IFITM3对小反刍兽疫病毒在山羊子宫内膜上皮细胞中增殖的调控效应

干扰素诱导跨膜蛋白3(interferon-induced transmembrane protein 3,IFITM3)在调控病毒感染过程中发挥重要作用,而其在小反刍兽疫病毒(peste des petits ruminants virus, PPRV)感染中是否发挥作用尚不明确。本研究旨在探究IFITM3对PPRV感染的影响。通过Western blot技术检测PPRV感染对山羊子宫内膜上皮细胞(endometrial epithelial cells, EECs)中IFITM3表达的影响,进一步通过基因过表达及敲降技术探究IFITM3对PPRV复制的影响。结果表明,PPRV感染山羊EECs后,PPRV N蛋白表达水平持续升高,而IFITM3的表达水平较未感染对照组呈先上升后下降趋势。在感染后2 h, IFITM3的表达极显著上调(P<0.001);感染后3～4 h, IFITM3的表达水平最大(P<0.000 1);感染后24 h, IFITM3表达水平下降且与对照组差异不显著。抑制IFITM3表达与对照组相比,PPRV感染后0～6 h,细胞内PPRV的N蛋白水平下降...     

羊口疮病毒在羔羊4种原代细胞中的增殖特性研究

为了解羊口疮病毒(ORFV)感染羔羊不同组织细胞的偏好性,分别体外培养肾外膜上皮细胞、口唇表皮细胞、肺上皮细胞和睾丸原代细胞,接种ORFV FX株病毒液,记录接种12、24、36、72 h时细胞病变特征,并根据Reed-Muench方法计算TCID50.结果显示,4种细胞均在不同时间点出现细胞变圆、出现空泡和细胞核凝集...     

干扰素刺激基因对羊口疮病毒在OFTu细胞中增殖的抑制效应

羊口疮病毒（ORFV）是重要的人兽共患病病原,不仅严重危害养羊业,而且威胁人类健康。干扰素刺激基因（stimulator of interferon genes,STING）作为细胞的DNA感受器,在机体天然免疫中起重要作用。为探索STING在ORFV感染中的作用及其对病毒复制的影响,本研究构建了ORFV感染羊胚胎鼻甲细胞（OFTu）的模型,分析了ORFV感染细胞后对STING及其相关基因的动态表达,探索了STING基因在干扰表达和过表达状态下对ORFV在细胞上增殖的影响。结果表明,ORFV感染OFTu细胞后,STING、cGAS、TBK1、IRF3、IRF7、IL-6、IFN-β、IL-1β和TNF-α的转录明显升高。OFTu细胞过表达STING可导致RIG-1、DDX41、IFI16、IRF3、IRF7、IL-6、TNF-α、IFN-α和IFN-β等基因转录上调。OFTu细胞在STING过表达状态下感染ORFV可介导TBK-1、IRF3、IFN-β和TNF-α的转录升高,抑制ORFV的复制;在STING表达干扰的状态下,ORFV感染OFTu细胞降低了TBK-1、IRF3、IFN-β和TNF-α的转录,增加了ORFV的复制。这表明STING蛋白能够增强抗病毒细胞因子的表达,抑制ORFV在OFTu细胞中的增殖,研究结果为深入理解STING在羊口疮病毒感染和复制中的作用提供了科学的理论依据,也为深入探索ORFV感染和致病的分子机制提供了基础数据。     

羊口疮病毒的研究进展

羊口疮(orf)病毒又称传染性脓疮(ecthyma contagiosum)病毒,属于痘病毒科副痘病毒属,能引起反刍动物和人发病,在我国西部地区流行比较广泛,是家畜疫病防控中重要的一种病原。     

羊口疮病毒威海株的分离鉴定

利用犊牛睾丸原代细胞对山东省威海市某奶山羊养殖场疑似羊口疮（Orf）山羊病料进行病毒分离,通过细胞病变观察、病毒滴度和聚合酶链反应（PCR）等方法对分离毒株进行鉴定。结果表明,研磨处理的病毒液接种于犊牛睾丸原代细胞后盲传至第5代出现病变,测得第6代分离株病毒滴度为106．5 TCID50／mL。扩增羊口疮病毒特异性 B2L 基因,发现该毒株与34个羊口疮毒株核苷酸序列同源性高达99％,包括福建株（KC588399．1、KC568398．1）。本研究成功分离鉴定了羊口疮病毒山东威海株。     

干扰素诱导跨膜蛋白IFITM3在黄羽肉鸡消化系统的分布

利用免疫组织化学技术探讨干扰素诱导跨膜蛋白IFITM3在鸡消化器官中的分布,同时运用RT-PCR和荧光定量PCR技术分析进一步分析肉鸡消化系统中各器官IFITM3蛋白的mRNA表达规律。免疫组化表明,IFITM3在鸡消化器官中均有分布,其中十二指肠IFITM3阳性细胞分布最多;RT-PCR和荧光定量PCR的结果与免疫组化的结果一致。本试验初步探明了IFITM3蛋白在黄羽肉鸡消化器官中的分布规律。     

羊口疮病毒内蒙古株的分离与初步鉴定

[目的] 确定内蒙古地区规模化养殖场发生的疑似羊口疮的病原。[方法] 无菌采集疑似羊口疮病料7份,经剪碎、研磨、离心等处理后接种山羊皮肤成纤维细胞（goat skin fibroblasts,GSFs）培养;对分离到的病毒毒株进行电镜观察;利用B2L基因引物进行特异性PCR鉴定,对获得的B2L基因序列测序,构建系统发育树,并进行同源性分析。[结果] 3份病料经GSFs培养48 h后出现明显病变,分离的病毒经负染后在电镜下观察呈卵圆形,病毒粒子长220~250 nm,宽125~200 nm,符合羊口疮病毒（orf virus,ORFV）粒子的形态特征,并将其命名为NM-ORFV-1株、NM-ORFV-2株、NM-ORFV-3株。分离株经B2L基因PCR鉴定获得1 137 bp的扩增产物,与预期一致;系统发育树及同源性分析显示,NM-ORFV-2株与NM-ORFV-3株遗传关系密切,处于同一分支,且与ORFV KP336704（中国）分离株的亲缘关系最近,同源性达到99.4%;NM-ORFV-1株与NM-ORFV-2株及NM-ORFV-3株处于不同分支,NM-ORFV-1株与NM-ORFV-2株的同源性为99.1%,与NM-ORFV-3株的同源性为99.0%,且与ORFV JQ904789（中国）疫苗株亲缘关系最近,同源性为99.6%。[结论] 内蒙古地区规模化养殖场疑似羊口疮病例的病原是ORFV。     

羊口疮病毒黑龙江省分离株的分离鉴定

为确定黑龙江大庆地区某羊场发生的疑似羊口疮(Orf)的病原,本研究采用MDBK细胞培养途径从病羊的口唇部位的痂皮病料中分离出1株病毒.通过电镜观察和IFA鉴定证明该分离株为Orf病毒,命名为OV/HLJ/04.其F1L基因核苷酸序列测定和进化分析显示,OV/HLJ/04与国内报道的山西株(HQ221964)的遗传关系密切,同源性达到98.2％.将该病毒分离株进行回归本动物试验,接种羔羊发生严重的羊口疮.     

Effects on ovine fetuses of exposure to ovine progressive pneumonia virus

Fetuses of 20 pregnant ewes at 4 gestational periods (45, 55, 85, and 100 days) were inoculated with ovine progressive pneumonia virus. Fourteen of 16 fetuses exposed to virus before gestational day 80 were either resorbed or expelled, whereas 10 of 15 fetuses exposed to virus after day 80 were normal at birth. Three of the 9 expelled fetuses and 1 of 2 newborn lambs had accumulations of lymphoid cells in the lungs. Virus was readily isolated from the tissues of expelled fetuses and newborn lambs. Lambs did not have precipitating antibody to the virus at birth, but 3 to 5 lambs had specific antiviral antibody at 18 months of age.     

Effects of EGF receptor ligands on fetal ovine myoblasts

Epidermal growth factor (EGF) receptors are widely distributed in mammalian tissues, including muscle. One ligand of these receptors, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is also strongly expressed in adult muscle. However, in vitro studies of EGF action in cultured muscle cells of different species have yielded conflicting results. The purpose of this study was to investigate the potential role of EGF and related factors in the growth and development of fetal ovine muscle. High affinity EGF receptors were detected on clonally purified ovine fetal myoblasts, using [(125)I] human EGF as a ligand (K(d) values of 47 and 54 pM in separate experiments). Competitive binding studies in mixed secondary cultures showed that EGF had the highest affinity for the fetal ovine receptor, followed by HB-EGF and transforming growth factor alpha (TGF-alpha). These ligands all stimulated DNA synthesis in clonally purified ovine myoblasts, with their relative potencies at 0.1 nM reflecting their receptor binding affinities. Maximal effects were seen at 1-10 nM. EGF (10 nM) did not significantly inhibit the differentiation of clonally purified fetal ovine myoblasts, although there was increased proliferation of nondifferentiating cells. Hence a variety of EGF receptor ligands have the potential to influence the proliferation ovine muscle cell precursors in utero, but it is unlikely that they promote differentiation.     

Maedi-visna virus infection of ovine mammary epithelial cells

The aim of this work was to perform a complete study of maedi-visna virus (MVV) infected mammary glands of naturally-infected sheep, and to determine if cells other than macrophages undergo a productive viral infection in this organ. Fifteen seropositive and two seronegative ewes were selected from MVV-infected flocks on the basis of clinical indurative mastitis and three sheep from an MVV-free flock. Within the mammary gland, MVV-positive cells were located by immunohistochemistry in the stroma and the epithelial alveolar barrier, most likely the ovine mammary epithelial cells (OMEC) of the acini. In situ hybridization confirmed these findings. Ultrastructural studies showed the presence of lentivirus-like particles budding off the cell surface in the alveolar barrier and also free in the acinar lumen. The presence of mammary histopathological lesions and MVV together with clear indications of productive infection (demonstration of a cytopathic effect in OMEC cultures and infection of co-cultures) were observed in the 15 seropositive and one of the seronegative sheep from the infected flock. These findings demonstrate that the OMEC were infected in vivo and probably underwent productive infection when studied ex-vivo. The OMEC of MVV-free sheep, which had subsequently been infected in vitro with MVV, also showed productive infection when challenged in vitro, confirming the replication of MVV in OMEC in vitro. The presence of MVV-infected OMEC in the mammary gland from infected animals, the productive infection in these OMEC and the release of lentiviral particles to the acinar lumen may have relevance in the pathogenesis and transmission of MVV infection.     

Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro

Myostatin is an important negative regulator of muscle growth and development. Natural mutations of the myostatin gene cause a double muscling phenotype in beef cattle, pigs and sheep. Therefore, it is feasible to produce a high growth domestic breed by generating a transgenic animal with a mutation, deletion or knockout of the myostatin gene. Our objective was to introduce a subtle mutation of G to A 281-bp upstream of the 3' untranslated region (3'UTR) end of the myostatin gene in Poll Dorset fetal myoblast cells in vitro. Fetal myoblast cells were isolated from fetuses at day 50 of gestation from Poll Dorset sheep and transfected with linear gene-targeting vector pMSTN-A using electroporation. We obtained seven gene-targeted cell colonies with homologous recombination, which were positive as confirmed by PCR, Southern blot. The Western blot analysis result demonstrated that the myostatin protein expression in positive colonies is lower than that of negative ones. These results strongly suggest that we successfully mutated the myostatin gene of Poll Dorset ovine fetal myoblast cells and the mutation can effectively downregulate the myostatin protein expression.     

Effects of incubation temperatures and bovine viral diarrhea virus immune serum on bovine turbinate cells persistently infected with a noncytopathogenic isolate of bovine viral diarrhea virus

Noncytopathogenic bovine viral diarrhea virus (BVDV) on initial inoculation in receptive cells induced a persistence in vitro at incubation temperatures of 33, 37, and 39 C. The mechanisms of this persistence were not the result of the induction of defective interfering particles or the selection of temperature-sensitive mutants, but were the result of a naturally occurring noncytopathogenic isolate of BVDV. Persistently infected cells were not freed of the infection by continuous passage in media containing homologous antibody. Persistently infected cells appeared to undergo an earlier senescence than did noninoculated cells of the same passage level and age. This phenomenon was reversed by renewal of the culture media.     

Effects of noncytopathic type 2 Bovine viral diarrhea virus on the proliferation of bone marrow progenitor cells

The purpose of this study was to investigate the effects of isolates of noncytopathic type 2 Bovine viral diarrhea virus (ncpBVDV-2) of high and low virulence on the proliferation of bone marrow progenitor cells. Holstein calves 6 to 7 mo old and BVDV-na?ve were inoculated intranasally with a BVDV isolate of high virulence (HV24515), a BVDV isolate of low virulence (LV11Q), or uninfected cell culture medium. Serial bone marrow and peripheral blood samples were collected before and after inoculation. Bone marrow mononuclear cells (BMMCs) were isolated and cultured for 5 d, and the mean number of colony-forming unit-granulocyte-macrophage (CFU-GM) colonies was determined. Tritiated (3H)-thymidine uptake by BMMCs was determined to indicate overall proliferative capacity. Virus isolation was done on concurrent samples of BMMCs and peripheral blood. Virus was isolated from BMMCs and peripheral blood buffy-coat cells as early as day 2 or 3 after inoculation. Neutropenia developed in both groups inoculated with a BVDV isolate. However, in the calves given LV11Q, neutrophil counts rebounded earlier in response to increased proliferation of BMMCs, whereas the response was delayed in calves given HV24515. Thymidine uptake was significantly increased (P = 0.0047) in BMMCs after inoculation compared with before inoculation in the calves given LV11Q but not in those given HV24515 or in the control calves. The median number of CFU-GM colonies was significantly decreased (P = 0.0164) after inoculation compared with before inoculation in the calves given HV24515, whereas there was no significant difference in the calves given LV11Q or in the control calves. The data support the hypothesis that the prolonged neutropenia observed in calves given HV24515 results at least in part from decreased proliferative capacity of bone marrow progenitor cells.     

钩藤散对衰老细胞增殖的影响

随着人们生活水平的不断提高,防衰老成为目前人类关注的焦点。钩藤散源于《本事方》,由钩藤、石膏、陈皮、麦冬、半夏、茯苓、人参、防风、生姜、甘草、菊花等11味药组成,为治疗肝厥头晕之证的经典方剂。笔者等以人胚肺二倍体成纤维细胞（2Bs）为模型,考察钩藤散对衰老细胞增殖的调控作用。     

Effects of intrauterine growth restriction during late pregnancy on the ovine fetal renal function and antioxidant capacity

This study investigated the effects of intrauterine growth restriction during late pregnancy on the ovine fetal renal function and renal antioxidant capacity. Eighteen ewes pregnant were randomly divided into control group (CG, ad libitum, 0.67 MJ ME·BW^−0.75·day⁻¹, n = 6), restricted group 1 (RG1, 0.18 MJ ME·BW^−0.75·day⁻¹, n = 6), and restricted group 2 (RG2, 0.33 MJ ME·BW^−0.75·day⁻¹, n = 6). At 140 days, the fetal blood, allantoic fluid and kidney tissue were collected to determinate fetal renal function and renal antioxidant capacity. The results showed that the fetal weight, kidney weight, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), aquaporin-2 (AQP-2) and aquaporin-3 (AQP-3), and total antioxidant capacity (T-AOC) in RG1 group were decreased compared with the CG (P < 0.05), but the contents of β2-Microglobulin (β 2-MG), cystatin C (Cys-C), filtered sodium excretion fraction (FENa), malondialdehyde (MDA), and hydroxyl radical (OH) in RG1 group were increased (P < 0.05). The impaired ovine fetal renal growth, antioxidant imbalance and dysfunction of glomerulus ultrafiltration, and the renal tubules reabsorption were induced by maternal malnutrition during late pregnancy.     

Immunomodulation by virulence proteins of the parapoxvirus orf virus

Three orf virus putative virulence proteins are described that exhibit immunomodulatory functions. The OVIFNR gene at the left terminus of the viral genome encodes an interferon resistance protein with homology to the E3L gene of vaccinia virus. OVIFNR functions by preventing a dsRNA-dependent kinase from inhibiting virus and cell protein synthesis as part of the interferon-induced anti-viral state within infected cells. The orf virus orthologue of the ovine interleukin-10 (vIL-10) gene is located at the right terminus of the viral genome. Both vIL-10 and host (ovine) IL-10 function in vitro as inhibitors of pro-inflammatory cytokine production by keratinocytes and macrophages, and both inhibit IFN-gamma production from activated peripheral blood lymphocytes. Both the orf virus vIL-10 and ovine IL-10 stimulate mast cell and thymocyte proliferation. In this respect the orf virus IL-10 differs from Epstein Barr virus IL-10 which does not exhibit cell proliferative activity. Finally, the orf virus GM-CSF inhibitory factor gene (GIF) at the right terminus of the viral genome encodes an inhibitor of GM-CSF that also binds IL-2. Together, these viral proteins are capable of inhibiting key components of the ovine anti-virus immune and inflammatory response.     

用犊牛睾丸细胞生产羊口疮疫苗时应注意的问题

在采用犊牛睾丸细胞生产羊口疮疫苗时，睾丸组织采集方法、存放温度、存放时间、制作中使用的营养液和血清、种毒标准、接收毒时间等因素均可影响羊口疮疫苗的产量及质量。