Pig δ Study On Cell Passage Weakening And Immunogenicity Of Coronavirus CHN-SC2015 Strain

Pig δ Coronavirus （PDCoV） is a new coronavirus that mainly causes vomiting,watery diarrhea,and death in piglets,posing a serious threat to the development of the pig industry.So far,there is no commercial vaccine approved for use in PDCoV,and vaccine development is an urgent need for the prevention and control of this disease.In this study,the PDCoV virulent strain CHN-SC2015 was used as the material to carry out the cultivation of cell passage attenuated strains,and the obtained attenuated strains were subjected to S gene and pathogenicity identification,as well as immunogenicity evaluation of the attenuated strains,laying a solid foundation for the development of a PDCoV attenuated vaccine.The main contents of this study include the following two aspects:1.Cell passage weakening and pathogenicity evaluation of CHN-SC2015 strainIn order to evaluate the biological characteristics,S genome changes,and pathogenicity changes caused by continuous passage of CHN-SC2015 onto foreign cells,this study conducted continuous passage on LLC-PK1 cells,and monitored virus titer changes every 10 generations,analyzed S gene changes,and selected P20,P80,P120,and P180 to orally infect 5-day old piglets to verify their pathogenicity.The results showed that the viral titer of CHN-SC2015 strain gradually increased during cell passage.Before P40generation,the viral titer increased rapidly,from 10^4.2 TCID₅₀/m L to 10^9.4 TCID₅₀/m L,and after P80,the viral titer remained relatively stable,maintaining between 10^8.5 TCID₅₀/m L and 10^10.4 TCID₅₀/m L,reaching the highest level in P160 generation(10^10.4 TCID₅₀/m L);No base deletion or insertion occurred in the S genome of different generations,but there were nucleotide and amino acid mutations,with a total of 33 amino acid mutations occurring;The pathogenicity results showed that P20 can cause significant diarrhea symptoms in piglets,with significant intestinal tissue lesions;The virulence of P80 and P120 decreased significantly,and one piglet in each group still suffered from diarrhea and pathological damage;The virulence of P180 has been completely weakened,and the infected piglets have been healthy throughout the entire process,with no significant intestinal lesions.2.Evaluation of immunogenicity of CHN-SC2015 cell attenuated strain（P180）in pigletsThis study further evaluated the immunogenicity of CHN-SC2015 attenuated pigs（P180）.P180 was selected to prepare an attenuated vaccine.Five 5-day old piglets were immunized with intramuscular injection twice.The first intramuscular injection was 10⁶TCID₅₀/m L（1 m L）,with an interval of 14 days.The second intramuscular injection was10⁶TCID₅₀/m L（2 m L）,and blood was collected every week（on the 0 th,7 th,14 th,21 st,and 28 th days after immunization）.ELISA was used to monitor Ig G and Ig A in piglets,and Plaque reduction neutralization test（PRNT）was used to monitor the level of neutralizing antibodies.After the end of the immune experiment（the 28 th day）,2 m L of tissue toxin(10⁸ TCID₅₀/m L)was used for evaluation of the attack protection experiment,and clinical symptoms,pathological changes,and fecal detoxification were monitored.The results showed that the Ig G antibody turned positive in the first week after immunization and reached a peak in the third week;Ig A antibody was positive in the first cycle and reached a peak in the fourth week,but the antibody level of 2 piglets did not significantly increase;The first circulating positive of neutralizing antibody,the neutralizing antibody level has been increasing during the immunization process until the end of the immunization experiment,and its highest antibody titer is 1:256 at the fourth week;The results of the challenge test showed that P180 could provide 100%immune protection,and all five immunized piglets were normal.No obvious pathological changes were found on autopsy,and no virus particles were found in the intestinal tract by immunohistochemistry;In the control group,there were typical intestinal lesions,severe lesions on pathological sections,and a large number of viral particles were observed by immunohistochemistry.In summary,the results indicate that P180 cell attenuated strain has very good immunogenicity and can induce strong neutralizing and protective effects,making it a candidate strain for developing attenuated vaccines.