Serological Investigation Of Influenza Viruses In Swine Herds And The Effects Of Amino Acid Changes At Hemagglutinin(HA) Protein Cleavage Site On Pathogenic Of H1N2 Subtype SIV

As known as ‘‘genetic mixing vessels’ ’,a number of subtypes of influenza A viruses of human and avian origin were isolated from pig herds around the world,including H1,H2,H3,H4,H5,H7,and H9.Different genotypes of influenza virus infecting pigs at the same time will lead to gene reassortment and promote the evolution of the virus,thus producing recombinant strains with pandemic potential.In order to investigate the epidemic situation of swine influenza virus,a total of 4920 serum samples were collected from pig farms in Guangdong,Fujian,Jiangxi and Hunan from July 2017 to November 2018,and the hemagglutination inhibition test(HI)was performed.It showed that H1 and H3 subtype influenza viruses were widely presented in pigs since the overall positive rate was 45.55%.The antibody positive rates against Eurasian avian-like H1N1,H3N2 SIV,Pdm09 H1N1,and Classic swine flu H1N1 virus were 31.42 %,19.51 %,14.39 %,and 6.16 %,respectively.EA H1N1 SIV became the dominant strain in swine herds.No H4,H5,H6,H7 influenza virus antibodies was detected.There were mixed infections of different subtype strains in pigs,among which EA H1N1 + Pdm09 H1N1 is 9.25%,EA H1N1 + H3N2 is4.04%,and Pdm09 H1N1+ H3N2 is 2.03%.There were differences in serum antibody positive rates in different regions.Fujian has the highest EA H1N1 serum antibody positive rate(P <0.05),which is 37.4 %.Henan has the highest Pdm09 H1N1 and H3N2 SIV antibody positive rates,which were 32.42 % and 21.0 %,respectively.SIV and Pdm09H1N1 infections occur in pig farms throughout the year.The average serum antibody positive rate of EA H1N1 SIV and H3N2 SIV from March to June was higher than that of annual average antibody positive rate,and the antibody positive rate of Pdm09 also reached peak in May.There were also differences in the serum antibody positive rate of pig herds at different stages,among which sows had the highest Pdm09 H1N1 serum antibody positive rate(P <0.01),and the boar has the highest EA H1N1 antibody positive rate(P <0.05).Interestingly,the antibody positive rate of H3N2 SIV in piglets and fattening pigs was higher than that in sows(P <0.01).These data provide basic references for prevention and control of influenza virus,and help better selecting the candidate vaccine strains.In order to investigate the effect of changes in the cleavage site on the pathogenicity and replication ability of swine influenza virus,we compared the amino acids at the P2 cleavage site at hemagglutinin of our previously isolated strains and all H1N1,H1N2 and H3N2 swine influenza viruses in China from the NCBI and GISAID databases.It shows that there are diversity in the amino acid distribution at the P2 position of the HA cleavage site of swine influenza viruses,in which the H1N1 SIV are S and A,mainly S;the H1N2 SIV are S,T and L,mainly S;the H3N2 SIV are T and N,mainly L.With close genetic distance between the isolated strain A / swine / Guangdong / DG2 / 2015(H1N2,The P2 cleavage site of HA protein is S)and A/swine/Guangdong/YJ28/2014(H1N2,The P2 cleavage site of HA protein is S),we constructed wild strain r/DG2,r/YJ28 and the mutant strain r / DG2-S328 L,r/YJ28 L328 S by reverse genetic technology.The results of the virus growth curve showed that the r / YJ28-328 L and r / YJ28-L328 S mutant strains were able to achieve similar peak titers in MDCK and A549 cells,although their replication capacity were different at some time points.The cleavage capacity of hemagglutinin test showed that when the P2 cleavage site was serine(S),the cleavage efficiency was higher than that of leucine(L).However,the mutation of HA-L328 S significantly increased the pathogenicity of r/ YJ28-L328 S to mice compared with r/YJ28,in contrast,the mutation of HA-S328 L significantly reduced the pathogenicity of r/ DG2-S328 L to mice compared with r/DG2.In conclusion,we found that the replacement of the H2 subtype influenza virus P2 cleavage site from serine to leucine maintain the growth ability of the strain in vitro,but weaken the Pathogenicity to mice.Our finding provide a new idea for studying the pathogenic mechanism of swine flu and provide a new strategy for the development of attenuated vaccines.