2012—2016年中国南方地区帕利亚姆血清群病毒的分离与序列特征分析

doi:10.11843/j.issn.0366-6964.2018.04.013

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (4): 761-770.doi: 10.11843/j.issn.0366-6964.2018.04.013

2012—2016年中国南方地区帕利亚姆血清群病毒的分离与序列特征分析

杨恒¹, 肖雷¹, 李占鸿¹, 孟锦昕¹, 杨振兴¹, 吕敏娜², 林栩慧², 廖德芳¹, 牛保生³, 李华春^1*

1. 云南省畜牧兽医科学院热带亚热带动物病毒重点实验室, 昆明 650224;
2. 广东省农业科学院动物卫生研究所, 广州 510640;
3. 云南省师宗县动物疫病预防控制中心, 师宗 655799

收稿日期:2017-09-12 出版日期:2018-04-23 发布日期:2018-04-19
通讯作者: 李华春,博士,研究员,主要从事蓝舌病病毒研究;E-mail:Li_huachun@hotmail.com
作者简介:杨恒(1978-),博士,副研究员,主要从事动物虫媒病毒研究,E-mail:yangheng2008.cool@163.com;肖雷(1971-),男,硕士,副研究员,主要从事牛羊虫媒病毒研究。
基金资助:
国家重点研发计划（2016YFD0500908）；公益性行业（农业）科研专项（201303035）；国家自然科学基金（31360621；31760744）

Sequence Analysis of Palyam Serogroup Virus Isolated in South China from 2012 to 2016

YANG Heng¹, XIAO Lei¹, LI Zhan-hong¹, MENG Jin-xin¹, YANG Zhen-xing¹, LÜ Min-na², LIN Xu-hui², LIAO De-fang¹, NIU Bao-sheng³, LI Hua-chun^1*

1. Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming 650224, China;
2. Guangdong Open Laboratory of Veterinary Public Health, Guangzhou 510640;
3. Animal Disease Control Center of Shizong County, Shizong 655799, China

Received:2017-09-12 Online:2018-04-23 Published:2018-04-19
Supported by:

摘要/Abstract

摘要：

旨在从牛、羊监控动物群中，对流行于我国的帕利亚姆血清群病毒（PALV）进行分离、鉴定与序列特征分析，明确我国流行毒株与国外毒株之间的关系。自监控动物采集的PALV阳性血液接种BHK-21细胞进行病毒分离；对所分离PALV毒株的Seg-7与Seg-2基因节段进行扩增、测序与序列分析；通过高分辨率琼脂糖凝胶电泳分析不同血清型PALV代表毒株基因组的“电泳带型”特征；通过免疫荧光与中和试验分析不同血清型PALV的阳性血清之间的交叉反应特性与中和活性。试验结果如下：2012—2016年，在云南、广西、广东共计分离出19株PALV；Seg-2与Seg-7测序与系统发育分析显示，分离毒株分属Chuzan virus（CHUV）、D’Aguilar virus（DAV）与Bunyip Creek virus（BCV）三种血清型，不同血清型毒株均与日本毒株具有最近的亲缘关系，而与澳洲、非洲毒株亲缘关系较远。病毒基因组电泳结果显示PALV基因组呈现“3-3-4”的电泳带型特征。免疫荧光结果显示针对CHUV的多克隆抗体可使感染CHUV、DAV与BCV的BHK-21细胞呈现特异性荧光。血清学中和试验显示，CHUV、DAV与BCV三种病毒的阳性血清之间无交叉中和保护作用。本研究报道了2012—2016年中国PALV的分离与Seg-2、Seg-7序列特征，并首次报道了PALV的DAV与BCV两种血清型病毒在我国的分离，研究结果将有助于掌握我国PALV的分布与遗传特征，为诊断试剂的开发、流行病学调查与致病性研究提供科学依据。

关键词: 帕利亚姆血清群病毒, 病毒分离, 病毒鉴定, 血清型, 序列分析, 交叉中和保护

Abstract:

The aim of this study was to isolate and identify the Palyam serogroup virus (PALV) prevalent in China from the sentinel herds and to clarify the relationship between the native strains and the foreign strains. For virus isolation, PALV-positive blood samples were collected from sentinel herds and blind passed on BHK-21 cells. Specific primers targeting Seg-2 and Seg-7 of PALV were designed for identification and sequence analysis of the isolated virus by RT-PCR amplification. Genomic RNA of the isolated virus was extracted from the infected cells and determined by high resolution agarose gel electrophoresis. Cross-reactivity and cross-neutralizing activity of positive sera from different serotypes of PALV were analyzed by immunofluorescence and serum neutralization test. The results were as follows:During 2012 to 2016, a total of 19 strains of PALV were isolated form Yunnan, Guangxi and Guangdong provinces. Seg-2 and Seg-7 sequencing and phylogenetic analysis showed that the Chinese strains of PALV belonged to Chuzan virus (CHUV), D'Aguilar virus (DAV) and Bunyip Creek virus (BCV) and showed closest genetic relationship with the Japanese strains, but showed relative far relationship with the Australian and African strains. High resolution agarose gel electrophoresis showed that the genomic dsRNA of different serotypes of representative PALV strains presenting "3-3-4" banding characteristics. Immunofluorescence results showed that BHK-21 cells infected with CHUV, DAV or BCV presented specific fluorescence when using polyclonal antibody against CHUV as detection antibodies. Serological neutralization tests showed that there was no cross-neutralization protection between the positive sera of CHUV, DAV and BCV viruses. This study reported the molecular characteristics of PALV isolated in China from 2012 to 2016. It's the first time, we reported the DAV and BCV isolated in China. These results will be helpful to better understand the distribution and genetic characteristics of Chinese PALV and provide basis for development of diagnosis reagent and carried out epidemiological and pathogenic research on PALV.

Key words: Palyam serogroup virus, virus isolation, virus identification, serotype, sequence analysis, cross-neutralization protection

中图分类号:

S852.659.4

杨恒, 肖雷, 李占鸿, 孟锦昕, 杨振兴, 吕敏娜, 林栩慧, 廖德芳, 牛保生, 李华春. 2012—2016年中国南方地区帕利亚姆血清群病毒的分离与序列特征分析[J]. 畜牧兽医学报, 2018, 49(4): 761-770.

YANG Heng, XIAO Lei, LI Zhan-hong, MENG Jin-xin, YANG Zhen-xing, Lü Min-na, LIN Xu-hui, LIAO De-fang, NIU Bao-sheng, LI Hua-chun. Sequence Analysis of Palyam Serogroup Virus Isolated in South China from 2012 to 2016[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 761-770.

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2012—2016年中国南方地区帕利亚姆血清群病毒的分离与序列特征分析

Sequence Analysis of Palyam Serogroup Virus Isolated in South China from 2012 to 2016

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