.25%)in 2016,which was 34.92%(95%CI: 32.22%-37.69%)in 2012.By contrast,that of EA-like virus showed rising trend and reached the highest(45.54%,95%CI: 42.41%-48.69%)in 2016.The sero-prevalence of H3 virus achieved the head in 2013,which was 27.21%(95%CI: 25.36%%-29.12%),and reached the bottom in 2016(8.93%,95%CI: 7.23%-10.87%).Furthermore,the results of serological test showed that the positive rates of antibody against to EA-like virus,which are 30.35%(95%CI: 28.97%-31.76%)and 27.02%(95%CI: 22.24%-32.22%)in Guangdong and Yunnan province,are significantly higher than that against to the other lineage viruses.The prevalence of EA-like virus in pig herds in Hunan and Jiangxi provinces are significantly lower than that of pdm/09,CS and H3 viruses.The positive rate of antibody against to H3 virus is 34.12%(95%CI: 29.37%-39.12%)and is significantly higher than that of pdm/09-,EA-and CS-like viruses,and the prevalence of antibody against to CS-like virus is 8.94%(95%CI: 7.74%-10.27%)and significantly lower than that of other lineages,indicating the regional epidemic of SIVs in southern China.Furthermore,On the other hand,a total of 4056 nasal swab samples were collectd from the breeding farms,abattoirs and live animal markets in Guangdong,Foshan,Dongguan and other cities in Guangdong province.14 novel SIVs were isolated in embryos and/or MDCK cells and categorized into H1N1,H1N2 and H3N2 subtypes.The results of phylogenetic analyses show that the internal genes reassorted from pdm/09-like PB2,PB1,PA and NP gene segments,pdm/09-and/or EA-like M gene segment and NTR-like NS gene segment.It is suggested that the pdm/09-like RNP genes has replaced the EA-and NTR-like internal genes that were formed in sorthern China since 2002,and has become prodominant SIV prevailing in pig herds.Moreover,the external genes of the isoaltes were assigned into EA and/or swine like H3N2 lineages,and multiple substitutions are observed in the HA and NA genes,indicating the antigenic shift of the novel isolates.14 isolates are categorized into 5 genotypes according to the composition of the genome.To investigate the virulence of the novel isolates,the viruses of 5 genotypes were inoculated into 6-week-old female Balb/c mice.The results show that the YJ4(genotype 1)and DG2(genotype 4)are considered to be the most pathogenic to mice,and the viruses are observed in the kidneys and brains,indicating the systematic infection.By contrast,the mice infected with FS4(genotype 5)present slight decrease of body weight,and the viruses can be detected in the lungs and turbinates,and not in the kidneys and brains.In order to identify relevant risk factors for IAV infection in pig farms,a detailed epidemiological research was conducted in Guangdong province.A total 153 pig farms were selected and the basic information were collected using standard questionnaires,including production practices,biosecurity practices,and IAV infection situation.Through detailed statistical analyses,the results show that there is a huge difference among the scale farms in producing and biosafety management in Guangdong Province,such as production record,the division of employees,regular disinfection,changing clothes and shoes before entering the field,changing clothing and boots in treatment,and so on.Furthermore,thedata collected were used to identify the risk factors for IAV infection through binary and multinomial logistic regression model.23 risk factors in Guangdong were identified,including years of operation,farm size,quarantine,contacting with dogs and poultry,wild birds entering piggeries and so on.Moreover,a prediction model was built for the risk of infection on IAVs,and the ROC analysis on the model showed that the area under the curve is 0.79(95%CI: 0.72-0.86),which has a certain accurary and can be used as a predicting model.The first H6N6 swine-orgin influenza virus was isolated and named as A/Swine/Guangdong/K6/2010(H6N6)in 2010,which is low virulent in mice.The mouse-adapted virus(GDK6-MA)was obtained after 12 times passage on mice and was observed highly pathogenic for mouse model.The differented expressed genes(DEGs)of the lung tissues of mice infected with GDK6-WT and GDK6-MA were estimated by high-throughput sequening using transcriptomes,and were classified using the significant enrichment analysis,GO functional classification analysis and KEGG pathwany significant enrichment analysis.The results showed that the DEGs in the lungs were enriched in cytokine response and respectors,chemokines,complemments,cell adhesion and inflammation response.Furthermore,the DEGs related to innate immune system were observed to different degrees of change.At first,the signal pathways in the lungs of Balb/c mice were activated after infecting with GDK6-WT and GDK6-MA viruses,including TLR3,TLR7 signal patways.On the other hand,the upregulation of the cytokines and respectors and the chemokines were found in the lungs of infected mice,including IL1,IL6,Cxcl9,Cxcl10 and so on.Moreover,the levels of upregulation of these genes in the GDK6-MA group were significantly higher than that of GDK6-WT group.The genes in the completmments and cell adhesion were observed to appear different levels of regulation,including C1 qa,C1qb,C1 qc genes of completmment and Cd84,Cd33,Cdh17 genes related to cell adhesion process.RT-q PCR was used for further analysis,and the results showed that the GDK6-WT and GDK6-MA could activated the signal pathways related to the inflammation response.Compared to the mice inoculated GDK6-WT,the genes in the lungs from GDK6-MA group showed significantly upregulated,thus increasing the host’s inflammatory response and pathogenicity of GDK6-MA viruses.