Replication of dengue-2 virus in Aedes albopictus mosquitoes. An electron microscopic study.

Sequential electronmicroscopic studies of Aedes albopictus mosquitoes infected with dengue-2 virus showed replication of virus particles and vesicular structures confined to cells having an active, rough, endoplasmic reticulum. Small granules not found on the surface of virions produced in mammalian cells were seen on the envelopes of the virus particles. There was no evidence of nuclear involvement as described in infected mouse neurones, and there was no apparent impairment of cellular function. Substantial viral replication was confined to cells of the salivary glands and nervous tissue, with lesser involvement of midgut, hemocytes, epidermal cells, fatbody, and foregut.

In vitro maturation and fertilization of swamp buffalo oocytes and their subsequent development.

The present experiment was carried out to evaluate the maturation, fertilization and subsequent embryo culture of swamp buffalo oocytes in vitro. The oocytes (n=273) were collected and morphologically graded based on the structure of cumulus-oocyte complexes as Grade 1 (compact, n=81), Grade 2 (expanded, n=70), Grade 3 (partially denuded, n=65) or Grade 4 (completely denuded, n=57). More than 60% of the in vitro matured oocytes co-cultured with capacitated spermatozoa demonstrated evidence of fertilization or cleavage to the 2-cell stage when either Grade 1 or 2 oocytes were used. The percentage of fertilized oocytes undergoing 2-cell stage cleavages from Grade 3 (53%) and Grade 4 (46%) groups was significantly lower (P<0.01) than that observed in the Grade 1 (64%) and Grade 2 (68%) groups. Development to the 6 to 8 cell stage substantiated fertilization of Grade 1 and 2 oocytes. These results demonstrated that swamp buffalo oocytes are capable of maturing in vitro, forming embryos, and developing at least to the 8-cell stage in culture medium alone.

Encapsidation defectiveness of herpes simplex virus type 2 during replication at acid pH condition.

The maximal yield of herpes simplex virus type 2 (HSV-2) grown at pH 6.5 decreased 10(2)-10(3) fold compared to that recovered at pH 7.5. Electron microscopic observation of the infected cells maintained at these 2 pH conditions indicated that approximately equal amounts of immature virions were synthesized 6 hours after infection. However, at 18 hours post infection the majority of viruses present in the nucleus of infected cells maintained at pH 6.5 were empty or partially cored capsids with some particles enveloped and present in the cytoplasm, whereas at pH 7.5 mature virions already appeared at the cytoplasmic membrane. Analysis of viral polypeptides by radioimmunoprecipitation indicated that the synthesis of p40, a family of polypeptides closely involved in viral DNA encapsidation, was significantly impaired in infected cells maintained at pH 6.5.

Morphological variation in pathogenic strains of Penicillium marneffei.

Penicillium marneffei is a dimorphic fungus known to be pathogenic to animals and man. The natural reservoir of this organism was known to be bamboo rats found in South Vietnam, Thailand and China. The first two human infections were reported in 1959 and 1973 from the United States. Up to 1984, five new cases of human penicillosis were reported from Thailand. Since then several more cases have been reported from different parts of the world mainly from the southern part of China. However, there are very limited mycological descriptions of this fungi. In this report, five Thai strains were studied for colonial morphology in comparison with Reference strain PLM 689. Variation in mycelial pigment was observed ranging from yellowish-green to orange with water soluble red pigment produced in every strain which can be seen early from the reverse side. Ultrastructural study by both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) was compared with that of the reference strain PLM 689. PLM 689 strain had only biverticillate penicilli, but all five strains from Thailand had both monoverticillate and biverticillate penicilli which occasionally appeared on the same branch. The conidia of the Thai isolates were oval in shape and 1.3-2 x 0.7-1.6 microns in size smaller than those of PLM 689 which were 2.5-4 x 2-3 microns. Phialides were also smaller and a little shorter but the number of phialides was similar to those of PLM 689 ranging 4-10 except for one strain which had 3-16 phialides. All Thai strains have stipes smaller and somewhat longer than those of PLM 689.(ABSTRACT TRUNCATED AT 250 WORDS)

Microscopic examination of Cryptosporidium oocysts in diarrhoeal stools.

Unconventional microscopic means for investigation of Cryptosporidium oocysts in patients' stools were explored in an attempt to obtain a more accurate diagnosis. The results showed that Nomarski interference contrast microscope provided clearer structures of oocysts in wet mount preparations than those under a normal light microscope and readily allowed distinction from yeast cells. Transmission electron microscopic study revealed that oocysts are thick walled and well sporulated. Their "untypical" appearance as seen by the light microscope resulted from sporozoites or the residuum that can be unfamiliar to some examiners. Electron microscopy provides definitive identification of Cryptosporidium spp. but Nomarski interference contrast microscopy was superior to bright field microscopy and may facilitate rapid diagnosis in routine fecal examination. The Ziehl-Neelsen modified acid fast technique was of value for differentiation and confirmation.

PIP: Histological studies were conducted with fecal specimens of Cryptosporidium oocysts, organisms that often cause fatal watery diarrhea in AIDS patients, to better distinguish them from yeasts. The specimens were from 3 patients with AIDS or suspected AIDS. The method used were bright field and Nomarski interference contrast microscopy of wet-mounted stools preserved in 10% formalin and stained by Giemsa and by the Neelsen modified acid-fast technique. Electron microscope sections were postfixed in osmium tetroxide. Ultra-thin sections were stained with and lead citrate before microscopic examination. Oocysts appeared under bright field microscopy as 3x4 mcm ellipsoidal bodies with a central large round granule, known as the residuum, and 1-4 granules. Interference contrast microscopy revealed banana-shaped sporozoites surrounding the residuum, clearly differentiating them from yeasts. Giemsa stains the sporozoites pale blue with purple dots, making it difficult to distinguish them from yeasts. Acid-fast stain turned the crescent-shaped sporozoites red and the residuum deep red, while yeasts stained blue. Cryptosporidium could easily by distinguished from yeast ultrastructurally by their double cell wall. Thus, interference microscopy and acid-fast staining are helpful to separate Cryptosporidium from similar sized yeasts, an alternative to intestinal biopsy which has formerly been required to diagnose this parasite in immune-compromised patients.

Ultrastructure of chordoma. A case report.

A 50-year-old male developed a sacro-coccygeal chordoma. Prior to the surgery, he had experienced back-pain, numbness of the right thigh and difficulty in voiding and defecation. Total excision of the mass was done and all symptoms were relieved. The light microscopic examination revealed a chordoma. The ultrastructural study was performed with particular interest in physaliferous cells. The fine structure disclosed the prominent associations of mitochondria and rough endoplasmic reticulum. The mitochondria showed irregularities in sizes and shapes but did not attenuate as much as previously reported. The vacuoles that were observed by light microscopy in physaliferous cells were both extra-cellular and intra-cellular and contained finely granular material of acid mucopolysaccharides probably of chondroitin type. The presence of both subplasmalemmal linear densities (SLD) and pinocytic vesicles was consistent with the histogenetic conviction that the tumor arose from mesodermal derivatives.

Dengue virus infection of mice: morphology and morphogenesis of dengue type-2 virus in suckling mouse neurones.

In dengue type-2 virus-infected neurones of suckling mice, formation of single-membrane vesicles is observed in the distended cisternae of the endoplasmic reticulum mostly of the perinuclear zone around 72 h after inoculation. Electron-dense 50-nm virus particles are arranged in chains in these distended cisternae; some form small crystalloid aggregates. Aberrant particles of different shapes are also seen in the distended cisternae about the same time that the virus particles appear. Parallel filamentous structures are occasionally observed in the cisternae that contain very few virions, either characteristic or aberrant. Increasing cytopathic changes are present after 75 to 96 h. There is an intense vesicular formation. Large numbers of virions and aberrant particles are seen either in the endoplasmic reticulum cisternae or smooth membrane vesicles. They are spread throughout the neurocytoplasm, extending into the dendrites. Dengue virions which are enclosed in fairly intact membrane-bound vesicles are released during cytolysis of the neurones. Morphogenesis of dengue virus type 2 is discussed.

Filamentous structures in dengue type 3 virus infected mouse neurones.

Dengue type 3 (H-87) virus was inoculated into suckling mouse brain and animals were sacrificed at 24 hr intervals. Parallel filamentous structures were found in infected neurones in close association with virus particles in the distended endoplasmic cisternae. They were usually arranged in a crystalloid pattern, oriented in different directions within the cisternae. Faint helical features were sometimes observed. These filamentous structures measured 15-25 nm in width and varied in length. Their possible involvement with viral material or a viral core is postulated.

Ultrastructural studies on dengue virus infection of human lymphoblasts.

Ultrastructural studies of dengue-2 virus-infected lymphoblastoid Raji cells showed that the virus induced an increase in the size of the rough endoplasmic reticula (RER) and that the replication of the virus was confined to the cisternae of these RER. The proliferating RER formed cytoplasmic inclusions that could be seen by light microscopy. This observation could be used as evidence of a cytopathogenic effect of dengue virus on infected Rajii cells in routine cultures. Accumulation of virions in the infected cells was minimal in comparison with other cell systems, however. Sporadic clusters of mature virions were often seen on the plasma membrane. These extracellular virions were distributed adjacent to the virus-bearing RER and were presumably released virions. Vertical transmission of the virus was evident in mitotic lymphoblasts. The replication pattern of dengue virus in lymphoblastoid cells suggests that efforts should be made to determine whether blast-transformed lymphocytes, numerous in secondary dengue infections, support dengue virus replication in vivo.