Cytopathogenic effect of African swine fever virus for pig monocytes: characterization and use in microassay.

A microculture assay is described for the titration of African swine fever virus (ASFV) using swine monocytes contained in mononuclear leucocyte (MNL) microcultures. Titration endpoints were determined by observing cytopathogenic effects (CPE) of ASFV infected monocytes with an inverted microscope at 40 X magnification. CPE was a late event following the detection of ASFV antigens in monocytes by radioimmune assay, immunofluorescence and hemadsorption. It began with the detachment, enlargement and rounding of monocytes which progressively formed into grape-like clusters of 3-20 or more cells which eventually lysed. The characteristic CPE was produced in monocyte microcultures by virulent, moderately virulent, Vero cell adapted, and nonhemadsorbing ASFV strains. The sensitivity and reproducibility of the CPE microassay was similar to that of the hemadsorption microassay.

Destruction and repair of mammary gland parenchyma of cows infected with foot-and-mouth disease.

Bovine mammary gland secretory epithelial cells show signs of infection as early as three days after exposure of lactating host dairy cows to pigs infected with foot-and-mouth disease. Isolated foci of necrotic alveoli, as observed by scanning electron microscopy, increased in area and number with time. Infection of individual milk synthesizing cells progressed to involve the entire secretory epithelium. Alveolar luminal contents, in contrast to those in control preparations, consisted of heavy concentrations of cellular debris and connective tissue fragments; few leukocytes were observed. Involutionary changes occurred 10 days after cow exposure. Significant evidence of repair in necrotic areas was present at 15 days, but limited repair occurred earlier. Characteristics of replacement secretory epithelium made it possible to differentiate it from older mammary gland parenchyma.

Foot-and-mouth disease virus-induced RNA polymerase is associated with Golgi apparatus.

Electrophoretic analysis of the Golgi apparatus isolated by differential centrifugation from radiolabeled cells infected with foot-and-mouth disease virus showed about 10 protein bands. The virus-induced RNA polymerase was identified by immunoprecipitation and electron microscope staining procedures. Pulse-chase experiments indicated that the polymerase passed through the Golgi apparatus in less than 1 h.

Monoclonal antibodies against African swine fever viral antigens.

Monoclonal antibodies specific for African swine fever (ASF) viral proteins of 14, 32, 73, 174, and 240 kDa were produced and characterized. Immunoelectron microscopy detected the 73 kDa but not the 14-, 32-, or 240-kDa proteins at the surface of the virion. The 32-kDa protein was detected by radioimmunoassay 2 hr after infection of porcine monocytes and Vero cells, was detected in the seven widely divergent ASFV isolates tested, and stained brilliantly virus-infected cells in indirect immunofluorescence suggesting that monoclonal antibodies directed against this protein may be useful in ASFV diagnosis. Two monoclonal antibodies detected heterogeneity between ASF viruses.

Correlation of surface and internal ultrastructural changes in cells infected with foot-and-mouth disease virus.

The surfaces of primary and continuous line cell cultures displayed the same sequence of morphological changes during the course of infection with foot-and-mouth disease virus. These changes could be classified into four broad stages: I) cells were flattened, closely attached to one another and microvilli appeared, II) cells rounded, microvilli began to disappear and the cells started to separate from one another by cytoplasmic strands, III) cells were discrete, rounded structures and IV) cells were rounded and had numerous attached buds, some of which contained virus. The internal changes included the appearance of increasing amounts of smooth membranous vacuoles lined with the viral induced RNA polymerase and the presence of buds, some with viral particles inside. While the different cell cultures showed similar internal and external changes as a result of infection, they responded to infection at different rates and contained subpopulations of resistant cells.

Association of foot-and-mouth disease virus induced RNA polymerase with host cell organelles.

The localization of foot-and-mouth disease viral-induced RNA polymerase has been determined in situ and in partially fractionated cell components by using polymerase antisera tagged with either peroxidase or ferritin. Electron microscopic examination revealed the polymerase to be heavily concentrated on membranes of the smooth membranous vacuoles (SMV) which are newly formed during infection and which were previously shown to be the site where newly synthesized viral RNA appeared. Polymerase antigen was also seen to be associated with the endoplasmic reticulum (ER), the assumed site of original synthesis, and to a lesser extent with mitochondria and the Golgi apparatus. There was no significant polymerase attachment to nuclear and plasma membranes.

Ultrastructural changes and antigen localization in tissues from foot-and-mouth disease virus-infected guinea-pigs.

Foot-and-mouth disease virus (FMDV)-induced ultrastructural changes in guinea-pig tongue, heelpad, mammary and liver tissues were examined using scanning and transmission electron microscopy. FMDV infection caused cell rounding and the release of virus in membrane limited vesicles in the animal tissues similar to that seen in other work in cell cultures. Microfilaments were present which may be responsible for cell rounding. Immunoperoxidase labeling revealed the attachment of the virus-infection associated (VIA) antigen to the smooth vacuoles of mammary and liver tissues, and to milk fat globules. The electron microscope immunoperoxidase procedure increased the sensitivity of detection sufficiently to allow the visualization of VIA antigen in tissues not previously shown to have the antigen. It is postulated that the release of the smooth vacuoles from the liver cells stimulates the animal's immune response to the VIA antigen.

Localization of foot-and-mouth disease--RNA synthesis on newly formed cellular smooth membranous vacuoles.

Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [3H] uridine. Both membrane formation and RNA synthesis became significant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the sites of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity.

Polyphosphate granules in encysted zoospores of Rozella allomycis.

Methods of ultracytochemistry and of X-ray energy dispersive analysis have been used to demonstrate that the "gamma-like" granules in encysted zoospores of the chytrid Rozella allomycis contain polyphosphate. The possibility that cysts contain two classes of polyphosphate granules which differ in structure, in function, and in origin is discussed.