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1.
《中兽医医药杂志》2015,(3)
通过Marc145细胞体外培养研究浒苔水煎提取物(Enteromorpha decoction,ED)对猪繁殖呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)的抑制作用。结果与仅接种病毒的对照组相比较,25%~50%浓度的ED对PRRSV均有显著抑制作用;ED处理Marc145细胞1、2、3 h均能显著降低PRRSV的滴度,三者之间差异不显著;ED对0.1 MOI和0.01 MOI接种量的病毒抑制作用无显著性差异;持续作用于Marc145细胞,ED可完全抑制PRRSV的增殖;PRRSV接种前用ED处理Marc145细胞较接种后ED处理更显著地降低了病毒滴度。细胞毒性试验显示,50%ED对Marc145细胞有轻微损伤作用,45%以下浓度则无损伤。 相似文献
2.
【目的】 研究纳米银（silver nanoparticles,AgNPs）体外抗猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）的作用,并初步分析其抗病毒作用机制,为PRRSV的防控提供新思路。【方法】 使用0.1875、0.375、0.75、1.5、3、6、12 μg/mL AgNPs处理Marc145细胞,采用CCK-8试剂盒评估AgNPs对Marc145细胞毒性,确定其安全浓度。通过显微观察、间接免疫荧光试验、病毒滴度测定、实时荧光定量RT-PCR方法评估AgNPs体外抗PRRSV感染Marc145细胞的效果。通过间接免疫荧光试验和实时荧光定量RT-PCR方法评估AgNPs对PRRSV的直接灭活效果。通过实时荧光定量RT-PCR方法分析AgNPs对不同感染复数（multiplicity of infection,MOI） PRRSV （0.0001~0.1）黏附和入侵Marc145细胞的影响,以及PRRSV感染Marc145细胞3、6、12、18和24 h后加入AgNPs对Marc145细胞增殖的影响。【结果】 AgNPs对Marc145细胞的最大安全浓度为1.5 μg/mL,0.375、0.75、1.5 μg/mL AgNPs均具有良好的体外抗PRRSV活性,0.375、0.75、1.5 μg/mL AgNPs均对PRRSV起一定灭活作用。AgNPs对不同MOI的毒株黏附和入侵Marc145细胞均有一定抑制作用,且不同时间（3、6、12、18、24 h）加入AgNPs对PRRSV增殖均有一定抑制作用。【结论】 AgNPs具有良好的体外抗PRRSV活性,体外抗PRRSV的作用机制包括直接灭活以及抑制病毒的黏附、入侵和增殖过程。 相似文献
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自噬参与多种疾病过程,且与病毒感染与增殖关系密切。为探究Marc145细胞感染猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)后自噬相关基因Beclin1、ATG5和ATG12mRNA转录差异及规律、PRRSV增殖与细胞自噬的联系;采用实时荧光定量PCR方法对自噬相关基因Beclin1、ATG5和ATG12 mRNA的转录进行测定,绘制了转录规律曲线图;并经实时荧光定量测定并绘制PRRSV在Marc145细胞中增殖规律图。结果显示,试验组自噬相关基因转录量明显高于对照组,表明PRRSV能诱导Marc145细胞自噬,且使Beclin1、ATG5和ATG12mRNA提前进入高转录状态;其转录规律与细胞病变、PRRSV增殖规律呈正相关。 相似文献
4.
旨在研究宿主热休克蛋白HSP90AB1对猪繁殖与呼吸综合征病毒(PRRSV)复制的影响。选用对PRRSV易感的MARC-145细胞系,探究过表达和干扰HSP90AB1对病毒复制的影响,利用激光共聚焦免疫荧光和免疫共沉淀验证HSP90AB1与PRRSV的非结构蛋白NSP2作用关系,利用双荧光素酶试验探究HSP90AB1和NSP2对NF-kB活性的影响。结果表明,过表达HSP90AB1后显著抑制病毒的复制,干扰HSP90AB1的表达后促进病毒的复制;免疫共沉淀结果和激光共聚焦免疫荧光说明,HSP90AB1和NSP2存在相互作用;双荧光素酶报告试验表明HSP90AB1能逆转NSP2对NF-κB活性的抑制作用。综上,HSP90AB1可能通过与PRRSV NSP2相互作用,拮抗NSP2对NF-kB活性的抑制来抑制病毒的复制和感染。 相似文献
5.
本研究旨在通过慢病毒载体构建稳定表达真核翻译起始因子5A(eukaryotic translation initiation factor 5A,eIF5A)的MARC-145细胞系并验证该细胞系对PRRSV感染的影响。全基因合成eIF5A序列，双酶切后构建慢病毒表达载体pLV-CMV-MCS-EF1a-Puro。将该载体和慢病毒包装质粒psPAX2与包膜蛋白质粒pMD2.G共转染HEK293T细胞，包装得到能表达eIF5A的慢病毒。将慢病毒转导至MARC-145细胞，经过嘌呤霉素筛选、细胞有限稀释法获得能稳定表达eIF5A的MARC-145细胞系。MTS试验证实构建的细胞系细胞活力无显著变化，病毒TCID50测定、实时荧光定量PCR、蛋白质免疫印迹和间接免疫荧光技术检测eIF5A稳定表达对PRRSV滴度、PRRSV N基因及N蛋白表达的影响，发现MARC-145-eIF5A细胞系能显著促进PRRSV的增殖。本研究成功构建了稳定表达eIF5A的细胞系MARC-145-eIF5A,PRRSV感染试验证实体外稳定表达eIF5A可以促进PRRSV在MARC-145细胞的... 相似文献
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《国外畜牧学(猪与禽)》2021,41(4)
为评价商品化支原体清除剂对猪繁殖与呼吸综合征病毒(PorcineReproductiveand RespiratorySyndromeVirus,PRRSV)污染支原体的净化效果,研究首先优化了Marc145细胞耐受支原体清除剂的工作浓度,确定最适工作浓度为25μg/mL,然后在PRRSV培养液中加入25μg/mL的支原体清除剂,盲传3代,不加支原体清除剂继续盲传3代,将最后一代PRRSV样品及后续3代盲传样品进行支原体PCR检测,结果显示成功清除了PRRSV中的支原体,并且PRRSV病毒效价与清除前无明显差异,最终得到了无支原体的纯净PRRSV,为PRRSV相关疫苗的生产及其他细胞或病毒污染支原体的清除提供了很好的理论支撑。 相似文献
8.
为了检测靶向猪繁殖与呼吸综合征病毒(PRRSV)的FnCas9-rgRNA敲除载体是否构建成功,试验先构建FnCas9-rgRNA敲除载体骨架并对其进行测序,然后将黄色荧光蛋白(EYFP)、PRRSV分别与各自的FnCas9-rgRNA敲除载体转染至HEK293、Marc145细胞内,通过流式细胞仪分析HEK293细胞内的荧光强弱及Marc145细胞的病变程度,并对FnCas9-rgRNA敲除载体的初步构建、载体活性进行研究。结果表明:FnCas9-rgRNA敲除载体骨架测序正确,流式细胞仪分析显示FnCas9-rgRNA敲除载体对HEK293细胞内EYFP基因的表达产生明显抑制作用,表达荧光的细胞数量明显下降,3种针对PRRSV设计的rgRNA分别与FnCas9表达载体共转染细胞,接种病毒后Marc145细胞病变程度明显降低。说明靶向PRRSV的FnCas9-rgRNA敲除载体构建方式正确且FnCas9-rgRNA敲除载体具有活性。 相似文献
9.
《畜牧兽医学报》2016,(11)
利用慢病毒表达技术将稳定持续抑制PRRSV复制的shRNA导入Marc-145细胞,建立稳定表达靶向抑制PRRSV复制的shRNA的Marc-145阳性细胞克隆,并从细胞模型水平阐明抑制PRRSV复制的关键靶基因的干扰效果。采用LR重组技术,将pENTR/U6/Nsp9-4、pENTR/U6/Nsp9-6及pENTR/U6/-CON分别与pDEST载体进行LR重组,获得表达骨架,重组后的表达载体在转染试剂介导下与已经优化的辅助质粒Vira PowerTM Packaging Mix共转染293-FT包装细胞,获得慢病毒样粒子,并用其感染Marc-145细胞,杀稻瘟菌素抗性筛选获得阳性细胞克隆,通过PCR、CPE、TCID50、Real-time PCR和间接免疫荧光试验等方法分别验证上述细胞株的稳定整合以及其表达的shRNA对PRRSV增殖的抑制效果。结果显示:优势干扰序列和无关序列被稳定整合在靶细胞基因组上,无论观察细胞病变效应、免疫荧光产生情况、致细胞半数感染量还是实时定量分析相对表达量,与正常Marc-145和整合有无关序列的两株阴性对照细胞相比,整合有NSP9-4和NSP9-6的两株细胞由于表达了靶向抑制PRRSV复制的shRNA,PRRSV对其易感性降低,而且差别显著,分别将其命名为Marc/pU6/NSP9-4和Marc/pU6/NSP9-6。经验证,本研究成功构建两株稳定表达靶向抑制PRRSV复制的shRNA细胞——Marc/pU6/NSP9-4、Marc/pU6/NSP9-6,同时获得一株表达无意义shRNA的Marc/pU6-CON辅助细胞,该细胞表达的shRNA具有明显抑制PRRSV复制的功能,不仅可以从细胞模型水平阐明抑制PRRSV复制的关键靶基因,也为PRRSV感染引起宿主细胞的变化以及对机体的致病机制等研究提供资料。 相似文献
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11.
Ding Z Li ZJ Zhang XD Li YG Liu CJ Zhang YP Li Y 《Veterinary immunology and immunopathology》2012,145(1-2):206-213
Viral infections usually result in alterations in the host cell proteome, which determine the fate of infected cells and the progress of pathogenesis. To uncover cellular protein responses in porcine reproductive and respiratory syndrome virus (PRRSV), infected pulmonary alveolar macrophages (PAMs) and Marc-145 cells were subjected to proteomic analysis involving two-dimensional electrophoresis (2-DE) followed by MALDI-TOF-MS/MS identification. Altered expression of 44 protein spots in infected cells was identified in 2D gels, of which the 29 characterised by MALDI-TOF-MS/MS included 17 up-regulated and 12 down-regulated proteins. Some of these proteins were further confirmed at the mRNA level using real-time RT-PCR. Moreover, Western blot analysis confirmed the up-regulation of HSP27, vimentin and the down-regulation of galectin-1. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected Marc-145 cells using proteomics to provide valuable information about the effects of PRRSV-induced alterations on Marc-145 cell function. Further study of the affected proteins may facilitate our understanding of the mechanisms of PRRSV infection and pathogenesis. 相似文献
12.
It is well documented that there is a delay in the development of effective immunity to porcine reproductive and respiratory syndrome virus (PRRSV) in infected and vaccinated pigs. This suggests that PRRSV might possess some inherent properties to evade host defense mechanisms during the early stage of infection. Dendritic cells (DCs) play a crucial role in the activation and control of T-cells in response to viral antigens. In this study, we investigated the phenotypic and functional property changes of bone marrow-derived immature DCs (BM-imDCs) that take place after infection by PRRSV. Results showed that BM-imDCs were permissive to PRRSV infection, as productive replication took place in these cells. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 is observed at 48 h following infection. Also at 48 h following PRRSV infection, a significant increase of IL-10 secretion by BM-imDCs was noticed. Results suggest that the inhibited expression of MHC I and the enhanced secretion of IL-10 by BM-imDCs after PRRSV infection might be among the strategies used by the virus to evade the host immune defenses. 相似文献
13.
Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are pathogens, which can significantly affect the swine industry worldwide. Field surveys suggest that simultaneous PRRSV and PCV2 infection is common in pigs. The objective of this study was to measure the changes in peripheral blood leukocyte subpopulations in piglets co-infected experimentally with PRRSV and PCV2, in order to analyze the synergistic influence of co-infection on the immune system. Changes in peripheral blood leukocyte subpopulations were systematically measured by flow cytometry (FCM). The levels of antibodies to PRRSV and PCV2 were detected by indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) and the indirect fluorescent antibody test (IFA), respectively. Serum viral loads were measured using real-time PCR. The results showed that piglets co-infected with PRRSV and PCV2 exhibited slower generation and lower levels of antibodies to PRRSV and PCV2, and increased amounts and a prolonged presence of both PRRSV and PCV2 in serum, in comparison to the piglets infected with either virus alone. The major finding in our study was that the total and differential leukocyte counts, including white blood cells (WBCs), monocytes, granulocytes and lymphocytes (T, B and NK cells, as well as T-cell subpopulations), dramatically decreased early during co-infection with PRRSV and PCV2 for about two weeks, in contrast with animals singly infected with either PRRSV or PCV2. These results suggest that PRRSV and PCV2 co-infection results in a synergistic decrease in immune cells in the peripheral blood of piglets. These data contribute to the understanding of the immunosuppressive effects resulting from PRRSV and PCV2 co-infection in pigs. 相似文献
14.
Chang HW Jeng CR Liu JJ Lin TL Chang CC Chia MY Tsai YC Pang VF 《Veterinary microbiology》2005,108(3-4):167-177
Two common viral pathogens of swine, namely, porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), were investigated in regard to their effects on monolayer cultures of swine alveolar macrophages (AMs). The purpose was to identify selected cellular changes and responses potentially associated with the clinical reactions of pigs infected with either or both of these viruses. Measurements included the (1) absolute and relative numbers of infected, viable, and apoptotic cells; (2) distribution of viral antigens; (3) levels of interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) produced and their association with the extent of virus-induced cytopathology. Four groups of AMs were studied, including mock-infected, PCV2 alone-infected (PCV2-A), PRRSV alone-infected (PRRSV-A), and PCV2 and PRRSV dually infected (PCV2/PRRSV) groups. The AMs of PCV2-A group had high antigen-containing rate without cell death. There was a marked increase in cell death and apoptosis in PRRSV-A group. However, a lower PRRSV-induced infectious rate, cell death, and apoptosis were seen in PCV2/PRRSV group. High levels of IFN-alpha production were detected in PCV2-infected groups, but not in mock-infected and PRRSV-A groups. The PRRSV-induced cytopathic effect (CPE) on MARC-145 cells or swine AMs was markedly reduced by pre-incubation of the cells with UV-treated or non-UV-treated supernatants of PCV2-infected AMs. In addition, the reduction in CPE was abolished when the supernatants of PCV2-infected AMs were pre-treated with a mouse anti-recombinant porcine IFN-alpha antibody. The results suggest that swine AMs were an important reservoir of PCV2; PCV2 infection reduced PRRSV infection and PRRSV-associated CPE in PCV2/PRRSV AMs; the reduction of PRRSV infection in AMs was mediated by IFN-alpha generated by PCV2 infection. The reduced PRRSV-associated CPE in AMs and increased pro-inflammatory cytokine production may lead to a more severe pneumonic lesion in those dually infected pigs. 相似文献
15.
Marek Sinkora John E Butler Kelly M Lager Hana Potockova Jana Sinkorova 《Veterinary research》2014,45(1)
Lymphocyte subsets isolated from germ-free piglets experimentally infected with swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were studied and the profile of these subsets among these three infections was monitored. Germ-free piglets were used since their response could be directly correlated to the viral infection. Because SIV infections are resolved even by colostrum-deprived neonates whereas PRRSV and PCV2 infections are not, SIV was used as a benchmark for an effectively resolved viral infection. PRRSV caused a large increase in the proportion of lymphocytes at the site of infection and rapid differentiation of B cells leading to a high level of Ig-producing cells but a severe reduction in CD2—CD21+ primed B cells. Unlike SIV and PCV2, PRRSV also caused an increase in terminally differentiated subset of CD2+CD8α+ γδ cells and polyclonal expansion of major Vβ families suggesting that non-specific helper T cells drive swift B cell activation. Distinct from infections with SIV and PRRSV, PCV2 infection led to the: (a) prevalence of MHC-II+ T cytotoxic cells, (b) restriction of the T helper compartment in the respiratory tract, (c) generation of a high proportion of FoxP3+ T cells in the blood and (d) selective expansion of IgA and IgE suggesting this virus elicits a mucosal immune response. Our findings suggest that PRRSV and PCV2 may negatively modulate the host immune system by different mechanisms which may explain their persistence.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-014-0091-x) contains supplementary material, which is available to authorized users. 相似文献16.
Maldonado J Segalés J Martínez-Puig D Calsamiglia M Riera P Domingo M Artigas C 《Veterinary journal (London, England : 1997)》2005,169(3):23-456
The objective of the present study was to determine the presence of recognised abortifacient viruses such as porcine reproductive and respiratory virus (PRRSV), Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2), in tissues from aborted fetuses and stillborn neonates in cases of late reproductive failure in swine. A total of 293 specimens (fetuses aborted in the last third of gestation and stillborn piglets) from 100 different cases of late-term abortions and premature farrowing from 15 different Spanish provinces were studied. PRRSV was detected in 9/100 cases by RT-PCR. Only 1/100 cases analysed (corresponding to a late-term aborted fetus with a negative PRRSV RT-PCR result) was positive for PCV2 by PCR. Neither ADV (monitored by viral isolation plus antigen detection) nor PPV (monitored by ELISA antigen capture test) infection was identified. The results suggest that PRRSV is one of the most important infectious agents, if not the most relevant one, associated with fetal infection leading to abortion or premature farrowing in Spain. Moreover, other viral pathogens such as ADV, PPV and PCV2 seem to have a minor impact on reproductive disease. 相似文献
17.
Inhibition of porcine reproductive and respiratory syndrome virus replication by short hairpin RNA in MARC-145 cells 总被引：3，自引：0，他引：3
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important contagious agents of swine in the world. The current vaccines cannot provide highly effective protection. In this study, the ability of specific short hairpin RNA directed against different genomic regions of PRRSV to inhibit virus replication in MARC-145 cells was examined. Seven plasmids expressing shRNA targeted to GP5 and nucleocapsid (N) protein coding region of PRRSV S1 strain RNA were constructed and delivered into MARC-145 cells. After infection, these cells, transfected with plasmids pSUPER-N3 or pSUPER-G1, showed a significant decrease in virus yield when compared to control cells, by detection using virus titers (TCID50), indirect immunofluorescence assay and real-time RT-PCR. The antiviral effect was sequence-specific and dose-dependent and could sustain for 96 h. Furthermore, by combination of treatment with plasmid pSUPER-N3 and pSUPER-G1, the viral inhibition cloud be significantly increased. In addition, the viral suppression efficiency by shRNA in previously infected cells was not significant different from that induced by shRNA before viral infection. It indicated that administration of the two different shRNA could have a synergistic effect. RNA interference targeting to the various regions of PRRSV might be a potential alternative virus control strategy. 相似文献
18.
猪繁殖与呼吸综合征病毒实验感染SPF猪外周血单核细胞和肺泡巨噬细胞早期凋亡细胞的观察 总被引：7，自引：0，他引：7
采用Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ／ＰＩ双染色法,用流式细胞仪检测了猪繁殖与呼吸综合征病毒（ＰＲＲＳＶ）实验感染ＳＰＦ猪不同时期外周血单核细胞和肺泡巨噬细胞感染Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ＾＋／ＰＩ＾－细胞群（早期凋亡细胞群）。结果显示,ＰＲＲＳＶ感染猪外周血单核细胞和肺泡巨噬细胞Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ＾＋／ＰＩ＾－细胞群的表达率均明显高于正常对照猪,感染后２４ｈ表达率达最高值。 相似文献
19.
Neumann EJ Grinberg A Bonistalli KN Mack HJ Lehrbach PR Gibson N 《Veterinary microbiology》2009,138(3-4):297-303
The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus. Virions of PRRSV contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2, GP3, GP4, and E. The GP5 is the major envelope proteins, which was involved in the formation and infectivity of PRRSV by coaction with other membrane proteins. Here, to determine the function of alone GP5 envelope protein in viral entry, we investigated the formation and infectivity of GP5-pseudotyped virus particles. By co-transfection of GP5 expression plasmids with murine leukemia virus (MuLV) based retroviral vectors (pHIT60, encoding MuLV Gag-Pol; pHIT111, encoding an MuLV genome with a β-galactosidase reporter gene) into 293 T cells and analysis of the culture medium using ultracentrifugation, Western blot, and infection assay. We observed that the GP5 envelope protein was incorporated into the MuLV retroviral vectors to generate an pseudotyped murine leukemia virus, which was infectious to PAM and Mack-145 target cells and displayed the same host range with wild-type PRRSV. The infection of the pseudotyped virus on PAM target cells is effectively neutralized by polyclonal antibodies specific for PRRSV or GP5. The results suggested that the GP5 protein may play a key role in the viral entry by interacting with the host cell receptor. The GP5-pseudotyped virus will be useful in the identification of the cellular receptor binding with GP5 protein. 相似文献
20.
Nielsen J Bøtner A Tingstedt JE Aasted B Johnsen CK Riber U Lind P 《Veterinary immunology and immunopathology》2003,93(3-4):135-151
It is well known that piglets congenitally infected with porcine reproductive and respiratory syndrome virus (PRRSV) can be viremic at birth, and that preweaning mortality due to secondary infections often increases during acute outbreaks of PRRS. Therefore, an immunosuppressive effect of in utero infection has been suggested. The aim of the present study was to characterise the changes of leukocyte populations in piglets surviving in utero infection with PRRSV. A total of 27 liveborn uninfected control piglets and 22 piglets infected transplacentally with a Danish strain of PRRSV were included. At 2 and 4 weeks of age, 21 of 22 (96%) and 7 of 14 (50%) examined infected piglets were still viremic, whereas PRRSV could not be detected in the six infected piglets examined at 6 weeks of age. Flow cytometry analysis was used to determine the phenotypic composition of leukocytes in peripheral blood and bronchoalveolar lavage fluid (BALF) of 2-, 4- and 6-week-old infected piglets and age-matched uninfected controls. The key observation in the present study is that high levels of CD8(+) cells constitute a dominant feature in peripheral blood and BALF of piglets surviving in utero infection with PRRSV. In BALF, the average high level of CD8(+) cells in 2-week-old infected piglets (33.4 +/- 12.6%) was followed by a decline to 7.3 +/- 3.0 and 11.1 +/- 3.0% at 4 and 6 weeks of age. BALF of control piglets contained 1.6 +/- 0.9, 2.3 +/- 1.8 and 1.9 +/- 0.5% CD8(+) cells, only. In peripheral blood, however, the average number of CD8(+) cells remained at high levels in the infected piglets throughout the post-natal experimental period (2.8 +/- 1.9, 2.9 +/- 1.8 and 3.2 +/- 1.7 x 10(6) CD8(+) cells/ml at 2, 4 and 6 weeks, respectively). In the controls, the average levels of CD8(+) cells were 0.9+/-0.2, 1.9 +/- 1.7 and 1.6 +/- 0.5 x 10(6)/ml, respectively. Furthermore, the numbers of CD2(+) , CD4(+)CD8(+) and SLA-classII(+) cells, respectively, in peripheral blood, together with the levels of CD2(+) and CD3(+) cells in BALF were increased in the infected piglets infected in utero compared to the uninfected controls.The kinetic analyses carried out in the present study reflect that in utero infection with PRRSV modulates immune cell populations in peripheral blood and BALF of surviving piglets. The observed changes are characterised by high levels of CD8(+) cells supporting an important role of these cells in PRRSV infection. The present results, however, do not support the existence of post-natal immunosuppression following in utero infection with PRRSV. 相似文献