Adsorption of norovirus and ostreid herpesvirus type 1 to polymer membranes for the development of passive samplers.

AIMS: This study was performed to develop a passive sampling methodology for the detection of two viruses in seawater in the area of shellfish production, the norovirus (NoV), a human pathogen implicated in gastroenteritis outbreaks linked to oyster consumption and the ostreid herpesvirus type 1 (OsHV-1), a virus associated with mass mortalities of Pacific oysters. METHODS AND RESULTS: Commercially, membranes were tested for their capacity to adsorb virus: zetapor, gauze, nylon, low-density polyethylene (LDPE) and polyvinylidene difluoride (PVDF). Laboratory exposures of membranes to contaminated water samples (stool, sewage, seawater) were performed. Our data show that the amount of NoV GII genome per membrane measured with qRT-PCR increased with the time of exposure up to 24 h, for all types of membranes except gauze. After 15 days of exposure, the amount of NoV GII per membrane continued to increase only for nylon and LDPE. The amount of OsHV-1 per zetapor membrane was significantly increased as soon as 4 h of exposure, and after 24 h of exposure for all types of membranes. Exposure of membranes to serial dilutions of various samples revealed that the amount of NoV GII and OsHV-1 per membrane is significantly higher in diluted samples. The detection of NoV and OsHV-1, respectively, with zetapor and PVDF membranes was found to be more efficient than the direct analysis of sewage and seawater. CONCLUSIONS: All membranes immersed in contaminated samples adsorbed NoV GII and OsHV-1. The amount of both viruses increased with the time of exposure. Zetapor and PVDF membranes seem to be more adapted to NoV GII and OsHV-1 detection respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Membranes tested will be used as passive samplers to improve the detection of virus in oyster production areas. Also, passive samplers could be a valuable tool for microbiome analysis with new generation sequencing.

Lessons from a transplantation of zebra mussels into a small urban river: An integrated ecotoxicological assessment.

It is often difficult to evaluate the level of contamination in small urban rivers because pollution is mainly diffuse, with low levels of numerous substances. The use of a coupled approach using both chemical and biological measurements may provide an integrated evaluation of the impact of micro-pollution on the river. Zebra mussels were transplanted along a metal and organic pollution gradient in spring 2008. For two months, mussels and water samples were collected from two sites every two weeks and analyzed for metal and PAH content as well as water physicochemical parameters. Diffusive gradients in thin film (DGT) were also used to assess levels of labile metals. Exposure of mussels to contaminants and potential impact were evaluated using physiological indices and various biomarkers including condition index (CI), defense mechanisms (glutathione-S-transferase: GST), digestive enzymes (amylase and cellulase) and genotoxicity (micronucleus test: MN and comet assay: CA). For most contaminants, the water contamination was significantly higher downstream. Bioaccumulation in zebra mussels was related to water contamination in the framework of the biodynamic model, which allowed us to take into account the biological dilution that was caused by the growth of soft tissue downstream. Thus, metal influxes were on average two times higher downstream than upstream in particular for Zn, Cr, Cu and Cd. Significant differences in condition index were observed (final CI was 0.42 ± 0.03 downstream and 0.31 ± 0.03 upstream) reflecting a better food availability downstream. Moreover a significant decrease of GST activity and digestive enzymes activity in the cristalline style was observed downstream. Interpreting this decrease requires considering not only micro-pollution but also the trophic status related to the water's physicochemistry. The MN test and the CA on gill cells highlighted genotoxicity in mussels transplanted downstream compared to upstream.

Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds.

An in situ study of the relationship between marine contamination and genotoxic effects was performed on female dab (Limanda limanda) collected from different sites in the eastern English Channel (France) known to be contaminated by polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs). DNA adducts in liver and DNA strand breaks in blood cells were determined respectively by the nuclease P1-enhanced post-labelling technique and an alkaline version of the comet assay. The extent of DNA base oxidation was also assessed for three of the six sampling sites in the study, using a comet assay in combination with a specific DNA repair enzyme, formamidopyrimidine glycosylase (Fpg).With Comet data, two groups of sites that seem in accordance with the pollution level have been distinguished. The extent of DNA strand breaks was higher in adult than juvenile female dab. From a technical point of view, comet assay sensitivity was affected by high intra-individual variability that accounted for nearly 70% of total variance (the site factor represented no more than 26%). The combined use of the comet assay and Fpg showed the presence of DNA oxidised bases in environmentally exposed dab.Although qualitative differences between the sampling sites were observed in DNA adduct profiles, no significant differences were found for total DNA adduct levels. DNA adducts did not appear to be associated with PAH exposure. Histopathological studies showed hepatic steatosis in most of the animals examined. Only one pre-cancerous lesion (an early stage of hyperplasia) was detected (associated frequency of 0.8%).

cDNA cloning and expression of two Ki-ras genes in the flounder, Platichthys flesus, and analysis of hepatic neoplasms.

The screening of a flounder cDNA library with a partial sequence of ras gene from flounder (exons 1 and 2) allowed the isolation of two complete cDNA sequences (ras1 and ras2) highly homologous to human Ki-rasb genes. ras1 and ras2 sequences have an homology of 77.3% indicating that they represent two distinct genes, which differ particularly in their 3 regions. ras1 and ras2 intron 1 sequencing revealed an homology of only 50%, confirming that they represent two different genes. Both genes encode for a 188 amino-acid protein, a size characteristic of Ki-rasb proteins. ras1 protein has the stronger homology to the human Ki-rasb protein (99% identity) and ras2 presents a 85.5% of homology. Two transcripts of respectively 2 and 2.8 kb were identified by northern blots with either ras1 or 2 probes. Preneoplastic and neoplastic livers collected from 14 flounder did not present any mutation on the ras2 gene.