盖塔病毒SC483株cDNA感染性克隆的构建

doi:10.3864/j.issn.0578-1752.2023.17.019

摘要/Abstract

摘要： 【背景】 盖塔病毒（Getah virus, GETV）是一种由蚊虫传播的病毒，分类学上属于披膜病毒科甲病毒属成员。该病毒宿主范围广，可感染猪、马、牛、狐狸等多种哺乳动物，人也可以感染，但在人群中造成的危害尚不知晓。动物感染主要临床表现为发热、皮疹、关节炎、繁殖障碍和死胎。GETV在世界范围内流行较为广泛，近年来在我国的流行呈上升趋势，2018年我国南方多个猪场爆发该病，GETV在畜禽养殖及公共卫生方面的危害渐渐受到人们的关注。目前，尚无用于预防和治疗盖塔病毒感染的商业化疫苗和药物。由于对GETV研究较少，其生物学特性、对不同物种的致病性和致病机制以及流行趋势在很大程度上均是未知的。【目的】 建立高效的GETV反向遗传操作平台，为深入研究GETV基因组结构与功能、致病机制以及开发新型疫苗奠定基础。【方法】 采取化学合成的方式人工合成了两端分别含有锤头状核酶（HamRz）和丁肝病毒核酶（HdvRz）序列的GETV SC483株基因组全长，并克隆至低拷贝pOK12-CMV载体中，从而获得含有GETV SC483株基因组全长cDNA克隆的重组质粒pGETV-SC483。将纯化的重组质粒pGETV-SC483转染BHK-21细胞进行病毒拯救。对拯救病毒进行连续传代、鉴定以及生物学特性分析，并对感染性克隆质粒pGETV-SC483在大肠杆菌中的遗传稳定性进行验证。【结果】 重组质粒pGETV-SC483转染BHK-21细胞后，48 h即可观察到GETV感染引起的典型细胞病变，获得的拯救病毒命名为rSC483。分别提取拯救病毒和亲本病毒基因组RNA，对病毒基因组进行RT-PCR扩增、Not I酶切以及序列测定。结果表明，拯救病毒不同于亲本病毒，其含有人为引入以消去Not I酶切位点的G4332A突变的分子标记。使用GETV特异性抗体作为检测抗体的间接免疫荧光试验和病毒粒子形态学电镜负染观察结果进一步表明，GETV拯救成功。噬斑形成试验和一步生长曲线试验结果表明，拯救病毒与亲本病毒具有相似的复制能力和增殖特性。另外，感染性克隆质粒pGETV-SC483在大肠杆菌DH5α中连续传代后的测序结果表明，该重组质粒具有良好的遗传稳定性。 【结论】 成功构建了稳定、高效的GETV SC483株全长cDNA感染性克隆，为GETV生物学特性及致病机理研究以及新型疫苗开发提供了技术平台。

关键词: 盖塔病毒, cDNA感染性克隆, 病毒拯救, 反向遗传操作系统

Abstract:

【Background】 Getah virus (GETV) is a mosquito-borne arbovirus, and belongs to the Alphavirus genus in the Togaviridae family. GETV has an extensive host range, which includes multiple mammals, such as pig, horse, cattle, fox and so on, although GETV is not known to cause disease in human. GETV infection often causes clinical symptoms, such as fever, urticarial rash, arthritis, reproductive disorders, and fetal death. GETV is widely prevalent in the world, and the prevalence in China is on the rise in recent years. In 2018, it suddenly broke out in many pig farms in southern China. The harm for livestock and poultry production and public health caused by GETV has got widespread attention. Currently, there is still no specific antiviral drug or vaccine against GETV. Due to the lack of research on GETV, its biological characteristics, pathogenicity, pathogenesis and epidemic properties are largely unknown. 【Objective】 The aim of this study was to develop a reverse genetics platform of GETV, which could provide a basis for investigating the structure and function of GETV genome, the genetic determinant of pathogenesis, and developing novel vaccine against the virus. 【Method】 The full-length genome of the GETV SC483 strain flanked by a hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequence was chemically synthesized and cloned into a low-copy plasmid pOK12-CMV to generate the recombinant plasmid pGETV-SC483, which was transfected into BHK-21 cells to rescue the virus. Then, compared with the parental virus, the rescued-virus was serially passaged, identified, and analyzed for biological characteristics. The genetic stability of the recombinant plasmid pGETV-SC483 in E. coli was also verified. 【Result】 The typical cell pathogenic effect (CPE) of GETV was observed in the cells after being transfected with the pGETV-SC483 for 48 h. The rescued GETV, namely rSC483, was successfully obtained, which had a G4332A mutation resulted in the deletion of a Not I restriction site to differentiate from the parental virus. The rescued virus was further verified by indirect immunofluorescence assay using GETV specific antibody as detection antibody and electron microscopic observation of virus particles. Furthermore, plaque phenotype and growth curve of the rescued virus were similar to that of its parental virus in BHK-21 cells. In addition, the infectious cloning plasmid pGETV-SC483 had good genetic stability in E. coli DH5α. 【Conclusion】 The successfully constructed full-length infectious cDNA clone of GETV SC483 strain provided a technical platform for the study of biological characteristics and pathogenesis of GETV as well as development of novel vaccine against GETV.

Key words: Getah virus, cDNA infectious clone, virus rescue, reverse genetics system

杜炳辰, 王铭, 刘春国, 王世达, 魏新宇, 路雅曼, 孙振钊, 刘在斯, 魏丽丽, 王靖飞, 杨德成. 盖塔病毒SC483株cDNA感染性克隆的构建[J]. 中国农业科学, 2023, 56(17): 3479-3486.

DU BingChen, WANG Ming, LIU ChunGuo, WANG ShiDa, WEI XinYu, LU YaMan, SUN ZhenZhao, LIU ZaiSi, WEI LiLi, WANG JingFei, YANG DeCheng. Construction of Infectious cDNA Clone of GETV SC483 Strain[J]. Scientia Agricultura Sinica, 2023, 56(17): 3479-3486.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.17.019

https://www.chinaagrisci.com/CN/Y2023/V56/I17/3479

图/表 7

图1

图2

图3

图4

图5

图6

图7

参考文献 32

[1]	KANAMITSU M, TANIGUCHI K, URASAWA S, OGATA T, WADA Y, WADA Y, SAROSO J S. Geographic distribution of arbovirus antibodies in indigenous human populations in the Indo-Australian archipelago. The American Journal of Tropical Medicine and Hygiene, 1979, 28(2): 351-363. doi: 10.4269/ajtmh.1979.28.351
[2]	NEMOTO M, BANNAI H, TSUJIMURA K, KOBAYASHI M, KIKUCHI T, YAMANAKA T, KONDO T. Getah virus infection among racehorses, Japan, 2014. Emerging Infectious Diseases, 2015, 21(5): 883-885. doi: 10.3201/eid2105.141975 pmid: 25898181
[3]	BROWN C M, TIMONEY P J. Getah virus infection of Indian horses. Tropical Animal Health and Production, 1998, 30(4): 241-252. doi: 10.1023/a:1005079229232 pmid: 9760716
[4]	L'VOV S D, GROMASHEVSKI'I V L, ARISTOVA V A, MOROZOVA T N, SKVORTSOVA T M, GUSHCHINA E A, PETROVA E S, L'VOV D K. Isolation of Getah virus (Togaviridae, Alfavirus) strains in North- Eastern Asia. Voprosy Virusologii, 2000, 45(5): 14-18.
[5]	LI X D, QIU F X, YANG H, RAO Y N, CALISHER C H. Isolation of Getah virus from mosquitos collected on Hainan Island, China, and results of a serosurvey. The Southeast Asian Journal of Tropical Medicine and Public Health, 1992, 23(4): 730-734.
[6]	KSIAZEK T G, TROSPER J H, CROSS J H, BASACA-SEVILLA V. Isolation of getah virus from nueva ecija Province, republic of the Philippines. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1981, 75(2): 312-313. pmid: 6117960
[7]	TURELL M J, O'GUINN M L, WASIELOSKI L P Jr, DOHM D J, LEE W J, CHO H W, KIM H C, BURKETT D A, MORES C N, COLEMAN R E, KLEIN T A. Isolation of Japanese encephalitis and Getah viruses from mosquitoes (Diptera: Culicidae) collected near Camp Greaves, Gyonggi Province, Republic of Korea, 2000. Journal of Medical Entomology, 2003, 40(4): 580-584. pmid: 14680130
[8]	LI Y Y, LIU H, FU S H, LI X L, GUO X F, LI M H, FENG Y, CHEN W X, WANG L H, LEI W W, GAO X Y, LV Z, HE Y, WANG H Y, ZHOU H N, WANG G Q, LIANG G D. From discovery to spread: The evolution and phylogeny of Getah virus. Infection, Genetics and Evolution, 2017, 55: 48-55. doi: 10.1016/j.meegid.2017.08.016
[9]	LIU H, ZHANG X, LI L X, SHI N, SUN X T, LIU Q, JIN N Y, SI X K. First isolation and characterization of Getah virus from cattle in northeastern China. BMC Veterinary Research, 2019, 15(1): 320. doi: 10.1186/s12917-019-2061-z pmid: 31488162
[10]	FUKUNAGA Y, KUMANOMIDO T, KAMADA M. Getah virus as an equine pathogen. Veterinary Clinics of North America: Equine Practice, 2000, 16(3): 605-617. doi: 10.1016/S0749-0739(17)30099-8
[11]	SHI N, LI L X, LU R G, YAN X J, LIU H. Highly pathogenic swine getah virus in blue foxes, Eastern China, 2017. Emerging Infectious Diseases, 2019, 25(6): 1252-1254. doi: 10.3201/eid2506.181983 pmid: 31107236
[12]	李楠, 何于雯, 左媛媛, 孟锦昕, 徐天刚, 王静林. 蠓虫源盖塔病毒(SZC30)结构基因分子生物学特征分析. 中国畜牧兽医, 2021, 48(4): 1149-1160.
	LI N, HE Y W, ZUO Y Y, MENG J X, XU T G, WANG J L. Analysis of molecular biological characteristics of structural genes of getah virus (SZC30) isolated from midge. China Animal Husbandry & Veterinary Medicine, 2021, 48(4): 1149-1160. (in Chinese)
[13]	WADA R, KAMADA M, FUKUNAGA Y, ANDO Y, KUMANOMIDO T, IMAGAWA H, AKIYAMA Y, OIKAWA M. Equine Getah virus infection: Pathological study of horses experimentally infected with the MI-110 strain. Nihon Juigaku Zasshi the Japanese Journal of Veterinary Science, 1982, 44(3): 411-418. pmid: 6290732
[14]	KAMADA M, ANDO Y, FUKUNAGA Y, IMAGAWA H, AKIYAMA Y. Studies on Getah virus-pathogenicity of the virus for horses. Experimental Reports of Equine Health Laboratory, 1981, 18: 84-93.
[15]	KUMANOMIDO T, WADA R, KANEMARU T, KAMADA M, HIRASAWA K, AKIYAMA Y. Clinical and virological observations on swine experimentally infected with Getah virus. Veterinary Microbiology, 1988, 16(3): 295-301. pmid: 2836997
[16]	IZUMIDA A, TAKUMA H, INAGAKI S, KUBOTA M, HIRAHARA T, KODAMA K, SASAKI N. Experimental infection of Getah virus in swine. The Japanese Journal of Veterinary Science, 1988, 50(3): 679-684. doi: 10.1292/jvms1939.50.679
[17]	SHIBATA I, HATANO Y, NISHIMURA M, SUZUKI G, INABA Y. Isolation of Getah virus from dead fetuses extracted from a naturally infected sow in Japan. Veterinary Microbiology, 1991, 27(3/4): 385-391. doi: 10.1016/0378-1135(91)90162-9
[18]	KUWATA R, SHIMODA H, PHICHITRASLIP T, PRASERTSINCHAROEN N, NOGUCHI K, YONEMITSU K, MINAMI S, SUPRIYONO, TRAN N T B, TAKANO A, SUZUKI K, NEMOTO M, BANNAI H, YOKOYAMA M, TAKEDA T, JITTAPALAPONG S, RERKAMNUAYCHOKE W, MAEDA K. Getah virus epizootic among wild boars in Japan around 2012. Archives of Virology, 2018, 163(10): 2817-2821. doi: 10.1007/s00705-018-3897-4
[19]	白志军, 彭翼飞, 林立辉, 田小东, 陈翠华, 方美玉, 徐春华, 蒋廉华, 陈唯军. 广东、海南两省人血清12种虫媒病毒抗体调查. 中国人兽共患病杂志, 2000, 16(1): 83-84.
	BAI Z J, PENG Y F, LIN L H, TIAN X D, CHEN C H, FANG M Y, XU C H, JIANG L H, CHEN W J. Prevalence of human antibody to arbovirus in Guangdong and Hainan Provinces. Chinese Journal of Zoonoses, 2000, 16(1): 83-84. (in Chinese)
[20]	梁勇, 张连山, 刘永为, 董运强, 王志强, 王峻巍, 梁国栋. 河北省涉县发烧样病人盖塔病毒IgM抗体筛查. 中国卫生检验杂志, 2010, 20(10): 2562-2563.
	LIANG Y, ZHANG L S, LIU Y W, DONG Y Q, WANG Z Q, WANG J W, LIANG G D. The screening of Getah virus IgM antibody to patients with fever from She County in Hebei Province. Chinese Journal of Health Laboratory Technology, 2010, 20(10): 2562-2563. (in Chinese)
[21]	梁勇, 张连山, 刘永为, 董运强, 王志强, 王峻巍, 梁国栋. 河北省涉县正常人群盖塔病毒感染状况筛查分析. 河北医药, 2010, 32(21): 3079-3080.
	LIANG Y, ZHANG L S, LIU Y W, DONG Y Q, WANG Z Q, WANG J W, LIANG G D. Screening and analysis of Gaeta virus infection in normal population in Shexian County, Hebei Province. Hebei Medical Journal, 2010, 32(21): 3079-3080. (in Chinese)
[22]	赵春生, 白志军, 彭翼飞, 赵文忠, 陈翠华, 方美玉, 田小东, 蒋廉华, 陈唯军, 刘建伟, 林立辉. 我国南方人鼠虫媒病毒血清流行病学调查. 中国公共卫生, 2001, 17(1): 64-66.
	ZHAO C S, BAI Z J, PENG Y F, ZHAO W Z, CHEN C H, FANG M Y, TIAN X D, JIANG L H, CHEN W J, LIU J W, LIN L H. The seroepidemiolgical investigation of arbovirus in humans and rats of the south of China. China Public Health, 2001, 17(1): 64-66. (in Chinese)
[23]	ZHAI Y G, WANG H Y, SUN X H, FU S H, WANG H Q, ATTOUI H, TANG Q, LIANG G D. Complete sequence characterization of isolates of Getah virus (Genus Alphavirus, Family Togaviridae) from China. The Journal of General Virology, 2008, 89(Pt 6): 1446-1456. doi: 10.1099/vir.0.83607-0
[24]	YANG T, LI R, HU Y, YANG L, ZHAO D, DU L, LI J, GE M, YU X. An outbreak of Getah virus infection among pigs in China, 2017. Transboundary and Emerging Diseases, 2018, 65(3): 632-637. doi: 10.1111/tbed.2018.65.issue-3
[25]	WANG N N, ZHAI X F, LI X L, WANG Y, HE W T, JIANG Z W, VEIT M, SU S. Attenuation of getah virus by a single amino acid substitution at residue 253 of the E2 protein that might be part of a new heparan sulfate binding site on alphaviruses. Journal of Virology, 2022, 96(6): e0175121. doi: 10.1128/jvi.01751-21
[26]	REN T W, MIN X L, MO Q R, WANG Y X, WANG H, CHEN Y, OUYANG K, HUANG W J, WEI Z Z. Construction and characterization of a full-length infectious clone of Getah virus in vivo. Virologica Sinica, 2022, 37(3): 348-357. doi: 10.1016/j.virs.2022.03.007
[27]	LU G, OU J J, JI J Z, REN Z X, HU X, WANG C Y, LI S J. Emergence of getah virus infection in horse with fever in China, 2018. Frontiers in Microbiology, 2019, 10: 1416. doi: 10.3389/fmicb.2019.01416 pmid: 31281304
[28]	REN T W, MO Q R, WANG Y X, WANG H, NONG Z R, WANG J L, NIU C X, LIU C, CHEN Y, OUYANG K, HUANG W J, WEI Z Z. Emergence and phylogenetic analysis of a getah virus isolated in southern China. Frontiers in Veterinary Science, 2020, 7: 552517. doi: 10.3389/fvets.2020.552517
[29]	REN X J, FU S H, DAI P F, WANG H Y, LI Y Y, LI X L, LEI W W, GAO X Y, HE Y, LÜ Z, CHENG J X, WANG G Q, LIANG G D. Pigsties near dwellings as a potential risk factor for the prevalence of Japanese encephalitis virus in adult in Shanxi, China. Infectious Diseases of Poverty, 2017, 6(1): 100. doi: 10.1186/s40249-017-0312-4 pmid: 28592296
[30]	左媛媛, 元正菊, 何于雯, 任炜杰, 孟锦昕, 王淑娟, 钟江国, 李楠, 王娟, 王静林, 徐天刚. 广州市及其周边地区马群中盖塔病毒血清流行病学调查. 中国动物检疫, 2021, 38(6): 35-39.
	ZUO Y Y, YUAN Z J, HE Y W, REN W J, MENG J X, WANG S J, ZHONG J G, LI N, WANG J, WANG J L, XU T G. Sero- epidemiological investigation on the GTV in horses in Guangzhou city and its surrounding areas. China Animal Health Inspection, 2021, 38(6): 35-39. (in Chinese)
[31]	黄耀伟, 李龙, 于涟. 人类及动物RNA病毒的反向遗传系统. 生物工程学报, 2004, 20(3): 311-318.
	HUANG Y W, LI L, YU L. The reverse genetics systems for human and animal RNA viruses. Chinese Journal of Biotechnology, 2004, 20(3): 311-318. (in Chinese)
[32]	祁小乐, 高宏雷, 高玉龙, 邓小芸, 步志高, 王晓燕, 王笑梅. 鸡传染性法氏囊病病毒Gt株感染性分子克隆的构建. 中国农业科学, 2007, 40(10): 2343-2349.
	QI X L, GAO H L, GAO Y L, DENG X Y, BU Z G, WANG X Y, WANG X M. Development of infectious molecular clones in infectious bursal disease virus Gt strain. Scientia Agricultura Sinica, 2007, 40(10): 2343-2349. (in Chinese)