表达PRRSV M蛋白重组腺病毒的构建及其免疫特性研究

本研究通过RT-PCR方法扩增猪繁殖与呼吸综合征病毒(PRRSV)S1株的M蛋白基因,将其克隆重组到人 血清5型腺病毒载体中,转染293细胞,制备重组腺病毒rAd-M。RT-PCR和IFA方法鉴定,结果表明rAd-M可表 达M基因的mRNA和M蛋白。纯化的rAd-M重组腺病毒经293细胞连续传25代,滴度稳定为107.8 TCID50／ mL。动物免疫试验结果表明,该重组腺病毒rAd-M能够刺激机体产生PRRSV的特异性抗体免疫和细胞免疫应 答反应,从而为PRRSV结构蛋白功能及其基因工程疫苗研究奠定了基础。     

MiR-506 inhibits PRRSV replication in MARC-145 cells via CD151

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine, which is caused by PRRS virus (PRRSV). CD151, one of PRRSV entry mediators, determines the cell susceptibility for PRRSV. Emerging evidence indicates that the host microRNAs (miRNAs) play key roles in modulating virus infection and viral pathogenesis. In the present study, targeting porcine CD151 miRNAs were identified, and their function during PRRSV infection in MARC-145 cells was further verified. We found that miR-506 could directly target porcine CD151 3′-UTR mRNA by luciferase reporter assay. Overexpression of miR-506 significantly decreased CD151 expression at both mRNA and protein levels. Furthermore, overexpression of miR-506 reduced cellular PRRSV replication and virus release in MARC-145 cells. Our results suggested that miR-506 could inhibit PRRSV replication by directly targeting PRRSV receptor of CD151 in MARC-145 cells. However, the molecular mechanisms of miR-506 and its function in vivo need further investigation.     

Oxidative stress and Nrf2 in the pathophysiology of diabetic neuropathy: old perspective with a new angle

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically detrimental pig pathogen that causes significant losses for the pig industry. The immunostimulatory effects of hemagglutinating virus of Japan envelope (HVJ-E) in cancer therapy and the adjuvant efficacy of HVJ-E have been previously evaluated. The objective of this study was to investigate the adjuvant effects of HVJ-E on immunization with killed PRRSV vaccine, and to evaluate the protective effects of this immunization strategy against virulent PRRSV infection in piglets. Next, the PRRSV-specific antibody response, lymphocyte proliferation, PRRSV-specific IL-2, IL-10 and IFN-γ production, and the overall protection efficacy were evaluated to assess the immune responses of the piglets. The results showed that the piglets inoculated simultaneously with killed PRRSV vaccine and HVJ-E had a significantly stronger immune response than those inoculated with killed PRRSV vaccine alone. Our results suggest that HVJ-E could be employed as an effective adjuvant to enhance the humoral and cellular responses of piglets to PRRSV.     

NSOM‐ and AFM‐based nanotechnology elucidates nano‐structural and atomic‐force features of a Y. pestis V immunogen‐containing particle vaccine capable of eliciting robust response

It is postulated that unique nanoscale proteomic features of immunogen on vaccine particles may determine immunogen‐packing density, stability, specificity, and pH‐sensitivity on the vaccine particle surface and thus impact the vaccine‐elicited immune responses. To test this presumption, we employed near‐filed scanning optical microscopy (NSOM)‐ and atomic force microscopy (AFM)‐based nanotechnology to study nano‐structural and single‐molecule force bases of Yersinia pestis (Y. pestis) V immunogen fused with protein anchor (V‐PA) loaded on gram positive enhancer matrix (GEM) vaccine particles. Surprisingly, the single‐molecule sensitive NSOM revealed that ～90% of V‐PA immunogen molecules were packed as high‐density nanoclusters on GEM particle. AFM‐based single‐molecule force analyses indicated a highly stable and specific binding between V‐PA and GEM at the physiological pH. In contrast, this specific binding was mostly abrogated at the acidic pH equivalent to the biochemical pH in phagolysosomes of antigen‐presenting‐cells in which immunogen protein is processed for antigen presentation. Intranasal mucosal vaccination of mice with such immunogen loaded on vaccine particles elicited robust antigen‐specific immune response. This study indicated that high‐density, high‐stability, specific, and immunological pH‐responsive loading of immunogen nanoclusters on vaccine particles could readily be presented to the immune system for induction of strong antigen‐specific immune responses.     

Purification of cell culture‐derived modified vaccinia ankara virus by pseudo‐affinity membrane adsorbers and hydrophobic interaction chromatography

A purification scheme for cell culture‐derived smallpox vaccines based on an orthogonal downstream process of pseudo‐affinity membrane adsorbers (MA) and hydrophobic interaction chromatography (HIC) was investigated. The applied pseudo‐affinity chromatography, based on reinforced sulfated cellulose and heparin‐MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo‐affinity MA and adsorption of virus particles onto the hydrophobic interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%. Biotechnol. Bioeng. 2010;107: 312–320. © 2010 Wiley Periodicals, Inc.     

高致病性猪繁殖与呼吸综合征病RT-PCR快速鉴别诊断方法的建立与临床应用

根据高致病性猪繁殖与呼吸综合征病毒(PRRSV)Nsp2基因的缺失信息,设计了3条特异性引物,以含高致病性PRRSV Nsp2基因的质粒pMDNSP2及普通PRRSV VR2332株RNA为模板,建立了快速诊断高致病性PRRSV RT-PCR方法.通过对临床组织病料的总RNA进行不同稀释倍数检测,结果表明该方法能从0.265 pg的总RNA中检测到PRRSV的基因,说明敏感性高.用该方法对猪瘟病毒(CSFV)、Ⅱ型圆环病毒(PCV-2)、伪狂犬病病毒(PRV),链球菌(Streptococcus)、副猪嗜血杆菌(Haemophilus parasuis)和大肠杆菌(Escherichia coli)同条件检测,结果都为阴性.进一步对36份疑似高致病性PRRSV临床组织病料细胞培养物、2株PRRSV商品活疫苗以及52个猪场所送检的184份临床样品进行了检测应用,结果36份疑似高致病性PRRSV临床组织病料的细胞培养物有5份样品为阳性且都为高致病性PRRSV,2株PRRSV商品活疫苗为普通PRRSV,52个猪场中有42个猪场(123份样品)呈阳性,其中只有1份为普通PRRSV.实验表明该方法能够准确地鉴别诊断高致病性PRRSV和普通PRRSV,且具有快速,敏感和特异的特点,具有临床实用性.     

Analysis of molecular variation in porcine reproductive and respiratory syndrome virus in China between 2007 and 2012

In the present study, 89 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-structural protein 2 (Nsp2) and glycoprotein 5 (GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discontinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007–2012 in China. Owing to the extensive use of representative vaccine strains, natural recombination events occurred between strains. Three isolates — HH08, DY, and YN-2011 — were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolutionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.     

Complete Genome Sequence of a Novel Variant Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Strain: Evidence for Recombination between Vaccine and Wild-Type PRRSV Strains

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of porcine reproductive and respiratory syndrome (PRRS), which can evolve continuously by random mutation or intragenic recombination. Here we report the complete genomic sequence of a PRRSV variant with nucleotide acid deletions and insertions in the nonstructural protein 2 (nsp2) gene and a possible recombination event between a modified live virus (MLV) vaccine strain and a prototype Chinese field strain.     

NADC30-like猪繁殖与呼吸综合征病毒样颗粒的制备与鉴定

【背景】猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)可以造成怀孕母猪的繁殖障碍及仔猪的呼吸系统疾病,近年来,NADC30-like谱系PRRSV已成为国内的优势流行毒株。【目的】研制针对NADC30-like谱系PRRSV的病毒样颗粒(virus-like particle,VLP)疫苗。【方法】将PRRSV NADC30-like毒株编码GP5蛋白开放阅读框5(open reading frame 5,ORF5)、ORF6(编码M蛋白)分别连接至pFastBac^TMDual载体P₁₀和P_H启动子下游多克隆位点,获得穿梭质粒pFB-30-ORF5及pFB-30-ORF6,酶切鉴定后,将ORF6基因插入到穿梭质粒pFB-30-ORF5 P_H启动子下游,构建穿梭质粒pFB-30-ORF5-OPF6。将上述3种穿梭质粒分别转化大肠杆菌DH10Bac感受态细胞,通过蓝白斑筛选及PCR鉴定重组杆粒。再将获得的重组杆粒转染至SF9昆虫细胞,发现细胞病变后收获病毒液,继续盲传3代,在透射电镜下观察是否有病毒样颗粒。用第3代病毒液感染SF9细胞后,分别用GP5蛋白、His-tag、Flag-tag单克隆抗体作为一抗,通过免疫电镜、间接免疫荧光(indirect immunofluorescence assay,IFA)、Western blotting鉴定重组蛋白。【结果】成功构建了3种穿梭质粒pFB-30-ORF5、pFB-30-ORF6和pFB-30-ORF5-OPF6,酶切鉴定正确。通过蓝白斑筛选及PCR验证后获得重组杆粒,分别命名为Bacmind-30-ORF5、Bacmind-30-ORF6和Bacmind-30-ORF5-ORF6。重组杆粒感染SF9细胞120h时出现明显的细胞病变,收获病毒液后,在透射电子显微镜可观察到大小为50nm左右呈现球形结构的VLPs。免疫电镜可以观察到胶体金颗粒结合在VLPs周围;IFA结果显示实验组均出现了明显绿色的特异性荧光灶;Western blotting结果表明,3种VLPs均出现特异性条带,并与预期大小一致。【结论】制备了3种NADC30-like谱系PRRSV的病毒样颗粒,为针对PRRSV新谱系流行株疫苗的研发奠定了基础。     

苜蓿花叶病毒提纯方法的改进*

用来自于白车根草（Trifolium repens）上的一个苜蓿花叶病毒分离物AMV－SY为材料,比较了3种以差速离心为主结合PEG沉淀和超速离心提纯病毒的方法,对提纯病毒进行紫外吸收测定、电镜检查和SDS－聚丙烯酰胺凝胶电泳检测的结果显示：以交替使用含有0.1mol/LEDTA和0.1mol/L MgSO4的磷酸缓冲液作为病毒悬浮介质的提纯程度最为理想,该方法提取苜蓿花叶病毒的得率为47.6mg/100g昆诺藜鲜病叶,该病毒分离物的外壳蛋白分子量为29kD。该方法的病毒得率较高、杂蛋白较少、病毒粒子完整,是比较理想的提纯方法。     

Development of a chimeric strain of porcine reproductive and respiratory syndrome virus with an infectious clone and a Korean dominant field strain

The K418 chimeric virus of porcine reproductive and respiratory syndrome virus (PRRSV) was engineered by replacing the genomic region containing structure protein genes of an infectious clone of PRRSV, FL12, with the same region obtained from a Korean dominant field strain, LMY. The K418 reached 10⁶ TCID₅₀/ml of viral titer with similar growth kinetics to those of parental strains and had a cross-reactive neutralizing antibody response to field serum from the entire country. The chimeric clone pK418 can be used as a practical tool for further studying the molecular characteristics of PRRSV proteins through genetic manipulation. Furthermore, successful construction of the K418 will allow for the development of customized vaccine candidates against PRRSV, which has evolved rapidly in Korea.     

A novel method for concentration of porcine reproductive and respiratory syndrome virus from the environmental samples using self-aggregating peptide-tagged CD151-binding capture

Environmental surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) represents a key issue in control of the disease. CD151 has recently been recognized as one of several receptors for PRRSV. We describe here a novel method for concentration of PRRSV from the environmental samples by CD151-binding capture. After fusion to self-aggregating peptide ELK16, the large extracellular loop (LEL) of porcine CD151 and its two segments (namely N63 and C63) were expressed in E. coli as protein aggregates. The three fusion proteins were purified to high purities by regular centrifugation and washing with Triton X-100. Viral binding assay showed that the C63-ELK16 protein, but not ELK16-N63 protein, had the specific binding affinity for PRRSV. The C63-ELK16 protein could bind to, and eluted from, PRRSV in pH-, temperature-, and time-dependent manners with a final virus recovery of 44.7%. By using PRRSV-spiked and experimentally infected pig fecal slurry samples, the C63-ELK16 binding capture-combined quantitative RT-PCR was shown to have higher detection sensitivity than the conventional RT-PCR. Although the viral RNA could be detected in the experimentally infected pig samples with or without C63-ELK16 binding capture, infectious PRRSV was not isolated without C63-ELK16 binding capture. Therefore, the CD151-binding capture method established offers sufficient recovery and quickness and will facilitate environmental PRRSV surveillance.

     

A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.     

Impact of a novel inactivated PRRS virus vaccine on virus replication and virus-induced pathology in fetal implantation sites and fetuses upon challenge

Preventing congenital infection is important for the control of porcine reproductive and respiratory syndrome (PRRS). Recently, in our laboratory, an inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine has been developed. Promising results in young pigs encouraged us to test the vaccine potency to prevent congenital infection. In the present study, the performance of this experimental inactivated vaccine was investigated in pregnant gilts. An advanced protocol was used to test the PRRSV vaccine efficacy. This protocol is based on recent insights in the pathogenesis of congenital PRRSV infections. Three gilts were vaccinated with an experimental PRRSV 07V63 inactivated vaccine at 27, 55, and 83 days of gestation. Three unvaccinated gilts were included as controls. At 90 days of gestation, all animals were intranasally inoculated with 10⁵ tissue culture infectious dose 50 (TCID₅₀) of PRRSV 07V63. Twenty days postchallenge animals were euthanized and sampled. The vaccinated gilts quickly developed virus neutralizing (VN) antibodies starting from 3 to 7 days postchallenge (1.0 to 5.0 log₂). In contrast, the unvaccinated gilts remained negative for VN antibodies after challenge. The vaccinated gilts had shorter viremia than the control gilts. Gross pathology (mummification) was observed in 8% of the fetuses from vaccinated gilts and in 15% of the fetuses from unvaccinated gilts. The number of fetuses with severe microscopic lesions in the fetal implantation sites (a focal detachment of the trophoblast from the uterine epithelium; a focal, multifocal, or full degeneration of the fetal placenta) was lower in the vaccinated (19%) versus unvaccinated (45%) gilts (P < 0.05). The number of PRRS-positive cells in the fetal placentae was higher in unvaccinated versus vaccinated gilts (P < 0.05). In contrast, the number of PRRS-positive cells in the myometrium/endometrium was higher in vaccinated versus unvaccinated gilts (P < 0.05). Fifty-seven percent of the fetuses from the vaccinated gilts and 75% of the fetuses from the unvaccinated gilts were PRRSV-positive. In conclusion, implementation of the novel experimental inactivated PRRSV vaccine primed the VN antibody response and slightly reduced the duration of viremia in gilts. It also reduced the number of virus-positive fetuses and improved the fetal survival, but was not able to fully prevent congenital PRRSV infection. The reduction of fetal infection and pathology is most probably attributable to the vaccine-mediated decrease of PRRSV transfer from the endometrium to the fetal placenta.     

Assembly of human severe acute respiratory syndrome coronavirus-like particles

Viral particles of human severe acute respiratory syndrome coronavirus (SARS CoV) consist of three virion structural proteins, including spike protein, membrane protein, and envelope protein. In this report, virus-like particles were assembled in insect cells by the co-infection with recombinant baculoviruses, which separately express one of these three virion proteins. We found that the membrane and envelope proteins are sufficient for the efficient formation of virus-like particles and could be visualized by electron microscopy. Sucrose gradient purification followed by Western blot analysis and immunogold labeling showed that the spike protein could be incorporated into the virus like particle also. The construction of engineered virus-like particles bearing resemblance to the authentic one is an important step towards the development of an effective vaccine against infection of SARS CoV.     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK-/gE-/GP5+expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV),based on the PRV genetically depleted vaccine strain TK-/gE-/LacZ+,scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene (the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK-/gE-/LacZ+,resulting in a PRV recombinant named TK-/gE-/GPSm+,which expressed the modified GPSm protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK-/gE-/GPSm+and TK-/gE-/GP5+expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK-/gE-/GPSm+against PRRSV were higher than that induced by TK-/gE-/GP5+.Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.     

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.     

猪繁殖与呼吸综合征病毒N基因和流感病毒HA基因的联合表达及应用

参照已公布的流感病毒血凝素基因(HA基因)及猪繁殖与呼吸综合征病毒(PRRSV)基因组序列,设计并合成一对引物P1、P2,以RT-PCR方法扩增出PRRSV的ORF7片段(约410bp),其中含HA基因主要核苷酸序列(33bp)。用BamH Ⅰ、Xho Ⅰ分别对扩增出的片段及pET32a质粒进行酶切,连接后构建了重组质粒pETHN并转化到BL21(DE3)宿主菌中诱导表达。用纯化后的表达产物与流感病毒血凝素单抗及乳胶建立了诊断猪繁殖与呼吸综合征(PRRS)的乳胶凝集试验。检测结果显示:该方法有良好的特异性及敏感性,与IDEXX公司FLISA检测试剂盒符合率达93.8％。