| Newcastle disease(ND) is considered as one of the most important animal diseases to pigeons throughout the world. The causative agent is Newcastle disease virus(NDV), also called Pigeon Paramyxovirus type â… (PPMV- â… ). PPMV- â… is an antigenic and host variant of chickens and antigenically and genetically distinguishable from other APMV-1 viruses, which forming a independent phylogenetic group Classâ…¡,genotype â…¥b.It is need to develop a novel and effective vaccine to prevent the infection and circulation of NDV in pigeons, for the shortage of special vaccine for PPMV-â… (Classâ…¡genotypeâ… ,â…¡are widely used in chikens).Reverse genetics manipulation become a significant method for the research of virus vaccine, for the quick obtain of chimeric viruses and attuenuated viruses.PI/CH/LHLJ/110822(ICPI= 1.19,MDT=63h) is a intermediate virulent genotype â…¥ of PPMV-â… isolated in Hei Longjiang province in 2011.A full-length c DNA of the complete 15,192-nt-long antigenome was already assembled to p UC18 vector and introduced a unique Nheâ… as genetic maker. An IN-FUSION PCR Cloning Kit was used to transferred the complete genome into low-copy POLTV5 vector, namely p OLTV5-0822 FL.Then, BSR T7/5 were co-transfected a mixture of the full-length and the NP-, P-, and L- helper plasmids of NDV V4 strain at ratio of 4:2:1:1 and obtain a rescued virus r0822. The rescued virus showed similar virulence and biological properties as parental virus, and keep the same characteristics in growth titer both in CEF and embryonated chicken eggs.The virulence of NDV is determined by F cleavage site.Therefore,we constructed gene substitution virus of La Sota F gene(Pi110822-LAF) and F cleavage site mutation virus(Pi110822-FCS) in our study. The ICPI value of Pi110822-FCS and Pi110822-LAF changed from 1.19 to 0.08 and MDT changed from 63 h to 168 h,which means the virulence changed from mesogenic to lentogenic. Pi110822-FCS and Pi110822-LAF were serially passed 20 times in embryonated egg allantonic cavity and the sequencing results confirmed the genetic stability of the mutations introduced in the F gene cleavage sites.In order to screening good immunogenicity to pigeons of PPMV-â… , we assessed the safety and immunogenicity.Subsequently, 3-day-old birds were infected through ED and IN with 0.1ml Pi110822-F CS stock solution, no morbidity and mortality were observed. The birds were challenged with genotype VII NDV/HLJ/01/06 strain.The immunity tests revealed that Pi110822-FCS and Pi110822-LAF were effectively in producing antibody.The mortality of Pi110822-FCS groups was 54.54%, while the Pi110822-LAF group was only 20%.The effectively reduced virus shedding from the vaccinated birds,it was end in 14 days latter. 20-day-old SPF chickens were immunized with inactivated Pi110822-FCS-based vaccines and the chicken exhibited HI titer of 7.5log2 in 14 d pi. The results shows the inactivated Pi110822-FCS-based vaccines can effectively stimulate the SPF chickens in producing antibody.We also conducted the immunity experiments in pigeons, but the effective results were not obtained for the absence of the SPF pigeons. The two attenuated virus are a promising vaccine candidate for the control of PPMV-â… . |
PDF Full Text Request