Preparation Of Hemagglutination Inhibition Test Antigen Of H3N2 And H5N1 Subtype Canine Influenza Virus

Canine influenza virus?CIV?belongs to the family Orthomyxoviridae,genus influenza virus A.CIV contain a segmented genome composed of eight single-stranded RNA molecules with negative polarity.Clinically,most dogs suffering from canine influenza exhibit a series of clinical symptoms similar to the symptoms of infectious tracheobronchitis.They are mainly characterized by fever,loss of appetite,elevated body temperature,depression,runny nose,cough and beating.Sneezing;occasionally severe pneumonia complications can occur.The hemagglutinin?HA?glycoproteins on the surface of the CIV envelope play an important role in the hemagglutination test and hemagglutination inhibition test?HI?.In recent years,canine influenza has been showing a certain trend in the world.The predominant subtypes are Equine-derived H3N8 subtype,poultry-derived H3N2 subtype,which cause severe respiratory diseases and even death in dogs.Pay attention.In this study,the following two strains of canine influenza strains were selected as the poisons:In 2014,H3N2 subtype CIV was isolated from dogs with influenza symptoms?A/canine/Guangzhou 04/2014?and in 2013 it suffered from severe respiratory diseases.The H5N1 subtype CIV isolated in dogs?A/canine/Guangdong/01/2013?;two strains were monitored through continuous passages,demonstrating their stable nature and excellent immunogenicity.Next,the following conditions were optimized for the two strains:optimization of the inactivation process of the two strains,optimization of the method for the treatment of the serum to be tested in the hemagglutination inhibition test,and discussion of the influence of different species of animal red blood cells on the hemagglutination inhibition test and preparation.The positive and negative control sera used in the hemagglutination inhibition test,the preparation of hemagglutination inhibition test antigens,and the best preservation method for inactivating antigens were finally calibrated to the standard of hemagglutination inhibition test procedures.The experimental results showed that the optimal chicken embryo proliferation condition of H3N2 subtype CIV was a virus dilution of 10^-4.Incubating at 37°C in a constant temperature environment for 48 h,the antigen inactivation condition was 0.025%concentration of?-propiolactone in Inactivated for 36 h under the condition of 4°C;the optimal proliferation condition of the chicken embryo of H5N1 subtype CIV was the dilution of virus 10^-7.Incubating at 37°C in a constant temperature environment for 48 h,the antigen inactivation condition was 0.025%concentration of?-propiolactone was inactivated at 4°C for 12 h.The optimal treatment method for the H3N2 and H5N1 test serum is the choose of receptor-destroying enzyme for the treatment,which can effectively eliminate the non-specific inhibitors that interfere with the accuracy of serum hemagglutination inhibition test.The hemagglutination inhibition test of H3N2 and H5N1subtype CIV can achieve the best results using chicken red blood cells.The positive control serum and negative control serum prepared experimentally have good specificity and sensitivity.The best method for the preservation of H3N2 and H5N1 antigens is vacuum freeze-drying,with a certain concentration of lyoprotectant?H3N2 lyoprotectant is 10%sucrose skim milk solution,H5N1 lyoprotectant is 5%sucrose skim milk solution?,certain the freeze-dried protective agent and the antigen?2:1 for H3N2,1:1 for H5N1?and a suitable lyophilization curve produced freeze-dried antigens with good physical condition and stable properties,which were H3N2 and H5N1 subtypes of canine influenza virus.Provides technical support for H3N2 and H5N1 subtype CIV hemagglutination inhibition test operations.