Cowdry Type Research Articles

Comparison of human cytomegalovirus growth in MRC-5 human fibroblasts, brain, and choroid plexus cells in vitro.

Cell cultures derived from human brain, choroid plexus, and human lung fibroblasts (MRC-5) were infected with the Towne strain of human cytomegalovirus (CMV). The cytopathic effect, beginning 24-48 hours after infection, was characterized by foci of enlarged rounded cells that spread slowly and eventually coalesced to destroy the entire monolayer within one week. Cowdry type A inclusion bodies and herpes virus nucleocapsids were seen in infected cells. CMV-specific antigen was demonstrated by immunofluorescence in fibroblasts and astrocytic cells of brain cultures and in cells of choroid plexus cultures as well as in MRC-5 fibroblasts. Despite these morphologic and immunochemical similarities the growth of CMV differed in cells of brain and choroid plexus origin as compared with MRC-5 cells. In brain and choroid plexus cell cultures most of the virus remained cell-associated throughout the observation period of one week, whereas in MRC-5 cells the CMV was found in both cell-associated and cell-free fractions of harvested material.

Nucleopathic changes in parvorirus-infected, cultured cells

Parvovirus replication is dependent upon progression of host cells through S phase of the cell cycle. Tritiated thymidine pulse-labeling was used to monitor host cellular DNA synthesis in order to study the development of nuclear alterations in bovine fetal spleen (BFS) cells infected with bovine parvovirus (BPV). Cultures of uninfected BFS cells synthesized DNA parasynchronously between 18 and 48 hr after planting. Cells were infected between 12 and 18 hr after planting to take advantage of the maximum number of S phase cells. Development of intranuclear inclusions in parasynchronous cells infected with four strains of BPV was studied by light and electron microscopy. Nucleopathic changes consisting of nucleolar stranding, increased creased basophilia and degranulation of the nucleoplasm were detected 12 hr post-infection (PI). A second early alteration was replacement of the nucleoplasm with a fine-grained homogeneous material by 18 hr PI. Cowdry type A and novel multifocal inclusions developed in cells with early changes beginning at 18 to 24 hr PI. Electron microscopic studies revealed discrete foci of empty virus capsids and virions in the nuclei which appeared to correspond with the foci in multifocal inclusions. These foci infer subnuclear compartmentalization of parvovirus replication. The uniqueness and striking morphology of multifocal inclusions have diagnostic significance.

Detection of herpes simplex virus infection of female genitalia by the peroxidase-antiperoxidase method alone or in conjunction with the Papanicolaou stain.

The sensitive peroxidase-antiperoxidase (PXAPX) method individually and in conjunction with the Papanicolaou (PAP) stain was used to detect herpes simplex virus (HSV) in specimens from human female genitalia. Initial studies using a model system of HSV-1 or HSV-2-infected Vero cells established (i) acetone as the most effective fixative, (ii) optimal dilutions of preimmunization and anti-HSV serum for differentiation of infected from noninfected cells, (iii) optimal concentration of 3,3'-diaminobenzidine tetrahydrochloride and H2O2 for maximal staining of infected cells with minimal background reaction, and (iv) removal of endogenous peroxidase with absolute MeOH. These various parameters, once established, were utilized in the PXAPX or PXAPX-PAP on human specimens from the vulva or cervix. In these specimens, examined by standard light microscopy, PXAPX-positive cells were dark brown with a single nucleus or multiple nuclei. By coupling the PAP to the PXAPX, detailed nuclear observations of PXAPX-positive cells were possible and revealed nuclear changes consistent with HSV infection, including syncytium formation, chromatin condensation, and an occasional Cowdry type A inclusion. The PXAPX and PXAPX-PAP correlated (r = 0.742) over a period of 72 h with HSV isolation from these samples.

Herpesvirus infection resembling Pacheco's disease in Amazon parrots

Herpesvirus infection was diagnosed in two parrots that died suddenly in a pet shop. Gross lesions were confined to reddening of the small intestinal mucosa. Histological examination of tissues from one specimen revealed a hepatitis and splenitis accompanied by intranuclear inclusions. Cowdry type A inclusions were observed in tissue culture and within the nuclei of chick embryo hepatocytes. Virus particles with a morphological similarity to herpesviruses were identified in electron microscope studies. The condition was considered to represent a case of Pacheco's parrot disease.

Herpes simplex type 2 encephalitis concurrent with known cerebral metastases

A 62-year-old woman developed neurologic deficits 7 months after pulmonary lobectomy for alveolar cell carcinoma of the lung. CT scan of the head demonstrated two metastases with marked peritumoral edema. Administration of Decadron, chemotherapy and 3,000 rad cranial radiation resulted in dramatic improvement of dysphasia and right hand paresis. Almost 2 months later, rhythmic, involuntary movements of the left hand developed. There was progression to multifocal seizures, grand mal seizures, postictal depression, status epilepticus, and coma, with death 9 days after onset of the movement disorder. Bronchoalveolar carcinoma was widely disseminated in lungs and bones, and as three metastases in brain. Bland "ischemic" necrosis in a pseudolaminar pattern was present in the neocortex. Innumerable Cowdry type A intranuclear inclusion bodies were seen in neurons, astrocytes, and oligodenodroglia. Immunofluorescence demonstrated Herpes simplex virus type 2 antigen and electron microscopy revealed virions with the morphology of the Herpes group. The case is significant for (1) the concurrence of intracranial metastases and Herpes simplex encephalitis, and (2) the causal agent, Herpes simplex virus type 2. The implication for the clinical neurocientist is the potential in a patient with systemic cancer, for the causation of neurologic complications by more than one factor or mechanism.

Herpesvirus salmonis : Characterization of a New Pathogen of Rainbow Trout

A new agent, provisionally designated Herpesvirus salmonis, was isolated from post-spawning rainbow trout (Salmo gairdneri) and studied primarily in the RTG-2 rainbow trout cell line. Infection of RTG-2 cells resulted in the formation of syncytia and Cowdry type A intranuclear inclusions. Replication occurred regularly at 5 and 10 degrees C, but was inconsistent at 15 degrees C, largely inhibited at 0 degrees C, and completely inhibited at 20 degrees C or higher. The virus was acid, heat, ether, and chloroform labile, but stable to freezing and thawing. It did not hemagglutinate. Viral DNA had a buoyant density of 1.709 g/cm(3) and a guanine-cytosine value of 50%. Hexagonal nucleocapsids had a diameter of 90 nm and were first seen in nuclei at 36 h. Enveloped forms measured about 150 nm and occurred both cytoplasmically and extracellularly. At 10 degrees C, a one-step growth culture required about 96 h; cell-associated virus peaked at about 10(5) PFU/ml and exceeded released virus by a factor of about 10.

Isolation and characterisation of herpes virus from turkeys.

Turkey herpesvirus (HVT) was isolated from the kidneys of 6 of 8 turkey poults from two flocks. The isolates were identified by syncytial-type of cytopathology, inhibition of plaque formation by 5-bromodeoxyuridine, formation of Cowdry type A intranuclear inclusions in cell cultures, and presence of herpes-type virions in negatively stained preparations and thin sections of infected cell cultures. One of these isolates inoculated into chickens proved apathogenic over an observation period of 10 weeks. Indirect immunofluorescence and serum neutralisation tests revealed serological relationship between these isolates and the strain NSW 1/70 of HVT. Staining of HVT-infected cell culture by Marek's disease herpesvirus (MDHV) antiserum showed intranuclear fluorescence but attempts to prepare HVT precipitating antigen or to demonstrate cross-precipiation between HVT and MDHV were unsuccessful.

Herpes simplex encephalitis: an autopsy case with isolation of type 1 herpes simplex virus

An adult case of herpes simplex encephalitis was studied after autopsy. Postmortem examination revealed necrotizing encephalitis associated with Cowdry type A intranuclear inclusion bodies in glial cells. Herper simplex virus type 1 was isolated from the removed brain. Herpes simplex virus antigens were detected diffusely in wide areas of the brain by immunofluorescent test and viral particles characteristic to herpes simplex virus were demonstrated by electron microscopy. There was an apparent discrepancy between severity of histological changes and distribution of virus antigen.

Sequential Immunofluorescence and Infectivity Studies on the Replication of Herpesvirus Saimiri in Owl Monkey Kidney Cells

Methods are described for the preparation and authentication of a highly specific antiserum against herpesvirus saimiri (HVS) capsid antigens. The antiserum was used in immunofluorescence tests to follow the development of capsid antigens in HVS-infected owl monkey kidney cells throughout the virus replication cycle in parallel with sequential titrations of virus infectivity in both cells and medium. Fluorescence was detected as a round or oval, bright green area of staining at the centre of the nucleus which was similar in outline to the Cowdry type A inclusion seen in HVS-infected cells stained by haematoxylin and eosin. The first detection of fluorescence towards the end of the eclipse phase of the virus growth cycle, and its abolition by the treatment of infected cultures with cytosine arabinoside confirmed the identity of HVS capsid antigens as late antigens. The failure to detect fluorescence in the cytoplasm of HVS-infected cells has brought to light a conflict between the site of accumulation of virus capsid antigens as determined by immunofluorescence and the finding, by electron microscopy, of cytoplasmic immature particles in intact cells during the early stages of the virus replication cycle. The significance of this discrepancy is discussed in relation to its possible existence for other members of the herpesvirus group.

Fulminant Herpes Simplex Hepatitis in an Adult: Report of a Case in Renal Transplant Recipient

A case of disseminated herpes simplex infection is reported in a 31-year-old renal transplant recipient. The patient presented with a unique clinical syndrome: high fever, severe sore throat with buccal and pharyngeal ulcerations, fulminant hepatitis, thrombocytopenia, and leukopenia. The patient died from hepatic failure, disseminated intravascular coagulopathy, and upper gastrointestinal bleeding. The diagnosis was made by positive herpes simplex virus culture from the throat, and was confirmed at autopsy by typical Cowdry's type A intranuclear inclusions in hepatocytes with positive herpes simplex virus culture from the liver. Review of the literature reveals that other reported cases have had very similar clinical findings, making disseminated herpes simplex infection with fulminant hepatitis a recognizable syndrome.

Identity of cowdry type B inclusions and nuclear bodies: Observations in reovirus encephalitis

This report describes the investigation of the pathogenesis of an unusual sequel of reovirus encephalitis in suckling rats. Intracerebral inoculation of reovirus types 1, 2, and 3 into suckling rats induced, in addition to viral cytoplasmic inclusions, distinctive intranuclear bodies corresponding most closely to those designated by Cowdry (1934) as type B inclusions. The latter were seen in cells free of reovirus inclusions and unlike the conventional reactions to these viruses, were readily observed in weanlings, were unassociated with inflammatory features, and persisted for extended periods without cytolytic effects. To test the hypothesis that another viral agent was responsible for production of these nuclear changes, attempts were made to induce this change by inoculation of tissue preparations obtained from infected animals beyond the period of active reovirus encephalitis. These were uniformly negative. Furthermore, strains of reovirus from various sources all induced this nuclear change. Electron microscopic studies of the choroid plexus yielded no evidence of a second virus but instead demonstrated that the inclusions were composed of granular aggregates, sometimes enclosed in lamellar membranes, characteristic of the structures called nuclear bodies. These studies expand the perspectives of the nature and pathogenesis of this cellular lesion and provide an optical indicator which facilitates its identification and study. They suggest that the well-known Cowdry B inclusion has as its electron microscopic counterpart, the nuclear body.

Herpes zoster myelitis. Evidence for viral invasion of spinal cord.

Left thoracic herpes zoster in a 59-year-old woman was succeeded by myelopathy ascending in a saltatory manner over ten weeks until death occurred by pulmonary embolus. Dexamethasone therapy was instituted ten days after the rash began. Pathologic findings were those of a unicentric and progressive gangliomyelopathy with cicatrization of the left T-8 dorsal root ganglion and complete necrosis of the spinal cord from T-8 to T-11 with serpentine extension caudally to L-5 and rostrally to C3. Cowdry type A intranuclear inclusion bodies and structures in glial cells consistent with nucleocapsids ofHerpesviruswere observed in cervical spinal cord.Herpesvirus varicella(VZ virus) was isolated and identified in the cervical spinal cord. The findings suggest that herpes zoster myelitis is mediated by direct viral invasion and cell lysis.

Chronic ulcerative keratitis caused by herpes simplex virus.

A 45-year-old white man had chronic ulcerative keratitis, believed to be herpetic, for two years. Penetrating keratoplasty was performed after a descemetocele had almost perforated the cornea. Microscopically, epithelial cells at the edges of the ulcer contained reddish-purple intranuclear inclusions consistent with Cowdry type A inclusion bodies, although they were not surrounded by a halo. Electron microscopic studies of the formalin-fixed, paraffin-embedded tissue revealed intranuclear, cytoplasmic, and extracellular viral particles that were morphologically typical of herpes simplex. A brief review of the literature establishes the practical application of electron microscopy as a diagnostic tool, especially when inclusion bodies suspected of viral origin are observed in tissues that have been stored in formalin or embedded in paraffin, even for a prolonged period of time.

Herpes simplex encephalitis: a light and electron microscopic study in a fatal case.

SUMMARYA fatal case was described of a 41–year‐old woman with a tentative clinical diagnosis of acute viral meningoencephalitis of nine days' duration. The neuropathologic findings by light microscopy resembled those of acute necrotizing encephalitis or herpes simplex encephalitis, including eosinophilic intranuclear inclusion bodies of Cowdry type A. Electron microscopic study of these inclusions demonstrated the presence of herpes‐type virus particles. Though the titer of herpes simplex virus examined on the sixth day of the illness was not elevated and the isolation of the virus was not successful, the case was regarded as herpes simplex encephalitis clinicopatholo‐gically.

Herpes simplex encephalitis in an anergic patient

A patient with Hodgkin's disease and impaired immunity in whom encephalitis developed due to herpesvirus hominis type 1 is described. The patient's clinical course was atypical, being slowly progressive over seven weeks beginning with right frontoparietal dysfunction but progressively involving both hemispheres and the brainstem. The pathologic features were similarly unusual, consisting of widespread neuronal destruction, astrocytic proliferation and Cowdry type A inclusion bodies. Absent were the typical inflammatory changes and characteristic hemorrhagic necrosis of the medial temporal and inferior frontal lobes. Intraventricular administration of cytosine arabinoside failed to alter his course. The patient's depressed immune system appears to have been responsible for the atypical nature of his illness. The variance of the illness suggests that in the more usual case of herpes simplex encephalitis both direct cytotoxicity of the virus and host immune responses contribute to the pathogenesis of the disease.

Subacute inclusion body encephalitis associated with myopathy.

Intranuclear inclusions suggesting intranuclear virus replication were found in sarcolemmal nuclei of the diaphragm and intercostal muscles of a patient with subacute encephalitis with Cowdry type A intranuclear inclusion bodies. Muscle fibers exhibited severe degenerative changes, including occasional stigmata of denervation. The morphological evidence favors interpretation of both the encephalitis and the myopathy as virus induced. This case provides support for the emerging concept of viral myopathy. Although myopathy in patients with latent encephalitis due to measles virus has not been stressed, the frequency of its occurrence has not been examined by systematic postmortem sampling of the voluntary musculature.

Herpesvirus ateles, a new lymphoma virus of monkeys.

A NEW herpesvirus, designated Herpesvirus ateles, has been isolated from a spider monkey (Ateles geofroyii) primary kidney culture. The culture was prepared from a mature male spider monkey (810–69) imported from Guatemala and housed in our laboratory for 12 months. The cell layer was obtained from the renal cortex by trypsinization using standard procedures. Growth medium was Eagle's minimum essential medium with 10% calf serum plus antibiotics. A well grown cell layer developed in 6–8 days. On day 11 the cell layer presented a large number of bizarre cells and polykaryocytes. On day 16 the cell layer was scraped and together with the culture fluid was collected as isolate No. 810 (H. ateles) and stored at 4° C, −86°C, and −176°C. H. ateles strain No. 810 had a titre of 104 TCID50/ml. in squirrel monkey foetal lung cell line No. 241, 104.5 TCID50/ml. in owl monkey kidney cell line No. 210, 103.5 TCID50/ml. in human embryonic lung cell line and 105.0 TCID50/ml. in rabbit kidney cell line at 14, 17, 23, and 30 days, respectively, after inoculation. All cell lines were prepared in our laboratory. The cytopathic effect was characterized by scattered foci of swollen and rounded cells, polykaryocytes and intranuclear inclusion bodies. The classical Cowdry type A inclusion body was observed in owl monkey kidney cultures. In human foetal lung, squirrel monkey kidney and rabbit kidney cell lines the inclusion bodies were small, measuring approximately 1 µm in diameter. The virus was inactivated by ether and heat (56° for 30 min) and inhibited by bromodeoxyuridine. The size was determined to be between 100 nm and 220 nm by filtration. Ultrastructural studies of infected tissue culture cells revealed typical herpesvirus particles (Fig. 1). These physical, chemical and morphological characteristics of the isolate place it in the herpesvirus group. Antisera to H. simplex, Herpes T., infectious bovine rhino tracheitis, sand rat nuclear inclusion agent, H. suis, ground squirrel agent, H. saimiri, spider monkey herpesvirus, and H. aotus failed to neutralize the infectivity of H. ateles1,2. Fluorescent antibody studies have also demonstrated that H. ateles is distinguishable from H. saimiri, OMKI 68–69, Herpes T. and H. simplex3. These data support the conclusion that H. ateles is a distinct member of the herpesvirus group.

Immunization against Marek's Disease

The turkey herpesvirus (HVT) and Marek's disease (MDHV) herpesvirus produce a similar virus-like cytopathic effect in monolayers of chick embryo fibroblasts. Discrete foci of rounded refractile cells and syncytial masses with some of the nuclei containing Cowdry type A inclusions were observed in affected monolayers. Electron micrographs indicate that both isolates are herpesviruses. The HVT appears to differ from MDHV in that the MDHV fluorescent antibody conjugate does not stain monolayers infected with HVT, nor does the MDHV antigen react with HVT antisera in the agar gel precipitin test. Continuous passage of MDHV in cell culture resulted in the loss of its oncogenic potential for the chicken, whereas HVT was found not to be oncogenic for chickens even with low cell-culture passages. One-day-old chicks vaccinated subcutaneously with either cell-free HVT, suspension of HVT in cell culture or attenuated MDHV were protected against MD when immunity was challenged by natural or artificial exposure. Lyophilized HVT or MDHV vaccines were found to be less effective than fresh live-virus vaccines. Investigations on the route of exposure indicate that these vaccines are most effective by the parenteral inoculation of these materials.

Use of Feathers in a Gel Precipitation Test for Marek’s Disease

THE skin was first described as being involved in Marek’s disease (MD) by Helmbolt et al. (1963). Since then, evidence has indicated this organ’s role in the transmission and pathogenesis of the disease. Beasley (1970) has transmitted MD with dander from MD infected chickens. Herpes type particles have been observed (Clanek, 1970; Nazerian and Witter, 1970) and infectious cell free extracts have been recovered (Calnek, 1970; Nazerian and Witter, 1970; Purchase, 1970) from the cutaneous follicular epithelium of MD infected chickens. Lapen et al. (1970), Calnek and Hitchner (1969) and Purchase (1970) have observed an immunofluorescent (IF) antigen associated with MD in the cutaneous follicular epithelium of MD infected fowl. Lapen et al. (1970) described Cowdry type A intranuclear inclusions in this epithelium and demonstrated they were part of the MD process.These observations suggested that the feather and associated follicular epithelium may contain sufficient MD associated antigen which could…

Observations on the Histopathology of Bovine Herpes Mammillitis

Summary Observations are recorded on the histology of experimental and natural lesions of bovine herpes mammillitis (BHM). A study was carried out on baby rats inoculated intradermally, and parallel observations were made in cattle with experimental and natural herpes mammillitis, and with generalized skin lesions following intravenous inoculation of BHM virus. In all these forms the histological changes were similar and were characterised in the epidermis by oedema, hydropic degeneration, vesiculation and cell infiltration. Cowdry Type A intranuclear inclusions were seen in either single or syncytial nuclei. Scabbing occurred early, and scab material showed only necrosis. Healing of the epidermis occurred under the scab and there was no permanent scar. Changes in the dermis consisted of oedema, hyperaemia and perivascular mononuclear cell infiltration.