Construction Of H3N2 Canine Influenza Virus HA Mutant Strains And Its Co-infection With Staphylococcus Pseudintermedius

Canine influenza virus belongs to influenza A virus,which is a enveloped,negative sense,single-stranded RNA viruse.It has been confirmed that H3N2 is a major subtype of CIV in China.CIV can cause respiratory diseases,but it is more likely to lead to secondary bacterial infection complications,such as pneumonia,pleural pneumonia,chronic bronchitis.HA fragments of influenza virus play a key role in the host range,replication capacity and pathogenicity of viral infection.In this study,based on complete eight gene fragments of A/canine/Jiangsu/06/2010(H3N2),we explored the effects of HA L222W and HA R326K mutations on CIV transmission and virulence.In addition,we established and optimized a in vitro coinfection model of CIV and Staphylococcus pseudintermedius(Sp)to investigate the effect of pre-infection of CIV on secondary infection of Sp in MDCK cells.1 Construction of site-directed mutant strains in hemagglutinin of H3N2 canine influenza virusIn order to investigate the role of mutations in HA gene of A/canine/Jiangsu/06/2010(H3N2),pBD-HA,PBD-HA L222W,pBD-HA R326K and pBD-HA L222W + R326K recombinant plasmids were constructed by site-directed mutagenesis.Using the eight plasmids reverse genetic operating system,recombinant plasmids of seven gene fragments,other than HA,from A/canine/Jiangsu/06/2010(H3N2)were transfected into MDCK and 293T cells,together with pBD-HA,pBD-HA L222W,pBD-HA R326K,pBD-HA L222W + R326K recombinant plasmids respectively.After that,four viruses were successfully rescued,named r-06,r-06/L222W,r-06/R326K and r-06/L222W +R326K.After eight consecutive passages and gene sequencing analysis,it was confirmed that the four viruses could be stably inherited,which provided the material basis for the subsequent study of the effects of 222 and 326 amino acid mutations in hemagglutinin of H3N2 canine influenza virus.2 The characteristics of site-directed mutant strains in hemagglutinin of H3N2 canine influenza virusIn order to study the effects of HA L222W and HA R326K mutations on the replication ability and pathogenicity of A/canine/Jiangsu/06/2010(H3N2),the characteristics of r-06/HA and r-06/HA L222W,r-06/HA R326K,r-06/HA L222W +R326K mutant strains were tested in vitro and in vivo.Plaque formation test and MDCK cell growth curve showed that compared with r-06/HA,the three site-directed mutant viruses had greater plaque,a shorter plaque-forming time and a higher virus titer.In addition,mutant strains could cause more cell apoptosis than r-06/HA.However,the virus growth curve on MDBK cells showed that the virus titer of r-06/HA was significantly higher than that of r-06/HA L222W and r-06/HA L222W + R326K at 24h.The results of the mouse test showed that the mutant strains had more weight loss and higher lung viral load as compared to r-06/HA.It is interesting to note that the results of r-06/HA L222W + R326K were between r-06/HA and r-06/HA L222W,r-06/HA R326K,both in vitro and in vivo.Chicken red cell binding and release test confirmed that r-06/HA L222W + R326K affected the balance of HA and NA.3 In vitro coinfection of canine influenza virus and Staphylococcus pseudintermediusTo investigate the coinfection of CIV and Sp in Madin-Darby canine kidney(MDCK)cells,a in vitro coinfection model of CIV and Sp was established and optimized.The experiment was divided into four groups:CIV-only group,Sp-only group,CIV/Sp group and control group.The results showed that,at 24 h of virus incubation,CIV/Sp group produced more significant cell injury than CIV-only or Sp-only group(P<0.001),with the cytotoxity of 39.7%.And,when compared with CIV-only group,the viral RNA loads in CIV/Sp group were significantly lower(P<0.001).Compared to the Sp-only group,bacterial adhesion and invasion levels for MDCK cells in CIV/Sp group were significantly increased at 4 h of bacterial incubation(P<0.001).The expression levels of proinflammatory cytokines in coinfected cells,such as interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)were significantly enhanced as compared to other three groups at 6h of bacterial incubation(P<0.001).In conclusion,HA L222W and HA R326K enhanced the replication ability and pathogenicity of H3N2 canine influenza virus,and the preincubation of CIV enhances Sp secondary infection of cells.