Ethanol inactivation of orthonairoviruses in ixodid ticks.

Ixodid ticks represent vectors and reservoirs for a broad range of zoonotic pathogens. Collected ticks from field studies are therefore usually stored in ethanol, which in higher concentrations effectively inactivates most of the known tick-borne pathogens. Although commonly practiced as gold standard for inactivation, hardly any scientific data demonstrate that ethanol sufficiently penetrates the comparatively thick cuticula of ticks. Therefore, Amblyomma hebraeum tick pools were stored for 21 days in ethanol (96%). Afterwards, the ethanol was removed and the ticks were homogenized. Quantitative 1H-NMR spectroscopic analysis was applied to determine the residual concentration of ethanol inside the ticks. 1H-NMR spectroscopic analysis revealed that ethanol constituted 28.3-42.6 mg of the total weight of three ticks in the pools (89.9-121.5 mg). In addition, the low-pathogenic Hazara orthonairovirus (HAZV) was used as a cell culture model for this study. The virus was exposed to ethanol concentrations between 0 and 60% and incubated under various temperature conditions for four time periods. Afterwards, the residual virus infectivity was determined by titration. Following ethanol exposure, HAZV did not grow in cells after 9 h of exposure to an ethanol concentration of 25%. These results demonstrate an extremely low ethanol resistance of the virus, which was generally in line with previously reported ethanol inactivation data for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). After prolonged storage and impregnation, comparable ethanol concentrations are achieved in the ticks, indicating the suitability of this inactivation method also for Bunyaviruses in ticks. At the very least, a massive virus inactivation can be assumed. Definitive proof of virus inactivation would require a bioassay of ethanol-treated infected ticks under appropriate biosafety conditions.

Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA-a pilot study to standardize techniques on which to base a detailed epidemiological survey.

A total of 356 clinical isolates of the encapsulated basidiomycetous fungus Cryptococcus neoformans var. neoformans, obtained from Australia, Argentina, Brazil, India, Italy, New Zealand, Papua New Guinea, South Africa, Thailand and the USA, were analyzed to lay the basis for a comprehensive evaluation of the global genetic structure of C. neoformans. Two polymerase chain reaction (PCR)-based typing techniques were standardized: PCR fingerprinting using a single primer specific to minisatellite or microsatellite DNA, and randomly amplified polymorphic DNA (RAPD) analysis using two combinations of three 20- to 22-mer random primers. Previous studies showed that the resultant profiles are reproducible and stable over time. Identical results were obtained in two different laboratories and by different scientists in the same laboratory. Both typing techniques separated the isolates into four major groups (VNI and VNII, serotype A; VNIII, serotype A/D; and VNIV, serotype D). The majority (78%) of isolates belonged to VNI, compared with 18% VNII, 1% VNIII and 3% VNIV. All US isolates could be differentiated by a unique, strain-specific PCR fingerprint or RAPD pattern in contrast to most of the non-US isolates, which showed a substantially higher degree of genetic homogeneity, with some clonality, in different parts of the world. Isolates obtained from the same patient at different times and from different body sites, had identical banding patterns. Both typing techniques should provide powerful tools for epidemiological studies of medically important fungi.