人细小病毒B19分子生物学研究进展

人细小病毒B19 (Human parvovirus B19,简称B19病毒),是目前为止已知能够感染并引起人类疾病的两种细小病毒科成员之一。B19病毒作为一种重要病原,能够引起如儿童传染性红斑、急性再障危象、胎儿水肿甚至死胎等疾病。文中从B19病毒基因型、病毒受体、基因组结构特点与复制、病毒转录与转录后调控、病毒非结构和结构蛋白特点与功能以及病毒诊断及抗病毒药物研究策略6个方面来综述B19病毒的最新研究进展,以期为B19病毒致病机制的深入研究与治疗诊断策略的制定提供参考。     

人博卡病毒感染的研究进展

细小病毒科属于DNA病毒,其中的细小病毒亚科可进一步分成五个种属,大多数细小病毒属主要感染家畜并致病。直到2005年,研究人员认为B19是唯一的一个对人类致病的细小病毒属病毒。但是2005年10月瑞典科学家Al-lander等从小儿下呼吸道感染分泌物中发现一种新的细小病毒,该病毒能引     

人细小病毒B19生物学特性研究进展

细小病毒B19是目前细小病毒家族中除人博卡病毒(HBoV)和新型细小病毒PARV4(Humanparvovirus 4)以外唯一可以引起人类疾病的病毒。它可能是多种疾病的致病因子,感染儿童可引起传染性红斑,感染成人可引起多发性关节病综合征,而对一些有免疫病,血液病的患者,B19感染可以引起严重的疾病,如慢性红细胞贫血,暂时性再障危象。病毒感染后对细胞的毒性是引起暂时性障碍危象和纯红细胞再障的直接原因。血液病患者中,B19病毒感染是引起暂时性障碍危象的主要病因,持续B19病毒感染会导致免疫缺损患者     

伊蚊浓核病毒三维结构的比较分析

细小病毒是目前已知的结构最小的病毒, 其宿主范围很广. 利用冷冻电镜和三维重构技术获得了伊蚊浓核病毒1.2 nm分辨率下的三维结构, 并利用计算机生物信息处理技术和氨基酸序列比对技术比较了该病毒和其他细小病毒的结构和序列的异同. 尽管均属于浓核病毒亚科, 但是无论从衣壳结构还是其蛋白质的氨基酸序列上, 伊蚊浓核病毒和其他昆虫的细小病毒都有很大差异. 相比之下, 伊蚊浓核病毒和人类B19细小病毒的衣壳蛋白相似性较大, 两者的结构蛋白和非结构蛋白一致性也高于伊蚊浓核病毒与其他昆虫的细小病毒的一致性. 此结果表明: 伊蚊浓核病毒和人类B19细小病毒有着密切关系, 前者可能来源于B19病毒的较近期变种.     

细小病毒B19诊断芯片的初步研究

初步探讨并制备细小病毒B19诊断芯片,进行实验室验证．用基因芯片点样仪将细小病毒B19诊断探针固定在特殊处理的玻片上,以细小病毒B19质粒重复检测．运用限制性显示(RD)技术,用Cy5标记的通用引物进行荧光标记,通过与基因芯片杂交,严谨洗涤,将非特异性的标记片段洗脱后,经扫描仪扫描,计算机解读．杂交结果显示,Cy5标记的探针均出现杂交信号,而阴性对照和空白对照的杂交信号均很弱：芯片检测具有高特异性、敏感性和可重复性．初步建立了较可靠的制备与检测细小病毒B19诊断芯片的方法,经验证诊断准确率高,假阳性率低．     

抗细小病毒B19-VP2单克隆抗体的建立

制备抗细小病毒B19－VP2单克隆抗体,用于检测人血清中的B19抗原,辅助诊断相关疾病;也可用于制备人类细小病毒基因工程疫苗。用纯化的基因工程表达的B19－VP2蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞和小鼠骨髓瘤Sp2/0细胞融合,有限稀释法克隆细胞。ELISA及IF证明抗体特异性。克隆筛选出4株细胞,并初步建立了检测B19－VP2抗原的双抗体夹心酶联免疫吸附试验,为双抗体夹心法检测B19抗原为临床相关疾病诊断提供了检测手段。     

非洲猪瘟病毒的分子生物学研究进展

非洲猪瘟是一类动物传染病,致死率高达100%,在我国虽未发现该病,但一旦发生会给畜牧养殖业带来巨大经济损失。文中概述了非洲猪瘟病毒的分类、形态、基因组特征、主要结构蛋白,以及分子生物学诊断技术的研究进展。为进一步研究该病毒的复制机理、毒力、致病机理及疫苗的开发提供参考依据。     

用巢式PCR检测育龄妇女人群人细小病毒B19感染的研究

目的调查人类细小病毒B19在武汉地区普通人群,尤其是育龄妇女中的感染状况。方法采集武汉地区2家医院的血液样本1700份,分为两组。以血清中提取的DNA为模板,进行巢式PCR扩增。结果第Ⅰ组（普通组,包括男性和女性）阳性检测率为4．50％,第Ⅱ组（妇女组）阳性检测率为8．33％。结论武汉地区育龄妇女的B19感染率高于普通人群,很有必要对孕妇进行诊断从而预防新生儿感染B19病毒。另外,由于巢式PCR具有灵敏、特异、简便等优点,适合于用来检测血液样本中的人细小病毒B19。     

新型冠状病毒SARS-CoV-2研究进展

在武汉发生的由新型冠状病毒SARS-CoV-2引发的人类冠状病毒病COVID-19,仅仅2个多月时间在我国及国际上70多个国家出现迅速传播,致病和死亡率高,人类生命受到了极大威胁。一些科学家火速投入研究,对SARS-CoV-2的来源和进化、形态特征和基因结构、感染和致病分子机制开展深入研究,取得了重大进展,为科学防控COVID-19提供了重要依据。根据上述研究的基础,文中对COVID-19病毒疫苗、抗体和抑制剂研发提出了设想,在研究防控COVID-19核心技术上具有一定的参考价值。     

细小病毒B19 Oligo探针设计

利用BLAST软件对细小病毒B19的序列进行序列比对,获得特异序列;利用生物学软件Oligo6.40设计特异性高、Tm值接近、长度均一的Oligo探针。结果获得了13条70bp的Oligo探针,用于芯片打印及细小病毒B19的检测。表明利用BLAST系统和生物学软件Oligo6.40设计细小病毒B19诊断芯片的探针是一种简便而有效的方法。     

Experimental infection of cynomolgus monkeys with simian parvovirus.

Simian parvovirus is a recently discovered parvovirus that was first isolated from cynomolgus monkeys. It is similar to human B19 parvovirus in terms of virus genome, tropism for erythroid cells, and characteristic pathology in natural infections. Cynomolgus monkeys were infected with simian parvovirus to investigate their potential usefulness as an animal model of human B19 parvovirus. Six adult female cynomolgus monkeys were inoculated with purified simian parvovirus by the intravenous or intranasal route and monitored for evidence of clinical abnormalities; this included the preparation of complete hematological profiles. Viremia and simian parvovirus-specific antibody were determined in infected monkeys by dot blot and Western blot assays, respectively. Bone marrow was examined at necropsy 6, 10, or 15 days postinfection. All of the monkeys developed a smoldering, low-grade viremia that peaked approximately 10 to 12 days after inoculation. Peak viremia coincided with the appearance of specific antibody and was followed by sudden clearance of the virus and complete, but transient, absence of reticulocytes from the peripheral blood. Clinical signs were mild and involved mainly anorexia and slight weight loss. Infection was associated with a mild decrease in hemoglobin, hematocrit, and erythrocyte numbers. Bone marrow showed marked destruction of erythroid cells coincident with peak viremia. Our findings indicate that infection of healthy monkeys by simian parvovirus is self-limited and mild, with transient cessation of erythropoiesis. Our study has reproduced Koch's postulates and further shown that simian parvovirus infection of monkeys is almost identical to human B19 parvovirus infection of humans. Accordingly, this animal model may prove valuable in the study of the pathogenesis of B19 virus infection.     

Human parvovirus B19 can infect cynomolgus monkey marrow cells in tissue culture.

The human pathogenic parvovirus B19 cannot be grown in standard tissue culture but propagates in human bone marrow, where it is cytotoxic to erythroid progenitor cells. We now show that parvovirus B19 can replicate in cynomolgus bone marrow. Cynomolgus monkeys may be a suitable animal model for pathogenesis studies of parvovirus B19.     

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02.

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.     

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.     

Human parvovirus B19: general considerations and impact on patients with sickle-cell disease and thalassemia and on blood transfusions

Human parvovirus B19 (B19V) is a small (22-24 nm) nonenveloped DNA virus belonging to the genus Erythrovirus (family Parvoviridae). Although it generally causes self-limiting conditions in healthy people, B19V infection may have a different outcome in patients with inherited hemolytic anemias. In such high-risk individuals, the high-titer replication may result in bone marrow suppression, triggering a life-threatening drop of hemoglobin values (profound anemia, aplastic crisis). To date there is no consensus concerning a B19V screening program either for the blood donations used in the hemotherapy or for high-risk patients. Moreover, questions such as the molecular mechanisms by which B19V produces latency and persistent replication, the primary site (sites) of B19V infection and B19V immunopathology are far from being known. This review summarizes general aspects of B19V molecular characteristics, pathogenesis and diagnostic approaches with a focus on the role of this pathogen in blood transfusions and in patients with some hemoglobinopathies (sickle-cell disease, thalassemia).     

人细小病毒B19与自然流产关系的研究

无菌留取 5 4例自然流产妇女和 43例妊娠无异常孕妇血清 ,用聚合酶链反应 (PolymeraseChainReaction ,PCR)检测的人细小病毒B19(HumanParvovirusB19,B19)DNA ,在自然流产组中人细小病毒B19DNA有 15例阳性 ,阳性率为 2 7.78%。正常对照组中 ,人细小病毒B19DNA有 2例为阳性 ,阳性率为 4.65 % ,用x2 检验 ,x2 =8.86,P <0 .0 1,两组有非常显著性差异。由此总结 ,人细小病毒B19感染可能是导致自然流产的原因之一     

In situ hybridization for the detection of human parvovirus B19 nucleic acid sequences in paraffin-embedded specimens

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a³⁵S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The³⁵S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelflife of the probe.     

In situ hybridization for the detection of human parvovirus B19 nucleic acid sequences in paraffin-embedded specimens

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a 35S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The 35S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelf-life of the probe.