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1.
ABSTRACT: The present study examined the immunological response of antigen presenting cells (APC) to genotype-I isolates of porcine reproductive and respiratory syndrome virus (PRRSV) infection by analysing the cytokine profile induced and evaluating the changes taking place upon infection on immunologically relevant cell markers (MHCI, MHCII, CD80/86, CD14, CD16, CD163, CD172a, SWC9). Several types of APC were infected with 39 PRRSV isolates. The results show that different isolates were able to induce different patterns of IL-10 and TNF-α. The four possible phenotypes based on the ability to induce IL-10 and/or TNF-α were observed, although different cell types seemed to have different capabilities. In addition, isolates inducing different cytokine-release profiles on APC could induce different expression of cell markers.  相似文献   

2.
This study was attempted to generate one Marc-145 cell line stably and highly expressing porcine CD163 (pCD163) and set the foundation for PRRSV isolation and vaccine production.CD163 was shown to be a cellular receptor capable of mediating infection of PRRSV non-permissive cell lines.The pCD163 gene was amplified by RT-PCR from porcine alveolar macrophages and cloned into the eukaryotic expression vector pCI-neo, then the positive plasmid pCI-pCD163 was transfected into Marc-145 cells.After selecting with G418 and subcloning for 3 times, Marc-145 cell line expressing pCD163 was established.IFA results indicated that the fluorescence of pCD163-Marc cells was significantly brighter than Marc-145 cells;Western blotting results indicated that the pCD163-Marc cells could express higher levels of CD163 and the expression level was 8.7 times higher than Marc-145 cells.The pCD163-Marc cell line could be stably passaged for 20 passages and the expression level of CD163 was similar with different passages, which would be a valuable tool for facilitating virus propagation and vaccine production.  相似文献   

3.
试验旨在构建一株高效表达猪CD163(pCD163)的Marc-145细胞系,为猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的临床分离和疫苗生产奠定基础。根据GenBank中序列设计引物从猪肺泡巨噬细胞(PAM)中扩增pCD163基因,将其插入真核表达载体pCI-neo构建真核表达质粒pCI-pCD163,将该重组质粒转染Marc-145细胞,通过G418筛选、单克隆化并扩大培养筛选获得表达pCD163的Marc-145细胞系,IFA、Western blotting鉴定其表达情况。IFA结果显示,构建的pCD163-Marc细胞系中荧光明显亮于普通Marc-145细胞;Western blotting结果显示,pCD163-Marc细胞系中CD163蛋白表达量约为对照Marc-145细胞中CD163蛋白表达量的8.7倍。且该细胞系可稳定传至20代,各代次之间表达量无差异。证明高效表达猪CD163的Marc-145细胞系构建成功。  相似文献   

4.
Swine leukocyte antigens (SLA) and a macrophage specific marker were monitored on porcine macrophages cultured with or without macrophage colony stimulatory factor (M-CSF) and on cells infected with African swine fever virus (ASFV). SLA expression was maximal either in the total cell extract or on the cell surface at 3-4 days of culture; after 4 days these values began to decrease. Fluorescence analyses of immunostained macrophages cultured with or without M-CSF indicated a major upward shift in the number of SLA Class I molecules on individual macrophages whereas for SLA Class II both a novel expression of Class II and an upward shift in the number of molecules per cell were evident. Infection of 3-day-old macrophage cultures with three different isolates of ASFV resulted in minor changes in surface expression of SLA Class I, SLA Class II, and macrophage markers. No differences in infection with ASFV was observed whether macrophages were SLA Class II positive or negative, nor was there blocking by anti-SLA Class I or Class II monoclonal antibodies of ASFV infection of cultured macrophages.  相似文献   

5.
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6.
Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism mainly for porcine alveolar macrophages (PAMs), but not for peripheral blood monocytes (BMo) in vivo. Previous research showed that only a few BMo became susceptible to PRRSV infection after 1 day culture. Porcine sialoadhesin (PoSn) and CD163 are identified to be the two main PRRSV receptors for binding and internalization. Both receptors are not expressed on BMo, or only expressed at low levels, which may explain why PRRSV cannot infect them. The relationship of BMo differentiation/aging, PRRSV receptor level, and susceptibility to PRRS virus infection has not been thoroughly investigated. In this study, BMo were successfully cultured with pig serum plus L929 cell culture supernatant. Our results showed that both the mRNA and protein expression levels of PoSn were significantly increased after 5-day culture. The mRNA level of CD163 was enhanced more than 20-fold after 1-day culture; CD163-positive BMo increased dramatically from about 2% after 2h- culture to about 50% after 96-h culture. Furthermore, cultured BMo became much more permissive to PRRSV infection, and the percentage of PRRSV-infected BMo was at least the same as PAMs, if not higher, when infected with CH-1a, the first PRRSV strain isolated in China, or HV, a highly virulent strain. Three other PRRSV strains including VR2332, and two classical Chinese isolates could also infect cultured BMo as well. Most importantly, PRRS virus was successfully isolated from 14 of 15 antibody-positive serum samples using cultured BMo. These results suggest that the enhanced susceptibility of cultured BMo to PRRS virus is coordinated with increased CD163 expression, but less related to the delayed (day 5) increased expression of PoSn. Thus, cultured BMo could be an alternative choice for PRRS virus isolation and identification.  相似文献   

7.
The aetiology of porcine post-weaning multisystemic wasting syndrome (PMWS) is poorly understood. Porcine circovirus type 2 (PCV-2) is an essential component of the experimental disease model for PMWS: however, evidence from experimental and field studies indicates that additional factors play a critical role in the aetiopathogenesis of PMWS. Current candidates include (1) immune stimulation (for example, via co-infection or vaccination), and (2) a novel infectious agent. A prospective, longitudinal case-control study was designed to investigate molecular triggers in leucocytes of neonatal piglets that may predispose to the development of PMWS. Blood samples were collected weekly from pigs (n=125) within five farms, from 1 week to 8 weeks of age: that is, before the appearance of clinical signs. Four colour flow cytometry was used to investigate changes in subsets of peripheral blood mononuclear cells, using monoclonal antibodies against the following cell associated markers; sIgG, CD3, MHCII dR, CD14, CD4a, CD8a, CD45RC, CD25, SWC3a, SWC8, CD163 and CD45. Sampling and laboratory analysis was supported by monitoring of clinical signs from 1 week to 20 weeks of age, or until disease supervened. At the conclusion of the study, 68 pigs (54%) were classified in Group 1 (no signs of clinical disease), 34 pigs (27%) in Group 2 (signs of clinical disease but not characteristic of PMWS), 17 pigs (14%) in Group 3 (suspect PMWS case) and 5 pigs (4%) in Group 4 (PMWS case). A single case of Porcine Dermatitis and Nephropathy syndrome (PDNS) was also diagnosed. Significant changes with age were demonstrated in clinically normal, neonatal pigs (Group 1), including an increase in B-cells and T-cells, and an increase in the proportion of total T-cells expressing MHCII. Within the T-cell subset, the proportion of CD8(+high) CD4(-) T-cells increased, in addition to the proportion of CD4(+) T-cells co-expressing CD8. Of the factors recorded, farm was found to have a highly significant effect on immune system development in the neonate. Comparison of Groups 1 and 4 cases identified significant differences between pigs which remained normal and those which subsequently developed PMWS. Pigs which went on to develop PMWS had a greater proportion of T-cells expressing MHCII in early life, higher mean intensity of expression of MHCII on T-cells, higher mean intensity of expression of MHCII on B cells and higher expression of CD25 on CD45RC(-) T-cells. These findings suggest that lymphocyte activation may be a key early event in the aetiology of PMWS.  相似文献   

8.
旨在初步探究非洲猪瘟病毒(African swine fever virus,ASFV)多基因家族MGF360-13L基因的功能。使用生物信息学软件对MGF360-13L基因进行分析;构建真核表达质粒pVAX1-MGF360-13L并转染至293T细胞进行表达;利用同源重组方法构建基因缺失毒株ASFV-ΔMGF360-13L,以相同的感染复数(MOI=0.01)在猪肺泡巨噬细胞(PAMs)中感染基因缺失毒株和亲本毒株(ASFV CN/GS/2018)后,利用红细胞吸附试验(HAD)计算病毒滴度并绘制体外生长曲线;以MOI=1将ASFV-ΔMGF360-13L和ASFV CN/GS/2018分别感染猪骨髓巨噬细胞(BMDM),利用qPCR和ELISA测定细胞炎性因子IL-1β、IL-6和TNF-α的表达水平。结果表明,MGF360-13L基因在ASFV基因Ⅱ型中相对保守,编码蛋白属于非分泌型、无跨膜区、亲水性好;构建的真核表达质粒pVAX1-MGF360-13L被293T细胞表达;成功获得MGF360-13L单基因缺失毒株ASFV-ΔMGF360-13L,与ASFV CN/GS/2018相比,其体外复制没有显著性差异;在ASFV-ΔMGF360-13L感染BMDM后,IL-1β、IL-6和TNF-α炎性细胞因子在转录和分泌水平都显著高于ASFV CN/GS/2018。本研究成功制备了ASFV的MGF360-13L基因缺失毒株,并证实了MGF360-13L基因作为ASFV复制非必需基因具有抑制炎性因子表达的功能,这为进一步注释ASFV MGF360-13L基因功能提供了线索。  相似文献   

9.
为建立检测猪繁殖与呼吸综合征病毒(PRRSV)允许细胞表面的Sn和CD163受体的方法,本研究设计针对Sn和CD163基因的特异性引物和荧光探针,建立了检测PRRSV Sn和CD163受体的荧光定量RT-PCR方法.结果显示,该方法在检测101 copies/μL～108 copies/μL模板范围内具有良好的线性关系.标准曲线的相关系数r值均大于0.996,扩增效率分别为100％和107％;该检测方法对Sn和CD163的检测下限均为10拷贝,敏感性高;批内重复试验和批间重复试验的变异系数均小于5％,具有良好的重复性.利用该方法对PRRSV感染肺泡巨噬细胞(PAM) 72 h后Sn和CD163受体mRNA的转录水平进行了检测,结果表明Sn和CD163受体的转录 水平显著上调.本研究为PRRSV病毒感染后两种主要受体变化趋势的研究提供了有效的检测方法.  相似文献   

10.
TLRs, Siglecs and CD163 are cell surface receptors that play an important role in immune response and sepsis. The objective of this study was to assess changes in the expression levels of several of these receptors (TLR2, TLR4, CD163, Siglec-1, Siglec-3, Siglec-5 and Siglec-10) on the surface of peripheral blood mononuclear cells from pigs with sepsis caused by Haemophilus parasuis. Flow cytometry was employed to analyze samples from an experimental infection and from cell cultures. A significant increase in CD163, TLR2 and Siglec-3 expression during infection was seen. However, in vitro exposure of peripheral blood monocytes to bacteria or sera from infected pigs did not increase the expression of these receptors. These changes may be due to recruitment of monocytes into the blood compartment in response to H. parasuis-induced sepsis.  相似文献   

11.
Evidence is presented to show that activation of endothelial and leucoyte adhesion molecules is a key step in transferring virus from infected leucocytes; and determines the restricted tissue tropism. A range of tissues from 2 experimentally infected mares in late pregnancy at 4 and 8 days after infection with EHV-1 were compared with those from normal pregnant and nonpregnant mares. Rabbit antisera to equine activated endothelial cell molecules were used to identify which tissues expressed these molecules in normal nongravid and gravid mares, and to investigate whether the range of tissues was altered in pregnant mares as a consequence of infection. The results indicated that the endothelium of the pregnant reproductive tract did express these molecules. In the 2 pregnant mares infected with EHV-1, the endothelial cells in the nasal mucosa also expressed these activated endothelial cell molecules. Therefore, the sites expressing these molecules closely correlated with those where virus infection of endothelial cells has been described and is consistent with experimental in vitro data, indicating that expression of these molecules is an essential stage in the transference of virus from leucocytes to endothelial cells.  相似文献   

12.
Bovine viral diarrhea virus (BVDV) has been detected in peripheral blood mononuclear cells (PBMCs) of immunocompetent animals, not being clear whether the development of a specific humoral immune response can prevent BVDV infection. The aim of this study was to evaluate the ability of non-cytopathic BVDV to replicate and produce infectious virus in PBMCs from calves pre-infected with BVDV and to elucidate the immunomodulatory effect of BVDV on these cells in an in vitro model. Quantification of virus was by quantitative PCR, while its replicative capacity and shedding into the extracellular environment was evaluated by viral titration. Apoptosis was assessed by flow cytometry analysis of annexin V and propidium iodide, and by expression of caspase-3/7. Flow cytometry was used to analyze the expression of CD14/CD11b/CD80, CD4/CD8/CD25, MHC-I/MHC-II and B-B2 markers. Our results showed that PBMCs from cattle naturally infected with BVDV were more susceptible to in vitro BVDV infection and showed a more severe apoptosis response than those from naïve animals. Non-cytopathic BVDV in vitro infection also resulted in a lack of effect in the expression of antigen presentation surface markers. All these findings could be related to the immunosuppressive capacity of BVDV and the susceptibility of cattle to this infection.  相似文献   

13.
非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用   总被引：1，自引：1，他引：0  
为制备非洲猪瘟病毒（ASFV）p62蛋白的特异性单克隆抗体,并初步应用于感染组织样品中ASFV抗原的免疫组化（IHC）检测,本研究以杆状病毒表达的非洲猪瘟病毒重组p62蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞进行融合获得杂交瘤细胞。结果显示：基于纯化的p62蛋白建立的间接ELISA方法对杂交瘤细胞进行筛选和亚克隆,获得了18株可稳定分泌抗非洲猪瘟病毒p62蛋白单克隆抗体的杂交瘤细胞株。经IFA检测,制备的单克隆抗体均与非洲猪瘟病毒反应,且不与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型等猪源常见病毒反应,特异性良好。抗体识别蛋白的鉴定结果显示,3株MAbs识别p35蛋白,15株MAbs识别p15蛋白。14株MAbs重链亚类为IgG1型,4株MAbs重链亚类为IgG2a,轻链均为κ链。利用18株MAbs对ASFV感染猪的肺、扁桃体、淋巴结等组织进行IHC检测,结果显示5株MAbs均能够与感染ASFV的组织发生特异性的免疫反应。本研究获得的非洲猪瘟病毒p62蛋白单克隆抗体可为非洲猪瘟病毒免疫学检测方法的建立及p62蛋白的结构功能等基础研究提供重要的生物材料。  相似文献   

14.
The pattern of porcine alveolar macrophage (AM) activation upon classical stimuli of two strains of African swine fever (ASF) viruses, an attenuated ASFV-BA71V and virulent ASFV-Georgia2007 were investigated. In an in vitro experiment ASFV-Georgia2007-infected AM showed M1 polarization pattern different from the one induced by classical stimuli. Altered morphology, appearance of binuclear cells, decreased synthesis of IFN-alpha as well as IFN-epsilon was observed compared with attenuated ASFV-BA71V, and decreased synthesis of IFN-omega compared with intact cells. However, CD68 level did not significantly differ between alveolar macrophage populations infected by ASFV-Georgia2007 and control group, while both LPS/IFN-gamma stimulation and non-pathogenic ASFV-BA71V virus increased the level of CD68 soluble receptor.AM infection with ASFV-Georgia2007 resulted in remarkable DNA proliferation whereas LPS/IFN-gamma and ASFV-BA71V induced less expressed DNA proliferation in activated cells. The higher value of nitric oxide was obvious in the cells infected with ASFV-BA71V, compared to ASFV-Georgia2007 and LPS/IFN-gamma activated cells.In conclusion, pattern of activation of alveolar macrophages induced by ASFV-Georgia2007 virus differs from the one expressed in LPS/IFN-gamma- and ASFV-BA71V-activated cells. ASFV-BA71V and LPS/IFN-gamma share similar antiviral response of porcine AM. Therefore we assume that wild type virulent ASFV can partially down regulate antiviral response of AM and conclude that evolutionary decrease of virulence in ASFV is related to alterations of control of the host cell antiviral response.  相似文献   

15.
Virus yields from porcine alveolar macrophages (AM) infected with African swine fever virus (ASFV) were greater and were achieved more rapidly, when inoculated at a high multiplicity of infection (MOI) than at low MOI. The difference was related to a lower percentage of cells becoming infected after low MOI inoculation. The reduced yields after low MOI were not caused by prolongation of the culture time, by bacterial endotoxins or by production of inhibitory substances by infected AM. Virus-infected AM were not susceptible to lysis in antibody-dependent cell mediated cytotoxicity (ADCC) assays and this was apparently due to a paucity of viral antigen expressed on the cell surface. Uninfected AM did not act as effectors in ADCC.Porcine bone marrow (PBM) cells were effective in mediation of ADCC and their activity was reduced after ASFV infection. Cells separated into adherent and non-adherent populations, depleted by carbonyl iron treatment or separated by Ficoll-Hypaque centrifugation, all showed effector activity in ADCC. The effector cells were not mature neutrophils or lymphocytes and were probably granulocytic precursors.  相似文献   

16.
Mononuclear phagocytes (MP) are cells of nonspecific immunity, playing an essential role in defense against bacterial pathogens. Although various MP subpopulations have been described in the pig, relations among these populations in vivo are unknown to date. The present study was aimed at describing porcine MP subpopulations infiltrating inflamed tissue of pigs under in vivo conditions. Actinobacillus pleuropneumoniae (APP) infection was used to induce an inflammatory response. CD172α, CD14, CD163, MHCII and CD203α cell surface molecules were used to identify MP by flow cytometry. Changes in MP subpopulations in the peripheral blood (PB) and bone marrow (BM) compartments along with the analysis of MP appearing in the inflamed lungs were assessed to elucidate the possible origin and maturation stages of the infiltrating MP. The MP population migrating to the inflamed lungs was phenotype CD14+ CD163+ CD203α+/− MHCII+/−. Concomitantly, after APP infection there was an increase in the PB MP CD14+ CD163+ CD203α− MHC II− population, suggesting that these cells give rise to inflammatory monocytes/macrophages. The CD203α and MHCII molecules appear on these cells after leaving the PB. In healthy animals, the BM MP precursors were represented by CD14− CD163− cells maturing directly into CD14+ CD163− that were then released into the PB. After infection, an altered maturation pathway of MP precursors appeared, represented by CD14− CD163− CD203α− MHCII− MP directly switching into CD14+ CD163+ CD203α− MHCII− MP. In conclusion, two different MP maturation pathways were suggested in pigs. The use of these pathways differs under inflammatory and noninflammatory conditions.  相似文献   

17.
Pigs with complete resistance to porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) have been produced by genetically knocking out the CD163 gene that encodes a receptor of the PRRSV for entry into macrophages. The objectives of this study were to evaluate associations of naturally occurring single nucleotide polymorphisms (SNPs) in the CD163 gene and in three other candidate genes (CD169, RGS16, and TRAF1) with host response to PRRSV-only infection and to PRRS vaccination and PRRSV/porcine circovirus 2b (PCV2b) coinfection. SNPs in the CD163 gene were not included on SNP genotyping panels that were used for previous genome-wide association analyses of these data. An additional objective was to identify the potential genetic interaction of variants at these four candidate genes with a mutation in the GBP5 gene that was previously identified to be associated with host response to PRRSV infection. Finally, the association of SNPs with expression level of the nearby gene was tested. Several SNPs in the CD163, CD169, and RGS16 genes were significantly associated with host response under PRRSV-only and/or PRRSV/PCV2b coinfection. The effects of all SNPs that were significant in the PRRSV-only infection trials depend on genetic background. The effects of some SNPs in the CD163, CD169, and RGS16 genes depend on genotype at the putative causative mutation in the GBP5 gene, which indicates a potential biological interaction of these genes with GBP5. In addition, genome-wide association results for the PRRSV-only infection trials revealed that SNPs located in the CDK5RAP2 or MEGF9 genes, near the TRAF1 gene, had suggestive effects on PRRS viral load, which indicates that these SNPs might contribute to PRRSV neuropathogenesis. In conclusion, natural genetic variants in the CD163, CD169, and RGS16 genes are associated with resistance to PRRSV and/or PCV2b infection and appear to interact with the resistance quantitative trait locus in the GBP5 gene. The identified SNPs can be used to select for increased natural resistance to PRRSV and/or PRRSV-PCV2b coinfection.  相似文献   

18.
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19.
为制备兔抗猪CD163多克隆抗体,采用RT-PCR方法从猪PAMRNA中扩增CD163胞质外区部分基因SRCR4-5,克隆至pGEX-6p-1原核表达载体,在IPTG诱导下获得约50000以包涵体形式表达的重组蛋白。WesternBlotting分析表明,表达产物能够被GST单抗所识别。用该重组蛋白免疫兔制备多抗血清,经间接ELISA检测,多抗血清效价可达10^5,流式检测发现可与Marc-145和PAM上的天然CD163分子相互作用。抗体阻断试验证明制备的多抗血清可以部分阻断PRRSV感染。本试验为进一步研究PRRSV与受体一CDl63相互作用机制打下了基础。  相似文献   

20.
Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) impairs local pulmonary immune responses by damaging the mucociliary transport system, impairing the function of porcine alveolar macrophages and inducing apoptosis of immune cells. An imbalance between pro- and anti-inflammatory cytokines, including tumour necrosis factor-α and interleukin-10, in PRRS may impair the immune response of the lung. Pulmonary macrophage subpopulations have a range of susceptibilities to different PRRSV strains and different capacities to express cytokines. Infection with PRRSV decreases the bactericidal activity of macrophages, which increases susceptibility to secondary bacterial infections. PRRSV infection is associated with an increase in concentrations of haptoglobin, which may interact with the virus receptor (CD163) and induce the synthesis of anti-inflammatory mediators. The balance between pro- and anti-inflammatory cytokines modulates the expression of CD163, which may affect the pathogenicity and replication of the virus in different tissues. With the emergence of highly pathogenic PRRSV, there is a need for more information on the immunopathogenesis of different strains of PRRS, particularly to develop more effective vaccines.  相似文献   

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