Farkas, K. | Search bioRxiv

bioRxiv2018

Two-step concentration of complex water samples for the detection of viruses

The accurate detection and quantification of pathogenic viruses in water is essential to understand and reduce the risk of human infection. In this paper, we validated a two-step method for the concentration of enteric viruses in water and wastewater samples. The method involves a tangential flow ultrafiltration step that reduces the sample volume of 1 - 10 L to approx. 50 mL, followed by secondary precipitation using polyethylene glycol 6000 that reduces the volume to 1 - 4 mL. For method validation, surface water samples were spiked with different concentrations of enteric viruses and viral recoveries in the concentrates exceeded 10% in all experiments. The method is suitable for water samples with high and low salinity and turbidity, allowing the accurate comparison of viral titers in a diverse range of water types. Furthermore, the method has the potential to concentrate other pathogens, e.g. bacteria or protozoa. Hence, the use of this method can improve the holistic assessment of risks associated with wastewater contaminated environments.\n\nO_LIModified tangential flow ultrafiltration method that reduces membrane clogging.\nC_LIO_LISecondary concentration with a beef extract-elution step to remove solid matter.\nC_LIO_LIThe two-step concentration method enables at least 10% recovery for different enteric viruses in difficult matrices.\nC_LIO_LIUsing the two-step concentration method 10,000x concentration can be achieved.\nC_LI\n\nSpecifications Table:\n\n\n\nO_TBL View this table:\norg.highwire.dtl.DTLVardef@12d4fceorg.highwire.dtl.DTLVardef@1d2880borg.highwire.dtl.DTLVardef@e1acf6org.highwire.dtl.DTLVardef@226492org.highwire.dtl.DTLVardef@1f8edf0_HPS_FORMAT_FIGEXP M_TBL C_TBL

microbiology↗

bioRxiv2018

Viromic analysis of wastewater input to a river catchment reveals a diverse assemblage of RNA viruses

Detection of viruses in the environment is heavily dependent on PCR-based approaches that require reference sequences for primer design. While this strategy can accurately detect known viruses, it will not find novel genotypes, nor emerging and invasive viral species. In this study, we investigated the use of viromics, i.e. high-throughput sequencing of the biosphere viral fraction, to detect human/animal pathogenic RNA viruses in the Conwy river catchment area in Wales, UK. Using a combination of filtering and nuclease treatment, we extracted the viral fraction from wastewater, estuarine river water and sediment, followed by RNASeq analysis on the Illumina HiSeq platform for the discovery of RNA virus genomes. We found a higher richness of RNA viruses in wastewater samples than in river water and sediment, and assembled a complete norovirus GI.2 genome from wastewater effluent, which was not contemporaneously detected by conventional qRT-PCR. To our knowledge, this is the first environmentally-derived norovirus genome sequence to be available from a public database. The simultaneous presence of diverse rotavirus signatures in wastewater indicated the potential for zoonotic infections in the area and suggested run-off from pig farms as a possible origin of these viruses. Our results show that viromics can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided appropriate and rigorous controls are included.\n\nImportanceEnteric viruses cause gastro-intestinal illness and are commonly transmitted through the faecal-oral route. When wastewater is released into river systems, these viruses can contaminate the environment. Our results show that we can use viromics to find the range of potentially pathogenic viruses that are present in the environment and identify prevalent genotypes. The ultimate goal is to trace the fate of these pathogenic viruses from origin to the point where they are a threat to human health, informing reference-based detection methods and water quality management.

microbiology↗