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

Abstract

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

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.

Figures/Tables 7

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References 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)

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