Evaluation of Aedes aegypti,Aedes albopictus,and Culex quinquefasciatus Mosquitoes Competence to Oropouche virus Infection

The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.     

Intracellular localisation of Mycobacterium marinum in mast cells

AIM: To study the bacteriocidal or bacteriostatic role of mast cells during infection with Mycobacterium. METHODS: Mycobacterium marinum (M. marinum) (BAA-535/M strain) was investigated for its ability to grow at a temperature relevant to the mammalian host. Primary mast cells were differentiated from bone marrows of mice, a human mast cell line (HMC-1) and a human monocytic cell line (MonoMac6) were maintained in culture. Mice were stimulated by intraperitoneal injection of heat-killed M. marinum to study cytochemically the degranulation of peritoneal mast cells. HMC-1 cells were stimulated with M. marinum to analyse mRNA expression for inflammatory reactant genes, while HMC-1 and primary mouse mast cells were infected with M. marinum to establish in parallel cell viability (lactate dehydrogenase release and cell counts) and viable mycobacterial counts. Flow cytometry was used to assess intracellular presence of fluorescein isothiocyanate labelled M. marinum after trypan blue quenching and to measure the extent of infection-induced apoptosis or necrosis in HMC-1. A GFP expressing recombinant M. marinum strain was used to assess intracellular location by fluorescence microscopy. Light microscopy of osmium tetroxide and Gram Twort stained sections of 0.5 μm and transmission electron microscopy were undertaken as sensitive methods. RESULTS: Since its isolation, M. marinum has adapted to grow at 37 °C. This study found that M. marinum infects HMC-1 cells and primary murine mast cells, where they survive, replicate, and cause dose dependent cell damage over the analysis period of up to 120 h. Amikacin was an effective aminoglycoside antibiotic to eliminate extracellular or membrane attached M. marinum in order to adequately quantify the intracellular bacterial loads. In vivo, intraperitoneal injection of heat-killed M. marinum led to the release of mast cell granules in mice. HMC-1 cells stimulated with M. marinum showed a biphasic pattern of increased mRNA expression for LL-37 and COX-2/TNF-α during 24 h of stimulation. In HMC-1, M. marinum localised to the cytoplasm whereas in primary mast cells, M. marinum were found in vacuoles. CONCLUSION: The effector role of mast cells in infection with M. marinum can be studied in vitro and in vivo.