Photodynamic Effect of some Phthalocyanines on Enveloped and Naked Viruses.

Activity of three photosensitizing phthalocyanine derivatives was tested for photodynamic inactivation towards two coated and two non-enveloped viruses - bovine viral diarrhea virus (BVDV), influenza virus A(H3N2), poliovirus type 1 (PV-1) and human adenovirus type 5 (HAdV5). In the case of coated viruses, a combination of virucidal and irradiation effects was registered by octa-methylpyridyloxy-substituted Ga phthalocyanine (Ga8) toward BVDV, whereas tetra-methylpyridyloxy-substituted Ga phthalocyanine (Ga4) revealed a marked photoinactivation only. No such effect was observed towards influenza A virus. In contrast, the photoinactivating potential of Ga4 and Ga8 marked very high values on naked viruses, especially on HAdV5, at which both the virucidal as well as the irradiation effects became combined.

The occurrence of five major Newcastle disease virus genotypes (II, IV, V, VI and VIIb) in Bulgaria between 1959 and 1996.

Partial sequence and restriction enzyme cleavage site analyses of the fusion protein gene were used to genotype 47 Newcastle disease virus strains isolated between 1959 and 1996 in Bulgaria. Viruses belonged to five major genotypes that appeared to be associated with epizootics characterized by temporal and/or geographical restrictions. Genotype IV viruses (responsible for the European branch of the first panzootic) dominated the scene up to the early 1980s, interspersed with sporadic outbreaks caused by genotype II (US strains causing pneumoencephalitis) viruses. Genotype V viruses (transmitted by psittacines from South America) were first shown in 1973 and persisted until the late 1980s. Genotype VI (earliest members from the Middle-East 1968/70 outbreaks) was represented by scattered isolations between 1974 and 1996. A genotype VIIb (recent Middle East epizootic) virus was isolated as early as in 1984. Newcastle disease epizootics in Bulgaria were highlighted by multiple infection with more than one genotype at any one time.

Fluorescent and electron-microscopy immunoassays employing polyclonal and monoclonal antibodies for detection of goose parvovirus infection.

Polyclonal antibodies (PAbs) raised in geese and eight mice hybridomas secreting monoclonal antibodies (MAbs) against the goose parvovirus (GPV) were prepared. They were used for development of immunofluorescence (IF) and immunoelectron-microscopic (IEM) techniques to demonstrate the GPV infection in infected organs and biological fluids. The GPV antigens were established by immunofluorescence within the nuclei and the cytoplasm of many infected cells of the chorioallantoic membrane of goose and Peckin duck embryos, liver and heart of mortally diseased goslings. By means of IEM it was possible to detect the GPV in native organ homogenate supernatants and allantoic fluids. All techniques used in the study could be successfully applied for rapid diagnosis of the GPV infection. The test systems on the basis of MAbs should, however, be preferred. By means of immunoblotting (IB) using PAbs and MAbs four viral proteins (VP) with MW 88, 77, 65 and 60 kDa were demonstrated. Contrary to the others the VP with MW 65 kDa was the most antigenically reactive though invisible in the SDS-PAGE and Coomassie-blue dye-stained preparations.

Detection and identification of the Newcastle disease virus infection by electron and immunoelectron microscopy.

Various electron microscopic (EM) and immunoelectron microscopic (IEM) techniques were used to demonstrate and identify the Newcastle disease virus (NDV) infection. By IEM, the number of virions in native allantoic fluids was increased 50-100 times in comparison with direct EM. The immunogold staining showed that a number of immunogold particles were specifically bound to the antigen determinants located on the virion surface and these results were much easier to interpret. The obtained results showed that the EM and IEM can be successfully employed for a precise and rapid detection of NDV as well as for identification of this infection among other viral or bacterial infections.

Kinetics and role of alpha 1-acid glycoprotein-dependent osmotic transport of water and ions in palmitoyl-L-oleoyl phosphatidylcholine liposomes.

alpha 1-Acid glycoprotein isolated from human blood plasma is known to influence cell permeability, although the mechanisms of this process are unclear. Here, the glycoprotein effects on the permeability of osmotically stressed phospholipid liposomes are studied as a model of membrane permeability. Liposomes containing glycoprotein were found to be osmotically sensitive to water and chloride salts of some monovalent (Na+, K+) and bivalent (Mg2+, Ca2+) ions. The permeations of these substances were determined by light-scattering measurements of the volume changes in liposomes after mixing with hyperosmotic solutions of chloride salts. The time courses of scattered light were recorded by means of stopped-flow spectrophotometry. Two processes were studied: the fast water outflow from liposomes and slower ion permeations through the lipid membrane. The second order permeation rate constants were determined at different glycoprotein concentrations for both processes. Values from 66 to 250 x 10(3) for water outflow and 2-500 M-1 sec-1 for the different ion permeations were obtained in order to characterize the permeations of solutes across the lipid membrane. The apparent activation energies also were calculated between 18 and 33 degrees C. The mercurial sulphydryl reagent pCMBS inhibited the ion permeations in the slow phase. When pCMBS was present in this phase, higher activation energies were obtained, indicating more difficult permeations. An interpretation of these results is that membrane permeability is mediated by aqueous pores. Membrane selectivity to monovalent metal ions also was demonstrated, but no correlation was observed between the ion radius of the corresponding metal cation and permeation rate constants. The discovery of non-specific pores in liposomes containing glycoprotein shows that they can serve as vehicles for the water and ions in the processes of passive transport through lipid membranes.

Electron- and immunoelectron-microscopic investigation on the rabbit haemorrhagic disease virus.

Electron- and immunoelectron-microscopic methods were used for detection and investigation of rabbit haemorrhagic disease virus (RHDV). The observed virus particles were uncoated, with an icosahedral symmetry, with a 35-40 nm dia and capsids built up by 32 capsomers with a central hollow part and a 3.5-5 nm dia. Particles with empty capsids could also be seen among them. Single virions visible in 5-fold symmetry, exhibited 10 projections on the surface. Virions with a 33-34 nm dia and cup-shaped surface depressions were a rare finding. Some of these particles were indistinguishable from the caliciviruses, but in most of them the cup-shaped depressions were single or no more then 2-3 in number, localized predominantly unilaterally or asymmetrically. Particles 25-29 nm large with full or empty capsids without capsomers could also be seen. Most probably they were some immature forms in the process of development of the RHDV.

Immunohistochemical localization of the rabbit haemorrhagic disease viral antigen.

Immunohistochemical investigations were carried out to determine organ and cellular localization of the rabbit haemorrhagic disease viral antigen (RHDVA). It was found in certain parenchymal liver cells near the interlobular septs and in some macrophages and pseudoeosinophils of all studied organs and blood. Whereas in morphologically preserved hepatocytes and macrophages the RHDVA accumulated in the nuclei, in cells with disintegrated nuclei it was distributed throughout the cytoplasm.