Viral sewage metagenomics

Consider the novel field of viral sewage metagenomics that can be used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency for enteric, respiratory and oncogenic viruses. The unbiased approach of Next Generation Sequencing (NGS) is better than traditional methods because of high sewage viral diversity and the continuous mutation of viral species.

The article entitled "Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing" by Hjelms? et al. highlights the 3 main challenges for viral sewage metagenomics and demonstrated that detection of viral pathogens depends on the method used for concentration and extraction of viral nucleic acids.

The efficiency to determine the viriome in sewage, of 4 commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and 4 extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS? miniMAG?, or PowerViral? Environmental RNA/DNA Isolation Kit) was evaluated.

Aspects studied included viral community composition, viral selectiveness, viral richness, viral pathogen detection, and viral contaminants. Extracted nucleotides were amplified with PCR and sequenced using the Illumina MiSeq platform.

Results from the Nucleospin RNA XS extraction kit were remarkably different from samples extracted with the three other kits attributed to the “on column DNase step” degrading the genomes of DNA viruses and the species rich bacteriophages. Personally, I think they should have used NucleoMag pathogen or NucleoMag DNA/RNA Water kit which are more appropriate for both DNA and RNA isolation from pathogens.

Nonetheless and probably most importantly, the Nucleospin RNA XS extraction kit had a consistent high viral specificity (3x that of PowerViral? Environmental RNA/DNA Isolation Kit and QIAamp Viral RNA Mini Kit). Public health important enteric RNA viruses as norovirus, rotavirus, and Hepatitis A and E virus were best detected when using the Nucleospin RNA XS extraction kit compared to the other tested extraction kits, especially in combination with organic flocculation with skim milk as concentration method. Noteworthy is that skim milk flocculation was also shown to be the method with the highest inhibition of qPCR.

Caution should be used when choosing concentration methods because some introduce inhibitors that can affect PCR amplification, quality of the prepared library, and subsequent virus detection.

“This theory was further supported by the qPCR results where extracts obtained from SMF had a high level of inhibition. Extraction with QIA has previously been shown to impair detection of HAdV in samples with high levels of organic matter. This was also the case in our study, where extraction with QIA inhibited HAdV detection in all cases except when combined with PEG concentration which both had the lowest starting volume (0.2 L) and an additional filtration step.”

The full article is available on open access here.