【作者】 仇旭升；

【导师】 刘秀梵；

【作者基本信息】 扬州大学 ， 预防兽医学， 2009， 博士

【摘要】 新城疫(Newcastle disease,ND)是一种严重的传染性疾病,能够感染多种禽类,给世界养禽业造成了巨大的损失。该病的病原新城疫病毒(Newcastle disease virus,NDV)属于副粘病毒科(Paramyxoviridae),副粘病毒亚科(Paramyxovirinae),禽腮腺炎病毒属(Avulavirus)。NDV的基因组由单负股RNA构成,目前已知的基因组长度有三种,15,186 nt、15,192 nt以及15,198 nt。基因组按照3′-NP-P-M-F-HN-L-5′的顺序编码6种病毒结构蛋白,分别是核蛋白(nucleoprotein,NP),磷蛋白(phosphoprotein,P),基质蛋白(matrix protein,M),融合蛋白(fusion protein,F),血凝素-神经氨酸酶蛋白(haemagglutinin-neuraminidase,HN),以及大分子蛋白(largeprotein,L)。除此之外,P基因还能够通过RNA编辑(RNA editing)机制在转录产物特定位点(UUUUUCCC)插入1个或2个非模板G,从而编码额外的两种非结构蛋白V和W。P基因编码的P蛋白能够辅助NP蛋白单体正确折叠,同时是RNA聚合酶的亚单位之一,在病毒基因组RNA的转录和复制中起着重要作用;V蛋白能够拮抗宿主细胞干扰素系统,对病毒的免疫逃避起着重要的作用。尽管如此,目前对NDVP基因的研究才刚刚开始,大部分数据来自于其它副粘病毒的研究结果,对于各种毒力NDV毒株P基因功能差异的研究尚未开始。本室测定了鹅源NDV强毒株ZJ1的全基因组序列,并在此基础上构建了该基因组cDNA的全长克隆以及分别表达该毒株NP、P以及L蛋白的3个真核表达质粒,通过病毒的体外包装技术,拯救出了具有感染性的病毒粒子,成功建立了鹅源NDV的反向遗传操作技术平台,这也为从病毒生物学特性的角度研究P基因提供了技术支持。1.基因Ⅲ型和Ⅳ型新城疫病毒全基因组测序及其在新城疫进化史中的重要地位分析根据EMBL/GenBank中已发表的基因Ⅲ型NDV全基因和基因Ⅸ型NDV基因片段设计一套全基因引物,将NDV全基因分为10个相互重叠的片段进行RT-PCR扩增。PCR产物连入pGEM-T easy载体测序后应用DNAStar软件进行遗传进化分析。我们测定了5株基因Ⅸ型NDV和2株基因Ⅲ型全基因组序列并进行遗传进化分析。实验数据显示,虽然两种基因型毒株同源性较高,但是基因Ⅸ型NDV NP基因内确实存在6 nt插入片段,基因全长为15,192nt,是与基因Ⅲ型NDV不同的毒株。鉴于基因Ⅸ型代表株F48E8的分离时间,可以确定早在上世纪三、四十年代就已经存在全长15,192nt的毒株。值得注意的是,常用的遗传进化分析以及限制性内切酶位点(RE)分析并不能有效的区分这两种基因型以及Ⅳ型,NP基因5’端非编码区核苷酸长度才是最可靠的遗传标志。最后,根据两个基因型毒株相近的分离时间和高度的同源性,我们推测最早有一群NDV毒株在遗传进化过程中分化为两支,保持15,186nt基因组长度的一支演变为基因Ⅲ型、Ⅳ型及相关毒株;而另外一支,在NP基因5′-NCR插入6个核苷酸,产生了15,192nt基因组长度的毒株,可能即为最早的基因Ⅸ型毒株,再逐步进化演变为后来的基因Ⅴ～Ⅷ型。2.新城疫病毒P/V/W基因编码产物的结构域分析及针对V蛋白C端结构域的抗体制备根据EMBL/GenBank上发布的NDV基因序列,分别设计针对基因Ⅱ、Ⅲ、Ⅵ和Ⅶ型以及class I NDV毒株P基因的引物。RT-PCR扩增后测序,从而获得5种基因型NDV P基因的正确序列。通过核苷酸序列预测P基因表达产物的氨基酸序列,并进行二级结构预测和三级结构模拟。实验结果发现,腮腺炎病毒属的猴副流感病毒V型(Simian virus 5,SV5)的V蛋白与NDV V蛋白的氨基酸序列同源性以及二级结构的相似性均较高,可以将其空间结构作为模板模拟NDV V蛋白以及P蛋白N端的空间结构。综合各种分析数据,可以确定P蛋白辅助N蛋白折叠的结构域位于N端前50aa内,其中20 aa-29 aa预测为一个潜在的疏水性α-螺旋;50 aa-131 aa结构紊乱;预测P蛋白螺旋卷曲结构位于221 aa-290 aa范围内,可能介导了P蛋白四聚体的形成以及与L蛋白的相互作用;预测NDV的X结构域位于291 aa-392 aa范围内,其中350 aa-392 aa区域内存在3个相连的α-螺旋,其二级结构与副粘病毒X结构域的C端亚单位结构相似,可能介导了P蛋白与衣壳化基因组的相互作用。V蛋白的C端结构域(C-terminal domain,CTD)的编码区域位于P蛋白132 aa-239 aa区域内,与螺旋卷曲结构部分重叠。最后,根据分析结果将基因Ⅶ型NDV毒株ZJ1 V蛋白CTD的序列插入pGEX-6p-1表达载体中诱导表达。表达产物通过切胶免疫的方法多次免疫小鼠,结果获得抗V蛋白CTD的多抗血清。利用间接免疫荧光鉴定多抗血清与病毒蛋白的反应情况,证明制备的抗CTD血清能够与NDV感染vero细胞作用,与正常Vero细胞不反应,为后续的研究工作奠定了基础。3.不同基因型新城疫病毒P基因对病毒毒力的影响利用BglⅡ酶切全长质粒pNDV/ZJ1,成功构建含有TVT载体、NP基因、P基因以及很少部分M基因和L基因片段的中间质粒pTX37。PCR分别分别扩增NDV基因Ⅱ型疫苗株LaSota、基因Ⅲ型中等毒力株JS/05/9/Go和基因Ⅶ型强毒株JS/05/5/Go的P基因,上游引物突变引入SmaⅠ的酶切位点,下游引物突变出ApaⅠ酶切位点。通过SmaⅠ和ApaⅠ将3株病毒的P基因替换进入中间载体pTX37,再利用BstZ17Ⅰ和XbaⅠ将重组质粒连入NDV基因组全长转录载体质粒。重组的3种NDV基因组全长转录载体质粒与3个辅助表达质粒pCI-L、pCI-NP和pCI-P共转染BSR-T7/5细胞,成功拯救出了P基因替换株RZJ-LP,RZJ-4P,RZJ-IP。通过MDT、ICPI、IVPI、细胞致病力以及生长曲线的检测,发现3株P基因替换株的毒力变现明显的差异:RZJ-LP和RZJ-4P在细胞上的生长滞后于亲本病毒ZJ1,细胞上清中的病毒含量比ZJ1低10倍左右;RZJ-4P和RZJ-LP的ICPI从ZJ1的2.54下降至2.33和1.51;RZJ-IP表现出了比ZJ1更强的感染力,感染细胞后最早出现病变的时间和完全破坏单层细胞的时间比ZJ1还要早12 h-24 h。3株P基因替换株的毒力强弱排序与其P基因来源毒株的毒力强弱排序相同,表明P基因是影响NDV毒力的因素之一。4.新城疫病毒P基因编码产物的结构域对病毒生物学活性的影响通过SmaⅠ和ApaⅠ的酶切位点,将基因Ⅱ型疫苗株LaSota P基因RNA编辑位点上游部分和下游部分分别替换进入基因Ⅶ型NDV ZJ1,成功拯救了具有嵌合P基因的两株重组病毒RZJ-LaN和RZJ-LaC。通过PCR在中间质粒pTX37的P基因RNA编辑位点后进行点突变引入终止密码子TAG,拯救V蛋白C端结构域缺失株RZJ-VS和荧光标记的RZJ-VS/GFP,重组病毒P蛋白的正常表达不受影响,而由+1G mRNA编码的V蛋白翻译到RNA编辑位点后即终止。通过MDT、ICPI、IVPI、细胞致病力以及生长曲线的检测,发现RZJ-LaN和RZJ-LaC的毒力比亲本病毒稍弱,但明显强于P基因替换株RZJ-LP,表明上一章中发现的RZJ-LP毒力大幅度下降是由P蛋白和V蛋白的多个结构域协同作用的结果,并非是单个结构域导致。在没有干扰素系统的Vero细胞上RZJ-LP、RZJ-LaN和RZJ-LaC生长曲线基本重合,而在原代细胞CEF、GEF上RZJ-LaN的增殖能力最强,RZJ-LaC最弱,RZJ-LaN感染细胞中病毒的最高含量比RZJ-LP高5～10倍,RZJ-LP比RZJ-LaC高5～10倍,表明ZJ1 V蛋白CTD的干扰素拮抗能力强于LaSota。V蛋白缺失株的生物学活性与相关报道完全相反,在细胞和鸡胚中的生长不受影响,接种Vero、CEF和GEF细胞以后,细胞病变以圆缩脱落为主,单层细胞完全破环的时间超过72 h。这种细胞病变过程与亲本病毒完全不同,暗示V蛋白的CTD结构域应该具有未知的功能。检测荧光发现,RZJ-VS/GFP和RZJ/GFP在以0.01MOI接种细胞后12 h都出现了很强的荧光,24 h后绝大多数单层细胞出现荧光,这表明ZJ1株的复制和传播能力非常的强,感染速度超过了细胞抗病毒免疫启动的速度,推测基因Ⅶ型NDV高效的增殖和传播能力也是其免疫逃避的机制之一。全文小结:(1)测定了2株基因Ⅲ型和5株基因Ⅸ型NDV的全基因组序列,证实两种基因型毒株虽然同源性较高,但是基因Ⅸ型NDV确实有6nt插入片段,基因全长为15,192 nt,是最早出现的15,192 nt基因组长度毒株。分析后推测,NDV毒株在遗传进化过程中分化为两支,保持15,186nt基因组长度的一支演变为基因Ⅲ型、Ⅳ型及相关毒株;而另外一支,在NP基因中插入6个核苷酸,产生了15,192nt基因组长度的毒株,可能即为最早的基因Ⅸ型毒株,再逐步进化演变为后来的基因Ⅴ～Ⅷ型。(2)通过对P基因表达产物的生物信息学分析,确定P蛋白和V蛋白结构域的大致位置。(3)通过反向遗传平台,替换基因Ⅶ型强毒ZJ1的P基因全基因或部分结构域,成功拯救5株重组病毒。3株P基因替换株的毒力强弱排序与其P基因来源毒株的毒力强弱排序相同,证实P基因是影响毒力的因素之一,同时发现P基因对毒力的影响是由多个结构域共同参与,而非单一结构域决定。(4)利用反向遗传平台,成功拯救2株V蛋白C端结构域缺失病毒,证实在相同病毒骨架下,ZJ1 V蛋白的干扰素拮抗能力强于LaSota。V蛋白C端结构域缺失株的生物学特性与亲本病毒差异不大,明显与NDV同类研究结果相反,推测基因Ⅶ型NDV高效的增殖和感染能力也是其免疫逃避机制之一。(5)细胞感染RZJ-VS以后,CPE明显改变,细胞以圆缩脱落为主,单层细胞完全破环的时间大幅度延长,暗示V蛋白的C端结构域应该具有未知的功能。更多还原

【Abstract】 Newcastle disease(ND)is one of the most serious infectious diseases of birds,and has been a major cause of economic losses in poultry industry.Its causative agent, Newcastle disease virus(NDV),is the sole member of avian paramyxovirus-1 (APMV-1),belonging to genus Avulavirus,subfamily Paramyxovirinae,family Paramyxoviridae,order Mononegaviriales.NDV possesses a negative-sense, single-stranded continuous RNA genome,which consists of 15,186 nt,15,192 nt or 15,198 nt.The genome of NDV contains six genes in the order of 3’-NP-P-M-F-HN-L-5’,encoding six viral proteins(nucleoprotein,phosphoprotein,matrix protein, fusion protein,haemagglutinin-neuraminidase and large protein,respectively).Like other members of the Paramyxovirinae,NDV edits its P gene by inserting one or two G residues at the conserved editing locus(UUUUUCCC)and transcribes three P-gene-derived mRNA species.The mRNAs encode the open reading frame(ORF)of P (unedited),the V ORF(with a+1 frameshift),and the W ORF(with a+2 frameshift).P protein associates with the soluble,monomeric form of N and prevents its illegitimate self-assembly onto cellular RNA.P protein also forms complexes with assembled form of N and L during transcription and replication.V protein,which is also transcribed from P gene,proved to be associated with viral Pathogenesis and functions as an alpha interferon antagonist.In 2000,NDV strain named ZJ1 was isolated and identified from goose flocks in Zhejiang province,and subsequently the complete genomic sequence of ZJ1 strain was determined.A transcription vector containing the full length cDNA of ZJ1 genome and three helper plasmids expressing NP,P or L protein respectively were constructed to establish the reverse genetics system for NDV ZJ1 strain.After cotransfecting the transcription vector together with three helper plasmids,highly virulent infectious NDV was successfully generated.This established reverse genetics system provides a powerful tool for the research of the P gene in this study.1.Full-length genome analysis of Newcastle diseases virus strains belonging to genotypeⅢandⅨreveals the common origin of recent epizootic viruses.Ten fragments from seven NDV strain were amplified and cloned into pGEM-T vector.Based on sequencing results,the entire genome sequences of five genotypeⅨstrains and two genotypeⅢstrains were determined.Alignment of the seven full-length sequences revealed the presence of the 6 nt insert in the 5’-noncoding region(NCR)of the NP gene of five genotypeⅨNDV isolates.In other words,the genome of genotype IX NDV was 15,192 nt in length,quite different from genotypeⅢstrains.The genome size of F48E8(F48E9)indicated that NDV strains with 15,192nt genome had emerged as early as 1940s,rather than 1960s when the infections of genotypesⅤandⅥstrains were found.Phylogenetic and genetic character analysis revealed that viruses belonging to genotypeⅨshared higher homology with early genotypesⅢandⅣstrains, although they possessed 6 nt insert in the NP gene,a derived characteristic of recent four genotypes.Based on epidemiological data and phylogenetic analysis,it is inferred that the insertion of 6 nt into the genome of a genotypeⅢstrain gave rise to genotypeⅨstrains,which could be the common origin of NDV strains belonging to genotypeⅤ-Ⅷ.2.Analysis of the domains of P gene-coded viral proteins and preparation of antiserum specific for C-terminal domain ofⅤproteinFragments of P gene from NDV strains belonging to genotypeⅡ,Ⅲ,Ⅵa,Ⅶd and classⅠwere amplified with 5 pairs of specific primers.The PCR products were cloned to pGEM-T vector and sequenced to determine the exact nucleotide sequence of P gene. Based on sequencing results,the amino acid sequences of P,Ⅴand W protein were predicted,and subsequently sent to internet for second structure prediction and modeling.It was predicted that the structure ofⅤprotein of simian virus 5(SV5),a member of Rubulavirus,was adequate for modelingⅤprotein or the N moiety part of P protein of NDV,due to the homology ofⅤproteins in amino acid and second structure. Structural investigations on the P gene-encoded proteins showed that anα-helix in the phosphoprotein N-terminal domain(PNT)from 20 aa to 29 aa was identified,which was conserved in all Paramyxoviruses,acting as a chaperone for newly synthesized N, and prevent it from bingding to non-viral RNA in the infected cells.The phosphoprotein C-terminal domain(PCT)was mapped for various protein-protein interactions required for viral transcription:the putative oligomerization domain(PMD)was in the region between aa 221 and aa 290;the putative C-terminal sub-domain ofⅩdomain was found beween aa 350 and aa 392,which was proved to be a N:RNA-binding domain of Paramyxovirus;the C-terminal domain(CTD)ofⅤprotein was found beween aa 132 and aa 239,partially overlapping with PMD.According to the results of second structure analysis,the CTD of P gene from ZJ1 was amplified and cloned into pGEX-6p-l for expression.Several BALB/c mice were immunized with the expression products for more than four times to produce anti-serum specific for CTD.The indirect fluorescence assay(IFA)confirmed that anti-CTD serum reacted with NDV-infected cells,rather than other normal cells.3.Rescued NDVs with P gene derived from diverse origins showed differences in biological characteristics,especially in pathogenicity. In order to construct a plasmid for P gene substitution,a fragment of ZJ1 cDNA from 3641 nt(BglⅡ)to 4748 nt(BstZ17Ⅰ)was amplied and ligated with BglⅡ-digested plasmid pNDV/Z J1,containing the full-length cDNA of NDV ZJ1.The newly constructed plasmid was identified and called pTX37.The fragments of P gene were amplified from three NDV strains as follows:lentogenic strain LaSota,belonging to genotypeⅡ;mesogenic strain JS/05/9/Go,belonging to genotypeⅢ;velogenic strain JS/05/5/Go,belonging to genotypeⅦ.The PCR products of three P gene were digested with Smal and Apal,and ligated into the digested pTX37 to replace the P gene of pTX37.Subsequently,the recombinant plasmid and pNDV/ZJl were digested with XbaI and BstZ17I,and the products of digestion were ligated together to construct recombinant full-length cDNA of ZJ1 with heterologous P gene.The transcription vectors containing recombinant full-length cDNA together with three helper plasmids expressing viral protein NP,P and L were cotransfected into BSR-T7/5 cells,expressing T7 RNA polymerase.Three recombinant NDV strains were successfully rescued and named RZJ-LP,RZJ-4P and RZJ-IP respectively.To compare the biological characteristics of rescued viruses and wild-type NDV strain ZJ1,the mean death time (MDT),intracerebral pathogenicity index(ICPI),intravenous pathogenicity index(IVPI) and viral growth kinetics test were carried out.The results showed that recombinant NDV strains with P from LaSota or JS/05/9/Go were less virulent than wild-type strain ZJ1.In Vero,CEF and GEF cells,RZJ-LP and RZJ-4P grew more slowly and produced 10-fold fewer infectious progeny compared to ZJ1.The IVPI of RZJ-LP was 1.51,much lower than 2.54 of ZJ1.The pathogenicity of the three recombinant viruses indicated that P gene played an important role in virulence.4.Rescued NDV ZJ1 strain with partial P gene replacement showed differences in biological characteristics.The N-terminal moiety and C-terminal moiety of P gene of LaSota was respectively subcloned into plasmid pTX37 to replace the counterpart of P gene of ZJ1.As described above,the recombinant plasmids were ligated into pNDV/ZJ1,and subsequently two recombinant ZJ1 strains with chimeric P gene were rescued,called RZJ-LaN and RZJ-LaC.Two point mutations were introduced after the RNA edition motif in the P gene of plasmid pTX37 to inhibit the expression of the C-terminal domain(CTD)of V protein,while the expression of phosphoprotein was not changed.The resultant plasmid was ligated into full-length cDNA of ZJ1 and subsequently two CTD-deficient NDVs, named RZJ-VS and RZJ-VS/GFP,were rescued.To compare the biological characteristics of rescued viruses and wild-type NDV strain ZJ1,pathogenicity tests of MDT,ICPI,IVPI and viral growth kinetics test were carried out.The IVPI of RZJ-LaN and RZJ-LaC was 2.26 and 2.46,a bit lower than that of ZJ1,indicating that all the domains rather than single domain of P gene contributed to the low-virulence of RZJ-LP. It is noted that RZJ-LP,RZJ-LaN and RZJ-LaC grew equally in Vero cells,a cell lineage lacking interferon system,while the growth of RZJ-LaN in CEF and GEF was faster than those of others and that of RZJ-LaC was the lowest.RZJ-LaN produced 100-fold fewer infectious progeny than RZJ-LaC in vitro,indicating that the antagonism of the CTD of ZJ1 was better than LaSota.Although the rescued strain RZJ-VS encoded a V protein without CTD,it grew well in cells and eggs,even the same as wild-type strain.The biological characteristics of RZJ-VS were contrary to the results of similar research on other strains.In indirect fluorescent assy(IFA),many fluorescent cells were observed in RZJ-VS/GFP or RZJ/GFP-infected cells 12 h post-infection,indicating that ZJ1 predominated in propagation,which could also be an immune evasion mechanism.In addition,the cytopathic effect(CPE)of RZJ-VS and RZJ-VS/GFP was obviously different from that of ZJ1,indicating that certain function of CTD might not be discovered.Conclusion:(1)The entire genome sequences of five genotypeⅨstrains and two genotypeⅢstrains were determined and analyzed.Alignment of the seven full-length sequences revealed that the genome size of genotypeⅨNDV isolates was 15,192 nt,quite different from genotypeⅢstrains.Based on epidemiological data and phylogenetic analysis,it is inferred that the insertion of 6 nt into the genome of a genotypeⅢstrain gave rise to genotypeⅨstrains,which could be the common origin of NDV strains belonging to genotypeⅤ-Ⅷ.(2)The amino acid sequences of P,V and W protein were predicted and sent to internet for second structure prediction and modeling.Finally,all the functional domains were roughly located in P gene.(3)By using reverse genetics technology,five recombinant ZJ1 strains were rescued, three of which contained heterologous P gene,the others containing chimeric P gene.The biological characteristics of recombinant strains showed that P gene play an important role in virulence.(4)By using reverse genetics technology,two recombinant ZJ1 strains encoding CTD-deficientⅤprotein were rescued.The biological characteristics of recombinant strains showed that CTD of ZJ1 was more powerful than that of LaSota.The CTD-lacking strains grew as well as parental strain,contrary to the results of similar research on other strains,indicating that ZJ1 predominated in propagation,which could also be an immune evasion mechanism.(5)The cytopathic effect(CPE)of RZJ-VS and RZJ-VS/GFP was obviously altered, indicating that certain function of CTD might not be discovered.更多还原