Absence of autophagy promotes apoptosis by modulating the ROS-dependent RLR signaling pathway in classical swine fever virus-infected cells

A growing number of studies have demonstrated that both macroautophagy/autophagy and apoptosis are important inner mechanisms of cell to maintain homeostasis and participate in the host response to pathogens. We have previously reported that a functional autophagy pathway is trigged by infection of classical swine fever virus (CSFV) and is required for viral replication and release in host cells. However, the interplay of autophagy and apoptosis in CSFV-infected cells has not been clarified. In the present study, we demonstrated that autophagy induction with rapamycin facilitates cellular proliferation after CSFV infection, and that autophagy inhibition by knockdown of essential autophagic proteins BECN1/Beclin 1 or MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) promotes apoptosis via fully activating both intrinsic and extrinsic mechanisms in CSFV-infected cells. We also found that RIG-I-like receptor (RLR) signaling was amplified in autophagy-deficient cells during CSFV infection, which was closely linked to the activation of the intrinsic apoptosis pathway. Moreover, we discovered that virus infection of autophagy-impaired cells results in an increase in copy number of mitochondrial DNA and in the production of reactive oxygen species (ROS), which plays a significant role in enhanced RLR signaling and the activated extrinsic apoptosis pathway in cultured cells. Collectively, these data indicate that CSFV-induced autophagy delays apoptosis by downregulating ROS-dependent RLR signaling and thus contributes to virus persistent infection in host cells.     

通过基因组定量研究猪瘟病毒在细胞中的增殖特性

应用间接免疫荧光、Real-time PCR和病毒感染滴度(TCID50)测定技术,分别从病毒抗原、病毒基因组RNA复制水平和病毒感染滴度变化3个方面,研究了猪瘟病毒(CSFV)在PK-15细胞中增殖的特点,用猪瘟病毒石门株感染96孔板培养的细胞,1×102个TCID50/孔,间接免疫荧光检测结果显示感染后8h能检测到被荧光抗体染色的感染细胞,随感染时间的延长,出现荧光的细胞数量逐渐增多,在感染后72h,几乎所有细胞均能出现荧光。Real-time PCR结果显示在细胞感染初期的8~24h,病毒的基因组RNA复制呈加速趋势,其拷贝数在感染后72h达到高峰。此外,在感染后8h能检测到病毒基因组负链RNA转录,不过负链RNA在病毒增殖过程中维持在较低的水平。TCID50测定结果表明CSFV的感染滴度增加趋势与基因组类似,在病毒感染8h后能检测到具有感染性的子代病毒,感染滴度在8~20h之间逐渐增长,24~48h之间增长速度稍减慢,在感染后48~52h达到高峰,能在72h之内维持较高的感染滴度。     

六种检测猪瘟病毒方法的比较

【目的】本研究旨在比较6种检测猪瘟病毒方法的优缺点。【方法】应用病毒分离、胶体金免疫层析试纸条、抗原捕捉ELISA、反转录-聚合酶链式反应(RT-PCR)、TaqMan荧光定量RT-PCR(RT-qPCR)和反转录-环介导等温扩增方法(RT-LAMP)等6种方法,分别对50份疑似猪瘟病料中的猪瘟病毒(Classical swine fevervirus,CSFV)进行检测。【结果】结果表明:RT-qPCR和RT-LAMP方法检出阳性样品数为13份,RT-PCR为11份,病毒分离为10份,抗原捕捉ELISA为9份,胶体金试纸条为8份;6种方法均检测为阳性8份,均为阴性37份。【结论】结果提示,在对猪瘟病毒进行检测时,RT-qPCR、RT-LAMP和RT-PCR由于其灵敏性高,可作为首选检测方法,但操作时需要避免假阳性的出现;病毒分离方法虽然操作繁琐,但结果准确,是确诊猪瘟必不可少的检测方法;抗原捕捉ELISA和胶体金试纸条检测时间较短,由于其敏感性较低所限,主要用于对畜群进行检测,不适合个体检测。     

猪瘟病毒C株共感染口蹄疫病毒影响口蹄疫病毒复制

【背景】猪瘟(Classical Swine Fever)是由猪瘟病毒(Classical Swine Fever Virus,CSFV)引起的猪高度接触性传染病,致死率极高。在临床中存在着CSFV与猪其他病原菌共感染的情况,例如CSFV与口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)的共感染。【目的】利用CSFV与FMDV共感染猪源宿主细胞,研究CSFV与FMDV共感染对FMDV病毒复制的影响。【方法】构建体外共感染细胞模型,在正常PK-15细胞上进行CSFV共感染FMDV实验,通过观察细胞病变效应(Cytopathic Effect,CPE)、实时荧光定量PCR(RT-qPCR)、Western Blot、间接免疫荧光检测CSFV和FMDV共感染及FMDV单独感染情况下FMDV复制水平的差异。利用RT-qPCR筛选鉴定能够影响FMDV复制的CSFV蛋白。【结果】CSFVC株共感染FMDV能够抑制FMDV的复制,而且灭活的CSFV同样抑制FMDV的复制。通过筛选鉴定出CSFV的C蛋白能够抑制FMDV复制。【结论】研究发现CSFV C株共感染FMDV能够抑制FMDV复制,而其C蛋白具有抑制FMDV复制的能力。     

Genetic variation in the 5' end and NS5B regions of classical swine fever virus genome among Japanese isolates.

Sixteen clinical strains of classical swine fever virus (CSFV) isolated in Japan were subjected to analyses of nucleotide sequence variations in the 5' end and NS5B regions of the genome. These isolates were divided into three genovars, CSFV-1, CSFV-2 and CSFV-3, based on palindromic nucleotide substitutions at the three variable loci in the 5' untranslated region (UTR). Phylogenetic trees constructed from nucleotide sequences in the 5'-UTR and NS5B gene indicated that the CSFV strains were divided into three clusters, I, II and III. CSFV strains included in clusters I, II and III were identical to those in the CSFV-1, CSFV-2 and CSFV-3 genovars, respectively.     

反向遗传学技术在猪瘟病毒研究中的应用

猪瘟目前在许多国家流行并对养猪业造成巨大损失。虽然常规疫苗(如中国猪瘟兔化弱毒疫苗,即C株)在猪瘟防控中发挥巨大作用,但近年来在猪瘟防控中出现的新情况,如非典型感染、持续性感染及免疫失败等;同时目前世界上许多国家正开展的猪瘟扑灭计划使得弱毒疫苗的应用受到很大限制。因此,加强猪瘟病毒在致病机理、传播机制等方面的研究以及加快新型猪瘟疫苗的开发是当务之急。近年来,反向遗传学技术的发展为猪瘟病毒基因功能研究和疫苗制备方面开辟了新思路。以下回顾了反向遗传操作技术在猪瘟病毒基因功能研究与标记疫苗株构建方面的研究进展,同时提出了该领域目前面临的问题,并对其未来发展方向进行了展望。     

表达猪圆环病毒2型Cap蛋白的重组猪瘟兔化弱毒疫苗株的构建与鉴定

猪瘟(CSF)是由猪瘟病毒(CSFV)引起的一种毁灭性传染病,给养猪业造成重大经济损失。猪瘟兔化弱毒疫苗(C株)是一株非常安全、有效的优秀弱毒疫苗,对各年龄和品种的猪都极其安全,同时对不同基因亚型的CSFV均能提供有效的免疫保护。在现地,CSFV和猪圆环病毒2型(PCV2)混合感染的现象时常发生,有必要研制针对这两种病毒混合感染的二价疫苗。本研究首次构建了表达PCV2 Cap蛋白的重组C株,并评价了其在体内外的特性。结果表明,该重组病毒与C株具有相近的体外增殖特征,能够稳定表达Cap蛋白,在家兔体内具有与C株相似的生物学表型,在免疫家兔后10 d,抗CSFV E2抗体全部转阳,然而抗Cap抗体未能转阳。本研究为进一步优化表达PCV2Cap蛋白的重组C株奠定了基础。     

An indirect ELISA of classical swine fever virus based on quadruple antigenic epitope peptide expressed in <Emphasis Type="Italic">E.coli</Emphasis>

In this study, a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV) E2 glycoprotein was expressed in E. coli to a obtain target protein. This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs. The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve (ROC) analysis based on 30 negative sera and 80 positive samples. The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination (IHA) test. The inter-assay and intra-assay coefficients of variation (CVs) for 16 sera were both ⩽6.8%. No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus (BVDV) antibodies was observed.     

特异寡聚核苷酸对猪瘟病毒在细胞中增殖抑制作用的研究

本实验探讨了寡聚核苷酸对CSFV复制的影响以及作为抗CSFV新型药物的可行性。实验结果表明针对CSFV5'um端非编码区NS3蛋白丝氨酸蛋白酶功能区的寡聚核酸对CSFV复制均有显著的抑制作用,而针对CSFV3' 端非编码区寡聚核苷酸仅有轻微抑制作用,5'端寡聚核苷酸具有最佳抑制作用,同时发现相应序列中正义聚核苷酸的作用要优于反义寡聚核苷酸;脂质体介导转染能显著提高寡聚核苷酸对CSF复制的抑制作用。这些初步结果也提示,CSFV5'端和3'端非编码区对CSFV复制的重要性和作用有所不同。     

新型猪瘟疫苗研究进展

猪瘟是由猪瘟病毒引起猪的一种急性、热性和高度接触性传染病.该病呈世界性分布,给世界养猪业造成了巨大的经济损失.目前,疫苗接种仍然是防控猪瘟的主要手段.虽然传统的猪瘟弱毒疫苗(如C株)安全有效,但猪瘟的临床表现发生了很大变化,呈现典型猪瘟和非典型猪瘟共存、隐性感染和持续感染并现,免疫失败的现象时有报道,且不能区分野毒感染和免疫接种.因此,研制安全、高效、能区分野毒感染和疫苗免疫动物(DIVA)的新型猪瘟疫苗极为必要.文中就近年来开发的核酸疫苗、病毒活载体疫苗、基于蛋白/肽的疫苗、基因缺失疫苗、嵌合瘟病毒疫苗等新型DIVA猪瘟疫苗作一综述.     

表达猪瘟病毒E2蛋白的BacMam病毒的构建及其免疫原性分析

含具有哺乳动物细胞活性的启动子的重组杆状病毒(BacMam病毒)可有效转导多种哺乳动物细胞,并被广泛用于开发新型非复制型载体疫苗．将水泡性口炎病毒G蛋白(VSV-G)基因插入多角体启动子下游,得到经修饰的杆状病毒转移载体,将对虾白斑综合症病毒(WSSV)ie1启动子控制下的猪瘟病毒E2基因表达盒插入此载体中,构建了BacMam病毒BacMam/G-ie1-E2,以其感染Sf9细胞和转导HeLa细胞,通过间接免疫荧光试验和Western blot分析检测E蛋白的表达,同时用BacMam病毒直接免疫小鼠,用检测猪瘟病毒抗体的间接ELISA方法检测免疫小鼠血清抗体,用基于CFSE和WST-8的淋巴细胞增殖试验评价其细胞免疫应答．结果显示,BacMam/G-ie1-E2能同时在昆虫细胞和哺乳动物细胞中高效表达E2蛋白,免疫小鼠能诱导产生针对猪瘟病毒的特异性抗体,免疫小鼠脾细胞经猪瘟病毒刺激后能诱导特异性的淋巴细胞增殖．这表明,由BacMam病毒介导的基因转移有望用于开发针对猪瘟病毒的非复制型载体疫苗．     

Synthesis and antiviral activity of a novel glycosyl sulfoxide against classical swine fever virus

A novel compound—2″,3″,4″,6″-tetra-O-acetyl-β-d-galactopyranosyl-(1→4)-2′,3′,6′-tri-O-acetyl-1-thio-β-d-glucopyranosyl-(5-nitro-2-pyridyl) sulfoxide—designated GP6 was synthesized and assayed for cytotoxicity and in vitro antiviral properties against classical swine fever virus (CSFV) in this study. We showed that the examined compound effectively arrested CSFV growth in swine kidney cells (SK6) at a 50% inhibitory concentration (IC₅₀) of 5 ± 0.12 μg/ml without significant toxicity for mammalian cells. Moreover, GP6 reduced the viral E2 and E^rns glycoproteins expression in a dose-dependent manner. We have excluded the possibility that the inhibitor acts at the replication step of virus life cycle as assessed by monitoring of RNA level in cells and culture medium of SK6 cells after single round of infection as a function of GP6 treatment. Using recombinant E^rns and E2 proteins of classical swine fever virus produced in baculovirus expression system we have demonstrated that GP6 did not influence glycoprotein production and maturation in insect cells. In contrast to mammalian glycosylation pathway, insect cells support only the ER-dependent early steps of this process. Therefore, we concluded that the late steps of glycosylation process are probably the main targets of GP6. Due to the observed antiviral effect accompanied by low cytotoxicity, this inhibitor represents potential candidate for the development of antiviral agents for anti-flavivirus therapy. Further experiments are needed for investigating whether this compound can be used as a safe antiviral agent against other viruses from unrelated groups.     

猪瘟DNA疫苗研究进展

自1990年DNA疫苗问世以来,已有许多研究者构建了不同类型的DNA疫苗。这些载体能诱发机体产生不同程度的特异性体液免疫和(或)细胞免疫。研究者们也在猪瘟DNA疫苗研究方面做出了很多努力并取得了一定的成果。以下从猪瘟DNA疫苗的构建和评价、佐剂在猪瘟DNA疫苗中的应用、猪瘟DNA疫苗与其他疫苗的联合应用以及目前猪瘟DNA疫苗存在的问题和解决途径等方面做了比较全面的阐述。     

猪瘟病毒Core蛋白核仁定位序列鉴定

黄病毒科病毒核衣壳蛋白的核仁定位在病毒颗粒包装与病毒复制中发挥重要作用。为鉴定黄病毒科的猪瘟病毒Core蛋白核仁定位序列,本研究构建了将Core蛋白、截短突变体和氨基酸位点突变体分别与增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP )融合的真核表达质粒,转染至PK15细胞后进行表达和定位分析,结果显示 Core蛋白核仁定位序列为PESRKKL,其关键氨基酸为R76K77,对理解猪瘟病毒Core蛋白结构与功能和为后续研究Core蛋白在病毒复制及颗粒包装中的作用有重要意义。     

Development of multiple elisas for the detection of antibodies against classical swine fever virus in pig sera

The major immunogenic proteins (Ems,E2 and NS3) of classical swine fever virus (CSFV) (Shimen strain) were expressed in E.coli and purified by affinity chromatography.The recombinant antigens were appl...     

非洲猪瘟病毒D1133L蛋白增加宿主波形蛋白磷酸化而促进病毒在猪巨噬细胞中的复制

非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)感染引起家猪和野猪的一种高死亡率的传染性疾病。ASFV具有庞大的基因组,其中非结构蛋白pD1133L被预测为其编码的6个解旋酶之一。本实验室应用免疫沉淀-质谱联用(immunoprecipitation-mass spectrometry, IP-MASS)技术筛选与pD1133L互作的宿主细胞蛋白,发现细胞波形蛋白(vimentin, VIM)为pD1133L互作的宿主蛋白之一,但尚不清楚宿主蛋白VIM对ASFV复制的影响。【目的】探究ASFV与VIM的相互调控作用,揭示VIM促进ASFV复制的机制。【方法】通过免疫共沉淀(co-immunoprecipitation, Co-IP)试验验证pD1133L与VIM存在互作关系;外源过表达VIM蛋白以及设计并合成VIM的siRNA探究VIM对ASFV复制的影响;利用Western blotting以及荧光定量PCR (quantitative real-time PCR, qPCR)方法检测ASFV对VIM蛋白水平以及转录水平的影响;通过Western blotting、间接免疫荧光试验(immunofluorescence assay, IFA)探究巨噬细胞感染ASFV后VIM磷酸化水平变化以及亚细胞定位变化情况;CCK-8试剂盒检测VIM磷酸化抑制剂KN-93处理的最佳浓度,并利用Western blotting以及IFA检测KN-93对VIM磷酸化、亚细胞定位以及对ASFV复制影响。【结果】VIM过表达促进ASFV复制,敲低VIM的表达则抑制ASFV复制;ASFV感染抑制VIM蛋白水平以及转录水平表达,且呈时间依赖性;ASFV感染后VIM发生磷酸化修饰且发生亚细胞定位改变,从而促进ASFV复制。【结论】证实了ASFV与宿主蛋白VIM之间的相互调控作用;初步确定ASFV感染后VIM受到ASFV pD1133L调控,亚细胞定位发生重排向核周聚集从而促进ASFV复制的机制。