Virus Detection in Sewage

With the appearance of COVID and polio the USA, the news has revealed that it is possible to detect and monitor certain viruses in municipal sewage. As a chemist I marvel at this. Sewage is a frightfully complex mixture of biological waste products along with many chemical cleaning products, detergents, grime and pharmaceuticals that go down the drain. How is it that one can collect enough intact virus particles from this fecal hell broth with enough purity to make a positive identification of genetic material?

A recent methodology is given in an article titled Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks and published in Appl Environ Microbiol. 2014 Nov; 80(21): 6771–6781, DOI: 10.1128/AEM.01981-14 by Maria Hellmér,^aNicklas Paxéus,^bLars Magnius,^cLucica Enache,^bBirgitta Arnholm,^dAnnette Johansson,^bTomas Bergström,^a and Heléne Norder^a,c. As you can see the work is from 2014 so this is not brand-spanking-new technology. It is interesting to note that the material used to sediment the viruses in this article was acidified powdered skim milk proteins. The article was found by a Google search and located at the NIH National Library of Medicine.

Why powdered skim milk? It could be that milk fat interferes with the process or the workers are just removing variables. More likely, it is because the widely available powdered milk that you buy at the grocery store is from skim milk. Dairy fat is too valuable for a business to squander and is used to make more profitable products like ice cream or whipping cream.

A more recent methodology has been reported in PLoS One. 2017; 12(1): e0170199. Published online 2017 Jan 18. doi: 10.1371/journal.pone.0170199. The article, by Mathis Hjort Hjelmsø,^#1,* Maria Hellmér,^#2 Xavier Fernandez-Cassi,³ Natàlia Timoneda,^3,4 Oksana Lukjancenko,¹ Michael Seidel,⁵ Dennis Elsässer,⁵ Frank M. Aarestrup,¹ Charlotta Löfström,^2,¤ Sílvia Bofill-Mas,³ Josep F. Abril,^3,4 Rosina Girones,³ and Anna Charlotte Schultz² and titled Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing. The article cites potential sedimentation substances as Iron(III) Chloride, powdered milk flocculation, PEG, and glass wool filtration. More extraction sources can be found online.

In the 2014 article above, the virus particles are extracted from the raw sewage onto acidified powdered skim milk proteins and amplified with quantitative polymerase chain reaction, qPCR. Powdered milk may seem strange but realize that virus particles can be removed by coagulation with metal ions, lime or with other polyelectrolytes, including proteins. The charge distribution on milk proteins will vary with acidity so these methods are very pH dependent. The viruses are naturally coated in proteins and thus will acquire surface charges varying with pH. The coagulation of proteins occurs when dissolved or suspended proteins irreversibly change their secondary structure by unfolding and condense to form a thicker solution or a solid form. The formation of cheese by acidification or solidifying a runny egg with heat are common examples of coagulation.

A 1973 review article by Gerald Berg in Bull World Health Organ. 1973; 49(5): 451–460, reviews methods for the removal of viruses from effluents, so knowledge of the sedimentation, or coagulation, of viruses in sewage has been around for a long while.

These articles are written by specialists in the field and may present considerable difficulty for a few readers. I would urge those so inclined to try to plow through the articles and pick up what you can. This holds true for all scientific papers. See what you can learn.