Effect Of Amino Acid Substitution At PB2 E627K And D701N Sites On The Pathogenicity Of H3N2 Subtype Canine Influenza Virus

Canine influenza virus?CIV?is the influenza A virus that can cause clinical symptoms such as fever,sneeze,cough,runny nose and loss of appetite in canines.Canines had been considered not to be infected with influenza virus for a long time.Since 2004,the case of canines were dead by CIV in Florida,USA,which had aroused people's concern for CIV.We isolated CIV H3N2 for the first time in 2010.CIV H3N2 is low pathogenic influenza virus,and all of them are avian-derived H3N2 influenza viruses by evolutionary tree analysis.After years of genetic evolution,they had been adapted to canines and steadily circulating in canines.Nowadays,lots of canines have become the companion animals that contract closely with human beings.CIV has become a potential factor for human public health.Therefore,it is significant for researching the CIV.The basis of the evolution of influenza virus in nature is the occurrence of gene mutations and recombination,and genetic mutations are more prone to occur.Studies had shown that there are some key amino acid sites in PB2 protein of influenza A virus,playing an important role in replication or pathogenicity of the influenza virus in mammals.Mutations in the E627K and D701N amino acids were the hot spots.Meanwhile,the effects of E627K and D701N substitutions are most certainly.To explore the effects of amino acid substitution at PB2 E627K and D701N sites on the pathogenicity of CIV H3N2.Site-directed mutagenesis technology was used to perform mutations on the pHW2000-PB2 plasmid of A/canine/Guangdong/02/2011?C/GD/02?reserve genetic system.Four rescue strains were obtained by reverse genetic technique:PB2 E627K,rC/GD/02-627K,PB2 D701N mutant strain rC/GD/02-701N,PB2E627K/D701N double-mutant strain rC/GD/02-627K/701N and original strain rC/GD/02.The biological characteristics showed that the original strain had a higher HA titer.The EID₅₀0 and TCID₅₀0 of three mutant strains were higher than those of rC/GD/02 strain.The growth curve of each mutant strains in MDCK cells showed a slight decrease in replication ability.Polymerase activity test results showed that mutant strains significantly enhanced the polymerase activity?P<0.05?,of which PB2 627K/701N mutation caused the most significant increase in polymerase activity?P<0.01?.The mice experiment showed that all rescue strains could injury the lungs and trachea of mice,but none of the strains caused the death of mice.Meanwhile,there was no significant difference among the strains on the body weight of mice.These results showed that neither mutation of PB2 E627K nor D701N could enhance the pathogenicity of CIV H3N2 in mice.The beagles experiment showed that all rescue strains did not cause beagles with high temperature,but could cause pneumonia with congestion,bleeding and lesions in lung.All the strains could lead lung,trachea and turbinate of canines to different extent of pathological tissue lesion.The antigen of CIV could also be detected.Each strain could cause the beagles to expelling toxin continuously for 9 days¹0 days.The rC/GD/02-627K and rC/GD/02-701N also showed a phenomenon of intermittent anal detoxification.At the same time,neutralizing antibodies began to be produced on the fifth day after infection and could persist in the blood for about100 days.However,there was no significant difference in pathogenicity among the all beagles that infected the each strain.Therefore,it was indicating that the mutation of PB2E627K and D701N did not enhance the pathogenicity of CIV H3N2 in beagles.In summary,we successfully rescued the CIV PB2 E627K,D701N,E627K/D701N mutant strains and compared their biological characteristics and polymerase activities.Mice and beagles could infect with these mutant strains,but amino acid substitution at PB2E627K and D701N sites on the pathogenicity of H3N2 subtype had no significant effect.Thus,this study provides a data basis for the prevention and control of CIV H3N2.Meanwhile,it provided a reference and method for the study of the pathogenicity of CIV.