WBT Sample Processing Methods Comparison

Another innovative aspect of our proposed sampling strategy includes a comparison between two virus concentration technologies. Concentration methods are critical for detecting viruses from environmental samples, as low viral particle loads in large volumes of wastewater require effective viral concentration strategies (11, 12, 45). SARS-CoV-2 is a non-segmented, positive-sense RNA virus ranging in size from 60 to 140 nm in diameter (14). It is also an enveloped coronavirus. The fact that it is enveloped is of significance in terms of sample concentration, as most methods for processing samples have been developed for viruses transmitted via fecal-oral routes, which tend to be non-enveloped and generally more persistent in the environment (66, 49). In terms of SARS-CoV-2, although enveloped, the outer membrane and nucleocapsid are one of the hardest protective layers among the coronaviruses (17), suggesting that capture of the virus is possible through intact membranes, in which case size-exclusion methods may be based upon the smallest size of the intact capsid (60 nm). Detection of SARS-CoV-2 in wastewater using traditional detection technologies (e.g., qRT-PCR) is often based upon measuring the nucleocapsid’s (N) protein, which has a size of 114 kDa (67) if cross-linked to its dimer. If the virus is no longer intact, size exclusion technologies that go to a much finer resolution (<100 kDa) would be necessary to capture the viral signal.

Sample concentration methods can be generally split into three categories: (1) virus adsorption-elution, (2) size exclusion using ultrafiltration, and (3) two-phase separation/partition precipitation. We will evaluate the first two of these technologies. Our focus is on technologies capable of capturing multiple classes of disease agents, whether they be protozoa, bacteria, or viruses. Among the categories of sample concentration, two are particularly adapted for capturing different classes of disease agents: (1) ultrafiltration, which is a method of size exclusion based upon very small molecular cutoff size filters (100k Da or less), and (2) virus adsorption-elution methodologies, among which electronegative membrane filtration has been the most widely studied and adopted for environmental measurements. Virus adsorption-elution can capture multiple classes of microbes through size exclusion of bacteria-sized microbes coupled with charge attraction of these same filters due to their electronegative charge. Viruses in water are positively charged through the addition of salts (e.g., MgCl2) and acidification of samples. The negative charge filters then attract the positively charged viruses in the aqueous phase through electrostatic interactions. Viruses on particles and larger-sized microbes are captured by size exclusion on the flat surface filters. A larger filter pore size (0.45 µm) allows for the passage of water even when the water is turbid. Such approaches for simultaneous capture of microbes from water using combinations of size-exclusion coupled with charge have been proposed by Bonilla et al. (9) and others (1,2). To date, only one comprehensive study (5) evaluated the efficiency of different sample concentration methods for enveloped viruses in wastewater (36). In the Ahmed et al. (5) study, raw wastewater samples were spiked with enveloped murine hepatitis virus and evaluated using both electronegative and ultrafiltration sample concentration methods. Results showed the highest recoveries using the electronegative filtration methods (preconditioning through the addition of MgCl2 only) and no prefiltration nor pre-centrifugation step simultaneously capturing virus from the solid phase through size exclusion and aqueous phase through charge attraction.

However, additional work is needed to further evaluate concentrations using background levels of SARS-CoV-2 in wastewater coupled with spiking experiments using model viruses. An innovative aspect of our proposed sample concentration approaches will be in confirming the recoveries of SARS-CoV-2 and model viruses via electronegative filtration and ultrafiltration. Model enveloped viruses will be used for spiking experiments (e.g., synthetic lentivirus and/or human heat-inactivated SARS-CoV-2) under BSL-2 biosafety conditions. Such data would complement the work of Ahmed et al. (5) to further confirm expected sample recoveries using ultrafiltration and/or electronegative filtration, a critical step in sample processing for virus detection and quantification.