Optimization Of Reverse Genetic Manipulation Platform Of Newcastle Disease Virus La Sota C5 Strain And Construction And Immunogenicity Evaluation Of Class I/II Chimeric Attenuated Strain

1.Establishment of reverse genetic manipulation for La Sota C5The genome of Newcastle disease virus(NDV)La Sota C5 strain was divided into 4 fragments to amplify the whole genome.Subsequently the 4 fragments and the other fragments were cloned into pMD-20T vectors step by step and obtained the transitive plasmid T-ABCD containing the main parts of the La Sota C5 genome sequence.T-ABCD and TVT-LT were digested by restriction enzyme Bgl II,the target fragments were purified and ligated,then the cDNA clone of NDV La Sota C5 strain was constructed successfully.Three helper plasmids and the TVT-La Sota C5 were co-transfected into BSR-T7/5 cells which can express T7 RNA polymerase.The transfected cells were collected at 72 h post-transfection and freeze-thawing for 3 times.The mixtures of the collected aliquots were inoculated into specific pathogen-free(SPF)chicken embryos.The HA titers of the collected allantoic fluid were detected according to the OIE standard method.The recused virus was confirmed by RT-PCR and gene sequencing analysis.The HA titers of the first passage of the allantoic fluid is 28,after 3 passages the H titres was elevated to 211.All the results indicated the NDV La Sota C5 strain reverse genetic system was successfully developed,which provided an advanced platform for the future research of NDV and the associated NDV-based vector vaccines development.2.The construction and rescue of Class I and Class II chimeric virusesThe M/F/HN gene of the NDV field strain JS10 was amplified using specific primers and cloned into the pCR2.1 T vector.By overlapping PCR,the restriction site of Afl II and Ssp I(?)M/F/HN gene was eliminated to obtain a plasmid T-AB2.Finally the Pac I and ASC I fragment of TVT-GLa Sota C5,was replaced by a corresponding fragment of T-AB2,resulting in another plasmid TVT-GLa Sota C5M/F/HN.The resultant plasmid TVT-GLa Sota C5 together with three helper plasmids,pCI-NP,pCI-P and pCI-L,were cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase.After inoculating the supernatant of the transfected cell culture into SPF chicken embryo,the new attenuated chimeric virus GLa Sota M/F/HN was successfully generated.According to the standard assays recommended by OIE,its mean death time(MDT)was more than 120h,EID50 was about 1010.2 EIDso/ml,HA titer was as high as 210 and intracerebral pathogenicity index(ICPI)was only 0.22,intravenous pathogenicity index(IVPI)was 0,indicating that the rescued virus was highly attenuated and suitable for a vaccine candidate.3.Comparison of the immune proprietary between GLa Sota M/F/HN and La SotaBoth rabbit antisera against chimeric NDV strain GLa Sota M/F/HN and NDV strain La Sota were prepared and evaluated by hemagglutination inhibition test(HI)and virus neutralization assay(VN)titers to different NDV strains.Neutralizing antibody titers of GLa Sota M/F/HN antiserum to the tested virulent viruses were 3-5 times higher than those of La Sota antiserum.GLa Sota M/F/HN antiserum showed more than 10-fold higher inhibition than La Sota antiserum to class I NDV strain 9a5b.VN assays of antisera were carried out in both chicken embryos and DF-1 cells.GLa Sota M/F/HN antiserum showed higher VN titers to different NDV strains tested than La Sota antiserum,especially for class I viruses.Chickens were vaccinated with 106 EID50 each of GLa Sota M/F/HN or La Sota,respectively,twenty one days later,challenged with class II virulent strain ZJ1.Obvious clinical signs or mortality can not be observed in two immunized and challenged groups.And virus shedding load from GLa Sota M/F/HN vaccinated group was lower than that in La Sota vaccinated group.Considering that i)GLa Sota M/F/HN showed broader cross-antigenicity among NDV strains than current vaccine strain La Sota;ii)GLa Sota M/F/HN vaccinated chickens were better protected for virus shedding than those vaccinated with La Sota.4.Comparison of the mucosal immunity between GLa Sota M/F/HN and La SotaIn this study,60 chickens were randomly divided into 3 groups:A,B and C.Chickens in group A and B were injected with 106 EID50 GLa Sota M/F/HN or La Sota,respectively.After immunization,five birds in each group were euthanized,and both the serum and the mucosal samples were collected and detected at the day post-immunization of 1d,4d,7d,14d,21d.An indirect ELISA assay was established to measure NDV specific antibodies IgM,IgG and SIgA.The results showed that:local mucosal immune responses can be induced in vaccinated group.But the GLa Sota M/F/HN immunized groups can induce stronger immune response than La Sota vaccinated group.The detection of immunoglobulin subclasses showed that:IgG was the main specific antibody in systemic immune responses,while mucosal immune responses mainly produced specific antibodies SIgA.