Eimeria bovis Macromeront Formation Induces Glycolytic Responses and Mitochondrial Changes in Primary Host Endothelial Cells

Eimeria bovis is an intracellular apicomplexan parasite that causes considerable economic losses in the cattle industry worldwide. During the first merogony, E. bovis forms large macromeronts with >140,000 merozoites I in host endothelial cells. Because this is a high-energy demanding process, E. bovis exploits the host cellular metabolism to fulfill its metabolic requirements. We here analyzed the carbohydrate-related energetic metabolism of E. bovis–infected primary bovine umbilical vein endothelial cells during first merogony and showed that during the infection, E. bovis–infected culture presented considerable changes in metabolic signatures, glycolytic, and mitochondrial responses. Thus, an increase in both oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were found in E. bovis–infected host cells indicating a shift from quiescent to energetic cell status. Enhanced levels of glucose and pyruvate consumption in addition to increased lactate production, suggesting an important role of glycolysis in E. bovis–infected culture from 12 days p.i. onward. This was also tested by glycolytic inhibitors (2-DG) treatment, which reduced the macromeront development and diminished merozoite I production. As an interesting finding, we observed that 2-DG treatment boosted sporozoite egress. Referring to mitochondrial activities, intracellular ROS production was increased toward the end of merogony, and mitochondrial potential was enhanced from 12 d p. i. onward in E. bovis–infected culture. Besides, morphological alterations of membrane potential signals also indicated mitochondrial dysfunction in macromeront-carrying host endothelial culture.

Experiment on obtaining an algologically pure culture of Tetraselmis viridis in non-sterile conditions

The possibility of obtaining an algologically pure culture of Tetraselmis viridis, grown on the Black Sea water in non-sterile conditions, was shown experimentally. Our experiments showed that at low initial population density of the culture after 1–3 days, there was an infection of the culture with blue-green species of microalgae (Oscillatoria sp.). Thrice repeated mechanical removal of blue-green microalgae cells by filtering the infected culture allowed obtaining an algologically pure culture of T. viridis. Under similar conditions of T. viridis cultivation, but with the initial addition of NaCl (15 g/l) to the nutrient medium aimed at increasing salinity to the Mediterranean level, there was no contamination of the culture.

Genome Sequence Analysis ofMycoplasmasp.HU2014, Isolated from Tissue Culture

The draft genome sequence of a novelMycoplasmastrain, designatedMycoplasmasp. HU2014, has been determined. The genome comprises 1,084,927 nucleotides and was obtained from a mycoplasma-infected culture of chicken DT40 cells. Phylogenetic analysis places this taxon in a group comprising the closely related speciesMycoplasma yeatsiiandMycoplasma cottewii.

Spindle Cell Conversion by Kaposi's Sarcoma-Associated Herpesvirus: Formation of Colonies and Plaques with Mixed Lytic and Latent Gene Expression in Infected Primary Dermal Microvascular Endothelial Cell Cultures

ABSTRACT Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.