| Newcastle disease(ND)is an acute,highly contagious infectious disease of poultry caused by Newcastle disease virus,with a high incidence and fatality rate,which has caused huge economic losses to the global poultry industry.The World Organization for Animal Health(WOAH)listed it as a legally reported animal disease,and China’s Ministry of Agriculture and Rural Affairs listed it as a Class II animal disease.Newcastle disease virus(NDV)can infect at least 236 species of poultry,of which domestic chickens and pearl chickens are the most susceptible.At present,the virus is seriously polluted in some areas of China,and there is a risk of virus mutation and new genotypes,and the epidemic and spread cannot be ignored.Vaccine immunization is the main means of prevention at present.According to the agricultural industry standard NY/T4045-2021 of the People’s Republic of China,the choice of vaccines is mainly attenuated live vaccine and inactivated vaccine,among which La Sota strain is the main strain to be prevented.goose parvovirus infection(GP),also known as gosling plague,is an acute or subacute septicemia infectious disease of goslings caused by goose parvovirus,often with septicemia,high incidence and fatality rate,and great harm to the production of geese.Among the commercial vaccines,live attenuated vaccines have good immune effect,but poor safety,and there are risks of strong virulence and dispersal.The production cost of inactivated vaccine is high,and it can not activate effective cellular immunity and local mucosal immunity,so the protection level is limited.Traditional vaccines have the problem of single protection,which is not matched with the circulating strains in some areas,and can not provide effective immune protection,and can not distinguish the antibodies against viral infection and vaccine immunity.The analysis of clinically suspected cases in Northeast China showed that the circulating strain of NDV was genotype VII.With the widespread occurrence of atypical symptoms of NDV,the expansion of the host infection range,and the migration of birds,there were reports that NDV was prevalent in waterfowl,and it was the same genotype Ⅶ as the dominant epidemic strain.In recent years,the isolation and identification of geese NDV strains in many areas showed that there was no characteristic variation in the prevalent NDV.Some studies have suggested that goose NDV is more harmful to the body by causing the release of a large number of inflammatory particles,and the recent strain isolated in Guangdong Province can spread rapidly between chickens and geese through contact,which is bound to have a strong impact on the goose farming industry.In order to implement the concept of modern green and safe breeding,promote the purification of gosling plague and goose paramyxovirus disease,and break the restrictions of these diseases on China’s goose breeding industry,it is imperative to develop new vaccines.This study used NDV VLPs as a platform to construct a new vaccine with "one injection and two prevention",and selected the popular ND commercial vaccine La Sota strain and the live gosling plague vaccine(SYG41-50)for comparative analysis,and finally obtained the virus-like particle vaccine with good immune effect:(1)The extracellular domain of NDV HN gene was replaced with the extracellular domain of gosling plague VP3 gene by the strategy of extracellular domain replacement;(2)The HN and M genes of NDV were combined with VP3 genes of GPV through the insect baculovirus expression system to form chimeric virus-like particles;(3)The immune effect of the new vaccine was evaluated through animal experiments.Through different immunization doses,immunization methods,etc.,the best vaccine groups were evaluated,and better immunization programs were formulated,providing new strategies for virus purification and green and safe breeding.1.Construction and identification of bivalent chimeric virus-like particlesAccording to the sequence information of goose parvovirus GD-01 strain(Gen Bank: DQ665790)registered in Gen Bank,the GPV VP3 gene was added with enzyme cleavage site and sent to the biological company for synthesis.The ECtodomain of HN gene of NDV NA-1 strain was replaced with the ectodomain of VP3 gene of GPV GD-01 strain by the ectodomain replacement strategy,and the ectodomain of VP3 gene was combined with the transmembrane region and ectodomain of HN gene by the overlap extension PCR technique,which was named VP3.After a large number of target genes were amplified by T vector,they were introduced into p Fast Bac 1 vector,identified by sequencing,transformed into the receptor state DH10 Bac,extracted recombinant rod-rbacmid-VP3 according to the reagent instructions,and then transfected into Sf9 cells separately to obtain r BV-VP3 recombinant baculovirus,and supernatant was collected after multiple passage.Recombinant baculovirus r BV-VP3 was constructed using the insect baculovirus expression system,and the recombinant baculovirus r BV-NA-1 HN,r BV-NA-1 M(preserved in laboratory)and r BV-VP3 were inoculated into sf9 cells in suspension culture with MOI=5.Supernatant was collected after 96 h culture at 26 ℃ and 180 r.They were identified by Western blot and transmission electron microscopy.The results showed that the target protein was correctly expressed and the VLPs were successfully assembled.The morphology of the VLPS was similar to that of wild NDV virions,all of which were nano-sized particles.In other words,a bivalent virus-like particle vaccine containing HN and M proteins of NA-1 strain and VP3 proteins of GD-01 strain was successfully constructed.2.Evaluation of immune effect of bivalent chimeric virus-like particlesThe immune effect of the virus-like particle vaccine was evaluated,and16-day-old goslings were selected for experiment.Goslings were first immunized twice,and control groups were set up for better analysis,including the commercial vaccine La Sota group,the live goslings plague vaccine(SYG41-50)group,and the PBS group.After two weeks of immunization,NDV NA-1 and GPV GD-01 strains were selected to attack the goslings.The observation results were as follows:(1)survival rate: no goslings died in the c VLPs group,and the goslings achieved complete protection;There were no deaths in the La Sota group.Gosling plague vaccine(SYG41-50)group died in part;(2)ND antibody level: There was no significant difference between the c VLPs group and the commercial vaccine La Sota strain group,but the antibody level of the c VLPs group began to decline after the challenge and then returned to the highest level,which was related to genotype matching;(3)GP antibody levels: The antibody levels of c VLPs group were similar to those of gosling plague vaccine(SYG41-50)group;(4)In vitro detoxification: c VLPs group had a shorter detoxification time in vitro than other vaccine groups.After the challenge,the goslings in the c VLPs group began to reduce detoxification on the 5th day and basically stopped detoxification on the 10 th day,while the goslings in the SYG vaccine group and the La Sota vaccine group still had detoxification on the 10 th day and stopped detoxification on the 14 th day.(5)In vivo viral load: The c VLPs group had a lower viral load than the other vaccine groups.With the increase of infection cycle,the virus replicates in the goslings and transfers to different tissues and organs.The amount of virus in the tissues and organs of the c VLPs group decreases on the 5th to 7th day,and the virus is completely cleared on the 14 th day.In the SYG vaccine group and the La Sota vaccine group,the amount of virus in the body began to decrease on the 7th to 10 th day,and the virus could still be detected in some organs on the 14 th day.To sum up,when the genotype matches the circulating strain,it can not only maintain a high antibody level and ensure the survival rate,but also reduce the viral load and shorten the detoxification time,which indicates that c VLPs is well expressed in goslings and can activate the immune response of goslings to resist the attack of strong virus,which is an ideal new vaccine.In the current situation that the epidemic has not been completely controlled,the development of the vaccine can lay a foundation for the elimination of wild poisons in farms and natural environments,and provide a new strategy for green and safe breeding. |
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