O型与A型口蹄疫重组标记疫苗病毒株的制备及鉴定

为研制针对我国边境地区(尤其是西南边境)流行的A型口蹄疫(FMD)的标记疫苗储备病毒株,本研究通过化学合成东南亚地区流行的A型口蹄疫疫毒(FMDV)A/VN/03/2009株的P1基因,将其插入含FMDV编码非结构蛋白3A aa91-aa105位缺失的O/HN/93病毒全长重组质粒p OFS/3A_(91-105)中,构建FMDV A型和O型嵌合的全长重组质粒p O/3A_(91-105)-AP1。该重组质粒经NotⅠ酶切线性化后转染于表达T7 RNA聚合酶的BSR/T7细胞中,拯救得到FMDV重组病毒。间接免疫荧光、RT-PCR和序列测定结果表明拯救的FMDV为正确的A型和O型间嵌合及3A aa91-aa105缺失的重组病毒。病毒蚀斑和一步生长曲线表明拯救病毒的感染性和复制能力稍低于其亲本病毒。该型间嵌合标记病毒的成功拯救为研制防控边境地区A型FMD的储备疫苗奠定了基础。     

O型口蹄疫病毒P1-2A3C基因在家蚕杆状病毒表达系统中的表达

口蹄疫是一种严重危害畜牧业生产的烈性传染病。为了促进O型口蹄疫病毒(FMDV)基因工程活载体疫苗的研制,选取O型FMDV编码序列中的衣壳蛋白前体P1-2A基因和蛋白酶3C基因,插入家蚕杆状病毒转移载体pVL1393中,构建重组载体pVL-P1-2A3C,并与线性化病毒Bm-BacPAK6 DNA共转染家蚕BmN细胞,获得重组病毒Bm-P1-2A3C。将重组病毒感染家蚕5龄幼虫,以双抗体夹心ELISA法和间接血凝方法检测血淋巴中的表达产物:目的蛋白在感染病毒后120 h的蚕血淋巴中表达量最高,抗原表达呈阳性的最大稀释倍数为1∶128。结果显示O型FMDV的P1-2A3C基因已在家蚕体内获得表达。     

BHK-21悬浮细胞培养条件筛选及口蹄疫病毒悬浮培养工艺研究

为了实现在幼地鼠肾细胞(BHK-21细胞)低血清全悬浮培养工艺下获得更高含量的口蹄疫病毒(FMDV),试验对比了4株不同来源的BHK-21悬浮细胞的培养特性,按体积1%、2%和5%分别接种O型、A型FMDV,接种后4,8,12,16,20,24小时取样进行146S、半数致死量(LD₅₀)对比研究,筛选形态良好、培养特性更稳定的BHK-21悬浮细胞株;以摇瓶悬浮培养筛选得到的BHK-21悬浮细胞株,使用O型FMDV O/HB/HK/99株、A型FMDV AF/72株,从接种剂量、活细胞密度(VCD)、病毒培养温度、病毒培养pH值、换液体积比例等工艺参数对病毒146S表达的影响进行了研究,并在7.5,50,500 L生物反应器中逐级放大,进行了O型FMDV O/HB/HK/99株、A型FMDV AF/72株各3个批次病毒液悬浮培养研究,确定O型FMDV O/HB/HK/99株、A型FMDV AF/72株接种BHK-21悬浮细胞的培养工艺。结果表明：筛选得到1株形态良好、培养特性更稳定的BHK-21-CP005悬浮细胞株;O型FMDV O/HB/HK/99株、A型FMD...     

口蹄疫病毒3ABC基因的扩增与序列分析

参考GenBank中发表的猪源O型口蹄疫病毒(FMDV)3ABC的基因序列,设计一对特异引物,分别以5株猪源O型FMDV流行毒株基因组RNA为模板,通过RT-PCR的方法获得3ABC基因,并克隆到pMD18-T载体中.测序结果显示:5株O型FMDV流行毒株的3ABC基因cDNA均为1 281bp,编码由427个氨基酸残基组成的多肽.同源性比较和系统发育树分析表明:5株O型FMDV流行毒株亲缘关系密切,属同一基因型.     

共表达O型FMDV衣壳蛋白和绿色荧光蛋白重组腺病毒的构建

为构建表达O型口蹄疫病毒(FMDV)的P12A3C基因及GFP基因的重组腺病毒rAdV-P12aEGFP2a3C,本研究以FMDV的2A基因序列为Linker,将报告基因EGFP插入FMDV的P12A与3C之间。重组腺病毒感染HEK-293细胞后可以观察到绿色荧光,表明EGFP蛋白获得表达。应用FMDV的VP2单克隆抗体4B2对重组病毒感染细胞进行western blot检测,反应条带与FMDV衣壳蛋白VP0和VP3的分子量大小相符,表明FMDV的完整衣壳蛋白和3C蛋白酶也均获得表达,而且EGFP的插入并未影响P1蛋白的表达和3C蛋白酶对P1的正确切割。重组腺病毒的生长特性分析表明,EGFP的插入也未影响该重组腺病毒的增殖特性。上述研究结果显示,表达FMDV衣壳蛋白P12A3C的重组腺病毒可以作为载体,以2A蛋白作为Linker表达一个小分子蛋白,为改进以腺病毒为载体的口蹄疫基因工程疫苗提供了新思路。     

一株Asia 1型口蹄疫病毒的全基因组序列测定及比较分析

参考GenBank上已发表的口蹄疫病毒（FMDV）全长基因组序列,设计了覆盖基因组全长的数对引物,通过RT-PCR方法对1株Asia1型（简称As01）FMDV进行分段克隆及序列测定,将测序结果利用软件进行拼接。结果表明,该毒株的FMDV基因组[不合poly（C）]全长8180nt,其中编码区为6987nt,5’和3’非编码区（UTR）分别为1078nt和95nt,3＇UTR之后为20nt的poly（A）尾巴。将As01株与其他FMDV参考毒株利用分子生物学软件进行多序列比对分析表明,As01株与Asia 1／Jiangsu／China／2005、Asia 1／Isr 1／3／63、ZB／CHA／58等Asia 1型FMDV遗传进化关系较为密切,而与O型和A型FMDV遗传关系较远。     

猪乙脑病毒WHe株的PrM-E、NS1、E-NS2A基因的克隆及序列测定

本文通过RT-PCR扩增了猪乙型脑炎病毒WHe株主要抗原基因NS1(约1.14 kb)、prM-E(约2.1 kb)、E-NS1-NS2A (约3.0 kb),并将NS1、E-NS1-NS2A、PrM-E基因克隆、测序.与Genbank发表的JEV基因序列进行序列分析,发现WHe株与其他12种典型的JEV强毒株的同源性在88.3 %～99.4 %之间.WHe株与K94P05株的同源性最低(88.3 %),与P3株的同源性最高(99.4 %),可认为WHe株是由P3株衍生而来的.在JEV基因组5'端389～3910的长3522 nt的决定JEV抗原性的C-prM-E-NS1-NS2A区中,WHe株与P3株仅有21个碱基不同,其中的14个单碱基突变为同义突变,另外7个为错义突变,导致相应的氨基酸序列(1173个)中的6个氨基酸发生了变异,其中的5个氨基酸变异出现在含有各关键的抗体中和决定簇的E蛋白内,另一个位于NS1蛋白内.     

禽传染性支气管炎病毒嗜肾毒株(X株)核衣壳基因的克隆和序列分析

根据已发表的禽传染性支气管炎病毒 (IBV) Beaudette株核衣壳基因序列 ,设计了 1对特异性引物 ,采用 RT-PCR方法 ,克隆了禽传染性支气管炎病毒嗜肾毒株 (X株 )的核衣壳基因 ,并测定了其序列。 X株 IBV与来自 Gen Bank的其他 10株 IBV相比 ,共存在 136个点突变 ,其中有 15处是连续突变区域 ,这些点突变总体呈散在分布。X株与其他毒株的核衣壳蛋白同源性在 90 .2 %～ 99.0 %之间 ,X株 IBV与同为肾型的 N株同源性最高。     

东北虎源犬瘟热病毒H基因生物信息学分析

从GenBank数据库下载犬瘟热病毒14种基因型H基因237株序列,与3株东北虎源犬瘟热病毒序列比对之后进行同源性和位点变异分析,最后构建系统发育树。研究发现:3株东北虎源犬瘟热病毒(KC579362、KC579363和KM386683)分别属于Arctic-like和Asia-1基因型,与其他基因型毒株的同源性为89.94%~100%之间,H基因序列1 632(G→A)、1 664(C→T)、1 729(G→A)和1 795(G→A)位点的突变可能是CDV跨宿主传播给东北虎的关键突变位点。Arctic-like和Asia-1型毒株对东北虎威胁最大,H基因变异位点可为研究东北虎源序列遗传变异特征提供分子生物学依据。     

口蹄疫病毒Hankou/99株基因组全序列测定与基因特征分析

本研究主要对口蹄疫病毒(FMDV) Hankou/99株全基因序列进行测定,通过基因序列分析,确定其基因型,丰富了FMDV基因库,为研究猪源FMDV的分子变异、感染性分子克隆及致病机理奠定基础.从感染FMDV Hankou/99株的细胞液中提取RNA,通过RT-PCR技术,获得猪FMDV Hankou/99分离株覆盖全基因组的5个cDNA片段(S、L、C、D、E),分别对这些片段进行克隆和序列测定.结果显示,FMDV Hankou/99株全基因组长8099 bp;5'NCR长1040 bp,开放阅读框长6966 bp;3'NCR长93 bp,其后是30个碱基的连续poly(A)结构.通过与参考株基因组结构比较分析,显示其在分类地位上属于O型FMDV,并与猪源FMDV毒株OLZ、TW/97同源性较高,特别是3A区域上都有30 bp的缺失.另外,通过与9个参考株的VP1系统发生树分析,显示其与OLZ、TW/97、O/Akesu/58、O/OMⅢ 4个毒株为同一基因型.     

A complex-trapping-blocking (CTB) ELISA, using monoclonal antibodies and detecting specifically antibodies directed against foot-and-mouth disease types A, O and C. I. Method and characteristics

A complex-trapping-blocking (CTB) enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies directed against foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, and C1 Detmold. For each strain two monoclonal antibodies directed against different antigenic sites of FMDV were used. The assay used either infectious, not inactivated antigen or inactivated antigen. We concluded that the CTB-ELISA was sensitive, type-specific, and more reproducible (P less than 0.05) than the serum neutralisation test (SNT). In addition, the test was easy to perform and results could be recorded within 3 hours. The cross-reactivity of bovine reference sera raised against the three FMDV strains was comparable in the CTB-ELISA and the SNT.     

Development and characterization of monoclonal antibodies against FMD virus type Asia-1 and determination of antigenic variations in the field strains

Twelve mouse monoclonal antibodies (MAbs) were developed against an Indian vaccine strain of foot and mouth disease virus (FMDV) type Asia-1 WBN 117/85. The MAbs were tested for their ability to bind to whole virus particle, trypsin-treated 146S (TT-146S) virus particle, sub-viral (12S and disrupted virus) antigens by ELISA and to neutralize virus infectivity in cell culture. Extensive characterization of MAbs revealed the existence of three different groups based on the binding of non-overlapping epitopes. Eight type Asia-1 specific MAbs (RF7, RF8, RD10, RE11, RC11, RC10/O, RB11 and RC10/M), which formed group 1 (G1), were found to bind a neutralizing, trypsin-sensitive (TS) and conformational epitope. Two MAbs (WB8 and WC3) in group 2 (G2) were found to bind a non-neutralizing, trypsin-resistant, conformational and 12S-specific epitope, which was intertypically conserved in all the four serotypes of FMDV (O, A, C and Asia-1) prevalent in India. Two MAbs (KG10 and KF10), which formed group 3 (G3), were found to be against a non-neutralizing, TS and conformational epitope, common to types Asia-1 and A. Members of G1 were IgG2a isotype, while those of G2 and G3 were IgG1 and IgG2b isotypes, respectively. Antigenic analysis of 31 FMDV type Asia-1 field isolates and two vaccine strains, using a panel of type Asia-1-specific MAbs, revealed antigenic similarity of the virus isolates tested and non-existence of neutralization escape mutants. The developed MAbs have practical utility, especially in the manufacture of FMD vaccine, diagnosis and FMDV characterization.     

Sequence analysis of the protein-coding regions of foot-and-mouth disease virus O/HK/2001

The nucleotide sequence of the protein-coding region of foot-mouth-disease virus (FMDV) strain O/HK/2001 was determined and compared with the sequences of other FMDVs that were registered in GenBank. The protein-coding region was 6966 nucleotides in length and encoded a protein of 2322 amino acid residues. Comparison of the nucleotide sequence and its deduced amino acid sequence with those of other isolates indicated that O/HK/2001 belonged to the Cathay topotype. A genomic coding region nucleotide sequence phylogenetic tree of several FMDV-O isolates showed that O/HK/2001 was most closely related to FMDV isolates found in Taiwan during 1997, and especially shared significant similarity to HKN/2002, suggesting that the virus causing outbreaks in Hong Kong was genetically most-closely related to that causing an outbreak of type O in Taiwan. Mutations in O/HK/2001 were revealed, including frequent substitutions in the VP1 and L proteins, and deletions involving 10 amino acid residues in the 3A protein. This study was undertaken to assess the regional variation of prevalent FMDV type O viruses and to establish a sequence database for FMDV molecular epidemiological investigation.     

Detection of Foot and Mouth Disease Virus by RT-PCR and Microplate Hydridization Assay Using Inactivated Viral Antigens

A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.     

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

口蹄疫多基因杆状病毒转移载体的构建

采用分步 PCR扩增连接的方法将口蹄疫病毒结构蛋白基因 P1 ,非结构蛋白基因 2 A、2 B、3 C蛋白酶和 3 D聚合酶按正确的读码框依次连接克隆入 p GEM-T载体。然后 ,将切取的目的基因亚克隆入杆状病毒转移载体 p Mel Bac B。构建成功的重组 p Mel Bac B/P1 2 X3 C3 D转移载体经测序鉴定 ,含有完整的目的基因表达盒 ,转移载体可与杆状病毒骨架载体进行昆虫 sf9细胞内同源重组 ,以产生重组杆状病毒     

云南边境O型口蹄疫监测及病毒VP1基因序列分析

口蹄疫是由口蹄疫病毒引起的主要侵袭偶蹄动物的一种急性热性高度接触性传染病。口蹄疫病毒为微RNA病毒科口蹄疫病毒属成员,存在7个不同血清型,病毒VP1蛋白抗原性差异是病毒血清型划分依据,而其编码基因(1D)核苷酸序列差异是同型病毒拓扑型(Topotype)或基因型鉴别依据。采用O/A/C/Asia-1多重RT-PCR技术,对2006年自云南边境地区采集的120份动物组织样品,进行口蹄疫病原监测,检出O型口蹄疫病毒阳性样品15份。对阳性样品中病毒VP1基因全序列进行扩增、纯化后,克隆至pMD18-T载体测序,并与已知代表性毒株进行比对及系统发育分析。结果发现:云南边境O型口蹄疫病毒阳性样品VP1基因核苷酸序列同源性介于77.3%~98.7%,可划分为3个不同的拓扑型或基因型:中东-南亚型(ME-SA)或泛亚型(PAN-Asia)、古典中国型(Cathay)、东南亚型(SEA)。部分样品VP1蛋白表位43位、154位关键性氨基酸位点存在变异。     

亚洲1型口蹄疫病毒RS株非结构蛋白P3区基因特征分析

经RT-PCR扩增得到一株口蹄疫亚洲1型流行毒株的非结构蛋白P3基因核苷酸序列,与其他代表性参考毒株的P3基因进行比较,分析该毒株P3区基因特征.结果表明,该毒株P3基因含有2 721个核苷酸,编码907个氨基酸;其中非结构蛋白3A的基因长度为459 bp,编码153个氨基酸;3个3B(VPg)基因长度分别是69、72、72 bp,分别编码23、24、24个氨基酸;3C基因长度为639 bp,编码213个氨基酸;3D基因长度为1 410 bp,编码470个氨基酸.氨基酸序列比较分析显示,3A基因C末端比其他基因更容易变异,根据3A基因构建的系统进化分析提示该流行毒株与YNBS/58和IND 321/01亲缘关系较近,属同一亚系谱.     

O型口蹄疫病毒结构蛋白VP1的原核表达与抗原性分析

研究分析了O型口蹄疫病毒(FMDV)结构蛋白VP1与当前猪FMDV疫苗血清的免疫反应性.将VP1基因克隆至原核表达载体pET32c,并在大肠埃希菌BL21中得到了表达,Western blot分析表明该重组蛋白与豚鼠O型FMDV标准阳性血清具有良好免疫反应性.目的蛋白经纯化后用ELISA分析其与猪疫苗血清的免疫反应性,结果显示该重组VP1蛋白(rVP1)只能与部分O型FMDV疫苗血清反应.推测当前使用的不同O型FMDV疫苗毒株在VP1重要中和抗原位点G-H环(134 aa～158 aa)与C末端(200 aa～213 aa)存在较大差异.     

中国鸭源H5N6 AIV血凝素和神经氨酸酶基因特征及遗传进化分析

为明确中国鸭源H5N6 AIV的HA和NA基因特征及其遗传变异情况,研究选取了NCBI流感病毒资源库里的32株具有完整ORF编码区的中国鸭源H5N6 AIV的HA和NA基因序列,利用分子生物学软件分析其HA和NA基因特征及其遗传进化情况。结果显示,中国鸭源H5N6 AIV的HA裂解位点有REKRRKR↓G、RERRRKR↓G和KEKRRKR↓G三种类型,HA有6个潜在的N-糖基化位点,其中第182、222、224位氨基酸依次为N、Q、G,具有与禽源唾液酸α-2,3受体结合的特性,然而HA发生了S123P/T、T156A、S223R氨基酸位点突变,部分毒株发生了I151T氨基酸位点突变;HA基因核苷酸遗传进化分析显示,中国鸭源H5N6 AIV遗传进化上均处于clade2.3.4.4分支,并进一步进化出Ⅰ和Ⅱ两个亚分支。部分中国鸭源H5N6 AIV NA第59~69位缺失11个氨基酸,中国鸭源H5N6 AIV NA基因遗传进化上分为两个大的分支,分别对应NA基因颈部缺失型和完整型两种类型;NA颈部完整型的NA有6个潜在的N-糖基化位点,NA颈部缺失型的NA由于第59~69位氨基酸的缺失,导致其第67位丢失了一个N-糖基化位点。研究为中国鸭源H5N6 AIV的生物学特性和遗传进化特征研究奠定基础。