感染性猪圆环病毒2型基因组DNA的分子克隆

本研究通过PCR扩增出猪圆环病毒2型(PCV—2)的全基因组(1768bp),克隆入pcDNA3载体的EcoR I酶切应点,获得含有PCV-2全基因组的重组质粒,命名为pcDNApcv2。将重组质粒大量扩增后,用EcoR I切出1768bp的PCV—2全基因组,在体外用T4DNA连接酶使其连接环化。用脂质体法将体外连接产物转染无PCV污染的PK—15细胞,经4次连续传代,用间接免疫荧光实验(IFA)及电镜观察证实已获得复制能力的PCV—2病毒。由此可见,本试验构建的环化的PCV—2全基因组DNA具有感染性。     

猪圆环病毒2型Cap基因单核苷酸缺失突变体的构建与生物学特性

猪2型圆环病毒(PCV2)是新出现的猪传染病—断乳仔猪多系统衰竭综合征病原.为了分析PCV2基因组核苷酸缺失突变对病毒复制的影响,以PCV2-HZ0201毒株基因组为模板,采用DpnⅠ定点突变技术,构建4个位于ORF2内的单核苷酸缺失突变体感染性克隆△1376PCV2,△1377PCV2,△1378PCV2和△1379PCV2.缺失突变体转染PK-15细胞后用间接免疫荧光检测病毒的拯救效果.结果显示,△1376PCV2感染性克隆能拯救出感染性病毒粒子,△1377PCV2,△1378PCV2和△1379PCV2感染性克隆不能拯救感染性病毒粒子,说明ORF2内1376位核苷酸的缺失不影响PCV2的复制.复制能力测定结果显示,△1376PCV2的复制能力比亲本病毒高10倍左右.促炎因子IL-6的转录本与血清浓度测定结果显示,△1376PCV2感染小鼠后的IL-6转录本与蛋白表达显著高于健康小鼠,且高于亲本病毒.这些数据首次揭示,ORF2内1376位核苷酸缺失的PCV2病毒粒子被提高了复制能力,同时促进了促炎因子IL-6的转录与表达.     

猪圆环病毒2型流行株的分离及其感染性克隆的构建

【目的】通过分离一株猪圆环病毒2型(PCV2)流行毒株,并构建其感染性克隆,为研究PCV2基因功能提供操作平台。【方法】通过PCR方法,从疑似患断奶仔猪多系统衰竭综合症(PMWS)的仔猪淋巴结中鉴定为猪圆环病毒(Porcine circovirus,PCV)2型阳性。把阳性病料接种PK-15细胞传代培养,在培养物中扩增出PCV2的全基因序列。对扩增出的全序列进行序列测定,并与GenBank中公布的5株广东PCV2分离株(GD-pz、GD-gj、GD-jm、GD-ss和GD-sz)进行同源性分析。通过EcoRⅠ和SalⅠ将PCV2全基因组序列克隆进pUC18载体中,获得含PCV2 GD-zq株全基因组单拷贝的重组质粒pPCV2-GD-zq,再通过SalⅠ和HindⅢ把另一个全长拷贝克隆进pPCV2-GD-zq质粒中,使PCV2 GD-zq株基因组DNA以头尾相接的双重复方式克隆进pUC18载体中,获得重组质粒pPCV2-2GD-zq。将pPCV2-2GD-zq DNA纯化和定量后转染PK-15细胞,拯救PCV2 GD-zq病毒。【结果】从PMWS感染的猪淋巴结中分离到了一株PCV2,命名为GD-zq株;序列分析结果显示,GD-zq株全基因组为1 767 bp,与GenBank中公布的5株广东PCV2分离株ORF1核苷酸一致性为97.1%-99.7%,编码氨基酸一致性为98.7%-100%;ORF2核苷酸一致性为93.2%-99.6%,编码氨基酸一致性为92.3%-99.1%;全基因一致性为96.0%-99.6%。pPCV2-2GD-zq质粒转染PK-15细胞后,其通过间接免疫荧光实验(IFA)能从转染细胞及其传代细胞中,检测到拯救出的病毒。【结论】分离了一株PCV2广东株GD-zq,成功构建了PCV2 GD-zq株的感染性克隆。     

嵌合型猪圆环病毒1-2型感染性DNA克隆的构建及其对小鼠的免疫原性

[目的]本研究旨在构建嵌合型猪圆环病毒1-2型感染性DNA克隆.[方法]利用PCR技术扩增PCV2 ORF2,克隆入缺失PCV1 ORF2的pSK-PCV1△ORF2中,得到pSK-sPCV1-2.该重组质粒中包含嵌合型PCV1-2全基因组,通过不完全酶切的方法,将嵌合型PCV1-2全基因组串联入pSK载体中,得到含串联双拷贝的嵌合型PCV1-2感染性DNA克隆.[结果]经序列测定,获得含串联双拷贝的嵌合型PCV1-2感染性DNA克隆.PCV1-2嵌合病毒接种BALB/c小鼠,用间接ELISA方法对接种后7、14、21、28、35、42 d的小鼠进行血清抗体检测.结果显示从接种后14 d开始,即有部分小鼠产生了针对PCV2 Cap蛋白的特异性抗体;至接种后42 d,几乎全部接种小鼠的血清均呈阳性.[结论]构建了PCV1-2型感染性DNA克隆,该嵌合病毒能够激发机体产生体液免疫应答.     

感染性猪圆环病毒2型基因组DNA的分子克隆

本研究通过PCR扩增出猪圆环病毒2型(PCV-2)的全基因组(1 768bp),克隆入pcDNA3载体的EcoRI酶切应点,获得含有PCV-2全基因组的重组质粒,命名为pcDNApcv2.将重组质粒大量扩增后,用EcoRI切出1 768bp的PCV-2全基因组,在体外用T4 DNA连接酶使其连接环化.用脂质体法将体外连接产物转染无PCV污染的PK-15细胞,经4次连续传代,用间接免疫荧光实验(IFA)及电镜观察证实已获得复制能力的PCV-2病毒.由此可见,本试验构建的环化的PCV-2全基因组DNA具有感染性.     

狂犬病病毒CVS-11株全序列测定及其感染性克隆的构建

[目的]测定狂犬病病毒标准攻击毒CVS-11株全基因组序列,构建CVS-11株全长cDNA感染性克隆.[方法]RT-PCR扩增CVS-11株全基因组得到有重叠的12个片段,分别克隆至平端载体pEASY-Blunt,测定CVS-11株全基因组核苷酸序列.用软件DNAMAN分析CVS-11全序列单一性酶切位点,设计引物,分4段扩增CVS-11全基因组,扩增产物经多步酶切、连接逐步插入至真核表达载体pcDNA3.1,获得全长质粒pcDNA3.1-CVS-11.pcDNA3.1-CVS-11与其辅助质粒pcDNA3.1-N、P、L、G共转染NA细胞,经免疫荧光染色、RT-PCR鉴定,拯救得到重组病毒rCVS-11.[结果]CVS-11全基因组序列由11 927个核苷酸组成,编码5个结构蛋白,结构基因排列同已知的其他狂犬病病毒一致.成功构建了CVS-11全长cDNA重组质粒pcDNA3.1-CVS-11和其辅助质粒pcDNA3.1-N、P、L和G.经共转染,成功拯救了重组病毒rCVS-11.[结论]CVS-11株感染性克隆的构建为从分子水平上进一步研究狂犬病病毒奠定了基础.     

嵌合猪圆环病毒PCV1-2的构建及其感染性初步鉴定

猪Ⅱ型圆环病毒(PCV2)是当前严重危害养猪业的重要病原之一。目前,世界上还没有有效疫苗用于该病毒的免疫预防。该研究利用PCR方法,将PCV2的ORF2基因替换猪Ⅰ型圆环病毒(PCV1)的ORF2基因,构建了以PCV1基因组为骨架的嵌合病毒(PCV1-2)分子克隆(pSK2PCV1-2)。将该分子克隆转染PK-15细胞并连续盲传5代,用RT-PCR方法可以在转染后盲传的细胞中检测到PCV1的ORF1 mRNA和PCV2的ORF2 mRNA,但检测不到PCV1的ORF2 mRNA和PCV2的ORF1 mRNA。间接免疫荧光检测显示在盲传第5代的细胞中有PCV2 ORF2蛋白的表达,表达蛋白主要分布于细胞核。该研究初步证实构建的PCV1-2分子克隆转染细胞后可以形成具有感染性的嵌合病毒,从而为更深入研究嵌合病毒生物学特性奠定了基础。     

猪繁殖与呼吸综合征病毒HuN4-F112弱毒株NSP2区表达猪瘟病毒E2基因表位的研究

建立共表达猪瘟病毒(Classic swine fever virus,CSFV)E2蛋白主要中和性抗原表位基因的猪繁殖与呼吸综合征病毒(PRRSV)反向遗传操作平台,为进一步研究PRRSV作为病毒载体提供实验数据。本研究在PRRSVHuN4-F112疫苗株感染性分子克隆的基础上,采用突变PCR技术将CSFV E2蛋白的主要中和性抗原表位基因插入HuN4-F112弱毒株PRRSV已鉴定出的两个NSP2蛋白的复制非必需区,经基因片段的克隆、拼接,构建了两株含有CSFV E2蛋白的主要中和性抗原表位基因的感染性PRRSV cDNA克隆psk-HuN4-F112-Δ508-532+E2和psk-HuN4-F112-Δ480-532+E2。用限制性内切酶SwaΙ分别将两株共表达的感染性克隆线性化,之后通过细胞外转录获得病毒RNA,用脂质体法分别将病毒RNA转染BHK-21细胞包装出病毒粒子,再分别将其转接到MARC-145细胞传代拯救出两株病毒。分别对两株拯救病毒进行RT-PCR扩增、酶切和序列分析鉴定。结果表明,两株拯救病毒含有不同于亲本病毒的分子标记(拯救病毒基因组的14 667位因同义突变产生的MluⅠ酶切位点)和插入基因序列。间接免疫荧光试验表明,CSFV E2蛋白主要中和性抗原表位基因均能在两株拯救的PRRSV中表达,且能够在其传代过程中稳定遗传。病毒生长特性结果比较显示两株拯救病毒与亲本病毒在MARC-145细胞上具有相似的增殖特性。本研究构建了两株共表达CSFV E2蛋白主要中和性抗原表位基因的PRRSV感染性克隆并获得了拯救病毒,且外源基因能在拯救病毒中稳定表达,为进一步开发新型PRRSV二联疫苗提供了有效的反向遗传操作平台。     

表达猪圆环病毒2型ORF2基因的重组脑心肌炎病毒的构建与鉴定

以脑心肌炎病毒(Encephalomyocarditis virus,EMCV)作为病毒载体,寻找基因组里理想的外源基因插入位点,为构建多价疫苗奠定基础。猪圆环病毒2型(Porcine circovirus type 2,PCV2)感染可引起断奶仔猪多系统衰竭综合征等多种疾病。脑心肌炎病毒(Encephalomyocarditis virus,EMCV)宿主谱广,能够天然感染多种哺乳动物。本研究采用EMCV NJ08毒株cDNA感染性克隆,将PCV2ORF2基因插入EMCV L蛋白基因第6位及第7位氨基酸之间,拯救获得重组EMCV rNJ08/Cap,拯救病毒含有分子遗传标记。Western Blot和IFA结果显示,该重组病毒成功表达Cap蛋白。重组病毒rNJ08/Cap在BHK-21细胞上生长曲线和病毒蚀斑大小与亲本病毒NJ08相似,证明EMCV可以作为表达外源基因的病毒载体,为PCV2和EMCV疫苗研究奠定了重要基础。     

Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. We report here the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2. The chimeric PCV1-2 clone contains the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1 genome. A reciprocal chimeric PCV2-1 DNA clone was also constructed by replacing the PCV2 capsid gene with that of PCV1 in the backbone of the PCV2 genome. The PCV1, PCV2, and chimeric PCV1-2 and PCV2-1 DNA clones were all shown to be infectious in PK-15 cells, and their growth characteristics in vitro were determined and compared. To evaluate the immunogenicity and pathogenicity of the chimeric infectious DNA clones, 40 specific-pathogen-free (SPF) pigs were randomly assigned into five groups of eight pigs each. Group 1 pigs received phosphate-buffered saline as the negative control. Group 2 pigs were each injected in the superficial inguinal lymph nodes with 200 micro g of the PCV1 infectious DNA clone. Group 3 pigs were each similarly injected with 200 micro g of the PCV2 infectious DNA clone, group 4 pigs were each injected with 200 micro g of the chimeric PCV1-2 infectious DNA clone, and group 5 pigs were each injected with 200 micro g of the reciprocal chimeric PCV2-1 infectious DNA clone. As expected, seroconversion to antibodies to the PCV2 capsid antigen was detected in group 3 and group 4 pigs. Group 2 and 5 pigs all seroconverted to PCV1 antibody. Gross and microscopic lesions in various tissues of animals inoculated with the PCV2 infectious DNA clone were significantly more severe than those found in pigs inoculated with PCV1, chimeric PCV1-2, and reciprocal chimeric PCV2-1 infectious DNA clones. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induces a specific antibody response to the pathogenic PCV2 capsid antigen but is attenuated in pigs. Future studies are warranted to evaluate the usefulness of the chimeric PCV1-2 infectious DNA clone as a genetically engineered live-attenuated vaccine against PCV2 infection and PMWS.     

A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas PCV1 is nonpathogenic. We previously demonstrated that a chimeric PCV1-2 virus (with the immunogenic capsid gene of PCV2 cloned into the backbone of PCV1) induces an antibody response to the PCV2 capsid protein and is attenuated in pigs. Here, we report that the attenuated chimeric PCV1-2 induces protective immunity to wild-type PCV2 challenge in pigs. A total of 48 specific-pathogen-free piglets were randomly and equally assigned to four groups of 12 pigs each. Pigs in group 1 were vaccinated by intramuscular injection with 200 microg of the chimeric PCV1-2 infectious DNA clone. Pigs in group 2 were vaccinated by intralymphoid injection with 200 microg of a chimeric PCV1-2 infectious DNA clone. Pigs in group 3 were vaccinated by intramuscular injection with 10(3.5) 50% tissue culture infective doses (TCID(50)) of the chimeric PCV1-2 live virus. Pigs in group 4 were not vaccinated and served as controls. By 42 days postvaccination (DPV), the majority of pigs had seroconverted to PCV2 capsid antibody. At 42 DPV, all pigs were challenged intranasally and intramuscularly with 2 x 10(4.5) TCID(50) of a wild-type pathogenic PCV2 virus. By 21 days postchallenge (DPC), 9 out of the 12 group 4 pigs were viremic for PCV2. Vaccinated animals in groups 1 to 3 had no detectable PCV2 viremia after challenge. At 21 DPC the lymph nodes in the nonvaccinated pigs were larger (P < 0.05) than those of vaccinated pigs. The PCV2 genomic copy loads in lymph nodes were reduced (P < 0.0001) in vaccinated pigs. Moderate amounts of PCV2 antigen were detected in most lymphoid tissues of nonvaccinated pigs but in only 1 of 36 vaccinated pigs. Mild-to-severe lymphoid depletion and histiocytic replacement were detected in lymphoid tissues in the majority of nonvaccinated group 4 pigs but in only a few vaccinated group 1 to 3 pigs. The data from this study indicated that when given intramuscularly in pigs, the attenuated chimeric PCV1-2 live virus, as well as the chimeric PCV1-2 infectious DNA clone, induces protective immunity against PCV2 infection and could potentially serve as an effective vaccine.     

A candidate inactivated chimeric vaccine PCV1-2 constructed based on PCV1 and PCV2 isolates originating in China and its evaluation in conventional pigs in regard to protective efficacy against PCV2 infection

A chimeric PCV1-2 clone containing the PCV2 capsid gene cloned into the backbone of the nonpathogenic PCV1 genome was recently generated based on PCV2 and PCV1 strains isolated in China. The efficacy of this available candidate inactivated vaccine was evaluated by subjecting conventional pigs to intramuscular immunization with the inactivated chimeric PCV1-2 virus, followed by challenge with wild-type PCV2 strain. By 35 days post-vaccination (DPV), all vaccinated pigs had developed seroconversion, having high indirect immunofluorescence assay (IFA) titers of antibody and neutralizing antibody against PCV2. By 21 days post-challenge, gross and microscopic lesions of lymph nodes and lungs in non-vaccinated but challenged pigs were significantly more severe than those found in the vaccinated group. PCV2 viral copy loads detected in the tracheobronchial lymph nodes or serum samples of vaccinated pigs were significantly smaller than those in non-vaccinated but challenged pigs (P ≤ 0.05). The results suggest that inactivated PCV1-2 is effective in inducing protective immunity against PCV2 infection.     

猪1型圆环病毒全基因组克隆及其感染性初步鉴定

猪圆环病毒(porcine circovirus,PCV)是由Tis-cher等[1]于1974年在PK-15细胞中发现,当时认为是一种细胞污染物,后被证实为一种新的病毒。病毒粒子为20面体对称,无囊膜,以滚环方式进行复制,可在PK-15细胞上生长但不引起细胞病变。其基因组是一种环状、单股副链DNA,与鸡贫血病毒(chicken anemia virus,CAV)、鹦鹉喙羽病毒(psittacine beak and feather disease circovirus,PBF-DAV)和人的TT病毒(transfusion transmittedvirus,TTV)同属圆环病毒科。猪圆环病毒有两种基因型即:PCV1和PCV2。前者广泛存在于猪源肾细胞中,在猪的组织…     

家养野猪源猪圆环病毒2型安徽株的分离鉴定及其全基因组序列分析

目的对安徽省临诊疑似PMW S家养野猪病例进行猪圆环病毒2型(PCV2)分离鉴定,并对分离株的全基因组进行克隆与序列分析。方法应用PK-15细胞进行PCV2的分离与增殖,根据PCR、IFA、电镜技术进行PCV2分离株的鉴定,克隆分离株的全基因组,并对序列进行分析。结果获得1株来自安徽家养野猪源PCV2分离株,命名为YZ0901。该毒株全基因组长为1 767 bp,属于PCV2b基因型。与GenBank已发表的国内外参考毒株的同源性介于93.9%~99.2%,与安徽分离毒株彼此之间的同源性介于93.4%~99.5%。结论安徽省家养野猪中存在PCV2感染,分离毒株与家猪源病毒差异不大,PCV2核苷酸序列比较稳定,其进化不存在明显的地域相关性,家养野猪源PCV2的基因型与当地PCV2流行株的基因型密切相关。     

猪2型圆环病毒原核表达产物的免疫原性测定

目的检测猪2型圆环病毒(PCV2)全基因组原核表达产物的免疫原性。方法根据PCV2JXL株序列(GenBank登录号AY491310),设计合成引物,利用多聚酶链式反应(PCR)从含有PCV2接种猪肾细胞PK15中扩增了Cap蛋白的氨基端片段(737-421nt),克隆入上游带有谷光苷肽-S-转移酶的原核表达载体pGEX-6p-1中,获得重组质粒pGEX-PCV737-421。PCV2Cap蛋白羧基端(426-37nt)和Rep蛋白已在过去的研究中分别融合表达。利用IPTG对3种重组大肠杆菌BL21进行诱导,进行聚丙烯酰胺凝胶电泳分析(SDS-PAGE)以及对PCV2阳性猪多抗血清的蛋白免疫印迹试验,在此基础上,将3种SDS-PAGE分离粗纯的重组表达产物碾碎后分别免疫BALB/c小鼠,用获得的抗鼠血清与PCV2病毒感染PK15细胞做间接免疫荧光抗体试验(IFA)。结果PCV2Cap蛋白的氨基端片段能在大肠杆菌中表达,且Cap蛋白羧基端和Rep蛋白的原核表达产物都能在免疫印迹试验中被PCV2阳性猪血清检测出特异性条带,重组蛋白免疫鼠血清可在IFA试验中观察到特异性的荧光分布,即检测到培养细胞中的病毒抗原。结论PCV2全基因组都可以用原核系统高效表达,且表达产物具有免疫原性。     

Effect of natural or vaccine-induced porcine circovirus type 2 (PCV2) immunity on fetal infection after artificial insemination with PCV2 spiked semen

The objectives of this study were to determine if vaccination against porcine circovirus type 2 (PCV2) or previous PCV2 infection of the dam are sufficient to prevent fetal infection when dams are artificially inseminated with PCV2-spiked semen. Nine sows (Sus domestica) were allocated into three groups of three dams each: The PCV2 naïve negative control Group 1 was artificially inseminated with extended PCV2 DNA negative semen during estrus, whereas the extended semen used in the vaccinated Group 2 (PCV2 vaccine was given 8 wk before insemination) and PCV2-exposed Group 3 (infected with PCV2 12 wk before insemination) was spiked with 5 mL of PCV2 inoculum with a titer of 10^4.2 tissue culture infectious dose (TCID₅₀) per milliliter at each breeding. The dams in the vaccinated and PCV2-exposed groups were positive for PCV2 antibody but negative for PCV2 DNA in serum at the time of insemination. Three negative control dams, two vaccinated dams, and three dams with previous PCV2 exposure became pregnant and maintained pregnancy to term. After artificial insemination, viremia was detected in one of three vaccinated dams and in two of three dams with previous PCV2 exposure. At farrowing, PCV2 infection was not detected in any piglets or fetuses expelled from the negative control dams or from dams with previous PCV2 exposure. In litters of the vaccinated dams, 15 of 24 live-born piglets were PCV2 viremic at birth, with 6 of 26 fetuses having detectable PCV2 antigen in tissues. In conclusion, vaccine-induced immunity did not prevent fetal infection in this sow model using semen spiked with PCV2.     

Effect of a porcine circovirus type 2 infection on embryos during early pregnancy

The aim of the present study was to assess the effects of porcine circovirus type 2 (PCV2) on porcine embryos and their receptor sows during the first 21 days of pregnancy. Hatched blastocysts exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15) and negative control embryos were transferred to PCV2-immune receptor sows at the 7th day of the cycle. Two weeks after transfer (D21), the receptor sows were euthanized and embryos were recovered. They were assessed macroscopically for viability and examined for viral antigen-positive cells by immunoperoxidase staining. The embryonic survival rate of the PCV2-exposed embryos (6.4%, 7 viable embryos out of 110 transferred) was significantly lower than the survival rate of the negative control embryos (65.4%, 34 viable embryos out of 52 transferred). All of the non-viable PCV2-exposed embryos (n=9) displayed immunohistochemical positive signals for PCV2-antigen in degenerated tissues. In the PCV2-exposed embryos that were categorized as viable at D21, small clusters (n=4) or no PCV2-positive cells (n=3) were detected. The pregnancy results of the receptor sows that received PCV2-exposed embryos (1/5) were considerably different from the negative control receptors (2/2), with 3 out of 5 sows displaying a regular return to oestrus. In conclusion, it can be stated that PCV2 can replicate in embryos and might lead to embryonic death. In a small proportion of embryos, PCV2 exposure does not have a detrimental effect on embryo development before D21.