Isolation And Identification Of Goose Paramyxovirus And Construction Of The Attenuated Virulent Mutant Strain

Goose Paramyxovirus Disease(GPMVD)is an acute,severe and highly contagious infectious disease,which was caused by Goose Paramyxovirus(GPMV).The mortality rate of infected gosling flocks with could reach up to 100%.The currently prevalent strains are gene type VII in China,whom antigenicity was different from the vaccine strains,gene type I and type II,and could not provide complete protection post-immunization.Therefore,to seek a currently popular gene type VII strain as a potential candidate has become an important practical significance in the aspect of preventing and treating the disease.In this study,a goose paramyxovirus was isolated from clinical samples on a goose farm of Heilongjiang.After serological diagnosis and isolation and identification,it was named the HEB-18 strain,and determined the level of its virulence.Experimental results showed that the hemagglutination value of the F3 HEB-18 strain allantoic fluid virus is 1:32,and its hemagglutination could be specifically inhibited by the chicken Newcastle disease virus antiserum,but not by the avian influenza virus H5 and H9 positive serum.The MDT of chicken embryos with the smallest amount of HEB-18 strain is 50.4h,the ICPI of 1-day-old chicks is 1.675 and the IVPI of 6 weeks old chicks is 2.34.These virulence indicators meets the criterion of strong virulence.Indirect immunofluorescence could observe specific green fluorescence in the inoculated group.Electron microscope observation showed that the virus particles were round with the diameter was about 200nm and owned a capsule structure.The virus was inoculated into BHK-21cells and passed continuously to the 10th generation.The target band was detected by PCR identification,and the TCID₅₀ was determined to be 10^-7.7/0.1ml.The whole genome of HEB-18 was amplified by RT-PCR in segments,inserted to p MD19-T simple for sequencing,and the whole genome sequence was obtained by splicing the sequencing results,and the genetic characteristics of the genome and F gene sequence were analyzed.The results indicated that the HEB-18 genome was 15192bp in length,and genetic evolution analysis showed that the HEB-18 strain belongs to gene type VII in the Class II branch.It is on the same branch and own the closest genetic relationship with the NA-1M strain.But it has a distant genetic relationship with the La Sota vaccine strain and V4 vaccine strain that are currently widely used in China.The deduced amino acid sequence of the F gene shows that the HEB-18 strain cleavage site is ¹¹²R-RQ-KR-F¹¹⁷.Combined with its virulence criterion,the isolated HEB-18 strain is a highly virulent gene VII Strain.Reverse genetic manipulation technology was used to clone the full-length c DNA of the HEB-18strain into the downstream of the CMV promoter of the p CI vector.A hammerhead ribozyme sequence(Ham Rz)and a hepatitis D virus ribozyme sequence(Hdv Rz)were introduced at both ends of the c DNA,and at the 7422nt site,A was mutated to C as a genetic marker through synonymous mutation.Point mutation technology was applied to co-mutate 6 bases,the amino acid sequence of the cleavage site112-117 of the F protein of the HEB-18 strain was mutated to the same sequence of the attenuated strain La Sota strain.In order to improve the efficiency of its gene translation,the auxiliary plasmids pc DNA3.1(+)-NP,pc DNA3.1(+)-P and pc DNA3.1(+)-L,was constructed by introduced the Kozak sequence(GCCACC)before the ORF.The full-length plasmid p CI-HEB-18 and the three helper plasmids pc DNA3.1(+)-NP,pc DNA3.1(+)-P and pc DNA3.1(+)-L were co-transfected into BHK-21 cells.And the recombinant virus r HEB-18 strain was obtained by a series identification such as indirect immunofluorescence,genetic marker detection and gene sequencing.The results of the determination of the pathogenicity of the recombinant virus showed that MDT>120h,ICPI was 0,and IVPI was 0.17.Compared with the parental virus HEB-18 strain,its pathogenicity is significantly reduced.The preparation of the GPMV r HEB-18 strain laid the foundation for the candidate strain of the attenuated goose paramyxovirus vaccine.