小反刍兽疫病毒感染性cDNA克隆的构建与病毒拯救

doi:10.11843/j.issn.0366-6964.2023.07.026

摘要/Abstract

摘要： 小反刍兽疫病毒（peste des petits ruminants virus，PPRV）是影响全球畜牧业的一种重要病原。本研究旨在建立稳定可靠的PPRV反向遗传操作平台，为解析PPRV致病机理、免疫逃逸机制、病毒复制机制等基础理论研究提供有效的技术平台，同时为开发更加安全、有效的新型疫苗奠定必要的前期基础。通过提取PPRV Clone9株的RNA，采用RT-PCR分6段扩增出PPRV反基因组cDNA，通过酶切、连接出PPRV Clone9株全长反基因组，获得pB-PPRV，同时构建表达PPRV核蛋白（nucleoprotein，N）、磷蛋白（phosphoprotein，P）和大蛋白（large protein，L）的3个辅助质粒。将pB-PPRV与辅助质粒通过脂质体转染BHK-T7细胞。转染3 d后，冻融3次，感染Vero细胞。传至第二代可观察到明显的细胞病变（CPE）。经间接免疫荧光、Western blot、RT-PCR和序列测定鉴定结果表明，拯救出具有感染性的病毒。拯救病毒（rPPRV-Clone9）在Vero细胞上可稳定传代，增殖动态与亲本病毒相似。本研究成功建立了PPRV Clone9株的反向遗传系统。

关键词: 小反刍兽疫病毒, 反向遗传, 病毒拯救

Abstract: Peste des petits ruminants virus (PPRV) is an important pathogen affecting the global livestock industry. The purpose of this research is to establish a stable and reliable PPRV reverse genetic manipulation platform, to provide an effective technical platform for the analysis of PPRV pathogenic mechanism, immune escape mechanism, virus replication mechanism and other basic theoretical research, at the same time, it will lay the necessary preliminary foundation for the development of safer and more effective new vaccines. The RNA of the PPRV Clone9 strain was extracted, and the PPRV antigenome cDNA was amplified by RT-PCR in six sections. The full-length antigenome of the PPRV Clone9 strain was digested and ligated to obtain pB-PPRV, and the PPRV nucleoprotein (N), three helper plasmids for phosphoprotein (P) and large protein (L). pB-PPRV and helper plasmids were co-transfected into BHK-T7 cells mediated by liposomes. Three days after transfection, Vero cells were infected by freezing and thawing three times. Significant cytopathic changes (CPE) were observed in the second generation. The results of indirect immunofluorescence, Western blot, RT-PCR and sequence determination showed that the infectious virus was rescued. The rescue virus (rPPRV-Clone9) was stably passaged on Vero cells, and the proliferation dynamics were similar to the parental virus. In this study, the reverse genetics system of PPRV Clone9 strain was successfully established.

Key words: peste des petits ruminants virus, reverse inheritance, virus rescue

中图分类号:

S852.65

王煜, 高月异, 高金源, 刘伟洁, 徐慧琳, 薛青红. 小反刍兽疫病毒感染性cDNA克隆的构建与病毒拯救[J]. 畜牧兽医学报, 2023, 54(7): 2956-2963.

WANG Yu, GAO Yueyi, GAO Jinyuan, LIU Weijie, XU Huilin, XUE Qinghong. Establishment of Reverse Genetics System of Peste des petits Ruminants Virus Clone9 Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2956-2963.

参考文献 31

[1]	KUMAR N, MAHERCHANDANI S, KASHYAP S K, et al.Peste des petits ruminants virus infection of small ruminants:A comprehensive review[J].Viruses, 2014, 6(6):2287-2327.
[2]	农业农村部, 海关总署.中华人民共和国农业农村部中华人民共和国海关总署公告第256号[J].中华人民共和国农业农村部公报, 2020(7):104-110.Ministry of Agriculture and Rural Affairs, General Administration of Customs.Ministry of Agriculture and Rural Affairs of the People's Republic of China announcement No.256 of the General Administration of Customs of the People's Republic of China[J].Gazette of the Ministry of Agriculture and Rural Affairs of the People's Republic of China, 2020(7):104-110.(in Chinese)
[3]	DIALLO A, MINET C, LE GOFF C, et al.The threat of peste des petits ruminants:Progress in vaccine development for disease control[J].Vaccine, 2007, 25(30):5591-5597.
[4]	董浩, 段小波.小反刍兽疫分子生物学研究进展[J].中国畜牧兽医, 2011, 38(10):135-138.DONG H, DUAN X B.Advance in molecular biology of peste des petits ruminants virus[J].China Animal Husbandry & Veterinary Medicine, 2011, 38(10):135-138.(in Chinese)
[5]	BAILEY D, BANYARD A, DASH P, et al.Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus[J].Virus Res, 2005, 110(1-2):119-124.
[6]	DIALLO A.Morbillivirus group:Genome organisation and proteins[J].Vet Microbiol, 1990, 23(1-4):155-163.
[7]	BANYARD A C, PARIDA S, BATTEN C, et al.Global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control[J].J Gen Virol, 2010, 91(12):2885-2897.
[8]	宋建德, 袁丽萍, 孙洪涛, 等.2015-2016年全球小反刍兽疫流行状况和防控[J].中国兽医杂志, 2017, 53(12):111-113.SONG J D, YUAN L P, SUN H T, et al.Global prevalence and prevention and control of PPR in 2015-2016[J].Chinese Journal of Veterinary Medicine, 2017, 53(12):111-113.(in Chinese)
[9]	刘永宏, 曹胜波, 赵丽, 等.中国小反刍兽疫疫情分析[J].西北农业学报, 2014, 23(9):19-26.LIU Y H, CAO S B, ZHAO L, et al.Analysis of epidemic Chinese peste des petits ruminants[J].Acta Agriculturae Boreali-Occidentalis Sinica, 2014, 23(9):19-26.(in Chinese)
[10]	BAO J Y, WANG Q H, LI L, et al.Evolutionary dynamics of recent peste des petits ruminants virus epidemic in China during 2013-2014[J].Virology, 2017, 510:156-164.
[11]	NIU B, LIANG R R, ZHANG S W, et al.Spatiotemporal characteristics analysis and potential distribution prediction of peste des petits ruminants (PPR) in China from 2007-2018[J].Transbound Emerg Dis, 2021, 69(5):2747-2763.
[12]	吴昊天, 满初日嘎, 王凤阳, 等.小反刍兽疫流行状况及分子诊断技术研究现状[J].动物医学进展, 2022, 43(1):122-126.WU H T, MANCHU R G, WANG F Y, et al.Epidemiological situation of research progress on molecular detection of peste des petits ruminants[J].Progress in Veterinary Medicine, 2022, 43(1):122-126.(in Chinese)
[13]	PARIDA S, MUNIRAJU M, MAHAPATRA M, et al.Peste des petits ruminants[J].Vet Microbiol, 2015, 181(1-2):90-106.
[14]	ZHAO H, NJEUMI F, PARIDA S, et al.Progress towards eradication of peste des petits ruminants through vaccination[J].Viruses, 2021, 13(1):59.
[15]	王煜.小反刍兽疫病毒Clone9株反向遗传操作系统的建立[D].北京:中国兽医药品监察所, 2022.WANG Y.Establishment of reverse genetics operation system of peste des petits ruminants virus Clone9 strain[D].Beijing:China Institute of Veterinary Drugs Control, 2022.(in Chinese)
[16]	RADECKE F, SPIELHOFER P, SCHNEIDER H, et al.Rescue of measles viruses from cloned DNA[J].EMBO J, 1995, 14(23):5773-5584.
[17]	BARON M D, BARRETT T.Rescue of rinderpest virus from cloned cDNA[J].J Virol, 1997, 71(2):1265-1271.
[18]	GASSEN U, COLLINS F M, DUPREX W P, et al.Establishment of a rescue system for canine distemper virus[J].J Virol, 2000, 74(22):10737-10744.
[19]	陈宗香, 张勇侠, 高雅丽, 等.麻疹疫苗株Schwarz反向遗传系统的建立[J].中国生物制品学杂志, 2021, 34(6):637-642.CHEN Z X, ZHANG Y X, GAO Y L, et al.Construction of a reverse-genetic system of measles vaccine virus Schwarz strain[J].Chinese Journal of Biologicals, 2021, 34(6):637-642.(in Chinese)
[20]	王煜, 孙淼, 陈延飞, 等.小反刍兽疫病毒反向遗传学研究进展[J].中国兽药杂志, 2022, 56(2):82-87.WANG Y, SUN M, CHEN Y F, et al.Research progress on reverse genetics of peste des petits ruminants virus[J].Chinese Journal of Veterinary Drug, 2022, 56(2):82-87.(in Chinese)
[21]	胡倩倩.表达GFP或BTV结构蛋白的小反刍兽疫疫苗株的构建及生物学特性硏究[D].南京:南京农业大学, 2012.HU Q Q.Construction and study of peste des petits ruminants vaccine strain virus expressing green fluorescence protein and bluetongue virus structural proteins[D].Nanjing:Nanjing Agricultural University, 2012.(in Chinese)
[22]	张勇侠, 李玫颖, 陈宗香, 等.麻疹病毒沪191株反向遗传系统的建立[J].病毒学报, 2017, 33(4):565-571.ZHANG Y X, LI M Y, CHEN Z X, et al.Construction of a reverse-genetics system of measles virus strain S191[J].Chinese Journal of Virology, 2017, 33(4):565-571.(in Chinese)
[23]	MUNIRAJU M, MAHAPATRA M, BUCZKOWSKI H, et al.Rescue of a vaccine strain of peste des petits ruminants virus:In vivo evaluation and comparison with standard vaccine[J].Vaccine, 2015, 33(3):465-471.
[24]	LIU H J, ALBINA E, GIL P, et al.Two-plasmid system to increase the rescue efficiency of paramyxoviruses by reverse genetics:The example of rescuing Newcastle Disease Virus[J].Virology, 2017, 509:42-51.
[25]	LIU H J, DE ALMEIDA R S, GIL P, et al.Comparison of the efficiency of different newcastle disease virus reverse genetics systems[J].J Virol Methods, 2017, 249:111-116.
[26]	PEETERS B P H, DE LEEUW O, KOCH G, et al.Rescue of Newcastle disease virus from cloned cDNA:Evidence that cleavability of the fusion protein is a major determinant for virulence[J].J Virol, 1999, 73(6):5001-5009.
[27]	LAWSON N D, STILLMAN E A, WHITT M A, et al.Recombinant vesicular stomatitis viruses from DNA[J].Proc Natl Acad Sci USA, 1995, 92(10):4477-4481.
[28]	LIU F X, ZHANG Y Q, LI L, et al.Rescue of eGFP-expressing small ruminant morbillivirus for identifying susceptibilities of eight mammalian cell lines to its infection[J].Virus Res, 2019, 261:60-64.
[29]	翟军军.小反刍兽疫病毒全长cDNA的构建及DNA疫苗的免疫学研究[D].兰州:中国农业科学院, 2012.ZHAI J J.Construction of Full-length cDNA of peste des petits ruminants Virus and Immunological study of the developed DNA vaccine[D].Lanzhou:Chinese Academy of Agricultural Sciences, 2012.(in Chinese)
[30]	刘振东.PPRV反向遗传系统的构建及重组病毒拯救[D].兰州:中国农业科学院, 2021.LIU Z D.Construction of PPRV reverse genetic system and rescue of recombinant virus[D].Lanzhou:Chinese Academy of Agricultural Sciences, 2021.(in Chinese)
[31]	李菊.PPRV N、C蛋白单克隆抗体的制备及C基因沉默的重组病毒拯救[D].兰州:西北民族大学, 2022.LI J.Preparation of monoclonal antibodies against N and C proteins of peste des petits ruminants virus and the rescue of the recombinant virus with C gene silence[D].Lanzhou:Northwest Minzu University, 2022.(in Chinese)