CYTOPLASMIC INCLUSIONS AND VIRUS-LIKE PARTICLES IN BLAST CELLS IN ACUTE LYMPHOBLASTIC LEUKEMIA

Cytoplasmic inclusions and virus-like particles are described in blast cells of peripheral blood from a 16-year-old female with acute lymphoblastic leukemia. Three kinds of inclusions were identified on electron microscopy. The first type of inclusion was single membrane-bounded vacuoles, some of which contained virus-like particles, the second was lysosome-like structures, and the third appeared to be of mitochondrial origin. Virus-like particles were round in shape and had a diameter of 26 to 58 nm. They consisted of an electrondense outer membrane and an electron-lucent core. At the present time the exact nature and significance of these virus-like particles still remain unclear.     

The occurrence and frequency of type C virus-like particles in placentas from patients with systemic lupus erythematosus and from normal subjects.

Type C RNA virus-like particles were found by electron microscopy in term placentas from 3 patients with systemic lupus erythematosus (SLE), 1 with probable SLE, and 2 normal patients. The virus-like particles were mainly of a budding or immature type located at or near the cell membrane of syncytiotrophoblasts in chorionic villi. Type C virus-like particles were not observed in the term placenta from a patient with chronic discoid LE, nor in early gestation specimens from 1 normal patient and 4 patients with SLE. The frequency of the Type C particles varied greatly: They were readily found in the patient with probable SLE, and only here were groups of budding particles observed. Type C particles were less numerous in one normal placenta and rare in the other positive placentas, both SLE and normal. Heretofore undescribed crystalline inclusions were found in the cytoplasm of chorionic villous endothelial cells from 3 patients with SLE and 1 with discoid LE. Tubuloreticular structures were observed in the maternal endometrium of 1 patient with SLE.     

Unusual cytoplasmic inclusions in blast cells in acute leukemia.

A 36-year-old man manifested the clinical signs of acute leukemia. Blast cells in the blood and bone marrow contained many large cytoplasmic inclusions. Cytochemical and ultrastructural studies indicated that the leukemic cells were of lymphocytic origin. Three kinds of inclusions were identified on electron microscopy: one type of inclusion contained virus-like particles; another resembled those in cases with hairy cell leukemia; and the third appeared to be autophagic in nature. The presence of the second type of inclusion in the blast cells and the relative chronicity of the clinical course suggest that this patient's disease is of lymphocytic origin, similar to hairy cell leukemia.     

Virus-like inclusions in the myocytes of the skeletal muscle in lateral amyotrophic sclerosis

Microscopic examination of musculus gastrocnemius biopsies was made in four cases of sporadic lateral amyotrophic sclerosis (LAS). The validity of the clinical diagnosis was confirmed by detected neurotrophic atrophy of the muscular fibers typical for LAS. Electron microscopic study revealed virus-like inclusions 200-450 nm in size in sarcoplasm of myocytes of all the patients. The inclusions consist of lined cells of hexagonal shape at the distance of 37-41 nm from each other. The inclusions resemble enteroviruses but are not identical to them both by size and structure of their elements. There were also specific ultrastructural changes of myocytes corresponding to viral infection. The above virus-like inclusions should be considered as specific structures formed as a result of metabolic shifts caused by productive action on the cell of infective or unknown factor.     

Small round virus-like particles associated with acute gastroenteritis in japanese children

Nine patients with acute gastroenteritis shed small round virus-like particles in the faeces, and seven of them developed serum antibody against those particles as judged by immune electron microscopy. The small round virus-like particle was measured at 29–32 nm in diameter and showed a fine spiky structure on the surface. The buoyant density of the small round virus-like particles was determined to be 1.37–1.40 gm/cm³ in cesium chloride density gradient. The small round virus-like particle differed morphologically from astrovirus and calicivirus, and was antigenically dissimilar to Norwalk agent and W agent. These observations suggest that the small round vims-like particle is one of the new gastroenteritis viruses or may be a new serotype of them. Attempts to cultivate the small round virus-like particles were unsuccessful.     

Potential spectrum of etiological agents of viral enteritis in hospitalized infants.

Fecal specimens were obtained from 1,160 infants and young children with acute nonbacterial gastroenteritis over a period of 2 years. A total of 100 specimens were obtained from age-matched asymptomatic controls. The specimens were examined for the presence of viruses by electron microscopy. Viruses or virus-like particles frequently associated with enteritis were detected in 27% (314 of 1,160) of the symptomatic patients. No viruses or virus-like particles were detected in the 100 control subjects. Rotavirus was detected in 73% (230 of 314) of the virus-positive samples. The mean age of rotavirus-positive patients was 11.5 months, although the patients ranged in age from 2 weeks to 5 years. Of the symptomatic patients, 45 (14%) exhibited small virus-like particles (15 to 40 nm) in the feces in the absence of any other detectable pathogen. Some of the virus-like particles observed in these patients appeared to be similar to astrovirus, and some appeared to be similar to the Otofuke agent or possibly minireovirus. Significantly, however, the mean age of infants with enteritis from whom these small virus-like particles were recovered was 4.5 months (range, 10 days to 19 months). Our findings confirmed the already-known fact that rotaviruses constitute the most important cause of viral enteritis in young children. In addition, small viruses may be an important cause of gastroenteritis in infants under 5 months of age.     

中国流行性出血热脏器标本病毒样颗粒属性的探讨

本文应用电镜观察了12例流行性出血热(EHF)尸体标本,其中7例(58%)出现了泡状病毒样颗粒。它为圆形或椭圆形,直径72～116nm,平均92nm;有包膜,没有核样体,表面粗糙或有微突。颗粒主要分布于网织细胞及心、肝实质细胞的核膜及线粒体,偶尔见于内质网和胞浆。泡状病毒样颗粒在大小和形态上,与洪涛等在培养细胞见到的EHF病毒以及其他学者观察的汉坦病毒和布尼亚病毒基本一致,为EHF病毒在人体脏器细胞的形态表现。     

Toga-like virus as a cause of fulminant hepatitis attributed to sporadic non-A, non-B

Virus-like particles (60-70 nm) with spiked surfaces budding into cell vacuoles and rod-shaped inclusions were detected in nuclei of hepatocytes from a British patient transplanted for sporadic non-A, non-B fulminant hepatitis (NANB-FHF), probably contracted in Kenya. Identical particles were seen in two successive grafts (days 2 and 10) at regrafting for recurrent FHF. Ultrastructural features resembled those of the RNA-containing arbovirus, Rift Valley fever virus, but serological markers against a representative panel for arboviruses (Togaviruses) and transmission in mice proved negative. The particles shared features with the different arboviruses seen in the hepatectomy specimen of a second patient with NANB-FHF, and in both patients an insect vector was implicated in the clinical history. The particles were identical in size to those of a third patient with NANB-FHF, who had remained in the United Kingdom. These findings, together with the recent report of isolation of an RNA-containing virus resembling the Togaviridae, in parenteral NANB, suggest that several exotic virus-like agents resembling the arboviruses may be involved in the aetiology of NANB, including in the sporadic forms of FHF in the United Kingdom.     

Virus-Like Particles in Australia Antigen-Associated Hepatitis: An Immunoelectron Microscopic Study of Human Liver

In a previous report (Huang SN: Hepatitis-associated antigen hepatitis: an electron microscopic study. Am J Pathol 64:483-500, 1971) liver biopsies of renal transplant patients who developed chronic progressive viral hepatitis associated with persistence of Australia antigenemia [Au(1)] while under immunosuppressive therapy were studied. Predominantly intranuclear 210-250 A spherical, virus-like particles were revealed in 12 of 13 biopsies examined by electron microscope. No such particles were found in biopsies from Australia antigen negative patients. To investigate the relationship of these virus-like particles to Australia antigen, 2 of the Au(1) hepatitis renal transplant patients were restudied 6 to 8 months later. Liver biopsy material was prepared for light microscopy, immunofluorescent microscopy, electron microscopy and immunoelectron microscopy. The specificity of the anti-Au(1) serum used in this study was ascertained by immunodiffusion and immunoelectrophoresis, and by immunoelectron microscopic studies of the antigen-antibody complex prepared in vitro by mixing the ferritin-conjugated anti-Au(1) reagent with the purified Au(1) particles. Electron microscopy of biopsies from liver not treated with antibody showed that virus-like particles persisted in liver cells. Immunofluorescent microscopy of teased liver biopsy suspensions showed nuclear and some cytoplasmic fluorescence, indicating the cellular localization of Au(1). The immunoelectron microscopic preparations showed agglutination of the virus-like particles and the presence of antibody coupled ferritin in the intranuclear and cytoplasmic particle agglutinates. No virus-like particles were seen in the biopsy from a control patient without Au(1) antigenemia, and results of the immunoelectron microscopic procedure were negative. Our observations that massive amounts of Au(1)-associated particles are located in liver cell nuclei of individuals with chronic active hepatitis strengthen the hypothesis of Blumberg et al that Au(1) is an infectious agent and/or the antigenic determinant of a hepatitis virus.     

Virus-like particles in an antarctic Aggregata sp.: I. Sporogonial stages

An unknown Aggregata sp. was found in the renal organ of an antarctic Benthoctopus sp., when it was inspected for the presence of dicyemid mesozoans. Merozoites invaded the renal epithelium, whereas sporogonial stages resided in the submucosal connective tissue. Interestingly, individuals of all developmental stages were found to be infected with hitherto unknown virus-like particles. These virus-like particles, spherical in shape, nonenveloped and measuring approximately 30 nm in diameter, could be observed in the nuclei as well as within the cytoplasm. Greater amounts of virus-like particles tended to be arranged in paracrystalline arrays. Although the infection was extensive, it obviously had no pathogenic effect on the parasites themselves. On the basis of the host specificity, size, morphology and histochemical analysis, which suggested the putative viral genome as RNA, a relationship with Totoviridae is assumed.     

CREUTZFELDT-JAKOB DISEASE WITH DEMONSTRATION OF VIRUS-LIKE PARTICLES

Two kinds of virus-like particles, doughnut-shaped particles and particles with dense core, were discovered in a brain tissue from a patient with Creutz-feldt-Jakob disease. The two types of virus-like particles are similar to immature type C and type C particles. Although no direct relationship between Creutzfeldt-Jakob disease and these virus-like particles can be established, these facts may suggest that viruses play some role in the pathogenesis of Creutzfeldt-Jakob disease.     

Intestinal fine structure in crohn's disease

Summary Resected intestines from eight patients with Crohn's disease and three control cases were investigated by transmission electron microscopy. Characteristic changes were observed in the mucosa of all Crohn's disease specimens, most typically an infiltration of numerous macrophages into the propria. These cells displayed large lysosomes with inclusions which were mainly dense, irregularly shaped and composed of aggregated particles and bizarre-shaped, myelin-like figures. Similar inclusions were also found in the lysosomes of the surface epithelial cells and the macrophages in the submucosa. This latter layer otherwise consisted of an oedematous, collagenous connective tissue. The muscularis appeared structurally unaffected. Qualitatively, the findings were almost identical in all patients with Crohn's disease, but varied quantitatively without any clear correlation with the clinical histories. Moreover, in all cases typical alterations were found not only in the macroscopically most clearly affected parts of the intestines, but also in grossly normal regions, close to the margins of resection.The analogy of the fine structural findings with those of granulomas produced by injection of bacteria into experimental animals suggests that a microbial invasion of the intestinal wall may have initiated the disease. It therefore seems reasonable to assume that the lysosomal inclusions we observed represent partly degraded bacteria. More occasionally, virus-like particles were found within the lysosomes of the epithelial cells and the macrophages in the underlying propria. In view of the diffuse spread of the alterations it seems possible that there exists a generalized defect in the barrier function of the intestine in Crohn's disease. This could lead to passage of bacteria and/or other agents into the mucosa, followed by an influx of inflammatory cells from the blood. Storage of non-degradable microbial components in macrophages could then be responsible for the initiation and propagation of a chronic inflammatory process, similar to that of other granulomatous disorders.     

Segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice

Insertion of a short-sized epitope at four different sites of yeast-expressed hamster polyomavirus major capsid protein VP1 has been found to result in the formation of chimeric virus-like particles. Here, we demonstrate that the insertion of 45 or 120 amino acid-long segments from the N-terminus of Puumala hantavirus nucleocapsid protein into sites 1 (amino acids 80-89) and 4 (amino acids 288-295) of VP1 allowed the highly efficient formation of virus-like particles. In contrast, expression level and assembly capacity of fusions to sites 2 (amino acids 222-225) and 3 (amino acids 243-247) were drastically reduced. Immunization of BALB/c mice with chimeric virus-like particles induced a high-titered antibody response against the hantavirus nucleocapsid protein, even in the absence of any adjuvant. The strongest response was observed in mice immunized with virus-like particles harboring 120 amino acids of hantavirus nucleocapsid protein. According to the immunoglobulin subclass distribution of nucleocapsid protein-specific antibodies a mixed Th1/Th2 response was detected. The VP1 carrier itself also induced a mixed Th1/Th2 response, which was found to be reduced in mice immunized with virus-like particles harboring 120 amino acid-long inserts. In conclusion, hamster polyomavirus VP1 represents a promising carrier moiety for future vaccine development.     

肝病毒表面抗原“a”决定簇病毒样颗粒的构建和鉴定

目的 构建一个以乙肝核心抗原为骨架的病毒样颗粒,乙肝表面抗原＂a＂决定簇呈现在该病毒样颗粒的表面.方法 乙肝adr血清型的HBsAg蛋白＂a＂抗原决定簇（aa139～148,CSKPTDGNCT）插入到HBcAg的第78位天冬氨酸和第79位脯氨酸之间,密码子优化之后基因合成,酶切后连接到表达载体上,在大肠埃希菌中进行表达,纯化后对蛋白进行电镜观察、ELISA检测抗原性、免疫兔后检测免疫原性.结果 构建得到预期的原核表达质粒,并在大肠埃希菌中表达,纯化后电镜观察为病毒样颗粒,ELISA检测证实具有良好的抗原性和免疫原性.结论 成功获得预期的带乙肝表面抗原＂a＂决定簇的病毒样颗粒,为其应用奠定基础.     

Viruses and virus-like particles detected in samples from diseased game birds in Great Britain during 1988

During 1988, 108 samples were received from game birds (78 from pheasants, 28 from partridges and two from quail) for virus isolation or detection; 89 being received during the June to August rearing period. The most common clinical signs resulting in the submissions were death, scour and stunting. Virus or virus-like particles were detected in 51 cases, 43 as a result of direct electron microscopy of gut contents, seven by agar gel precipitin test for the presence of Marble spleen disease antigen and one by isolation, of a rotavirus. Particles observed by electron microscopy were: rotavirus - 15, adenovirus - 1, reovirus - 1, enterovirus - 1, 'fimbriated' virus-like particles - 10, rod-shaped virus-like particles - 19, On three occasions more than one type of particle was seen in the same sample.     

Generating enveloped virus-like particles with in vitro assembled cores

Alphaviruses are comprised of a nucleocapsid core surrounded by a lipid membrane containing glycoprotein spikes. Previous work demonstrated that in vitro assembled core-like particles are similar in structure to the nucleocapsid core in the native virus. Here we demonstrate that in vitro assembled core-like particles can be inserted into viral glycoprotein-expressing cells to generate enveloped virus-like particles. These virus-like particles bud from cells like native virus, are similar in size to the native virus, and can enter cells to release the contents of the core-like particle into the cytoplasm of the cell. Virus-like particles can be used to infect cells with biological and non-biological cargoes. The generation of enveloped virus-like particles containing an in vitro core and in vivo synthesized glycoproteins has applications for gene and drug delivery, medical imaging, and also basic mechanistic studies of virus assembly.     

SARS尸检组织的病理变化和超微结构观察

目的 研究严重急性呼吸综合征(SARS)尸检组织的临床病理和超微结构特征。方法 对1例SARS死亡患者做即刻肺穿刺和12h后尸检,进行病理形态和超微结构的观察;用Macchiavello法做病毒包涵体染色;并对淋巴结、脾脏、结肠、小肠及骨髓组织行CD20、CD45RO(UCHL-1)、CD4、CD8、CD68、CD34免疫组织化学标记。结果 SARS肺的主要病变为急性弥漫性全小叶性间质性炎,可见肺泡腔内透明膜形成和增生及脱落的肺泡上皮,偶见胞质内病毒包涵体样结构,病毒包涵体染色阳性,肺内小血管增生、扩张,呈血管炎性改变。淋巴结、脾脏结构破坏,淋巴滤泡消失,脾小体萎缩,淋巴细胞明显减少,组织细胞增生;结肠、小肠孤立和集合淋巴结淋巴滤泡消失;骨髓增生减低,巨核细胞增多。免疫组织化学染色：淋巴结、脾脏B细胞CD20弥漫散在阳性,CD45RO(UCHL-1)散在阳性,CD4辅助T细胞显著减少,CD8毒性T细胞稍增加,CD4／CD8比例明显小于0．5。电镜观察：肺泡内的单核巨噬细胞、肺泡上皮胞质内可见病毒样颗粒,大小80～160nm,有光晕或花环状包膜。结论 肺部明显急性弥漫性全小叶性间质性炎,肺泡腔透明膜形成,肺外淋巴造血系统明显损害,尤T细胞明显;内脏器官出血、坏死和血管炎改变等为急性SARS的形态特征;肺内所见病毒样颗粒可能为新型冠状病毒,推测其为此次SARS流行的主要病原体。     

Microbial structures in a patient with sporadic non-A, non-B fulminant hepatitis treated by liver transplantation

Double-shelled virus-like particles (60 nm) and long cytoplasmic tubular structures were found in the cytoplasm of hepatocytes from areas of collapsed and regenerating areas of hepatectomised liver in a 13-year-old boy who received a liver graft for fulminant hepatitis attributed to sporadic non-A, non-B hepatitis. The patient died on the ninth postoperative day from acute graft failure. Although virus-like particles were not found, instead, gram-negative rods were identified in the necrotic graft and the most likely cause of death was a gram-negative septicaemia with a Shwartzman-like reaction localized to the liver.     

Ultrastructure of bone marrow in chicks inoculated with chicken anaemia agent (MSB1-TK5803 strain)

Ultrastructural changes of the bone marrow in chicks inoculated with chicken anaemia agent (CAA: MSB1-TK5803 strain) and age-matched controls are described. Erythropoiesis in the lumen of the intravascular spaces (sinuses) of unaffected bone marrow were classed into four consecutive stages according to cell morphology: proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts and erythrocytes. In the early stage of infection, haematopoietic cells showed irregular plasma membrane, vacuolisation, formation of pseudopods and electron-opaque regions in the cytoplasm as well as intranuclear inclusions consisting of fine granular or homogeneous materials. Aggregation of virus-like particles, of 14 nm in diameter, was rarely observed in the degenerative haematopoietic cells. Subsequently, an increase in numbers of macrophages with engulfed degenerative and necrotic haematopoietic cells and plasma cells with prominent Golgi complex and well developed rough endoplasmic reticulum was observed in the anaemic stage. Although immature cells were rarely seen on day 12 of postinoculation (pi), active erythropoiesis resumed on day 20 pi or later.     

Subcellular Immunolocalization of Porcine Circovirus Type 2 (PCV2) in Lymph Nodes from Pigs with Post-weaning Multisystemic Wasting Syndrome (PMWS)

Post-weaning multisystemic wasting syndrome (PMWS) is one of the most significant porcine diseases worldwide. The causative agent is porcine circovirus type 2 (PCV2), the smallest virus known to infect animals. Data related to the structural and ultrastructural aspects of this infectious disease are sparse and there is little knowledge of the subcellular localization of PCV2 and its replication in the tissues of pigs naturally affected by PMWS. The present study describes the cellular localization of PCV2 in the lymph nodes of pigs affected by PMWS by application of immunolabelling techniques for light and transmission electron microscopy (TEM). PCV2 particles were exclusively detected in histiocytes. Ultrastructural alterations including marked dilatation of rough endoplasmic reticulum and swelling of mitochondria were associated with PCV2-labelled intracytoplasmic inclusions (ICIs) with recognizable virions. Within the ICIs icosahedral virus-like particles were specifically labelled with a PCV2 capsid antibody, whereas particles with a granular appearance were not labelled. Colocalization studies with confocal microscopy and double immunolabelling with TEM indicated a close relationship between virus and the mitochondria, suggesting that these organelles may play an important role in the replication of PCV2. The present findings further support the hypothesis that virus replicates within the histiocytes of lymph nodes.