Epidemiology Investigation Of Swine Influenza And Genetic Variation Analysis Of The Viruses Isolated From Ningxia

Swine influenza(SI),caused by influenza A viruses,is an acute infectious respiratory disease.The primary clinical manifestations in pigs are fever and acute respiratory distress,such as nasal discharge,cough,and dyspnea,which are similar to human,with low mortality but high morbidity,however,influenza has serious consequences in pigs due to the increased time needed to attain slaughter weight,which will cause great economic losses.Pigs are susceptible to both avian and human influenza A viruses,and they have been proposed to be the “intermediate host” and“mixing vessel” for generation of pandemic virus through reassortment.In order to prevent and control swine influenza,predict the pandemic influenza in human,the surveillance of swine influenza virus(SIV)and the study on the genetic evolution of SIV are very meaningful acts.In this study,920 serum samples and 920 double nostril swabs were collected from 25 pig farms in 11 cities of Ningxia and tested by serological and pathogenic diagnosis methods.The results of serological detection showed the antibody positive rates of H1N1 and H3N2 SIV were 21.2% and 2.6%,and the antibody positive rate of H1N1+H3N2 SIV was 0.65%.The results of pathogenic detection revealed 128 samples were positive and the positive rate was 13.9%.Then,in 2013 to 2014,some other samples collected from pig farms,slaughter houses and free-range households in22 cities of Ningxia were detected.The results of serological detection showed that for 1710 serum samples,the antibody positive rate of H1 SIV(11.81%)was much higher than that of H3 SIV(0.46%),the H1 and H3 doubel antibody positive rate was0.35%,and the antibody positive rates of H5 and H9 influenza virus were 0.06% and0.18%,respectively.The results of pathogenic detection revealed among the 4230 double nostril swabs,588 samples were positive and the positive rate was 13.9%.These results indicated H1N1 SIV was the predominant virus in pigs in Ningxia,while the mixed infenction of H1N1 and H3N2 SIV and inapparent infection alsoexisted.In order to study the genetic and evolutionary characteristics,the M2 genes of 5H1N1 SIV and 4 H3N2 SIV isolates were phylogenetically analyzed.The results showed among the 5 H1N1 SIVs,4 isolates belonged to North America H1N1 SIV,while 1 isolates belonged to human-like H1N1 SIV.The four H3N2 isolates all belonged to human-like H3N2 SIV,and had higher homology with viruses isolated from Guangdong and Fujian.In order to investigate the genetic characteristics of H1N1 SIVs,the genomes of 5 H1N1 viruses isolated in 2011 were amplified,the viral evolution and critical amino acid mutations were analyzed.The results showed the five H1N1 isolates had genetic diversity,one virus belonged to human-like H1N1 SIV,two viruses belonged to classical H1N1 SIV,and another two were reassortant viruses with PB2,PB1,PA,NP and NS gene originating from North America triple reassortant SIV,and the other three genes(HA,NA,M)deriving from classical H1N1 SIV.The amino acid differences were identified in the receptor bingding sites and antigenic sites in the five viruses,and three viruses had PB2 E627 K.In order to meet the needs of SIV detection,two RT-PCR methods used to detect SIVs or differentiate H1 and H3 subtype,and one pyrosequencing method were developed.The size of amplified product was 231 bp for the universal RT-PCR which could detect all the SIVs,while the second RT-PCR method could identify H1,H3,N1,N2 subtype simultaneously.The two RT-PCR methods both had good specificity and repeatability.The pyrosequencing method used the amplified products of univrersal RT-PCR,and the sequencing area covered the sites associated with amantadine resistance in M2 gene,which could be used in quick identification of drug resistance.In this study,we carried out systematic surveillance of SIV in Ningxia,and investigated the genetic evolution and critical amino acid mutations of some isolates.These results enriched the epidemiological database of SIV,deepened the understanding of current situation of SI in Ningxia,and had great theoretical significance to the prediction and control of SI in China.