靶向TLR9基因siRNA的筛选及其对BHV-1增殖规律的影响

为研究牛胚胎气管（bovine embryonic tracheal,EBTr）细胞Toll样受体9（Toll-like receptors 9,TLR9）在牛疱疹病毒Ⅰ型（bovine herpesvirus type 1,BHV-1）感染的天然免疫反应中的作用,本试验利用小分子干扰RNA（siRNA）技术,以TLR9为靶向分别设计并合成3条siRNA干扰序列,用SYBR GreenⅠ实时荧光定量PCR技术检测应用siRNA-TLR9干扰后TLR9基因的表达变化,筛选出最佳的siRNA-TLR9干扰序列,继而转染EBTr细胞使其TLR9基因沉默后感染BHV-1,用TaqMan实时荧光定量PCR方法检测BHV-1不同时间点的增殖变化。结果显示,转染48 h后,与阴性对照组相比,siRNA-TLR9A、siRNA-TLR9B和siRNA-TLR9C分别对TLR9 mRNA表达量下调了60.90%、24.05%和40.75%。筛选出siRNA-TLR9A片段在12~72 h可以显著抑制TLR9 mRNA表达（P < 0.05）。用该片段转染EBTr细胞再感染BHV-1后,6~72 h siRNA-TLR9A组BHV-1 DNA拷贝数低于对照组。本试验成功筛选出了特异性的抑制EBTr细胞TLR9基因的siRNA序列,并证明抑制TLR9的表达可降低BHV-1的增殖能力。     

BHV-1感染牛巨噬细胞对CD300a转录的影响

为探究抑制性受体CD300a在牛Ⅰ型疱疹病毒(bovine herpesvirus 1,BHV-1)免疫调控机制中的作用,分离鉴定了1株BHV-1内蒙古株,并建立CD300a基因的荧光定量PCR方法,检测BHV-1感染牛单核巨噬细胞后0,6,12,24,48,72 h 6个时间点以及空白对照组CD300a基因的转录变化。将β-actin基因作为内参,使用双标准曲线法计算攻毒组与对照组CD300a基因各时间段的相对表达量,分析CD300a在攻毒前、后的表达差异。结果显示,BHV-1分离株在MDBK细胞上出现典型细胞病变效应,测得其病毒滴度为10^7.23 TCID₅₀/mL。本研究建立的CD300a基因荧光定量PCR检测方法,CD300a基因和β-actin基因标准品的Ct值与模板浓度之间均具有良好的线性关系,溶解曲线均为特异单峰,最低检测限均为10拷贝/μL,具有良好的灵敏性,可以准确检测出BHV-1攻毒后巨噬细胞内的CD300a基因表达量。经计算,攻毒组CD300a表达量均高于对照组,其中,6,12,24 h的表达差异倍数显著高于0,72 h...     

高致病性禽腺病毒4型中国流行株在LMH细胞中的增殖规律

为研究禽腺病毒4型(FAd V-4)中国流行株在鸡肝癌上皮细胞系(LMH)中的生长特性和增殖规律,分别利用SYBR Green I实时荧光定量PCR和病毒滴度测定方法检测FAd V-4感染LMH细胞后12、24、36、48、60、72、84、96和120 h的基因组复制和病毒粒子增殖动态。结果显示,FAd V-4中国流行株在LMH中能够很好增殖,不同时间点的病毒滴度与病毒基因组拷贝数呈正相关,病毒收获的最佳时间为感染后60 h。研究结果为防控鸡肝炎-心包积液综合征疫苗的研发和生产提供一定的依据和参考。     

牛坏死杆菌对EPH4细胞的增殖抑制和诱导凋亡作用

为探究牛坏死杆菌对小鼠乳腺上皮细胞系(EPH4细胞)增殖和凋亡的影响,本研究将坏死杆菌(牛A25菌株)以感染复数(MOI)为100感染EPH4细胞,用EdU细胞增殖法检测细胞增殖率,用Hoechst染色以及DNA Ladder检测细胞凋亡,并用实时荧光定量PCR(RT-qPCR)检测凋亡基因的mRNA相对表达量变化。EdU检测结果显示,坏死杆菌可抑制EPH4细胞增殖;Hoechst染色以及DNA Ladder检测结果均显示,坏死杆菌可促进细胞凋亡的发生;RT-qPCR结果显示,与对照组相比,坏死杆菌感染EPH4细胞2 h时,促凋亡基因Bax、Bax/Bcl-2的mRNA相对表达量显著上调(P<0.05),感染4 h时,促凋亡基因Caspase-3和Caspase-9的mRNA相对表达量极显著上调(P<0.001),感染6 h时,促凋亡基因AIF的mRNA相对表达量极显著上调(P<0.001)。由此可见,牛坏死杆菌能够抑制EPH4细胞增殖且诱导细胞凋亡,为进一步研究坏死杆菌致病机制提供了相关数据支持。     

牛轮状病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立及应用

旨在建立一种SYBR GreenⅠ实时荧光定量PCR(RT-qPCR)检测牛轮状病毒(BRV)的方法,并采用针对BRV vp6基因序列保守区设计的引物,使用RT-qPCR方法进行目的基因扩增,优化反应条件,制备阳性标准品,建立标准曲线,确定重复性、敏感性和特异性,与常规PCR方法比较.结果显示,建立的RT-qPCR方法...     

关岭牛MyoD Ⅰ基因在不同组织和时期中的表达分析

为研究MyoDⅠ基因在关岭牛不同组织表达量的差异及在肌肉发育过程中的表达情况,试验采用实时荧光定量PCR技术对关岭牛背最长肌、大腿肌、心脏、肝脏、脂肪、小肠6个组织的MyoDⅠ基因相对表达量进行检测。同时分析关岭牛胎儿、18月龄、30月龄时期背最长肌组织中MyoDⅠ基因的表达规律。结果显示,MyoDⅠ基因在背最长肌中表达量最高,在出生后的关岭牛肌肉组织中也具有较高的表达量,且随着出生时间的推移呈上升趋势。推测可能由于背最长肌是肌肉富集地方,因此基因表达量较高,随着年龄增大,肌肉丰满度增加,基因表达量也随着增加。     

马疱疹病毒1型SYBR Green Ⅰ荧光定量PCR检测方法的建立

为建立马疱疹病毒Ⅰ型(EHV-1)的检测方法,本研究以EHV-1 gB基因的一段保守区域(1207 bp～1509 bp)作为检测的目的片段设计引物,通过对其反应条件的优化,建立了特异性检测EHV-1的SYBR Green I 荧光定量PCR方法.实验结果表明:该方法检测目的基因的灵敏度下限为10拷贝/μL,比常规PCR方法高100倍;与马疱疹病毒4型(EHV-4)及其他马传染病病原体无交叉反应;组内及组间的变异系数均小于2％.该方法检测速度快及高敏感性的特点为马鼻肺炎的防制提供了有力保障,同时也为进一步开展马鼻肺炎相关的研究提供了有效的辅助检测方法技术.     

牛冠状病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及初步应用

针对牛冠状病毒(BCoV)N基因保守序列设计合成了1对特异性引物,以体外转录法制备的cDNA标准品为模板,建立了检测BCoV的SYBR GreenⅠ实时荧光定量PCR方法。在1个反应体系中模板最低检测限为7.8 copies/μL,比普通RT-PCR更加灵敏;与牛主要消化道和呼吸道病毒病病原均无交叉反应;批内、批间重复变异系数均低于1.5%;应用定量PCR检测了39份疑似临床病料,阳性检出率为51.28%(20/39),普通RT-PCR为41.03%(16/39)。结果表明,该方法特异性强、稳定性好、准确率高,本方法将为BCoV的临床诊断和流行病学调查提供技术保障。     

袋鼠疱疹病毒1型实时荧光PCR方法的建立

为实现袋鼠临床样本中疱疹病毒1型(Macropodid herpesvirus 1,MaHV-1)的出入境快速检测,基于MaHV-1的gB基因序列设计特异引物和TaqMan探针,建立了一种MaHV-1荧光PCR检测方法。试验结果显示,该方法只对MaHV-1 gB基因呈现特异性扩增,与禽传染性喉气管炎病毒、伪狂犬病毒和牛传染性鼻气管炎病毒不发生交叉反应,对阳性标准质粒对照(pCR-MaHV-1-gB)的最低检测限为8个拷贝数/反应。该方法的组内和组间试验的Ct值变异系数介于0.17%～0.96%之间,具有良好的重现性。试验结果表明,本研究建立的实时荧光PCR方法可用于袋鼠MaHV-1的病原学检测。     

牛疱疹病毒gD-PCR鉴别检测方法的建立

建立一种PCR技术,既能快速检测疱疹病毒1型成员牛传染性鼻气管炎病毒(bovine infectious rhinotracheitisvirus,IBRV),又能区分牛疱疹病毒5型和伪狂犬病毒。根据基因库中牛传染性鼻气管炎病毒的gD基因序列,应用primer 5.0软件设计了gD PCR引物,建立PCR方法,反应条件是:94℃预变性5min,94℃1min,58℃1min,72℃1min,30个循环,72℃7min,4℃5min。该方法能从IBRV阳性样本和参考毒株中扩增出372bp的目的片段,而从同属的牛疱疹病毒5型中扩增出440bp和206bp两条目的片段,从同属的伪狂犬病毒中扩增出303bp的目的片段,但不能从非疱疹病毒属成员猪呼吸与繁殖综合征病毒中扩增出目的条带。该PCR检测IBRV的灵敏度可达1PFU/mL以上。鉴于其灵敏度高、特异性好,可望在牛疱疹病毒感染快速鉴别检测方面发挥重要作用。     

Bovine herpesvirus type 1 abortions detected by a semi-nested PCR in Brazilian cattle herds

Bovine herpesvirus type 1 (BHV-1) was investigated by a semi-nested polymerase chain reaction (SN-PCR) and by MDBK cell culture virus isolation in organ fragments from 55 aborted fetuses collected from beef and dairy cattle herds with history of reproductive problems in the North of Paraná State, Brazil. A 425 bp amplicon of the BHV-1 glycoprotein D gene was detected in 14 (25.4%) aborted fetuses. BHV-1 was isolated in MDBK cells from the tissue of 5 (9.1%) fetuses. The specificity of positive results was evaluated by Restriction Fragment Length Polymorphism (RFLP) with Bgl I restriction of DNA amplified by SN-PCR, and by virus-neutralization and immunofluorescence with rabbit anti-BHV-1 polyclonal antibodies for virus isolated in cell culture. The results of this work demonstrate the importance of using other diagnostic techniques, like SN-PCR, for BHV-1 detection in organ fragments from aborted fetuses and the high frequency of this virus in reproductive failures in Brazilian cattle herds.     

鲤科疱疹病毒2型TaqMan荧光定量PCR检测方法的建立

为建立快速和敏感的检测鲤科疱疹病毒2型(CyHV-2)的方法,本研究根据CyHV-2 DNA聚合酶基因序列合成引物和TaqMan探针,建立了CyHV-2荧光定量PCR检测方法.结果显示,以重组质粒为标准品建立的标准曲线在5拷贝/μL～5×108拷贝/μL具有良好的线性关系,相关系数为0.999;其最低检出量为5拷贝/μL,比普通PCR的敏感度高100倍.该方法仅对CyHV-2的靶基因序列进行扩增,而对锦鲤疱疹病毒、流行性造血器官坏死病病毒、病毒性出血性败血症病毒、传染性胰脏坏死病病毒及鲤春病毒血症病毒核酸扩增结果均为阴性.组内和组间重复试验变异系数的平均值分别为0.5％和0.3％,具有良好的重复性.与常规PCR相比较,该方法具有快速、敏感、特异及高通量检测等优点,适用于对CyHV-2的快速检测.     

一例犊牛呼吸道疾病的病原学诊断

[目的] 2023年8月,天津某牛场暴发以呼吸道症状为突出特征的传染病。为了确定病因,对发病犊牛进行相关病原的病原学诊断。[方法] 采集病死犊牛肺部病料,采用PCR方法对牛副流感病毒3型（BPIV-3）、牛呼吸道合胞体病毒（BRSV）、牛疱疹病毒1型（BHV-1）、牛腺病毒3型（BAdV-3）、牛分枝杆菌（Mycobacterium bovis）、牛肺炎克雷伯菌（Bovine Klebsiella pneumoniae）、牛支原体（Mycoplasma bovis）7 种牛呼吸道病原进行检测。[结果] 样本检测出BHV-1、BAdV-3共2 种病原,存在混合感染。其他病原未检出。[结论] 本研究证实BHV-1、BAdV-3在该牛场中存在和流行,为犊牛呼吸道疾病防控提供参考。     

Dynamics of infection and immunity in a dairy cattle population undergoing an eradication programme for Infectious Bovine Rhinotracheitis (IBR)

Several countries within the European Union (EU) have successfully eradicated Infectious Bovine Rhinotracheitis (IBR), while others (e.g. Germany) are making efforts to achieve IBR-free status. EU member states IBR eradication programmes must meet Community legislation requirements that ban breeding farms from purchasing positive animals, from using whole-virus IBR vaccines, and from inseminating cows with semen from positive bulls. A follow-up study from 2002 to 2005 was carried out in the province of Trento (Italy), where a compulsory programme for IBR eradication was started in 1998. IBR outbreaks (identified on the basis of seroconversion of sentinel animals) were concentrated in larger positive herds. A higher incidence was recorded between 2003 and 2004. An association between markedly high temperatures in the summer of 2003 and virus reactivation has been suggested but is yet to be confirmed. The practice of driving cattle to common alpine pastures for the summer season did not play a significant epidemiological role in IBR transmission. Premising that only seronegative animals are allowed to enter dairy farms, animal movement increases the infection risk to a moderate extent. The long-term persistence of IBR antibodies was more pronounced in animals positive for antibodies to the glycoprotein E (gE). Scattered seroconversions, occurring mostly in positive herds, require careful interpretation in order to avoid overestimating the incidence of the infection at herd level.     

Bovine herpesvirus 5 BICP0 complements the bovine herpesvirus 1 homolog

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related (82% amino acid identity) but differ strongly in neuropathogenesis. The immediate-early gene for BICP0 is less conserved (70% amino acid identity) and may contribute to a dissimilar phenotype. A peculiar difference is a guanosine hexamer in the BICP0-1 gene which aligns with only five guanosines in the BICP0-5 gene and therefore results in a frameshift in the latter open reading frame. Thus, the C-terminal amino acid sequence (residues 643–676 of BICP0-1 vs. 655–720 of BICP0-5) is completely different. We introduced the BICP0-5 frameshift into the BoHV-1 genome cloned as a bacterial artificial chromosome (BoHV-1 BAC) using the Red recombination system with galK selection and counterselection. Transfection of MDBK cells with the resulting BAC produced recombinant virus that replicated like wild type BoHV-1 in vitro. Attempts to exchange the entire BICP0-1 gene by the BoHV-5 homolog using the same approach failed repeatedly. Therefore, we cotransfected purified BICP0⁻/galK⁺-BoHV-1 BAC DNA with a recombination plasmid coding for BICP0-5 with or without a HA tag into MDBK cells. BoHV-1 recombinants expressing the respective proteins were characterized. In vitro, all recombinants grew to similar titers as the parental viruses, which demonstrates that BICP0-5 compensates for the growth defect of BICP0⁻/galK⁺-BoHV-1 and functionally complements BICP0-1 of BoHV-1. We conclude that BICP0 may be suitable to positively select BoHV-1 recombinants with deletions or insertions of additional genes of interest.