Construction And Biological Characteristics Of NDV LaSota-CCL19 Strain

Newcastle disease virus(NDV)is a highly contagious and pathogenic disease in chickens.NDV infection can induce the expression of antiviral interferon-responsive genes,resulting in the up-regulation of cytokines such as CCL19,CCL21,and CCL4 after NDV infection.Effector leukocytes to enhance cell-mediated responses,but whether the same effect exists after CCL19 recombination into NDV vaccine strains has not yet been reported.Therefore,in this study,a chimeric strain of CCL19 and La Sota was constructed,and its biological characteristics were studied to evaluate the newly constructed strain more comprehensively.1.The effect of CCL19 on M,HN transcription and HN protein expression of NDV La Sota strain.Through in vitro cell experiments,the laboratory-constructed eukaryotic expression vector pCMV-HA-CCL19 was transfected into DF-1 cells to overexpress CCL19,and DF-1 cells were inoculated with NDV La Sota-GFP virus(LS-GFP for short),and then qPCR and Western Blot were used to detect the effect of overexpression of CCL19 on the transcription and protein expression of M and HN in La Sota.The results showed that the chemokine CCL19 interacted with NDV infection,and overexpression of CCL19 significantly promoted the transcription of M and HN and the expression of HN protein in La Sota at 48 h(P<0.01).2.Construction of NDV LS-CCL19.The primers for the whole genomes of CCL19 and La Sota were designed by Primer premier software,the pLS vector and the target gene CCL19were amplified from the pLS-GFP and pET30a-CCL19 plasmids by PCR,and then the pLS and the target gene CCL19 were seamlessly cloned by In-Fusion.CCL19,and the recombinant plasmid pLS-CCL19 was screened and identified.After that,the helper plasmids P,NP,L and pLS-CCL19 were co-transfected into BHK-21 cells containing MVA-T7 RNA polymerase system to rescue the recombinant virus NDV La Sota-CCL19(LS-CCL19 for short),and the recombinant virus was inoculated into SPF chicken embryos.Multiplication and collection of allantoic fluid virus,detection of CCL19 and F genes in the allantoic fluid virus gene by PCR,identification of the whole genome of the recombinant virus,confirming that the gene sequence is correct,and then using the hemagglutination test to detect the activity of the recombinant virus LS-CCL19,determined that the recombinant virus LS-CCL19 was successfully rescued.3.Biological properties of NDV LS-CCL19.The collected LS-CCL19 and LS-GFP allantoic fluid were diluted by doubling and the virus titer of LS-CCL19 was 8?9 log2,and the virus titer of LS-GFP was 5 log2 by hemagglutination test.LS-CCL19(0.01 MOI)and LS-GFP(1MOI)were inoculated into DF-1 cells,respectively,and the activity and proliferation of the virus were detected.The results showed that both LS-CCL19 and LS-GFP could infect DF-1 cells normally,and consistent with the NDV proliferation law;LS-CCL19 and LS-GFP allantoic fluid were inoculated into SPF chick embryos by gradient dilution,the EID₅₀of LS-CCL19 calculated by Reed-Muench was 10^-10.46/mL,and the EID₅₀of LS-GFP was 10^-7.625/0.1 mL,the EID₅₀of the commercial vaccine(La Sota)was 10^-9.38/mL;LS-CCL19 and LS-GFP measured according to the OIE detection standard were MDT?120 h,ICPI=0.After challenge with NDV LS-CCL19 and NDV LS-GFP,only a small amount of virus was detected in the lung tissue at the early stage of infection,and the alveoli were not damaged by pathological section,indicating that LS-CCL19and LS-GFP are attenuated strains.After the commercial vaccine group(106 EID50),LS-CCL19A group(106 EID50),and LS-CCL19 B group(105 EID50)were immunized with 7-day-old SPF chickens,10 chickens in each group were given the primary immunization by nasal drip.Booster immunization was performed after 21 d.After each immunization,chicken wing inferior venous blood was collected 3 times every 7 d to obtain antibody serum.The hemagglutination inhibition test was used to detect the antibody titer.The HI test results showed that the LS-CCL19 antibody titer gradually increased.It increased to be stable,the highest antibody titer was 2.89±0.46 log2,and it had good humoral immunogenicity..Conclusion:The results of in vitro experiments confirmed that overexpression of CCL19 significantly promoted the transcription of M and HN and the expression of HN protein in La Sota;the NDV LS-CCL19 strain was successfully constructed,and it was an attenuated strain with good biological characteristics and humoral immunity originality.It provides an experimental basis for further research on the cellular immunogenicity and novel vaccine of NDV LS-CCL19 strain.