鸡胚免疫新城疫疫苗后宿主法氏囊转录组学分析

doi:10.16431/j.cnki.1671-7236.2024.04.031

摘要/Abstract

摘要： 【目的】通过转录组学分析鸡胚免疫新城疫病毒(Newcastle disease virus,NDV) TS09-C株后宿主法氏囊内免疫关键基因及信号通路分子转录差异,探究免疫后宿主法氏囊内免疫相关分子的调控反应。【方法】将60只18胚龄SPF鸡胚随机分为免疫组和对照组,30枚/组,免疫组将NDV TS09-C株通过羊膜腔接种鸡胚,剂量为10^3.0 EID₅₀/枚,注射体积为0.1 mL,对照组以同样的方法接种等体积PBS。检测免疫后不同时间点肺脏、胸腺、脾脏和法氏囊组织的病毒载量。选取病毒载量最高的法氏囊组织进行转录组学测序后,筛选免疫相关差异表达基因并分析其参与的主要功能及通路。【结果】免疫后1 d法氏囊组织中即可监测到病毒,免疫后3 d病毒载量达到最高。将免疫后3 d的法氏囊进行转录组学分析,与对照组相比,免疫组有453个基因上调、98个基因下调,其中免疫相关基因有36个上调、10个下调。免疫相关差异表达基因主要富集于自噬和细胞因子-细胞因子受体相互作用的免疫相关通路。差异表达基因蛋白互作分析显示,自噬通路中富集的BCL2与其他免疫相关基因有较多互作关系,且在B细胞活化、分化、免疫系统发育等功能注释中均有参与。【结论】本研究结果揭示了鸡胚免疫NDV TS09-C株后法氏囊内调控B细胞活化、分化的潜在靶向基因及通路,为进一步研究该毒株鸡胚免疫后B淋巴细胞调控的分子机制提供新的信息。

关键词: 新城疫病毒(NDV); TS09-C株; 鸡胚免疫; 法氏囊; 转录组分析

Abstract: 【Objective】 The aim of this experiment was to analyze the transcriptional differences of immune key genes and signaling pathway molecules in host’s bursa of Fabricius after in-ovo immunization with Newcastle disease virus TS09-C strain in chicken embryos using transcriptomics,and explore the immune related molecular regulatory responses in the host’s bursa of Fabricius after immunization.【Method】 Sixty 18 embryo SPF chicken embryos were randomly divided into immunization group and control group,with 30 embryos per group.The immunization group inoculated NDV TS09-C strain into chicken embryos through the amniotic cavity,the dose was 10^3.0 EID₅₀/piece and the injection volume was 0.1 mL,while the control group was inoculated with equal volume PBS using the same method.The viral load of lung,thymus,spleen and bursa of Fabricius tissue was detected at different time points after immunization.The bursa of Fabricius tissue with the highest viral load was selected for transcriptome sequencing and analysis.Immune related differentially expressed genes were screened and the main functions and pathways they involved were analyzed.【Result】 The virus could be detected in bursa of Fabricius tissue 1 day after immunization,and the viral load reached the highest level 3 days after immunization.Transcriptome analysis was performed on the bursa of Fabricius 3 days after immunization.Compared with control group,there were 453 up-regulated genes and 98 down-regulated genes in immunization group,including 36 up-regulated genes and 10 down-regulated genes related to immunity.Immune related differentially expressed genes were mainly enriched in the immune related pathways of autophagy and cytokine-cytokine receptor interactions.Differentially expressed gene protein-protein interaction analysis showed that BCL2 enriched in the autophagy pathway interacted with other immune related genes,and was involved in functional annotation such as B cell activation,differentiation,and immune system development.【Conclusion】 The results of this study revealed the potential target genes and pathways regulating the activation and differentiation of B cells in bursa of Fabricius of chicken embryo immunized by NDV TS09-C strain,and provided new information for further study of the molecular mechanism of B lymphocyte regulation after in-ovo vaccination by NDV TS09-C strain.

Key words: Newcastle disease virus (NDV); TS09-C strain; in-ovo vaccination; bursa of Fabricius; transcriptome analysis

中图分类号:

S852.65

蒋立人, 冯贺龙, 姜鑫瑞, 王梓晨, 张高峰, 商雨, 曾哲, 姚伦, 汪宏才, 罗青平, 田光明, 温国元. 鸡胚免疫新城疫疫苗后宿主法氏囊转录组学分析[J]. 中国畜牧兽医, 2024, 51(4): 1632-1641.

JIANG Liren, FENG Helong, JIANG Xinrui, WANG Zichen, ZHANG Gaofeng, SHANG Yu, ZENG Zhe, YAO Lun, WANG Hongcai, LUO Qingping, TIAN Guangming, WEN Guoyuan. Transcriptomic Analysis of Host’s Bursa of Fabricius After in-ovo Vaccination with Newcastle Disease Vaccine[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1632-1641.

参考文献

[1] HU Z,HE X,DENG J,et al.Current situation and future direction of Newcastle disease vaccines[J].Veterinary Research,2022,53(1):99.
[2] GALLILI G E,BEN-NATHAN D.Newcastle disease vaccines[J].Biotechnology Advances,1998,16(2):343-366.
[3] GANAR K,DAS M,SINHA S,et al.Newcastle disease virus:Current status and our understanding[J].Virus Research,2014,184:71-81.
[4] SOKALE A,ZHAI W,POTE L,et al.Effects of coccidiosis vaccination administered by in ovo injection on the hatchability and hatching chick quality of broilers[J].Poultry Science,2017,96(3):541-547.
[5] 樊三玲,李丽,罗青平,等.鸡胚免疫疫苗的研究现状及展望[J].湖北农业科学,2018,57(S2):154-157. FAN S L,LI L,LUO Q P,et al.Research progress and prospect of in-ovo vaccine for chickens[J].Hubei Agricultural Sciences,2018,57(S2):154-157.(in Chinese)
[6] NEGASH T,AL-GARIB S O,GRUYS E.Comparison of in ovo and post-hatch vaccination with particular reference to infectious bursal disease.A review[J].The Veterinary Quarterly,2004,26(2):76-87.
[7] WEN G,LI L,YU Q,et al.Evaluation of a thermostable Newcastle disease virus strain TS09-C as an in-ovo vaccine for chickens[J].PLoS One,2017,12(2):e0172812.
[8] MAST J,NANBRU C,DECAESSTECKER M,et al.Vaccination of chicken embryos with escape mutants of La Sota Newcastle disease virus induces a protective immune response[J].Vaccine,2006,24(11):1756-1765.
[9] FENG H,YAO L,ZENG Z,et al.Identification of embryonic chicken proteases activating Newcastle disease virus and their roles in the pathogenicity of virus used as in ovo vaccine[J].Journal of Virology,2023,97(5):e0032423.
[10] COOPER M D,RAYMOND D A,PETERSON R D,et al.The functions of the thymus system and the bursa system in the chicken[J].The Journal of Experimental Medicine,1966,123(1):75-102.
[11] RIBATTI D,TAMMA R,ELIEH ALI KOMI D.The morphological basis of the development of the chick embryo immune system[J].Experimental Cell Research,2019,381(2):323-329.
[12] GLICK B,CHANG T S,JAAP R G.The bursa of Fabricius and antibody production[J].Poultry Science,1956,35(1):224-225.
[13] MATSUDA H,BITO Y.Different effects of bursectomy of chickens on immune response to Newcastle disease virus and Salmonella Pullorum antigens[J].Poultry Science,1973,52(3):1042-1052.
[14] JIN H,KONG Z,MEHBOOB A,et al.Transcriptional profiles associated with Marek’s disease virus in bursa and spleen lymphocytes reveal contrasting immune responses during early cytolytic infection[J].Viruses,2020,12(3):354.
[15] XU H,LIN S,ZHOU Z,et al.New genetic and epigenetic insights into the chemokine system:The latest discoveries aiding progression toward precision medicine[J].Cellular & Molecular Immunology,2023,20(7):739-776.
[16] SCOTT T R.Our current understanding of humoral immunity of poultry[J].Poultry Science,2004,83(4):574-579.
[17] WANG X,WU J,HU S,et al.Transcriptome analysis revealed the roles of long non-coding RNA and mRNA in the bursa of Fabricius during pigeon (Columba livia) development[J].Frontiers in Immunology,2022,13:916086.
[18] LUNA M,RODRÍGUEZ-MÉNDEZ A J,LUNA-ACOSTA J L,et al.Expression and function of chicken bursal growth hormone (GH)[J].General and Comparative Endocrinology,2013,190:182-187.
[19] LUNA M,RODRÍGUEZ-MÉNDEZ A J,BERUMEN L,et al.Immune growth hormone (GH):Localization of GH and GH mRNA in the bursa of Fabricius[J].Developmental and Comparative Immunology,2008,32(11):1313-1325.
[20] SUN Y,YU S,DING N,et al.Autophagy benefits the replication of Newcastle disease virus in chicken cells and tissues[J].Journal of Virology,2014,88(1):525-537.
[21] CLARKE A J,SIMON A K.Autophagy in the renewal,differentiation and homeostasis of immune cells[J].Nature Reviews Immunology,2019,19(3):170-183.
[22] HARDWICK J M,SOANE L.Multiple functions of BCL-2 family proteins[J].Cold Spring Harbor Perspectives in Biology,2013,5(2):a008722.
[23] EGUCHI Y,EWERT D L,TSUJIMOTO Y.Isolation and characterization of the chicken bcl-2 gene:Expression in a variety of tissues including lymphoid and neuronal organs in adult and embryo[J].Nucleic Acids Research,1992,20(16):4187-4192.
[24] FENG X L,ZHENG Y,ZONG M M,et al.The immunomodulatory functions and molecular mechanism of a new bursal heptapeptide (BP7) in immune responses and immature B cells[J].Veterinary Research,2019,50(1):64.
[25] TRAPP-FRAGNET L,SCHERMULY J,KOHN M,et al.Marek’s disease virus prolongs survival of primary chicken B-cells by inducing a senescence-like phenotype[J].PLoS Pathogens,2021,17(10):e1010006.
[26] JÓZEFIAK A,LARSKA M,POMORSKA-MÓL M,et al.The IGF-1 signaling pathway in viral infections[J].Viruses,2021,13(8):1488.
[27] HU B,LI H,ZHANG X.A balanced act:The effects of GH-GHR-IGF1 axis on mitochondrial function[J].Frontiers in Cell and Developmental Biology,2021,9:630248.