Construction Of Infectious Clone And Virus Rescue Of Attenuated Genotype ? Newcastle Disease Virus

Newcastle disease(ND),caused by Newcastle disease virus(NDV),is an highly contagious,acute and severe infectious avain disease,which are listed as an infectious disease that must be reported by the World Organization for Animal Heath.Our country lists it as a type of animal epidemic.Although NDV has only one serotype,there are multiple genotypes.At present,the epidemic strain in China is genotype ? of C lass ?.The widely used vaccine strains B1,Clone30,La Sota and V4 belonged to genotypes I and II,which cannot effectively prevent and control genotype VII strains.Therefore,the new vaccines consistent with the genotypes of current epidemic strains should be developed to deal with the prevention and control of current ND.In this study,a strain named HB of NDV was sequenced.Based on the whole genome sequence,the reverse genetic operation platform of HB strain was established and the F protein was modified,which are aimed to provide conditions for the development of attenuated genotype VII vaccine.A strain of Newcastle disease virus was detected from clinical sample in an affected chicken farm in llebei Province,named HB.10 pairs of overlapping primers were designed based on the genomic characteristics of NDV.Then the whole genome sequence of HB strain was amplified The result showed that the whole genome of the HB strain was 15192 bp,the 3' leader sequence was 55 nt,and the 5' trailing sequence was 114 nt.The six structural proteins encoded are matrix protein(M),fusion protein(F),hemagglutinin-neuraminidase protein(HN),nucleocapsid protein(NP),phosphoprotein(P)and RNA-dependent RNA polymerase Protein(L),the order of genome sequence is 3'-NP-PMF-HN-L-5'.Phylogenetic analysis showed that the HB strain belonged to the genotype ? strain of Class ?,had high homology with the duck-derived strains Md/CH/LGD/1/2005,Fujian/FP/02,and BP01(99.1%?99.2%),and they gathered with the same cluster.While HB strain had low homology with the V4,Mukteswar and La Sota vaccine strains widely used in China(82.9%?85.6%),which belonged to the different cluster.The phylogenetic tree constructed based on the the F gene showed that the HB strain belonged to the subgenotype Vlld strain,was the dominant subgenotype strain among the current epidemic strains in China.When compared with the sequences of other strains,we found that 4,16 and 2 amino acid mutations appeared in the signal peptide,heptapeptide repeat region and tra ns membrane domain of the F protein.In the functional domain and epitopes of the HN protein,we found 1 and 5 amino acid mutations,respectively.The mutations at these key sites are related to the fusion activity and immune evasion of the virus.Using reverse genetic manipulation technology,the full-length cDNA of HB strain is divided into two parts:external gene plasmid(M,F and HN)and internal gene plasmid(NP,P and L).By synonymous mutation,the base A at position 3100 of the Lprotein was mutated to T as a genetic marker to distinguish it from wild virus.The overlap PCR technology was used to mutate the 112-117 amino acid sequence of the F protein into the La Sota strain cleavage site sequence.Then,three helper plasmids PCI-NP,PCI-P,and PCI-L were constructed.The external gene plasmid,internal gene plasmid and three helper plasmids were co-transfected into BSR-T7/5 cells.The rescued virus rHB was identified by CPE,sequencing of molecule label and indirect immunofluorescence,which revealed ed that rHB can cause typical cytopathy(CPE)on BHK-21 cells and the genetic marker of rescued virus can be stably inherited and the virus was successfully rescued.Analysis of the biological characteristics of rescued virus showed that rHB strain and parent virus had similar proliferation characteristics on BHK-21 cells.The successful construction of infectious clones of NDV-HB strain can provide a technical platform for further research for the pathogenic mechanism ofNDV and the development of new genetically engineered vaccines.