Isolation And Identification Of PPMV-1 Immunogenicity And Construction Of Full-length CDNA Clone

Pigeon paramyxovirus 1(PPMV-1)infection,also known as pigeon Newcastle disease(ND),is a highly contagious disease caused by Newcastle disease virus(NDV)in pigeons.The disease has become one of the most harmful diseases to the pigeon industry in China due to its high infectiousness,high transmission rate and high mortality rate.In this study,clinical samples of pigeons and wild birds in Guangxi collected between2020 and 2023 were detected and virus isolated,and the isolates were further subjected to whole-genome sequencing,genetic evolutionary analysis and virulence determination.Since there is no commercial pigeon Newcastle disease vaccine available in China,this study chose to use a strongly virulent isolate for the production of inactivated vaccine with oil emulsion,to test its immunogenicity and immunoprotective efficacy,and to construct a full-length c DNA clone for the viral genome to lay the foundation for the construction of its reverse genetic operating system and the subsequent development of an antigen-matched vaccine for pigeon Newcastle disease.1.Isolation and identification,full-length genome sequencing and genetic evolutionary analysis of PPMV-1In this study,1289 clinical samples of pigeons and wild birds from some cities in Guangxi were investigated epidemiologically for PPMV-1.6 positive samples were detected from pigeon samples in farms and live bird markets,and 2 strains of PPMV-1 were successfully isolated and named as GXN1 and GXN2.whole genome sequencing results showed that the two isolates were 15192 nt in length and their genome structure was3’-NP-P-M-F-HN-L-5’,which was consistent with the NDV genome,and the F protein cleavage site sequence was 112R-R-Q-K-R-F117,which was consistent with the strong virulent strain.Genetic evolutionary analysis showed that the two isolates belonged to the same genetic subtype VI.2.1.1.2.2 and were genetically distant from traditional vaccine strains such as La Sota,B1,Clone30 and V4.The F protein of the two isolates had more mutations in the signal peptide region,and some amino acid sites were mutated in the fusion peptide region and two heptapeptide repeats;a total of six amino acid sites were mutated in the neutralization epitope of the HN protein,two of which were present in a linear antigenic epitope345-353 of the HN protein.2.Virulence determination and immunogenicity study of the isolatesThe mean death time(MDT)and intracerebral pathogenicity index(ICPI)of the chick embryos of the two isolates were determined using WOAH standards,and the results showed that GXN1 was a strongly virulent strain and GXN2 was a weakly virulent strain.The crosshemagglutination inhibition test between La Sota and GXN1 strains showed that the antigenic correlation coefficient "R" value was 0.67,indicating the antigenic difference between the two strains.The inactivated oil emulsion vaccine of GXN1 strain was prepared in accordance with the Chinese veterinary pharmacopoeia,and the 30-day-old pigeons were vaccinated by subcutaneous neck injection,and the second immunization was conducted 14 days after the first vaccination.The HI potency of GXN1 group is slightly higher than the inactivated vaccine and live vaccine groups.In the viral challenge and protection test,2/10 pigeons died in the La Sota live vaccine group and 1/10 pigeon died in the inactivated vaccine group,while there was no significant abnormality in the GXN1 inactivated vaccine group.3.Construction of full-length c DNA clone of GXN1 strainThe rigorous plasmid p BR322 was used as the cloning vector,and the auxiliary element T7 promoter,the core sequence of Hdv Rz and T7 terminator were introduced into the vector.The full-length c DNA of GXN1 strain was divided into five fragments for amplification,and the five fragments were cloned into the transcriptional vector in three times by homologous recombination technology.The full-length c DNA clone of GXN1 strain based on T7 promoter was successfully constructed after verification by sequencing,which laid the foundation for the subsequent virus rescue work and the construction of its reverse genetic operating system.