猪流行性腹泻病毒细胞适应毒LJB-03株在三种Vero细胞上增殖效果的比较
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摘要
为探究猪流行性腹泻病毒(PEDV)细胞适应毒LJB-03株在3种Vero细胞上的增殖特性,分别以正常Vero细胞、适应无血清培养基的Vero细胞和稳定表达跨膜丝氨酸蛋白酶2 (TMPRSS2)的Vero/TMPRSS2细胞(繁殖PEDV时无须添加胰酶)为宿主细胞,繁殖PEDV LJB-03株,观察这3种细胞接毒后的病变特点和病变时间,利用空斑法比较其增殖特性,并以间接免疫荧光和电镜技术鉴定病毒的增殖。结果表明,PEDV LJB-03株在正常Vero细胞和适应无血清培养基的Vero细胞上增殖时需添加的胰酶最佳质量浓度为7ug/m L;在这3种细胞上增殖时的最佳感染复数均为0.01;PEDV LJB-03株在3种细胞上效价达到峰值的时间均为36 h,其中在正常Vero细胞上病毒效价最高为7.17×10~7PFU/m L,在Vero/TMPRSS2细胞上病毒效价为3.33×10~7PFU/m L,在无血清Vero细胞上的病毒效价为2.85×10~7PFU/m L。综上所述,正常Vero细胞上PEDV LJB-03株的病毒效价可达其他2种细胞的2倍以上,但在培养病毒时正常Vero细胞需添加胰酶和血清,因此,适应无血清培养基的Vero细胞与Vero/TMPRSS2细胞在培养PEDV LJB-03株时更具有潜力。
In order to compare the proliferation characteristics of porcine epidemic diarrhea virus(PEDV) LJB-03 strain on 3 kinds of Vero cells, PEDV LJB-03 strain was propagated on the normal Vero cells, the Vero cells adapted to serum-free medium, and the Vero/TMPRSS2 cells stably expressing transmembrane serine protease 2(TMPRSS2) without the addition of trypsin. The characteristics and time of cytopathic effect of these three kinds of cells were observed, then the proliferation characteristics of the virus were analyzed by plaque assay. The virus was identified by indirect immunofluorescence assay and electron microscopy. The results showed that the optimal concentration of trypsin was 7ug/m L when LJB-03 strain was proliferated in the normal Vero and the Vero cells adapted to serum-free medium;the best multiplicity of infection(MOI) were both 0.01 when proliferated on the three kinds of cells.The virus titer reached peak at 36 hours post infection.Among these peaks,the virus titer on the normal Vero cells was the highest,which was 7.17 ×10~7 PFU/m L;followed by Vero/TMPRSS2 cells,the virus titer reached 3.33 ×10~7 PFU/m L;virus titer in the Vero cells adapted to serum-free medium was 2.85×10~7 PFU/m L.In summary,the virus titer of PEDV LJB-03 strain on the normal Vero cells was 2 times higher than the other two cells,but serum and trypsin were needed when proliferating this virus on the normal Vero cells. Therefore,the Vero cells adapted to serum-free medium and the Vero/TMPRSS2 cells have more potential in the propagation of PEDV LJB-03 strain.
In order to compare the proliferation characteristics of porcine epidemic diarrhea virus(PEDV) LJB-03 strain on 3 kinds of Vero cells, PEDV LJB-03 strain was propagated on the normal Vero cells, the Vero cells adapted to serum-free medium, and the Vero/TMPRSS2 cells stably expressing transmembrane serine protease 2(TMPRSS2) without the addition of trypsin. The characteristics and time of cytopathic effect of these three kinds of cells were observed, then the proliferation characteristics of the virus were analyzed by plaque assay. The virus was identified by indirect immunofluorescence assay and electron microscopy. The results showed that the optimal concentration of trypsin was 7ug/m L when LJB-03 strain was proliferated in the normal Vero and the Vero cells adapted to serum-free medium;the best multiplicity of infection(MOI) were both 0.01 when proliferated on the three kinds of cells.The virus titer reached peak at 36 hours post infection.Among these peaks,the virus titer on the normal Vero cells was the highest,which was 7.17 ×10~7 PFU/m L;followed by Vero/TMPRSS2 cells,the virus titer reached 3.33 ×10~7 PFU/m L;virus titer in the Vero cells adapted to serum-free medium was 2.85×10~7 PFU/m L.In summary,the virus titer of PEDV LJB-03 strain on the normal Vero cells was 2 times higher than the other two cells,but serum and trypsin were needed when proliferating this virus on the normal Vero cells. Therefore,the Vero cells adapted to serum-free medium and the Vero/TMPRSS2 cells have more potential in the propagation of PEDV LJB-03 strain.
引文
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[19]CHEN Q,LI G W,STASKO J,et al.Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States[J].Journal of Clinical Microbiology,2014,52(1):234-243.
[20]EVEN M S,SANDUSKY C B,BARNARD N D.Serum-free hybridoma culture:ethical,scientific and safety considerations[J].Trends in Biotechnology,2006,24(3):105-108.
[21]张香玲,张生琰,孙燕,等.无血清培养Vero细胞及其传代稳定性分析[J].中国生物制品学杂志,2017,30(1):68-73.(in chinese)ZHANG Xiang-ling,ZHANG Sheng-yan,SUN Yan,et al.Serum-free culture and stability during subculture of Vero cells[J].Chinese Journal of Biologicals,2017,30(1):68-73.(in Chinese)
[22]KLUGE S,ROUROU S,VESTER D,et al.Proteome analysis of virus-host cell interaction:rabies virus replication in Vero cells in two different media[J].Applied Microbiology and Biotechnology,2013,97(12):5493-5506.
[23]TAN K Y,TEO K L,LIM J F,et al.Serum-free media formulations are cell line-specific and require optimization for microcarrier culture[J].Cytotherapy,2015,17(8):1152-1165.
[2]SUN D,WANG X Y,WEI S,et al.Epidemiology and vaccine of porcine epidemic diarrhea virus in China:a mini-review[J].Journal of Veterinary Medical Science,2016,78(3):355-363.
[3]PENSAERT M B,BOUCK P D.A new coronavirus-like particle associated with diarrhea in swine[J].Archives of Virology,1978,58(3):243-247.
[4]XUAN H,XING D,WANG D.Study on the culture of porcine epidemic diarrhea virus adapted to fetal porcine intestine primary cell monolayer[J].Chinese Journal of Veterinary Science,1984,4(3):202-208.
[5]CARVAJAL A,LANZA I,DIEGO R,et al.Evaluation of a blocking ELISA using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies[J].Journal of Veterinary Diagnostic Investigation,1995,7(1):60-64.
[6]FAN B C,JIAO D,ZHAO X N,et al.Characterization of Chinese porcine epidemic diarrhea virus with novel insertions and deletions in genome[J].Scientific Reports,2017,7:44209.
[7]FAN J H,ZUO Y Z,LI J H,et al.Heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in China[J].Virus Genes,2012,45(1):113-117.
[8]GAO X,LI D L,ZHAO J Y,et al.Complete genome sequence of porcine epidemic diarrhea virus from an outbreak in a vaccinated farm in Shandong,China[J].Genome Announcements,2016,4(4):e00619-16.
[9]徐丽华,李军星,苏菲,等.2011-2017年浙江省猪病毒性腹泻病的流行病学调查[J].中国兽医科学,2018,48(5):625-630.XU Li-hua,LI Jun-xing,SU Fei,et al.Epidemiological investigation on the viral diarrhea in pigs in Zhejiang Province during 2011-2017[J].Chinese Veterinary Science,2018,48(5):625-630.(in Chinese)
[10]潘溪.猪流行性腹泻病毒的分离鉴定及ELISA检测方法的初步建立[D].北京:中国农业科学院研究生院,2015.PAN Xi.Isolation of Porcine Epidemic Diarrhea Virus and Establishment of ELISA Methods for Antibody Detection[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2015.(in Chinese)
[11]HOFMANN M,WYLER R.Propagation of the virus of porcine epidemic diarrhea in cell culture[J].Journal of Clinical Microbiology,1988,26(11):2235-2239.
[12]SHIRATO K,MATSUYAMA S,UJIKE M,et al.Role of proteases in the release of porcine epidemic diarrhea virus from infected cells[J].Journal of Virology,2011,85(15):7872-7880.
[13]施雯.丝氨酸蛋白酶对猪流行性腹泻病毒S蛋白的作用及其对病毒繁殖的影响[D].哈尔滨:东北农业大学,2017.SHI Wen.The effects of typeⅡtransmembrane serine proteases(TTSPs)on the spike protein of porcine epidemic diarrhea virus and viral replication[D].Harbin:Northeast Agricultural University,2017.(in Chinese)
[14]黄祯祥,田鹏,李玉英.医学病毒学基础及实验技术[M].北京:科学出版社,1990:130-141.HUANG Zhen-xiang,TIAN Peng,LI Yu-ying.Medical Virology Basics and Experimental Techniques[M].Beijing:Science Press,1990:130-141.(in Chinese)
[15]KUSANAGI K,KUWAHARA H,KATOH T,et al.Isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate[J].The Journal of Veterinary Medical Science,1992,54(2):313-318.
[16]PARK J E,CRUZ D J,SHIN H J.Trypsin-induced hemagglutination activity of porcine epidemic diarrhea virus[J].Archives of Virology,2010,155(4):595-599.
[17]LEE S,KIM Y,LEE C.Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112[J].Virus Research,2015,208:215-224.
[18]OKA T,SAIF L J,MARTHALER D,et al.Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene[J].Veterinary Microbiology,2014,173(3-4):258-269.
[19]CHEN Q,LI G W,STASKO J,et al.Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States[J].Journal of Clinical Microbiology,2014,52(1):234-243.
[20]EVEN M S,SANDUSKY C B,BARNARD N D.Serum-free hybridoma culture:ethical,scientific and safety considerations[J].Trends in Biotechnology,2006,24(3):105-108.
[21]张香玲,张生琰,孙燕,等.无血清培养Vero细胞及其传代稳定性分析[J].中国生物制品学杂志,2017,30(1):68-73.(in chinese)ZHANG Xiang-ling,ZHANG Sheng-yan,SUN Yan,et al.Serum-free culture and stability during subculture of Vero cells[J].Chinese Journal of Biologicals,2017,30(1):68-73.(in Chinese)
[22]KLUGE S,ROUROU S,VESTER D,et al.Proteome analysis of virus-host cell interaction:rabies virus replication in Vero cells in two different media[J].Applied Microbiology and Biotechnology,2013,97(12):5493-5506.
[23]TAN K Y,TEO K L,LIM J F,et al.Serum-free media formulations are cell line-specific and require optimization for microcarrier culture[J].Cytotherapy,2015,17(8):1152-1165.