珠江上游地区蚊虫乙型脑炎病毒核酸检测及PrM和E基因序列分析

doi:10.16431/j.cnki.1671-7236.2017.02.005

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (2): 336-343.doi: 10.16431/j.cnki.1671-7236.2017.02.005

珠江上游地区蚊虫乙型脑炎病毒核酸检测及PrM和E基因序列分析

吕顺燕^1,2, 王静林¹, 李楠¹, 胡骑¹, 何于雯¹, 赵德宏¹, 陈红宇¹, 李华春¹

1. 云南省畜牧兽医科学院云南省热带亚热带动物病毒重点实验室, 昆明 650224;
2. 云南省昆明市东川区动物疫病预防控制中心, 昆明 654100

收稿日期:2016-06-25 出版日期:2017-02-20 发布日期:2017-02-25
通讯作者: 王静林, 李华春 E-mail:Wangjl107@163.com;li_huachun@hotmail.com
作者简介:吕顺燕(1979-), 女, 云南东川人, 本科, 兽医师, 研究方向:动物疫病预防控制, E-mail:759445528@qq.com
基金资助:
国家自然科学基金（31460669）；国家公益性（农业）行业专项（201303035）

Nucleotide Detection of Mosquito Japanese Encephalitis Virus and Sequence Analysis of PrM and E Genes in Upper Pearl River Region

LV Shun-yan^1,2, WANG Jing-lin¹, LI Nan¹, HU Qi¹, HE Yu-wen¹, ZHAO De-hong¹, CHEN Hong-yu¹, LI Hua-chun¹

1. Yunnan Tropical and Subtropical Animal Viral Disease Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming 650224, China;
2. Dongchuan Center for Animal Disease Control and Prevention, Kunming 654100, China

Received:2016-06-25 Online:2017-02-20 Published:2017-02-25

摘要/Abstract

摘要：

试验旨在了解珠江上游地区乙型脑炎病毒（JEV）主要传播媒介蚊虫的种类、分布及携带JEV基因型和分子特征。2013年7月在珠江上游地区师宗县采集蚊虫和库蠓标本，并对蚊虫进行分类鉴定；同时采用黄病毒属引物对采集到的蚊虫和库蠓标本进行检测，阳性标本用JEV PrM和E基因特异引物进行扩增、测序，然后采用生物信息学软件进行序列分析。在4个采集点共采集蚊虫3 705只，分别属于库蚊、按蚊、伊蚊和阿蚊4个属的7种蚊虫，牛圈以中华按蚊、骚扰阿蚊为优势蚊种，羊圈以中华按蚊、骚扰阿蚊为优势蚊种，猪圈以三带喙库蚊、中华按蚊和骚扰阿蚊为优势蚊种。采用JEV PrM和E基因特异引物对采集到蚊虫和库蠓标本分162批进行扩增、测序，采用生物信息学软件进行序列分析。结果显示，4株蚊虫携带的病毒均为基因Ⅰ型JEV，与疫苗株SA14-14-2 E蛋白存在15个氨基酸差异位点，在8个影响病毒毒力的重要位点上未发生突变。以上结果提示珠江上游地区存在多种蚊虫种类，三带喙库蚊和中华按蚊是该地区的优势蚊种，三带喙库蚊是当地JEV的主要传播媒介，在当地的媒介中存在基因Ⅰ型JEV流行，而流行株的毒力并未降低。

关键词: 乙型脑炎病毒; 蚊虫分类; 库蠓; PrM基因; E基因

Abstract:

This experiment was conducted to understand mosquito species, distribution of the main vector of Japanese encephalitis virus (JEV), JEV genotype and molecular characteristics in upper Pearl River region. We collected mosquito and Culicoides specimen in Shizong county, upper Pearl River region in July 2013, and these specimens were classified and identified. Meanwhile, JEV nucleotide were detected using PrM and E genes specific primers in 162 pools of mosquito and Culicoides specimen, and the positive specimens were sequencd and analyzed by bioinformatics software. Total 3 705 mosquitoes were collected from 4 collection spots, and the mosquitoes belong to 7 species in 4 generas: Culex, Anopheles, Aedes and Armigeres. In cattle pens, Anopheles Sinensis and Armigeres subalbatus were the dominant mosquito species. In sheep pens, Anopheles Sinensis and Armigeres subalbatus were the dominant mosquito species. And in pig pens, Culex tritaeniorhynchus, Anopheles Sinensis and Armigeres subalbatus were the dominant mosquito species. 4 of 162 pools mosquito and Culicoides were positive detecting by using PrM and E genes specific primers. Sequence analysis of JEV PrM and E genes results showed that 4 strains virus from Culex tritaeniorhynchus samples belonged to genotype Ⅰ JEV, and existing 15 amino acid difference sites between vaccine strain SA14-14-2 in the E gene protein, and 8 important sites which affected virus virulence did not mutant. These results suggested that there were many mosquito species in upper Pearl River region, and Culex tritaeniorhynchus, Anopheles Sinensis were dominant mosquito species in this region, and Culex tritaeniorhynchus was the main media of JEV in local region, genotypeⅠ JEV was popular in local media, and virulence of the pandemic strain did not reduce.

Key words: Japanese encephalitis virus; classification of mosquito; Culicoides; PrM gene; E gene

中图分类号:

R384.1

吕顺燕, 王静林, 李楠, 胡骑, 何于雯, 赵德宏, 陈红宇, 李华春. 珠江上游地区蚊虫乙型脑炎病毒核酸检测及PrM和E基因序列分析[J]. 《中国畜牧兽医》, 2017, 44(2): 336-343.

LV Shun-yan, WANG Jing-lin, LI Nan, HU Qi, HE Yu-wen, ZHAO De-hong, CHEN Hong-yu, LI Hua-chun. Nucleotide Detection of Mosquito Japanese Encephalitis Virus and Sequence Analysis of PrM and E Genes in Upper Pearl River Region[J]. , 2017, 44(2): 336-343.

参考文献

[1] 高晓燕. 乙型脑炎病毒IgM抗体检测方法的建立及其应用[D]. 北京:中国疾病预防控制中心, 2006.
[2] Weaver S C, Reisen W K. Present and future arboviral threats[J]. Antiviral Research,2010,85(2): 328-345.
[3] Ricklin M E, Garcìa-Nicolàs O, Brechbühl D, et al. Japanese encephalitis virus tropism in experimentally infected pigs[J]. Vet Res, 2016,47:34.
[4] Desingu P A, Ray P K, Patel B H, et al. Pathogenic and genotypic characterization of a Japanese encephalitis virus isolate associated with reproductive failure in an Indian pig herd[J].PLoS One,2016,11(2):e0147611.
[5] Gurav Y K, Bondre V P, Tandale B V,et al. A large outbreak of Japanese encephalitis predominantly among adults in Northern region of West Bengal, India[J]. J Med Virol, 2016,88(11):2004-2011.
[6] Dwibedi B, Mohapatra N, Rathore S K, et al. An outbreak of Japanese encephalitis after two decades in Odisha, India[J]. Indian J Med Res, 2015,142 (Suppl):S30-S32.
[7] Hanna J N, Ritchie S A, Phillops D A, et al. An outbreak of Janpanese encephalitis in the Torres Strait, Australia, 1995[J]. Med J Aust,1996,165:256-260.
[8] Zheng Y, Li M, Wang H, et al. Japanese encephalitis and Japanese encephalitis virus in mainland China[J]. Rev Med Virol, 2012,22(5):301-322.
[9] Li M H, Fu S H, Chen W X, et al. Genotype Ⅴ Japanese encephalitis virus is emerging[J]. PLoS Negl Trop Dis, 2011,5(7):e1231.
[10] 张海林.云南省虫媒病毒研究进展[J].中国媒介生物控制杂志,2004,15(5):410-414.
[11] Kuno G. Universal diagnostic RT-PCR protocol for arboviruses[J].J Virol Methods, 1998, 72(1): 27-41.
[12] Wang J L, Zhang H L, Fu S H, et al. Distribution of mosquitoes and mosquito-borne arboviruses in Yunnan province near the China-Myanmar-Laos border[J]. American Journal of Tropical Medicine and Hygiene, 2011,84(5):738-746.
[13] 董学书.云南蚊类志(上卷)[M].昆明:云南科技出版社,1993.
[14] 张海林,米竹青,张云智,等. 云南省边境地区蚊虫自然感染乙脑病毒的研究[J]. 中国媒介生物学及控制杂志,2002,13(2):101-104.
[15] 张海林,自登云,施华芳,等. 云南三带喙库蚊分布特点及自然感染乙脑病毒的调查[J]. 中国媒介生物学及控制杂志,1999,3:192-194.
[16] 张海林, 自登云, 龚正达. 云南省乙脑病毒宿主和媒介研究[J].中国预防兽医学报, 2000,22(2): 81-83.
[17] Wang J L, Pan Xiao L, Zhang H L, et al. Japanese encephalitis viruses from bats in Yunnan, China[J]. Emerging Infectious Diseases,2009,15(6):943-946.
[18] 王静林,张海林,周济华,等.云南省乙型脑炎病毒基因分型研究[J].中华实验临床病毒学杂志,2008,22(2):87-90.
[19] 杨起饶,张嘉玉,刘行知,等. 云南省昭通地区鸟类乙脑病毒分离与鉴定[J]. 中国人兽共患病杂志,1991, 1: 52-54.
[20] Solomon T,Ni H,Beasle D W, et al. Originan evolution of Japanese encephalitis virus in Southeast Asian[J]. J Virol, 2003,77(5):3091-3098.
[21] Phan T N, Marial del C P, Vuong Duc C, et al. Shift in Japanes encephalitis virus (JEV) genotype circulating in Northern Vietnam implication for frequent introductions of JEV from Southeast Asia to East Asia[J]. J Gen Virol, 2004,85(Pt 6):1625-1631.
[22] Yoshikawa A, Nabeshima T, Inoue S, et al. Molecular and serological epidemiology of Japanese encephalitis virus (JEV) in a remote island of western Japan: An implication of JEV migration over the East China Sea[J]. Trop Med Health, 2016, 44(3):8.
[23] 王环宇,付士红,李晓宇,等.我国首次分离到基因型Ⅰ乙脑病毒[J].中华微生物学和免疫学杂志,2004, 24(11):843-849.
[24] Pan X L, Liu H, Wang H Y, et al. Emergence of genotypeⅠof Japanese encephalitis virus as the dominant genotype in Asia[J]. J Virol, 2011,85(19):9847-9853.
[25] Wang H Y, Takasaki T, Fu S H, et al. Molecular epidemiological analysis of Japanese encephalitis virus in China[J]. J Gen Virol, 2007,88(Pt 3):885-894.
[26] 李晓宇,宋宏,付士红,等.中国流行性乙脑病毒分子生物学特性研究[J]. 病毒学报, 2004,20(3): 200-209.
[27] Cao L, Fu S, Gao X, et al. Low protective efficacy of the current Japanese encephalitis vaccine against the emerging genotype 5 Japanese encephalitis virus[J]. PLoS Negl Trop Dis, 2016, 10(5):e0004686.
[28] Zhao Z J, Date T, Li Y, et al. Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone[J]. J Gen Virol, 2005, 86(Pt 8): 2209-2220.
[29] Ni H, Chang G J, Xie H, et al. Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14[J]. J Gen Virol, 1995, 76(Pt 2): 409-413.
[30] Kolaskar A S, Kulkarni-Kale U. Prediction of three-dimensional structure and mapping of conformational epitopes of envelope glycoprotein of Japanese encephalitis virus[J]. Virology,1999, 261(1): 31-42.
[31] 丛晓燕,吴家强,王怀忠,等. 猪乙型脑炎病毒E基因的克隆与序列分析[J].中国畜牧兽医,2009,36(9):71-73.
[32] 宋子域,吴科榜,吴丽丽,等.流行性乙型脑炎快速血清学诊断方法研究进展[J].中国畜牧兽医,2015,42(12):3392-3396.
[33] 厚华艳,丁玲玲,吕茂杰,等. 猪乙型脑炎病毒E蛋白结构域Ⅲ原核表达和抗原性分析[J]. 中国畜牧兽医,2016,43(4):934-939.

珠江上游地区蚊虫乙型脑炎病毒核酸检测及PrM和E基因序列分析

Nucleotide Detection of Mosquito Japanese Encephalitis Virus and Sequence Analysis of PrM and E Genes in Upper Pearl River Region

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