Molecular Mechanism Of Porcine Deltacoronavirus Entry And Immune Protection Evaluation Of Vaccine

Porcine deltacoronavirus?PDCoV?is a newly identified pathogen belonging to the Coronavirus family,a member of the?coronavirus genus.PDCoV can infect all days old pigs which showing diarrhea,vomiting and dehydration.Vaccine immunization,especially passive immunization in sows,is the most effective method for preventing and controlling virus infection,such as Porcine epidemic diarrhea virus?PEDV?,Transmissible gastroenteritis virus?TGEV?,Porcine rotavirus?PoRV?and PDCoV.At present,PDCoV vaccine is not available.Therefore,the preparation of inactivated vaccine and immune evaluation are particular important for the prevention and control of PDCoV.In this study,the pathogenicity of PDCoV isolate NH P10 was first studied in 5-day-old SPF pigs.The results showed that typical clinical symptoms after the piglets were orally infected with PDCoV(10⁴ TCID₅₀,1 mL/pig).Thinning intestinal wall was observed and fulled with pale yellow liquid.HE staining showed that the intestinal villi of jejunum and ileum showed obvious atrophy,shortening and shedding at 72 h after infection.The jejunum and ileum are the main sites of PDCoV infection.Different from the previous reports,the clinical symptoms of vomiting did not occur after infection with the PDCoV NH strain.Pathogenicity experiments provide a challenge model for studying the protection efficiency of passive immunity on newborn piglets.At the same time,this study investigated the molecular mechanism of PDCoV entry in IPI-2I cells.The results showed that PDCoV can infect cells through two pathways,a trypsin-mediated one at the cell surface and another facilitated by cathepsins in the endosome.Further studies have shown that viral entry via the cell surface pathway is 10¹00 fold higher than that of the endomesome pathway.After viral infection,the expression of CTSL and CTSB was increased in vivo and in vitro,accompanied with the increase of the enzyme activity of CTSB in vitro.To evaluate the protection effect of passive immunity of inactivated PDCoV vaccine,all sows were immunized 40 days prior to delivery and a booster was administered 20 days before delivery at Houhai point.Five days after delivery,the piglets were challenged with PDCoV virulent virus.The passive immunoprotective efficiency was evaluated according to clinical pathogenesis,pathological damage and virus release.The results suggested that the inactivated PDCoV vaccine can induce IgG and neutralizing antibody responses in the serum and milk of sows.Piglets can obtain the antibodies by drinking milk and the challenge experments showed that the inactivated vaccine can provide 87.1%passive immune protection rate for piglets.In addition to passive immunization to provide passive immune protection for piglets,active immunization can increase the immune level of pigs and reduce the risk of PDCoV infection.First,we simulated natural infections,the 25-day-old SPF pigs were orally infected with PDCoV strain NH and investigated the antibody response after infection.The results showed that PDCoV infection caused caused clinical symptoms of diarrhea in 25-day-old weaned pigs at fifth day.Antibody response showed that antibody levels increased rapidly after the third oral infection and high levels of IgA were detected.To evaluate the effects of active immunization,we adopted three immunization strategies:inactivated vaccine twice vaccination?K+K?;live vaccine first vaccination,and booster with an inactivated vaccine?L+K?;live vaccine twice vaccination?L+L?.The results showed that neutralizing antibody became positive two weeks after immunization by the three immune strategies,and the antibody level increased rapidly after booster immunization.The level of neutralizing antibody in the K+K immunized group was higher than the other two groups.Furthermore,two weeks after immunization,the K+K immunized group can detect high levels of IFN-?in serum,indicating that inactivated vaccine immunization can also activate cellular immune responses.The results of challenge experiments showed that the protective effect of K+K immunized group was higher than the other two groups.The above results indicate that immunization twice with an inactivated vaccine can induce a better neutralizing antibody response and provide protection for pigs.