宿主细胞基因序列插入亚洲1型口蹄疫病毒导致病毒对牛的致病性减弱的研究
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摘要
1株Asia 1型口蹄疫疫苗毒株在BHK21悬浮培养细胞中传代时与宿主BHK21细胞m RNA重组,导致VP1第206位与第207位氨基酸之间插入了VDLV序列,该插入突变使得疫苗免疫效果大幅下降,不能产生有效保护。比较发现,含12 nt插入序列的变异毒株(Vac-Asia1-VDLV)与亲本毒株(Asia 1/HN/06)在BHK21细胞上的生长曲线存在较大差异,变异毒株可感染CHO-K1,pgsA-745,pgsB-618,pgsD-677细胞,毒价可达到1×105PFU/mL以上,而亲本毒不能感染CHO类细胞并产生蚀斑。动物试验结果显示,乳鼠半数致死量(LD50)与亲本毒株相比变化不明显。但牛感染试验表明,大剂量(1×107LD50)接种牛舌面,Vac-Asia1-VDLV不引起体温反应和其它临床症状,荧光定量PCR检测鼻拭子和血液时未发现病毒,而Asia 1/HN/06引起典型的口蹄疫症状,鼻拭子和血液中检出病毒。上述试验结果表明,与亲本毒相比,变异毒株Vac-Asia 1-VDLV对牛的致病性明显降低。
An Asia 1 foot-and-mouth disease vaccine strain was recombined with the host BHK21 m RNA during passage in suspension cell culture,resulting in the insertion of VDLV sequence between amino acids 206 and 207 of VP1.The insertion mutation deteriorated the immune effect of the vaccine and failed to protect the challenged animals.It was found that the growth curve of the recombination strain(Vac-Asia 1-VDLV) which contains the 12-nt insertion sequence was significantly different from that of the parent strain(Asia 1/HN/06) in BHK21 cells.The recombination strain could infect CHO-K1,pgs A-745,pgs B-618,and pgs D-677 cells with a titer of more than 1×105PFU/ml,while the parent strain could not infected CHO cells and developed plaques.There was no obvious difference of median lethal dose(LD50)between the Vac-Asia1-VDLV and parental strain based on animal experiments.However,the cattle pathogenicity experiment showed that the high-dose(1×107LD50) inoculation of cattle tongue did not increase body temperature and cause other clinical symptoms.No virus was detected in nasal swab and blood samples by QRT-PCR.Asia 1/HN/06 caused typical clinical symptoms of foot-and-mouth disease,and virus was detected in nasal swabs and blood sample.Therefore,the results showed that the pathogenicity of the variant Vac-Asia 1-VDLV to bovine was significantly lower compared to parent virus.
An Asia 1 foot-and-mouth disease vaccine strain was recombined with the host BHK21 m RNA during passage in suspension cell culture,resulting in the insertion of VDLV sequence between amino acids 206 and 207 of VP1.The insertion mutation deteriorated the immune effect of the vaccine and failed to protect the challenged animals.It was found that the growth curve of the recombination strain(Vac-Asia 1-VDLV) which contains the 12-nt insertion sequence was significantly different from that of the parent strain(Asia 1/HN/06) in BHK21 cells.The recombination strain could infect CHO-K1,pgs A-745,pgs B-618,and pgs D-677 cells with a titer of more than 1×105PFU/ml,while the parent strain could not infected CHO cells and developed plaques.There was no obvious difference of median lethal dose(LD50)between the Vac-Asia1-VDLV and parental strain based on animal experiments.However,the cattle pathogenicity experiment showed that the high-dose(1×107LD50) inoculation of cattle tongue did not increase body temperature and cause other clinical symptoms.No virus was detected in nasal swab and blood samples by QRT-PCR.Asia 1/HN/06 caused typical clinical symptoms of foot-and-mouth disease,and virus was detected in nasal swabs and blood sample.Therefore,the results showed that the pathogenicity of the variant Vac-Asia 1-VDLV to bovine was significantly lower compared to parent virus.
引文
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[20] DE LOS SANTOS T,DE AVILA BOTTON S,WEIBLEN R,et al.The leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mRNA and blocks the host innate immune response[J].Journal of Virology,2006,80(4):1906-1914.
[21] PACHECO J M,GLADUE D P,HOLINKA L G,et al.A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle[J].Virology,2013,446(1-2):260-267.
[2] JAMAL S M.,FERRARI G,AHMED S,et al.Molecular characterization of serotype Asia 1 foot-and-mouth disease viruses in Pakistan and Afghanistan;emergence of a new genetic Group and evidence for a novel recombinant virus[J].Infection,Genetics and Evolution,2011,11,2049-2062.
[3] ZOU X,ZHU Y,BAO H,et al.Recombination of host cell mRNA with the Asia 1 foot-and-mouth disease virus genome in cell suspension culture[J].Archives of Virology,2018,Sep 19.[Epub ahead of print].
[4] KLEIN J.Understanding the molecular epidemiology of foot-and-mouth-disease virus[J].Infection,Genetics and Evolution,2009,9(2):153-161.
[5] ALAM S M,AMIN R,RAHMAN M Z,et al.Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus(FMDV)serotype Asia 1[J].Advances and Applications in Bioinformatics Chemistry,2013,6:37-46.
[6] BROWN F.Problems with BHK 21 cells[J].Developments in Biological Standardization,1998,93:85-88.
[7] ZHAO Q,PACHECO J M,MASON P W.Evaluation of genetically engineered derivatives of a Chinese strain of foot-and-mouth disease virus reveals a novel cell-binding site which functions in cell culture and in animals[J].Journal of Virology,2003,77(5):3269-3280.
[8] JOSHI R,CHANDRASEKAR S,PAUL S,et al.Growth kinetics and immune response of chimeric foot-and-mouth disease virus serotype'O'produced through replication competent mini genome of serotype Asia 1,63/72,in BHK cell lines[J].Virus Research,2013,173(2):299-305.
[9] LEWIS-ROGERS N,MCCLELLAN D A,CRANDALL K A.The evolution of foot-and-mouth disease virus:impacts of recombination and selection[J].Infection,Genetics and Evolution,2008,8(6):786-798.
[10] LEE K N,OEM J K,PARK J H,et al.Evidence of recombination in a new isolate of foot-and-mouth disease virus serotype Asia 1[J].Virus Research,2009,139(1):117-121.
[11] KHATCHIKIAN D,ORLICH M,ROTT R.Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into the haemagglutinin gene of an influenza virus[J].Nature,1989,340(6229):156-157.
[12] CHARINI W A,TODD S,GUTMAN G A,et al.Transduction of a human RNA sequence by poliovirus[J].Journal of Virology,1994,68(10):6547-6552.
[13] RUIZ-SAENZ J,GOEZ Y,TABARES W,LOPEZ-HERRERA A.Cellular receptors for foot and mouth disease virus[J].Intervirology,2009,52(4):201-212.
[14] WANG G,WANG Y,SHANG Y,et al.How foot-and-mouth disease virus receptor mediates foot-and-mouth disease virus infection[J].Virology Journal,2015,12:9.
[15] YANG B,YANG F,ZHANG Y,et al.The rescue and evaluation of FLAG and HIS epitope-tagged Asia 1 type foot-and-mouth disease viruses[J].Virus Research,2016,213:246-254.
[16] ZHU Y,ZOU X,BAO H,et al.Insertion site of FLAG on foot-and-mouth disease virus VP1 G-H loop affects immunogenicity of FLAG[J].Journal of Integrative Agriculture,2018,17(7):1655-1666.
[17] LIAN K,YANG F,ZHU Z,et al.The VP1 S154D mutation of type Asia1 foot-and-mouth disease virus enhances viral replication and pathogenicity[J].Infection,Genetics and Evolution,2016,39:113-119.
[18] BISWAL J K,SUBRAMANIAM S,RANJAN R,et al.Partial deletion of stem-loop 2 in the 3'untranslated region of foot-and-mouth disease virus identifies a region that is dispensable for virus replication[J].Archives of Virology,2016,161(8):2285-2290.
[19] KLOC A,DIAZ-SAN SEGUNDO F.Foot-and-mouth disease virus 5'-terminal S fragment is required for replication and modulation of the innate immune response in host cells[J].Virology,2017,512:132-143.
[20] DE LOS SANTOS T,DE AVILA BOTTON S,WEIBLEN R,et al.The leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mRNA and blocks the host innate immune response[J].Journal of Virology,2006,80(4):1906-1914.
[21] PACHECO J M,GLADUE D P,HOLINKA L G,et al.A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle[J].Virology,2013,446(1-2):260-267.