Amiloride inhibition of human cytomegalovirus replication.

Amiloride, an inhibitor of Na+/H+ exchange, interfered with cytomegalovirus (CMV) DNA synthesis, blocked the formation of nuclear inclusions, and reduced CMV infectious yields. The reduction of CMV infectious yields was concentration dependent with an ED90 of 46 microM. Amiloride at a concentration of 150 microM reduced CMV yields by about 100-fold. Reduction of infectious yields appeared to be related to interference with the formation of nuclear inclusions and to inhibition of CMV DNA synthesis. Nuclear inclusions were much reduced in size and demonstrated poorly defined cellulae in the amiloride-treated cells. CMV DNA synthesis was inhibited by approximately 70% when cells were treated with 150 microM amiloride. The reduction in CMV yields could not be related to the reported inhibitory effect of amiloride on protein synthesis. In amiloride (150 microM)-treated, CMV-infected cells, late, yet not immediate-early or early, protein synthesis was markedly decreased relative to untreated, CMV-infected cells. Accordingly, CMV DNA synthesis and the replication of CMV may be related to Na+ entry through an amiloride-sensitive pathway.     

PCR-RFLP analysis of cytomegalovirus infections associated with bone marrow transplantation in Japanese children.

In order to investigate the longitudinal molecular epidemiology of cytomegalovirus (CMV) infections associated with bone marrow transplantation (BMT) in Japanese children, we analyzed 36 CMV strains from 11 cases. Three regions (DNA polymerase, glycoprotein H, and immediate-early regions) of CMV DNA were amplified by polymerase chain reaction (PCR), and amplified products were each digested with two restriction enzymes, followed by electrophoresis. These restriction fragment length polymorphism (RFLP) analyses allowed the differentiation of 36 strains into 13 genotypes. Each patient excreted his or her own CMV with distinct genotype over the study period of up to one year. CMVs of two different genotypes were recovered during a one-month study from one recipient, who received a peripheral blood stem cell transplantation. Although the majority of patients and donors were CMV-seropositive before BMT, multiple CMV infections might not be common and the reactivation of latently infected CMV might be prominent in Japanese children receiving transplants.     

DNA base changes induced following in vivo exposure of unadapted, adapted or ada- Escherichia coli to N-methyl-N'-nitro-N-nitrosoguanidine

The adaptive response is one of the major repair pathways in Escherichia coli that removes DNA alkylation damage. To investigate the role of the adaptive response in mutagenesis, the E. coli gpt forward mutation assay system was used to determine the mutation spectrum of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in MNNG-adapted and unadapted GP120 (wild-type) and unadapted PJ5 (ada-5) cells. We observed that 34/37 mutations in the unadapted GP120 cells, 38/40 mutations in the adapted GP120 cells, and 10/10 mutations in the PJ5 cells were GC----AT transitions. The remaining 3/37 mutations in the unadapted GP120 cells were large insertions. The remaining 2/40 mutations in the adapted GP120 cells were transversions with one a GC----CG and the other an AT----CG. A surrounding sequence specificity of mutagenesis was observed for the GC----AT transitions in both the unadapted (GP120 and PJ5) and adapted (GP120) cells, with 70% of the unadapted PJ5, 68% of the unadapted GP120, and 61% of the adapted GP120 mutations occurring at the middle G of the sequence 5'--GG(A or T)--3'. Both strains also displayed a statistically significant preference for mutagenesis at guanine bases in the non-transcribed strand. The overall distribution of mutated sites in the gpt gene in adapted and unadapted cells was similar, although the rate of mutations at certain sites appeared different. These minor differences could result from either non-uniform repair of alkylation damage at different sites on the DNA, or altered processing of the alkylated bases to mutations in the adapted state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural and cytochemical studies of the fate of unsecreted collagen precursors after administration of colchicine to mice

The administration of colchicine disrupts the normal organization of the Golgi complex and blocks the secretion of collagen precursors in periodontal ligament fibroblasts of the mouse. The fate of the unsecreted collagen precursors contained in Golgi-derived saccules and newly formed dense bodies was followed by electron microscopy. A progressive condensation of saccule content along with phase separation of electron-dense and electron-lucent material was observed. Fusion of saccules with dense secretory bodies gave rise to larger inclusions (zebra bodies; ZB) filled with a combination of electron-dense and electron-lucent material. In some ZB, these materials appeared to polymerize into fibrillar units. The fibrillar units stained with silver methenamine like normal collagenous fibrils. These results suggest that unsecreted collagen precursors accumulate in vesicular compartments within which partial polymerization can occur. This finding may explain some reports of intracellular collagenous fibrils in fibroblasts of pathologically altered connective tissues.     

Schistosoma mansoni: development of the cercarial glycocalyx

The development of the cercarial glycocalyx of Schistosoma mansoni was studied by transmission electron microscopy and immunofluorescence light microscopy employing antibodies raised against extracted and chromatographed glycocalyx. By electron microscopy, cercariae present in the brood chamber of daughter sporocysts were surrounded by an electron-dense granular and fibrillar matrix. This material appeared structurally distinct from the glycocalyx which was coarsely fibrillar and located only on the surface of organisms that had developed a final tegument. The thickness of the glycocalyx apparently increased with the maturation of the tegument, since teguments that had many spines also had the thickest glycocalyx. Immunofluorescent staining of frozen sections of infected snail hepatopancreas showed that glycocalyx antigens were present on the surface of the cercariae and not in the matrix within the brood chamber or in snail tissues. Immunofluorescent staining of isolated larval cercariae showed staining of some but not all parasites with partially elongated tails. These studies suggest that the glycocalyx develops late in cercarial development (late in Stage 6 or in Stage 7 of Cheng and Bier), is made by the cercariae themselves, and is not a product of either the sporocyst wall cells or snail hepatopancreas.     

Functional interaction of nuclear domain 10 and its components with cytomegalovirus after infections: cross-species host cells versus native cells

Species-specificity is one of the major characteristics of cytomegaloviruses (CMVs) and is the primary reason for the lack of a mouse model for the direct infection of human CMV (HCMV). It has been determined that CMV cross-species infections are blocked at the post-entry level by intrinsic cellular defense mechanisms, but few details are known. It is important to explore how CMVs interact with the subnuclear structure of the cross-species host cell. In our present study, we discovered that nuclear domain 10 (ND10) of human cells was not disrupted by murine CMV (MCMV) and that the ND10 of mouse cells was not disrupted by HCMV, although the ND10-disrupting protein, immediate-early protein 1 (IE1), also colocalized with ND10 in cross-species infections. In addition, we found that the UL131-repaired HCMV strain AD169 (vDW215-BADrUL131) can infect mouse cells to produce immediate-early (IE) and early (E) proteins but that neither DNA replication nor viral particles were detectable in mouse cells. Unrepaired AD169 can express IE1 only in mouse cells. In both HCMV-infected mouse cells and MCMV-infected human cells, the knocking-down of ND10 components (PML, Daxx, and SP100) resulted in significantly increased viral-protein production. Our observations provide evidence to support our hypothesis that ND10 and ND10 components might be important defensive factors against the CMV cross-species infection.     

Origin and formation of different types of vacuoles induced by the multiplication of the alphavirus Sindbis virus in various cell systems

Spherule-containing vacuoles and nucleocapsid-bearing vacuoles (cytopathic vacuoles types 1 and 2 respectively of Grimley et al. 1968) induced by Alphavirus Sindbis were studied in brains from newborn mice, chicken embryo fibroblasts, and two lines of tumoral glial cells from muridae. Endoplasmic reticulum (ER) elements and finely granular electron-dense material also seen in contact with nucleocapsids seemed to be involved in the formation of the classical single-membrane spherule-containing vacuoles. A second type of spherule-containing vacuoles were characterized by their double membrane and an amorphous electron-dense content and were probably derived from mitochondria. Nucleocapsid-bearing vacuoles were formed from modified ER elements and seemed to be linked to excessive synthesis of viral material. Such ER alterations were not observed in RG6 cells. In these cells, there were only spherule-containing vacuoles, while nucleocapsids were seen associated with the cytoplasmic membrane only.     

All is fair in virus-host interactions: NK cells and cytomegalovirus

The infection of mice with mouse cytomegalovirus (MCMV) as a model of human cytomegalovirus (HCMV) infection has been particularly informative in elucidating the role of innate and adaptive immune response mechanisms during infection. Millions of years of co-evolution between cytomegaloviruses (CMV) and their hosts has resulted in numerous attempts to overwhelm each other. CMVs devote many genes to modulating the host natural killer (NK) cell response and NK cells employ many strategies to cope with CMV infection. While focusing on these attack-counterattack measures, this review will discuss several novel mechanisms of immune evasion by MCMV, the role of Ly49 receptors in mediating resistance to MCMV, and the impact of the initial NK cell response on the shaping of adaptive immunity.     

Fine structure of nucleoli in micronucleated cells

The correlation between the number of nucleolus organizing regions (NOR) on metaphase chromosomes and the number of nucleoli was studied in normal and micronucleate cells. Many micronuclei, but not all, were able to form complete nucleoli with fibrillar and granular RNP components and fibrillar centers. Micronuclei which failed to form complete nucleoli often contained multiple electron-dense bodies of fibrillar material. These structures, which were much smaller than nucleoli, reacted with nucleolus-specific antibodies and the Ag-As method in the same way as complete nucleoli, but lacked fibrillar centers and granular RNP components. The data suggest that these nucleolus-like ‘blobs’ contain nucleolar material which, following mitosis, has been enclosed in micronuclei which do not contain nucleolus organizing chromosomes. No evidence was found for the activation of latent NORs not expressed in mononucleate cells.     

The 2b proteins of Cucumber mosaic virus generally have the potential to differentially induce necrosis on Arabidopsis

Plant viral symptoms are rarely explained by direct molecular interaction between a viral protein and a host factor, but rather understood as a consequence of arms race between host RNA silencing and viral silencing suppressors. However, we have recently demonstrated that the 2b protein (2b) of Cucumber mosaic virus (CMV) HL strain could bind to Arabidopsis catalase that is important in scavenging cellular hydrogen peroxide, leading to the induction of distinct necrosis on Arabidopsis. Because we previously used virulent strains of subgroup I CMV in the study, we here further analyzed mild strains of subgroup II CMV, which share 70 to 80% sequence homology with subgroup I, to understand whether the necrosis induction is a general phenomenon to compromise host defense system mediated by catalase in the pathosystem of any CMV strains and Arabidopsis. Based on the results, we concluded that 2bs of subgroup II could also bind to catalase, resulting in decrease in catalase activity and weak necrosis on Arabidopsis. Because the 2b-catalase interaction did not prevent CMVs from spreading, it may eventually operate in favor of CMV.     

Similarities and Differences in the Development of Laboratory Strains and Freshly Isolated Strains of Herpes Simplex Virus in HEp-2 Cells: Electron Microscopy

HEp-2 cells infected with two laboratory strains (mP and MP) and two freshly isolated strains (F and G) of herpes simplex virus were fixed at intervals between 4 and 50 hr postinfection and sectioned, and were then examined with the electron microscope. These studies revealed the following. (i) All four strains caused identical segregation of nucleoli and aggregation of host chromosomes at the nuclear membrane. (ii) The development of MP virus could not be differentiated from that of its parent mP strain. (iii) There were quantitative differences between laboratory (mP) and freshly isolated (F) type 1 strains. Thus, cells infected with F contained numerous nuclear crystals of nucleocapsids and relatively few cytoplasmic structures containing enveloped nucleocapsids. Conversely, cells infected with mP or with MP virus contained numerous cytoplasmic structures with enveloped nucleocapsids and relatively few nuclear crystals of nucleocapsids. (iv) There were qualitative differences between type 2 strain (G) isolated from genital lesions and type 1 strains. Thus, cells infected with the G strain contain numerous filaments in nuclei and unenveloped and partially enveloped nucleocapsids in the cytoplasm. Of particular interest is the finding that cytoplasmic membranes in apposition to nucleocapsids were thickened and bent as if they were enveloping the particle. The significance of the qualitative differences in the development of the four strains is discussed.     

Replication of dengue, yellow fever, St. Louis encephalitis and vesicular stomatitis viruses in a cell line (TRA-171) derived from Toxorhynchites amboinensis

The replication of seven arboviruses in a cell line (TRA-171) derived from a nonhematophagous mosquito was studied. Four serotypes of laboratory adapted and three serotypes of unadapted dengue viruses replicated in the TRA-171 cell line, inducing syncytia. The sensitivity of TRA-171 cells to dengue virus infection was comparable to that of Aedes albopictus or A. pseudoscutellaris cells. Yellow fever, St. Louis encephalitis, and vesicular stomatitis viruses also replicated. All four serotypes of dengue viruses could be plaque assayed with TRA-171 cell cultures.     

Human cytomegalovirus infects Caco-2 intestinal epithelial cells basolaterally regardless of the differentiation state

Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.     

鸭病毒性肠炎病毒强毒株的形态发生学与超微病理学研究

应用透射电镜和超薄切片技术,研究鸭病毒性肠炎病毒(duck enteritis virus,DEV)CH强毒株人工感染成年鸭后,病毒在宿主细胞内的形态发生及各组织器官的超微结构变化.结果表明,感染后不同时间剖杀及发病后死亡鸭的肝、肠、脾、胸腺、法氏囊等组织器官中,均观察到典型的疱疹病毒粒子.病毒主要的靶细胞为淋巴细胞、网状内皮细胞、成纤维细胞、巨噬细胞、血管内皮细胞、肠道上皮细胞、肠道平滑肌细胞和肝细胞等.DEV的核衣壳有空心型、致密核心型、双环型和内壁附有颗粒型四种形态,存在胞核和胞浆两种装配方式.病毒核衣壳可在核内获得皮层,通过核内膜获得囊膜成为成熟病毒;也可通过内外核膜进入胞浆,在其中获得皮层,然后在各种质膜上获得囊膜,最后成熟病毒释放到细胞外.伴随着病毒的复制、装配和成熟,细胞中出现多种核内和胞浆包涵体、核内致密病毒核酸颗粒、微管和中空短管以及胞浆内膜包裹的电子致密小体、双层管等病毒相关结构.超微研究表明,组织细胞有坏死和凋亡两种变化.坏死细胞肿胀甚至破裂,线粒体肿胀空泡化,粗面内质网扩张,核糖体脱落,有的细胞器甚至完全崩解,染色质或固缩或溶解.凋亡细胞则染色质聚集,胞浆凝聚深染,细胞膜上有大量空泡,并有凋亡小体形成.细胞坏死与凋亡往往同时存在,疾病发生过程中,脾、胸腺、法氏囊以及小肠固有层中的淋巴细胞凋亡数量明显增多.