Isolation And Identification,Genomic Sequencing And Analysis Of The Pigeon Paramyxovirus Type Ⅰ And Development Of The Real-time Fluorescence Quantitative RT-PCR Detection Methods Of The Related Factors Of Cell Apotosis Pathway

Pigeon Newcastle disease（ND）is the most common viral infectious disease in pigeon industry.The disease is mainly caused by the strain of pigeon paramyxovirus type 1（PPMV-1）which has been identified as a variant of Newcastle disease virus（NDV）.In recent years,the disease occurred and spread among pigeons in many places in China,causing great economic losses to pigeon industry.Therefore,isolation and identification,genome sequencing and molecular genetic variation analysis of PPMV-1 will be carried out in this study,which is of great significance for understanding the epidemic situation,evolutionary trend of the virus and ND prevention and control The main research contents and results of this paper are as follows:1.Isolation and identification of PPMV-1,sequencing and molecular evolution analysis of its F geneIn this study,the clinical samples suspected outbreaks of pigeon Newcastle disease were collected from various rigions of Guangxi during 2019-2020 to conduct isolation and identification of PPMV-1 and determination the F gene sequence and molecular evolution analysis of the isolates were also done.The results showed that a total of six PPMV-1 strains were isolated and identified from the diseased pigeon flocks,named pi/CH/GX1001/2019,pi/CH/GX1101/2019,pi/CH/GX1201/2019,pi/CH/GX1202/2019,pi/CH/GX0108/2020 and pi/CH/GX0114/2020.The F gene sequencing results showed that the length of the coding region of the F gene of the six virus isolates was 1662 nucleotides（nt）,encoding 553 amino acids（aa）.The F protein cleavage site of the isolates was ¹¹²RRQKRF¹¹⁷,which possesses the virulent NDV molecular characteristics.Then,the determined virus sequences were combined with the gene sequences of the reference strains in the Gen Bank database to construct separate data sets for phylogenetic,molecular clock and geographic phylogenetic analysis of the PPMV-1 strain by Bayesian phylodynamic analysis.The results of molecular evolutionary analysis showed that pi/CH/GX1201/2019 and pi/CH/GX1202/2019 belonged to sub-genotype VI.2.1.1.2.1（former VIj）,while pi/CH/GX1001/2019,pi/CH/GX1101/2019,pi/CH/GX0108/2020 and pi/CH/GX0114/2020 belonged to sub-genotype VI.2.1.1.2.2（former VIk）,and these two sub-genotypes are the dominant genotypes of domestic PPMV-1 strains.Molecular clock results indicated that genotype VI PPMV-1 strains began to show evolutionary divergence around the1960s.The most recent common ancestor（t MRCA）of sub-genotype VI.2.1.1.2.1 and VI.2.1.1.2.2 strains can be traced back to 1989.The results of the geographic phylodynamic analysis provide a basis for the speculation of the traceability analysis of NDV in China.The two sub-genotypes of NDV,VI.2.1.1.2.1 and VI.2.1.1.2.2,may have evolved independently from a common ancestor from Europe（e.g.,Italy or Belgium）that was first introduced to China after continuous independent evolution.2.Determination of whole gene sequences and genetic variation analysis of four representative PPMV-1 strainsFour representative strains of PPMV-1 in Guangxi,pi/CH/GX1202/2019which belong to VI.2.1.1.2.1 sub-genotype based on the F gene sequence and pi/CH/GX1001/2019,pi/CH/GX1026/2016,pi/CH/GX1101/2019 which belong to VI.2.1.1.2.2,were selected for doing plaque-purification,and the whole genome of the virus was sequenced to do genetic evolutionary analysis.The research on the characteritics of the virus strains and the prevalence and genetic variation pattern of PPMV-1 strains in Guangxiat the molecular level would provide theoretical basis for the prevention and control of pigeon Newcastle disease.The results showed that four purified PPMV-1 virus strains were obtained after three rounds of purification on BHK-21 cells.The full-length RNA nucleotides of the four PPMV-1 strains were 15192 nt,all containing six open reading frames,3’-NP-P-M-F-HN-L-5’,encoding six structural proteins:nucleoprotein,phosphoprotein,matrix,fusion（F）,hemagglutinin-neuraminidase,and large polymerase protein,respectively.The results of phylogenetic evolutionary tree analysis of the whole gene sequences showed that the four PPMV-1 strains belonged to two sub-genotypes,with pi/CH/GX1202/2019belongingtosub-genotypeVI.2.1.1.2.1,pi/CH/GX1001/2019,pi/CH/GX1026/2016 and pi/CH/GX1101/2019 belonged to sub-genotype VI.2.1.1.2.2,and the typing results were consistent with those based on F gene sequence typing.The similarities of nucleotides of the whole gene sequences of the four PPMV-1 isolates were 92.3%-98.3%.The F protein of the four virus strains was conserved in the heptapeptide repeat region（A and B）,with mutations in the signal and fusion peptide regions;the HN protein glycosylation site and the cysteine residue site were also conserved,and only pi/CH/GX1101/2019（H199R）and pi/CH/GX1202/2019（I352V）had a site change in the each neutralization epitope.3.Establishment of real-time fluorescence quantitative RT-PCR methods for apoptosis pathway-related factors of PPMV-1-infected chickensIn this study,five differentially expressed genes in the apoptotic pathway（TRIM25,CAS8,CDKN1A,BIRC2,IKBα）were screened by transcriptome analysis of the PPMV-1-infected chickens with RNA-Seq technology in our research group,and five pairs of specific primers were designed to amplify the corresponding five target genes from chicken peripheral blood lymphocytes,respectively.The recombinant plasmids were successfully constructed as standards,and the fluorescence quantitative RT-PCR standard curves were established,while sensitivity,specificity and reproducibility tests were performed.The results showed that the standard curves of RT-qPCR for cytokines were well correlated;the lysis curves showed a single lysis peak,and the standard deviations and coefficients of variation of intra-and inter-batch replicate tests for each gene were in accordance with the requirements.The present study successfully established five real-time fluorescence quantitative RT-PCR（RT-qPCR）detection methods for five apoptotic factors,which provides technical support for rapid,accurate and quantitative analysis of m RNA transcript levels of apoptotic pathway-related factors,and provides a good basis for further study of the intrinsic immune mechanism of PPMV-1infection in chickens.In summary,six PPMV-1 isolates from Guangxi were isolated and identified in this study,and it was found that sub-genotypes VI.2.1.1.2.1 and VI.2.1.1.2.2 subtype were mainly prevalent in Guangxi pigeons.Phylogenetic analysis and traceability analysis of the isolates were carried out by Bayesian method.At the molecular level,the variation of genetic diversity of PPMV-1isolates in China was revealed,and it was confirmed that the PPMV-1 genetype VI strain in China was probably introduced from Belgium or Italy and evolved independently from a common ancestor after it was first introduced into China.Four Guangxi representative PPMV-1 strains,pi/CH/GX1202/2019,pi/CH/GX1001/2019,pi/CH/GX1026/2016 and pi/CH/GX1101/2019,were purified and analyzed for their genetic variation.The genotyping results of PPMV-1 were consistent with those based on the F gene sequence of the virus,which enriched the biological information of NDV strain.The study also successfully established five real-time fluorescence quantitative RT-PCR detection methods for differentially expressed genes in apoptotic path in chicken.This study provides a theoretical basis and technical support for exploring the epidemiology of NDV,enriching the information base of whole genome of the virus,and understanding the pathogenesis and immune response of the virus.It also provides an important reference for the prevention and control of ND.