B亚型禽偏肺病毒B1分离株感染性克隆的构建与鉴定

doi:10.11843/j.issn.0366-6964.2025.02.028

摘要/Abstract

摘要：

B亚型禽偏肺病毒(avian metapneumovrus, aMPV)可引起鸡肿头综合征，造成免疫抑制，引发严重的继发感染并导致鸡生产性能下降。建立国内aMPV分离株的反向遗传学系统对病毒研究及开发针对性疫苗具有重要意义。将病毒基因组扩增片段分别测序，通过序列拼接获得了完整的aMPV B1分离株基因组序列。根据病毒基因组序列中的酶切位点，将基因组分4段依次连入pTH载体，获得病毒基因组质粒pTH-B1。在病毒基因组的第7 474位引入同义突变，沉默原有Sal Ⅰ酶切位点以作为遗传标记，在pTH-B1质粒M与F基因之间引入PmeⅠ酶切位点，并利用该位点插入EGFP基因，获得质粒pTH-B1EGFP。将病毒N、P、M2.1基因的表达盒共同克隆到pCI-Neo表达载体中，获得辅助质粒pCI-NPM2.1。将L基因单独克隆到pCI-Neo表达载体中，获得辅助质粒pCI-L。将pTH-B1质粒和pTH-B1EGFP质粒分别与两种辅助质粒共转染BSR/T7细胞，然后将转染细胞与Vero细胞共培养并连续传代。结果显示：通过细胞病变(CPE)和绿色荧光观察，以及N基因和遗传标记检测、间接免疫光试验(IFA)和Western blot共同验证，表明构建的rB1株和rB1-EGFP株拯救成功。rB1株和rB1-EGFP株与亲本毒株的增殖水平和趋势相似。本研究通过三质粒拯救系统获得了B亚型aMPV B1株的感染性克隆，M和F基因间的非编码区可插入基因表达序列，为进一步探究B亚型aMPV的致病机理和疫苗研发奠定了必要基础。

关键词: B亚型禽偏肺病毒, 感染性克隆, 三质粒, 病毒拯救

Abstract:

Avian metapneumovirus (aMPV) subtype B is associated with the development of swollen head syndrome in chickens, which can lead to immunosuppression, severe secondary infections, and a subsequent decline in the productive performance of the poultry. The establishment of a reverse genetics system for domestic aMPV isolates is of considerable significance for advancing virological research and facilitating the development of targeted vaccines. The amplicons of the viral genome were sequenced individually, and the complete genome sequence of the aMPV B1 strain was deduced through sequence assembly. The genome was divided into four segments based on enzyme cleavage sites and sequentially inserted into the pTH vector to construct the viral genome plasmid pTH-B1. A synonymous mutation introduced at position 7 474 of the viral genome to silence the Sal I cleavage site as a genetic marker. A Pme I cleavage site was introduced between the M and F genes of the pTH-B1 plasmid, and the EGFP expression cassette was inserted using this site to obtain the plasmid pTH-B1EGFP. The expression cassettes of the viral N, P, and M2.1 genes were co-cloned into the pCI-Neo expression vector, yielding the auxiliary plasmid pCI-NPM2.1. The L gene was individually cloned into the pCI-Neo expression vector, yielding the auxiliary plasmid pCI-L. The pTH-B1 and pTH-B1EGFP plasmids were co-transfected with the two auxiliary plasmids into BSR/T7 cells, respectively. Subsequently, the transfected cells were co-cultured with Vero cells and continuously passaged for further analysis. The successful rescue of the rB1 and rB1-EGFP strains was confirmed through the observation of cytopathic effects (CPE) and green fluorescence, along with the detection of vrial N gene and genetic marker, indirect immunofluorescence assay (IFA) and Western blot. The proliferation levels and trends of the rB1 and rB1-EGFP strains were found to be similar to those of the parental virus strain. This study successfully obtained the infectious clone of the subtype B avian metapneumovirus B1 strain using a three-plasmid rescue system. The intergenic non-coding region between the M and F genes is capable of accommodating gene expression sequences, which lays a necessary foundation for further investigation into the pathogenesis of subtype B aMPV and the development of vaccines.

Key words: avian metapneumovirus subtype B, infectious clone, a three-plasmid rescue system, viral rescue

中图分类号:

S852.659.5

于泽坤, 姜承源, 袁洪兴, 周生, 段笑笑, 李彦, 宋勤叶. B亚型禽偏肺病毒B1分离株感染性克隆的构建与鉴定[J]. 畜牧兽医学报, 2025, 56(2): 788-802.

YU Zekun, JIANG Chengyuan, YUAN Hongxing, ZHOU Sheng, DUAN Xiaoxiao, LI Yan, SONG Qinye. Construction and Identification of Infectious Clone of the Avian Metapneumovirus of Subtype B Strain B1[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 788-802.

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引物名称 Primer name	引物序列(5′→3′) Sequence	片段长度/bp Size
FAF	$\underline{{\rm{ACGACTCACTATAGG}}}$GACGAGAAAAAAACGCATTCAAGTCACAATAG	2 405
FAR	TGCCTCAGATTCACCGCTCGAG
FBF	$\underline{{\rm{AGGATCAAAGCTCGA}}}$GCGGTGAATC	5 485
FBR	$\underline{{\rm{TATGGTCGGCCTATAA}}}$TGCAAGACCCAATTGC
FCF	$\underline{{\rm{TATAGGCCGACCATAC}}}$CTAAAGGATGAC	3 118
FCR	$\underline{{\rm{ATTTGTAATAGATTTGG}}}$TACCTGCTATC
FDF	$\underline{{\rm{GGTGAATCTGAGGCA}}}$GTAGTTAACATGATAGCAGGTAC	2 987
FDR	$\underline{{\rm{GATGCCATGCCGACC}}}$CACGGCAAAAAAACCGTATTCAATAC
B1-Pme I1F	GGGACAAGTGTTTAAACTGAGTAATTAAAAAATATGGGGCAAGTAAAATGTACCTC	8 483
B1-Pme I1R	$\underline{{\rm{ATTGGCTCTAGCACG}}}$ACTAACT
B1-Pme I2F	$\underline{{\rm{CGTGCTAGAGCCAAT}}}$TGTGGAG	8 515
B1-Pme I2R	TACTCAGTTTAAACACTTGTCCCATTTTTTTTATTAAACTACAGAAGAATAGTAGAC
B1-EGFPF	$\underline{{\rm{ATGGGACAAGTGTTT}}}$ATGGTGAGCAAGGGCGAGG	757
B1-EGFPR	$\underline{{\rm{ATATTTTTTAATTACTCAGTTT}}}$TTACTTGTACAGCTCGTCCATGCCG
EGFP基因检测F	TTCAGCGTGTCCGGCGAG	735
EGFP基因检测R	TCTTGTATCTTCCCGCTGGC
LF	$\underline{{\rm{GGCTAGCCTCGAGAATTC}}}$GCCACCATGGACCCATCCAGTGAGC	6 057
LR	$\underline{{\rm{GCGGCCGCCCGGGTCGAC}}}$CTATTTTGTGCTCAGTATGTACCCTGT
NPM2.1-NF	$\underline{{\rm{ATAGCGATAAGGATC}}}$TAGTTCATAGCCCATATATGGAGTTCCGC	2 380
NPM2.1-NR	$\underline{{\rm{ATAATCAAGTAGAGG}}}$TTTTACTTGCTTTAAAAAACCTCC
NPM2.1-PF	$\underline{{\rm{CCTCTACTTGATTATT}}}$GACTAGTTATTAATAGTAATCAATTACGGGGTCA	2 149
NPM2.1-PR	$\underline{{\rm{CACCATACGCGGATC}}}$GGTGCGGGCCTC

引物名称 Primer name	引物序列(5′→3′) Sequence	片段长度/bp Size
N基因检测F	GCAGACAATGTGGAACGAACTGC	475
N基因检测R	TTAGCTCTGCCTGCACAGACACATG
SalⅠ检测F	ACCAGGTAGGAACTTACAATCCTAG	1 331
SalⅠ检测R	AGTAGCTTTATAATCTTGAGCACTC
qB1F	AATAGTCCTCAAGCAAGTCCTCAGA	135
qB1R	TGTTGTAATTTGACCTGTTCTACACT
qB1-probe	FAM-CTGGTGTTATCAGCCTTAGGCTTGACGCT-BHQ

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