| Influenza A virus had a wide host range and can cause acute respiratory diseases in humans,poultry,pigs,dogs,cats,horses and other animals.Avian infuenza virus(AIV)belonged to Orthomyxoviridae A influenza virus genus,which posed a serious threat to poultry industry and human safety.Avian influenza virus could be divided into highly pathogenic avian influenza virus(HPAIV)and low pathogenic avian influenza virus(LPAIV).Among the low-pathogenic avian influenza virus,H9N2 subtype was widely distributed and has the great harm to poultry breeding.In recent years,many domestic and foreign scholars have studied the biological characteristics of H9N2 subtype AIV.They found many amino acid sites that affect the antigenic changes,but the evolution of antigenic properties of H9N2 subtype AIV was still unknown.At the same time,studies have shown that some host long non-coding RNA(LncRNA)could regulate the replication process of influenza virus.However,the number of known functional LncRNAs were limited.Therefore,studying the antigenic variation of H9N2 subtype AIV and exploreing the interaction mechanism between influenza virus and host LncRNA,were of great value for the development of influenza virus vaccine and the interaction between influenza virus and host.Based on the background,the main contents of this study are as follows:1.Amino acid variation and antigenic of the H9N2 stype AIVIn this study,a total of 1856 full-length sequences of the HA protein of the domestic H9N2 subtype avian influenza virus from 1994 to 2016 were downloaded from the NCBI.Then the genetic evolution analysis was performed to determine that the current domestic main epidemic branch is clade h9.4.2.5.The processed 1856 amino acid sequences were analyzed,A/Chicken/Guangxi/55/2005(GX55)was used as the template,and the amino acid mutation rate of HA1 protein was greater than 20% as the standard,and the 20 mutated positions were screened out.point.By drawing a three-dimensional structure diagram to remove 3internal amino acid positions,the remaining 17 amino acid positions for the next step: 72,109,135,146,149,150,162,163,180,182,183,198,202,216,217,236,238 amino acid positions(H9 numbering).Based on the condition that the proportion in nature is greater than 1%,a total of 19 kinds of mutation combinations were screened out.The GX55-p HH21 recombinant vector was constructed,and the 19 combinations of mutations were completed on this basis.The 12-plasmid reverse genetic system was used for virus rescue.Cross-hemagglutination inhibition tests were performed on these mutant strains with some classic vaccine strains and strains isolated from the laboratory in recent years to draw an antigenic map.The results showed the influence of antigenic distance between the current H9N2 strains and the mutant recombinant viruses.At the same time,it was found that there were 2 mutant recombinant viruses showing a tendency to switch from the old antigen group to the new antigen group.In addition,this study continued to conduct temporal and regional analysis of the above 1856 sequences,we found that the mainstream branch of H9N2 subtype AIV in China from 1994 to 2006 was relatively diversified.However,since 2007,the mainstream branch of H9N2 subtype AIV in China was clade h9.4.2.5 appearing as a single popular branch.Therefore,we defined the strains isolated around 2007 as the old strain group and the new strain group,then we analyzed the amino acid sequence differences between the old and new strain groups.As a result,it was found that there were 15 amino acid positions difference between the new and old strains.Interestingly,except for the 180 positions,the variant forms of the other positions were highly consistent with the Mut-2 mutant strain.Furthermore,we selected the representative strain GX/55 from the old antigenic group,the representative strain A/Chicken/Jiaxing/19166/2019(19166)from the new antigenic group,and Mut-2 virus to prepare inactivated vaccines.14 day after immunized,each group of chickens were infected with GX/55 strain and 19166 strain.As a result,it was found that the vaccine prepared by the Mut-2 virus can prevent the infection of prevalent strains to a certain extent.This study reveals the genetic evolution characteristics and antigenicity of the domestic H9N2 subtype AIV,and provides a theoretical basis for the prevention of the H9N2 subtype AIV.2.Mechanism of LncRNA regulating influenza virus replicationThe purpose of this study was to explore and screen the host long non-coding RNA(LncRNA)caused by influenza A virus infection which can regulate virus replication.Firstly,we grind the lung tissues of mice in the virus infected group and the negative control group to extract total RNA.Then we reverse transcribed it into c DNA for sequencing.According to the criteria of p value<0.05 and fold change >20,a total of 32 candidate genes were screened according to the multiple of difference for further verification.Then we used quantitative real-time PCR(qRT-PCR)to verify the expression trend,and we found that there were 20 candidate genes were consistent with the transcriptome sequencing results.Then we constructed these candidate genes into pcDNA3.1(+)vector,and the transfection experiment was carried out in mouse lung epithelial cells(Mle-12).24 h after overexpression,A/WSN/1933(H1N1)was infected with a dose of MOI=0.5,and the cell supernatant was collected 24 h after virus infection,then functional verification was performed by hemagglutination test.The verification results showed that one of the candidate genes(ID: TCONS＿00373113)can significantly inhibit the replication of WSN virus.The virus titer of the collected samples was titrated by the plaque assay,and the results werw consistent with the trend of hemagglutination results.Therefore,we concluded that the candidate LncRNA is an antiviral LncRNA,and we named it as SAAL based on its regulatory mechanism.In order to further explore the antiviral mechanism of SAAL,we carried out the prediction analysis of LncRNA-m RNA co-expression,and then we screened out 8candidate target genes according to the size of co-expression correlation and the role of mRNA for the next step verification.Through qRT-PCR experiments,it was found that the transcription level of Serpina3i was significantly up-regulated after SAAL overexpressed.Therefore,it can be concluded that there was a co-expression relationship between Serpina3i and SAAL.In order to verify the function of Serpina3i,we constructed a Serpina3i-pcDNA3.1 overexpression plasmid and synthesized a small interfering RNA(si-Serpina3i).Then we found that overexpression of Serpina3i inhibited the replication of WSN,meanwhile,knocking down of Serpina3i promoted the replication of WSN in Mle-12 cells.These studies proved for the first time that Serpina3i could inhibit influenza virus replication.In order to explore the relationship between Serpina3i and SAAL,we performed SAAL supplementation after knocking down Serpina3i,then we found that supplementing SAAL under the premise of knocking down Serpina3i did not inhibit the replication of WSN.Therefore,we concluded that SAAL upregulated the transcription level of Serpina3i thus inhibited the replication of influenza virus,and Serpina3i was a key regulatory gene of SAAL.Later,in order to further study the antiviral mechanism of SAAL and Serpina3i,we overexpressed SAAL and Serpina3i in Mle-12 cells.24 h later,we infected WSN virus.Then we collected cell lysate at 6 h and 12 h after infection,qRT-PCR results showed that whether SAAL or Serpina3i was overexpressed caused a significant up-regulation of the transcription level of IFN-β;then we tested the transcription levels of several classic ISGs and found that the same results.In summary,we have discovered a novel LncRNA: SAAL,which can increase the transcription level of Serpina3i to cause the up-regulation of IFN-β and ISGs,thereby inhibiting the replication of influenza virus.This study provided a new target and new mtheoretical basis for studying the interaction between virus and host. |
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