Rescue Of Canine Influenza Virus CIV-GFP And Its Analysis Of Biological Characteristics In Vitro And In Vivo

Canine influenza virus(CIV)is a member of the influenza A virus,influenza virus genus,Orthomyxoviridae.The main species in the canine population are the CIV H3N8 derived from equine influenza virus and the CIV H3N2 derived from avian influenza virus.The dogs infected by CIV can show clinical symptoms such as elevated body temperature,coughing,depression,and reduced food intake.Severe dogs can develop secondary infections and eventually die.As the number of pets increases,the opportunities for dogs to be in contact with human beings have increased.As a"natural mixer"for influenza viruses,dogs may be a potential threat to human health after being infected with influenza.Therefore,the study of CIV is of great significance to public health.Currently,the research of the CIV's marking in cells or in vivo dynamics tracking after infection is empty.The reason is that we don't have a recombinant virus with marker tracking that is function genetically stable and easy to express.Green fluorescent protein(GFP)emits green fluorescence at a wavelength of 510 nm after being excited by 395 nm near-ultraviolet light or 470 nm blue light under Ca²⁺catalysis.This reaction requires neither the participation of the enzyme nor the substrate.The photoprotein is non-toxic to the cells,has a stable structure,and not only has a long-lasting effect,but can be directly detected inside the cells.So,the use of GFP protein to achieve accurate localization of virus in vivo or cells and to track the dynamic process after infection has broad application prospects in viral clinical research.but there is still no research on the application of GFP labeling to CIV.The experiment is to insert a GFP gene into the NS protein of CIV H3N2,and introduce two silent mutations at the No.6 site and No.504 site of the NS frame to prevent splicing of NS fragment m RNA.Then,we add a 2A-self-proteolytic cleavage site to modify the NS1-GFP and NEP expressing as single polyproteins.The CIV-GFP are rescued using the 8-plasmid reverse genetics system.The rescued virus has no significant difference in the characteristics of HA titer,EID₅₀ and TCID₅₀ from the wild strain,and the rescued virus can infect MDCK cells and express GFP protein,and basically fits the growth curve of CIV on MDCK cells.Although CIV-GFP may gradually lose GFP during multi-cycle replication on MDCK cells,we also found that nearly 10%of the virus populations were positive for GFP after 4 generations.We consider that the marking of the virus can be done by CIV-GFP in the initial generations.After the recused virus infected BALB/c mice,the clinical symptoms were consistent with the symptoms of CIV infected mice.Fluorescent markers were observed in the cryosections of lung tissues,and the imaging of living bodies could mark the virus in mice.In summary,this experiment successfully rescued the CIV-GFP carrying the GFP gene,and its characteristics and pathogenicity are not significantly different from the original strain.The GFP gene can be stably expressed after infection of MDCK cells and BALB/c mice,and plays a role in tracking the dynamics of the labeled virus.This experiment provides methods and materials for basic research and pathogenicity research of CIV H3N2,and provides a basis for the development of CIV vaccine.