Field Evaluation of Improved Methods to Detect Infectious Group F Adenoviruses in Source Water

Adenoviruses (Ads) are non-enveloped respiratory and enteric viruses containing a dsDNA genome. Group F ads 40 and 41 are of particular interest due to their persistence and abundance in ambient waters. Therefore, it is important to develop, verify, and apply a sensitive, specific, and efficient method to detect and quantify infectious adenoviruses in environmental samples of water and sewage. In this study the proposed method for infectivity assay is a combination of cell culture and mRNA reverse transcription (RT)-PCR for the viral hexon gene. In addition, the use of a housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), as an internal control for mRNA expression was used to evaluate the performance of this method. Source water samples of 20 L were collected three times from six water treatment plants (WTP) and concentrated to small volumes suitable for molecular analysis by hollow fiber ultrafiltration (HFUF) for primary concentration and polyethylene glycol (PEG) precipitation, chloroform extraction, and ultracentrifugation for secondary concentration. Because ads 40 and 41 are difficult to culture, newly developed transactivated 293 cell lines (293 CMV and 293 RAS) were compared to the STDG293 cell line to examine the levels of detectable viral mRNA expression and the incubation time required. The overall results indicated that infectious ads can be successfully detected from environmental source water and sewage samples using the new transactivated and standard cell lines. The housekeeping gene, GAPDH, as a positive control for the performance of CC/mRNA RT-PCR was found to be effective.Master of Science in Public Healt

Whole Genome Sequencing of Enteroviruses Species A to D by High-Throughput Sequencing: Application for Viral Mixtures

Human enteroviruses (EV) consist of more than 100 serotypes classified within four species for enteroviruses (EV-A to -D) and three species for rhinoviruses, which have been implicated in a variety of human illnesses. Being able to simultaneously amplify the whole genome and identify enteroviruses in samples is important for studying the viral diversity in different geographical regions and populations. It also provides knowledge about the evolution of these viruses. Therefore, we developed a rapid, sensitive method to detect and genetically classify all human enteroviruses in mixtures. Strains of EV-A (15), EV-B (40), EV-C (20), and EV-D (2) viruses were used in addition to 20 supernatants from RD cells infected with stool extracts or sewage concentrates. Two overlapping fragments were produced using a newly designed degenerated primer targeting the conserved CRE region for enteroviruses A-D and one degenerated primer set designed to specifically target the conserved region for each enterovirus species (EV-A to -D). This method was capable of sequencing the full genome for all viruses except two, for which nearly 90% of the genome was sequenced. This method also demonstrated the ability to discriminate, in both spiked and unspiked mixtures, the different enterovirus types present