| Newcastle disease viruses?NDV?is one of the important pathogens in the poultry industry.It can cause Newcastle disease?ND?in pigeon,also known as pigeon paramyxovirus type 1?PPMV-1?infections.In the last ten years,the disease often broke out in pigeon flocks in China,and caused significant economic losses to the pigeon industry.To investigate the prevalence of Newcastle disease in pigeon in Guangxi,the differences in pathogenicity of different genotype NDVs in pigeons and differences in transcriptomics,in this study,isolation and identification of pigeon-origin NDV epidemic strains was carried out.The pathogenicity test in pigeons with three NDV strains of different genes and transcriptome sequencing of infected pigeons and verification of the results of sequencing.The main research contents and results are as the following.1.In this study,the clinical samples of suspected Newcastle disease in pigeon occurred in the recent winter were collected and detected,and then isolation and identification of the virus was carried out.The results showed that a pigeon-origin isolate of Newcastle disease virus,named Pi/CH/GX1220/2019,was isolated and identified.The results of cloning and gene sequence analysis of the F gene of the strain showed that the amino acid motif of the protein cleavage site was 112RRQKRF117,which accords with the characteristic of the virulent NDV;The Pi/CH/GX1220/2019 isolate belongs to the sub-genotype VI.2.1.1.2.1?VIj?NDV;The genetic evolution analysis of the complete F gene sequences of the isolate and epidemic strains of PPMV-1 isolated in recent year in Guangxi region indicated that they had the close relationships.The molecular time-dynamic study showed that the strain might derive from Guangdong.2.The genotype VI of NDV is the dominant genotype of infected host of pigeon,and the ND viruses of genotype VII are still the main epidemic strains in chicken flocks in China.The genotype VIII NDV has been isolated again in China in the past 10 years,however,the pathogenicity of genotype VIII NDV in pigeons has not been studied.To explore the pathogenicity of three different genotype NDVs,including genotype VI,VII and VIII strians in pigeons,the experimental infections in pigeon were done with genotype VI pigeon-origin strain pi/CH/GXP22/2000,genotypeVIIpigeon-originstain pi/CH/GXP40/2001 and genotype VIII NDV from cockfight.The results showed that all the pigeons were sick in the three virus-infected groups after infection.The histopathological results of the affected pigeons showed hemorrhage and necrosis of the spleen,infiltration and necrosis of inflammatory cells in the pancreas and caecum tonsils.Viral load can be detected at 3 dpi and 7 dpi from the spleens of the three groups of infected pigeons at 3 and 7 days post-infection;the morbidity and pathological changes of the pi/CH/GXP22/2000 infected group are mild and no death,and with low level of viral load in spleen,while the pi/CH/GXP40/2001 infection group had the most severe clinical sign and lesions.3.At present,there are few studies on the differences in the transcriptome of pigeons infected with NDV of different genotypes,and there is a lack of transcriptome analysis data of pigeons infected with genotype VIII.Therefore,in order to explore the pathogenic mechanism of pigeons infected with different genotypes of NDV,in this study,RNA sequencing?RNA-Seq?technology was employed to to analyze the transcriptome of spleen tissues of infected-pigeons by the above three different genotypes of NDV at 0,3 and 7 days post-infection?dpi?.The results showed that compared with the control group,the spleen tissues of the three infected groups were enriched with more differential expression genes at 7 dpi than 3 dpi.The up-regulated differential expression genes in the spleen of three NDV-infected pigeon groups were mainly inflammatory response factors:IL1B,IL18,SOCS1,etc.;and interleukin family related genes such as IL1R2,IL22R2,I17EL,IL7RA,IL2RA,etc.;interferon and its related protein genes IFI6,IFIT5,MX,ISG15,OASL,etc.;chemokines CCL3,CCL19,CCL21,etc.;and other immune response,apoptosis related genes such as RIG-I,TLR22,BIRC1,RSAD2,?defensin 7,etc.The down-regulated genes are mainly related to metabolism,such as GLR,GLUL,PLA2G,PA21B,CBPB1,etc.GO enrichment analysis found that most of them were related to molecular function,and KEGG pathway analysis found that the differential expression genes in the spleen of NDV-infected pigeon were mainly involved in metabolic related pathways and immune response.The genotype?NDV pi/CH/GXP22/2000 strain is easier to activate immune response in pigeon.Although the immune response activated by genotype?NDV pi/CH/GXP40/2001 strain and genotype?NDV game-fowl/CH/GXGB2011/2011 strain in pigeon is late,but the response is stronger.Meanwhile,we successfully established real-time fluorescence quantitative RT-PCR detection methods for pigeon RIG-I-like signaling pathway and related factors of apoptosis pathway,including pigeon RIG-I,TRIM25,MITA,IKK?,I?B?,Apaf1,BIRC2 and GADD45B gene.The results of transcriptomics verified by these methods are basically identified with that of transcriptome sequencing.In summary,a sub-genotype VI.2.1.1.2.1?VIj?pigeon-derived NDV strain was isolated and identified,and the genetic evolution relationship and source of the virus strain were analyzed in this study.The three NDV strainspi/CH/GXP22/2000,pi/CH/GXP40/2001and game-fowl/CH/GXGB2011/2011of different genotypes were compared for pathogenicity in pigeon and it was found that pathogenicity of pi/CH/GXP40/2001 was the highest.Transcriptomics analysis of the spleen of NDV-infected pigeons revealed the main differential genes.These differential expressed genes related to viral infections are mainly involved in metabolic-related pathways and immune responses.It provides data for exploring the molecular epidemiological research,pathogenic mechanism and immune response of NDV,and provides a reference for the prevention and control of NDV. |
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