Cellular processes essential for African swine fever virus to infect and replicate in primary macrophages

The macrophage (Mø) is an essential immune cell for innate immunity. Such cells are targeted by African swine fever virus (ASFV). The early phases of infection with ASFV have been previously characterized in non-leukocyte cells such as Vero cells. Here, we report on several additional key parameters that ASFV utilizes during the infection of primary Mø. Related to virus infection, we established that receptor-mediated endocytosis of the virus by Mø is not the exclusive means of entry to infect the host cells. Analysis of the ensuing processes identified divalent cation-dependent activities to be particularly important, relating to the virus requirement for microtubule assembly needed for endocytic and endosomal processing. Actin-dependent endocytosis and endocytic flux involving microtubule activity are also implicated, pointing to entry via phagocytosis. Subsequently, the virus avoids terminal degradation by circumventing mature lysosome activities, including autophagosome–lysosome delivery. Nevertheless, the replicative cycle is apparently dependent on certain lysosomal functions, i.e. activities sensitive to propylamine are essential for the virus, whereas vinblastine- and leupeptin-sensitive functions only partially influence viral replication. The present work has identified cellular processes essential for ASFV to infect and replicate in the macrophage. These findings will improve our understanding of the cellular pathways employed by viruses infecting immune scavenger cells.     

A new natural spiro heterocyclic compound and the cytotoxic activity of the secondary metabolites from Juniperus brevifolia leaves

A new natural spiro compound 3,4-dehydrotheaspirone and the known arctiol [1β,6α-dihydroxy-4(14)-eudesmene] were isolated from Juniperus brevifolia. Arctiol is reported for the first time in the Juniperus genus. Their structures were established by 1D, and 2D NMR and MS spectra. Antimicrobial and cytotoxic activities of 1 and several secondary metabolites ,,,,,,,,, previously isolated by our group from J. brevifolia were evaluated and some SAR has been established. The 18-hydroxydehydroabietane (4) displayed great antiproliferative activity against cancer cell lines tested, namely HeLa, A-549 and MCF-7. Compound 4 also presented a significant bactericidal effect against Bacillus cereus at different concentrations tested.     

Heat expanded starch-based compositions

A heat expansion process similar to that used for expanded bead polystyrene was used to expand starch-based compositions. Foam beads made by solvent extraction had the appearance of polystyrene beads but did not expand when heated due to an open-cell structure. Nonporous beads, pellets, or particles were made by extrusion or by drying and milling cooked starch slurries. The samples expanded into a low-density foam by heating 190-210 degrees C for more than 20 s at ambient pressures. Formulations containing starch (50-85%), sorbitol (5-15%), glycerol (4-12%), ethylene vinyl alcohol (EVAL, 5-15%), and water (10-20%) were studied. The bulk density was negatively correlated to sorbitol, glycerol, and water content. Increasing the EVAL content increased the bulk density, especially at concentrations higher than 15%. Poly(vinyl alcohol) (PVAL) increased the bulk density more than EVAL. The bulk density was lowest in samples made of wheat and potato starch as compared to corn starch. The expansion temperature for the starch pellets decreased more than 20 degrees C as the moisture content was increased from 10 to 25%. The addition of EVAL in the formulations decreased the equilibrium moisture content of the foam and reduced the water absorption during a 1 h soaking period.     

Reaction between hydroxycinnamic acids and anthocyanin-pyruvic acid adducts yielding new portisins

Three new anthocyanin-derived pigments were found to occur in a 2-year-old Port red wine. Their structures were elucidated through LC/DAD-MS and NMR analysis and were found to correspond to a pyranoanthocyanin moiety linked to substituted cinnamyl substituents. The structures of these compounds are very similar to the one already reported for portisins, with a phenolic moiety replacing the catechin moiety. The newly formed anthocyanin-derived compounds display a bathochromic shift of the lambdamax ( approximately 540 nm) when compared with their anthocyanin-pyruvic acid adduct precursor (lambdamax = 511 nm), which may be due to the extended conjugation of the pi electrons in the structures of those pigments. Studies performed in model solutions helped to clarify the formation mechanism of these pigments that can result from the nucleophilic attack of the olefinic double bond of a hydroxycinnamic acid to the eletrophilic C-10 position of the anthocyanin-pyruvic acid adduct, followed by the loss of a formic acid molecule and decarboxylation. The chromatic characterization of these malvidin-3-glucoside-derived compounds revealed a higher resistance to discoloration against the nucleophilic attack by water and bisulfite when compared to malvidin-3-glucoside that is almost converted into its colorless hemiacetal form. However, the resistance to discoloration of these new pigments is not as high as the one reported for catechin-derived portisins. This could be explained by the presence of a smaller group (hydroxycinnamyl group), which does not protect so efficiently the chromophore against nucleophilic attack at the C-2 position. The occurrence of these pigments in red wine highlights new chemical pathways involving anthocyanin-pyruvic acid derivatives as precursors for the formation of new pigments in subsequent stages of wine aging that may contribute to its color evolution.     

Comparison of 11 herpesvirus isolates from tortoises using partial sequences from three conserved genes

Herpesviruses are an important cause of epidemic disease in tortoises. There are at least two serologically distinct herpesviruses capable of infecting tortoises. Methods for the diagnosis of herpesvirus infections in tortoises include virus isolation and a number of different PCRs. We have compared 11 virus isolates collected from various species in different countries over several years using sequences from three different viral genes. During this study we used four different PCR protocols described for the diagnosis of herpesvirus infections in tortoises. The protocols used included two based on portions of the DNA polymerase gene, one targeting the UL5 homologue, and one targeting the UL39 homologue. Comparison of the methods showed that the tortoise herpesvirus-specific protocols were all serotype specific. Sequences of the obtained amplicons were compared with one another and with sequences of herpesviruses available in GenBank. The sequence alignments showed that the tortoise herpesviruses were most closely related to members of the subfamily Alphaherpesvirinae. They also showed that the tortoise isolates could be clearly divided into two genogroups.     

Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.     

Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil

The viability of a fungal formulation using the nematode-trapping fungus Duddingtonia flagrans was assessed for the biological control of horse cyathostomin. Two groups (fungus-treated and control without fungus treatment), consisting of eight crossbred mares (3–18 years of age) were fed on Cynodon sp. pasture naturally infected with equine cyathostome larvae. Each animal of the treated group received oral doses of sodium alginate mycelial pellets (1 g/(10 kg live weight week)), during 6 months. Significant reduction (p < 0.01) in the number of eggs per gram of feces and coprocultures was found for animals of the fungus-treated group compared with the control group. There was difference (p < 0.01) of 78.5% reduction in herbage samples collected up to (0–20 cm) between the fungus-treated group and the control group, during the experimental period (May–October). Difference of 82.5% (p < 0.01) was found between the fungus-treated group and the control group in the sampling distance (20–40 cm) from fecal pats. During the last 3 months of the experimental period (August, September and October), fungus-treated mares had significant weight gain (p < 0.01) compared with the control group, an increment of 38 kg. The treatment with sodium alginate pellets containing the nematode-trapping fungus D. flagrans reduced cyathostomin in tropical southeastern Brazil and could be an effective tool for biological control of this parasitic nematode in horses.     

Canine distemper virus-induced depletion of uninfected lymphocytes is associated with apoptosis

Canine distemper virus (CDV), a negative stranded RNA morbillivirus, causes a multisystemic disease in dogs, which is associated with a severe immune suppression. The aim of the study was to examine the influence of early CDV infection on leukocyte depletion, lymphopenia and virus-induced cell death in dogs infected with a virulent CDV strain. From 10 infected dogs, peripheral blood leukocytes were harvested periodically, phenotyped and analyzed for CDV antigen content and apoptosis using Annexin V-FITC and propidium iodide labeling. CDV infection induced a severe CD3+ T cell and CD21+ B cell depletion in all animals at 3 days post-infection (d.p.i.). For dogs with severe distemper, developing virus persistence in the lymphoid tissue and central nervous system, this lymphopenia lasted until the end of the experiment. Increased levels of lymphocyte apoptosis were found at 3 d.p.i., and monocyte apoptosis at 6 d.p.i. This was more prominent in the group of animals with severe distemper. At 3 d.p.i. no leukocyte infection was detectable indicating that the early lymphocyte depletion and apoptosis was not a direct consequence of virus infection. Taken together, our results demonstrate that CDV-induced lymphopenia is an early event and that the degree of lymphocyte depletion correlates with the severity of disease and virus persistence in the lymphoid tissue and central nervous system.