Generation And Immunological Characteristics Evaluation Of A Recombinant Genotype â…¦ NDV Strain NA-1as A Viral Vector Based On CMV Promoter

Newcastle disease virus, also known as avian Paramyxovirus type-1virus,distributes all over the world and endanger the global poultry industry seriously. NDVhas a single serotype, but many genotypes. Epidemiological studies revealed thatgenotype VII NDV strains were circulating predominantly worldwide while othergenotypes appeared sporadically. However, the genotype II lentogenic vaccine strainsisolated more than a half century ago remain in current and widespread use to controlthe ND outbreaks. The differences in their antigenicity and genotypes between thepresently circulating strains and commercial vaccine strains are considered the mainreasons why commercial vailable vaccines do not completely prevent virulent NDVinfection and virus shedding and transmission following challenge. Even in thevaccinated birds virulent NDV strains are still isolated frequently. The development ofreverse genetics especially ideal recovery system will provide a powerful tool for theanalysis of the relationship between NDV structures and their functions as well aspathogenicity mechanism. And it also offers a new direction for developing genotypeVII NDV candidate attenuated vaccines and vaccine vectors which are matched withthe prevalent NDV strains.This study take the goose-origin genotype VII NDV isolate NA-1as the researchobject, a new more efficiently and simply T7-RNA polymerase-free recovery systemfor NA-1was developed based on the eukaryotic cell RNA polymerase II. And on thisbasis the virulent naturally-occurring F protein cleavage site motif of NDV NA-1wasmodified to an avirulent motif as that of the avirulent strain LaSota, resulting agenotype VII NDV candidate attenuated vaccine and vaccine vector rmNA-1. Then,using red fluorescence protein and green fluorescence protein gene as report genes weconstructed a recombinant NDV expressing two foreign proteins to explore thepotential of recombinant NDV serve as multivalent vaccine and development of tumor therapeutic vector carrying multiple immune molecules. What is more, using thismodified goose-origin genotype VII NDV as an avirulent, safe, and effective vaccinevector, a recombinant NDV rmNA-VP3, expressing exogenous protective gene VP3of Goose parvovirus was generated and its biological characterization andimmunological characteristics as a bivalent vaccine against GPV infection and ND ingoslings were evaluated. The main contents of this paper are as follows:I. Development of a reverse genetics system for NDV NA-1strainbased on CMVpromoterAccording to the genome sequence information published on Genbank ofgoose-origin genotype VII NDV NA-1, seven overlapping PCR fragments of theentire viral genome was divided. Each fragment was then cloned with virus-specificprimer pairs, sequenced, and assembled according to a stepwise cloning strategy.Then, the assembled full-length cDNA flanked by the hammerhead ribozymesequence (HamRz) and the hepatitis delta virus ribozyme sequence (HdvRz)antigenome sequences was inserted into the mammalian expression vector pCI-neounder the control of CMV promoter. The resulting full-length clone was designatedpCI-NA-1. Meanwhile the ORFs of the N, P and L genes were amplified withsequence-specific primers and cloned into the vector pcDNA3.1under the control ofCMV promoter, too. The resultant helper plasmids were designated as pcDNA-NP,pcDNA-P and pcDNA-L. The infectious viruses were recovered by co-transfectingthe full-length cDNA clone and supporting plasmids into BHK-21cells. And then arecombinant NDV expressing the green fluorescent protein (GFP) as a reporter genewas generated, which provide reliable basis for preparing a new genotype VII NDVlive vector to develop bivalent or multivalent vaccine for the future studies.II. Generation of a live-attenuated goose-origin genotype VII NDV NA-1strainby reverse geneticsTo generate a genotype VII NDV candidate attenuated vaccine strain or vaccinevector to match the genotype and antigenicity with currently circulating strains, themulti-basic cleavage site motif RRQKR↓F at the F protein cleavage site of NDVNA-1strain was changed to the dibasic motif GRQGR↓L as that of the avirulentstrain LaSota, resulting in a mutant virus named rmNA-1based on the efficient andsimple reverse-genetic system established previously. Pathogenicity tests showed thatthe modified NDV rmNA-1was is highly attenuated, and are classified as avirulent. The growth kinetics of the NDV rmNA-1was similar with that of the parental virusstrain rNA-1. And the modified sequence at the cleavage sites were preserved andstably maintained after10passages in chicken embryos. No reverse mutation or othermutation occurred. These results will lay the foundation for preparing a new genotypeVII NDV live-attenuated vaccine candidate and vaccine vector.III. Generation of a recombinant Newcastle disease virus expressing twofluorescent protein and evaluation its biological characterizationTo explore the potential of a recombinant NDV expressing two foreign proteinsserve as multivalent vaccine and the possibilities for developing tumor therapeuticvector carrying multiple immune molecules, the genes encoding the red fluorescenceprotein mCherry and green fluorescence protein GFP were linked together by InternalRibosome Entry Site (IRES) constituting a single NDV transcription cassette and wasinserted between the P and M genes of the NDV rmNA-1, resulting in therecombinant NDV rmNA-mCh-GFP expressing the two reporter genes. The redfluorescence protein mCherry and green fluorescence protein GFP were expressedsimultaneously in cells infected with rmNA-mCh-GFP as well as the proteins of NDVNA-1. The recombinant virus rmNA-mCh-GFP has similar chicken embryo growthpatterns compared to parental rmNA-1. Although the maximal viral titer ofrmNA-mCh-GFP was approximately25-fold lower than that of the parental virusrmNA-1, it still could get to considerable growth titers. These results indicated thatthe recombinant NDV has the potential to serve as live viral vector to express two ormore exogenous proteins.VI. Generation of a recombinant Newcastle disease virus expressing VP3proteinof Goose parvovirus and evaluation its immunogenicityThe open reading frame (ORF) of VP3gene of GPV was amplified and insertedbetween the P and M genes of the NDV rmNA-1, resulting in the recombinant NDVrmNA-VP3expressing the VP3protein of GPV. The expression of VP3protein wasconfirmed by indirect confocal immunofluorescencestaining and western blot analysis.Comparing with the parental strain, the pathogenicity of recombinant NDVrmNA-VP3was lower, and still kept the genetic stability. The VP3gene was stablymaintained after serial passage10times in10-day-old embryonated SPF chicken eggsand the mutations introduced in the F gene cleavage site were preserved. Therecombinant virus rmNA-VP3has similar chicken embryo growth patterns compared to parental rmNA-1and wild type rNA-1, but the highest titer of rmNA-VP3wasapproximately20-fold lower than that of the parental virus rmNA-1and the maximalviral titer of rmNA-VP3came later because of the insertion of foreign gene VP3. Thevaccination studies in goslings showed that the recombinant NDV rmNA-VP3hasgood immunogenicity and during the experimental period, the birds inoculatedappeared healthy without any signs of vaccine side-effects. The recombinant NDVrmNA-VP3has the potential to serve as a bivalent live vaccine against gosling plagueand ND since it could induce substantial neutralizing antibodies against both NDVand GPV simultaneously. And furthermore the NDV rmNA-1strain could be used as avaccine vector to express exogenous protein without impacting its immunogenicity.