脊髓灰质炎病毒重组体的构建及其抗原性状分析

以脊髓灰质炎病毒（以下简称脊灰病毒）为载体构建的重组体活病毒可以做为探讨脊灰病毒的抗原结构和特性的有益手段。在构建重组有甲型肝炎病毒小片段抗原多肽的重组脊灰病毒基础上,分析了脊灰病毒VP1上中和抗原位点I的空间构象特点,并探讨了插入的外源抗原片段对其空间结构的可能影响。     

脊髓灰质炎病毒缺陷型载体瞬时表达系统Ⅰ的构建

为探索可表达较大片段外源基因的脊灰病毒重组载体,以HBV-S基因置换脊灰病毒的P1基因,同时以另一途径提供脊灰病毒P1结构蛋白,经转染入Hela细胞中形成缺陷型重组病毒颗粒,此病毒可以感染新的细胞,并表达其重组的外源基因,但不产生子代病毒.实验结果表明,这种瞬时表达系统的构建,为脊灰病毒缺陷型重组载体用于基因转导技术打下基础.     

脊髓灰质炎病毒缺陷型载体瞬时表达系统Ⅰ的构建

为探索可表达较大片段外源基因的脊灰病毒重组载体,以ＨＢＶＳ基因置换脊灰病毒的Ｐ１基因,同时以另一途径提供脊灰病毒Ｐ１结构蛋白,经转染入Ｈｅｌａ细胞中形成缺陷型重组病毒颗粒,此病毒可以感染新的细胞,并表达其重组的外源基因,但不产生子代病毒。实验结果表明,这种瞬时表达系统的构建,为脊灰病毒缺陷型重组载体用于基因转导技术打下基础。     

脊髓灰质炎病毒缺陷型载体瞬时表达系统I的构建

为探索可表达较大片段外源基因的脊灰病毒重组载体,以ＨＢＶ－Ｓ基因置换脊灰病毒的Ｐ１基因,同时以另一途径提供脊灰病毒Ｐ１结构蛋白,经转染入Ｈｅｌａ细胞中形成缺陷型重组病毒颗粒,此病毒可以感染新的细胞,并表达其重组的外源基因,但不产生子代病毒。实验结果表明,这种瞬时表达系统的构建,为脊灰病毒缺陷型重组载体用于基因转导技术打下基础。     

病毒工厂：病毒复制的关键场所

多种病毒的复制和组装过程需要在被称为"病毒工厂"的特殊结构内完成。随着研究水平的不断提高,研究者已经在一定程度上揭示了病毒工厂的形成过程及结构。病毒入侵细胞后,能够招募细胞和病毒成分从而形成病毒组装和成熟的场所,细胞膜结构和细胞骨架能够参与该结构的形成,且部分病毒形成的病毒工厂还需线粒体提供能量。除上述特征外,病毒工厂的结构及形态会随病毒复制阶段的不同而不断变化。本文将对病毒工厂的结构、细胞器的招募、病毒工厂结构的变化及大分子物质的运输进行综述。     

脊髓灰质炎病毒中Ⅰ9株基因组克隆及其部分结构分析

以脊髓灰质炎病毒RNA为模板,反转录合成了长链ds-cDNA。将合成的脊灰病毒中I9株ds—cDNA片段重组到质粒pAT153上,获得了约占脊灰病毒中I9基因组95％以上的cDNA克隆。对克隆的中I9cDNA部分片段作了限制性内切酶图谱分析和部分核酸的序列分析,发现脊灰病毒中I9部分核酸顺序有所改变。     

中山市2011年健康儿童脊髓灰质炎、麻疹和乙型肝炎抗体水平监测

目的了解中山市1~14岁健康儿童脊髓灰质炎(脊灰)病毒、麻疹病毒和乙型肝炎(乙肝)病毒抗体水平状况,为维持无脊灰、消除麻疹和控制乙肝提供依据。方法采用多阶段整群抽样方法,随机选择220名1~14岁健康儿童抽取静脉血,采用ELISA法检测麻疹病毒抗体Ig G、乙肝病毒表面抗原和抗体;用中和试验检测脊灰(Ⅰ、Ⅱ、Ⅲ)型病毒中和抗体。结果脊灰、麻疹和乙肝病毒抗体阳性率分别为91.36%、93.18%和66.36%,脊灰病毒Ⅰ、Ⅱ、Ⅲ型和麻疹病毒抗体几何平均滴度(GMT)分别为1∶215.90、1∶119.05、1∶31.40和1∶1 254.45;乙肝病毒表面抗原阳性率为1.36%。结论中山市1~14岁健康儿童对脊灰病毒具有较高的免疫水平,已形成对脊灰病毒有效的免疫屏障;乙肝得到有效控制;但麻疹未能形成有效的免疫屏障。     

口服口服脊髓灰质炎疫苗国家引入IPV

背景 脊髓灰质炎及其控制脊髓灰质炎(脊灰)是脊灰病毒1、2和3型引起的一种急性传染病,经人→人接触(粪→口途径)传播,疫苗前时代,所有儿童均暴露于脊灰病毒,1／200的易感儿童暴露后发生麻痹型脊灰。     

贵州省贞丰县疫苗衍生脊髓灰质炎病毒循环的病毒学分析

为及时发现和阻断疫苗衍生脊髓灰质炎（脊灰）病毒（vaccine-derived polioviruses, VDPVs）循环、脊灰野病毒的输入和能够引起临床脊灰的其它脊灰疫苗相关病毒,做好贵州省无脊灰状态下脊灰病毒学监测工作,采用病毒分离、鉴定与核苷酸序列测定和分析方法,对贵州省贞丰县及周围10个县2003～2004年报告的急性弛缓性麻痹（AFP）病例及2004年接触者粪便标本的病毒学监测的结果进行了分析. 对收集到的105例AFP病例和47例密切接触者的278份便标本进行了病毒学监测,结果从66例中共分离到肠道病毒（EV）66株,阳性率为43.4%,其中脊灰病毒（PV）29例,分离率为19.1%,非脊灰肠道病毒（NPEV）37例,分离率为24.3%.29例PV经中国疾病预防控制中心病毒病预防控制所国家脊灰实验室鉴定,24例为疫苗相似株,5例为脊灰Ⅰ型VDPVs,这5例均为2004年从贞丰县所分离到.贞丰县及周围县EV阳性检出率（43.4%）高于2003～2004年全省水平（22.9%～24.6%）,2004年PV分离率比2003年高达2.6倍,29株PV中单个Ⅰ型占34.5%,明显高于往年（2000～2002年全省平均4.1%）.本研究提示,Ⅰ型VDPVs在贞丰县引起了循环（cVDPVs）,通过口服脊灰疫苗强化免疫已经阻断cVDPVs的传播.人群中PV和NPEV带毒率明显增高,非VDPVs引起的临床符合脊灰病例不容忽视;应加强脊灰病毒学监测数据的分析和早期疫情预警工作.     

Ⅱ类囊膜病毒膜融合的分子机制

病毒囊膜与宿主细胞膜的膜融合是囊膜病毒入侵的重要过程,病毒囊膜融合糖蛋白的一系列结构变化引发此过程.综述了Ⅱ类囊膜病毒、弹状病毒及疱疹病毒融合蛋白结构与功能研究的最新进展,介绍了软件分析并定位融合蛋白功能区域的方法.Ⅱ类病毒与Ⅰ类病毒融合蛋白的融合前结构不同,但融合后结构(发夹三聚体结构)相似.弹状病毒与疱疹病毒的融合蛋白集合了Ⅰ/Ⅱ类融合蛋白的某些特征,但其结构变化及融合过程各不相同,被归为新型融合蛋白.上述研究为基础设计的以病毒融合过程为靶标的抑制子,可为抗病毒新药的研制提供新思路.     

Cold-adapted poliovirus mutants bypass a postentry replication block.

In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.     

Entry of poliovirus into cells is blocked by valinomycin and concanamycin A

Poliovirus contains a virus particle devoid of a lipid envelope that does not require an intact pH to enter into susceptible cells. Thus, the blockade of pH gradient generated in endosomes is not sufficient to impede the translocation of poliovirus particles to the cytoplasm, suggesting that translocation takes place at the plasma membrane. Measuring both viral protein synthesis and eIF4G-1 cleavage mediated by poliovirus protease 2A has been used to monitor productive entry of poliovirus into cells. Translation of the input poliovirus RNA produces enough 2A(pro) to cleave eIF4G-1, providing a sensitive assay to estimate poliovirus RNA delivery to the cytoplasm followed by its translation. Combination of concanamycin A, a vacuolar proton-ATPase inhibitor, and valinomycin, an ionophore that promotes K(+) efflux from cells, powerfully prevented poliovirus infection. Moreover, modifying the ionic conditions of the culture medium (increasing the concentration of K(+) and decreasing the concentration of Na(+)), together with concanamycin A, also significantly interfered with poliovirus entry. These findings suggest that poliovirus RNA requires an intact concentration of K(+) ions inside the cells to be uncoated and to gain access to the cytoplasm. In addition, the actual contribution of concanamycin A (as well as other inhibitors of endocytosis) to the total inhibition of productive poliovirus entry points to the idea that at least some percentage of polioviral subparticles translocates from the endosomes.     

Entry of poliovirus into cells does not require a low-pH step.

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.     

Inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles.

The entry of animal viruses into cells is associated with permeabilization of the infected cells to protein toxins such as alpha-sarcin (C. Fernández-Puentes and L. Carrasco, Cell 20:769-775, 1980). This phenomenon has been referred to as "the early permeabilization by animal viruses" (L. Carrasco, Virology 113:623-629, 1981). A number of inhibitors of poliovirus growth such as WIN 51711 6-(3,4-dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile (DEPC) and Ro 09-0410 specifically block the uncoating step of poliovirus but have no effect on attachment or entry of poliovirus particles into cells. These agents are potent inhibitors of the early permeabilization induced by poliovirus to the toxin alpha-sarcin. Thus, the uncoating of poliovirus is required for the permeabilization of cell membranes to proteins. The increased entry of labeled heparin promoted by virus entry is not blocked by these agents, indicating that poliovirus binds to its receptor and is internalized along with heparin in endosomes in the presence of WIN 51711, DEPC, or Ro 09-0410. We conclude that the delivery to the cytoplasm of some molecules that coenter with virion particles does not take place if the uncoating process is hindered.     

Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry

Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-beta-cyclodextrin (MbetaCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MbetaCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MbetaCD. We further demonstrate that treatment with MbetaCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.     

Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5'' nontranslated region and involvement of a cellular 57-kilodalton protein.

Translation of poliovirus RNA is initiated by cap-independent internal entry of ribosomes into the 5' nontranslated region. This process is dependent on elements within the 5' nontranslated region (the internal ribosomal entry site) and may involve novel translation factors. Systematic mutation of a conserved oligopyrimidine tract has revealed a cis-acting element that is essential for translation in vitro. The function of this element is related to its position relative to other cis-acting domains. This element is part of a more complex structure that interacts with several cellular factors, but changes in protein binding after mutation of this element were not detected in a UV cross-linking assay. A 57-kDa protein from the ribosomal salt wash fraction of HeLa cells was identified that binds upstream of the oligopyrimidine tract. Translation of poliovirus mRNA in vitro was strongly and specifically inhibited by competition with the p57-binding domain (nucleotides 260 to 488) of the 5' nontranslated region of encephalomyocarditis virus, indicating a probable role for p57 in poliovirus translation. p57 is likely to be identical to the ribosome-associated factor that binds to and is necessary for the function of the internal ribosomal entry site of encephalomyocarditis virus RNA.     

The clathrin endocytic pathway in viral infection.

How important is the clathrin-dependent endocytic pathway for entry of viruses into host cells? While it is widely accepted that Semliki Forest virus (SFV), an enveloped virus, requires this pathway there are conflicting data concerning the closely related Sindbis virus, as well as varying results with picornaviruses such as human rhinovirus 14 (HRV 14) and poliovirus. We have examined the entry mode of SFV, Sindbis virus, HRV 14 and poliovirus using a method that identifies single infected cells. This assay takes advantage of the observation that the clathrin-dependent endocytic pathway is specifically and potently arrested by overexpression of dynamin mutants that prevent clathrin-coated pit budding. Using HeLa cells and conditions of low multiplicity of infection to favor use of the most avid pathway of cell entry, it was found that SFV, Sindbis virus and HRV 14 require an active clathrin-dependent endocytic pathway for successful infection. In marked contrast, infection of HeLa cells by poliovirus did not appear to require the clathrin pathway.     

Inhibition of poliovirus RNA synthesis by brefeldin A.

Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit poliovirus replication 10(5)- to 10(6)-fold. BFA does not inhibit entry of poliovirus into the cell or translation of viral RNA. Poliovirus RNA synthesis, however, is completely inhibited by BFA. A specific class of membranous vesicles, with which the poliovirus replication complex is physically associated, is known to proliferate in poliovirus-infected cells. BFA may inhibit poliovirus replication by preventing the formation of these vesicles.     

New (fluorescent) light on poliovirus entry

To initiate infection, poliovirus must release its RNA genome into the cytoplasm of a target cell, a process called 'uncoating'. How this occurs has remained uncertain, despite studies over several decades. Two new studies re-address the question of poliovirus entry. The results suggest that poliovirus enters different cells by different mechanisms, and point to a role for virus-induced intracellular signals in the process.     

The antigenic structure of poliovirus

We have solved the structure of the Mahoney strain of type 1 and the Sabin (attenuated vaccine) strain of type 3 poliovirus by X-ray crystallographic methods. By providing a three-dimensional framework for the interpretation of a wealth of experimental data, the structures have yielded insight into the architecture and assembly of the virus particle, have provided information regarding the entry of virus into susceptible cells, and defined the sites on the virus particle that are recognized by neutralizing monoclonal antibodies. Thus locating mutations in variants selected for resistance to neutralizing monoclonal antibodies has defined three antigenic sites of the surface of the virion, and provided clues as to the mechanisms by which viruses escape neutralization. Finally, comparison of the structures of the two strains, together with analysis of sequences of many poliovirus strains, have begun to define the structural changes associated with serotypic differences between polioviruses.