口蹄疫病毒入侵宿主细胞研究进展

口蹄疫病毒(FMDV)是小RNA病毒科,口蹄疫病毒属的典型成员,是一种基因组大约含有8 400个核苷酸的无囊膜单股正链RNA病毒。大量研究发现识别细胞表面受体并侵入细胞是FMDV感染宿主细胞非常重要的环节;对FMDV而言,利用哪种受体就决定了利用哪种內吞路径。近年来在口蹄疫病毒入侵宿主细胞方面进行了大量研究,在一定程度上解释了口蹄疫病毒感染机制方面的问题,为解决实际生产问题提供了重要依据。对前期工作进行阶段性总结,为后期深入研究口蹄疫病毒致病机制和探索更有效的防治措施提供参考。     

整联蛋白与口蹄疫病毒感染

整联蛋白是一类重要的细胞表面分子,除介导细胞与胞外基质及细胞间的粘附外,还对细胞的识别、生长和分化具有重要作用。FMDV对细胞的侵染依赖于宿主细胞受体,整联蛋白作为口蹄疫病毒侵入细胞的关键决定簇,在致病机制、组织和器官嗜性中具有重要作用。本文就整联蛋白的结构、功能及在FMDV感染中的作用等方面进行了综述,以期阐明FMDV侵染细胞的机制。     

VHS蛋白,一种具有mRNA剪切活性的多种功能蛋白质

单纯疱疹病毒UL41基因编码的病毒宿主关闭蛋白(VHS蛋白)是一种核酸酶,具有。RNA剪切活性．可引起宿主细胞蛋白质合成的快速关闭。通过干扰IFN-α／β介导的抗病毒免疫反应、降低宿主细胞MHCI和MHCII类分子的表达、减少免疫系统中病毒抗原的提呈以及抑制宿主先天免疫反应等,VHS蛋白在α疱疹病毒的发病机制和免疫逃避过程中发挥重要作用。     

口蹄疫病毒受体的结构与功能研究进展

口蹄疫病毒（FMDV）感染过程中的特异性受体是FMDV识别,结合宿主细胞的分子基础,研究FMDV受体的结构与功能对防治口蹄疫具有重要的理论意义和应用价值。本论述了近年来关于整联蛋白αvβ3,αvβ6和硫酸乙酰肝素（HS）三种FMDV受体的结构与功能的研究进展。     

DEAD-box RNA解旋酶家族在天然免疫应答信号通路中的作用

病毒入侵宿主细胞时,宿主细胞启动抑制病毒复制的免疫机制。同样,病毒也会利用多种手段去逃避先天免疫感应机制的监测以及宿主细胞对外来者的降解,同时还会操纵宿主细胞为自身的增殖提供便利。DEAD-box解旋酶家族是一类存在于宿主细胞中的功能蛋白,它们在转录、剪接、mRNA的合成和翻译等多种细胞过程中起着关键作用。该家族成员拥有识别RNA的能力以及参与多个细胞过程,所以它们可以以多种方式影响病毒感染宿主细胞后引起的天然免疫应答。本文就近年来有关于DEAD-box RNA解旋酶在天然免疫方面的研究进行综述,以期为相关研究提供材料支撑。     

长链非编码RNANRAV在抗病毒应答中对干扰素刺激基因(ISG)转录的调控作用

<正>天然免疫是宿主抵抗病毒的第一道防线。病毒侵染宿主后,宿主的病原识别受体(PRR)鉴别出病毒的核酸或蛋白质组分,激活抗病毒天然免疫应答。一系列天然免疫信号通路被PRR激活,通路下游的转录因子随之活化并进入细胞核,核内各种免疫相关基因依次开始转录调控。首先,干扰素大量表达并被分泌至细胞外,随后上百种干扰素刺激基因(ISG)转录表达。这些ISG蛋白分布至细胞内外,通过多种机制抵御病毒的感染复制,以清除机体内的病毒。长链非编码RNA(Long non-coding RNA,lnc RNA)是一类长度大于200nt、不具有蛋白编码特性的RNA分子。近年来大量实验证据表明,长链非编码RNA在细胞     

口蹄疫病毒受体研究进展

口蹄疫病毒感染宿主细胞的第一步是病毒与被感染细胞表面的某种受体结合,在这种受体的介导下,病毒颗粒才能进入细胞内。细胞受体是决定口蹄疫病毒宿主特异性和组织特异性的主要因素之一。口蹄疫病毒受体的研究对于揭示口蹄疫病毒的致病免疫机理具有重要价值。就近年来已发现的αvβ1、αvβ3、αvβ6、αvβ8四种整联蛋白和硫酸乙酰肝素受体作一综述。     

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.     

早幼粒白血病蛋白核体与病毒感染

早幼粒白血病蛋白核体（promyelocytic leukaemia nuclear bodies,PML-NBs）是哺乳动物细胞中普遍存在的一种动态的细胞核亚结构,参与DNA损伤与修复、细胞衰老与凋亡、基因表达调控以及肿瘤发生与抑制等多种重要的细胞活动。研究表明,PML-NBs也是多种病毒入侵细胞的作用靶点。PML-NBs通过介导细胞固有免疫反应或者作为细胞干扰素信号通路元件参与宿主细胞的抗病毒防御活动。该文以几种DNA和RNA病毒为例,综述了在病毒感染过程中PML-NBs与病毒的相互作用以及这些相互作用的功能意义,从而揭示PML-NBs在抵御病毒感染和免疫反应中的重要作用,并提出运用病毒单分子实时示踪（Single-virus Tracking）这一新技术深入研究PML-NBs在病毒感染中作用的可行性。     

病毒逃逸宿主免疫反应机制的研究进展

先天性免疫反应是宿主抵御病毒感染的第一道防线,也是激活适应性免疫的基础,其在宿主清除病毒的免疫反应中发挥关键作用,成为当前免疫学研究的热点。在先天性免疫反应中,病毒感染细胞后主要通过模式识别受体识别病毒入侵,进而产生干扰素和一系列细胞因子来抵抗病毒入侵或清除病毒;而在适应性免疫中,机体主要通过T细胞和B细胞特异性识别入侵的病毒并将其清除。与此同时,病毒为了能够更好地在宿主细胞中获得生存,进化了多种可逃逸宿主免疫系统的机制。现将主要针对于病毒逃逸宿主免疫反应的一系列机制进行阐述。     

用口蹄疫病毒非结构蛋白区分注苗动物和自然感染动物研究进展

口蹄疫是由口蹄疫病毒引起的偶蹄类动物烈性传染病,疫苗接种是防治口蹄疫暴发的主要措施之一,而要控制口蹄疫流行,首先要将病毒感染动物从疫苗接种群体中区分开来。以口蹄疫病毒非结构蛋白（NSP）为抗原,检测动物体内的NSP抗体是一种很好的区分感染动物和疫苗免疫动物的诊断方法,许多实验室开展了相关研究,并取得了一定的成绩。     

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.     

Chimeric virus-like particles elicit protective immunity against serotype O foot-and-mouth disease virus in guinea pigs

Foot-and-mouth disease (FMD) is an acute and highly contagious disease caused by foot-and-mouth disease virus (FMDV) that can affect cloven-hoofed animal species, leading to severe economic losses worldwide. Therefore, the development of a safe and effective new vaccine to prevent and control FMD is both urgent and necessary. In this study, we developed a chimeric virus-like particle (VLP) vaccine candidate for serotype O FMDV and evaluated its protective immunity in guinea pigs. Chimeric VLPs were formed by the antigenic structural protein VP1 from serotype O and segments of the viral capsid proteins (VP2, VP3, and VP4) from serotype A. The chimeric VLPs elicited significant humoral and cellular immune responses with a higher level of anti-FMDV antibodies and cytokines than the control group. Furthermore, four of the five guinea pigs vaccinated with the chimeric VLPs were completely protected against challenge with 100 50% guinea pig infectious doses (GPID₅₀) of the virulent FMDV strain O/MAY98. These data suggest that chimeric VLPs are potential candidates for the development of new vaccines against FMDV.     

Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. The disease is highly contagious and FMD virus (FMDV) replicates and spreads extremely rapidly. Outbreaks in previously FMD-free countries, including Taiwan, the United Kingdom, and Uruguay, and the potential use of FMDV by terrorist groups have demonstrated the vulnerability of countries and the need to develop control strategies that can rapidly inhibit or limit disease spread. The current vaccine, an inactivated whole virus preparation, has a number of limitations for use in outbreaks in disease-free countries. We have developed an alternative approach using a genetically engineered FMD subunit vaccine that only contains the portions of the viral genome required for virus capsid assembly and lacks the coding region for most of the viral nonstructural (NS) proteins including the highly immunogenic 3D protein. Thus, animals inoculated with this marker vaccine can readily be differentiated from infected animals using diagnostic assays employing the NS proteins not present in the vaccine and production of this vaccine, which does not contain infectious FMDV, does not require expensive high-containment manufacturing facilities. One inoculation of this subunit vaccine delivered in a replication-defective human adenovirus vector can induce rapid, within 7 days, and relatively long-lasting protection in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, but did not develop antibodies against viral NS proteins demonstrating the ability of FMD subunit vaccinated animals to be differentiated from infected animals. To stimulate early protection prior to the vaccine-induced adaptive immune response we inoculated swine with the antiviral agent, type I interferon, and induced complete protection within 1 day. Protection can last for 3-5 days. The combination of the FMD marker vaccine and type I interferon can induce immediate, within 1 day, and long-lasting protection against FMD. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread this strategy may reduce the number of animals that need to be slaughtered during an outbreak.     

Foot and mouth disease virus polyepitope protein produced in bacteria and plants induces protective immunity in guinea pigs

The goal of this project was to develop an alternative foot and mouth disease (FMD) vaccine candidate based on a recombinant protein consisting of efficient viral epitopes. A recombinant gene was designed that encodes B-cell epitopes of proteins VP1 and VP4 and T-cell epitopes of proteins 2C and 3D. The polyepitope protein (H-PE) was produced in E. coli bacteria or in N. benthamiana plants using a phytovirus expression system. The methods of extraction and purification of H-PE proteins from bacteria and plants were developed. Immunization of guinea pigs with the purified H-PE proteins induced an efficient immune response against foot and mouth disease virus (FMDV) serotype O/Taiwan/99 and protection against the disease. The polyepitope protein H-PE can be used as a basis for developing a new recombinant vaccine against FMD.     

The importance of being short: the role of rabies virus phosphoprotein isoforms assessed by differential IRES translation initiation

The rabies virus (RV) phosphoprotein P is a multifunctional protein involved in viral RNA synthesis and in counteracting host innate immune responses. We have previously shown that RV P gene expression levels can be regulated by using picornavirus internal ribosome entry site (IRES) elements. Here we exploited a particular feature of the foot-and-mouth disease virus (FMDV) IRES, namely, preferential initiation at a downstream initiation codon, to address the role of N-terminally truncated RV phosphoproteins usually generated in RV-infected cells through ribosomal leaky scanning. Recombinant RVs in which P synthesis was directed by the poliovirus or FMDV IRES produced full-length P (P1) or a truncated form (P2), as the dominant product, respectively. While the P2 overexpressing virus showed attenuated growth in interferon-incompetent cells, it was superior to the P1 overexpressing virus in preventing expression of host interferon-stimulated genes. This indicates that in RV infected cells the availability of the truncated P2 protein is critical for viral resistance to interferon.     

乙型肝炎病毒特异性细胞毒性T淋巴细胞在病毒清除过程中的作用

在乙型肝炎病毒(HBV)感染过程中,适应性免疫与病毒的致病和清除密切相关。一般认为,体液免疫产生的抗体可以清除外周循环的病毒颗粒,从而阻止病毒在宿主体内的传播,细胞免疫主要清除被感染细胞中的病毒。HBV特异性的细胞毒性T淋巴细胞(CTL)在抑制HBV复制过程中发挥着重要的作用。CTL在肝内主要通过分泌γ干扰素抑制病毒,同时,当CTL识别HBV抗原后,HBV特异性CTL募集抗原非特异性炎症细胞对肝组织浸润,造成肝细胞的损伤。对CTL抗病毒作用进行深入研究,将为乙型肝炎的治疗开辟新的途径。     

Interplay between Kaposi's sarcoma-associated herpesvirus and the innate immune system

Understanding of the innate immune response to viral infections is rapidly progressing, especially with regards to the detection of DNA viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) is a large dsDNA virus that is responsible for three human diseases: Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. The major target cells of KSHV (B cells and endothelial cells) express a wide range of pattern recognition receptors (PRRs) and play a central role in mobilizing inflammatory responses. On the other hand, KSHV encodes an array of immune evasion genes, including several pirated host genes, which interfere with multiple aspects of the immune response. This review summarizes current understanding of innate immune recognition of KSHV and the role of immune evasion genes that shape the antiviral and inflammatory responses.     

Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.