Analysis of Cryptococcus Extracellular Vesicles.

Extracellular vesicles (EVs) are produced by all domains of life. In fungal pathogens, they participate in virulence mechanisms and/or induce protective immunity, depending on the pathogenic species. EVs produced by pathogenic members of the Cryptococcus genus mediate virulence, antifungal resistance, as well as humoral and cell-mediated immunity. The isolation of cryptococcal EVs has been laborious and time-consuming for years. In this chapter, we detail a fast protocol for the isolation and analysis of EVs produced by members of the Cryptococcus genus.

Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus

BACKGROUND The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

Genetic and biological characterisation of Zika virus isolates from different Brazilian regions

BACKGROUND Zika virus (ZIKV) infections reported in recent epidemics have been linked to clinical complications that had never been associated with ZIKV before. Adaptive mutations could have contributed to the successful emergence of ZIKV as a global health threat to a nonimmune population. However, the causal relationships between the ZIKV genetic determinants, the pathogenesis and the rapid spread in Latin America and in the Caribbean remain widely unknown. OBJECTIVES The aim of this study was to characterise three ZIKV isolates obtained from patient samples during the 2015/2016 Brazilian epidemics. METHODS The ZIKV genomes of these strains were completely sequenced and in vitro infection kinetics experiments were carried out in cell lines and human primary cells. FINDINGS Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. Infection kinetics experiments were carried out with mammalian cell lines A549, Huh7.5, Vero E6 and human monocyte-derived dendritic cells (mdDCs) and insect cells (Aag2, C6/36 and AP61) and suggest that some of these mutations might be associated with distinct viral fitness. The clinical isolates also presented differences in their infectivity rates when compared to the well-established ZIKV strains (MR766 and PE243), especially in their abilities to infect mammalian cells. MAIN CONCLUSIONS Genomic analysis of three recent ZIKV isolates revealed some nonsynonymous substitutions, which could have an impact on the viral fitness in mammalian and insect cells.

Avaliação da produção de citocinas pelos macrófagos J774. G8 após o tratamento com bioterápicos / Evaluation of cytokine production by J774. G8 macrophages after treatment with biotherapics

Strains of macrophages, such as murine J774.G8 macrophages, are susceptible to influenza A infection [1]. One of the responses to viral infection involves the production of various types of immunostimulatory cytokines by infected cells [2].In all cases, there were no significant differences compared to control groups. However, the production of TNF-? detected in macrophages treated by intact and inactivated biotherapics presented a tendency to increase after infection. In fact, similar results were previously detected in other experiments conducted only with the intact biotherapic [3]. The release of the cytokine MCP1 in all experimental situations presented a tendency to decrease after the viral infection when compared to untreated macrophages. No statistically significant difference was detected in the production of IL 12 and IL 10. These experiments will be repeated to confirm the data obtained.(AU)