The Molecular Mechanism Of NA Protein Influencing Virus Replication For H3N2 Subtype Canine Influenza

Since 2007,A Asia's new kind of H3N2 subtype canine influenza virus?CIV?has been widely prevalent in our country,causing an acute infectious disease in dogs-canine influenza.In the study,H3N2 CIV originally derived from H3N2 subtype avian influenza virus,and then evoloved to infect dogs.In the preliminary experiments,the results demonstrated that H3N2 CIV could transmit among dogs via air,and also infect other mammals,such as mice and guinea pigs,but not avian species.However,the pathogenicity mechanism of the H3N2 CIV is not clear.It has been reproted that HA and NA play important roles in the pathogenicity of influenza virus.Thus,avian-origin HA or NA single gene exchanged homologue gene of H3N2 CIV,and then rescued those recombinant viruses with the backbone of H3N2 CIV.These viruses infected with BALB/c mice to determin the key genes and amino acids for H3N2 CIV replication,and then investigated the molecular mechanism.The concrete contents were below:1,Determine the key genes for H3N2 CIV caused illness.In the early study,two highly homology viruses were selected.One was canine influenza virus which caused dog illness A/canine/influenza virus/01/2010?H3N2??renamed C1?,and another was avain influenza virus A/duck/shanghai/06/2009?H3N2??renamed D6?,which did not cause dog illness.These two virues were rescued and recalled rC1 and rD6 respectively.The HA or NA gene singly exchanged with that of C1,and the recombinant viruses with the backbone of C1 were rescued successfully which named as C1-D6HA and C1-D6NA separately.These viruses were inoculated intranasally with 5-week old BALB/c mice in a dose of 10⁶EID_50.The result demonstrated that C1-D6NA could not hardly replicate in the lungs of mice although parent rC1 replicated well.Meanwhile,pathological histology observation found that only lungs of mice infected with rC1 or C1-D6HA had obvious pathological changes,but those infected with C1-D6NA were not.These results suggested that NA gene might be the key gene for CIV caused illness.2,Determine the key amino acids for H3N2 CIV caused illness.Compared a large number of sequences of NA proteins for H3N2 CIV and H3N2 avian influenza viruses with softeware of Lasergene 7.0,and focused on 4 amino acid residues.Acorrding to these positions,constructed 4 pBD mutation plasmids,and obtained their corresponding recombinant viruses which named as,C1-NA?L24M?,C1-NA?K54E?,C1-NA?S154P?and C1-NA?G430R?.In mice pathogenicity experiments,the result demonstrated that CIV-NA?K54E?and CIV-NA?S154P?could not replicate in the lungs of mice,but parental rC1 virus could replicate well.This result was the same as that replicated in the Madin-Darby canine kidney?MDCK?.Thus 54K and 154S of NA protein might be the key amino acids for C1.3,The molecular mechanism for H3N2 CIV caused illness.The main funtion for NA protein is enzymatic activity.Thus,in order to confirm whether 54K and154S caused the change of NA enzymatic activity,and then influence the pathogenicity of H3N2 CIV,rC1 and their NA mutation viruses were amplified by SPF?Specific pathogen free?embrynated chicken eggs,and then detected NA enzymatic activity.The result demonstrated that the enzymatic activities of C1-NA?K54E?and C1-NA?S154P?were lower than those of rC1,C1-NA?L24M?and C1-NA?G430R?.In a word,54K and 154S could obviously enhance the NA enzymatic acitivities.In order to confirm the increae of NA emzymatic activity influences the release of viruses,the two experiments of virus elution from chicken erythrocytes and progeny viruses releasing from MDCK were performed.The results of these two trials indicated that C1-NA?K54E?and C1-NA?S154P?could obviously decrease the viruses releasing from cellular membranes than rC1 and other 2 recombinant viruses.Therefore,54K and 154S can enhance NA enzymatic activities,and improve the releasing ablitity of virus from cellular membranes,and then are better for H3N2 CIV to replicate and infect dogs,which provides some theory basis for the effective control of H3N2 canine infuenza.