猪圆环病毒2型与猪细小病毒共感染对猪肺泡巨噬细胞吞噬功能及其干扰素表达水平的影响

【目的】分析猪圆环病毒2型(PCV2)与猪细小病毒(PPV)共感染对猪肺泡巨噬细胞(PAM)吞噬功能及其干扰素表达水平的影响,为进一步阐明猪断奶后多系统衰减综合征的发病机制提供实验依据。【方法】将48头5周龄健康仔猪随机分为PCV2组、PPV组、PCV2/PPV组和对照组,每组12头。PCV2和PPV组经口鼻途径分别接种PCV2或PPV,PCV2/PPV组同时接种PCV2和PPV,对照组接种细胞营养液。感染后3、7、14和35d(dpi)从每组随机选择3头剖杀,检测PAM的存活率、吞噬活性及其α1型干扰素(IFN-α1)、γ干扰素(IFN-γ)mRNA的表达水平。【结果】PCV2组和PCV2/PPV组PAM的存活率于3、7、14dpi均低于对照组,35dpi均与对照组PAM的存活率接近,PCV2与PCV2/PPV两组之间差异不显著(P>0.05);3个病毒感染组PAM的吞噬功能均显著下降(P<0.01),其中PCV2/PPV组的吞噬功能低于PCV2组(P<0.05);PCV2/PPV组PAM中IFN-α1和IFN-γ mRNA的水平持续低于PCV2、PPV组和对照组的水平(P<0.01)。【结论】PCV2与PPV共感染没有引起PAM的活力进一步下降,但导致其吞噬功能及IFN-α1和IFN-γmRNA的表达水平持续降低。     

人类冠状病毒调节宿主抗病毒天然免疫分子机制

SARS冠状病毒和正在全球流行的猪源H1N1型流感病毒等人类新发呼吸道病毒对人类生命健康构成严重威胁.人类重要呼吸道病毒与宿主抗病毒天然免疫的关系是近年来研究热点.SARS冠状病毒等很多RNA病毒能够编码某种蛋白质,抑制干扰素表达以及干扰素介导的抗病毒信号通路.人类冠状病毒木瓜样蛋白酶(papain-like protease,PLP)利用其自身去泛素化酶(DUB)活性,使干扰素表达通路中重要调节蛋白发生去泛素化,从而抑制干扰素信号传导.同时,PLP蛋白酶通过阻碍干扰素表达信号通路中最新发现的重要调节蛋白ERIS(也称MITA/STING)二聚化,使其失活并丧失激活干扰素通路的功能,这些发现对于阐明人类重要呼吸道病毒对宿主细胞抗病毒天然免疫反应的调节作用及其机制具有重要意义,为人类新发病毒致病机理、免疫防治以及抗病毒药物研究提供新的思路.     

猪流行性腹泻病毒与宿主抗病毒天然免疫抑制

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)能引起猪腹泻等肠道疾病,属于α属冠状病毒,它的爆发给很多国家养猪业造成了严重的经济损失。2010年以来,PEDV感染在中国出现大规模爆发,一种突变型PEDV也于2013年在美国出现并迅速传播。 RNA病毒能够通过Toll样受体通路3（TLR3）和RIG-I样受体通路（RLR）诱导I型干扰素的产生。但以往的研究表明,PEDV感染能抑制I型干扰素的合成。近年来有关PEDV调节宿主天然免疫应答的研究取得了很大进展。PEDV主要通过编码作为干扰素拮抗剂的病毒蛋白以及隐藏病毒自身病原相关分子模式（PAMP）等两种方式逃逸宿主天然免疫应答。目前已报道,PEDV非结构蛋白1可通过降解CBP阻碍干扰素调节因子3(IRF-3)组装成增强子复合体;木瓜蛋白酶样蛋白酶可通过其去泛素化酶活性阻断天然免疫信号通路传递;3C样蛋白酶可通过剪切NEMO发挥干扰素拮抗剂活性;核衣壳蛋白通过结合TBK1抑制I型干扰素产生。PEDV也可通过合成加帽酶隐藏其病原相关分子dsRNA来避免激活天然免疫通路。PEDV抗病毒天然免疫机制阐明为研究PEDV感染免疫和致病机制提供了重要的理论依据,为研发抗PEDV新型疫苗和药物提供了基础。     

表达猪圆环病毒Ⅱ型ORF2基因的重组猪伪狂犬病病毒的免疫原性

【目的】为研制预防猪圆环病毒II型(PCV2)感染的重组伪狂犬病病毒(PRV)活载体疫苗。【方法】将PCV2 ORF2基因插入到PRV通用载体pG中,利用脂质体LipofectamineTM2000试剂盒将重组转移质粒pGO与猪PRV弱毒HB98株DNA共转染猪睾丸(ST)细胞,通过3轮蚀斑纯化重组病毒。将重组病毒、商品化PCV2灭活苗及DMEM培养液分别免疫6周龄雌性昆明小鼠,4周后加强免疫1次,首免后第8周用PCV2强毒NY株对小鼠进行攻毒。【结果】成功获得表达ORF2基因的重组病毒PGO,首免重组病毒后小鼠体内抗PCV2的ELISA抗体水平很低,二免后小鼠PCV2特异的ELISA抗体水平明显升高,并且重组病毒组能够激发PCV2特异的淋巴细胞增殖效应。攻毒试验表明重组病毒组和PCV2灭活疫苗组均能有效抵抗PCV2强毒攻击。【结论】表明表达ORF2基因的重组病毒PGO具有良好免疫原性。     

表达猪圆环病毒II型ORF2基因的重组猪伪狂犬病病毒的免疫原性

摘要:【目的】为研制预防猪圆环病毒II型(PCV2)感染的重组伪狂犬病病毒(PRV)活载体疫苗。【方法】将PCV2 ORF2基因插入到PRV通用载体pG中,利用脂质体LipofectamineTM 2000试剂盒将重组转移质粒pGO与猪PRV弱毒HB98株DNA共转染猪睾丸(ST)细胞,通过3轮蚀斑纯化重组病毒。将重组病毒、商品化PCV2灭活苗及DMEM培养液分别免疫6周龄雌性昆明小鼠,4周后加强免疫1次,首免后第8周用PCV2强毒NY株对小鼠进行攻毒。【结果】成功获得表达ORF2基因的重组病毒PGO,首免重组病毒后小 鼠体内抗PCV2的ELISA抗体水平很低,二免后小鼠PCV2特异的ELISA抗体水平明显升高,并且重组病毒组能够激发PCV2特异的淋巴细胞增殖效应。攻毒试验表明重组病毒组和PCV2灭活疫苗组均能有效抵抗PCV2强毒攻击。【结论】表明表达ORF2基因的重组病毒PGO具有良好免疫原性。     

MicroRNA-98促进猪圆环病毒2型在3D4/21细胞中的复制

【目的】通过高通量测序的方法获得PCV2感染3D4/21细胞的miRNAs表达谱,并探讨miRNA-98在PCV2复制中的作用。【方法】本研究以猪肺泡巨噬细胞系3D4/21细胞为细胞模型,对PCV2感染过程中的3D4/21细胞进行miRNAs差异表达分析,筛选与病毒复制相关的特异性miRNAs,并探讨其在PCV2复制中的作用。【结果】经高通量测序,获得PCV2感染3D4/21细胞的miRNAs表达谱,结合实验室前期研究筛选获得miRNA-98。实验表明,miRNA-98的表达量随PCV2感染时间的延长而持续升高,其变化趋势与Cap蛋白表达变化基本一致,由此推测miRNA-98与PCV2复制正相关。过表达miRNA-98可显著上调Cap蛋白的表达量和PCV2的复制。进一步的研究表明,miRNA-98参与调节宿主免疫相关细胞因子的表达和PCV2的复制。【结论】miRNA-98可通过调节免疫相关细胞因子的表达调控宿主免疫功能,帮助PCV2逃逸宿主免疫,促进PCV2在3D4/21细胞中的复制。这些发现不仅为深入了解PCV2与宿主之间的关系提供了新视角,还有望为猪圆环病毒相关疾病的防控提供新的抗病毒策略。     

表达猪圆环病毒2型ORF2基因的重组猪繁殖与呼吸综合征病毒的构建与鉴定

猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)和猪圆环病毒2型(Porcine circovirus type 2,PCV2)是目前危害世界养猪业的两种重要病原。本研究采用PCR方法构建PRRSV疫苗株TJM-F92全长cDNA克隆载体pCMV-TJM,并在其ORF7和3′UTR之间插入AflⅡ/MluⅠ酶切位点和转录调控序列TRS6,构建获得pCMV-TJM-TRS表达载体。将PCV2ORF2基因插入该载体AflⅡ/MluⅠ位点,获得重组质粒pCMV-TJM-Cap。将pCMV-TJM、pCMV-TJM-TRS和pCMV-TJM-Cap分别转染Marc-145细胞,拯救获得3种重组病毒rTJM、rTJM/TRS和rTJM/Cap。基因测序列、酶切鉴定、Western blot、间接免疫荧光和病毒生长特性结果显示,3种重组PRRSV病毒都含有特征性分子标记,在Marc-145细胞增殖特性与亲本病毒相似;rTJM/Cap传至第8代,仍含有外源Cap基因,病毒感染细胞能有效表达PCV2Cap蛋白,从而为PRRSV致病机制和PRRSV-PCV2疫苗研究奠定了重要基础。     

抗病毒天然免疫通路中IRF-3调控新机制

干扰素调节因子-3(interferon regulatory factor-3,IRF-3)是IRF家族中重要 转录因子之一,在调控干扰素(interferon, IFN)基因表达和抗病毒天然免疫反应中具有重要作 用. 最新发现的MITA (mediator of IRF-3 activation, 又称STING/ERIS)蛋白是宿主抗病 毒天然免疫反应中的一种重要调节分子. 病毒侵染时,MITA与IRF-3相互作用,特异性激活 IRF-3,并募集TANK结合激酶1（TANK binding kinase 1, TBK1）与IFN通路中的线粒体抗 病毒信号蛋白MAVS(mitochondrial anti-viral signaling protein)形成复合物,且MITA可 被TBK1磷酸化,诱导Ⅰ型IFN及IFN刺激基因(interferon stimulate genes, ISG)的表达 ,诱发抗病毒天然免疫反应. 同时还发现,泛素连接酶RNF5（ring finger protein 5）可对MITA 发生泛素化修饰从而抑制其对IRF-3活化,实现对宿主抗病毒天然免疫反应负调节作用. 本 室研究发现,严重性急性呼吸系统综合症冠状病毒(severe acute respiratory syndrome co ronavirus, SARS-CoV）和人类新型冠状病毒（human coronavirus NL63, HCoV-NL63）的 木瓜样蛋白酶（papain-like protease, PLP）利用其特有的去泛素化酶（deubiquitinase, DUB）活性,通过宿主细胞泛素-蛋白酶体信号系统对IRF-3的泛素化等翻译后修饰进行调节 ,从而成为该种病毒逃逸机体抗病毒防御系统主要手段之一.     

ANXA1促进Ⅰ型干扰素表达抑制口蹄疫病毒的复制

【目的】研究膜联蛋白A1(Annexin A1,ANXA1)对Ⅰ型干扰素(I-IFN)表达及口蹄疫病毒(FMDV)复制的影响。【方法】开展过表达及Knockdown实验,检测ANXA1对FMDV复制的影响。利用双荧光素酶报告系统检测ANXA1对ISRE和IFN-β启动子元件活化的影响。双荧光素酶报告系统鉴定ANXA1调控Ⅰ-IFN通路活化靶分子。Western blotting检测ANXA1对干扰素调解因子3(IRF3)磷酸化的影响。Real-time PCR检测ANXA1对干扰素刺激基因(ISGs)的影响。【结果】过表达ANXA1显著抑制FMDV的复制;下调ANXA1表达促进FMDV复制(P0.01或P0.05);ANXA1促Ⅰ型干扰素通路活化,呈现剂量依赖性(P0.01)。ANXA1显著增强IRF3的磷酸化,促进ISGs的表达(P0.01或P0.05)。【结论】ANXA1促进Ⅰ-IFN表达,抑制FMDV的复制。     

Dengue Virus Targets the Adaptor Protein MITA to Subvert Host Innate Immunity

Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓⁹⁶G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.     

PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-β induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle’s Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-β induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.     

猪圆环病毒2型Cap蛋白在烟草叶绿体中的表达

猪圆环病毒2型(porcine circovirus type 2,PCV2)可读框2(open reading frame 2,ORF2)基因编码具有多个抗原表位的病毒衣壳(Cap)蛋白,能在机体内有效引发免疫反应并产生中和抗体,是预防和治疗猪圆环病毒病(porcine circovirus-associated disease,PCVAD)的主要抗原。但Cap蛋白高昂的生产成本限制了它的广泛应用。为了探索Cap蛋白新的生产途径,本研究选择烟草叶绿体为表达平台,构建了ORF2抗原基因烟草叶绿体表达载体pNK-a03,并通过基因枪法(gene biolistics)将表达载体导入烟草叶绿体中。转化植株经PCR和RT-PCR检测,证实ORF2抗原基因已整合进烟草叶绿体基因组中且正常转录。蛋白质印迹检测和ELISA分析表明,Cap蛋白在烟草叶绿体内获得有效表达,具有免疫活性。此外,种子萌发研究结果显示,转基因烟草植株F1代具有aadA抗性,目的基因可正常进行遗传。这些研究结果为探索Cap蛋白的新型表达途径提供了理论基础。     

安徽省部分地区2016–2018年猪圆环病毒2型遗传进化分析

【背景】猪圆环病毒(Porcinecircovirus,PCV)可以引起断奶仔猪多系统衰竭综合症(Postweaningmultisystemicwastingsyndrome,PMWS)。【目的】了解安徽省部分地区猪圆环病毒2型(PCV2)的遗传变异情况。【方法】采用PCR技术,对2016-2018年间安徽省部分地区猪场疑似PMWS感染的组织样品共计31份进行PCV2检测和全基因扩增,并通过DNAStar等生物信息学软件对所得到的PCV2毒株基因序列进行遗传进化分析。【结果】所得的8株PCV2安徽株与GenBank上已发表的国内外参考毒株相比较,核苷酸相似性为92.8%-99.0%,ORF2及其推导的氨基酸序列相似性分别为85.8%-99.6%和82.5%-100%。遗传进化树分析结果显示8株安徽株中有1株PCV2a,2株PCV2b,5株PCV2d,未发现PCV2c、PCV2e和PCV2f基因型。此外,通过对ORF2基因编码的氨基酸序列分析,发现各基因型Cap蛋白氨基酸序列上的位点具有其独特性。【结论】近年来PCV2在安徽地区的猪群中感染较为普遍,其中PCV2d基因型的感染病例增多,逐渐成为安徽地区的优势流行株。本研究为安徽地区的PCV2防控提供了一定的参考依据。     

Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice

The Cap protein of porcine circovirus type 2 (PCV2) that serves as a major host-protective immunogen was used to develop recombinant vaccines for control of PCV2-associated diseases. Growing research data have demonstrated the high effectiveness of flagellin as an adjuvant for humoral and cellular immune responses. Here, a recombinant protein was designed by fusing a modified version of bacterial flagellin to PCV2 Cap protein and expressed in a baculovirus system. When administered without adjuvant to BALB/c mice, the flagellin-Cap fusion protein elicited stronger PCV2-specific IgG antibody response, higher neutralizing antibody levels, milder histopathological changes and lower viremia, as well as higher secretion of cytokines such as TNF-α and IFN-γ that conferred better protection against virus challenge than those in the recombinant Cap alone-inoculated mice. These results suggest that the recombinant Cap protein when fused to flagellin could elicit better humoral and cellular immune responses against PCV2 infection in a mouse model, thereby acting as an attractive candidate vaccine for control of the PCV2-associated diseases.     

Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway during Porcine Circovirus Type 2 Infection Facilitates Cell Survival and Viral Replication

Virus infection activates host cellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which regulates diverse cellular activities related to cell growth, survival, and apoptosis. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), a major causative agent of postweaning multisystemic wasting syndrome, which is an emerging and important swine disease, can transiently induce the PI3K/Akt pathway in cultured cells at an early step during PCV2 infection. Activation of the PI3K/Akt signal was also induced by UV-irradiated PCV2, indicating that virus replication was not required for this induction. Inhibition of PI3K activation leads to reduced virus yield, which is associated with decreased viral DNA replication and lower virus protein expression. However, inhibition of PI3K activation greatly enhanced apoptotic responses as evidenced by the cleavage of poly-ADP ribose polymerase and caspase-3 as well as DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining during the early stage of PCV2 infection. Furthermore, the pancaspase inhibitor zVAD.fmk alleviated the reduction in Akt phosphorylation levels by inhibiting PI3K activation, indicating that the signaling promotes cell survival and thereby favors viral replication. These results reveal that an antiapoptotic role for the PI3K/Akt pathway induced by PCV2 infection to suppress premature apoptosis for improved virus growth after infection, extending our understanding of the molecular mechanism of PCV2 infection.     

Enhancement of the immunogenicity of a porcine circovirus type 2 DNA vaccine by a recombinant plasmid coexpressing capsid protein and porcine interleukin‐6 in mice

The development of effective vaccines against porcine circovirus type 2 (PCV2) has been accepted as an important strategy in the prophylaxis of post‐weaning multisystemic wasting syndrome; a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, DNA vaccines usually induce weak immune responses. In this study, it was found that the efficacy of a DNA vaccine expressing Cap protein was improved by simultaneous expression of porcine IL‐6. A plasmid (pIRES‐ORF2/IL6) separately expressing both Cap protein and porcine IL‐6 was constructed and compared with another plasmid (pIRES‐ORF2) expressing Cap protein for its potential to induce PCV2‐specific immune responses. Mice were vaccinated i.m. twice at 3 week intervals and the induced humoral and cellular responses evaluated. All animals vaccinated with pIRES‐ORF2/IL6 and pIRES‐ORF2 developed specific anti‐PCV2 antibodies (according to enzyme‐linked immunosorbent assay) and a T lymphocyte proliferation response. The percentages of CD3⁺, CD3⁺CD8⁺, and CD3⁺CD4⁺ subgroups of peripheral blood T‐lymphocytes were significantly higher in mice immunized with pIRES‐ORF2/IL6 than in those that had received pIRES‐ORF2. After challenge with the virulent PCV2 Wuzhi isolate, mice vaccinated with pIRES‐ORF2/IL6 had significantly less viral replication than those vaccinated with pIRES‐ORF2, suggesting that the protective immunity induced by pIRES‐ORF2/IL6 is superior to that induced by pIRES‐ORF2.