Ultrastructural characteristics and morphogenesis of porcine reproductive and respiratory syndrome virus propagated in the highly permissive MARC-145 cell clone.

A Québec reference strain of PRRSV (IAF-KLOP) was successfully propagated in MARC-145 cells, a highly permissive cell clone to PRRSV derived from the MA-104 cell line. Purified extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter, with an isometric core about 25-30 nm. By indirect immunofluorescence, detection of viral antigens within the cytoplasm was possible as soon as 6 h.p.i. Nucleocapsids, budding at smooth endoplasmic reticulum (ER), and enveloped viral particles that tended to accumulate in the lumen of ER or Golgi vesicles, were the main features of the viral morphogenesis. The virus apparently was released by exocytosis.

Detection of bovine coronavirus and type A rotavirus in neonatal calf diarrhea and winter dysentery of cattle in Quebec: evaluation of three diagnostic methods.

The use of direct electron microscopy, enzyme-linked immunosorbent assay, and protein A-gold immunoelectron microscopy for the identification of bovine coronavirus and type A rotavirus were examined. Two hundred and forty-nine samples from diarrheic calves and winter dysenteric cattle from seven geographic areas in Quebec were examined for the presence of viruses by direct electron microscopy of negatively stained preparations. In addition, all the samples were analyzed by enzyme-linked immunosorbent assay, and a random selection of 47 samples were also analyzed by protein A-gold immunoelectron microscopy. Thirty-nine percent of samples examined by direct electron microscopy contained viral particles; bovine coronavirus and type A rotavirus were the most common viruses involved. Overall agreement between any two of the methods used compared favorably with results obtained by others using similar methods. The presence of coronavirus and rotavirus in fecal samples obtained from neonatal calves and the presence of coronavirus in samples from winter dysenteric adult cattle suggested their etiological roles in the respective diseases. Furthermore, results from protein A-gold immunoelectron microscopy of coronavirus-like particles implied that a different coronavirus or some other viruses might be involved in these diseases. Finally, the efficiency of direct electron microscopy, enzyme-linked immunosorbent assay and protein A-gold immunoelectron microscopy as diagnostic tools is discussed.

Porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of Quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome.

Cytolytic and noncytolytic strains of the porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in primary cultures of porcine alveolar macrophages (PAM) from lung homogenates of stillborn fetuses or blood samples of dyspneic piglets collected from Quebec pig farms having experienced acute or chronic outbreaks of PRRS. Serological identification of the virus was confirmed by indirect immunofluorescence and indirect protein A-gold immunoelectron microscopy using reference antiserum prepared from experimentally-infected specific pathogen free (SPF) piglets and monoclonal antibodies (MoAbs) directed against the p15 nucleocapsid (N) protein of the reference ATCC-VR2332 isolate. Intracytoplasmic enveloped viral particles that tended to accumulate into cytoplasmic vesicles were observed in the infected PAM; no budding was demonstrated at the level of the cytoplasmic membrane. The extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter (averaged diameter of 50 particles was 58.3 nm), with an isometric core about 25-30 nm. Buoyant density of the virus in CsCL density gradients was estimated to 1.18-1.20 g/mL. No hemagglutinating activity was demonstrated. Analysis of semipurified virions of isolate IAF-exp91 by radioimmunoprecipitation (RIPA) and Western immunoblotting experiments, using reference rabbit and porcine hyperimmune sera, revealed four major viral proteins, a predominant 15 kD N protein and three other proteins with predicted M(r_ of 19, 26 and 42 kD. Progeny viral particles produced in PRRSV-infected PAM in the presence of tunicamycin lacked the 42 kD protein, thus confirming its N-glycosylated nature. Immunoprecipitation experiments using the anti-ATCC-VR2332 MoAbs confirmed the close antigenic relationships between Quebec and American reference isolates of PRRSV.

Ultrastructural study of mouse thymus virus replication.

With the exception of thymocytes, no other cell types have been reported to be involved in mouse thymus virus (MTV) infection. The ultrastructure of thymuses of mice infected with MTV were examined. The earliest sign of infection was detected 5 days p.i.; lymphocytes, epithelial and phagocytic (macrophages) reticular cells were shown to be affected. Viral particles and filamentous structures were present in both the nucleus and the cytoplasm of these cells. At more severe stages of cellular necrosis, 6 and 7 days p.i., cytoplasmic granulation as well as loss in definition of cytoplasmic organelles became apparent. This was followed by nuclear degradation and aggregation of cells. After 9 days p.i. necrotic lesions were still observed but viral particles were no longer detectable. This study provides evidence of the susceptibility of macrophages to MTV.

Swine reproductive and respiratory syndrome in Québec: Isolation of an enveloped virus serologically-related to Lelystad virus.

Sera were collected from convalescent sows and sick piglets from six pig farms in southern Quebec that have experienced outbreaks of the so-called porcine reproductive and respiratory syndrome. By indirect immunoperoxidase, a few of these sera (4 of 14) (28.6%) were found to be positive for antibody to the Lelystad virus, whereas by indirect immunofluorescence 30 of 36 (83.3%) were positive for antibody to the antigenically-related American isolate ATCC-VR2332. Pregnant sows inoculated intranasally with filtered homogenates prepared from the lungs of necropsied piglets obtained from a seropositive farm developed fever, inappetence, and reproductive failure characterized by stillbirths and various stages of mummification. Lesions of interstitial pneumonia were induced in experimentally-infected specific pathogen-free piglets. A virus, having morphological and biological characteristics of viruses assigned to the family Togaviridae, was isolated from lung tissues of experimentally-infected animals; it could only be propagated in primary cultures of porcine alveolar macrophages. Identification of the virus was confirmed by indirect immunofluorescence using a monoclonal antibody directed against the nucleocapsid protein of the ATCC-VR2332 isolate and porcine sera that were found positive for antibody to both the Lelystad and ATCC-VR2332 isolates.

Mouse thymic virus infection: ultrastructural and immunocytochemical studies of infected thymus cells.

Only limited attention has been paid to the cell population that is affected in the course of Mouse Thymic Virus (MTV) infection. In the present study, thymic cells of newborn mice infected with MTV were examined for general ultrastructural and immunocytochemical characteristics. The earliest sign of infection was detected 5 days after inoculation. Lymphocytes, epithelial reticular cells, macrophages, and lymphoepithelial cell complexes (thymic nurse cells) were affected. Viral particles and filamentous structures were present in both the nucleus and the cytoplasm of these cells. At more advanced stages of cellular necrosis, 6 to 7 days post-infection, cytoplasmic granulation and loss in definition of cytoplasmic organelles became apparent. This was followed by nuclear degradation and cellular aggregation. The selective effect of MTV on lymphocyte subpopulations was also observed. Two populations of infected lymphocytes were identified by single and double immunogold labelling employing monoclonal antibodies and different sizes of gold particles. CD4+8+ and CD4+8- lymphocytes were found to be selectively lysed by MTV.

Electron microscopy of mouse thymic virus.

Thymuses of mice infected with mouse thymic virus (MTV), a herpesvirus, were examined with the electron microscope. Damage was manifested by distortions of cells, nuclei, and other organelles, with cellular aggregation apparent at advanced stages of necrosis. Infected cells also contained MTV particles and filaments. Negative staining showed that naked MTV capsids were icosahedral in shape with diameters ranging from 95 to 110 nm. Enveloped capsids appeared spherical and ranged in diameter from 125 to 165 nm. Immunogold labeling revealed the presence of antigenic sites around both naked and enveloped capsids, and also on the 10 nm filaments. The use of electron microscopy and immunohistochemical techniques as diagnostic tools to demonstrate MTV infection could prove valuable for the detection of the infection and the study of its pathogenesis.

Regulation of cyclic nucleotides in retinal photoreceptors. An ultracytochemical approach on the role of cyclases.

The distribution of cyclases in retinal photoreceptors of dark- and light-adapted brook trout was studied by means of a cytochemical method (lead precipitation). It confirms earlier reports that retinal photoreceptors contain high levels of cyclic nucleotides, and that cAMP predominates in cones and cGMP in rods. There is an apparent difference in the level of the cyclases with the adaptive states. In addition, the catalytic unit of cyclase is interlamellar in cones. In rods, adenylate cyclase is intradiscal, while the location of guanylate cyclase varies with the adaptive state. The variation of cyclase with adaptation indicates that this enzyme has a role in the process of visual transduction.