The samples taken in the field will be subject to laboratory investigations for:
- The Identification and quantification of nucleic acids of noroviruses, hepatitis A and E viruses, human adenoviruses and sapoviruses (herein referred to as ‘the target viruses’)
- The elution of intact and infectious viral particles.
- The enrichment of potentially harmful viruses for further analysis.
The aims and approaches are summarised below:
Optimization of viral recovery from environmental samples
We aim to evaluate and optimise standard methods for the recovery of target viruses from wastewater effluent and shellfish as well as water and sediment from the fresh, estuarine and marine environment.
Although previous studies have focused on the extraction of specific members of our target viruses from a subset of the environmental matrices described here, there are combinations of viral target vs. environmental matrix that have not previously been addressed.
We will compare the efficiency of traditional extraction processes and compare outcomes with novel approaches. For sensitive quantification, quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR) will be used, targeting a representative sequence of the genomes of target viruses.
Sample preparation for metagenomics analysis
Metagenomics is defined as “the direct genetic analysis of genomes contained within an environmental sample”. Viral genomes consist of either double stranded (ds) or single stranded (ss) RNA or DNA molecules and genomic libraries for both can be produced from each environmental sample. To produce a paired end library for sequencing:
- The complement for each harvested DNA strand will be synthesised to produce dsDNA from ssDNA viruses, whilst doubling the numbers of dsDNA molecules.
- Viral RNA will be converted to DNA by reverse transcription and then made into ds complimentary DNA (cDNA).
A DNA and an RNA library will be produced from each environmental sample and barcoded samples sequenced in pools, which will enable the generation of sequence datasets in numbers and depths of coverage that have only recently become possible.
Exploring viral infectivity in environmental samples
Current viral diagnostic methods rely on genome detection by PCR as most enteric viruses cannot be routinely cultured. A key question is whether the viral genomes detected by PCR originate from viable viruses capable of human infection. Virus inactivation by environmental stressors act via the disruption of the viral genome and/or capsid protein.
In the absence of culture, the available options are to attempt to determine the integrity of virus genome, and/or capsid, as a marker for infectivity, to supplement PCR. An alternative is to use a surrogate cultivatable virus, as an index for infectivity. Our approach is to establish possible methods for determining virus integrity (both genome and capsid approaches) and to evaluate these using laboratory infectivity models. FRNA bacteriophage, a candidate surrogate, will be investigated in parallel and performance compared with the infectivity models.