Epidemiological Investigation For Swine Influenza Virus(SIV) In Southern China And The Mechanism Research Of Anti-influenza Viurs For Diammonium Glycyrrhizinate(DG)

Swine influenza(SI)is an acute and infectious respiratory disease in swine that caused by swine influenza virus(SIV).To date,three subtypes SIV(H1N1,H1N2,and H3N2)circulating in swine herds in worldwide,and different subtypes SIV can be divided into different lineages.What's more,SIV can be reassortmented between different subtypes or lineages.Because pig have a special receptor distribution characteristic on respiratory tract,it can be infected with avian influenza virus and human influenza virus,and then generate new reassortment virus in swine.Thus,swine is considered to playing as a “mixer” for influenza virus and plays an important role in transmission chain between avian,swine and human.In order to know about the epidemic and infection sutation,as well as the mutation,reassortment of SIV in swine herds in Southern China,the epidemiology and serological survey of SIV was conducted.Three SIV strains were identified from 236 lung samples and 143 nasal swabs,named A/Swine/Guangxi/NK/2016,A/Swine/Guangdong/ZSBS/2017,A/Swine/Fujian/NP/2017.The sequencing results of genome for three strains showed that they are H1N1 subtype SIV,and the amino acids identical of HA protein each other is between 96.6% to 97.9%.Phylogenetic analysis showed that they are all belongs to Eurasian-like avian(EA)lineage,and all of them was reassortment with pdm09 H1N1 SIV.For strain A/Swine/Guangxi/NK/2016,the HA and NA gene belongs to EA lineage,PB2,PB1,PA,NP and M gene belongs to pdm09 lineage,while NS gene belongs to classical swine influenza(CS)lineage.For strain A/Swine/Guangdong/ZSBS/2017,HA,NA,NP and NS gene belongs to EA lineage,while PB2,PB1,PA and M gene belongs to pdm09 lineage.For strain A/Swine/Fujian/NP/2017,HA,NA,M and NS gene belongs to EA lineage,while PB2,PB1,PA and NP belongs to pdm09 lineage.These results indicate that the reassortment between EA lineage and pdm09 lineage is common in swine herds.In addition,the glycosylation site at position 23 of HA protein is missing in strain A/Swine/Guangxi/NK/2016.Serological survey for SIV was conducted and 3265 serum samples that collected from Southern China were detected by HI test,the result shows that H1N1(including EA lineage and pdm09 lineage)and H3N2 subtype SIV co-ciculating in swine herds.The positive rate for H1N1 EA lineage,H1N1 pdm09 lineage and H3N2 subtype SIV is 27.53%,20.98 and 34.85%,respectively.In addition,antibody for H9N2 subtype influenza virus was also detected from surem samples and the positive rate is 0.64%,which indicate that the infection of H9N2 in swine herds have occured.But no samples was positive to H5N1 and H7N9 subtype influenza virus.Co-infection with different subtype or lineage SIV was also found and the co-infection is common in swine herds,the co-infection rate between EA and pdm09 lineage is 11.3%,while the co-infection rate between H1N1 EA line and H3N2 SIV is 10.01%.The antibody titer for EA,pdm09 and H3N2 SIV can be reached to 1:1280,while the highest titer for H9N2 is only 1:160,which indicte H9N2 may be not adaptation for swine.We also found that the epidemic of SIV is seasonal and mainly epidemic in winter.Because the coinfection and reassortment of SIV,further monitor for SIV in swine herds should be conducted and provide scientific basis for SIV prevention.Finally,we have studied the anti-influenza virus effect of Diammonium Glycyrrhizinate(DG)and its mechanism.Our results shows DG can inhibit the replication of influenza virus and the antivirus effect is dose-dependece,DG can significant inhibit the replication of influenza virus when the concentration of DG is 250?g/mL.Subsequently,we have studied the effect of DG for the replication cycle of influenza virus,the results shows DG have no effects for early stage of the replication cycle(binding and entry stage),and DG mainly inhibit late stage of the replication cycle(replication stage).In addition,we also compared the mRNA express level for several cytokines(including IFN-??IFN-??IFN-? ?TNF-??IL-6 and IL-8)in DG treatment group and control group,and we found the mRNA express level for IFN-? and TNF-? is significantly difference between groups that treatment with DG and without DG.The mRNA express level for IFN-? in group treatment with DG is 1.7 times higher than that in control group,while the mRNA express level for TNF-? in group treatment with DG is 2.4 times lower than that in control group.Thus,we conjecture that DG have immunomodulatory functions,it can induce the production of IFN-? to defense the infection of influenza virus.On the other hand,it also can reduce the express level of proinflammatory cytokine,such as TNF-?,to mitigate inflammatory response and extenuate the damage that cause by severe inflammatory response.Our results indicate that DG can be as a potential anti-influenza virus agent.