Isolation And Identification Of Swine Influenza Virus Of EA H₁N₁ In Guangdong Province And Replicability And Pathogenicity Of EA And Pdm/09 Recombinant Strain

Pig is one of the important hosts of influenza virus.Porcine respiratory epithelial cells have both ?-2,3 and ?-2,6 sialic acid receptors,which not only can infect human influenza virus but also can infect avian influenza virus.Pig plays a role of “mixing vessels” in the process of mutation,recombination and cross-host transmission of influenza virus,which has important public health significance.The predominant subtypes of swine influenza virus in China are H1 and H3,of which the Eurasian avian-like H1N1(EA H1N1)virus is widely endemic in China,established a stable genetic lineage,endangering human and animal health.In this study,1170 swine nasal swabs were collected from slaughterhouses and farms and from September 2016 to June 2017.Six swine influenza viruses were isolated from SPF chicken embryos and MDCK cells and the isolation rate was 0.5%.All the isolates were identified as EA H1N1 isolates and 6 isolates with EA,pdm/09 and NTR branching genes.The results of genetic evolution showed that the surface genes HA and NA was originated from the EA branch,that the internal segment RNP gene was derived from pdm/09,that the M segment belonged to the two branches of pdm/09 and EA,and the NS segment belonged to the NTR branch.According to the attribution of M genes,they are divided into two genotypes.MS315,MS493 and 52 belong to the same genotype M gene from pdm/09,FS17,MS285 and SS11 belong to the same genotype M gene from the EA branch.According to the nucleotide homology analysis of HA gene and the cross-detection of mouse serum antibody,the nucleotide sequences of HA gene of six isolates were analyzed by nucleotide homology with Sw/GD/104 and Sw/GX/18.The SS11,FS17,MS285,MS315 and MS493 isolates belong to the same Sw/GX/18 antigen class and isolate 52 belongs to the antigen type Sw/GD/104.In addition,the results of cross-reaction between the serum antibodies of the isolates showed that the isolate 52 had a lower cross-reactivity(<40)with the antigens of the other isolates and a higher antigen cross-protection between the remaining isolates.As a result,the results of the nucleotide analysis are same.The analysis of the molecular sequences of isolates showed that the HA cleavage sites of the six isolates were located in PSIQSR?GL,which was typical of low pathogenic influenza virus.There were 6 potential glycosylation sites in HA gene of isolate 52,and 5 potential glycosylation sites in all other 5 isolates and less one glycosylation site at position 184.There were individual site substitutions among the isolates at the amino acid sites in the antigen-binding region and the receptor-binding region,in which isolate 52 had nonsynonymous significance with the other 5 isolates in the antigen-binding region of Sa,Sb,Ca1 and Ca2 Amino acid substitutions.The replacement of these amino acids may prompt the reassortment of swine influenza viruses to alter receptor and antigenic properties.Six of the isolates showed that P224 S mutation in the PA protein,and all the six isolates of PB2 protein had mutations of T271 A,GQ590/591 SR,and none of the six isolates has the E627 K and D701 N mutation.In addition,there is a neuraminidase inhibitor resistance mutation H274 Y on the NA protein.There is a mutation of the anti-adamantane resistance site S31 N on the M2 protein.Animal experiments showed that virus titres can be detected both in the lungs and in the nose of mice after challenge,and isolated strains can be efficiently replicated in mice with different antigen types and genotypes that are different in mice disease ability.Isolate 52 showed the strongest virulence in mice,followed by an average body weight which was loss of about 20% for MS493 and FS17,and a slight weight was loss in mice in MS285 and SS11 groups.The type of antigen 52 belongs to Sw/GD/104 which is different from the other 5 isolates,which may be the reason why isolates 52 being more virulent.This study provides a basic research on the molecular characteristics of evolutionary evolution and the pathogenicity of swine influenza viruses of different EA H1 NA genotypes and antigenic types.In order to study the virulence and replication ability of EA and pdm/09 recombinant viruses,reverse genetic technology was used for fragment replacement to construct recombinant viruses.The results showed that HA and four fragments constituting RNP complex can improve the replication ability of recombinant strains on MDCK cells,but the replication ability can not be restored to the parental strain YJ4.By replacing the HA and the RNP complex 4 fragments,their recombinant strains replicate in MDCK cells restoring to the level which is close to the parent strain YJ4.Animal experiments in mice also showed that the replacement of HA fragments and RNP complexes made the recombinant strains approach the level of the parent strain YJ4.The polymerase activity results showed that PB2 and PB1 from YJ4 could enhance the activity of polymerase.PB2,PB1,PA and NP fragments were knocked out respectively.The results showed that the ability to replace PB1 and PB2 fragments on MDCK was significantly reduced,and the pathogenic capacity of mice was also reduced significantly.The replacement of PA and NP fragments did not obviously reduce their replication and pathogenicity,which is similar to r1057+HA.+RNP.In the analysis of the amino acid site of the parent strain,there is D222 G substitution on the YJ4 HA fragment,so that the strain can be combined with the alpha-2,and the 6 receptor can be combined with the alpha-2,3 receptor.The replacement of T588 I on the YJ4 PB2 fragment requires further validation of its single amino acid site.In the present study,it was shown that the HA fragment of EA and the PB1 and PB2 fragments of pdm/09 can enhance the replication and pathogenicity of the recombinant strain.