Dynamic Distribution of Classical Swine Fever Virus in vivo After Infection by Intermediate Virulent Strains
SUN JunXiang1, ZHANG QianYi1, XU HeMin2, WANG TuanJie1, XU Lu1, ZOU XingQi1, ZHU YuanYuan1, LI Cui1, XIA YingJu1, XU Yuan1, CHEN Kai1, ZHANG YuJie1, ZHAO QiZu1, WANG Qin1通讯作者:
第一联系人:
责任编辑: 林鉴非
收稿日期:2018-04-18接受日期:2018-05-28网络出版日期:2018-11-01
| 基金资助: |
Received:2018-04-18Accepted:2018-05-28Online:2018-11-01
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孙骏翔, 张乾义, 徐和敏, 王团结, 徐璐, 邹兴启, 朱元源, 李翠, 夏应菊, 徐嫄, 陈锴, 张玉杰, 赵启祖, 王琴. 猪瘟病毒中等致病力毒株在体内的动态分布[J]. 中国农业科学, 2018, 51(21): 4146-4156 doi:10.3864/j.issn.0578-1752.2018.21.013
SUN JunXiang, ZHANG QianYi, XU HeMin, WANG TuanJie, XU Lu, ZOU XingQi, ZHU YuanYuan, LI Cui, XIA YingJu, XU Yuan, CHEN Kai, ZHANG YuJie, ZHAO QiZu, WANG Qin.
0 引言
【研究意义】猪瘟（classical swine fever,CSF）是由猪瘟病毒（classical swine fever virus,CSFV）引起的猪的高度接触性、致死性传染病[1],对全球养猪业造成严重危害[2]。目前我国猪瘟的感染类型主要是亚急性感染及慢性感染,但因发病缓慢或不易察觉,给我国的养猪业带来极大损失[3],造成这种感染类型的主要病原是中等致病力猪瘟毒株[4]。研究亚急性猪瘟感染后病毒RNA在体内的动态分布有助于阐述亚急性病程中CSFV的复制分布规律,为当前CSF的早期诊断和预防提供技术保障。【前人研究进展】国内外研究多集中在动物体感染过程中病毒蛋白分布,且已经证实病毒蛋白载量与组织嗜性及病程密切相关[5]。猪瘟慢性感染病程中扁桃体、颌下淋巴结、肝脏、心肌等组织的病毒RNA动态载量研究已经证实了扁桃体等淋巴器官是CSFV侵入宿主的主要器官[6]。但是CSFV侵入宿主后对其他组织的嗜性及病毒核酸在组织中具体结构的定位依然不清楚,因此需要进行深入的研究。ViewRNA ISH是一种基于原位杂交发展起来的可以定位RNA的技术。目前,该技术已经在生物学方面有了广泛应用,在神经生物学方面,ViewRNA ISH与微流体芯片技术共同验证了Eef1a1、Ctnnb1、Nrxn3等转录本存在于轴索中[7],表明成熟的皮质轴突包含一些特殊的细胞骨架、蛋白运输及线粒体维护等转录本。在寻找肿瘤治疗靶点的研究中,应用此技术对外周血循环肿瘤细胞（circulating tumor cells,CTCs）进行RNA的原位杂交,鉴定出Wnt 2在CTCs中高表达,可作为治疗靶点,抑制肿瘤的转移[8]。ZHANG等[9]应用ViewRNA ISH观察到CSFV感染PK15后,病毒 RNA在体外吸附和进入靶细胞时间早于0.5 小时。这些研究说明了ViewRNA ISH对细胞中特定RNA进行定位和检测具有很好的可行性。【本研究切入点】但是,在猪瘟亚急性感染病程中,病毒 RNA在感染猪组织器官中的分布研究还未见报道,本研究应用ViewRNA ISH在核酸水平观察CSFV在组织中的分布情况,同时应用IHC、H.E染色验证ISH的观测结果。【拟解决的关键问题】在构建病程持续28 d的亚急性猪瘟感染动物模型的基础上,采用ViewRNA ISH对中等致病力CSFV感染后免疫器官、消化系统、呼吸系统等各组织中病毒RNA的分布动态进行系统研究,对阐明中等致病力CSFV的致病机理具有重要的科学意义。1 材料与方法
试验于2016年7月至2017年5月在中国兽医药品监察所,国家/OIE猪瘟参考实验室完成。1.1 试验材料
试验用猪：购自河北保定某猪场,选取10 kg左右,30日龄,健康断奶大白仔猪。抗体检测试剂盒：CSFV抗体检测试剂盒由中国兽医药品监察所国家/OIE猪瘟参考实验室提供;猪繁殖与呼吸综合征病毒（PRRSV）抗体检测试剂盒、猪伪狂犬病病毒（PRV）抗体检测试剂盒、牛病毒性腹泻病毒（BVDV）抗体检测试剂盒均购自IDEXX公司;猪细小病毒（PPV）抗体检测试剂盒购自PrioNics公司;猪圆环病毒（PCV）抗体检测试剂盒购自科前生物制品有限责任公司;口蹄疫病病毒（FMDV）抗体检测试剂盒深圳绿诗源生物技术有限公司。
病原检测试剂：CSFV、PRRSV、PRV、PPV、PCV（I型和II型）、BVDV、FMDV病原检测引物由Invitrogen公司合成;FQ-PCR主要试剂：SuperScriptTMIII 反转录酶购自Invitrogen公司;Taq HS DNA聚合酶,10×PCR Buffer等购自宝生物工程（大连）有限公司;核酸提取试剂盒购自宝生物工程（大连）有限公司。
CSFV ViewRNA ISH试剂：CSFV 特异性探针、ACTB探针的合成以及试剂盒的组装由美国Affymetrix公司完成。
IHC试剂：CSFV E2蛋白单克隆抗体WH303由英国APHA实验室赠送;羊抗鼠IgG购自康为世纪;显色液购自美国Vector Laboratories。
其他试剂：无水乙醇、二甲苯、甲醛、氨水、过氧化氢、柠檬酸钠等购自北京化学试剂有限公司。
Thermobrite ViewRNA ISH仪,美国雅培公司。
1.2 试验方法
1.2.1 探针的设计 根据GenBank公布的CSFV SM 株（AF333000）设计ISH探针序列,对猪看家基因β-actin（ACTB）（AK237086）的mRNA序列设计特异性探针作为内参。CSFV探针序列：
P1：GGA CTA GCA AAC GGA GGG ACTA GCC GTA GTG GCG AGC TCC CTG GGT GGT CTA AGT CCT GAG TAC AGG ACA GTC GTC AAT AGT TCG ACG TGA GCA GGA GC;
P2：TAT GAT TTA TTG CAA GCC CAG AGGTAC GGT ATA G AA GAC GGG ATA AAT ATC ACC AAA TCC T;
P3：AGG TGG TCA GAC AAC ACT TCT AGT TAC ATG CCG GGG AGA AAT ACA ACC ACA ATCCTA GCT AAA ATG GCC ACA AGG TTA GAT TCC AGT GGT GAG AGG GGT ACC ATA GCA TAT GAG AAA GCA GTA GCA TTC AGC TTC CTG CTG ATG TAC T。
ACTB探针序列：
ACTB：TTC CTT CCT GGG TAT GGA ATC CTG TGG CAT CCA CGA AAC TAC CTT CAA CTC AAT CAT GAA。
1.2.2 试验用猪的筛选 试验前采集断奶仔猪的全血,对其进行CSFV、PRRSV、PRV、PPV、PCV（I型和II型）、BVDV、FMDV 8种重要猪病病原和抗体的筛查。选取病原、抗体全部阴性猪作为本次实验动物。
1.2.3 猪瘟亚急性感染模型的构建和样本采集 试验用猪共18头随机分成2组：试验组16头,每头猪颈部肌肉注射1.5×103.66TCID50的CSFV中等致病力毒株（HeBHH1/95）;对照组2头,颈部肌肉注射等量无菌生理盐水,将两组实验动物隔离饲养。从感染前一天进行体温测定,参照文献[10]的方法进行临床打分。在感染后第1、3、6、10、13、20、24、28 天（day post infection, dpi）剖杀试验猪,采集十二指肠、胰腺、脾脏、回盲瓣、肾脏、肺脏等6种组织器官保存于多聚甲醛中,制成石蜡切片,备用。
1.2.4 感染组织中 CSFV ViewRNA ISH方法优化 切片经过脱蜡水化、1%蛋白酶K处理组织、预杂交液处理组织、探针杂交、细胞核蓝染后使用光学显微镜观察。根据Affymetrix ViewRNA ISH说明书提供的蛋白酶孵育时间范围（5—20 min）及组织预处理液煮沸时间范围（5—20 min）,采用正交试验对1.2.3制备的切片优化反应条件、时间。采用优化后的条件对切片进行ViewRNA ISH方法检测。
1.2.5 猪瘟亚急性感染组织间接IHC检测法 参考朱长康[11]CSFV间接IHC检测法对1.2.3制备的切片进行病毒定位检测,采用Vector显色液显色观察。
1.2.6 猪瘟亚急性感染组织H.E染色 参考文献[12]的H.E染色法对1.2.3制备的切片进行H.E染色。
2 结果
2.1 猪瘟亚急性感染模型的构建
根据文献[10]的方法（0—30分）,对CSFV中等致病力毒株（HeBHH1/95）感染猪体后进行临床记分：1—5 dpi感染猪临床症状得分较低,少于5分;6—10 dpi感染猪临床症状记分迅速增加;11—26 dpi感染猪临床得分一直维持在15分左右,直到28 dpi临床记分达到最高值20分,结果见图1。感染后每天两次对猪进行体温测定,取其算术平均值：潜伏期内感染猪体温正常;8—10 dpi感染猪体温呈上升趋势;13—24 dpi患畜体温总体呈现热稽留,持续在40℃左右;此后体温开始回落,至濒死前体温回落至39.5℃左右,对照组体温一直维持在（39±0.5）℃,结果见图2。根据感染猪临床记分、体温测定指标及病程,成功构建了CSFV亚急性感染动物模型。图1
新窗口打开|下载原图ZIP|生成PPT图1CSFV中等致病力毒株（HeBHH1/95）感染猪体临床打分图
Fig. 1Clinical scores after infection of CSFV HeBHH1/95 strain
图2
新窗口打开|下载原图ZIP|生成PPT图2CSFV中等致病力毒株（HeBHH1/95）感染猪体温变化情况
Fig. 2Change of body temperatures after infection of CSFV HeBHH1/95 strain
2.2 CSFV ViewRNA ISH方法优化
参考组织形态及CSFV RNA信号强度,各组织器官的预处理液的煮沸时间及蛋白酶的消化时间优化结果如表1Table 1
表1
表1各组织器官的预处理液煮沸时间及蛋白酶消化时间
Table 1
| 组织名称 Tissue | 预处理液处理时间 Preconditioning liquids work time (min) | 蛋白酶消化时间 Proteinase K work time (min) |
|---|---|---|
| 肺脏Lung | 10 | 20 |
| 十二指肠 Duodenum | 10 | 10 |
| 回盲瓣 Ileocecal valve | 10 | 10 |
| 胰腺Pancreas | 10 | 10 |
| 脾脏Spleen | 20 | 10 |
| 肾脏Kidney | 20 | 20 |
新窗口打开|下载CSV
2.3 猪瘟亚急性感染组织器官CSFV ViewRNA ISH、IHC与H.E染色的检测
ViewRNA ISH结果显示,1 dpi十二指肠的杯状细胞周围检测到病毒 RNA,整个病程中,肠绒毛病毒 RNA逐渐增多;IHC结果显示,1 dpi检出病毒蛋白,整个病程中,病毒蛋白含量及分布与CSFV ViewRNA ISH相似。10—13 dpi 病毒 RNA信号及蛋白信号强度显著增加;H.E结果显示,1 dpi肠绒毛出现出血和淋巴浸润,随着病程延长,肠绒毛柱状上皮细胞脱落（图3）。图3
新窗口打开|下载原图ZIP|生成PPT图3HeBHH1/95感染后十二指肠ViewRNA ISH、IHC与H.E染
a表示ViewRNA ISH检测结果,其中CSFV的RNA靶序列被染成红色,细胞核被染成蓝色。b表示IHC检测结果,其中CSFV的靶蛋白被染成玫红色。c表示H.E染色结果.用注射生理盐水猪做阴性对照。IV：肠绒毛;GC：杯状细胞
Fig. 3Duodenum ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×)
“a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. IV: intestinal villus; GC: goblet cell
ViewRNA ISH结果显示,1 dpi胰腺的腺泡出检测到RNA信号,随着病程延长,CSFV在腺泡及朗格罕岛的含量逐渐增加;IHC结果显示,1 dpi可以检测到病毒蛋白阳性信号存在于腺泡,在整个病程中病毒蛋白均集中于腺泡及朗格罕岛;H.E结果显示,3 dpi腺泡周围出血,10 dpi后腺泡结构不清（图4）。
图4
新窗口打开|下载原图ZIP|生成PPT图4HeBHH1/95感染后胰腺ViewRNA ISH、IHC与H.E染色
a表示ViewRNA ISH检测结果,其中CSFV的RNA靶序列被染成红色,细胞核被染成蓝色。b表示IHC检测结果,其中CSFV的靶蛋白被染成玫红色。c表示H.E染色结果。用注射生理盐水猪做阴性对照。PA：腺泡;PI：朗格罕岛
Fig. 4Pancreas ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×)
“a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. PA: Pancreas acinar; PI: Pancreatic islet
ViewRNA ISH结果显示,1 dpi回盲瓣的李氏隐窝及固有层检测到病毒RNA信号,随着病程延长,CSFV含量在该位置逐渐增加;IHC结果显示,1 dpi未见病毒蛋白阳性信号,3 dpi可以检测到病毒蛋白阳性信号存在于固有层和李氏隐窝,随着病程延长,病毒蛋白含量逐渐增加;H.E结果显示,3 dpi固有层有炎性细胞浸润同时伴随充血,10—28 dpi,杯状细胞萎缩凋亡（图5）。
图5
新窗口打开|下载原图ZIP|生成PPT图5HeBHH1/95感染后回盲瓣ViewRNA ISH、IHC与H.E染色
a表示ViewRNA ISH检测结果,其中CSFV的RNA靶序列被染成红色,细胞核被染成蓝色。b表示IHC检测结果,其中CSFV的靶蛋白被染成玫红色。c表示H.E染色结果.用注射生理盐水猪做阴性对照。LP：固有层;LC：李氏隐窝
Fig. 5Ileocecal valve ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×)
“a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. LP: Lamina propria; LC: Crypt of Lieberkuhn
ViewRNA ISH结果显示,3 dpi脾脏椭圆体周围有少量病毒RNA,6 dpi动脉周围淋巴鞘出现病毒RNA阳性信号,20—28 dpi边缘区的病毒RNA含量大量增加;IHC结果显示,3 dpi在椭圆体周围有病毒蛋白阳性信号,6 dpi动脉周围淋巴鞘及边缘区出现病毒蛋白信号;H.E结果显示,3 dpi脾脏动脉周围淋巴鞘边缘区域开始出现部分红细胞,并且有少量炎性细胞浸润,20 dpi后脾脏呈现弥漫性出血,并且脾脏组织结构难以辨认（图6）。
图6
新窗口打开|下载原图ZIP|生成PPT图6HeBHH1/95感染后脾脏ViewRNA ISH、IHC与H.E染色
a表示ViewRNA ISH检测结果,其中CSFV的RNA靶序列被染成红色,细胞核被染成蓝色。b表示IHC检测结果,其中CSFV的靶蛋白被染成玫红色。c表示H.E染色结果.用注射生理盐水猪做阴性对照。E：椭圆体;PALS：动脉周围淋巴鞘;MZ：边缘区
Fig. 6Spleen ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×)
“a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. E: Ellipsoid; PALS: Periarterial lymphatic sheath; MZ: Marginal zone
ViewRNA ISH结果显示,1 dpi肾脏的肾小球、肾小管检测到病毒阳性信号,10 dpi 病毒信号主要集中在肾小管和肾小球;IHC结果显示,1 dpi在肾小管中检测到病毒蛋白阳性信号,3 dpi发现肾小球存在病毒蛋白,随着病程延长,病毒蛋白集中在肾小管;H.E结果显示,1—6 dpi肾小球毛细血管充血,肾小管间隙可见少量出血,在10—13 dpi出血更为严重,并出现少量炎性细胞,20—28 dpi肾小球囊腔缩小,部分几乎不可见,呈现纤维化（图7）。
图7
新窗口打开|下载原图ZIP|生成PPT图7HeBHH1/95感染后肾脏ViewRNA ISH、IHC与H.E染色
a表示ViewRNA ISH检测结果,其中CSFV的RNA靶序列被染成红色,细胞核被染成蓝色。b表示IHC检测结果,其中CSFV的靶蛋白被染成玫红色。c表示H.E染色结果.用注射生理盐水猪做阴性对照。G：肾小球;RT：肾小管
Fig. 7Kidney ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×)
“a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. G: glomerulus; RT: renal tubular
ViewRNA ISH结果显示,3 dpi肺脏的细支气管附近检测到病毒RNA,整个病程中,病毒 RNA含量略有增加;IHC结果显示,3 dpi检出病毒蛋白,整个病程中,病毒蛋白含量均较低;H.E结果显示,在3 dpi肺间质增厚充血并出现少量炎性细胞,支气管内可见出血,在6—13 dpi血管内可见大量血细胞、少量脱落的组织细胞和其他分泌物,并伴有炎性细胞浸润,20—28 dpi,肺间质充血明显缓解（图8）。
图8
新窗口打开|下载原图ZIP|生成PPT图8HeBHH1/95感染后肺脏ViewRNA ISH、IHC与H.E染色
a表示ViewRNA ISH检测结果,其中CSFV的RNA靶序列被染成红色,细胞核被染成蓝色。b表示IHC检测结果,其中CSFV的靶蛋白被染成玫红色。c表示H.E染色结果.用注射生理盐水猪做阴性对照。B：细支气管;LI：肺间质
Fig. 8Lung ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×)
“a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining.Negative control was done by injection of saline pig. B: Bronchiole; LI: Lung interstitium
3 讨论
ViewRNA ISH是一种新型原位杂交技术,融合了传统原位杂交技术与荧光原位杂交（FISH）技术的优点[13]。通过设计目的基因的“Z”型结构特异探针,利用“Z”型探针底部与经蛋白酶消化后暴露的靶RNA杂交,之后加入标记引物与相应的“L”型标记探针,使得一个转录样本便可产生8 000倍的放大效应。ViewRNA ISH基于特殊的探针设计与专有的信号放大方法[14],在准确性、灵敏度、重复性等方面较常规原位杂交技术都有大幅提高[15],可以在单细胞中对多个目标基因的低拷贝乃至单拷贝RNA做出检测。ViewRNA ISH具有操作周期短,一天内可以完成的优点。相对于不易获取抗体的免疫组化和免疫荧光试验,ViewRNA ISH针对目标基因或序列,可以进行灵活的探针设计,新探针设计一周内可完成,实现了缩短试验周期的目标。因此,通过设计CSFV RNA与β-actin RNA探针并优化了CSFV ViewRNA ISH方法,为研究猪瘟亚急性感染后体内病毒RNA和蛋白的分布情况,有助于阐述亚急性病程病毒的复制分布规律,为猪瘟的早期诊断和预防奠定重要基础。根据猪瘟病程长短、临床症状等可将猪瘟分为急性型、亚急性型、慢性型等不同类型。其中亚急性型猪瘟一般病程达21—30 d[2],患畜精神沉郁,食欲下降或停食,体温一般升高至40.5℃左右,便秘、腹泻交替出现。亚急性型猪瘟动物模型的构建主要与病毒致病力和接种剂量有关,本研究应用1.5×103.66TCID50的中等致病力毒株HeBHH1/95成功构建出与上述症状相似的亚急性动物模型。
本试验为保证检测结果的可靠性,对猪瘟亚急性感染猪的组织器官连续切片后在相似视野下进行CSFV ViewRNA ISH、IHC和H.E进行检测,发现CSFV ViewRNA ISH在1 dpi即可检测出回盲瓣的固有层存在病毒 RNA,而IHC和H.E在3 dpi才可在回盲瓣中检测到病毒蛋白和组织的病变,这表明ViewRNA ISH有很好的灵敏性且试验周期要短于IHC和H.E,是一种快速、灵敏的原位检测病毒RNA的方法。应用CSFV ViewRNA ISH发现淋巴细胞核内同样存在病毒RNA,这与张玉杰等[16]CSFV在PK15细胞中定位结果一致,提示病毒在细胞中的增殖需要宿主细胞核的参与。
脾脏是机体最大的免疫器官[17],但对淋巴组织有嗜性的CSFV却没有在1 dpi脾脏中检测到病毒RNA。首先脾脏是产生IgM的主要场所,在感染初期IgM对及时清除病毒发挥着关键作用,且脾脏中存在大量的自然杀伤细胞[18]、单核巨噬细胞[19]、树突状细胞等免疫细胞[20],这些免疫细胞在先天性免疫阶段发挥着重要抗病毒作用;其次脾脏没有输入淋巴管,也没有淋巴窦,不利于病毒随淋巴循环进入脾脏;这可能是本研究构建的猪瘟亚急性感染早期脾脏中病毒RNA信号较弱不易检出的原因。肺脏是CSFV嗜性较弱的器官,20 dpi后肺间质充血明显缓解,且病毒阳性信号减弱。CSFV在宿主体内感染的靶细胞主要是骨髓源细胞[21],如巨噬细胞,肺泡巨噬细胞能够吸收大部分进入呼吸道的颗粒物质,但是它既不能够迁移到相关的淋巴结[22],也没有抗原递呈功能[23],所以CSFV不易在肺脏中复制传播。CSFV在体内的靶细胞不仅有巨噬细胞,同时还有血管内皮细胞[24],CSFV侵入血管内皮细胞后导致血管通透性增加,因此在H.E染色中各组织出现以充血、出血、淤血为主的病理变化。CSFV对中胚层组织尤其是造血器官及血管有着特殊的亲和力[25],肾脏不是免疫器官,却在1 dpi检测到病毒 RNA及蛋白,这可能与肾脏是中胚层分化而来的器官有关系[26],病毒主要位于肾小管上皮及肾小球等具有分泌功能的部位。消化管与外界直接相连,病毒等病原微生物易随着食物进入消化系统,且胃肠黏膜组织中25%是淋巴组织,这有利于嗜免疫细胞的CSFV在消化系统中传播复制。在胰腺的腺泡、十二指肠的肠绒毛及黏膜下层、回盲瓣的黏膜下层等具有分泌功能的部位同样有大量病毒聚集。
结合以上分析发现,CSFV虽然是一种亲免疫细胞的病毒,但是由于先天性免疫等因素,在亚急性感染早期,CSFV没有在脾脏大量复制。同时这几种组织在13 dpi时出现了病毒RNA及蛋白均明显增多的现象,在猪瘟慢性感染病程中,病毒在前期同样增长缓慢[27],这是由于机体先天性免疫系统发挥了重要作用。例如,CSFV感染能够诱导IL-1β的释放[28],而IL-1可以引起机体发热[29],这或许也是感染早期体温迅速升高的原因,而到了感染中期,宿主的白细胞数量明显减少,造成对病毒的抑制作用减弱[30],导致在13 dpi CSFV含量迅速增加。这进一步说明在感染早期由于先天性免疫的作用,CSFV在脾脏的增殖复制存在一定阻力。从13 dpi后的病程中CSFV在各组织内开始大量繁殖,并广泛分布在组织内的各个部位,CSFV不仅在像脾脏动脉周围淋巴鞘等免疫细胞聚集区大量复制增殖,还会在胰腺的腺泡、肾脏的肾小管等具有分泌功能的结构处增殖复制。
4 结论
采用优化的CSFV ViewRNA ISH能在1 dpi检测到猪瘟亚急性感染猪回盲瓣组织中的病毒 RNA,较IHC能更早检出病毒的存在;采用CSFV ViewRNA ISH、ICH方法对各个组织中病毒RNA进行定位,结果与H.E染色检测到的组织损伤部位一致,表明ViewRNA ISH具有较高的灵敏度,适用于感染早期CSFV在嗜性组织中RNA动态分布研究;研究还表明猪瘟亚急性感染早期病毒RNA通常在腺泡、肾小管等结构及脾脏的椭圆体周围复制,感染中期在各组织中广泛分布且大量增殖,感染后期病毒主要富集在淋巴结和有分泌功能的部位,表明病毒对分泌结构如腺泡也存在明显嗜性。参考文献 原文顺序
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被引期刊影响因子
DOI:10.1007/s00705-016-3065-7URLPMID:27695950 [本文引用: 1]
Abstract Since the first rescue of a recombinant Newcastle disease virus (rNDV) in the late 1990s, many more rNDVs have been rescued by researchers around the world. Regardless of methodology, the main principle behind rescue of the virus has remained the same, i.e., the formation of a functional replication complex by simultaneously providing the full-length viral RNA and the viral NP, P and L proteins. However, different strategies have been reported for the insertion of the full-length genome into a suitable transcription vector, which remains the most challenging step of the rescue. Moreover, several systems have been published for provision of the DNA-dependent RNA polymerase, which is needed for transcription of viral RNA (vRNA) from the transfected plasmid DNA. The aim of this article is to consolidate all of the current cDNA assembly strategies and transcription systems used in rescue of rNDV in order to attain a better understanding of the advantages and disadvantages of each approach.
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[本文引用: 2]
DOI:10.1016/S0378-1135(00)00138-3URLPMID:10785321 [本文引用: 1]
A historical and current perspective is given of classical swine fever and its impact on pig production in different regions of the world. Data were obtained from a variety of sources including returns to the Office International des Epizooties, official government reports, other published material and local information through personal contacts. The disease has been recognized for about 170 years and efforts to control it by official intervention began in the nineteenth century. Despite this it remains a lingering problem in many parts of the world where it has both, an economic impact on swine production and a constraining effect on trade due to the measures necessary to prevent spread.
URLPMID:5773219 [本文引用: 1]
Cheville NF, Mengeling WL.
DOI:10.1186/1743-422X-8-201URLPMID:3107811 [本文引用: 1]
Background Classical swine fever (CSF), caused by the Classical swine fever virus(CSFV), is an Office International des Epizooties (OIE) notifiable disease. However, we are far from fully understand...
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DOI:10.1523/JNEUROSCI.6130-08.2009URLPMID:3632375 [本文引用: 1]
Abstract Using a novel microfluidic chamber that allows the isolation of axons without contamination by nonaxonal material, we have for the first time purified mRNA from naive, matured CNS axons, and identified the presence of >300 mRNA transcripts. We demonstrate that the transcripts are axonal in nature, and that many of the transcripts present in uninjured CNS axons overlap with those previously identified in PNS injury-conditioned DRG axons. The axonal transcripts detected in matured cortical axons are enriched for protein translational machinery, transport, cytoskeletal components, and mitochondrial maintenance. We next investigated how the axonal mRNA pool changes after axotomy, revealing that numerous gene transcripts related to intracellular transport, mitochondria and the cytoskeleton show decreased localization 2 d after injury. In contrast, gene transcripts related to axonal targeting and synaptic function show increased localization in regenerating cortical axons, suggesting that there is an increased capacity for axonal outgrowth and targeting, and increased support for synapse formation and presynaptic function in regenerating CNS axons after injury. Our data demonstrate that CNS axons contain many mRNA species of diverse functions, and suggest that, like invertebrate and PNS axons, CNS axons synthesize proteins locally, maintaining a degree of autonomy from the cell body.
DOI:10.1038/nature11217URLPMID:3408856 [本文引用: 1]
Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases1. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device2for efficient capture of CTCs from an endogenous mouse pancreatic cancer model3and subjected CTCs to single molecule RNA sequencing4, identifyingWnt2as a candidate gene enriched in CTCs. Expression ofWnt2in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensityin vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of Map3k7 (Tak1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed enrichment for Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.
DOI:10.1186/s12985-017-0734-4URLPMID:5395781 [本文引用: 1]
Classical swine fever (CSF) is a highly contagious fatal infectious disease caused by classical swine fever virus (CSFV). A better understanding of CSFV replication is important for the study of pathogenic mechanism of CSF. With the development of novel RNA in situ Hybridization method, quantitatively localization and visualization of the virus RNA molecular in cultured cell or tissue section becomes very important tool to address these pivotal pathogenic questions. In this study, we established ViewRNA ISH method to reveal the dynamic distribution of CSFV RNA in PK15 cells. We designed several specific probes of CSFV RNA and reference gene β-actin for host PK15 cells to monitor the relative location of CSFV RNA and house-keeping gene in the infected cells. After determining the titer of reference strain CSFV (HeBHH1/95) with the 50% tissue culture infective dose (TCID50), we optimized the protease K concentration and formalin fixation time to analyze the hybridization efficiency, fluorescence intensity and repeatability. In order to measure the sensitivity of this assay, we compared it with the fluorescent antibody test (FAT) and immunohistochemical(IHC) method. Specificity of the ViewRNA ISH was tested by detecting several sub genotypes of CSFV (sub genotype 1.1, 2.1, 2.2 and 2.3) which are present in China and other normal pig infectious virus (bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine circovirusII(PCV-2). The lowest detection threshold of the ViewRNA ISH method was 10618, while the sensitivity of FAT and IHC were 10615and 10614, respectively. The ViewRNA ISH was specific for CSFV RNA including 1.1, 2.1, 2.2 and 2.3 subtypes, meanwhile, there was no cross-reaction with negative control and other viruses including BVDV, PPV, PRV and PCV-2. Our results showed that after infection at 0.5 hpi (hours post inoculation, hpi), the CSFV RNA can be detected in nucleus and cytoplasm; during 3–9 hpi, RNA was mainly distributed in nucleus and reached a maximum at 12hpi, then RNA copy number was gradually increased around the cell nucleus during 24–48 hpi and reached the peak at 72hpi. To our knowledge, this is the first to reveal the dynamic distribution of medium virulence CSFV RNA in PK15 cells using the ViewRNA ISH method. The sensitivity of the ViewRNA ISH was three to four orders of magnitude higher than that of FAT and IHC methods. The specificity experiment showed that the ViewRNA ISH was highly specific for CSFV and no cross-reaction occurred to negative control and other pig infectious virus. This assay is more suitable for studying the CSFV RNA life cycle in cell nucleus. The results proved that CSFV RNA enters into PK15 cells earlier than 0.5hpi, relative to the eclipse period of cytoplasm is 6–9 hpi and CSFV RNA has ever existed in nucleus.
DOI:10.1099/0022-1317-79-12-2981URLPMID:9880012 [本文引用: 2]
Cytopathogenicity of (CSFV) depends on the presence of defective particles containing a subgenomic (sg) with a defined deletion. In a previous report we described the spontaneous generation of this sg and therefore of cytopathogenic (cp) CSFV in porcine kidney cell cultures persistently infected with CSFV. Frequently, some cells survived the and could be further propagated. They remained positive for viral antigen and continued to shed complete virus and in most cases also defective virus particles. SK-6 cells that had survived the ((surv)cells) were used to investigate these findings further. In contrast to persistently infected cells that had not experienced a , (surv) cells were protected from the when superinfected with cp CSFV or with cp virus. Similarly, cells which were rescued and further propagated after with cp CSFV also proved to be protected from the CSFV-induced . When either virus obtained from (surv) cells that had spontaneously lost the sg or virus from which defective particles had been removed was used to establish persistently infected cells, these cells were also protected from the after with cp CSFV. These findings suggest that the virus contained in (surv) cells confers on the cell the ability to resist the CSFV-induced . However, when naive cells were infected with supernatants from (surv)cells that contained defective virus particles, the reappeared within three to five virus passages, indicating that the sg retained its cytopathogenic potential.
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DOI:10.1016/j.jcv.2015.06.085URL [本文引用: 1]
Enteroviral infections are common, affecting humans across all age groups. RT-PCR is widely used to detect these viruses in clinical samples. However, there is a need for sensitive and specific in situ detection methods for formalin-fixed tissues, allowing for the anatomical localization of the virus and identification of its serotype. The aim was to design novel enterovirus probes, assess the impact of probe design for the detection and optimize the new single molecule in situ hybridization technology for the detection of enteroviruses in formalin-fixed paraffin-embedded samples. Four enterovirus RNA-targeted oligonucleotide RNA probes – two probes for wide range enterovirus detection and two for serotype-targeted detection of Coxsackievirus B1 (CVB1) – were designed and validated for the commercially available QuantiGene ViewRNA in situ hybridization method. The probe specificities were tested using a panel of cell lines infected with different enterovirus serotypes and CVB infected mouse pancreata. The two widely reactive probe sets recognized 19 and 20 of the 20 enterovirus serotypes tested, as well as 27 and 31 of the 31 CVB1 strains tested. The two CVB1 specific probe sets detected 30 and 14 of the 31 CVB1 strains, with only minor cross-reactivity to other serotypes. Similar results were observed in stained tissues from CVB –infected mice. These novel in-house designed probe sets enable the detection of enteroviruses from formalin-fixed tissue samples. Optimization of probe sequences makes it possible to tailor the assay for the detection of enteroviruses on the serotype or species level.
DOI:10.3969/j.issn.1008-0589.2011.10.11URL [本文引用: 1]
为了对体外培养细胞感染的不同马传染性贫血病毒(EIAV)株进 行特异的鉴别检测,本研究基于一种新的RNA原位杂交-ViewRNA技术,通过设计合成针对EIAVFDDV13和EIAVUK3病毒株基因组RNA的 特异性探针,并结合信号放大技术和荧光标记技术,同时通过激光共聚焦显微镜进行观察,建立了可时两种不同株的EIAV基因组RNA进行细胞内原位检测的方 法.研究结果表明,两组探针能够特异地与感染细胞中EIAVFDD13和EIAVUK3的病毒RNA相结合,可在两种不同EIAV株共感染的宿主细胞中实 现对两种毒株的有效鉴别和定位检测.该方法为进一步研究EIAV与宿主细胞间相互作用提供了有效手段.
DOI:10.3969/j.issn.1008-0589.2011.10.11URL [本文引用: 1]
为了对体外培养细胞感染的不同马传染性贫血病毒(EIAV)株进 行特异的鉴别检测,本研究基于一种新的RNA原位杂交-ViewRNA技术,通过设计合成针对EIAVFDDV13和EIAVUK3病毒株基因组RNA的 特异性探针,并结合信号放大技术和荧光标记技术,同时通过激光共聚焦显微镜进行观察,建立了可时两种不同株的EIAV基因组RNA进行细胞内原位检测的方 法.研究结果表明,两组探针能够特异地与感染细胞中EIAVFDD13和EIAVUK3的病毒RNA相结合,可在两种不同EIAV株共感染的宿主细胞中实 现对两种毒株的有效鉴别和定位检测.该方法为进一步研究EIAV与宿主细胞间相互作用提供了有效手段.
DOI:10.3864/j.issn.0578-1752.2016.12.015URL [本文引用: 1]
【目的】为研究CSFV RNA在体外感染细胞中的分布及定位,建立了一种准确、敏感的RNA可视化原位杂交技术。【方法】本研究通过比对Gen Bank中公布的CSFV、BVDV和BDV全序列,避开BVDV和BDV的同源区,设计了CSFV RNA及内参基因β-actin的特异探针。以CSFV中等致病力毒株（He BHH1/95）为参考毒株,在PK15细胞中培养病毒,加入RNA可视化原位杂交的特异探针和相应试剂,采用荧光共聚焦显微镜进行成像观察。通过综合分析观测结果、荧光强度、重复性等因素,采用正交试验优化了对原位杂交过程中具有重要影响的蛋白酶K浓度和甲醛固定时间,建立了CSFV RNA可视化原位杂交技术,并与FAT方法比较该技术灵敏度;用我国目前流行的CSFV 1.1、2.1、2.2、2.3基因亚型及BVDV、PPV、PRV和PCV-2病毒进行特异性试验。最终,以CSFV强致病力毒株（SM）接种PK15细胞,病毒感染后0.5、1、3、6、8、10、14、18、24、36、48、72、96h（hours post inoculation,hpi）取样,每个时间点2个重复,采用CSFV RNA可视化原位杂交技术进行检测。为佐证病毒蛋白在细胞中的定位及分布,同时采用FAT方法对SM株E2蛋白在PK15细胞中的表达情况进行动态研究。【结果】采用该技术在荧光共聚焦显微镜下可观察到CSFV RNA在细胞中的定位;当蛋白酶K浓度为1：1 000、甲醛固定时间为30min时为最优反应条件;灵敏度试验表明该技术对病毒的检测极限为10-8/200μL,比FAT高3.5个数量级;特异性试验结果显示该探针能与CSFV 1.1、2.1、2.2、2.3亚型结合,与BVDV、PPV、PCV-2、PRV无交叉反应。采用该技术对CSFV RNA感染后在靶细胞中的定位与分布研究结果显示：0.5hpi在胞核和胞浆均能检测到RNA,0.5—6hpi RNA主要分布于胞核内并在核内富集;10hpi胞浆内RNA逐渐增多,胞核内RNA逐渐减少,24hpi RNA主要集中在胞浆内细胞核周围;36hpi核外RNA大量聚集增多,72hpi达到峰值;96hpi RNA总量有17
DOI:10.3864/j.issn.0578-1752.2016.12.015URL [本文引用: 1]
【目的】为研究CSFV RNA在体外感染细胞中的分布及定位,建立了一种准确、敏感的RNA可视化原位杂交技术。【方法】本研究通过比对Gen Bank中公布的CSFV、BVDV和BDV全序列,避开BVDV和BDV的同源区,设计了CSFV RNA及内参基因β-actin的特异探针。以CSFV中等致病力毒株（He BHH1/95）为参考毒株,在PK15细胞中培养病毒,加入RNA可视化原位杂交的特异探针和相应试剂,采用荧光共聚焦显微镜进行成像观察。通过综合分析观测结果、荧光强度、重复性等因素,采用正交试验优化了对原位杂交过程中具有重要影响的蛋白酶K浓度和甲醛固定时间,建立了CSFV RNA可视化原位杂交技术,并与FAT方法比较该技术灵敏度;用我国目前流行的CSFV 1.1、2.1、2.2、2.3基因亚型及BVDV、PPV、PRV和PCV-2病毒进行特异性试验。最终,以CSFV强致病力毒株（SM）接种PK15细胞,病毒感染后0.5、1、3、6、8、10、14、18、24、36、48、72、96h（hours post inoculation,hpi）取样,每个时间点2个重复,采用CSFV RNA可视化原位杂交技术进行检测。为佐证病毒蛋白在细胞中的定位及分布,同时采用FAT方法对SM株E2蛋白在PK15细胞中的表达情况进行动态研究。【结果】采用该技术在荧光共聚焦显微镜下可观察到CSFV RNA在细胞中的定位;当蛋白酶K浓度为1：1 000、甲醛固定时间为30min时为最优反应条件;灵敏度试验表明该技术对病毒的检测极限为10-8/200μL,比FAT高3.5个数量级;特异性试验结果显示该探针能与CSFV 1.1、2.1、2.2、2.3亚型结合,与BVDV、PPV、PCV-2、PRV无交叉反应。采用该技术对CSFV RNA感染后在靶细胞中的定位与分布研究结果显示：0.5hpi在胞核和胞浆均能检测到RNA,0.5—6hpi RNA主要分布于胞核内并在核内富集;10hpi胞浆内RNA逐渐增多,胞核内RNA逐渐减少,24hpi RNA主要集中在胞浆内细胞核周围;36hpi核外RNA大量聚集增多,72hpi达到峰值;96hpi RNA总量有17
URL [本文引用: 1]
URL [本文引用: 1]
DOI:10.1182/blood-2006-09-046201URLPMID:17244679 [本文引用: 1]
Abstract Tumor growth promotes the expansion of myeloid suppressor cells. An inverse correlation between natural killer (NK) cell activation and myeloid suppressor cell (MSC) expansion in tumor-bearing patients and mice prompted us to investigate the role of MSCs in controlling NK antitumor cytotocixity. After adoptive transfer to naive recipients, CD11b(+)Gr-1(+) MSCs freshly isolated from spleens of tumor-bearing mice but not naive mice were able to inhibit NK cell cytotoxicity. An in vivo imaging analysis indicates that the removal of tumors resulted in a significant increased ability (P < .05) in NK cell cytotoxicity to eliminate injected YAC-1 cells from the lungs. Fluorescence-activated cell sorter (FACS) analysis of the composition of lung leukocytes further indicates that the removal of tumors also leads to the reduction of MSCs accumulated in the lung. These data suggest that MSCs suppress NK cell cytotoxicity. The inhibition of NK cell cytotoxicity is cell-cell contact dependent. Inhibition of perforin but not granzyme B production was responsible for MSC-mediated inhibition of NK cytotoxicity. Western blot analyses further suggests that MSCs suppress IL-2-mediated NK cell cytotoxicity by affecting the activity of Stat5.
URLPMID:3307443 [本文引用: 1]
Macrophages (M phi s) are an important component of the immune response and mediate numerous other functions. Phenotypic and functional subsets of circulating monocytes have been described, but few similar studies have analyzed M phi s in human tissues. By use of immunohistochemical techniques and a large number of monoclonal antibodies, the presence and distribution of phenotypic subpopulations of M phi s and dendritic cells in human spleen were assessed. The results of this study show that different subsets of M phi s and dendritic cells are present in the spleen and that some of these occupy discrete microanatomic locations. In the red pulp (RP) certain groups of antigens are expressed by different proportions of uniformly distributed M phi s in the cords. On the other hand, some antigens are present on M phi s that form clusters of variable size within the red pulp. M phi s in the splenic marginal zone (MZ) share some antigens with red pulp M phi s, but in addition express CR3, Mo-2, 61D3, and 63D3. These antigens are found on only a few RP M phi s. MZ cells expressing one antigen shared with RP M phi s (Leu-3a,b) and one present largely on the MZ cells (63D3) form clusters around small vessels; these structures resemble the so-called splenic ellipsoids that may play a role in the trapping of circulating antigens. Phagocytic M phi s (tingible body M phi s) of the white pulp follicular germinal centers were also shown to differ from RP and MZ cels with respect to the expression of the antigens detected by anti-FcR, Leu-M3, Mo-2, 25F9, and anti-CR3. The unique topographical and surface antigenic features of dendritic cells were confirmed by this study. Furthermore, these cells were found to share a number of antigens with RP, MZ, and white pulp M phi s, which suggests that they may be derived from a common progenitor. The presence of phenotypic subpopulations and variation in distribution among human splenic phagocytic cells and dendritic cells may be indicative of functional specialization.
DOI:10.3389/fimmu.2014.00059URLPMID:3925839 [本文引用: 1]
Dendritic cells (DC) in the spleen are highly activated following intravenous vaccination with a foreign-antigen, promoting expansion of effector T cells, but remain phenotypically and functionally immature after vaccination with a self-antigen. Up-regulation or suppression of expression of a cohort of pancreatic enzymes 24–7265h post-vaccination can be used as a biomarker of stimulatory versus tolerogenic DC, respectively. Here we show, using MUC1 transgenic mice and a vaccine based on the MUC1 peptide, which these mice perceive as a self-antigen, that the difference in enzyme expression that predicts whether DC will promote immune response or immune tolerance is seen as early as 4–865h following vaccination. We also identify early production of IL-10 as a predominant factor that both correlates with this early-time point and controls DC function. Pre-treating mice with an antibody against the IL-10 receptor prior to vaccination results in DC that up-regulate CD40, CD80, and CD86 and promote stronger IFNγ+ T cell responses. This study suggests that transient inhibition of IL-10 prior to vaccination could improve responses to cancer vaccines that utilize self-tumor antigens.
DOI:10.1006/viro.2000.0361URLPMID:10873748 [本文引用: 1]
Granulocytopenia, a hematological hallmark of classical swine fever, is partially responsible for the suppression of innate immune defenses during classical swine fever. The present report demonstrates that this depletion was apparent as early as 3 days postinfection (p.i.). Both mature peripheral and bone marrow neutrophils were affected, whereas immature neutrophils increased absolutely in the periphery and coincidentally immature myeloid progenitors in the bone marrow. These data suggest that a pathogenic relationship exists between these compartments. The central event was not the arrest of hematopoietic cell proliferation or of the mobilization process, but instead apoptosis and possibly also necrosis were shown to play a role. This increase in apoptotic and dead cells was detected as early as 1 3 days p.i. In contrast, viral RNA in bone marrow hematopoietic cells (BMHC) was first detected 5 days p.i., and significant amounts of infected BMHC were detected only 7 days p.i., with the major target being the myeloid compartment. The increased caspase-3 activity observed supported a role for apoptotic cell death. Furthermore, the elevated caspase-9 activity indicated the involvement of the mitochondrial apoptotic pathway. Taken together, the results demonstrate that granulocytopenia and bone marrow atrophy are mediated by hematopoietic cell death and that indirect virus ost-mediated mechanisms are likely to be responsible.
DOI:10.1016/j.immuni.2006.03.008URLPMID:16618595 [本文引用: 1]
Although alveolar macrophages are normally quiescent to prevent damaging the alveoli, in this issue of Immunity, Takabayshi et al. (2006) demonstrate that alveolar macrophages can self-regulate their function on demand to mount an appropriate immune response.
DOI:10.4049/jimmunol.176.6.3578URLPMID:16517726 [本文引用: 1]
Abstract We investigated the fate of latex (LX) particles that were introduced into mice intranasally. Macrophages acquired the vast majority of particles and outnumbered LX particle-bearing airway dendritic cells (DCs) by at least two orders of magnitude. Yet alveolar macrophages were refractory to migration to the draining lymph node (DLN), and all transport to the DLN could be ascribed to the few LX(+) airway DCs. Upon macrophage depletion, markedly greater numbers of DCs were recruited into the alveolar space. Consequently, the number of DCs that carried particles to the DLN was boosted by 20-fold. Thus, a so far overlooked aspect of macrophage-mediated suppression of airway DC function stems from the modulation of DC recruitment into the airway. This increase in DC recruitment permitted the development of a robust assay to quantify the subsequent migration of DCs to the DLN. Therefore, we determined whether lung DCs use the same molecules that skin DCs use during migration to DLNs. Like skin DCs, lung DCs used CCR7 ligands and CCR8 for emigration to DLN, but the leukotriene C(4) transporter multidrug resistance-related protein 1 did not mediate lung DC migration as it does in skin, indicating that pathways governing DC migration from different tissues partially differ in molecular regulation.
DOI:10.1186/1297-9716-45-48URLPMID:24758593 [本文引用: 1]
Infection of domestic swine with the highly virulent Shimen strain of classical swine fever virus causes hemorrhagic lymphadenitis and diffuse hemorrhaging in infected swine. We analyzed patterns of gene expression for CSFV Shimen in swine umbilical vein endothelial cells (SUVECs). Transcription of the vascular endothelial growth factor (VEGF) C gene (VEGF-C) and translation of the corresponding protein were significantly up-regulated in SUVECs. Our findings suggest that VEGF-C is involved in mechanisms of acute infection caused by virulent strains of CSFV.
DOI:10.1354/vp.40-2-157URLPMID:12637755 [本文引用: 1]
Twenty pigs were inoculated with a virulent isolate (Quillota strain) of classical swine fever (CSF) virus to determine the chronological development of lesions in bone marrow. Histopathologic, ultrastructural and immunohistochemical (detection of viral antigen gp55, myeloid-histiocyte antigen, CD3 antigen, and FVIII-rag), and morphometric techniques were employed. Viral antigen was detected from 2 days postinfection (dpi) in stromal and haematopoitic cells, and severe atrophy related to apoptosis of haematopoitic cells was observed. Megakaryocytes (MKs) did not show significant changes in number, but there were important qualitative changes including 1) increased numbers of cloud-nuclei MKs, microMKs, apoptotic MKs, and atypical nucleated MKs and 2) decreased number of typical nucleated MKs. Morphometric study of these cells showed a decrease in cytoplasmic area. MK infection was detected from 2 dpi, but in a small percentage of cells. Myeloid cells showed quantitative changes, with an increase in granulocyte numbers. Apoptosis of lymphocytes and viral infection of erythroblasts were also observed. The main changes in stroma were depletion of T lymphocytes in the middle phase of the experiment and macrophages. Viral infection was also observed in these cells. MK lesions suggest dysmegakaryocytopoiesis, which would aggravate the thrombocytopenia already present and could be responsible for it. Granulocyte changes would lead to the appearance of circulating immature forms, whereas lymphocyte apoptosis in bone marrow would contribute to lymphopenia.
DOI:10.1681/ASN.2013080831URLPMID:24357672 [本文引用: 1]
Abstract Human pluripotent stem cells (hPSCs) can generate a diversity of cell types, but few methods have been developed to derive cells of the kidney lineage. Here, we report a highly efficient system for differentiating human embryonic stem cells and induced pluripotent stem cells (referred to collectively as hPSCs) into cells expressing markers of the intermediate mesoderm (IM) that subsequently form tubule-like structures. Treatment of hPSCs with the glycogen synthase kinase-3 inhibitor CHIR99021 induced BRACHYURY(+)MIXL1(+) mesendoderm differentiation with nearly 100% efficiency. In the absence of additional exogenous factors, CHIR99021-induced mesendodermal cells preferentially differentiated into cells expressing markers of lateral plate mesoderm with minimal IM differentiation. However, the sequential treatment of hPSCs with CHIR99021 followed by fibroblast growth factor-2 and retinoic acid generated PAX2(+)LHX1(+) cells with 70%-80% efficiency after 3 days of differentiation. Upon growth factor withdrawal, these PAX2(+)LHX1(+) cells gave rise to apically ciliated tubular structures that coexpressed the proximal tubule markers Lotus tetragonolobus lectin, N-cadherin, and kidney-specific protein and partially integrated into embryonic kidney explant cultures. With the addition of FGF9 and activin, PAX2(+)LHX1(+) cells specifically differentiated into cells expressing SIX2, SALL1, and WT1, markers of cap mesenchyme nephron progenitor cells. Our findings demonstrate the effective role of fibroblast growth factor signaling in inducing IM differentiation in hPSCs and establish the most rapid and efficient system whereby hPSCs can be differentiated into cells with features characteristic of kidney lineage cells. Copyright 2014 by the American Society of Nephrology.
DOI:10.1089/vim.2017.0010URLPMID:28514189 [本文引用: 1]
Abstract The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor- mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN- was upregulated slightly at 5 and 9 dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.
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DOI:10.1016/j.immuni.2005.09.015URL [本文引用: 1]
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