羊口疮病毒作为免疫调节剂在疾病治疗中的研究进展

羊口疮病毒(ORFV),也称接触传染性脓疱皮炎病毒,为痘病毒科脊索动物痘病毒亚科副痘病毒属中的成员,是世界各地绵羊和山羊的常见病原体。ORFV可通过多种不同的策略来影响免疫细胞,进而操控宿主的免疫应答。灭活的ORFV具有免疫调节作用,可作为预防和治疗的免疫调节剂,用于感染性疾病、慢性病毒病、肿瘤、肝纤维化等的治疗。我们对ORFV在临床前期或临床治疗中的研究进展进行简要综述。     

羊口疮病毒干预宿主细胞泛素-蛋白酶体系统的分子机制

羊口疮病毒(Orf virus,ORFV)感染细胞过程中为了对抗宿主细胞的抗病毒作用,依靠种群长期培育的一系列功能性基因,如干扰素抗性基因、Bcl-2样基因和细胞周期蛋白抑制物基因等,遏制宿主细胞免疫清除和免疫调节作用。同时,ORFV也利用某种机制干预细胞泛素-蛋白酶体系统(Ubiquitin-proteasome system,UPS)的信号调控途径,有效抑制胞内信号传导和CD8+T细胞活化,以庇护病毒粒子成熟和释放。本文综述了细胞UPS功能与病毒干预机制的内在联系及其相关元件的关键作用,探讨ORFV在选择压力下,主动采取的抑制宿主细胞免疫应答和设计胞内免疫逃逸的进化趋势。     

羊口疮024基因的表达、多抗制备及亚细胞定位

目的克隆表达羊口疮病毒(Orf virus,ORFV)024基因,制备多克隆抗体,检测其免疫原性,并对其进行亚细胞定位分析。方法根据GenBank中羊口疮病毒024基因序列设计引物,进行PCR扩增。将其亚克隆至表达载体,构建原核重组质粒pGEX-6P-1-024以及真核重组质粒pEGFP-N1-024。将原核重组质粒转化Rosetta (DE3)细胞,IPTG诱导表达,SDS-PAGE鉴定024蛋白的表达情况。纯化后的024蛋白免疫BALB/c雌鼠,获得血清并制备多克隆抗体,进行Western-blot分析。将构建的真核质粒转染MDBK细胞,24h后通过免疫荧光观察其在细胞中的表达及亚细胞定位。结果 SDS-PAGE结果表明,羊口疮病毒024基因在Rosetta (DE3)细胞中正确表达,大小为59kDa。Western-blot结果表明024蛋白能与制备的多抗发生特异性反应,具有良好的反应原性。荧光显微镜下结果表明024蛋白转染MDBK细胞后主要在细胞质中表达。结论本实验为后续深入研究ORFV 024基因功能和致病机制奠定了基础。     

羊口疮病毒119蛋白表达、纯化和多克隆抗体的制备

[目的]克隆表达羊口疮病毒ORFV119蛋白,以纯化重组蛋白为免疫原制备鼠多克隆抗体,并鉴定抗体的特性。[方法]PCR扩增ORFV119基因,克隆入原核表达载体p ET-28a(+)中构建重组质粒p ET28a-119。经双酶切和测序正确后,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定融合蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,之后目的蛋白免疫BALB/c小鼠,制备ORFV119多克隆抗体并对其通过中和实验进行鉴定。[结果]成功构建重组质粒p ET28a-119,在大肠杆菌BL21中ORFV119融合蛋白以部分可溶形式表达。可溶性目的蛋白纯化后作为免疫原制备鼠多克隆抗体,抗体效价达1∶12 800,中和实验显示该多抗具有保护作用,可减轻宿主细胞在病毒感染时的病变效应(中和效价66 ND50/m L)。[结论]成功诱导表达、纯化ORFV119蛋白并制备其多克隆抗体,为深入研究ORFV感染、发病机理及羊口疮疾病的临床诊断奠定基础。     

人巨细胞病毒博弈宿主免疫进展

人巨细胞病毒(HCMV)感染在人群中极其普遍,病毒一旦侵入机体,将长期存在于体内,且具有潜伏-活化的生物学特性。在病毒与宿主共同进化的漫长过程中,病毒靶向性的产生了多种免疫逃避机制,通过编码病毒自身免疫调节分子,参与调控机体主要组织相容性复合体、细胞免疫、体液免疫、细胞因子及趋化因子等方面的功能,以躲避宿主的免疫杀伤作用。HCMV的免疫调节基因被认为在病毒的致病机制中扮演重要角色。本文将对近年来有关HCMV的免疫调控机制研究作一综述,从病毒编码的免疫调节分子功能的角度并结合本实验室的相关研究成果,探讨病毒与宿主免疫的相互作用过程,从病毒干预宿主免疫关键分子作用的角度映射机体对抗病毒的免疫机理。     

痘病毒介导的NF-κB信号通路的调控

痘病毒是人和多种动物痘病的病原体,作为嗜上皮型的DNA病毒,在进化过程中,痘病毒特定的基因产物作用于机体免疫调控系统,调节免疫反应的进程和作用方式,进而完成其感染细胞、复制和繁殖的生物学过程。NF-κB信号通路是机体免疫系统重要的信号转导调节系统,各种痘病毒采用自身特殊的策略作用于这一免疫调节的重要靶系统,应对机体对其免疫清除和免疫反应。对痘病毒介导的宿主免疫调节的深入研究有助于研发新型疫苗和治疗性制剂。本文对近十年来各种痘病毒编码的多种目标蛋白参与宿主NF-κB信号通路调控的分子机制进行综述。     

羊口疮病毒蛋白ORFV035的表达、纯化和多克隆抗体的制备

克隆表达羊口疮病毒蛋白ORFV035,并制备其多克隆抗体,为后续对病毒复制、装配、形态发生和成熟过程的研究奠定基础。PCR扩增羊口疮病毒ORFV035基因,将其与质粒pET-30a(+)经Bam HⅠ和HindⅢ双酶切后连接,构建重组质粒pET30a-035。重组质粒经双酶切和测序鉴定,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,纯化后目的蛋白免疫小鼠,制备多克隆抗体并对其进行鉴定。成功构建了重组质粒pET30a-035,在大肠杆菌BL21中以包涵体形式高效表达。包涵体洗涤、溶解后进行Ni柱纯化,得到纯度较高的ORFV035-his融合蛋白。以纯化蛋白免疫小鼠获得多克隆抗体。Western blot检测显示该多抗可以识别天然ORFV035蛋白。成功诱导表达、纯化ORFV035蛋白并制备ORFV035多克隆抗体。     

痘病毒宿主范围因子及其作用机制研究进展

痘病毒是已知病毒中基因组最大、宿主谱最广和危害最严重的人兽共患病病原之一。随着病原和宿主互作的分子生物学、功能基因组学以及免疫学相关技术的发展,痘病毒宿主范围因子及其相关作用机制被相继发现和研究,这为痘病毒的组织嗜性和宿主特异性的阐明奠定了分子基础。本文将通过对脊椎动物重要痘病毒属主要成员的宿主范围因子及其作用机制的介绍,以加深对痘病毒的分子遗传演化、组织嗜性和跨宿主种间感染机制的理解和认识。     

抗羊口疮病毒ORFV118蛋白单克隆抗体的制备、鉴定及其应用

制备抗羊口疮病毒118(ORFV118)重组蛋白的单克隆抗体并鉴定其生物学特性。构建ORFV118原核表达重组质粒pET33b-ORFV118,将重组质粒转化大肠杆菌BL21,诱导表达出ORFV118重组蛋白;利用Ni-NTA亲和层析法纯化ORFV118重组蛋白;以纯化蛋白为抗原免疫小鼠,通过杂交瘤技术制备单克隆抗体;利用间接ELISA法、Western blot、免疫组化等方法分别对单抗特异性、效价、亚型以及ORFV118蛋白的功能进行研究。获得了3株稳定分泌单克隆抗体的细胞株,分别命名为1A2,3B5,5D10,它们诱生的小鼠腹水效价分别为110 000,16 400,18 000。其中效价最高的1A2抗体亚型为IgG1,能特异性结合其免疫原蛋白、真核表达产物以及病羊皮肤组织中的ORFV118蛋白。免疫组化结果显示单抗1A2染色局限于皮肤表皮层角化细胞及浸润至皮下组织的炎症细胞,与病毒侵染上皮组织层的特性相符。制备的单抗1A2能特异性识别ORFV118。深入研究单抗1A2将为了解ORFV118的生物学特性,为羊传染性脓疱病的诊断、预防与治疗提供可能和新思路。     

A novel Bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus ORF virus

Apoptotic cell death forms part of the host defense against virus infection. We tested orf virus, a member of the poxvirus family, for the ability to inhibit apoptosis and found that orf virus-infected cells were fully resistant to UV-induced changes in cell morphology, caspase activation, and DNA fragmentation. By using a library of vaccinia virus-orf virus recombinants, we identified an orf virus gene (ORFV125) whose presence was linked with the inhibition of apoptosis. The 173-amino-acid predicted protein had no clear homologs in public databases other than those encoded by other parapoxviruses. However, ORFV125 possessed a distinctive C-terminal domain which was necessary and sufficient to direct the protein to the mitochondria. We determined that ORFV125 alone could fully inhibit UV-induced DNA fragmentation, caspase activation, and cytochrome c release and that its mitochondrial localization was required for its antiapoptotic function. In contrast, ORFV125 did not prevent UV-induced activation of c-Jun NH2-terminal kinase, an event occurring upstream of the mitochondria. These features are comparable to the antiapoptotic properties of the mitochondrial regulator Bcl-2. Furthermore, bioinformatic analyses revealed sequence and secondary-structure similarities to Bcl-2 family members, including characteristic residues of all four Bcl-2 homology domains. Consistent with this, the viral protein inhibited the UV-induced activation of the proapoptotic Bcl-2 family members Bax and Bak. ORFV125 is the first parapoxvirus apoptosis inhibitor to be identified, and we propose that it is a new antiapoptotic member of the Bcl-2 family.     

Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus

Bovine papular stomatitis virus (BPSV) and orf virus (ORFV), members of the genus Parapoxvirus of the Poxviridae, are etiologic agents of worldwide diseases affecting cattle and small ruminants, respectively. Here we report the genomic sequences and comparative analysis of BPSV strain BV-AR02 and ORFV strains OV-SA00, isolated from a goat, and OV-IA82, isolated from a sheep. Parapoxvirus (PPV) BV-AR02, OV-SA00, and OV-IA82 genomes range in size from 134 to 139 kbp, with an average nucleotide composition of 64% G+C. BPSV and ORFV genomes contain 131 and 130 putative genes, respectively, and share colinearity over 127 genes, 88 of which are conserved in all characterized chordopoxviruses. BPSV and ORFV contain 15 and 16 open reading frames (ORFs), respectively, which lack similarity to other poxvirus or cellular proteins. All genes with putative roles in pathogenesis, including a vascular endothelial growth factor (VEGF)-like gene, are present in both viruses; however, BPSV contains two extra ankyrin repeat genes absent in ORFV. Interspecies sequence variability is observed in all functional classes of genes but is highest in putative virulence/host range genes, including genes unique to PPV. At the amino acid level, OV-SA00 is 94% identical to OV-IA82 and 71% identical to BV-AR02. Notably, ORFV 006/132, 103, 109, 110, and 116 genes (VEGF, homologues of vaccinia virus A26L, A33R, and A34R, and a novel PPV ORF) show an unusual degree of intraspecies variability. These genomic differences are consistent with the classification of BPSV and ORFV as two PPV species. Compared to other mammalian chordopoxviruses, PPV shares unique genomic features with molluscum contagiosum virus, including a G+C-rich nucleotide composition, three orthologous genes, and a paucity of nucleotide metabolism genes. Together, these data provide a comparative view of PPV genomics.     

Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against lethal herpesvirus challenge infection in mice

Orf virus (ORFV; Parapoxvirus ovis) was used to develop a novel vector system for the generation of effective and safe live vaccines. Based on the attenuated ORFV strain D1701-V, recombinants were produced that express the glycoproteins gC (D1701-VrVgC) or gD (D1701-VrVgD) of the alphaherpesvirus of swine, pseudorabies virus (PRV). Expression of gC and gD was also demonstrated on the surface of recombinant virus-infected murine cells that do not produce infectious ORFV. Single or combined immunization with the ORFV recombinants protected different mouse strains of a host species nonpermissive for ORFV against a fulminant, lethal PRV challenge infection equal to immunization with PRV live vaccine. Most notably, even a single immunization with D1701-VrVgC was protective, whereas two applications of D1701-VrVgD were required for immune protection. The higher protective capacity of D1701-VrVgC correlated with the induction of a strong specific humoral immune response. This suggestion was supported by transfer experiments using sera from recombinant-immunized mice, which resulted in partial gC but not gD antibody-mediated protection of the na?ve recipients. Remarkably, immunization of different immune-deficient mice demonstrated that the application of the PRV gC-expressing recombinant controlled the challenge infection in the absence of either CD4(+) or CD8(+) T cells, B cells, or an intact perforin pathway. In contrast, D1701-VrVgD-immunized mice lacking CD4(+) T cells exhibited reduced protection, whereas animals lacking CD8(+) T cells, B cells, or perforin resisted the challenge infection. The present study demonstrates the potential of these new vector vaccines to efficiently prime both protective humoral and cell-mediated immune mechanisms in a host species nonpermissive for the vector virus.     

Selective induction of apoptosis in antigen-presenting cells in mice by Parapoxvirus ovis

Viruses have evolved numerous mechanisms to avoid host immune reactions. Here we report a mechanism by which Parapoxvirus ovis (PPVO) interferes with antigen presentation. PPVO (orf virus) causes orf, an acute skin disease of sheep and goats worldwide. Importantly, PPVO can repeatedly infect its host in spite of a vigorous inflammatory and host immune response to the infection. We demonstrate in a mouse system that PPVO induces apoptosis in a significant number of antigen-presenting cells after intraperitoneal injection using the CD95 pathway, thus preventing a primary T-cell response. We also show that PPVO induces a compensatory activation of the immune system. Our results may help to explain the phenomenon that natural PPVO infections in sheep occur repeatedly even after short intervals. They also suggest that the combination of immunosuppressive and immunostimulatory mechanisms is an effective survival strategy that might be used in other viruses as well.     

The novel ORFV protein ORFV113 activates LPA-p38 signaling

Viruses have evolved mechanisms to subvert critical cellular signaling pathways that regulate a wide range of cellular functions, including cell differentiation, proliferation and chemotaxis, and innate immune responses. Here, we describe a novel ORFV protein, ORFV113, that interacts with the G protein-coupled receptor Lysophosphatidic acid receptor 1 (LPA₁). Consistent with its interaction with LPA₁, ORFV113 enhances p38 kinase phosphorylation in ORFV infected cells in vitro and in vivo, and in cells transiently expressing ORFV113 or treated with soluble ORFV113. Infection of cells with virus lacking ORFV113 (OV-IA82Δ113) significantly decreased p38 phosphorylation and viral plaque size. Infection of cells with ORFV in the presence of a p38 kinase inhibitor markedly diminished ORFV replication, highlighting importance of p38 signaling during ORFV infection. ORFV113 enhancement of p38 activation was prevented in cells in which LPA₁ expression was knocked down and in cells treated with LPA₁ inhibitor. Infection of sheep with OV-IA82Δ113 led to a strikingly attenuated disease phenotype, indicating that ORFV113 is a major virulence determinant in the natural host. Notably, ORFV113 represents the first viral protein that modulates p38 signaling via interaction with LPA₁ receptor.     

Heterogeneity among Orf Virus Isolates from Goats in Fujian Province,Southern China

Orf virus is a parapoxvirus that causes recurring contagious ecthyma or orf disease in goat, sheep and other wild and domestic ruminants. Infected animals show signs of pustular lesions on the mouth and muzzle and develop scabs over the lesions. Although the infection is usually cleared within 1–2 months, delayed growth and associated secondary infections could still impact the herds. Orf virus can also infect humans, causing lesions similar to the animals in pathological histology. Prior infection of orf virus apparently offers little protective immunity against future infections. Several gene products of orf virus have been identified as responsible for immunomodulatory functions. In our recent study of orf virus isolates from an area along the Minjiang River in northern Fujian Province, we found a high heterogeneity among isolates from 10 farms within a 120-kilometer distance. Only two isolates from locations within 1 km to each other had same viral genes. There is no correlation between the geographical distance between the corresponding collection sites and the phylogenetic distance in ORFV011 or ORV059 genes for any two isolates. This finding suggests that there are diverse populations of orf virus present in the environment. This may in part contribute to the phenomenon of recurring outbreaks and heighten the need for better surveillance.     

Appearance of the bona fide spiral tubule of ORF virus is dependent on an intact 10-kilodalton viral protein

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.     

Characterization of parapoxviruses circulating among wild Japanese serows (Capricornis crispus)

We antigenically and molecularly compared 5 parapoxvirus isolates and 7 viral DNA samples from clinical lesions of Japanese serows with 3 viruses from sheep and goats. All isolates from Japanese serows except one, Ishikawa-S, reacted with six monoclonal antibodies to orf virus (ORFV). Restriction endonuclease analysis using amplified viral DNA showed the ORFV-specific pattern in all samples except Ishikawa-S, which showed a bovine papular stomatitis virus (BPSV)-specific pattern. Partial nucleotide sequences of the envelope genes were determined and those of all samples from Japanese serows and sheep except Ishikawa-S were completely identical and also had high identities with the goat virus. These findings suggest that parapoxvirus infection in Japanese serows might be mainly caused by ORFV and accidentally by BPSV. The envelope gene sequenced here seems to be conserved in Japanese ORFVs.