PCV2与PRRSV体外共感染对肺泡巨噬细胞系炎症反应的影响

为了探讨猪圆环病毒2型(Porcine circovirus type 2,PCV2)与猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)共感染后调控炎症反应的机制,选用稳定表达CD163的PAM细胞系3D4/21,分别设PCV2、PRRSV、PCV2+PRRSV(接种PCV2后12 h接种PRRSV)共感染组和对照组,利用间接免疫荧光检测(indirect immuno fluorescene assay,IFA)方法检测PCV2和PRRSV在PAM-CD163上的阳性率,采用RT-PCR检测IL-6、IL-8、IL-1β、IL-10表达量的动态变化。结果显示PCV2和PRRSV均能感染PAM-CD163;PCV2单独感染PAM-CD163,早期促进了IL-6、IL-8、IL-1β的表达,晚期促进IL-10表达;PRRSV单独感染PAM-CD163能在晚期促进IL-1β、IL-10的表达;与PCV2单独感染PAM-CD163相比,PCV2+PRRSV共感染促进了IL-10表达并且持续增加;与PRRSV单独感染PAM-CD163相比,PCV2+PRRSV共感染促使IL-6、IL-8、IL-1β的表达量较早出现增加,并且持续增加。可见两种病毒之间存在一定的相互作用,共同调节机体免疫系统。本研究结果有助于解析PRRSV与PCV2共感染调控炎症反应的机理。     

猪繁殖与呼吸综合征病毒和猪圆环病毒2型共感染对外周血单个核细胞促炎细胞因子mRNA表达的影响

将猪繁殖与呼吸综合征病毒（PRRSV）和猪圆环病毒2型（PCV2）单感染和共感染6周龄健康仔猪,采用Real-ti me PCR技术对外周血单个核细胞（PBMC）中IL-1β、IL-6、IL-8和TNF-α等促炎细胞因子的mRNA表达进行定量分析。结果表明,病毒感染后,PRRSV感染组、PCV2感染组IL-1β、IL-6、IL-8和TNF-α的mRNA表达水平均上调,其中PRRSV感染组的IL-6、IL-8显著上调,PCV2感染组的IL-6、IL-8和TNF-α显著上调;PRRSV/PCV2共感染组仅有IL-8和TNF-α的mRNA表达水平上调且差异显著;并且,PRRSV/PCV2共感染组IL-1β、IL-6和IL-8的mRNA表达水平均低于单独感染组,仅TNF-α的mRNA表达水平显著高于单感染组。结果提示,TNF-α的过量表达可能在PRRSV和PCV2协同致病机制中扮演重要角色。     

免疫复合物对猪繁殖与呼吸综合征病毒诱导的PAM细胞因子转录的影响

本试验通过研究免疫复合物影响猪繁殖与呼吸综合征病毒(PRRSV)在巨噬细胞内增殖的因素,来进一步探讨免疫复合物经FcγR介导的PRRSV感染机制.本研究将含200 TCID50 PRRSV病毒液与等体积的终浓度为1.30 mg· mL-1的猪IgG-兔抗猪IgG复合物分别先后和同时接种于PAM细胞,分别于12、24、36、48 h收集细胞液,同时设PRRSV感染对照组、免疫复合物对照组和健康细胞对照组,利用建立的相对荧光定量PCR和绝对荧光定量PCR方法分别检测巨噬细胞中IFN-α、IL-10和TNF-α的mRNA转录水平及PRRSV的RNA水平,并进行定量分析,同时采用ELISA方法检测健康细胞中IFN-α的蛋白水平.结果显示,在感染后12～36 h期间,免疫复合物能够抑制PRRSV诱导的IFN-α的mRNA水平,TNF-α mRNA水平在感染后12 h被促进,而在24～36 h期间被抑制.在感染后48 h,免疫复合物能促进IFN-α和TNF-α的mRNA水平,而在12～48 h期间均能抑制IL-10的mRNA水平,此外,免疫复合物在感染12、24和36 h时间段内均能明显促进病毒的增殖,但在48 h后不显著.健康细胞中IFN-α的蛋白水平在培养12～48 h之内呈“降-升-降”的趋势.结果表明,IFN-α蛋白的表达与其mRNA的转录不同步.在PRRSV感染初期,免疫复合物能抑制PRRSV诱导的抗病毒因子的mRNA水平,病毒的复制被促进,而在感染后期,抗病毒因子达到一定的浓度后发挥抗病毒作用,在一定程度上抑制病毒的复制.而TNF-α的转录规律不明显,表明免疫复合物可能同时与激活型和抑制型FcγR结合来共同调节PRRSV诱导的细胞因子的转录.     

NADC30-like PRRSV和PCV2d共感染与继发感染PCV2抗体阳性断奶仔猪的致病性

为了确定近年来国内主要流行的猪繁殖与呼吸综合征病毒(PRRSV)NADC30-like谱系与猪2型圆环病毒(PCV2d亚型)共感染与继发感染后对猪的致病作用,使用本实验室分离的NADC30-like谱系PRRSV与PCV2d毒株感染PCV2抗体阳性断奶仔猪。选取30只5周龄PCV2抗体阳性断奶仔猪,随机均分为6组,分别为PBS、PCV2、PRRSV、PCV2-PRRSV、PRRSV-PCV2和Co-PRRSV-PCV2感染组。记录各组临床指征(体温、体质量、临床症状和存活率);检测特异性抗体及TNF-α、IFN-γ、IL-4和IL-10共4种细胞因子的变化;并对主要组织脏器的病理变化进行分析。结果显示,各感染组在感染PRRSV后均出现体温升高、体质量减轻、咳喘、腹泻、精神沉郁等临床症状;Co-PRRSV-PCV2、PRRSV-PCV2和PRRSV组表现出更为严重的肉样变和淋巴细胞浸润;先感染PCV2可以延迟PRRSV特异性抗体的产生;PRRSV组表现出高水平的TNF-α和IL-10;NADC30-like PRRSV与PCV2d共感染与继发感染PCV2抗体阳性仔猪会出现严重的临床症状和...     

猪圆环病毒2型感染对猪瘟弱毒疫苗接种猪免疫应答的影响

为探索猪圆环病毒2型(PCV2)感染对猪瘟(CSF)弱毒疫苗接种猪免疫应答的影响,将20头28 d断奶仔猪随机分为V-I、I-V、V和C4组,5头·组-1.V-I组在接种CSF弱毒疫苗后2d感染PCV2;I-V组在感染PCV2 后2d接种CSF弱毒疫苗;V组只接种CSF弱毒疫苗;C组为空白对照组.共免疫2次,间隔21 d.免疫后定期检测血清CSFV特异性抗体水平、外周血淋巴细胞(PBLC)增殖活性和PBLC中IFN-γ、IL-2、IL-4和IL-10 mRNA的表达水平.结果显示:在CSF弱毒疫苗免疫前或免疫后感染PCV2,均会导致CSFV抗体水平低下,血清抗体阳转率下降,PBLC增殖活性降低.初次免疫后,V-I与I-V组的PBLC内IFN-γ、IL-2、IL-4和IL-10 mRNA的表达量严重不足,其中V-I组的表达量最低.加强免疫后V-I与I-V组的PBLC内IFN-γ与IL-10的表达失衡,IL-2和IL-4的表达缺乏,其中I-V组的细胞因子表达失衡和缺乏更严重.上述研究表明,CSF弱毒疫苗免疫前或免疫后感染PCV2均会影响机体的体液和细胞免疫应答水平,导致PBLC内细胞因子的表达严重抑制和紊乱.     

猪繁殖与呼吸综合征病毒TJM92株对猪肺泡巨噬细胞相关因子mRNA表达量的影响

用灌洗法从健康仔猪分离猪肺泡巨噬细胞(PAM)。将PAM和TJM92-PRRSV混合孵育作为试验组,用10%RPMI-1640与PAM孵育作为对照组。分别孵育0 h、6h、12 h、18h和24h收获PAM,用qRT-PCR检测相关因子mRNA表达量。结果显示:与对照组相比,试验组TLR2、IL-10、IL-1α、IL-1β和TNF-αmRNA表达量先升后降,最后恢复上升。MHCⅡ和CD40 mRNA表达量先降后升。结论:TJM92-PRRSV可以诱导PAM相关因子mRNA表达量整体短暂下调然后迅速恢复。本试验结果为进一步研究PRRSV对免疫功能影响提供有益参考。     

猪繁殖与呼吸综合征病毒和猪圆环病毒2型共感染对外周血单个核细胞凋亡细胞因子mRNA表达的影响

将猪繁殖与呼吸综合征病毒(Porcine reproductine and respiratory syndrome virus,PRRSV)和猪圆环病毒2型(Porcine circovirus type 2,PCV2)单感染和共感染6周龄健康仔猪,采用real-time PCR技术对外周血单个核细胞(PBMC)中的病毒栽量以及Fas、FasL、TNFR1和TNF-α等凋亡细胞因子mRNA表达水平进行检测,采用流式细胞术对PBMC的凋亡比率进行检测.结果显示,PRRSV/PCV2共感染组PBMC中PRRSV和PCV2载量、PB-MC凋亡比率均显著高于PRRSV感染组或PCV2感染组.所有病毒感染组的Fas、FasL、TNFR1和TNF-α的mRNA表达水平均显著上调,并且PRRSV/PCV2共感染组的表达水平均显著高于单感染组.结果表明,Fas/FasL、TNFR1/TNF-α表达水平的显著上调可能在PRRSV和PCV2协同诱导凋亡机制中扮演重要角色.     

RNAi沉默藏猪TGF-β1对PRRSV病毒复制和免疫的效应研究

转移生长因子-β1(Transforming growth factor beta 1,TGF-β1)在猪繁殖与呼吸综合征病毒(PRRSV)感染宿主细胞的过程中发挥重要作用。基于此本篇通过RNAi技术从体外沉默藏猪外周血单核细胞(Tp-PBMCs)中TGF-β1基因表达并随即感染PRRSV为出发点,探究TGF-β1表达受抑对PR R SV复制、细胞活性以及免疫应答的影响。研究表明,随着TGF-β1基因表达的抑制,Tp-PBMCs中PRRSV复制被明显抑制,抑制率高达99.0%;细胞活性升高约150%~160%;并成功激活多个抗病毒免疫相关基因转录,其中白细胞介素-2(interleukin-2,IL-2)、α干扰素(interferon-alpha,IFN-α)、γ干扰素(interferon-gamma,IFN-γ)、α肿瘤坏死因子(tumor necrosis factoralpha,TNF-α)、Toll样受体-3(toll-like receptor 3,TLR-3)的转录水平明显升高;而白细胞介素-8(interleukin-8,IL-8)、白细胞介素-10(interleukin-10,IL-10)的mRNA水平明显降低。由此可见,用RNAi技术沉默TGF-β1基因表达不仅能抑制PRRSV的复制,还能激活免疫应答进一步抑制病毒感染的发生,从而为控制藏猪感染PRRSV提供了一条新的途径。     

白细胞介素-10对口蹄疫病毒感染小鼠T细胞增殖及其表达TNF-α、IFN-γ和IL-2的影响

白细胞介素-10(IL-10)增高是口蹄疫病毒(FMDV)感染过程中显著特征之一。本研究旨在探讨IL-10对FMDV感染小鼠外周血T细胞增殖及其表达效应功能相关细胞因子的影响。采用CCK-8和流式细胞术分别检测小鼠外周血T细胞增殖和T细胞表达效应功能相关细胞因子(TNF-α、IFN-γ和IL-2)。结果显示,与对照小鼠相比,FMDV感染小鼠(感染12、24、36和48 h)外周血T细胞对刀豆蛋白A刺激的增殖均显著下降(P<0.05或P<0.01);FMDV感染小鼠的外周血CD4⁺T细胞表达TNF-α和IL-2均显著下降(均P<0.01),CD8⁺T细胞表达TNF-α、IFN-γ和IL-2也显著下降(P<0.01或P<0.000 1)。体内阻断IL-10/IL-10R信号或者敲除IL-10均能显著恢复FMDV感染小鼠外周血T细胞的增殖(P<0.05或P<0.01),但不影响CD4⁺和CD8⁺T细胞表达TNF-α、IFN-γ和IL-2。本研究首次揭示FMDV能抑...     

高致病性猪繁殖与呼吸综合征病毒非结构蛋白NSP1对猪体免疫抑制作用

为了研究高致病性PRRSVNSP1蛋白的免疫作用,本研究将高致病性PRRSVNSP1重组腺病毒（rAd—NSP1）接种体外培养的猪肺泡细胞（PAM）,用实时荧光定量PCR和ELISA方法分别检测IFN-γ和IL-10水平,结果为rAd—NSP1接种PAM细胞72h后可显著降低细胞上清中IFN-γ的水平,而IL-10的含量显著提高。将rAd—NSP1接种无PRRSV感染的30日龄商品仔猪,分别检测其外周血液淋巴细胞增殖作用和IFN-γ与IL-10的水平,结果显示,NSP1可显著减低淋巴细胞增殖和IFN-7的表达,同时诱导产生较强的IL-10反应。采用无PRRSV感染的30日龄商品仔猪免疫猪瘟疫苗后1周接种rAd—NSP1,结果猪瘟抗体的水平明显低于wtAd组（P〈0．05）,证明高致病性PRRSVNSP1蛋白具有免疫抑制作用。     

Pseudorabies virus, porcine parvovirus, and porcine enterovirus interactions with the zona pellucida of the porcine embryo

Porcine embryos (n = 93) were incubated on cell monolayers that had been previously inoculated with pseudorabies virus, porcine parvovirus (PPV), or each of 2 porcine enteroviruses. After 2, 24, or 48 hours of incubation, the embryos were fixed in glutaraldehyde and examined by electron microscopic procedures. It was found that pseudorabies virus adsorbed to the zona pellucida (ZP) and entered sperm tracks in the ZP. The PPV and both enteroviruses entered pores in the ZP and were associated with sperm that were at or near the outer surface of the ZP. In addition, PPV was seen enmeshed in cellular debris on the outer surface of the ZP. Evidence of a productive viral infection of the blastomeres of the embryos was not found.     

猪MSTN基因敲除载体的构建及细胞筛选

构建猪肌肉生长抑制素(Myostatin,MSTN)基因的打靶载体并获得敲除MSTN基因的猪胎儿成纤维细胞.以Puro为正筛选基因,白喉毒素-A(DT-A)为负筛选基因.将同源长臂和同源短臂分别插入Puro基因的两侧.同源长短臂分别为4 294 bp和1 015 bp,定点敲除MSTN基因的部分内含子2和部分外显子3.采用FugeneHD 转染法将打靶载体转入37 d的猪胎儿成纤维细胞中,转染后的细胞采用嘌呤霉素筛选.结果显示,成功构建了对猪MSTN基因部分区域进行敲除的打靶载体,共得到48个具有药物抗性的细胞克隆,经PCR检测,获得2个正确同源重组的细胞克隆.     

多重PCR检测猪细小病毒和猪伪狂犬病病毒的研究

根据GenBank上已发表的猪细小病毒（Porcine parvovirus，PPV）的VP2基因序列和猪伪狂犬病病毒（Porcine pseudorabies virus，PRV）的gH基因序列，设计合成了两对特异引物，分别建立了PPV和PRV的单项PCR诊断方法，通过对扩增条件的筛选，最终成功地建立了PPV和PRV的复合PCR诊断方法，即利用一次PCR反应，可同时扩增PPV的751bp和PRV355bp的特异性片段，而扩增猪圆环病毒Ⅱ型（PCV．2）及相应的培养细胞（PK-15）核酸结果均为阴性，对PPV和PRV的最低检出量分别为100Pg和10Pg的DNA。该方法适合对PPV和PRV的联合检测和鉴别诊断。     

猪卵母细胞与猪、水牛及食蟹猴精子的胞质内显微受精

为探讨猪精子、水牛精子及食蟹猴精子分别注入到猪卵母细胞后原核形成及早期胚胎发育情况,利用屠宰场收集的猪卵母细胞,经体外成熟44～48h后,进行胞质内显微受精(ICSI)操作。试验1:体外培养18～19h,用Ho-echst33342荧光染色,检查原核形成情况。试验2:进行体外培养,ICSI后2d检查分裂率,7d记录囊胚率。试验1结果显示:在原核形成率上,猪同种显微受精,原核形成率(50.10%)显著高于注入水牛精子(37.06%)及食蟹猴精子(37.48%),且差异显著(P0.05),注入水牛精子和食蟹猴精子间差异不显著。试验2结果显示:猪同种显微受精所得的分裂率(86.58%)和囊胚率(29.93%)与水牛精子注入(70.98%,18.48%)、食蟹猴精子注入(78.69%,16.92%)差异显著(P0.05)。结果表明,水牛精子、食蟹猴精子分别注入到猪卵母细胞后,观察到雌雄原核和精子解聚;水牛精子注入猪卵母细胞与食蟹猴精子注入猪卵母细胞,经体外培养发育到囊胚。     

猪细小病毒与猪伪狂犬病毒二重SYBR GreenⅠ实时荧光PCR方法的建立与检测

本研究旨在建立一种能快速、灵敏、同时检测出猪细小病毒与猪伪狂犬病毒的基于SYBR GreenⅠ实时荧光定量PCR方法。参照GenBank中登录的相关基因序列,设计了2对引物分别用于扩增PRV gH基因与PPV NS1基因的部分片段。将测序正确的PRV gH基因与PPV NS1基因片段克隆入pGEM-T Easy载体,转化大肠杆菌DH5α,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBR GreenⅠ荧光定量PCR标准曲线和熔解曲线,并对其灵敏性、特异性和重复性进行验证。结果表明,猪细小病毒与猪伪狂犬病毒荧光定量PCR的标准曲线的Tm值分别为80.9℃和86.5℃,熔解曲线特异,灵敏度分别可达248拷贝/μL和160拷贝/μL,是普通PCR检测方法的100倍。本次建立的猪细小病毒与猪伪狂犬病毒荧光定量PCR检测方法实现了2种病毒的同时检测,能够对PRV、PPV混合感染的临床病料进行快速诊断。     

The survey of porcine teschoviruses, porcine circovirus and porcine transmissible gastroenteritis virus infecting piglets in clinical specimens in China

A multiplex PCR assay was developed and evaluated for its ability to simultaneously detect three viral infections of swine. Specific primers were carefully selected from articles published for each of the following three viruses: porcine circovirus type II (PCV2), porcine teschovirus (PTV) and porcine transmissible gastroenteritis virus (TGEV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 168 bp (PTV) and 499 bp (TGEV). The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 6.60?×?10², 8.43?×?10² and 7.30?×?10² copies for PCV2, PTV and TGEV, respectively. Among 127 samples which were collected from Heilongjiang, Jilin, Henan and Guangxi provinces, the single infection of PCV2, PTV and TGEV was 99.21, 46.88 and 65.35 %, respectively, and co-infection of the three viruses was 26.77 %. In conclusion, the multiplex PCR has the potential to be useful for routine molecular diagnosis and epidemiology.     

猪γ干扰素的体外表达及其多克隆抗血清的制备

从健康仔猪前腔静脉无菌采血,分离外周血单个核细胞,提取细胞总RNA,用猪γ干扰素(IFN-γ)基因特异性引物通过RT-PCR扩增猪IFN-γ的全长cDNA序列,将其克隆到pMD18-T载体中,再亚克隆到pUC18和表达载体pET-30a中,经测序发现其序列与GenBank报道的IFN-γ基因序列基本一致.将重组质粒pET-30a-IFN-γ转化大肠杆菌BL21,于37℃经IPTG诱导表达,IFN-γ基因获得表达,表达产物以包涵体形式存在,占菌体总蛋白的39%.经SDS-PAGE及Westernblot分析表明,表达的融合蛋白分子量约为25 ku.将包涵体用8 mol/L脲变性,用ProBondTM Purification Systerm纯化,经透析复性,得到纯化的目的蛋白,含量可达0.3 mg/mL.将复性蛋白免疫新西兰白兔三次,制备了高滴度的猪IFN-γ抗血清.本实验表达和纯化了猪IFN-γ,并制备兔抗猪IFN-γ的抗血清,为下一阶段关于猪γ干扰素重组蛋白其应用奠定了基础.     

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (PCV) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of PCV (which recognises both PCV-1 and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twenty-eight of the 30 Spanish pigs and all three American pigs had PCV in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyer's patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that PCV is present in tissues of almost all pigs affected by PDNS, and PCV has to be considered as a possible agent involved in the pathogenesis of the syndrome.     

Association of porcine circovirus 2 with porcine respiratory disease complex

A retrospective study was performed on natural cases of porcine respiratory disease complex (PRDC) to determine the association and prevalence of PRDC with porcine circovirus 2 (PCV2) and other co-existing pathogens in Korea. Histologically, alveolar septa were markedly thickened by infiltrates of mononuclear cells. Moderate to marked multifocal peribronchial and peribronchiolar fibrosis were present and often extended into the airway lamina propria. Among the 105 pigs with PRDC, 85 were positive for PCV2, 66 were positive for porcine reproductive and respiratory syndrome virus (PRRSV), 60 were positive for porcine parvovirus (PPV), and 14 were positive for swine influenza virus (SIV). There were 80 co-infections and 25 single infections. A co-infection of PCV2 with another additional bacterial pathogen is frequently diagnosed in PRDC. The combination of PCV2 and Pasteurella multocida (38 cases) was most prevalent followed by PCV2 and Mycoplasma hyopneumoniae (33 cases). The consistent presence of PCV2, but lower prevalence of other viral and bacterial pathogens in all pigs examined with PRDC, has led us to speculate that PCV2 plays an important role in PRDC.