Supercoiled circular DNA of an insect granulosis virus

The DNA of the granulosis virus of the Indian meal moth, Plodia interpunctella, was characterized by physical chemical and electron microscopic techniques. Twenty-five percent of the DNA extracted from purified virus was isolated as supercoiled circular molecules. The remaining 75% consisted of relaxed circular molecules. These molecular forms were indicated by the production of two radioactive bands during sedimentation of (3)H-labeled granulosis virus DNA in alkaline sucrose gradients or in equilibrium density gradients of neutral cesium chloride/propidium iodide. Electron microscopic visualization of the DNA that banded at the higher density in the latter gradients revealed supercoiled structures whereas that of DNA that banded at the lower density demonstrated relaxed circular molecules. The superhelical molecules were converted to relaxed circles by treatment with pancreatic DNase.The molecular weight of the viral DNA was calculated to be 81 x 10(6) by sedimentation in neutral sucrose and 78 x 10(6) by sedimentation in alkaline sucrose. The molecular weight estimated from length measurements in electron micrographs was 76 x 10(6). The buoyant density of the granulosis virus DNA was 1.703 g/cm(3) and that of its insect host DNA was 1.697 g/cm(3). Equilibrium sedimentation in cesium chloride and thermal denaturation indicated G + C contents of 44% and 39% for the viral and host DNA, respectively.     

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.     

原位分子杂交检测上海真厉螨与柏氏禽刺螨体内肾综合症出血热病毒的研究

目的：提供革螨传播肾综合征出血热病毒（HFRSV）的分子生物学证据。方法：用上海真厉螨与柏氏禽刺螨叮咬HFRSV接种乳小鼠，取叮咬后d10及d100以上的上海真厉螨与柏氏禽刺螨冰冻切片，用地高辛标记HFRSVcDNA核酸探针原位分子杂交检测其体内病毒RNA。结果：叮咬野鼠型（76－118株）接种乳小鼠后d10上海真厉螨检出病毒RNA，在细胞浆、细胞核中及核膜上呈细密颗粒状或全浆阳性，见于生殖器官细胞、胃及支囊上皮细胞及脑皮质细胞；叮咬家鼠型（UR株）病毒接种乳小鼠后d12螨亦检出病毒RNA，病毒的细胞组织分布与野鼠型相同；分别检测野鼠型螨31 只和家鼠型23 只, 其中阳性分别为17 只和10 只; 叮咬野鼠型病毒接种乳小鼠后d132, 家鼠型d102螨仍能检出病毒RNA。柏氏禽刺螨叮咬野鼠型病毒接种乳小鼠d10, 检测螨20只, 其中阳性12 只, 病毒RNA 主要分布于生殖器官细胞内。结论: 上海真厉螨与柏氏禽刺螨均可经叮咬获得HFRSV , 上海真厉螨可分别感染野鼠型和家鼠型HFRSV , 野鼠型在螨群体内至少可维持132 d, 家鼠型102 d, 具有贮存两型病毒的作用, 是HFRSV 的适宜媒介和贮存宿主, 在动物宿主间交叉传播两型HFRSV 中起重要作用。     

Monoclonal antibodies specific for Hantaan virus.

Six hybridoma cell line producing monoclonal antibodies to Hantaan virus were established by fusion of NS-1 mouse myeloma cells with spleen cells of mice immunized with Hantaan virus strain 76-118. The specificity of these monoclonal antibodies was established by immunoblotting analysis and immunofluorescence. Five of the clones reacted with antigens on the cell surface and in the cytoplasm, and one clone reacted with a determinant expressed only in the cytoplasm of the infected cells. Two of the clones produced antibodies that reacted with a Mr 50,000 polypeptide in virus-infected cellular extracts and purified virus preparations. The monoclonal antibodies were used to examine the antigenic relationship among Hantaan virus strains and between Hantaan virus and Prospect Hill virus and the virus of nephropathia epidemica. Three antibodies were capable of distinguishing between the Lee strain and the 760-118 strain of Hantaan virus and three additional antibodies reacted with determinants shared by both virus strains. None of the six reacted with Prospect Hill virus or the virus of nephropathia epidemica.     

Leukemia Viruses Associated with Mouse Myeloma Cells

Myeloma cells derived from BALB/c and C3H mice show evidence of infection by a murine leumemia virus. The immunoglobulin-producing myelomas secrete an RNA-containing virus with a density of 1.20 to 1.22 gm/cm(3). RNA with a sedimentation coefficient of 74 S in 0.1 M sodium sodium chloride has been isolated from secreted virus particles and has a base composition similar to that found for other murine leukemia virus RNA. An intracellular virus particle has been partially purified and has a density of 1.29 to 1.32 gm/cm(3). Both extracellular and intracellular virus particles contain the leukemia virus group-specific antigen.     

Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins

SUMMARY. Hepatitis C virus (HCV) in highly infectious sera has been shown to be predominantly associated with low-density lipoproteins. To determine whether the association is specific to low-density lipoproteins (LDL) or very low-density lipoproteins (VLDL), we fractionated HCV-containing plasma by a column chromatographic procedure known to separate these classes. Hepatitis C virus RNA detected by polymerase chain reaction (PCR) was associated primarily with the very low-density (VLDL) fraction. However, it could not be ruled out that virus-associated LDL may have eluted with this fraction. Hepatitis C virus virions isolated from sera having sufficient titre for visualization by electron microscopy are generally coated with antiviral antibodies, therefore we utilized the lipid association to isolate antibody-free virions. Very low-density lipoproteins were isolated by ultracentrifugal flotation and then treated with deoxycholate to release the virions. These were then isolated in a highly purified form by centrifugation in a sucrose gradient. The 1.10–1.11 gml^-1 region of the gradients contained 60–70 nm particles. Particles with similar surface structure but having a diameter of only 30–40 nm constituted about 30% of the total. The latter may represent defective interfering particles. The identity of both small and large particles with HCV virions and associated particles was confirmed by their trapping on grids by an anti-HCV E2 monoclonal antibody, and by their aggregation by rabbit antiserum to an amino-terminal peptide of E1. Thus, both E1 and E2 epitopes are displayed on the surface of intact HCV virions.     

Cell-free synthesis of rat and rabbit gastric proton pump

Ribonucleic acid was isolated from the fundic gastric mucosae of rats and rabbits by cesium chloride centrifugation of guanidine isothiocyanate-denatured mucosal homogenates, and poly A+ RNA was recovered from the pellets by oligodeoxythymidine column selection. When added to rabbit reticulocyte lysates, this poly A+ RNA stimulated [35S]methionine incorporation into trichloroacetic acid-precipitable material. Fluorographic analysis of the lysates showed protein synthesis to be dominated by polypeptides with molecular weights from 40,000 to 50,000, presumably prepepsinogen isoforms. Immune precipitation of the lysates with monoclonal antibodies directed against the gastric H+,K+-adenosine triphosphatase yielded bands at 94 kilodaltons and more diffuse banding at 180 kilodaltons. Further purification of the poly A+ RNA on sucrose gradients eliminated prepepsinogen messenger RNA; nascent H+,K+-adenosine triphosphatase synthesized by purified messenger RNA consisted of polypeptides with molecular weights between 88,000 and 94,000. The study indicates that cell-free translation of gastric mucosal messenger RNA may provide a useful model for analysis of gastric H+,K+-adenosine triphosphatase biosynthesis and processing.     

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

Bunyaviridae is a large family of viruses that have gained attention as "emerging viruses" because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or "spikes," were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry.     

Breakdown of HeLa cell DNA mediated by vaccinia virus

Breakdown of HeLa cell DNA begins within 90 min after infection with vaccinia virus at a multiplicity of infection of 2-plaque-forming units per cell, and ends about 7.5 hr after infection. HeLa cell DNA is degraded to a uniform size of 1 to 2 x 10(7) daltons, as judged by alkaline sucrose sedimentation analysis. The rate of host-cell DNA degradation by vaccinia virus increased directly with the multiplicity of infection. Sedimentation patterns in neutral and alkaline sucrose gradients of viral DNA from infected cells, as well as from partially purified virions, indicated that two size classes of DNA were present. Class 1 DNA sediments like T4 DNA in neutral gradients and has a molecular weight twice that of T4 DNA in alkaline gradients. Class II DNA sediments as a molecule of lower molecular weight than T4 DNA in both types of gradients. Infection of prelabeled HeLa cells with vaccinia virus did not result either in formation of trichloroacetic acid-soluble radioactivity or, upon purification of the virions, radioactivity associated with class I DNA, indicating that vaccinia virus does not reutilize HeLa cell DNA.     

抗柯萨奇病毒A16型单克隆抗体的制备和应用

目的 制备抗CA16病毒的特异性单克隆抗体,评价单克隆抗体在ELISA和细胞免疫化学染色中的作用,并利用单抗建立CA16快速检测胶体金免疫层析法并对其进行初步评价方法 CA16病毒接种RD细胞大量培养,氯化铯密度梯度超速离心纯化病毒颗粒,透射电镜鉴定纯化产物。福尔马林灭活病毒颗粒后免疫Balb/c小鼠,利用细胞融合技术制备稳定分泌抗CA16单抗的杂交瘤细胞株。ELISA和免疫荧光试验分别对单抗特性进行分析。根据单抗反应特性,分析单抗在细胞ELISA、细胞免疫组化中的应用,并利用特异性抗CA16单抗建立CA16胶体金免疫层析快速检测法结果 氯化铯密度梯度离心纯化病毒颗粒电镜观察显示,病毒颗粒为二十面体立体对称球形结构,大小均匀。经免疫小鼠,获得1株稳定分泌抗CA16单克隆抗体的杂交瘤细胞株(mAb CA16-19),该单抗为IgG2a亚型,细胞免疫荧光表明,单抗与RD宿主细胞内的CA16病毒颗粒结合。细胞ELISA显示,随着病毒滴度增加吸光度也增加。细胞免疫化学染色分析表明,单抗可与CA16病毒感染的RD结合,而不与EV71感染RD细胞和正常RD细胞反应。因此,该单抗可用于细胞ELISA和细胞免疫化学染色。同时,依据前期制备的抗CA16单抗(mAb CA16-10),并结合此次制备的mAb CA16-19共同建立胶体金免疫层析试纸条,该试纸条不与EV71和柯萨奇病毒B型交叉反应,最低检测限为6.25×10^6.25TCID₅₀/0.1 mL结论 利用氯化铯密度梯度离心纯化出CA16病毒颗粒,筛选出1株特异性抗CA16单抗,此株单抗可用于细胞免疫荧光试验、细胞ELISA和细胞免疫化学染色试验。并以单抗为原材料建立了CA16快速检测胶体金免疫层析法,为CA16病毒感染的快速诊断提供帮助。