H9N2亚型AIV NA基因多聚腺苷酸化信号位点对病毒复制的影响

doi:10.3969/j.issn.1004-1524.2022.10.07

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (10): 2132-2137.DOI: 10.3969/j.issn.1004-1524.2022.10.07

H9N2亚型AIV NA基因多聚腺苷酸化信号位点对病毒复制的影响

罗钰雯(), 夏静(), 李念灵, 李永新, 申玉玺, 李淑芸, 陈雯, 樊顺怡, 崔敏, 黄勇

四川农业大学动物医学院,四川成都 611130

收稿日期:2021-02-20 出版日期:2022-10-25 发布日期:2022-10-26
通讯作者: 夏静
作者简介:*夏静,E-mail: xiajing1028@163.com
罗钰雯(1997—),女,四川雅安人,硕士研究生,主要从事鸡传染病与分子生物学研究。E-mail: Yuwen_Luo@126.com
基金资助:
四川农业大学双支计划(2021993061)

Effect of polydenosine signal site of H9N2 AIV NA gene on virus replication

LUO Yuwen(), XIA Jing(), LI Nianling, LI Yongxin, SHEN Yuxi, LI Shuyun, CHEN Wen, FAN Shunyi, CUI Min, HUANG Yong

College of Veterinary Medicine, Sichuan Agricultural University,Chengdu 611130,China

Received:2021-02-20 Online:2022-10-25 Published:2022-10-26
Contact: XIA Jing

摘要/Abstract

摘要：

H9N2亚型禽流感病毒NA基因vRNA 5'端的多聚腺苷酸化信号位点具有多样性,突变发生时,该位点失去与聚合酶的结合能力或结合能力减弱,阻碍多聚腺苷酸化过程,影响病毒mRNA的转录过程。为探究H9N2亚型禽流感病毒NA基因5'端多聚腺苷酸化信号位点处5个或6个连续的尿嘧啶(U₅或U₆)碱基对病毒生物学特性的影响,以一株四川分离株A/Chicken/China/Sichuan/CQY/2014(H9N2)为骨架的反向遗传系统构建突变株。结果显示,含U₅的H9N2突变株(rCQYU₅-H9N2)的血凝效价、TCID₅₀和EID₅₀滴度均高于含U₅的H9N2突变株(rCQYU6-H9N2),但生长曲线无显著差异,表明H9N2 NA基因5'端多聚腺苷酸化信号位点U₅或U₆结构均不影响病毒的拯救,但U₅有助于提高病毒在鸡胚与细胞中的复制滴度。本实验研究结果可为H9N2亚型禽流感病毒的复制机理等研究提供参考。

关键词: 禽流感病毒, 反向遗传, 多聚腺苷酸化

Abstract:

The polyadenylation signal site at the 5' end of the NA gene vRNA of H9N2 subtype avian influenza virus is diverse. When mutated, this site loses its binding effect with polymerase or leads to weakened binding ability, it blocks the polyadenylation process and affects the transcription process of viral mRNA.To explore the effect of 5-terminal polyadenylation signal sites (five or six uracil bases, U₅ or U₆ ) of NA gene on the H9N2 subtype of avian influenza virus rescue, a series of mutants were constructed with a reverse genetic technology based on a A/Chicken/China/Sichuan/CQY/2014 (H9N2) strain which was isolated in Sichuan Province. The results showed that both of the hemagglutination titer, TCID₅₀ and EID₅₀ of rCQYU₅-H9N2 strain were higher than those of rCQYU₆-H9N2 strain, while the growth curve had no significant difference. The results indicated that the structure of U₅ and U₆ at the 5' terminal of the H9N2 NA gene had no effect on the rescue of the virus, while the five consecutive uracil bases were more favorable for the titer of the H9N2 AIV on MDCK cells and chicken embryo. The results of this experimental study provided a certain reference for the research on the replication mechanism of H9N2 subtype avian influenza virus.

Key words: influenza virus, reverse genetic technology, polyadenylation

中图分类号:

S852.65

罗钰雯, 夏静, 李念灵, 李永新, 申玉玺, 李淑芸, 陈雯, 樊顺怡, 崔敏, 黄勇. H9N2亚型AIV NA基因多聚腺苷酸化信号位点对病毒复制的影响[J]. 浙江农业学报, 2022, 34(10): 2132-2137.

LUO Yuwen, XIA Jing, LI Nianling, LI Yongxin, SHEN Yuxi, LI Shuyun, CHEN Wen, FAN Shunyi, CUI Min, HUANG Yong. Effect of polydenosine signal site of H9N2 AIV NA gene on virus replication[J]. Acta Agriculturae Zhejiangensis, 2022, 34(10): 2132-2137.

图/表 5

表1 同源重组法构建重组质粒的引物

Table 1 Primers for construction of recombinant plasmids via homologous recombination

基因 Gene	上游引物 Forward primer(5'→3')	下游引物 Reverse primer(5'→3')
PB2	GACCTCCGAAGTTGGGGGGGAGCGAAAGCAGGTCAAATATATTCA	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGTCGTTTTTAAAC
PB1	GACCTCCGAAGTTGGGGGGGAGCAAAAGCAGGCAAACCATTTGA	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGCATTTTTTCATG
PA	GACCTCCGAAGTTGGGGGGGAGCGAAAGCAGGTACTGATTCAAA	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGTACTTTTTTGGA
NP	GACCTCCGAAGTTGGGGGGGAGCAAAAGCAGGGTAGATAATCACTC	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGGTATTTTTC
NS	GACCTCCGAAGTTGGGGGGGAGCAAAAGCAGGGTGACAAA	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGGTGTTTT
M	GACCTCCGAAGTTGGGGGGGAGCAAAAGCAGGTAGATGTTT	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGTAGTTTTTTAC
HA	GACCTCCGAAGTTGGGGGGGAGCAAAAGCAGGGGAATTTCA	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGGTGTTTTT
NAU₅	GACCTCCGAAGTTGGGGGGGAGCAAAAGCAGGAGTGAAAA	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGAGTTTTTTCTA
NAU₆	GACCTCCGAAGTTGGGGGGGAGCAAAAGCAGGAGTGAAAA	TTTTGGGCCGCCGGGTTATTAGTAGAAACAAGGAGTTTTTTTCTA

图1 A/Chicken/China/Sichuan/CQY/2014(H9N2) 毒株全基因RT-PCR结果 M1, DL2000 DNA Marker; M2, DL5000 DNA Marker; NS~PB2,引物NS~PB2的PCR扩增产物。

Fig.1 A/Chicken/China/Sichuan/CQY/2014(H9N2) strain whole-genome RT-PCR result M1, DL2000 DNA Marker; M2, DL5000 DNA Marker; NS-PB2,PCR products of primer NS-PB2.

图2 rCQY-H9N2在 MDCK 细胞培养中的细胞病变 A,正常 MDCK 细胞;B,接种500 μL 的rCQYU5-H9N2在48 h MDCK 细胞 CPE;C,接种500 μL 的rCQYU6-H9N2在48 h MDCK 细胞 CPE;D,接种500 μL 的CQY-H9N2 在48 h MDCK细胞CPE。

Fig.2 Cytopathy of rCQY-H9N2 in MDCK cell culture A, Normal MDCK cells; B, MDCK cells were inoculated with 500 μL rCQYU5-H9N2 influenza virus CPE at 48 h; C, MDCK cells were inoculated with 500 μL rCQYU6-H9N2 influenza virus CPE at 48 h;D, MDCK cells were inoculated with 500 μL CQY-H9N2 influenza virus CPE at 48 h.

表2 病毒生物学特性

Table 2 The biological characteristics of virus

毒株 Strain	血凝效价 Hemagglutination titers(log₂)	半数细胞培养物感染量 TCID₅₀/mL^-1	鸡胚半数感染量 EID₅₀/mL^-1
CQY-H9N2	9.0	10^5.75	10^7.30
rCQYU₅-H9N2	9.0	10^5.76	10^7.30
rCQYU₆-H9N2	7.5	10^4.50	10^6.55

图3 病毒在鸡胚和MDCK细胞中的生长曲线 A, 病毒在MDCK细胞中的生长曲线;B,病毒在鸡胚中的生长曲线。拷贝数以单位体积(1 μL)核酸拷贝数量的指数对数值表示。

Fig.3 Growth curves of virus on chicken embryo and MDCK cells A,Growth curves of virus on MDCK cells; B, Growth curves of virus on chicken embryo. Copy number was expressed as the logarithm of the number of nucleic acid copies per unit volume (1 μL).

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H9N2亚型AIV NA基因多聚腺苷酸化信号位点对病毒复制的影响

Effect of polydenosine signal site of H9N2 AIV NA gene on virus replication

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