猪瘟病毒非结构蛋白NS5A与Beclin1相互作用并促进病毒增殖

为探究宿主蛋白Beclin1在猪瘟病毒(classical swine fever virus, CSFV)非结构蛋白NS5A激活细胞自噬反应过程中的作用及具体分子机制,本研究在感染CSFV及表达NS5A蛋白的ST细胞中,利用qRT-PCR方法检测Beclin1、PI3K/Akt通路相关因子表达变化情况;利用激光共聚焦、Co-IP及GST-pulldown等方法研究Beclin1与NS5A相互作用关系;通过在ST细胞中过表达或敲低Beclin1,研究其对CSFV复制的影响。结果表明,ST细胞感染猪瘟病毒或外源表达NS5A蛋白,Beclin1转录和蛋白表达水平均显著升高,且PI3K/Akt通路相关因子表达水平与之呈正相关。此外,CSFV NS5A蛋白与Beclin1蛋白在细胞中存在共定位且具有相互作用。最后,作者发现在细胞中过表达Beclin1,对CSFV复制起到明显促进作用;反之,利用siRNA敲低Beclin1后,抑制PI3K/Akt通路活化,CSFV增殖表现出明显抑制效应。以上结果表明,Beclin1蛋白对CSFV复制具有促进作用,其机制是通过与NS5A的相互作用调控PI3K/Ak...     

Characterization of the C-terminal sequence of NS5A necessary for the assembly and production of classical swine fever virus infectious particles

Recent studies show that classical swine fever virus (CSFV) NS5A is an essential replicase component, but it is not known how NS5A participates in viral particle production. In this study, deletion and substitution mutations were introduced into the C-terminus of CSFV NS5A. The efficiency of Core protein release and extracellular and intracellular infectivity levels were assessed and NS5A–Core interaction was investigated. These results suggested that CSFV NS5A was a key factor for the assembly of infectious CSFV particles. The C-terminal sequence from amino acids 478 to 487 and amino acids S481 and T482 were necessary for CSFV assembly and production. The effect of NS5A on CSFV assembly and production might be related to NS5A–Core interaction. T482 was found to be conserved in the C-terminus of NS5A proteins of pestiviruses and hepatitis C virus (HCV), therefore suggesting that it might be important for these virus assembly and production.     

Characterization of classical swine fever virus associated with defective interfering particles containing a cytopathogenic subgenomic RNA isolated from wild boar

Classical swine fever virus (CSFV) strain WB82, isolated from a wild boar in 1982, induced a distinct cytopathic effect (CPE) in primary swine testicle cell culture and in most of the porcine cell lines. This strain of CSFV was found to be composed of two biotypes. cytopathogenic (cp) CSFV, as a minor population, and noncytopathogenic (noncp) CSFV, as a major population. The noncp CSFV (designated strain WB82/E+) was obtained by biological cloning, and it showed the exaltation of Newcastle disease virus phenomenon. In Northern blot analysis and RT-PCR assay, CSFV RNA with a subgenomic (sg) length was detected in addition to full-length viral RNA only in the cells in which a CPE had been revealed. These RNAs represent the genomes of typical defective interfering (DI) particles because of the strict dependence on a complementing helper virus and interference with replication of the helper virus. The sg RNA, which exhibits the genomes of the DI particles, lacked the nucleotides of the viral genomic region from Npro to NS2 (4764 bases). When extracted sg RNA was transfected to the cells infected with the WB82/E+ strain, a distinct CPE was observed. Interestingly, the CPE was observed in cells infected with other heterologous noncp CSFV ALD and GPE- strains by sg RNA transfection. The results suggested that these noncp CSFVs act as helper viruses for the replication of sg RNA (DI particles). It was also shown that the cytopathogenicity of strain WB82 is caused by apoptosis.     

甲型流感病毒NS1蛋白研究进展

甲型流感病毒(Influenza A virus,IAV)是引起每年季节性流感及偶尔的流感大流行的主要病原体,严重危害公共卫生和社会经济.IAV属于分节段的单股负链RNA病毒,其基因组编码多种不同的蛋白质,其中非结构蛋白1(non-structural protein 1,NS1)是IAV的调节宿主免疫应答和病毒致病性...     

牛病毒性腹泻病毒非结构蛋白NS3的研究进展

非结构蛋白NS3是致细胞病变型牛病毒性腹泻病毒的分子标记蛋白,它具有多种生物学功能。研究结果表明,NS3在病毒非结构蛋白的加工、成熟、基因组复制与转录过程中以及宿主细胞致细胞病变效应的产生中起重要作用。文章总结了NS3的生物学特性及其相关功能,为阐明NS3的致细胞病变作用机理奠定基础。     

猪瘟病毒石门株NS3蛋白的原核表达及其抗体检测间接ELISA的建立

为配合猪瘟新型疫苗的研发,建立了猪瘟病毒NS3蛋白抗体检测间接ELISA,以期达到有效区分新型疫苗免疫猪与自然感染猪(包括常规疫苗接种猪)的目的。以接种猪瘟病毒(CSFV)石门株的PK-15细胞为模板提取总RNA,经特异性PCR扩增获得长度为2 049bp的CSFV NS3基因,将其克隆至插入了具有自聚集自切割功能短肽的原核表达载体pET-32a(+),在大肠埃希菌Rosetta(DE3)中优化表达CSFV石门株NS3基因。Western blot分析表明重组蛋白NS3具有反应原性。将纯化的重组蛋白NS3作为包被抗原建立检测CSFV NS3抗体的间接ELISA,以美国爱德士(Idexx)猪瘟病毒抗体检测试剂盒抗体检测结果为标准,对502份血清样品进行检测。结果表明,所建立方法的特异性为96.9%,敏感性为89.7%,总符合率为95.8%,为猪瘟新型疫苗的推广应用提供了血清学检测方法。     

N蛋白磷酸化位点突变影响猪繁殖与呼吸综合征病毒的复制及亚基因组的转录

为探究猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）的N蛋白磷酸化修饰是否对病毒的复制及亚基因组的转录产生影响，将XH-GD PRRSV N蛋白的已知磷酸化位点Ser-105和Ser-120分别或同时突变为Ala，拯救突变病毒，命名为A105、A120、A105-120，测定其生长曲线和亚基因组的转录水平。结果显示：突变病毒在MARC-145的病毒复制速率与滴度显著低于亲本毒株（P<0.01或P<0.05），而在原代PAMs复制速率降低（P<0.01），不影响病毒最终的滴度。突变毒株的基因组RNA（genomic RNA，gRNA）转录水平显著降低（P<0.01或P<0.05），磷酸化位点突变能显著降低长链亚基因组mRNA（subgenomic RNA，sgmRNA）2、3的转录（P<0.01或P<0.05），但短链sgmRNA4、5表达量随时间呈增加趋势。以gRNA表达量为基准对各亚基因组表达量进行整体分析，发现N蛋白磷酸化显著影响了sgmRNA4和sgmRNA5的表达（P<0.01或P<0.05）。结果提示，N蛋白磷酸化位点突变影响PRRSV的复制及亚基因组转录。     

Absence of protection from West Nile virus disease and adverse effects in red legged partridges after non-structural NS1 protein administration

The red-legged partridge (Alectoris rufa) is a competent host for West Nile virus (WNV) replication and highly susceptible to WNV disease. With the aim to assess in this species whether the inoculation of non-structural protein NS1 from WNV elicits a protective immune response against WNV infection, groups of partridges were inoculated with recombinant NS1 (NS1 group) or an unrelated recombinant protein (mock group), and challenged with infectious WNV. A third group received no inoculation prior to challenge (challenge group). The NS1 group failed to elicit detectable antibodies to NS1 while in the mock group a specific antibody response was observed. Moreover, no protection against WNV disease was observed in the NS1 group, but rather, it showed significantly higher viral RNA load and delayed neutralizing antibody response, and suffered a more severe clinical disease, which resulted in higher mortality. This adverse effect has not been observed before and warrants further investigations.     

青蒿素与牛病毒性腹泻病毒NS5B蛋白的分子对接预测及其抗病毒作用

【目的】 采用分子对接以及体外试验确定青蒿素对牛病毒性腹泻病毒（Bovine viral diarrhea virus,BVDV）复制的抑制作用,为抗病毒药物和制剂开发提供新思路。【方法】 以BVDV-NS5B蛋白为作用靶点,通过PDB数据库检索蛋白三维晶体结构,并对其结构进行适当修饰处理;检索TCMSP数据库中的青蒿素结构,应用Autodock软件将两者进行分子对接及结合能打分。随后采用CCK-8试剂盒测定青蒿素对MDBK细胞的最大安全浓度;将选定浓度的青蒿素进行梯度稀释,采用先加病毒后加药物、先加药物后加病毒、中药和病毒同时作用的3种不同加药方式进行药物抗病毒试验,确定对病毒的最佳药物抑制浓度、预防浓度和杀灭浓度。应用实时荧光定量PCR法检测3种药物作用方式下BVDV的拷贝数,进一步明确药物对BVDV复制的作用。【结果】 分子对接数据表明青蒿素与BVDV-NS5B存在相互作用,结合自由能为-28.6748 kJ/mol。青蒿素在MDBK细胞上的最佳药物安全浓度为100 μmol/L。3种作用方式下青蒿素浓度为100 μmol/L时均可有效影响BVDV的复制,青蒿素对BVDV的抑制作用最为明显。【结论】 青蒿素可与BVDV-NS5B蛋白靶点互作,并能在MDBK细胞上有效抑制BVDV的复制,本研究为抗BVDV中药筛选奠定了基础。     

Suppression of bovine viral diarrhea virus replication by small interfering RNA and short hairpin RNA-mediated RNA interference

Bovine viral diarrhea virus (BVDV) is a ubiquitous viral pathogen that affects cattle herds' worldwide causing significant economic loss. The current strategies to control BVDV infection include vaccination (modified-live or killed) and control of virus spread by enhanced biosecurity management, however, the disease remains prevalent. With the discovery of the sequence-specific method of gene silencing known as RNA interference (RNAi), a new era in antiviral therapies has begun. Here we report the efficient inhibition of BVDV replication by small interfering (siRNA) and short hairpin RNA (shRNA)-mediated gene silencing. siRNAs were generated to target the 5' non-translated (NTR) region and the regions encoding the C, NS4B and NS5A proteins of the BVDV genome. The siRNAs were first validated using an EGFP/BVDV reporter system and were then shown to suppress BVDV-induced cytopathic effects and viral titers in cell culture with surprisingly different activities compared to the reporter system. Efficient viral suppression was then achieved by bovine 7SK-expressed BVDV-specific shRNAs. Overall, our results demonstrated the use of siRNA and shRNA-mediated gene silencing to achieve efficient inhibition of the replication of this virus in cell culture.     

Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein N^pro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE^-, N^pro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE^- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE^- vaccine virus in pigs, the IRF3-degrading function of N^pro was not recovered. Therefore, we examined whether restoring the ability of N^pro to block IFN-α/β induction of both the avirulent and moderately virulent GPE^--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in N^pro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional N^pro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional N^pro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.     

Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.     

Genetic and biological comparison of tick-borne encephalitis viruses from Hokkaido and far-eastern Russia

We compared the biological properties of Oshima 5-10 (tick-borne encephalitis [TBE] virus isolated in Hokkaido, Japan) and Sofjin-HO (Far-Eastern subtype TBE virus) including plaque formation, virus replication and virus protein synthesis in BHK-21 cell cultures to reveal strain differences. We also determined the complete nucleotide sequences of both strains and compared the deduced amino acid sequences. Plaques of Oshima 5-10 were smaller than those of Sofjin-HO. Virus titers in culture fluid of Oshima 5-10 were 1/100 of those of Sofjin-HO at 9 and 12 hr after infection. Less viral protein and RNA syntheses of strain Oshima 5-10 was observed than with Sofjin-HO. Genetic analysis revealed 1.4% of amino acids to differ with Sofjin-HO. No difference between the two strains was detected in the motif sequence of the viral enzyme, cleavage sites of viral protein or glycosylation sites of NS1.     

Development of a triplex TaqMan real-time RT-PCR assay for differential detection of wild-type and HCLV vaccine strains of classical swine fever virus and bovine viral diarrhea virus 1

In this study, genomic sequences of pestiviruses available in GenBank were aligned to design three primer pairs and TaqMan probes: two targeting the NS5A region of the viral genome of classical swine fever virus (CSFV) for the differentiation of wild-type CSFV and hog cholera lapinized vaccine (HCLV) vaccine, and one targeting the 5'-untranslated region of bovine viral diarrhea virus 1 (BVDV-1). With these primers and probes, a triplex TaqMan real-time RT-PCR assay was developed for differentiating wild-type CSFV, the HCLV strain, and BVDV-1. The detection limit of the assay was 4.5 TCID(50) for wild-type CSFV, 10 TCID(50) for HCLV-strain CSFV, and 3.2 TCID(50) for BVDV-1. The triplex real-time RT-PCR had at least 98% (248 samples) agreement with other RT-PCR methods. The assay provides a sensitive tool for simultaneous detection and differentiation of wild-type CSFV and HCLV from BVDV-1.     

Inability of NS1 protein from an H5N1 influenza virus to activate PI3K/Akt signaling pathway correlates to the enhanced virus replication upon PI3K inhibition

Background

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, activated during influenza A virus infection, can promote viral replication via multiple mechanisms. Direct binding of NS1 protein to p85β subunit of PI3K is required for activation of PI3K/Akt signaling. Binding and subsequent activation of PI3K is believed to be a conserved character of influenza A virus NS1 protein. Sequence variation of NS1 proteins in different influenza A viruses led us to investigate possible deviation from the conservativeness.

Results

In the present study, NS1 proteins from four different influenza A virus subtypes/strains were tested for their ability to bind p85β subunit of PI3K and to activate PI3K/Akt. All NS1 proteins efficiently bound to p85β and activated PI3K/Akt, with the exception of NS1 protein from an H5N1 virus (A/Chicken/Guangdong/1/05, abbreviated as GD05), which bound to p85β but failed to activate PI3K/Akt, implying that as-yet-unidentified domain(s) in NS1 may alternatively mediate the activation of PI3K. Moreover, PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, did not suppress but significantly increased the replication of GD05 virus.

Conclusions

Our study indicates that activation of PI3K/Akt by NS1 protein is not highly conserved among influenza A viruses and inhibition of the PI3K/Akt pathway as an anti-influenza strategy may not work for all influenza A viruses.     

Immunological properties of recombinant classical swine fever virus NS3 protein in vitro and in vivo

Classical swine fever (CSF) is a highly contagious and often fatal disease of pigs characterised by fever, severe leukopenia and haemorrhages. With vaccines having an importance in disease control, studies are seeking improved protein-based subunit vaccine against the virus (CSFV). In this respect, recombinant viral NS3 protein was analysed for its immunopotentiating capacity, particularly in terms of cytotoxic immune responses. NS3 was effective at inducing in vitro responses, quantified by lymphoproliferation, IFN-gamma ELISPOT, flow cytometric detection of activated T cell subsets, and cytotoxic T cell assays. Peripheral blood mononuclear cells from CSFV-immune pigs could be stimulated, but not cells from na?ve animals. In addition to the IFN-gamma responses, induction of both CD4+ T helper cell and CD8+ cytotoxic T cells (CTL) were discernible--activation of the latter was confirmed in a virus-specific cytolytic assay. Attempts were made to translate this to the in vivo situation, by vaccinating pigs with an E2/NS3-based vaccine compared with an E2 subunit vaccine. Both vaccines were similar in their abilities to stimulate specific immune responses and protect pigs against lethal CSFV infection. Although the E2/NS3 vaccine appeared to have an advantage in terms of antibody induction, this was not statistically significant when group studies were performed. It was also difficult to visualise the NS3 capacity to promote T-cell responses in vivo. These results show that NS3 has potential for promoting cytotoxic defences, but the formulation of the vaccine requires optimisation for ensuring that NS3 is correctly delivered to antigen presenting cells for efficient activation of CTL.     

日本乙誓脑炎病毒NS1基因及其疫苗的研究进展

日本乙型脑炎是由日本乙型脑炎病毒引起的严重的人畜共患传染病。乙型脑炎病毒的三种糖基化蛋白PrM、E和NS1是其主要免疫保护性抗原,其中PrM和E蛋白是日本乙型脑炎病毒的结构蛋白,其结构和相关疫苗研究的报道较多。NS1蛋白是日本乙型脑炎病毒的非结构蛋白,其主要参与病毒复制的早期阶段,推测可能参与病毒组装和释放。可诱导补体依赖性溶细胞反应,不能产生中和抗体,能诱发机体在非中和性抗体存在的情况下产生保护性免疫。在接种乙脑病毒的细胞的细胞内、细胞表面及上清液中均含有大量的NS1蛋白。对小鼠接种NS1蛋白或NS1蛋白特异性抗体,均能让小鼠产生对乙脑病毒感染的保护性免疫作用。本文主要就乙型脑炎病毒非结构基因NS1及其免疫疫苗的研究进展进行综述,为其更进一步深入研究IEV NS1基因及其相关疫苗奠定基础。     

Molecular characterization of RNA and protein synthesis during a one-step growth curve of bovine viral diarrhoea virus in ovine (SFT-R) cells

The aim of this study was to determine the kinetics of noncytopathic bovine viral diarrhoea virus (BVDV) multiplication and synthesis of BVDV specific RNA and proteins in ovine cells (SFT-R) during a one-step growth curve. The virus titre and RNA level were determined by focus-forming assay and real time RT-PCR. The RNA synthesis was detected by Northern blot while synthesis of E2 and NS3 proteins was assayed by immunohistochemistry and Western blot. The results showed that synthesis of viral RNA is initiated at 4 h, NS3 and E2 proteins are detectable at 6-7 h and the replication cycle is complete at 10-12 h. Additionally, we provide evidence that NS2-3 protein was cleaved in ovine cells early during infection and in proliferated leukocytes of acutely infected sheep. This study showed that synthesis of BVDV RNA and proteins in ovine cells occurs at similar times as found in bovine cells.