Studies On The Isolation And Identification,Animal Models And Therapeutic Antibodies Of The Feline Calicivirus Guangxi Epidemic Strains

Feline calicivirus?FCV?is a highly contagious pathogen.It can cause common oral and respiratory tract infections in cats,mainly manifested as clinical symptoms such as oral ulcers,rhinitis,conjunctivitis and upper respiratory tract diseases.The lethal rate of virulent strains is more than 50%,seriously endangering the health of cats.Due to the widespread prevalence of FCV,resulting in the continuous generation of mutant strains,the protective efficacy of existing vaccines is also declining,so it is of great significance to carry out research on FCV genetic evolution analysis and therapeutic drugs.At present,there is still no clinically effective drugs to treat cats infected with FCV,and it is necessary to develop safe and effective therapeutic drugs.Ig G accounts for about 75%of the total immunoglobulin.It is the antibody with the highest content in the serum produced during the humoral immune response.It has specific functions such as neutralizing antigen,activating complement and binding receptors.Therefore,it is the first choice for passive immunotherapy.In antiserum passive immunotherapy,in order to overcome the non-specific reaction caused by the binding of complete antibody molecules to cells with Fc receptors and allergic reactions caused by its antigenicity,Fab fragments or F?ab'?₂ were commonly used in place of intact antibody molecules.The selected strains with strong virulence,high price,and high homology with domestic popular strains in recent years were subjected to complete gene sequence determination and establishment of animal models.At the same time,prepared FCV inactivated vaccines to immunize animals,prepared high-efficiency F?ab'?₂ antibodies and carried out purification research,laid the foundation for the development of FCV therapeutic antibodies.This study consists of three stages:1.FCV isolation and identification,genetic evolution analysis and pathogenicity testIn this study,55 suspected FCV samples collected from 2017 to 2019 in Nanning City Pet Hospital,Cat House,Flower and Bird Market were detected by RT-PCR method,and 10positive samples were obtained,with a positive rate of 18.2%.After the positive samples were inoculated with fetal kidney passage cells?F81?,5 cases showed typical cytopathic effect?CPE?caused by FCV.Through sequencing and BLAST comparison,it was determined to be FCV,and a total of 5 FCV epidemic strains were obtained,named GXN1,GXN2,GX2019,GXN4 and GXN5.By measuring the TCID₅₀of 5 epidemic strains,3 of them were selected as high prices strains for genetic evolution analysis.It was found that the three isolates were in different evolutionary branches from the domestic vaccine strain 255 and the foreign vaccine F9 strain currently in clinical use,and there was a risk of vaccine immunization failure.Animal pathogenicity test found that GX2019 strain was the most pathogenic.Used for the next step of research.2.Complete gene sequence determination of FCV-GX2019 and establishment of animal modelsAccording to the conventional method,design 3 pairs of primers to amplify the whole gene of GX2019,and obtain the target gene sequence with a size of 7687 bp by spliced.The whole gene of GX2019 strain was uploaded to Genbank,and the accession number obtained was MK867378.Genetic evolution analysis found that this strain had the closest genetic relationship with the Jilin isolate CH-JL4.Compared with vaccine strain 255?domestic strain?and F9?foreign strain?,some key amino acid sites related to antigenicity had changed,which may lead to a reduction in vaccine protection.In order to build animal models,diluted the virus to different prices and inoculated by nasal drip route cats and established animal models.The results showed that after infecting cats with different doses of FCV-GX2019 strain,the cats in the challenge group showed clinical symptoms such as fever,increased eye corner secretions,tongue ulcers,and weight loss.The swab was tested positive for FCV on the 3rd day of infection,7 days later,there was a death situation in the test cats.After 14 days ofclinical and pathological changes of FCV such as alveolar wall thickening,alveolar erythrocyte and inflammatory cell exudation observed by histopathological section,which confirmed the establishment of the animal models.3.Preparation and purification of anti-FCV immunoglobulin F?ab'?₂A large number of GX2019 virus were cultured and TCID₅₀ was determined.After inactivated the virus with formaldehyde,mixed it with Tween-80 to make an aqueous phase.White oil,Siben-80 and aluminum stearate were boiled to make an oil phase,and mixed the aqueous phase with a homogenizer to make Oil-inactivated vaccine.After safety testing,rabbits were immunized subcutaneously,and horses were injected intramuscularly into the neck and buttocks.Serum was collected to determine the serum neutralization prices and serum Ig G antibody content.Ammonium precipitation method was used to purify Ig G,and the concentration of FCV-Ig G was measured.Then,the sample obtained by enzymolysis was purified through Protein A+G chromatography column by affinity chromatography to separate F?ab'?₂ fragments,measured the concentration of immunoglobulin F?ab'?₂,calculated the recovery rate of F?ab'?₂,and verified by SDS-PAGE electrophoresis and safe sterility test.