Experimental canine adenovirus glomerulonephritis: histological,immunofluorescence and ultrastructural features of the early glomerular changes.

Eighteen 16-week-old dogs were inoculated i.v. with a virulent strain of canine adenovirus and the renal glomeruli were examined by light, electron (transmission and scanning) and immunofluorescence microscopy at intervals from 24 h to 7 days after inoculation of virus. From 2 days onwards intranuclear inclusion bodies and cell-associated viral antigen were detected in mesangial, endothelial and circulating mononuclear cells. This was accompanied by swelling and, in some cases, necrosis of these cells with partial or complete occlusion of capillary loops. In 5 dogs examined on days 5-7 after inoculation of virus, granular deposition of IgG, C3 and viral antigen was observed in mesangial areas and electron dense deposits were found in the mesangial matrix.     

Studies on the site of replication of vesicular stomatitis virus

Summary The cellular site of synthesis of vesicular stomatitis virus was studied through the use of actinomyciu D, mitomycin C, ultraviolet light irradiation, immunofluorescence and electron microscopy. Following the various treatments, virus infectivity was assayed by tube titrations and plaque assays. Chick embryo cells, African green monkey kidney cells and the WI-38 strain of human embryonic lung cells were used in the study. The results obtained indicate that the replication of VSV is not DNA dependent and that the replicative process takes place in the cytoplasm. This is supported by the results of immunofluorescent tracing and electron microscopy. In the former case no nuclear localization of viral antigen was demonstrable throughout the replicative cycle of the virus. Electron microscopy also revealed that virus particles were located exclusively in the cytoplasm of both antibiotic treated infected cells as well as in infected but untreated cells. The virus particles in antibiotic treated cells appeared normal and in larger number than in untreated cultures.A visiting scientist from the Nigerian Federal Government.     

Growth of an autonomously replicating parvovirus (feline panleukopenia): Kinetics and morphogenesis

Summary Feline embryo (FEmb) cell cultures, in which 90 percent of cells were dividing (cycling), were synchronized, by serum deprivation, to the degree that 88 per cent of the cells divided within a 12 hour period. When such cultures were infected with feline panleukopenia virus (FPV) at a multiplicity of infection of 5.7, a maximum level of cell associated virus was attained 28 hours post-infection (p.i.). There was a tendency for virus to remain cell associated in that cell lysis did not begin until 40 hours p.i.The genesis of FPV inclusion bodies was studied by light microscopy. Inclusions were intranuclear, weakly basophilic and Feulgen positive; they were first observed 8 hours p.i., and increased to be present in 90 percent of cells by 40 hours. Mitosis was markedly inhibited in FPV infected monolayers.The earliest changes observed by electron microscopy of infected cells were the presence of virus particles within nuclei, progressive chromatin margination, and nucleolar changes involving apparent segregation of the fibrillar and granular components. Virus particles measured 20 nm in diameter, and appeared either uniformly electron dense or possessed a dense margin and a pale center; many of the latter contained a single, central, dark spot. Virions ultimately became closely packed in all areas unoccupied by other nuclear components. In some nuclei a linear arrangement of virions was noted, but paracrystalline arrays were not seen.Other changes observed in infected nuclei included the presence of nucleolar remnants sometimes in the form of solid or hollow bodies comprised of nucleolar granules or filaments; distension of the space between the two membranes of the nuclear envelope; and the presence of aggregates of abnormal, electron dense material within the nucleus. Discontinuities of the plasma membrane and swelling of cytoplasmic organelles were commonly seen in cells showing advanced nuclear changes, but at least the inner membrane of the nuclear envelope generally remained intact. The characteristic, well defined inclusions of light microscopy were not observed by electron microscopy, and thus probably represented a preparation (shrinkage) artifact.With 11 Figures     

Ultrastructural studies of the envelopment and release of guinea pig herpes-like virus in cultured cells

The process of envelopment and release of guinea pig herpes-like virus was examined in both infected guinea pig kidney and thymus tissue culture cells by electron microscopy. The majority of the nucleocapsids were enveloped by budding into nuclear vacuoles; some were enveloped by budding from the inner nuclear membrane. Budding into cytoplasmic vacuoles was also seen. Many enveloped virus particles inside the nuclear vacuoles were pear shaped with a tail-like structure. Approximately 23% of pear-shaped virus particles were seen in the infected thymus fibroblastic cells, but only 6% were found in the infected epithelial cells. The envelopes of all nuclear enveloped virus particles appeared as smooth membranes, while the majority of particles exhibiting fuzzy and thick dense envelopes were seen in the cytoplasm or extracellular space. The average diameter of the cytoplasmic or extracellular enveloped virus particles was approximately 167 nm, and the average diameter of the nuclear enveloped virus particles was about 146 nm.Data also showed that mature nuclear virus particles were first released into perinuclear cisterna and then traveled through cytoplasmic channels to the extracellular space.     

Rapid diagnosis of infectious bursal disease infection by immunofluorescence on clinical material

Direct immunofluorescence on impression smears of bursa of Fabricius and direct electron microscopic examination of bursal specimens were compared with virus isolation in chick embryo fibroblasts (CEF) and embryonated eggs for the diagnosis of infectious bursal disease (IBD). In chicks experimentally infected with a virulent strain of virus, immunofluorescence was a more sensitive method of demonstrating infection than direct electron microscopy or virus isolation. In chicks experimentally infected with an avirulent strain of virus, immunofluorescence and virus isolation in CEF were equally sensitive methods of demonstrating infection and more sensitive than direct electron microscopy or virus isolation in embryonated eggs. In two field surveys immunofluorescence was, overall, more sensitive than virus isolation and direct electron microscopy and gave a good correlation with histopathological diagnosis of IBD.     

Adenovirus infection of the large bowel in HIV positive patients.

AIMS: To describe the microscopic appearance of adenovirus infection in the large bowel of human immunodeficiency virus (HIV) positive patients with diarrhoea. METHODS: Large bowel biopsy specimens from 10 HIV positive patients, eight of whom were also infected with other gastrointestinal pathogens, with diarrhoea were examined, together with six small bowel biopsy specimens from the same group of patients. Eight of the patients had AIDS. The biopsy specimens were examined by light microscopy performed on haematoxylin and eosin stained and immunoperoxidase preparations, the latter using a commercially available antibody (Serotec MCA 489). Confirmation was obtained with electron microscopy. RESULTS: The morphological appearance of cells infected with adenovirus showed characteristic nuclear and cellular changes, although the inflammatory reaction was non-specific. Immunoperoxidase staining for adenovirus was sensitive and specific, and the presence of viral inclusions consistent with adenovirus was confirmed by electron microscopy. CONCLUSIONS: The light microscopic features of adenovirus infection are distinctive and immunocytochemistry with a commercially available antibody is a sensitive and specific means of confirming the diagnosis. Further studies of the role of adenovirus in causing diarrhoea in these patients are indicated.     

MORPHOLOGY OF THE WARTHIN-FINKELDEY GIANT CELLS IN MONKEYS WITH EXPERIMENTALLY INDUCED MEASLES

The lymphoid tissues of 9 monkeys infected experimentally with wild type measles virus were examined by light and electron microscopy. Multinucleated giant cells of the Warthin-Finkeldey type were found 7 to 11 days after virus inoculation. The giant cells occurred mostly in the germinal center of lymphatic follicles, where they underwent degeneration and disappeared rapidly. Lymphoid and reticular types of giant cells were recognized. The ultrastructural evidence suggested that some of the nuclei contained in giant cells were formed by an aberrant nuclear cleavage. The majority of giant cells, however, were postulated to arise from virus-mediated cell fusion, although direct evidence of cell fusion was not seen. Peculiar nuclear changes, in which some nuclei and fragments of the outer nuclear membrane were contained in a common outer membrane of the nuclear envelope, were observed in all lymphoid giant cells. Both cytoplasmic and intranuclear inclusions were seen to be composed of viral nucleoprotein strands. The former were detected in all giant cells of both types, and the latter in occasional nuclei, providing the direct evidence that giant cell formation resulted from replication of the virus.     

Comparative study of mouse brains infected with Japanese encephalitis virus by intracerebral or intraperitoneal inoculation

The brains of mice infected with Japanese encephalitis (JE) virus by intracerebral inoculation (IC), intraperitoneal inoculation with sham intracerebral inoculation (IP+sIC), and intraperitoneal inoculation (IP) were studied by light and electron microscopy. The mortality rates and mean survival days were 100% and 4.8 days for the IC group, 92% and 9.0 days for the IP+sIC group, and 58% and 13.4 days for the IP group. Accordingly, the brain samples of sick mice were examined by light and electron microscopy 4 days post-inoculation (p.i.) for the IC group, 7 days p.i. for the IP+sIC group and 12 days p.i. for the IP group. In light microscopy, the mouse brains in the IC group showed little inflammatory change with only mild generalized glial-cell proliferation and mononuclear cell infiltration. In electron microscopy, however, a majority of neurons in the brain were seen to be infected with virus that replicated exclusively in the neuronal secretory system, including rough endoplasmic reticulum (RER) and the Golgi apparatus. In contrast, light microscopic observation of the brains from the IP+sIC and the IP groups showed prominent inflammatory changes with leucocytic infiltration and perivascular cuffing. Neuronal degeneration and neuronophagia were also prominent. In electron microscopy, neurons were infected in the same manner as in the IC group, but showed more advanced degenerative changes with marked cytoplasmic rarefaction and frequent neuronal disintegration. Mononuclear cells were frequently found in direct contact with degenerating and disintegrating neurons. The results showed that (a) the basic process of JE virus replication in brain neurons was present in the three groups of mice, (b) in the peripherally inoculated mice the process was accompanied by inflammatory reaction with resultant neuronal destruction, and (c) breach in the blood-brain barrier at the time of peripheral viral inoculation played an important role in the viral invasion of the CNS.     

A morphologic study of FL cells infected with para-influenza 3 virus

Summary The sequential changes in FL cells infected with para-influenza 3 virus under conditions in which persistent infection develops were studied by light microscopy, specific immuno-fluorescence and electron microscopy. Hemadsorption in this system was also examined by electron microscopy. The giant multinucleated cell, the characteristic initial cytopathic effect, was shown to be truly syncytial. Specific immunofluorescence appeared in syncytia in the nuclei and at the nuclear membrane; later when the giant cells had broken down and proliferating pleomorphic, spindle-shaped and rounded cells appeared, fluorescence was most pronounced at the plasma membranes. Virus particles which arise from the plasma membranes were first seen along the plasma membrane of otherwise unchanged cells just outside the syncytia. There was extensive virus release at the borders of spindle-shaped cells and in the region of virus release the cytoplasm was finely filamentous. The virus particles ranged in size from 100 to 160 m. They were seen to have a limiting membrane with an inner structure consisting of several dense, round profiles. There was also an outer amorphous, ill-defined layer. Definite changes in the ultrastructure of the nucleus were infrequent but small granules in the nuclear matrix with diameters of 11 to 17 m were of special interest. Both virus-and cyto-hemadsorption were seen but the pattern differed from that seen with influenza virus.This study was aided by grant P 145 B from The American Cancer Society, Inc. and by grant H-3493 (Cl) from The National Heart Institute, National Institutes of Health.Deceased 1960.     

Venezuelan Equine Encephalomyelitis in an Adult Animal Host: An Electron Microscopic Study

Adult white mice were inoculated intravenously with a virulent strain of Venzuelan equine encephalomyelitis virus. They were sacrificed sequentially, and cerebral tissue was collected for light and electron microscopic examination and virus titer determination. Viruses were first seen on the fifth day postinoculation infecting and arising from endothelial cells in the cerebrum. Subsequently, oligodendrocytes became infected, giving rise to mature virions. At this time in the infection, a particular electron-dense cell, probably representing a glial cell type, phagocytized mature virions. This resulted in autoinfection, as seen by viral growth in and destruction of these dense cells at a later stage of the infection.     

Coxsackievirus group B replication in cultured fetal baboon aortic smooth muscle cells

All six coxsackievirus B (CVB) serotypes replicated to various extents in fetal baboon aortic smooth muscle cells (SMC) in culture. CVB3 and CVB4 replicated to the highest titers and induced no cytopathology at the level of light microscopy. Maximum yields of CVB3 were produced between 12 and 24 hr postinoculation. Up to 15% of SMC cells became infected, as determined by immunofluorescence assays with anti-CVB3 antiserum, yet overall cell division in infected cultures did not differ from infected SMC cultures. Electron microscopy of CVB3-inoculated SMC cultures revealed changes in some cells: viruslike particles, secondary lysosomes containing dense bodies, and peripheral nuclear chromatin condensation. CVB3 replicated well in SMC passages up to the eighth, but did not replicate in eleventh-passage cells. Because of the cardiotropic and myotropic potential of this virus and its ability to replicate in aortic SMC with associated ultrastructural alterations, CVB3 (and other CVB) should be further examined as an etiologic agent(s) that could induce atherosclerosis.     

Investigation of immunoperoxidase-labelled rotavirus in tissue culture by light and electron microscopy

A tissue culture-adapted strain of bovine rotavirus, grown in calf kidney monolayers, has been examined by light and electron microscopy after immunoperoxidase labelling. Some of the characteristic problems associated with pre-embedding methods have been demonstrated. Preparative techniques involving pretreatment of infected cells with a detergent followed by a detergent/fixative combination have enabled virus antigen to be labelled while maintaining satisfactory ultrastructural preservation.     

Histopathology and immunohistochemistry of renal lesions due to avian infectious bronchitis virus in chicks uninoculated and previously inoculated with highly virulent infectious bursal disease virus.

A nephropathogenic strain of infectious bronchitis virus (IBV) was inoculated intra-tracheally into 14-day-old specific-pathogen-free chicks or ones previously inoculated with highly virulent infectious bursal disease virus (IBDV) at 7 days of age. The renal lesions were examined histopathologically and immunohistochemi-cally at intervals up to 30 days post-inoculation. The mortality was 20% in the IBDV + IBV-inoculated group, but not in the IBV-inoculated one. Swollen and pale kidneys due to IBV infection were more severe and of longer duration in dually infected chicks. At the early stage of infection, the histopathological changes in the kidneys were similar in both groups, but the ducto-tubular damage was more severe in the dually infected chicks. At the late stage of infection, the renal lesions were characterized by chronic interstitial nephritis with formation of lymphoplasmacytic nodules in IBV-inoculated chicks and by chronic active nephritis which consisted of tubular degeneration, lymphoid cell reaction and interstitial fibrosis in IBDV + IBV-inoculated ones. More IBV antigen-positive cells persisted longer in the kidneys of dually infected chicks than in those of IBV-inoculated ones.     

Replication of the Trichoplusia ni granulosis and nuclear polyhedrosis viruses in cell cultures

Newly established Trichoplusia ni (cabbage looper) embryonic cell lines were infected with T. ni granulosis virus (TnGV) and T. ni nuclear polyhedrosis virus (TnSNPV). Infection of cultured cells with TnGV was ascertained by peroxidase-antiperoxidase staining, DNA slot-blot hybridization, and transmission electron microscopy. Initially, 15 cell lines supported TnGV replication, the percentage of infected cells ranging from 1 to 50%.However, susceptibility of the 15 cell lines to TnGV infection either decreased or was lost within 20 to 25 passages from the initial primary culture. Infection of cells with TnSNPV was determined by phase contrast and electron microscopy. TnSNPV infected 29 36 cell lines tested, the percentage of infected cells ranging from 1 to 60%.     

Detection of Lucké herpes virus antigens in infected frog pronephric cells

Summary A frog pronephric cell line, infected with herpes virus derived from Lucké renal carcinomas ofRana pipiens was examined for the presence of Lucké herpes virus antigens. Non-infected pronephric cells were controls. Antiserum to purified Lucké tumor herpes virus was applied in blind tests to the normal and virus infected cells. Both cytoplasmic and nuclear fluorescence was found in the herpes virus infected cells after indirect immunofluorescence with the antiserum. Infected cells cultivated at the optimum growth temperature of 25° C or maintained at 9° C, a temperature inducive to herpes virus replication, showed equivalent fluorescence reactions. No fluorescence was found in the normal pronephric cell line. Examination of parallel herpes infected cells showed cytopathic effect in monolayers by light microscopy, and nuclear or cytoplasmic immunofluorescence. Electron microscopic examination revealed proviral elements in nuclei and sparsely scattered herpes virus coincident with cytoplasmic fluorescence.With 5 Figures     

Differences in the morphology of herpes simplex virus infected cells: I. Comparative scanning and transmission electron microscopic studies on HSV-1 infected HEp-2 and chick embryo fibroblast cells.

Infection with herpes simplex virus type 1 (HSV-1) induces different morphological changes in different cell lines. This is demonstrated by comparative scanning (SEM and transmission (TEM) electron microscopic investigations of cell cultures prepared under identical conditions. SEM of HSV-1 infected HEp-2 cells reveals a slightly altered cell surface: only the number of the microvilli is reduced. Large amounts of released virions are detectable adhering to the outer plasma membrane. Ultra-thin sections show typical virus maturation steps in the nuclei (formation of nucleocapsids and virus budding from the inner lamella of the nuclear membrane) and in the cytoplasm (egress of enveloped nucleocapsids through membranous structures). HSV-infected primary chick embryo fibroblast (CEF) cells are characterized by crumpled and rough surfaces without virus particles adhering to the membrane. Ultra-thin sections exhibit atypical virus maturation with many unenveloped nucleocapsids within the cytoplasm. The distribution of HSV-induced antigen(s) on the surface of the infected cells is identical in the two cell systems as determined by the peroxidase labelling technique. The c.p.e. (as seen by phase contrast light microscopy) is similar in both HEp-2 and CEF cells: both fusion and rounding up is induced in the infected cells.     

Interaction of Nocardia asteroides with Rabbit Alveolar Macrophages: Association of Virulence, Viability, Ultrastructural Damage, and Phagosome-Lysosome Fusion

In vitro-maintained rabbit alveolar macrophages were infected with three strains of Nocardia asteroides. It was found that N. asteroides GUH-2 was resistant to macrophage killing, while N. asteroides 14759 was intermediate in resistance, and N. asteroides 10905 had little resistance to killing by macrophages. These observations correlated well with the data on relative virulence previously determined in mice. To establish the intracellular events leading to these differences, we determined the occurrence of phagosome-lysosome fusion in infected macrophages by both electron and fluorescent microscopic methods. It was found that the virulent strain GUH-2 inhibited phagosome-lysosome fusion; the intermediately virulent strain, 14759, partially inhibited fusion; and the less-virulent strain, 10905, was unable to inhibit fusion. In addition, electron microscopy of infected macrophages demonstrated that cells of the virulent strain, GUH-2, were not damaged, and only some of the cells of the intermediately virulent strain, 14759, were damaged, while most of the cells of the less virulent strain, 10905, exhibited considerable cellular destruction. These data indicated a direct correlation between the virulence of these organisms and their resistance to killing by alveolar macrophages, their lack of macrophage-induced ultrastructural damage, and their ability to inhibit phagosome-lysosome fusion. Thus, it appears that inhibition of phagosome-lysosome fusion in alveolar macrophages may be one of the mechanisms of pathogenicity of virulent strains of N. asteroides.     

An electron microscopic study of the surface structures and hemadsorption on chick embryo cells infected with rabies virus

Summary Characteristic alterations at the surface of chick embryo cells infected with the HF-TC strain of rabies virus and the binding sites of hemadsorption were studied employing both scanning and transmission electron microscopes. The initial alteration of the cell surface structure revealed by scanning electron microscopy was an appearance of elongated and reticulated microvilli on the 2nd day after virus inoculation. On the 3rd day, numerous bullet-shaped virions could be seen budding as single, tetrapod-like structures and as radial projections both from the perikarya and microvilli. Thereafter, elongation of microvilli, formation of numerous blebs in various sizes, disappearance of filopodia, and rounding up of infected cells were observed as characteristic cytopathic effects by rabies virus infection.The attachments of goose erythrocytes to the infected cells occured in two forms. The one was adsorption of erythrocytes to the cell surface involving microvilli and filopodia in the absence of detectable virus, and the other was adsorption of erythrocytes to the virus particles budding from cell surface. The former could be seen from the early stage of infection through the end of observation period, while the latter was observed only on and after the 3rd day after virus inoculation. These findings were also confirmed with transmission electron microscopy.With 10 Figures     

Scanning electron microscopical observations on the cytopathology of porcine enteroviruses in PK-14 cells.

Examination by scanning electron microscopy (SEM) of PK-15 cells infected with each of the two cytopathogenic types of porcine enterovirus revealed changes similar to those observed by light microscopy. However, differences between the two types could be detected at an earlier stage of the infection. Cells infected with T80 virus (type I) were rounded, with surface microridges, while V13 (type II) infected cells were characterized by cytoplasmic protrusions.     

Morphological features of Murray Valley encephalitis virus infection in the central nervous system of Swiss mice

We have examined the histological and ultrastructural features of CNS infection with Murray Valley encephalitis (MVE) virus in mice inoculated with a virulent parental strain (BH3479). Light microscopic examination revealed neuronal necrosis in the olfactory bulb and hippocampus of MVE-infected brains by 5 days post-infection (pi). Electron microscopy of these regions showed endoplasmic reticulum membrane proliferation, and tubular and spherical structures in the cisternae of the endoplasmic reticulum, Golgi complex and nuclear envelope. At seven to eight days pi, infected neurones exhibited chromatin condensation and extrusion, nuclear fragmentation, loss of segments of the nuclear envelope, reduced surface contact with adjacent cells and loss of cytoplasmic organelles. This cell injury was particularly noticeable in the proximal CA3 and distal CA1 regions of the hippocampus. The inflammatory cell profile consisted of macrophages, lymphocytes and especially neutrophils, and many of these inflammatory cells were apoptotic. High mortality rates in the BH3479-infected population of mice correlated with the intense polymorphonuclear and mononuclear leucocyte inflammatory infiltrate in the CNS.