鸡传染性支气管炎病毒M41株全基因组序列测定及致病性分析

doi:10.11843/j.issn.0366-6964.2025.08.034

摘要/Abstract

摘要：

为系统评价传染性支气管炎病毒(infectious bronchitis virus，IBV)M41株传代过后是否生物特性会发生改变，我们将国家兽医微生物菌(毒)种保藏中心1979年从法国引进的IBV M41株(保藏编号CVCC AV1511，简写AV1511-1979)在SPF鸡上连续传代5次，评估其在基因组变异、排毒规律、组织嗜性等方面的差异。采用Illumina高通量测序方法测序传代前后基因组序列，并建立了IBV攻毒模型，使用RT-PCR、RT-qPCR、气管环纤毛摆动试验等方法从病毒含量、临床症状观察、组织嗜性等方面评价其传代前后差异情况。结果表明AV1511-1979株在SPF鸡上连续传代5代后全基因组未发生变异，但是与其他来源的Mass株在1a、5a、N蛋白区域差异较大；攻毒模型中试验鸡出现精神沉郁、张口呼吸和呼吸啰音等明显临床症状，剖检发现气管环纤毛摆动状况不佳或停止摆动；RT-qPCR结果显示病毒在感染鸡的气管、肺脏、肾脏、腺胃、十二指肠、盲肠扁桃体内均可复制，尤其在气管、肾脏、腺胃中病毒载量较高，传代前后的病毒载量在统计学上无显著差异。研究表明：IBV M41株AV1511-1979株在SPF鸡上传代5代内生物特性稳定，不发生改变，作为IBV灭活疫苗生产用种毒、IBV疫苗检验用标准强毒株，我们的工作为生产种毒代次控制、疫苗评价提供了参考。

关键词: 鸡传染性支气管炎病毒, M41株, SPF鸡, 传代, 生物特性

Abstract:

In order to systematically evaluate whether the biological characteristics of infectious bronchitis virus (IBV) M41 strain will change after serial passages, we continuously passaged the IBV M41 strain (CVCC AV1511, abbreviated as AV1511-1979) introduced from France by the National Center for Veterinary Culture Collection in 1979 on SPF chickens for five times to evaluate its differences in genome variation, virus shedding, and tissue tropism. Illumina high-throughput sequencing method was used to sequence the genome sequences before and after passage, and an IBV challenge model was established. RT-PCR, RT-qPCR and tracheal ring cilia swing test were used to evaluate the differences before and after passage in tems of virus content, clinical symptom observation and tissue tropism. The results showed that the whole genome of the AV1511-1979 strain did not mutate after 5 generations of continuous passage on SPF chickens, but it was quite different from Mass strains from other sources in 1ab, 5a, 4b and N protein regions. In the virus challenge model, the inoculated chickens showed obvious clinical symptoms such as depression, mouth breathing and respiratory rales. The ciliary activity of the tracheal ring was examined by autopsy, and it was found that the ciliary activity was poor or stopped. The results of RT-qPCR showed that the virus could be replicated in the trachea, lung, kidney, glandular stomach, duodenum and cecal tonsil of infected chickens, especially in the trachea, kidney and glandular stomach. There was no significant difference in viral load before and after passage. The results showed that the biological characteristics of the IBV M41 strain, AV1511-1979, were stable and did not change within 5 generations on SPF chickens. As a standard virulent strain for the production of IBV inactivated vaccine and IBV vaccine testing, our work provides a reference for the generation control of production viruses and vaccine evaluation.

Key words: avian infectious bronchitis virus, M41 strain, SPF chickens, passage, biological characteristics

中图分类号:

S852.659.6

赵靖玉, 李丹, 张兵, 张乾义, 张锦华, 宋亚芬, 杨承槐. 鸡传染性支气管炎病毒M41株全基因组序列测定及致病性分析[J]. 畜牧兽医学报, 2025, 56(8): 3967-3975.

ZHAO Jingyu, LI Dan, ZHANG Bing, ZHANG Qianyi, ZHANG Jinhua, SONG Yafen, YANG Chenghuai. Genome Sequencing and Pathogenicity Analysis of Infectious Bronchitis Virus M41 Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3967-3975.

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引物名称 Primer names	引物序列(5′→3′) Primer sequences (5′→3′)	片段大小/bp Size
IBV-F	ATGTCTATCGCCAGGGAAATGTC	266
IBV-R	GCTCTAACTCTATACTAGCCTA
Mass-F	AGTTTCAATTGCTTACGGTCCT	360
Mass-R	AATAGCCAAACCTGCGTCT

编号 No.	GenBank登录号 GenBank accession No.	编号No.
编号 No.	GenBank登录号 GenBank accession No.	1	2	3	4	5
1	AV1511-1979	*	99.9	92.2	92.2	92.2
2	ON713866.1	99.6	*	92.2	92.2	92.1
3	DQ834384	97.8	97.6	*	99.9	99.9
4	MK937830	97.6	97.5	99.0	*	99.9
5	AY851295	97.8	97.6	100.0	99.9	*

编码区 CDS	最大同一性 The most percent identity
1a	89.1
5a	89.4
N蛋白	90.7
1b	93.5
3a	94.8
5b	95.2
m蛋白	96.2
E蛋白	96.9
S蛋白	97.7
3b	99.5

毒株 Strain	临床观察结果^a Clinical observation results						血清转阳结果^d Results of seroconversion
毒株 Strain	3 dpi	5 dpi	7 dpi	9 dpi	11 dpi	13 dpi	14 dpi
F1-E1 AV1511-1979	18/30^b (60%)^c	26/27 (96%)	24/24 (100%)	14/14 (100%)	7/14 (50%)	3/14 (21%)	3/14 (21%)
F5-E1 AV1511-1979	20/30 (67%)	25/27 (93%)	20/24 (83%)	14/14 (100%)	7/14 (50%)	3/14 (21%)	5/14 (36%)

毒株 Strain	F1-E1 AV1511-1979			F5-E1 AV1511-1979
毒株 Strain	前段 Proximal segment	中段 Middle segment	后段 Distal segment	前段 Proximal segment	中段 Middle segment	后段 Distal segment
1	0、0、0	0、0、0、0	0、0、0	0、0、0	0、0、0、0	0、0、0
2	2、2、0	0、0、0、0	1、0、0	0、0、0	0、0、0、0	0、0、0
3	0、0、0	0、0、0、0	0、0、0	0、0、0	0、0、0、0	0、0、0
4	1、0、0	0、0、0、0	0、0、0	0、0、0	0、0、0、0	0、0、0
5	0、0、0	0、0、0、0	0、0、0	0、0、0	0、0、0、0	0、0、0
6	1、1、1	0、0、1、1	2、1、0	0、0、0	0、0、0、0	0、0、0
7	0、0、1	1、1、2、0	2、2、0	0、0、0	0、0、0、0	0、0、0
8	0、0、0	1、0、0、0	1、1、0	0、0、0	0、0、0、0	0、0、0