Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs

Replication of porcine circovirus type 2 (PCV2) in pigs, as measured by spliced capsid mRNA (Cap mRNA) and viral DNA, was investigated following experimental infection. Peripheral blood mononuclear cells (PBMCs), and tissue from bronchial lymph nodes (BLN), inguinal lymph nodes (ILN), tonsils, lungs, liver, kidneys, spleen and thymus from infected pigs on different days post-infection (DPI) were assessed. PCV2 replication differed dramatically between tissues from the same infected pig. The virus actively replicated in most tested tissues at 14DPI in association with increased PCV2 associated lesions and PCV2 antigen levels, although no clinical signs correlated with PCV2 associated disease were observed in infected pigs during the course of the study. The PCV2 Cap mRNA was detected only at 13DPI in PBMCs from infected pigs, suggesting replication of the virus in circulating blood is transient and not a major site for PCV2 replication in vivo. Evaluation of the Cap mRNA and viral DNA synthesis in T and B lymphocyte and monocyte populations from PBMCs and BLN at various intervals post-inoculation revealed replication of PCV2 in all cell subpopulations; however, viral replication in B lymphocytes was greater than observed in mononuclear cells isolated from BLN at 14DPI indicating that B lymphocytes may be an important cell population for PCV2 replication. These findings further our understanding of the cell types permissive for PCV2 replication and the pathogenesis of PCV2 infection in vivo.     

NO自由基对猪圆环病毒2型复制的体外抑制作用研究

试验旨在探讨NO自由基(NO·)对猪圆环病毒2型(PCV2)复制的抑制作用。硝普钠(SNP)和抗坏血酸(VC)联合加入培养基中可产生NO·,而单独使用SNP产生NO的形式为NO^+。本研究采用MTT法测定药物的细胞毒性,通过Griess反应测定供体药物产生NO的水平。分别用SNP、VC、SNP+VC和对照药物乙酰青酶胺(NAP)处理PK-15细胞,6 h后接种1 MOI的PCV2病毒液,72 h后收获培养上清和细胞,通过间接免疫荧光试验(IFA)、Western blotting分析、病毒效价和实时荧光定量PCR试验检测病毒增殖水平。结果显示:SNP、VC和SNP+VC对PK-15细胞的最大安全浓度分别为500、62.5和31.25μmol/L;60μmol/L SNP和30μmol/L SNP+30μmol/L VC两种供体产生的NO量极显著高于非药物处理组和对照药物NAP处理组(P<0.01);与阳性对照组相比,60μmol/L SNP+30μmol/L VC处理组中的PCV2毒价和DNA拷贝数极显著下降(P<0.01),且PCV2 Cap蛋白的表达也受到明显抑制,而SNP和VC单独处理组上述指标无显著变化(P>0.05)。以上结果表明,SNP+VC产生的NO·对PCV2增殖有显著抑制作用,SNP产生的NO^+不影响PCV2增殖,NO体外抑制PCV2增殖主要是通过NO·实现。     

Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages

Monocyte/macrophage lineage cells are major target cells of porcine circovirus type 2 (PCV2). From tissues of field pigs suffering from postweaning multisystemic wasting syndrome (PMWS), both intracytoplasmic and intranuclear PCV2 signals, including antigens and nucleic acid, were easily detected in the monocyte/macrophage lineage cells. However, there was a high incidence of intracytoplasmic PCV2-positive signals, but lack of intranuclear signals and PCV2 replication in these cells in vitro. Concurrent infection with bacteria and activation of immune system are suggested to promote viral replication. In this study, lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA) were used to stimulate PCV2-inoculated alveolar macrophages (AMs). A decrease in intracytoplasmic but increase in intranuclear PCV2-positive signals, including antigens and nucleic acid, were detected in LPS-treated PCV2-inoculated AMs, but not in PMA-treated cells. Additionally, the replication product corresponding to PCV2 spliced major capsid protein (Cap) mRNA and a significant elevation in PCV2 titer were demonstrated in the LPS-treated PCV2-inoculated AMs. The results imply that Gram-negative bacterial co-infection in PCV2-infected pigs may be an important factor in promoting PCV2 replication and contributing, at least partially, to the full development of PMWS.     

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products

The effect of Fasciola hepatica excretory-secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2(+), CD4(+), CD8(+), gammadeltaTCR(+) or CD21(+) cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 microg per well) or suboptimal (0.25 microg per well) doses and with FhESPs at several doses (1.25-20 microg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 microg per well. These cells were incubated with or without monocytes (CD14(+) cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 microg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21(+) lymphocytes (B lymphocytes), CD14(+) cells (monocytes) and gammadeltaTCR(+) cells were not stimulated by ConA. T lymphocyte subsets (CD2(+), CD4(+) or CD8(+) cells) proliferation was decreased by FhESPs at 5 microg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.     

外泌体介导PCV2通过Toll7/8/9-MyD88-NF-κB信号通路诱导淋巴细胞分泌IL-10

以接种PCV2的PK-15细胞所分泌的外泌体(P-Exo)孵育的脾脏淋巴细胞为研究对象,采用Western blot、间接免疫荧光、ELISA和qPCR等方法,分析外泌体对脾脏淋巴细胞IL-10分泌的影响及其信号通路.结果显示,P-Exo呈杯状,平均直径为50?150 nm,表达标志蛋白CD81和TSG101,包含PC...     

Deletion of the single putative N-glycosylation site of the porcine circovirus type 2 Cap protein enhances specific immune responses by DNA immunisation in mice

The immunogenicity of a putative N-linked glycosylation site located at amino acids 143-145 (N143YS) of the porcine circovirus 2 (PCV2) Cap protein was investigated. Eukaryotic vectors expressing wild-type PCV2 Cap (pCap) and N-linked glycosylation site mutant Cap (pCap-m) were constructed and the immunogenicity of these proteins was determined following DNA vaccination in BALB/c mice. pCap-m elicited significantly higher Cap-specific T lymphocyte proliferative activity, percentage of CD8(+) T cells, ratio of immunoglobulin (Ig) G2a:IgG1 and levels of interferon-γ compared to pCap (P<0.05). These results indicate that deletion of the N-glycosylation site in the PCV2 Cap protein enhances specific immune responses and may have a role in Cap-based DNA vaccines with enhanced immunogenicity.     

Induction of proinflammatory cytokines and caspase-1 by leptin in monocyte/macrophages from holstein cows

The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.     

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication.     

Monocyte-derived dendritic cells enhance cell proliferation and porcine circovirus type 2 replication in concanavalin A-stimulated swine peripheral blood lymphocytes in vitro

Dendritic cells (DCs) are professional antigen presenting cells cooperating with other immune cells for the activation of innate and adaptive immune responses. The objective of the present study was to investigate the replication activity of porcine circovirus type 2 (PCV2) in DCs and/or lymphocytes during their cross talk and its possible mechanism. Two models were set, herein. Swine blood monocyte (Mo)-derived DCs (MoDCs) or peripheral blood lymphocytes (PBLs) were inoculated with PCV2 prior to their co-cultivation. Bacterial lipopolysaccharide (LPS) and concanavalin A (Con A) were used to stimulate MoDCs and PBLs, respectively. During 6 days of cultivation, a high PCV2 antigen-containing rate without detectable intranuclear signals and a slight but significant increase in the copy number of PCV2 genome were detected in PCV2-inoculated MoDCs. The presence of LPS alone or PCV2-free PBLs, however, had no effect on the location of PCV2 antigens or copy number of PCV2 genome in PCV2-inoculated MoDCs. On the contrary, active PCV2 replication occurred in Con A-stimulated PCV2-inoculated PBLs. When compared with blood Mos, MoDCs induced significantly higher cell proliferation and intensified PCV2 replication in Con A-stimulated PCV2-inoculated PBLs, for which direct contact between MoDCs and lymphocytes was required. Among the cytokines secreted by Con A-activated PBLs, interleukin (IL)-2, but not IL-4 or interferon-γ, could induce cell proliferation and PCV2 replication in PCV2-inoculated PBLs. The findings suggest that although MoDCs support only limited PCV2 replication in themselves, their accessory cell function is required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes.     

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

Previously, it was shown that modulation of the immune system enhances porcine circovirus type 2 (PCV2) replication in pigs. In the present study, the effect of the mitogen concanavalin A (ConA) on PCV2 replication was investigated. Since ConA induces T-lymphocyte activation and initiates the production of interferon-gamma (IFN-gamma), a cytokine that enhances PCV2 replication in porcine epithelial and monocytic cell lines in vitro, it was examined if the effects observed with ConA were mediated by IFN-gamma. In an in vitro study, ConA but not IFN-gamma enhanced PCV2 replication in peripheral blood mononuclear cells (PBMC). Up to 2.08% and 0.96% of PBMC were antigen positive for PCV2 strains 1121 and Stoon-1010, respectively, and a low virus production was observed. PCV2-infected PBMC were identified as CD4(+) (40%), CD8(+) (54%) and IgM(+) (11%). In a subsequent in vivo study, caesarean-derived colostrum-deprived piglets were injected with ConA or IFN-gamma 12h before inoculation and every 3 days for 9 days after inoculation with strain 1121. PCV2 was isolated from inguinal lymph node biopsies from 10 days post-inoculation (dpi) in ConA-treated pigs and from 15dpi in non-treated and IFN-gamma-treated pigs. ConA increased PCV2 replication levels, but disease was not observed. Half of the ConA-treated and IFN-gamma-treated pigs showed a delayed humoral immune response, but this delay did not result in increased PCV2 replication in these pigs. These experiments demonstrated that ConA enhances PCV2 replication in PBMC in vitro and in lymphoid tissues in vivo.     

Characterization of porcine circovirus type 2 (PCV2) infection in swine lymphocytes using mitogen-stimulated peripheral blood lymphocytes from healthy PCV2-carrier pigs

Information regarding the susceptibility of swine lymphocytes to PCV2 is rather limited. To further explore and characterize the PCV2 infection in swine lymphocytes, an in vitro model using concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) obtained from clinically healthy PCV2-carrier pigs was introduced. It was found that the PCV2 antigen-containing rate was below 2% in PBLs from healthy PCV2-free pigs following treated simultaneously with Con A and PCV2. However, significantly higher PCV2 antigen- and nucleic acid-containing rates could be seen in Con A-stimulated PBLs from clinically healthy PCV2-carrier pigs. Prior to Con A treatment, both of the PCV2 antigen- and nucleic acid-containing rates in PBLs from healthy PCV2-carrier pigs were less than 1%; however, they reached 22.1+/-5.7% by flow cytometry and 27.1+/-6.5% by in situ hybridization, respectively, at 4-day post-incubation with Con A. Phenotyping of PCV2 antigen-containing cells revealed that PCV2-positive cells could be detected in both T and B lymphocyte populations within which IgM-positive B lymphocytes appeared to have a relatively higher positive rate. The Con A-stimulated PBLs also displayed a significantly higher viral load by the measurement of either PCV2 DNA copy number or viral titer when compared with the non-treated PBLs from healthy PCV2-carrier pigs. The results indicate that PBLs, especially IgM-bearing B lymphocytes, are indeed susceptible to PCV2 infection and PCV2 is capable of replicating in dividing lymphocytes. This activation-induced replication may explain in part the pathogenesis of lymphoid depletion in PMWS-affected pigs.     

小鼠感染猪圆环病毒2型的超微结构病变观察

为探讨猪圆环病毒2型（PCV2）对小鼠超微结构的病理损伤及其病毒在细胞内的复制,20只6周龄的昆明小鼠随机平均分成2组（即A组和B组）,A组小鼠经腹腔注射PCV2细胞培养物0.1mL/只（含病毒1 000TCID50）,B组以同样的方式和剂量注射无菌细胞培养液作为对照。于PCV2感染后14d,处死所有小鼠,取其组织做电镜观察和PCV2PCR检测。结果显示,在电镜下,所有PCV2感染鼠的超微结构病变基本一致,主要表现为淋巴器官、心脏、肝脏、肺脏、肾脏、脑、肠等脏器实质细胞凋亡或坏死,细胞内线粒体水肿、内质网扩张,间质毛细血管淤血、炎症细胞浸润,并在脾脏、胸腺、淋巴结的淋巴细、巨噬细胞,以及肝细胞,肾足细胞,脑神经细胞的胞浆或胞核内发现病毒包涵体。同时通过PCR检测,所有PCV2感染鼠的组织均可检出PCV2DNA。B组（对照组）小鼠除在淋巴结可见极少数淋巴细胞凋亡外,其他组织均无任何超微结构病变;同时,所有组织也未检出PCV2DNA。由此说明PCV2可在昆明小鼠多脏器实质细胞内复制,并诱导细胞凋亡。     

猪圆环病毒2型复制及其影响因素

猪圆环病毒2型（PCV2）DNA的复制是滚环复制,Rep和Rep′蛋白是PCV复制所必需的蛋白质,核心元件A*TA*TAC为必需的结合位点。复制的影响因素包括病毒内部因素如病毒基因内的CpG寡核苷酸和干扰素反应元件、病毒黏附受体(硫酸乙酰肝素和硫酸软骨素B乙糖胺)和病毒内吞作用（小GTP酶和肌动蛋白）;外部因素如DL-硒蛋氨酸和刀豆素A。抑制PCV2复制的措施有猪营养的全面,干扰RNA技术,疫苗的使用等。     

ORF1 but not ORF2 dependent differences are important for in vitro replication of PCV2 in porcine alveolar macrophages singularly or coinfected with PRRSV

The objective of this study was to investigate cytokine expression and in vitro replication of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in pulmonary alveolar macrophages (PAMs) emphasizing PCV2 open-reading frame (ORF) origin (PCV2a or PCV2b) and PRRSV strain. Chimeric PCV2 viruses composed of different combinations of ORF1 and ORF2 of PCV2a or PCV2b (chimera PCV2a-2b and chimera PCV2b-2a) were constructed and five different PRRSV isolates were utilized: Type 1 (SD 01-08) or type 2 (NC16845b, VR-2332, MN-184, JA-142). PAMs were infected singularly or with combinations of PCV2b, PCV2a, chimera PCV2a-2b, and chimera PCV2b-2a, and one of the five PRRSV isolates. Real-time PCR was used to test PAMs (PCV2 mRNA) and supernatants (PRRSV RNA, PCV2 DNA, PCV2 mRNA) harvested at 24, 48, 72 and 96h post inoculation (hpi). Levels of IFN-γ, TNF-α and IL-10 were determined by quantitative ELISAs. PCV2 replication in PAMs was limited to groups inoculated with PCV2 strains containing ORF1 of PCV2a (PCV2a, chimera PCV2a-2b). Furthermore, in supernatants, PCV2 mRNA was only detected in groups coinfected with PRRSV regardless of strain at 48hpi supporting an enhancing effect of PRRSV on PCV2 infection. Changes in cytokine levels were minimal and associated with PRRSV strain for TNF-α. In summary, in vitro differences in PCV2 replication in PAMs inoculated with different PCV2-PRRSV combinations were independent of PCV2 ORF2 origin with minimal effects of concurrent PRRSV infection perhaps indicating that PCV2-specific changes in ORF1 may be more important than those in ORF2.     

C-terminal heat shock protein 70 of Mycobacterium tuberculosis as a molecular adjuvant for DNA vaccination with the porcine circovirus type 2 ORF2 (capsid) gene in mice

Mycobacterium tuberculosis heat shock protein 70 (HSP70) and the peptide binding C-terminal portion of HSP70 (amino acids 359-610; HSP70c) exert an adjuvant effect when used in vaccines. To enhance the immunogenicity of a DNA vaccine against porcine circovirus type 2 (PCV2), recombinant plasmids encoding the PCV2 ORF2 (capsid) gene fused to full length hsp70 (pCA-TCH) or truncated C-terminal hsp70c (pCA-TCHc) were constructed. Immunisation of mice with pCA-TCHc induced higher serum immunoglobulin G antibody levels, stronger T helper 1 immune responses and lower PCV2 viral titres following challenge than immunisation with pCA-TCH or Cap plasmids only.     

Leptin inhibits mitogen-induced proliferation of peripheral T lymphocytes from Holstein cows

This study examined the mitogenic response of bovine peripheral T lymphocytes to leptin, a pleiotropic hormone regulating food intake and energy expenditure. Leptin alone slightly suppressed proliferation of T lymphocytes in the presence of concanavalin A (ConA). Leptin also inhibited proliferation of T lymphocytes induced by anti-CD3 antibody. ConA treatment activated some protein kinases, including p44/p42(MAPK) and Akt/PKB, while anti-CD3 antibody treatment increased mRNA expression of suppressor of cytokine signalling (SOCS) 3, interferon (IFN)gamma, interleukin (IL) 2 and IL4 in T lymphocytes. Leptin alone increased only SOCS3 mRNA expression. Simultaneous treatment with mitogens and leptin enhanced IFNgamma mRNA expression but decreased IL2 mRNA expression, without any synergistic effect on phosphorylation of protein kinases or mRNA expression of SOCS3 and IL4. These results suggest that leptin modulates bovine T lymphocyte functions.     

Preparation of Monoclonal Antibodies against Porcine Circovirus Type 2 Cap Protein

The structural protein Cap encoded by ORF2 of porcine circovirus type 2 (PCV2) was expressed in genetic engineering recombinant bacteria and used as the immunogen after purification.Five hybridoma cell lines against PCV2 Cap protein named as 3D12,4D5,4B9,4C9 and 4G10,respectively,were developed after fusion between SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with purified recombinant PCV2 Cap protein.Except the heavy chain type of 4D5 was identified as IgG2b,others were identified as IgG1;The light chains were all kappa.In Western blotting assay,all the monoclonal antibodies (mAbs) couldn't specifically recognize the 47 ku recombinant PCV2 Cap protein,but showed strong specific fluorescence in PCV2 infected PK15 cells in IFA,which indicated that all the mAbs recognized comformational epitope.The neutralization test showed that all the mAbs had neutralization activity.These results laid the foundation for further study of the structure and function of PCV2 ORF2 gene,and establishment of the method for diagnosing PCV2 rapidly and exactly.     

SHIV-KB9病毒中国恒河猴细胞适应株的制备

试验旨在研究体外制备SHIV-KB9病毒中国恒河猴细胞适应株,在细胞水平和中国恒河猴体内评价其生物学特性。 试验将SHIV-KB9半长质粒连接后转染CEMx174细胞,转染上清与正常恒河猴PBMCs共培养。定期测定培养液中的P27抗原水平。当病毒复制达高峰期时收集培养上清,分装并冻存,测定病毒RNA载量和TCID₅₀。静脉感染中国恒河猴,研究该批次SHIV-KB9在体内的病毒学、免疫学指标变化及变异情况,分析其基本的生物学特性。结果表明,本研究共制备了95 mL SHIV- KB9病毒原液,病毒载量为2.678×10⁵ 拷贝/mL,TZM-bl细胞测定病毒的TCID₅₀为3.16×10³/mL,gp120序列分析表明病毒未发生变异。10倍稀释的3个不同浓度的SHIV-KB9均静脉成功感染中国恒河猴,引起外周血CD4⁺/CD8⁺大幅下降。因此,此次制备的SHIV-KB9细胞适应株生物学特性稳定,适合作为毒种库建立SHIV-KB9/中国恒河猴模型。     

猪圆环病毒2型Cap蛋白单克隆抗体的制备

本研究利用已构建的基因工程重组菌表达猪圆环病毒2型(porcine circovirus type 2,PCV2)ORF2编码的Cap蛋白,纯化后作为免疫原,免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合。经间接免疫荧光试验(IFA)筛选及有限稀释法进行3次亚克隆后,本试验最终获得5株稳定分泌抗PCV2 Cap蛋白单克隆抗体的杂交瘤细胞,分别命名为3D12、4D5、4B9、4C9和4G10。其中4D5为IgG2b亚型,其余4株单克隆抗体均为IgG1亚型,轻链类型均为κ。Western blotting鉴定结果表明获得的5株单克隆抗体均不能特异性的识别47 ku重组PCV2 Cap蛋白;对病毒感染细胞进行IFA试验,结果显示5株单克隆抗体均特异性的识别病毒抗原,表明5株单克隆抗体识别的抗原表位均为构象表位。中和试验结果表明,5株单克隆抗体均有中和活性。本试验结果为进一步探索ORF2基因的结构、功能及建立快速准确的诊断方法奠定了基础。