中国三株牛结节性皮肤病病毒的分离鉴定及其GPCR基因分析

doi:10.11843/j.issn.0366-6964.2024.12.025

摘要/Abstract

摘要：

为了解我国牛结节性皮肤病(lumpy skin disease, LSD)田间毒株流行情况及遗传进化情况并观察其生物学特征，本研究对临床疑似发生牛结节性皮肤病的牛皮肤结节样本进行PCR检测、病毒分离、免疫荧光鉴定、电镜观察、GPCR基因测序和遗传进化分析。结果表明：利用原代羊睾丸细胞(PLT)分离得到3株牛结节性皮肤病病毒(LSDV)，分别命名为LSDV/Heilongjiang/2022株、LSDV/Jiling/2022株和LSDV/Jiangxi/2022株，对三株病毒进行一步生长曲线测定，前96 h病毒增长速度最快，96~120 h时滴度最高，之后病毒生长速度开始减缓，接种96 h后滴度最高，三株病毒分别可达到10^6.0 TCID₅₀·mL^-1、10^5.3 TCID₅₀·mL^-1和10^5.1 TCID₅₀·mL^-1。透射电镜下病毒粒子呈椭圆形，病毒直径为200~300 nm。将三株病毒GPCR基因序列与中国各地区以及近年其他国家流行毒株进行遗传进化分析，三株分离株与中国各地区毒株均处于同一分支上，说明中国各地区牛结节性皮肤病的流行有很强的相关性。本研究成功从黑龙江、吉林及江西三省牛皮肤结节病料中分离出三株LSDV，丰富了我国LSDV的流行病学及病原学数据，并对LSDV的持续监测与新型疫苗研发工作有参考意义。

关键词: 牛结节性皮肤病病毒, 分离鉴定, 生物学特征, 遗传进化分析

Abstract:

This study was designed to understand the prevalence and genetic evolution of field strains of lumpy skin disease (LSD) in cattle in our country, and determine the biological characteristics of lumpy skin disease virus (LSDV). This study conducted PCR testing, virus isolation, immunofluorescence identification, electron microscopy observation, GPCR gene sequencing, and genetic evolution analysis on skin nodule samples from cattle suspected of having lumpy skin disease. The results indicate that the disease affecting cattle in three provinces in our country is Lumpy Skin Disease. Three LSDV strains were isolated using primary lamb testicular cells (PLT) and named LSDV/Heilongjiang/2022, LSDV/Jiling/2022, and LSDV/Jiangxi/2022. The growth curve of the three viruses showed the fastest growth rate in the first 96 hours, with the highest titer at 96 to 120 hours. Afterward, the virus growth rate started to slow down, and the highest titer was reached 96 hours after inoculation, the titers for the three strains were 10^6.0 TCID₅₀·mL^-1, 10^5.3 TCID₅₀·mL^-1, and 10^5.1 TCID₅₀·mL^-1, respectively. Under transmission electron microscopy, virus particles appeared elliptical, with a diameter between 200 and 300 nm. Genetic evolution analysis of the GPCR gene sequences of the three strains, along with prevalent strains in various regions of China and other countries in recent years, showed that the three isolated strains and the prevalent strains in various regions of China are on the same branch. This indicates a strong correlation in the prevalence of Lumpy Skin Disease in various regions of China. This study successfully isolated three strains of LSDV from skin nodules of cattle in Heilongjiang, Jilin, and Jiangxi provinces, enriching the epidemiological and pathogenic data of LSDV in China. It holds significant importance for the continuous monitoring of LSDV and the development of new vaccines.

Key words: lumpy skin disease virus, isolation and identification, biological characteristics, phylogenetic analysis

中图分类号:

S852.659.1

周祉玉, 杜吉革, 莘若兰, 张嘉雯, 潘晨帆, 印春生, 陈小云, 朱真. 中国三株牛结节性皮肤病病毒的分离鉴定及其GPCR基因分析[J]. 畜牧兽医学报, 2024, 55(12): 5620-5630.

ZHOU Zhiyu, DU Jige, XIN Ruolan, ZHANG Jiawen, PAN Chenfan, YIN Chunsheng, CHEN Xiaoyun, ZHU Zhen. Isolation and Identification of Three Lumpy Skin Disease Viruses in China and Their GPCR Gene Analysis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5620-5630.

图/表 7

表 1

表 2

LSDV参考分离株"

登录号 Login number	毒株名称 Strain names	国家 Country	登录号 Login number	毒株名称 Strain names	国家 Country	登录号 Login number	毒株名称 Strain names	国家 Country
OL542833	LSDV/Russia/Tyumen/2019	Russia	OM803091	LSDV China/GD02/2020	China	MW699032	LSDV/Russia/ Dagestan/2015 75 passage	Russia
MH646674	LSDV/Russia/Saratov/2017	Russia	OM803092	LSDV China/GX01/2020	China	MW656253	LSDV/280-KZN/RSA/2018	South Africa
OM530217	LSDV/Russia/Saratov/2019	Russia	OM984485	LSDV XJ201901	China	MW030512	Mutant LSDV clone Neethling-RIBSP(TK-)EGFP	Kazakhstan
MN636843	LSDV LSD-148-GP-RSA-1997	South Africa	ON152411	LSDV72/PrachuapKhiriKhan/ Thailand/2021	Thailand	MT643825	LSDV 210LSD-249/BUL/16	Bulgaria
AF409138	LSDV Neethling vaccine LW 1959	South Africa	OP508345	LSDV China/Xinjiang/Cattle/ Aug-2019	China	MT130502	LSDV Neethling-RIBSP vaccine	Kazakhstan
KX764643	LSDV SIS-Lumpyvax vaccine	South Africa	OP654649	LSDV/China/SiC/2021	China	MN995838	LSDV pendik	Turkey
KX764644	LSDV Neethling-Herbivac vaccine	South Africa	OP752701	LSDV/FJ/CHA/2021	China	MN642592	LSDV Kubash/KAZ/16	Kazakhstan
KX764645	LSDV Neethling-LSD vaccine-OBP	South Africa	OP922506	LSDV/CHINA/FJ2/2021	China	MH893760	LSDV/Russia/Dagestan/2015	Russia
MG972412	LSDV Cro2016	Croatian	OP985536	LSDV/MZGD/2020/China	China	KY829023	LSDV Evros/GR/15	Greece
MK441838	LSDV Herbivac LS	South Africa	OQ253250	LSDV/Thailand	Thailand	KY702007	LSDV SERBIA/Bujanovac/2016	Serbia
MT134042	LSDV/Russia/Udmurtiya/2019	Russia	OQ253254	LSDV 144240/64	Thailand	KX894508	LSDV 155920/2012	Israel
MW435866	LSDV SA-Neethling	South Africa	OQ267777	LSDV 65F12959	Thailand	AF409137	LSDV NW-LW isolate Neethling Warmbaths LW	South African
MW656252	LSDV/Haden/RSA/1954	South Africa	OQ349695	LSDV/Thailand/PraChuap KhiriKhan/2021	Thailand	KX683219	LSDV KSGP 0240	Kenya
OM793604	LSDV 33-KZN RSA 1977	South Africa	OQ511520	LSDV/Thailand/Trang/2022	Thailand	MN072619	LSDV Kenya	Kenya
OM793605	LSDV Hoffmeyer RSA 1958	South Africa	OQ555660	LSDV/CHINA/HLJ/2022	China	MW631933	LSDV LSD	Morocco
OM793606	LSDV Potter RSA 1958	South Africa	OR232413	LSDV Indonesia 2022 S1	Indonesia	NC 003027	LSDV NI-2490	Kenya
OM793607	LSDV Fourie-FS RSA 1959	South Africa	OR347834	LSDV 138156/64	Thailand	OK422494	LSDV/Cattle/India/2019/ Ranchi-1/P50	India
OM793608	LSDV Neethling-WC RSA 1957	South Africa	OR735987	LSDV 65A14015	Thailand	OP297402	LSDV-WB/IND/19	India
OM793609	LSDV Vaccine LW-1959 1988	South Africa	PP145892	LSDV MVZT2725	Malaysia	OP688128	LSDV V392.1	Bangladesh
OR134848	LSDV/Serbia/6402/2016	Serbia	PP053747	LSDV LSD50 NIB	Bangladesh	OP688129	LSDV V395.1	Bangladesh
MT992618	LSDV KZ-Kostanay-2018	Kostanay	OR393173	LSDV/2022/Anand/N9	India	OR194148	LSDV/Kurgan/2018	Russia
MW355944	LSDV China/GD01/2020	China	OR393172	LSDV/2022/Anand/N8	India	OR393169	LSDV/2022/Jamnagar/N3	India
MW732649	LSDV/HongKong/2020	China	OR134849	LSDV/Serbia/7695/2016	Serbia	OR393171	LSDV/2022/Surat/N7	India
MZ577075	LSDV 20L70 Dinh-To/VNM/20	Vietnam	OR134845	LSDV/Serbia/4592/2016	Serbia	OR393174	LSDV/2019/Ranchi/P50	India
MZ577076	LSDV 20L81 Bang-Thanh/ VNM/20	Vietnam	OR134843	LSDV/North Macedonia/5000/2016	North Macedonia	OR393175	LSDV/2021/Banswara	India
OM033705	LSDV/Thailand/YST/2021	Thailand	OR134842	LSDV/Greece/715/2015	Greece	OR393176	LSDV/2022/Camel	India
OM105589	LSDV/China/XJ01/2019	China	OR134836	LSDV/Albania/4770/2016	Albania	OR602866	LSDV Karnataka 2020 (1)	India
OM793602	LSDV Russia Tomsk 2020	Russia	OR134832	LSDV/Albania/1000/2017	Albania	OQ588787	LSDV/02/KASH/IND/2022	India
OM793603	LSDV Russia Khabarovsk 2020	Russia	OQ606832	LSDV/IND-2022/NIAB-PVNRTVU	India	ON010590	LSDV Neethling-RIBSP/7C	Kazakhstan

表 2

图 1

图 2

图 3

图 4

图 5

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引物名称 Primer names	引物序列(5′→3′) Primer sequences	预扩增条带/bp Preamplified band/bp
LSDV-F	TCCGAGCTCTTTCCTGATTTTTCTTACTAT	192
LSDV-R	TATGGTACCTAAATTATATACGTAAATAAC	192
GPCR-F	TTATCCAATGCTAATACTACCA	1 146
GPCR-R	ATGAATTATACTCTTAGCACA	1 146

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