Adenovirus infection enhances in vitro adherence of Streptococcus pneumoniae.

Viruses are thought to facilitate bacterial infections of the respiratory tract, but the mechanisms are poorly understood. The present study analyzed the effect of adenovirus on bacterial adherence to human respiratory tract epithelial cells. The human lung carcinoma cell line A549 was infected with adenovirus of types 1, 2, 3, 4, 5, and 9. At a multiplicity of infection of 75 particles per cell, cytopathic effects occurred in 75 to 100% of the cells within 48 h. The virus-infected cells were harvested at various times after infection and analyzed for the ability to bind strains of Haemophilus influenzae and Streptococcus pneumoniae. Adenovirus (types 1, 2, 3, and 5) commonly causing respiratory tract infections increased the binding of adherent S. pneumoniae strains to the cells. This effect was not seen for other adenovirus types. Adenovirus infection did not change the adherence of cells of poorly adhering strains of S. pneumoniae or H. influenzae. The increase in adherence of S. pneumoniae could be inhibited by the DNA synthesis inhibitor cytosine arabinofuranoside, which is known to block the late phase of the adenovirus infection. When electron microscopy was used, there was no evidence that virus particles bound directly to bacteria. Adherence was not affected by pretreatment of the cells with virus particles or viral proteins. This suggested that adenovirus infection upregulated receptors for S. pneumoniae. The increased attachment may be one mechanism by which viruses precondition the respiratory mucosa for bacterial infection.     

Ultrastructural study of viral replication in human fetal intestinal organ culture

Summary Human fetal intestinal organ cultures were employed to study the growth patterns of various viruses and resultant cytoarchitectural changes using electron microscopic techniques. Representative strains of adenovirus, adenovirusassociated virus, herpes simplex virus, poliovirus, and echovirus were chosen for the study. The intracellular localization of these viruses was demonstrated, and the occurrence and rate of cellular destruction were observed. The difficulty of visualizing smaller viruses represented was emphasized. The use of these techniques to detect certain fastidious viruses of the gastrointestinal tract was suggested, since human fetal intestinal organ culture provides differentiated cell types which may be important for the initiation and support of viral replication.     

Thin-layer chromatography of lipids in adenovirus-treated human erythrocytes

Summary Lipid changes of human erythrocytes treated with adenovirus type 8, 9 and 10 have been studied with thin-layer chromatography. It has been established that each of the virus types induced several qualitative changes, which were manifested either in the disappearance of spots or in the development of new ones, as well as in the changes of Rf values and staining characteristics. These changes varied with the individual types and were different from those observed with influenza virus. The decrease of aminophosphatides and other amino acid-containing lipids characterized the effect of type 8 adenovirus. Erythrocytes treated with type 9 adenovirus displayed the highest number of spots, while the decrease of unsaturated lipids and neuraminic acid characterized the effect of type 10 adenovirus.     

Adenovirus and intranuclear inclusions in appendices in intussusception.

AIMS: To examine the light and electron microscopic features of appendices removed at the time of surgical reduction of intussusception in children; and to confirm that the viral inclusions seen in some of them are due to adenovirus. METHODS: A series of 39 appendices from cases of intussusception and 15 control appendices were reviewed. Light microscopic examination of haematoxylin and eosin stained sections was performed on all of them and one appendix with large numbers of inclusions was examined by electron microscopy. Non-isotopic in situ hybridisation using a biotinylated DNA probe was carried out on sections of appendix from 30 of the cases of intussusception and from the 15 controls. RESULTS: Light microscopic examination showed viral inclusions in 19 of the appendices from the cases of intussusception and in none of the controls. Electron microscopic examination showed viral particles with the typical features of adenovirus. Most of the appendices with viral inclusions in the haematoxylin and eosin stained sections also contained adenovirus DNA as shown by in situ hybridisation. CONCLUSIONS: Viral inclusions seen in appendices from cases of intussusception are caused by adenovirus. Adenovirus DNA was not demonstrable in appendices from cases of intussusception without viral inclusions and the aetiological factors involved in intussusception in these children remain unknown.     

Persistent infection of human adenovirus type 5 in human monocyte cell lines.

Adenovirus infection of human monocyte hybridoma cell lines and the fusion partner U937 was investigated. Adenovirus adsorbed poorly to these cells as well as primary human alveolar macrophages. The virus-binding experiments showed a 100-fold reduction in apparent viral binding to these cells compared to the permissive HeLa cells. Adsorption of adenovirus to these cells could be enhanced by preincubation of adenovirus with its antiserum. Following entry into the cells amplification of adenovirus DNA was detected starting at 2 days postinfection but few mature virus particles were produced. The infected cultures survived the infection and continued to grow for more than a year. In these chronically infected cultures, linear adenovirus DNA persisted up to 200 copies per cell and a small amount of mature virus was produced. Infectious center assay and cell cloning experiments showed that the majority of the cells in the chronically infected cultures harbor adenovirus genome. These results indicate that restriction of replication of human adenovirus type 5 at the late phase results in persistent infection of U937 and the human monocyte hybridoma cell lines.     

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.     

Viral infection in placenta relevant cells – a morphological and immunohistochemical cell culture study

Viral infections in pregnancy are known to cause fetal malformation, growth restriction, and even fetal death. Macroscopic placental examination usually shows slight and unspecific changes. Histology may show secondary, non‐specific tissue reaction, i.e. villitis with lymphocytic invasion. Primary specific morphology characteristics are known for some virus, like cytomegalovirus, parvovirus, and herpes simplex, however many viral infections show non‐specific changes. Placenta relevant cells as human first trimester trophoblasts HTR8/SVneo, primary human umbilical vein endothelial cells (HUVEC), and primary human embryonic fibroblasts were examined following infection with commonly occurring virus like adenovirus and enterovirus. Morphology in routine stained sections and virus‐specific immunostains were studied 4, 8, 24, 48, 72 h after infection. Nuclear enlargement was seen in the infected cells. A specific diagnosis of adenovirus or enterovirus infection, however, was not possible without specific immunostains.     

Detection of adenovirus in patient specimens by indirect immune electron microscopy.

Immune electron microscopic procedures for the detection of adenovirus type 7 directly in throat swab specimens from patients are described. Nineteen of 25 throat swab samples, known to be positive for adenovirus type 7 by isolation of virus from tissue culture, were shown to contain aggregates of adenovirions coated with antibody. Sensitivity tests of the immune electron microscopic method showed that as few as 16 to 32 tissue culture infective doses of virus could be detected by the direct immune electron microscopic technique. It was also demonstrated that aggregation of virus-antibody complexes could be further enhanced by use of anti-immunoglobulin G sera (indirect immune electron microscopic procedures). These results demonstrate that examination of patient specimen by immune electron microscopic procedures is a feasible and rapid method for adenovirus detection and suggest that it could be applied as a routine laboratory procedure for the diagnosis of other virus infections.     

Genetic expression of human adenoviruses in simian cells. Evidence for interserotypic inhibition of viral DNA synthesis

Most simian cells are permissive for SV40 and adenovirus-SV40 hybrids but nonpermissive for human adenoviruses, and the defect has been shown to take place at the level of processing of late viral mRNAs (Klessig and Grodzicker, 1979). Viral DNA synthesis and virus progeny production were studied in simian cells infected with different adenovirus serotypes. Adenoviruses belonging to oncogenic subgroups A and B (Ad31 and Ad3) failed to replicate their DNA in CV1 cells, whereas DNA replication occurred for all the other serotypes. Co-infection of CV1 cells with SV40 and Ad3 (or Ad31) resulted in the inhibition of SV40 DNA synthesis, as well as cellular DNA synthesis. The inhibition was not related to adenovirus DNA replication, since SV40 did not complement the Ad3/Ad31 replication defective function. Similar results were obtained in coinfected BSC and MK2 simian cell lines. Inhibition of Ad2ND1 DNA synthesis and gene expression also occurred in co-infection of simian cells with nondefective Ad2ND1 hybrid and defective Ad3/Ad31. In permissive human cell lines (HeLa or KB) co-infected with Ad2 and Ad3 (or Ad31), a dominant, inhibitory effect of Ad3 (or Ad31) over Ad2 was also observed. The inhibition appeared to function stoichiometrically and not catalytically, and to involve early adenovirus gene products. In both simian and human cells a hierarchy of dominance appeared between serotypes belonging to different subgroups. The degree of inhibitory effect occurred in the following decreasing order: Ad3 and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad31 (A), Ad2 and Ad5 (C).     

Comparison of replication of adenovirus type 2 and type 4 in human lymphocyte cultures.

Adenovirus type 2 was capable of replicating in purified lymphocyte cultures from human adenoid specimens. Phytohemagglutinin stimulation enhanced the replication of virus. Viral titers of 103 to 104 50% tissue culture infective doses per ml were reached after 4 to 8 days. Only 1 to 3 per 106 cells were found to produce virus. In contrast, there was no evidence that lymphocyte cultures could support the replication of adenovirus type 4. The life span of cultures infected with type 2 or 4 was not reduced. The possibility that lymphocytes infected with virus play a role in initiating natural, persisting adenovirus infections of human adenoids is discussed.     

Relationship of Adenovirus to Lymphocytes in Naturally Infected Human Tonsils and Adenoids

Purified lymphocytes from human tonsil and adenoid specimens were cultured with and without phytohemagglutinin. Adenovirus was isolated from lymphocytes of 8 of 90 specimens tested. With one exception, it was necessary to culture the lymphocytes before infectious virus could be detected. Phytohemagglutinin stimulation enhanced the recovery of virus. The results suggest that lymphocytes in tonsils and adenoids may be naturally infected with adenovirus and that, in positive cultures, at least 1 of every 10(7) cells harbors virus or viral precursor at initiation of the cultures. Adenovirus was demonstrated directly in fresh suspensions of unpurified cells from tonsils and adenoids in seven cases. In five of these cases, at least 1 of every 10(6) cells contained infectious virus. Adenovirus was isolated from 61 (62%) of 98 tonsil and adenoid specimens by the conventional method of tissue fragment culture after various periods of cultivation. The viruses isolated were of serotypes 1, 2, 5, and 6.     

Changes in Morphology and Cellular Replacement of Surface Epithelia in Human Gastric Mucosa Affected by Different Pathological Changes: A Scanning Electron Microscopical Study of Human Gastric Biopsy Material

The surface epithelium of normal gastric mucosa shows a constant turnover, migrating from the proliferation zone at the neck of the gastric glands to the mucosal surface. The cytologic changes associated with the concomitant cellular differentiation were so far studied in sections by light and electron microscopy only. With these methods neither the changes of surface morphology associated with cellular maturation nor the shedding of surface epithelia can be recorded. With the scanning electron microscope it is possible to study especially these changes of the mucosal surface plus those of the single surface epithelia over a wide range of magnification and with a sufficient depth of focus.In this study therefore an attempt was made to correlate some scanning electron microscopic findings concerning these changes of mucosal and cellular surface with the results of parallel light microscopic examinations. For this purpose double biopsies of human gastric mucosa were taken during routine endoscopic examination. One of these biopsies was processed for light microscopic examination after formaldehyde fixation. The other was fixed in buffered glutaraldehyde solution and 1 % OS04 solution and thereafter the material was processed for scanning electron microscopic study by dehydration against an increasing acetone gradient, critical point drying and sputter coating of the dry specimens which were mounted on metal stubs with conductive silver.Some aspects of surface morphology of the mucosa and the single cells in correlation to different types of gastritis are demonstrated and the morphological expressions of cytological degeneration and shedding from the mucosal surface are commented on.     

Enhancement of adenovirus infection in WI-38 and AGMK cells by pretreatment of cells with 5-iododeoxyuridine.

Adenovirus replicates inefficiently in WI-38 and AGMK cells. Treatment of these cells with 5-iododeoxyuridine (IdU) prior to infection enhances the yield of adenovirus by 10- to 1000-fold. IdU-pretreated cultures demonstrate an increased percentage of adenovirus T-antigen-positive cells in comparison to untreated controls. IdU treatment has no effect on virus adsorption, and in AGMK cells IdU enhancement is additive to SV40 enhancement of adenovirus yield. Thus, IdU treatment relieves a restriction to virus growth that occurs prior to T-antigen synthesis. IdU pretreatment has no effect on virus growth in HEK, a permissive cell type. Incorporation of IdU into cellular DNA is required for enhancement.     

Diagnosis of fastidious enteric adenoviruses 40 and 41 in stool specimens

Thirty-five stool specimens, collected over a 14-week period from pediatric gastroenteritis patients and shown to contain adenovirus by electron microscopy, were inoculated onto 293 and HeLa cells. Virus isolates were characterized by serum neutralization and restriction endonuclease cleavage analysis of viral DNA from infected cells. Adenovirus was isolated upon primary inoculation of 293 cells from all 35 specimens shown to contain adenovirus by electron microscopy. Fastidious adenoviruses 40 and 41 (Ad40 and Ad41) were found in 17 (49%) of the stool specimens, and 4 of these specimens contained a conventional species (Ad1, Ad1, Ad18, Ad31) as well as Ad40. This was first manifest by the observation that four of the isolates which initially grew only in 293 cells acquired the capacity to grow in HeLa cells upon subsequent passage. In each case, the conventional species was undetectable by DNA analysis in the original inoculum but was selected in 293 cells and became the only one detectable by the second passage. Four other specimens, containing Ad1 or Ad31 alone, failed to grow initially in HeLa cells but did grow in 293 cells. The results of this study demonstrate therefore that (i) 293 cells are more sensitive than HeLa cells for the isolation of conventional as well as fastidious enteric adenovirus species and (ii) identification of viruses from patient specimens should involve minimal passage of the virus in cell culture, as a single passage can result in misdiagnosis of the virus associated with the infection.     

Infection and direct injury in human hepatocyte explants and a hepatoblastoma cell line due to hepatiticomimetic (non-hepatitis) viruses

Hepatitis is caused by hepatitis viruses, but hepatitis or hepatocellular enzyme abnormalities is sometimes associated with infection by the hepatiticomimetic viruses. The direct and indirect effects of infection with hepatiticomimetic viruses were examined in two human hepatocyte systems. Poliovirus, adenovirus, and herpes simplex virus (HSV) induced cytopathology in Hep G2 cells. Measles virus caused no change in hepatocytes. Poliovirus infection did not affect cellular protein synthesis, and the peak of hepatocellular enzyme release coincided with the peak of virus release. The increase in adenovirus protein synthesis correlated with the decrease of transferrin synthesis, and enzyme release was not prominent. HSV induced viral protein synthesis with enhanced processing and inhibition of synthesis of alpha1-antitrypsin. The peak of enzyme release was later than the peak of virus release. In primary hepatocytes, poliovirus, adenovirus, and induced extensive cytopathology and enzyme release, and VZV caused cytopathology and significant but minute enzyme release. The ratio of lactate dehydrogenase to aspartate aminotransferase release was larger in poliovirus infection in both hepatocytes than in HSV or VZV infection. Although poliovirus and adenovirus are released by cytolysis and HSV and VZV are secreted by exocytosis of cytoplasmic vacuoles, enzyme release was independent of the type of virus release. Adenovirus showed strong cytotoxicity but did not modify the membrane nor cause enzyme release. Enzyme release was associated with modification of the surface membrane due to apoptosis with poliovirus and necrosis with HSV. Consequently hepatocellular injury by viral infection did not reflect the amount or pattern of hepatocellular enzyme release.     

Electron microscopic study of nuclear and nucleolar changes in cells infected with canine distemper virus (CDV)

Summary The nuclear changes occurring in dog kidney cells infected with a virulent and an attenuated strain of canine distemper virus were examined by electron microscopy. No ultrastructural evidence for intranuclear virus replication was observed even though large eosinophilic nuclear inclusions were found in up to 34% of infected cells examined by light microscopy. This discrepancy between light and electron microscopic findings was discussed in relation to the possible structure of the nuclear inclusions. Marked morphological changes were noted in the nucleoli of up to 12% of cells infected with the virulent strain, CDV/BR, involving complete segregation of the granular and fibrillar components. The nucleolar lesions were similar to those induced by chemical inhibitors of cellular nucleic acid metabolism.     

Liver necrosis,adenovirus type 2 and thymic dysplasia

Summary An association between adenovirus and liver cell damage, although suspected, has not been frequently demonstrated in the human. In this presentation the necropsy of a two month old female infant with thymic dysplasia will be reported who died with liver necrosis in the absence of pulmonary changes. Sections of the liver showed numerous Feulgen-positive intranuclear inclusions with a distinct mosaic-like pattern on high magnification. Adenovirus was isolated from the liver and identified as type 2 by hemagglutination-inhibition and neutralisation tests. Electronmicroscopy of the liver showed considerable multiplication of the virus in most of the liver cells. The characteristic para-crystalline array of the virions could frequently be seen. The appearance of the virion, its size, and cellular distribution was typical of an adenovirus.This appears to be the first instance in which adenovirus has actually been shown by electron-microscopy in the human liver, and the third instance in which liver necrosis due to adenovirus was associated with thymic dysplasia.This work was supported by Canadian Federal Public Health Research Grant No. 602-7-97 (to Dr. J. Embil). Presented at the Meeting of the Canadian Association of Pathologists, Sherbrooke, Quebec, 1972.     

Synthesis, cellular localization, and quantification of penton-dodecahedron in serotype 3 adenovirus-infected cells

Adenovirus penton is a non-covalent complex composed of the penton base and fiber proteins, localized at the twelve vertices of the icosahedral virus capsid. In cells infected by adenovirus serotype 3 (Ad3), penton is found not only in the virus capsid but also self-assembled in dodecahedra formed through interactions between the twelve penton bases. In this study, the intracellular trafficking of penton proteins from the cytoplasm to the nucleus has been followed, and the nuclear re-arrangement induced by viral infection has been observed by electron microscopy of ultrathin sections. The amount of dodecahedra has been assessed in relation to the number of Ad3 infectious virions produced during the Ad3 replication cycle. It appears that dodecahedra are produced in a large excess over viral infectious particles and that they are located intranuclearly along the nuclear membrane of Ad3-infected cells at late times of infection.     

Applications of electron microscopy to diagnostic pulmonary pathology

Viruses and other possible causative agents should be sought light and electron microscopically in all cases of ill-defined diseases including "sarcoid." Ideally, tissue should be prepared for electron microscopic examination as soon as a specimen is obtained; however, when this has not been done, tissue preserved in formalin solution can be used. Viruses, some bacteria, and other agents suspected on the basis of light microscopic findings can be verified electron microscopically by reprocessing paraffin-embedded tissue from areas that show smudge cells, focal necrosis with atypical cellular proliferation, and nuclear inclusions. Electron microscopically, all dying cells show swelling and rupture of cellular organelles and membranes; reactive changes include proliferation of branching tubules and paracrystalline and other types of proteinaceous precipitates (inclusions) in both the nucleus and cytoplasm. Qualitative and quantitative changes of cellular organelles, fibrils, microvilli, and intercellular junctions reflect hyperplasia, metaplasia, or dysplasia of the cell and may enable identification of the diseases, e.g., desquamative interstitial pneumonia. In various conditions, basal laminae become irregular, disruptive, or reduplicated following epithelial necrosis and regeneration. Electron microscopic evidence of immunologic damage to basal lamina and cells and immuno-electron-microscopic features of the lung in general require further studies. Electron microscopic features of transbronchial biopsy specimens may be diagnostic in cases of alveolar proteinosis, histiocytosis X, and amyloidosis. Ultrastructural abnormalities of cilia are common; primary ciliary defects are rare. Finally, light microscopic, scanning electron microscopic, and x-ray energy-dispersive spectrometric examinations of paraffin-embedded sections appear most practical for the pathologic evaluation of cases of pneumoconiosis.