Evaluation Of Immune Effect Of Recombinant Newcastle Disease Virus RmNA-VP3 Expressing Goose Parvovirus VP3 Gene

Newcastle disease（ND）and Goose parvovirus（GP）are serious damages to the goose industry in China caused by Newcastle disease virus（NDV）and Goose parvovirus（GPV）,respectively.Both diseases have high mortality rates.Vaccine is an effective means to prevent and control ND and GP.However,with the continuous evolution of the virus,changes in the epidemic trend,and the strengthening of national defense control,the traditional traditional ND and GP attenuated vaccine strains can no longer meet the needs of epidemic prevention.NDV has only one serotype,but there are many genotypes.The current NDV epidemic is dominated by genotype VII strains.However,most commercial ND vaccine strains are genotypes II and III,which are more homologous to genotype VII strains.Low,genetic distance is relatively long.When the genotype NDV virus attacks poultry,traditional vaccines cannot provide complete protection,and can isolate NDV virus strains from immunized poultry,which is not conducive to the prevention and purification of ND.Therefore,the use of vaccines that match the genotype of the epidemic strain is an inevitable trend in the prevention and control of ND.GPV has strict species specificity,and only goose embryos or duck duck embryos can be used in the production of attenuated vaccines,which results in a lot of inconvenience and cost in the production of the vaccines,and there is a risk of latent infection in the use of attenuated vaccines.The development of a new GP vaccine is inevitable.In order to make up for the shortcomings of traditional vaccines and to be able to prevent and control ND and GP at the same time,in the early stage of this laboratory,the goose-derived gene type VII NDV NA-1 strain was used as the research object,and the NDV reverse genetic operation platform was constructed.Weakly mutated the amino acid sequence of the F protein cleavage site of the NDV virulent strain NA-1strain into the corresponding sequence of the weak virus strain LaSota F protein,constructed a recombinant NDV rmNA-1,and inserted the VP3 protein genome of the main protective antigen of GPV on this basis.A recombinant NDV rmNA-VP3expressing the GPV VP3 gene was constructed.This study evaluated the stability,safety,and clinical immune effect of recombinant NDV rmNA-VP3 to determine whether it has potential as a new generation of ND and GP vaccine candidate strains.Ⅰ.Detection of genetic stability of recombinant NDV rmNA-VP3In order to test the genetic stability of recombinant NDV rmNA-VP3,rmNA-VP3 was serially passaged in chicken embryos and chickens.The rmNA-VP3was continuously passaged to the 25th generation in SPF chicken embryos.After amino acid sequence analysis,the amino acid sequence of the cleavage site of the rmNA-VP3 F protein did not undergo a strong mutation;rmNA-VP3 was passaged in chickens for 5 consecutive times.According to amino acid sequence analysis,no strong mutation occurred in the cleavage site of F protein.The results showed that rmNA-VP3 can be stably inherited in chicken embryos for at least 25 generations and in chickens for at least 5 generations,indicating that recombinant NDV rmNA-VP3has a relatively high Good stability.Ⅱ.restructuring NDV rmNA-VP3 laboratory safety testSafety is one of the important indicators for investigating vaccine quality.In this study,the safety of recombinant NDV rmNA-VP3 was evaluated by a single dose multiple vaccination test and a single overdose vaccination test.Recombinant NDV rmNA-VP3 was inoculated with a dose of 10⁶EID₅₀（0.2mL）into the 16-day-old chicks by subcutaneous injection in the neck.Two weeks later,the same dose was used for the same inoculation method.The chicks were clinically observed for two weeks after inoculation.It was found that the goslings behaved normally,and no pathological changes were found in necropsy tissues and organs.The 16-day-old goslings were inoculated with 10 times the regular inoculation dose.The goslings were clinically observed for two weeks after inoculation.The goslings behaved normally,and there was no pathological change in the tissues and organs under autopsy.It was confirmed that the recombinant NDV rmNA-VP3 is safe.Ⅲ.Evaluation of immune effect of recombinant NDV rmNA-VP3In order to evaluate the clinical protective effect of the recombinant NDV rmNA-VP3,the recombinant NDV rmNA-VP3 was immunized twice to 16-day-old and 30-day-old goslings,and two weeks later,the genotype NDV virulent strain NA-strain and GPV virulent strain were used.The challenge test was performed,and the ND commercial vaccine LaSota group,the GP commercial vaccine SYG _41-50 group,and the PBS control group were set up to further evaluate the clinical protective effect of rmNA-VP3 by comparison.Survival observations showed that rmNA-VP3 was able to provide 100%protection to goslings.ND antibody monitoring results show that the antibody levels of the rmNA-VP3 group and the LaSota vaccine group are almost the same,but the ND antibody level of the rmNA-VP3 group will first decrease and then rise to the highest level after challenge.This is due to the genotype of the vaccine strain and the virulent strain.Due to a match.GP antibody levels showed that the antibody levels in the rmNA-VP3 group were similar to those in the SYG 41-50 group.Detoxification tests found that after challenge,the geese in the rmNA-VP3 immunized group had less detoxification,and the detoxification time was earlier than that in the ND and GP commercial vaccine groups.Examining the virus distribution in the goose except the goose found that the virus distribution in the rmNA-VP3 immunized group was less and the virus clearance was faster in the body.This shows that when the genotype of the NDV vaccine strain matches the epidemic strain,the vaccine protection effect is more ideal.At the same time,the VP3 gene is well expressed in the goslings,which can induce the immune response of the goslings and help the goslings to resist the attack of GPV.