Research Of Inactivated Vaccine Of GEL01 Adjuvant To H3N2 Subtype Canine Influenza Virus

Canine influenza?CI?is a canine respiratory disease caused by the Canine influenza virus?CIV?of the Orthomyxoviridae family.The main clinical symptoms of sick dogs showed fever,runny nose,cough,sneezing,depression,loss of appetite and so on.The number of dogs in the world has about 4 billion,about 150 million pet dogs in China,as one of humanity's most important companion animals,most likely as a potential intermediate host for the spread of influenza virus to humans.At present,it has been found that H1N1,H3,H5,H9 and H10 subtypes CIV can infect dogs.H3N2 subtype CIV is the main epidemic strain until now.Since H3N2 CIV was first discovered in China in 2006,Asia is pandemic in 2015 and has spread to the United States and the world.However,only the H3N8 and H3N2 subtype CIV inactivated vaccine approved by the United States for the CIV vaccine has been researched and only the unlisted H3N2 subtype CIV vaccine has been developed in Korea.As of 2018,there is no effective vaccine research on H3N2 subtype CIV in China.Therefore,the development of H3N2 subtype CIV vaccine has great significance for the prevention and control of canine influenza virus.In this study,four CIV strains A/canine/Guangdong/01/2014?H3N2??GD1401?,A/canine/Guangdong/02/2014?H3N2??GD1402?,A/canine/Guangdong/03/2014?H3N2??GD1403?and A/canine/Guangdong/04/2014?H3N2??GD1404?isolated from animal hospital in 2014 winter were inoculated with SPF chicken embryos for subculturing.The results showed that only GD1404 strains HAU stabilized at 2⁷²⁸ titer in the eighth generation until ten generations.HA and NA gene sequence analysis of the original virus homology was 100%,can be identified as vaccine candidate strains.And it was used as F1strain in SPF chicken embryo passage for the establishment of species library.The F5progeny in seed banks was used to infected 10-week-old Beagle dogs to establish the challenge model for vaccine research.The results showed that the best route of infection was intranasal inoculation and the minimum infection dose of puppies was 10³ 50%egg-infectious dose(EID₅₀).The vaccine strain was inactivated at 4?⁸?and 100 rpm shaking at different concentrations and times by three different inactivators:formaldehyde,diethyleneimine?BEI?and?-propiolactone?BPL?.The optimal inactivation conditions were obtained from the degree of inactivation and the hemagglutination titer before and after inactivation with BPL inactivator at a final concentration of 0.025%for 48 h.Inactivated GD1404 strain was mixed with GEL01 adjuvant according to the ratio of92:8 to prepare the inactivated vaccine,and the ten-week beagle dogs were subjected to animal safety experiment and challenge experiment.Experiments show that the inactivated vaccine is safe and effective for dogs.The HI antibody was 1:40 on the 7th day after the first immunization,and the HI antibody reached the highest on the 14th day after the challenge with the CIV,which was 1:5120.And the clinical symptoms of CIV did not appear after the challenge,the body temperature was also maintained at normal levels,and only one canine nasal swab virus titer in the immune group appeared detoxification on the2nd day.In this study,GD1404 strain was selected as the vaccine strain.Through the optimization of inactivation process and the establishment of a scientific CI clinical symptom score standard,a safe and effective GEL01 adjuvant inactivated vaccine was prepared,which is the public health significance of CIV provide an important basis.