Epidemiological Investigation Of New Canine Influenza Virus And Experimental Vaccine Construction

Canine influenza was a disease that caused respiratory symptoms in dogs.It was first occurred in the United States during 2004.After that,an epidemic of canine influenza was also found in our country among 2005.Further studies have found that the H3N8 line of the American dog flu epidemic was caused by cross-infecting dogs with equine influenza virus and undergoing adaptive mutation.In China and South Korea,however,the canine influenza epidemics were directly cross-infected dogs by avian H3N2 influenza virus and spread among dogs.After 2016,the United States and China had successively discovered the prevalence of the noval H3N2 subtype canine influenza virus.Thus,it was urgent to understand the prevalence of canine influenza viruses in our country to evaluate the cross-species infection capacity,public health risks of the new H3N2 subtype canine influenza viruses and develop new vaccines.In 2014 to 2019,1185 swab samples from dogs with respiratory symptoms were collected from 4 provinces in China.We isolated 5 strains of canine influenza virus.The complete genome sequencing found that the 5 viruses selected were all noval H3N2subtype canine influenza viruses,and it was highly homologous with the avian H3N2subtype.Evolutionary analysis suggested that the canine influenza viruses circulating in China,South Korea and the United States all belonged to the same evolutionary branch group after 2017,which indicating that the avian influenza virus interspecies transmission from the A/Canine/Jilin/Qiaoke01/2017(H3N2)was a noval type of canine influenza virus strains represented by replacing the original.Subsequently,we analyzed 3579 sera from 27 provinces and cities,including pet dogs,free-range dogs,and stray dogs.The resul(?)ts demostrated that the virus had been widespread in our country and it was the most widespread among stray dogs.The positive rate of antibody in pet dogs with better breeding and management conditions were able to reach 18.89%.In order to evaluate the infectious ability and public health risk of the new canine influenza virus,the infection and transmission ability of the virus to canine,avian and mammalian model(mice and guinea pigs)were respectively carried out.Infection and dissemination experiments found that dogs were highly susceptible to novel H3N2canine influenza viruses,and viruses were highly transmitted in dogs.The replication ability of the virus infection in SPF-chickens was extremely low,and virus replication was not detected in ducks,as well as virus was not found to be transmitted between avian.Theses findings suggested that the virus had lost the ability to retransmit back to the poultry through adaptation in the dog herd.Mammalian model infection and transmission results found that the novel H3N2 canine influenza virus was able to directly infect mice and guinea pigs without adaptation and could be spread horizontally in the latter through contact mode,which suggested that the virus had certain public health risks.Additionally,we constructed novel H3N2 canine influenza virus vaccine candidate virus based on the PR8/31 backbone by using reverse genetics technology.After the cultivation of full suspension MDCK cells,the blood coagulation was reached 2048,and the viral TCID₅₀is 10^10.5/m L.It was 4 times that of the chicken embryo,which can improve the production of vaccines,and cost savings.And then,a vaccine antigen with HA value of 8192 was prepared through inactivation concentration and purification.Furthermore,we obtained mouse-adapted virus strains that were able to cause mouse death and established noval canine influenza virus mouse lethal infection model through continuous passage of mice.Through the evaluation of this model,it was found that the HI antibody in mice reached 2048 after a single immunization,which was able to resist the lethal infection of the new canine influenza virus.Furthermore,it was proved that 2560 HAUs antigen was able to provide complete protection and the immunization duration of dogs reached 180 days through a series of experiments(such as target animal minimum immunization dose and efficacy study,dog immunization duration detection,immune protection evaluation,etc.,),which could be offered safety and effective immune protection for dogs.This study proved that the novel canine influenza virus had become the main epidemic strain in the dog population among our country,and the risk of the virus returning to the poultry population was low,but there were public health risks of infecting humans and other mammals.Thus,it was necessary to keep monitoring and attention.Through the research of candidate vaccines,we prepared virus seed-bank based on the reverse genetic platform,explored the production process,and established mouse alternative evaluation model.These had laid the platform foundation and technical support for the declaration and application of canine influenza vaccine.