QUANTIFICATION OF INFECTIOUS ADENOVIRUSES IN RIVER WATER

International audienceIntroductionAdenoviruses are double-stranded DNA viruses involved in a wide range of illnesses. All adenovirus types are excreted in faeces leading to a potential transmission through ingestion, inhalation or direct contact with contaminated water. To date, a majority of the data on environmental water samples have been obtained with molecular methods only, resulting in the lack of information on the infectious status of these adenoviruses. Integrated cell culture-PCR (ICC-PCR) based on the combination of cell culture with PCR allows detecting infectious adenoviruses more rapidly than conventional cell culture assays and is applicable on environmental water samples (Wyn-jones et al., 2011). In a previous work we showed that ICC-qPCR could be used to detect infectious adenoviruses in wastewater and river water, within two days (Ogorzaly et al., 2013). Moreover, ICC-qPCR could also be used as a quantitative method as suggested by the results obtained with laboratory strains of adenovirus (Li et al., 2009).ObjectivesThe present work aimed to show that ICC-qPCR could be used for the quantification of naturally occurring infectious adenoviruses in surface water. Moreover a response period below two days was expected.MethodsHuman adenovirus type 2 (HAdV-2; HPACC, NCPV#213) propagated in 293A cells (Life Technologies, R705-07) was used for the evaluation of ICC-qPCR as a quantitative method. Over a 3-month period (January-March 2016), 10 surface water samples were then collected in the Meurthe River in the urban area of Nancy located in the north-east of France. The adenoviruses naturally present in the water samples (20 L) were concentrated with the glass wool method (Wyn-Jones et al., 2011). Samples positive for adenovirus genome were then analysed by ICC-qPCR. For this purpose, the standard curve obtained for HAdV-2 was analysed simultaneously to each river water sample. The infectivity titre of adenovirus in river water estimated by ICC-qPCR was expressed as equivalent to the most probable number of cytopathic unit per litre (eqMPNCU/L).ResultsFirst, we generated a standard curve corresponding to the quantity of DNA detected in cells 24 h post-infection in function of the quantity of infectious adenovirus type 2 inoculated on cells. A linear response was observed from 1 x 105 to 10 eqMPNCU/reaction and a sensitivity of 10 eqMPNCU/L was expected on river water samples.Second, the river samples were analysed by qPCR and ICC-qPCR methods. Seven river water samples (n=10) were positive for adenovirus genome resulting in a mean concentration of 2.5 x 103 ± 2.74 x 103 genome copies/L. Among these samples, infectious adenoviruses were detected by ICC-qPCR in five samples resulting in a mean concentration of 1.67 x 101 ± 1.05 x 101 eqMPNCU/L. Globally, infectious adenoviruses represented 0.2 % to 2.1 % of the total adenoviruses detected by qPCR.ConclusionsICC-qPCR can be used as a quantitative method. For this purpose, a standard adenovirus having known titre is used as a reference and adenovirus DNA is quantified 24 h post-infection. Moreover, ICC-qPCR allows the quantification of infectious adenoviruses naturally present in river water. This method could improve notably future monitoring studies on infectious adenoviruses in the environment