Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains.

High occurrence of hepatitis E virus in samples from wastewater treatment plants in Switzerland and comparison with other enteric viruses.

Hepatitis E virus (HEV) is responsible for many enterically transmitted viral hepatitides around the world. It is currently one of the waterborne diseases of global concern. In industrialized countries, HEV appears to be more common than previously thought, even if it is rarely virulent. In Switzerland, seroprevalence studies revealed that HEV is endemic, but no information was available on its environmental spread. The aim of this study was to investigate -using qPCR- the occurrence and concentration of HEV and three other viruses (norovirus genogroup II, human adenovirus-40 and porcine adenovirus) in influents and effluents of 31 wastewater treatment plants (WWTPs) in Switzerland. Low concentrations of HEV were detected in 40 out of 124 WWTP influent samples, showing that HEV is commonly present in this region. The frequency of HEV occurrence was higher in summer than in winter. No HEV was detected in WWTP effluent samples, which indicates a low risk of environmental contamination. HEV occurrence and concentrations were lower than those of norovirus and adenovirus. The autochthonous HEV genotype 3 was found in all positive samples, but a strain of the non-endemic and highly pathogenic HEV genotype I was isolated in one sample, highlighting the possibility of environmental circulation of this genotype. A porcine fecal marker (porcine adenovirus) was not detected in HEV positive samples, indicating that swine are not the direct source of HEV present in wastewater. Further investigations will be necessary to determine the reservoirs and the routes of dissemination of HEV.

New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses.

Rhinoviruses and enteroviruses are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus evolution, we screened persons with respiratory illnesses by using rhinovirus-specific or generic real-time PCR assays. We then sequenced the 5 untranslated region, capsid protein VP1, and protease precursor 3CD regions of virus-positive samples. Subsequent phylogenetic analysis identified the large genotypic diversity of rhinoviruses circulating in humans. We identified and completed the genome sequence of a new enterovirus genotype associated with respiratory symptoms and acute otitis media, confirming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identified recombinants among circulating rhinoviruses and mapped their recombination sites, thereby demonstrating that rhinoviruses can recombine in their natural host. This study clarifies the diversity and explains the reasons for evolution of these viruses.