一株牛蛙源虹彩病毒的分离及鉴定

用鲤鱼表皮瘤细胞系(epithelioma papulosum cyprini cell line,EPC)从福建省某美洲牛蛙养殖场分离到一株病毒FJ049。感染病毒的EPC呈现细胞圆缩、颗粒增多、脱落等特征性病变。间接免疫荧光检测结果表明,感染FJ049的EPC细胞与虹彩病毒单克隆抗体反应并出现特异性的胞浆荧光。采用PCR对虹彩病毒主衣壳蛋白(major capsid protein,MCP)基因保守区域进行扩增,扩增出531 bp的特异性基因片段。MCP基因同源性和遗传进化分析结果表明分离株FJ049与沼泽绿牛蛙虹彩病毒RGV9808的核苷酸同源性最高,为99.8%,属于虹彩病毒科蛙病毒属。     

Expression of Survivin,Bcl‐2 and Bax Proteins in the Domestic Cat (Felis catus) Endometrium During the Oestrus Cycle

Apoptosis has been shown to be an important regulator of endometrium function. To clarify the regulation of apoptosis in the cat endometrium during the normal oestrus cycle, the expressions of the apoptosis‐related proteins (Bcl‐2 and Bax) and their correlation to the inhibitor of apoptosis protein Survivin were analysed using immunohistochemistry. The TUNEL technique (TdT‐mediated dUTP nick end labelling) was also used to detect DNA fragmentation characteristic of apoptotic cells. The results demonstrated that TUNEL labelling is not effective for the detection of apoptosis in cat endometrium. Survivin was expressed in the luminal and glandular epithelial cells of cat endometrium during all phases of the oestrus cycle. Survivin was localized in both the cytoplasm and nuclei of superficial and deep uterine gland cells during the luteal phase, while only cytoplasmic staining was observed during the follicular and anoestrus phases. Bax immunoreactivity in the cytoplasm of luminal and glandular epithelial cells as well as the smooth muscle cells of blood vessels was weak in the anoestrus phase. Compared with anoestrus, the intensity of Bax immunostaining was moderate in the follicular phase and increased dramatically in the luteal phase. Bcl‐2 immunostaining in the cytoplasm of luminal and glandular epithelial cells was moderate in the anoestrus phase. During the early follicular phase, cytoplasmic Bcl‐2 immunostaining was detected mostly in glandular epithelial cells. In the mid‐follicular phase, in glands, the amount of Bcl‐2 protein increased progressively from the superficial to the deep layer. In contrast, the expression of Bcl‐2 decreased in the secretory phase, being very low or absent in the mid‐ and late luteal phases. The overall results suggest that Survivin, Bax and Bcl‐2 proteins may cooperatively contribute to cell apoptosis and cell proliferation in the cat uterus during the oestrus cycle.     

犬呼肠病毒3型在BHK-21细胞上增殖的研究

采用酶标ＳＰＡ法进行了犬呼肠３ 型病毒在ＢＨＫ２１ 细胞上定位的研究。结果表明：接毒后６ ｈ 即可在胞浆见到絮状阳性反应,２４ ｈ 后呈强阳性着染;Ｈ．Ｅ．染色在胞浆内可见强嗜酸性着染或嗜酸性包涵体,提示可能是病毒感染引起的特征性病变,感染后细胞病变发展较快,６０ ｈ 细胞大部脱落;病毒血凝滴度结果也证实了细胞病变与病毒增殖有关。     

MDCK细胞感染犬传染性肝炎病毒后的超微结构观察

MDCK细胞感染犬传染性肝炎病毒(ICHV)BJ—1株后6h未见明显改变;12～18h胞核肿大,核内形成黑色颗粒样球状体;24h胞核内偶见病毒粒子,36h许多细胞内含病毒、病毒副晶体、形态各异的包涵体和多种板层样结构;48h感染细胞核膜以破损或出芽方式将成熟病毒粒子释入胞浆.     

Comparative Study of Proliferation and Apoptosis Characteristics of Different Virulence of Aleutian Mink Disease Virus in CRFK Cells

This study was aimed to compare the proliferation and apoptosis characteristics of different virulence of Aleutian mink disease virus(AMDV) in feline kidney cell (CPFK). CRFK cells were infected separately with the standard strain of AMDV-G and the wild strains of AMDV-DL124, AMDV-DL125, AMDV-QD2, AMDV-QD3 and AMDV-ZJ3. Indirect immunofluorescence,Real-time quantitative PCR and TCID₅₀ were used to detect the replication and expression of the viruses in the cells, at the same time the apoptosis induced by the viruses was detected. The results of IFA showed that CRFK cells which infected separately by wild strains appeared the fluorescence after 12 h of infection. With the extension of the infection time, the fluorescence of cells increased, but the fluorescence of AMDV-G was later than the wild strains, almost all of the cells were appeared the fluorescence after 72 h of infection. The Real-time quantitative PCR results showed that the tendency of genome replication of different virus were approximately the same. The genome replication began at 3 h after the AMDV-DL125 infection,the rapid increase of genome replication of AMDV-G occurred at 24 h after the infection, the genome replication of whole viruses reached a peak after 72 h of infection. The results of TCID₅₀ showed that the latent period of viruses infection were 0 to 12 h after infection, AMDV-G reached the peak after 60 h of infection. However, the wild strains at 48 to 72 h could maintain a higher infection titer and reached peak at 72 h after infection, then decreased with the cell disintegration. Apoptosis detection results which analysis by SPSS 23.0 statistical analysis software showed that, compared with control group,cell apoptosis was significantly higher after 2 to 12 h of cells infected by wild strains induced (P<0.05), cell apoptosis of AMDV-G was significantly lower than wild strains, however, the time of cell apoptosis induced by AMDV-G was longer,the apoptosis was still significant after 24 h of infection. But the apoptosis caused by virus infection was mostly concentrated in 2 to 12 h after infection. The results would provide some references for the study on the culture and identification and pathogenic mechanism of AMDV.     

不同毒力水貂阿留申病毒在猫肾细胞中的增殖特性及凋亡特性的比较研究

试验旨在对不同毒力水貂阿留申病毒（Aleutian mink disease virus,AMDV）在猫肾细胞（feline kidney cell,CRFK）中的增殖规律及其诱导细胞凋亡情况进行比较研究。将标准毒株AMDV-G及分离到的野毒株AMDV-DL124、AMDV-DL125、AMDV-QD2、AMDV-QD3、AMDV-ZJ3接种CRFK细胞,应用间接免疫荧光、实时荧光定量PCR、TCID₅₀测定技术研究病毒在细胞中的复制及表达情况,同时检测病毒诱导的细胞凋亡情况。间接免疫荧光结果显示,5株野毒株荧光着色趋势差异不大,均在感染后12 h出现荧光,随感染时间延长荧光增多,AMDV-G荧光出现时间比野毒株晚,但病毒感染后72 h几乎所有细胞均出现荧光;实时荧光定量PCR结果显示,基因组复制趋势大致相同,AMDV-DL125感染后3 h复制开始,AMDV-G感染后24 h复制才开始并呈快速增长趋势,但感染后72 h均达到峰值。TCID₅₀检测结果表明,0~12 h为病毒感染潜伏期,AMDV-G感染后60 h达到峰值,野毒株均在感染后72 h达到峰值,但是6株病毒均能在感染后48~72 h维持较高的感染滴度,其后随细胞崩解而降低。SPSS 23.0统计软件分析凋亡检测结果显示,与对照组相比,野毒株感染细胞后2~12 h诱导细胞凋亡差异显著（P<0.05）,AMDV-G诱导细胞凋亡差异明显低于野毒株,但是诱导细胞凋亡时间较野毒株长,在感染后24 h仍对细胞凋亡有较明显的诱导作用,但是各病毒诱导的细胞凋亡主要集中在2~12 h。该结果为AMDV的培养、鉴定及致病机理研究提供一定参考。     

Selective apoptotic behaviour of bovine herpesvirus 1 in an epithelial-like microenvironment

Apoptosis seems to play an important role in the pathogenic profile of bovine herpesvirus 1 (BHV-1) infection. Nitric oxide (NO) is also important as a signal molecule. In this study, apoptosis was selectively induced in HEp-2 cells in the early stage [1-3 h postinfection (PI)] of BHV-1 multiplication, and this apoptotic process was realised through the caspase-8, and partially through the caspase-3, pathway. BHV-1 infection inhibited staurosporine- (SS-) induced apoptosis only if the SS was added at 6 h PI. The results of this study showed that the 'NO-apoptosis' relation was realised through the caspase-8 pathway ('outer membrane receptor' pathway) at a later stage of infection in apoptosis induced by BHV-1 + SS. Our previous report (Yazici et al., 2004) and this study together showed that BHV-1 might induce and inhibit cell-type-specific pathways of apoptosis.     

Study on Proliferation and Apoptosis of EBL Cells Induced by P48 Protein of Mycoplasma bovis

The aim of this study was to investigate the effect of P48 protein on proliferation and apoptosis of embryonic bovine lung (EBL) cells.In this study,samples which co-incubated with P48 protein and EBL cells in different concentrations at different time points were collected,the proliferation rate of the cells was detected by MTT method,and the changes in the nuclear morphology of EBL cells were observed by DAPI staining method.Meanwhile,flow cytometry was used to detect the apoptosis rate of EBL cells induced by P48 protein.Real-time quantitative PCR was used to detect the changes in mRNA level of apoptotic marker genes,and Western blotting tested the Bax and Beclin-1 protein expressions.The results showed that under the condition of 72 h and 10 μg/mL of protein concentration treatment,P48 extremely significantly inhibited EBL cells proliferation (P<0.01),while 0.1 and 0.5 μg/mL protein concentration had no inhibitory effect (P>0.05).The nuclear morphology showed no significant change after protein induction for 12 h,but wrinkled and condensed at 24 h.The nucleus was fragmented,and a sprouted apoptotic body was appeared at 48 and 72 h.Apoptosis related genes expression showed no obvious increase at 2 and 12 h at mRNA level,but gradually increased at 24,48 and 72 h,and it showed a time-dependent manner.Accordingly,the expression of apoptosis marker proteins Bax and Beclin-1 significantly increased.Flow cytometry analysis showed that the apoptosis rate of EBL cells induced by P48 protein was 48.44%.In conclusion,P48 recombinant protein of Mycoplasmas bovis inhibited the proliferation of EBL cells and promoted their apoptosis,which provided reference for revealing the pathogenic mechanism of Mycoplasmas bovis.     

牛支原体P48蛋白诱导EBL细胞增殖和凋亡的研究

试验旨在研究牛支原体P48蛋白对胎牛肺（embryonic bovine lung,EBL）细胞增殖和凋亡的影响。收集不同时间段、不同浓度P48蛋白与EBL细胞共孵育的样品,通过MTT法检测细胞增殖率;DAPI染核法观察EBL细胞核形态变化;流式细胞技术检测该蛋白诱导EBL细胞的凋亡率;实时荧光定量PCR检测凋亡标志物的mRNA相对表达变化;Western blotting方法检测Bax和Beclin-1蛋白表达水平。结果显示：当作用时间为72 h,P48蛋白浓度在10 μg/mL时,对EBL细胞增殖有极显著的抑制作用（P<0.01）,在0.1和0.5 μg/mL时对EBL细胞的增殖的抑制作用不显著（P>0.05）;经蛋白诱导12 h细胞核形态未有明显变化,24 h细胞核形态发生皱缩和凝聚,48和72 h细胞核发生碎裂;凋亡标志基因mRNA表达在2和12 h没有明显提高,在24、48、72 h有显著提高,与作用时间呈正相关,同时凋亡相关蛋白Bax和Beclin-1的表达随之显著提高。流式细胞技术结果显示P48蛋白诱导EBL细胞凋亡率为48.44%。综上表明,牛支原体P48重组蛋白能够抑制EBL细胞的增殖,促进细胞凋亡,为进一步揭示牛支原体的致病机制提供参考依据。     

Invasion and Replication of Photobacterium damselae subsp. piscicida in Fish Cell Lines

Abstract

The objective of this study was to evaluate the ability of Photobacterium damselae subsp. piscicida to invade and replicate within different fish cell lines. Channel catfish ovary (CCO), fathead minnow (FHM), and epithelioma papillosum cyprini (EPC) cell lines were all susceptible to invasion and supported replication of P. damselae in an in vitro invasion assay in which extracellular growth was controlled with gentamicin. The number of bacteria recovered from EPC and CCO cells increased significantly after 6, 12, and 18 h, indicating that P. damselae replicated within those cells. There was also a significant increase in EPC cells at 3 h. Although the number of bacteria recovered from FHM cells increased slightly after 3 and 6 h, it declined at 12 and 18 h. The decline, however, was due to the early release of bacteria from FHM cells associated with a greater susceptibility to initial infection, which resulted in early cell lysis and subsequent exposure to gentamicin in the media. Using light and electron microscopy, we observed the invasion of bacteria as early as 30 min after infection. Intracellular bacteria were initially contained in small, close-fitting vacuoles that developed into large, clear, spacious vacuoles over time. There was no evidence of bacteria free in the cytoplasm. The intracellular location of P. damselae was confirmed by means of transmission electron microscopy with ruthenium red staining to discriminate between the extra- and intracellular spaces. This is the first report that provides evidence of intracellular replication of P. damselae in cell lines.     

传染性法氏囊病病毒变异E株感染鸡细胞凋亡的研究

研究了传染性法氏囊病病毒（IBDV）变异E株人工感染28日龄SPF雏鸡后鸡法氏囊淋巴细胞的凋亡情况。电镜观察和DNA电泳分析结果表明,IBDV感染后12～48h,雏鸡法氏囊淋巴细胞出现典型细胞凋亡的形态学特征和生化特征;经流式细胞计检测和荧光染色观察,统计学分析表明,IBDV感染后24～48h,雏鸡法氏囊淋巴细胞凋亡数量显著增加（P<0.05或P<0.01）。试验结果揭示IBDV变异E株人工感染可以诱导雏鸡法氏囊淋巴细胞凋亡。     

Features of cell degeneration and death in hepatic failure and systemic lymphoid depletion characteristic of porcine circovirus-2-associated postweaning multisystemic wasting disease

Tissue section replicates from lymphoid tissues and livers of gnotobiotic swine were examined by immunohistochemistry for the colocalization of porcine circovirus-2 (PCV-2) nucleocapsid and terminal deoxynucleotidyl transferase (TdT)-mediated incorporation of biotinylated nucleotides (UTP) onto the 3'-exposed hydroxyl groups (nick end labeling) nuclear deoxyribonucleic acid (TUNEL), a marker for apoptosis. Single- and dually stained replicates from uninfected controls, subclinically affected PCV-2-infected gnotobiotic pigs, PCV-2-infected piglets immunosuppressed with cyclosporine (Cys), and PCV-2-infected piglets with post-weaning multisystemic wasting syndrome (PMWS) were evaluated. Thymuses were used as positive controls for apoptosis absent PCV-2, tissue sections from dogs given hyperthermic stress were examined as positive controls for induced TUNEL. Tissues from heat-stressed dogs contained TUNEL-positive cell nuclei in both lymphoid tissues and liver, TUNEL was greatest shortly after the delivery of the hyperthermic insult. In uninfected control and subclinically affected PCV-2-infected gnotobiotic pigs, rare hepatocytes and lymphoid cells were TUNEL positive, the frequency of these was similar to that seen in uninfected controls. In PMWS-affected and Cys-treated PCV-2 piglets, the only consistent strongly positive TUNEL signal was contained within the cytoplasm of virus-positive phagocytic mononuclear cells. In phagocytes, some PCV-2 inclusions were TUNEL positive. Collectively, these data indicate that apoptosis is not the primary mechanism of lymphoid depletion and hepatocyte loss in PMWS. Apoptosis associated with systemic viral diseases may be attributable to pyrexia rather than direct or indirect effects of viruses on target cells.     

Antiproliferative effects and apoptosis induction by probiotic cytoplasmic extracts in fish cell lines

Probiotic bacteria are known to exert a wide range of beneficial effects on their animal hosts. Control of intestinal homeostasis, inflammation suppression and a reduction in the incidence of cancer all rely on the antiproliferative potential of probiotics. In this paper, we assess the antiproliferative activity of probiotics in two teleost fish cell lines SAF-1, a fibroblast cell line and EPC, an epithelioma from carp. Cells were grown in the presence of cytoplasmic extracts obtained from two bacterial strains, Lactobacillus delbrüeckii subsp. lactis (LDL) and 51M6. Proliferation and apoptosis were measured after 4, 24, 48 or 72h in culture by the crystal violet or by double staining flow cytometry assays, respectively. Generally, LDL had stronger effects on cell growth than 51M6. Moreover, SAF-1 cells were more susceptible to growth inhibition than EPC cells. Apoptosis took place following growth inhibition, especially when LDL extracts were used. The results are discussed in terms of the biological significance of probiotic bacteria that naturally occur on the fish mucosal surfaces with an emphasis on how dose and species specificity may be determinant factors.     

猪圆环病毒Ⅱ型Rep基因在PK15细胞中的表达及特性

为研究猪圆环病毒Ⅱ型(PCV2)Rep基因在PK15细胞中的表达特性，通过PCR方法克隆了PCV2杭州株(HZ0201)Rep基因全长945bp片段，与真核表达栽体pCI—neo构建为重组质粒pCI-PCV2-Rep。pCI-PCV2-Rep质粒转染PK15细胞后48h，通过RT-PCR可检测到PCV2 Rep mRNA的转录；用猪PCV2多抗血清作间接免疫荧光试验，可检测到Rep基因表达产物。在表达量低的细胞中，PCV2 Rep蛋白主要位于PK15的细胞浆，在表达量高的细胞中，细胞浆和细胞核中均含有大量的Rep蛋白，表明Rep对PK15细胞的细胞浆和细胞核的亲嗜性没有明显差别。     

副猪格拉瑟菌5型细胞致死性膨胀毒素CdtB破坏猪呼吸道上皮屏障的机制

旨在探究副猪格拉瑟菌(Glaesserella parasuis,GPS)突破猪呼吸道上皮屏障引起系统性感染的机制。通过超速离心和密度梯度离心提取副猪格拉瑟菌外膜囊泡(outer membrane vesicles, OMVs),OMVs经SDS-PAGE显示，蛋白质条带分布在55～100 ku,经透射电子显微镜(TEM)观察，OMVs的粒径在100～200 nm,纳米粒子直径分析(NTA)结果显示，样品在100～200 nm处的粒子数目最多。进而用制备的HbpA及OmpP2多克隆抗体对OMVs及不含OMVs的细菌上清进行Western blot验证，证实所提取的样品为外膜囊泡，且进一步结果证明细胞致死性膨胀毒素(CDT)在GPS培养物中主要以OMVs的形式存在。用OMVs或CdtB处理猪气管上皮细胞(swine tracheal epithelial cells, STEC)36 h,检测STEC中cleaved-caspase3、ZO-1和Occludin的蛋白表达水平，并用FITC-葡聚糖(FD-4)检测STEC单层细胞的细胞旁通透性。结果发现，OMVs与CdtB处理后凋亡相关蛋...     

水貂阿留申病毒诱导猫肾细胞(CrFK)凋亡

为研究水貂阿留申病毒(AMDV)在体外诱导猫肾细胞(CrFK)凋亡情况,用AMDV-G株感染CrFK细胞,采用CCK-8法检测CrFK细胞感染后的细胞存活率,用AO/EB染色法检测CrFK细胞核的形态变化,用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率,并通过分光光度计法检测细胞凋亡执行分子Caspase-3活性。结果显示,AMDV-G株感染可导致CrFK细胞增殖率下降,产生明显的形态学凋亡特征;流式细胞术检测结果显示,AMDV-G株感染可引起CrFK细胞凋亡并随着感染时间的延长凋亡率增加,同时Caspase-3活性显著升高。上述结果表明,AMDV-G株在体外能够诱导CrFK细胞凋亡,为进一步研究AMDV致病机理奠定基础。     

PRV感染三叉神经节细胞对PI3K/Akt信号通路的影响及其调控凋亡

以小鼠三叉神经节(TG)原代细胞为基础,应用实时荧光定量PCR(q-PCR)、Western blot等方法检测伪狂犬病病毒(pseudorabies virus,PRV)(MOI=1)感染TG细胞后不同时间点PI3K、Akt基因的转录水平和蛋白表达情况.PRV感染TG细胞后,利用PI3K特异性抑制剂LY294002处...     

Influence of environmental temperature on experimental infection of redfin perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss) with epizootic haematopoietic necrosis virus, an Australian iridovirus

SUMMARY Experimental transmission of epizootic haematopoietic necrosis virus (EHNV) to adult redfin perch Perca fluviatilis and juvenile rainbow trout Oncorhynchus mykiss was undertaken at different water temperatures using intraperitoneal (IP) and bath inoculation. Redfin perch were highly susceptible to EHNV by both routes of infection. Bath inoculation with as few as 0.08 TCID₅₀. _mL^-1 was lethal. The incubation period in redfin perch was about 11 days at a water temperature of 19–21°C but was longer at colder temperatures and disease did not occur at temperatures below 12°C. The longest incubation period recorded in redfin perch was 28 days. Rainbow trout were not susceptible to infection by bath inoculation but the disease was reproduced after IP inoculation with 10^5.6 TCID₅₀ at water temperatures ranging from 8–21°C. The incubation period was 3–10 days at 19–21°C, but was up to 32 days at 8–10°C. Persistent infection with EHNV was detected by virus isolation in a clinically unaffected rainbow trout after 63 days. The implications of these findings in the understanding of the epidemiology of EHNV infection are discussed.     

Pathogenesis of chicken-passaged Newcastle disease viruses isolated from chickens and wild and exotic birds

The pathogenesis of six Newcastle disease virus (NDV) isolates recovered from chickens (Ckn-LBM and Ckn-Australia) and wild (Anhinga) and exotic (YN parrot, pheasant, and dove) birds was examined after the isolates had been passaged four times in domestic chickens. Groups of 10 4-wk-old specific-pathogen-free white leghorn chickens were inoculated intraconjunctivally with each one of the isolates. The infected birds were observed for clinical disease and were euthanatized and sampled at selected times from 12 hr to 14 days postinoculation or at death. Tissues were examined by histopathology, by immunohistochemistry (IHC) to detect viral nucleoprotein (IHC/NP), and by in situ hybridization to detect viral mRNA and were double labeled for apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ([TUNEL] or IHC/caspase-3) and viral nucleoprorein (IHC/NP). Birds infected with the three low virulence viruses (Ckn-LBM, YN parrot, and Ckn-Australia) did not develop clinical disease. Microscopic lesions were observed only at the inoculation site and in organs of the respiratory system. The detection of viral nucleoprotein (N) was restricted to the inoculation site. The pheasant and dove isolates were highly virulent for chickens with marked tropism for lymphoid tissues, confirmed by the presence of large numbers of cells positive for viral N protein and viral mRNA. Viral N protein was detected early in the cytoplasm of cells in the center of the splenic ellipsoids. The apoptosis assays (TUNEL and IHC/caspase-3) showed increased apoptosis in the splenic ellipsoids as well. Apparently, apoptosis is an important mechanism in lymphoid depletion during NDV infection.     

B7+CTLA4+ T cells engage in T-T cell interactions that mediate apoptosis: a model for lentivirus-induced T cell depletion

Apoptosis in lymph node (LN) T cells of feline immunodeficiency virus (FIV)-infected cats is associated with cells co-expressing B7.1 and B7.2 costimulatory molecules, and their ligand CTLA4. To study the possibility of B7.1/B7.2-CTLA4 mediated T-T interactions and the predicted induction of T cell apoptosis in vitro, costimulatory molecules were up-regulated on CD4+ and CD8+ T cells by mitogen stimulation. B7.1 expression on in vitro stimulated CD4+ and CD8+ cells increased within 24h; B7.2 and CTLA4 expression increased after 48-72 h. Apoptosis, as analyzed by terminal deoxynucleotidyl transferase (transferase nick end labeling, TUNEL)-based staining followed by three color flow cytometric analysis, correlated to the cells expressing B7 and/or CTLA4. Blocking experiments revealed that CD4+ and CD8+ T cell apoptosis could be significantly inhibited with anti-B7 antibodies. As FIV infection results in immune activation with a T cell phenotype similar to that of the in vitro activated T cells, the data support the hypothesis that the chronic expansion of B7+CTLA4+ LN T cells in infected cats allows for T-T cell interactions resulting in T cell depletion and eventually the development of AIDS.