Implications of long-term sample storage on the recovery of viruses from wastewater and biobanking.

Wastewater-based monitoring has been widely implemented worldwide for the tracking of SARS-CoV-2 outbreaks and other viral diseases. In many surveillance programmes, unprocessed and processed wastewater samples are often frozen and stored for long periods of time in case the identification and tracing of an emerging health threat becomes necessary. However, extensive sample bioarchives may be difficult to maintain due to limitations in ultra-freezer capacity and associated cost. Furthermore, the stability of viruses in such samples has not been systematically investigated and hence the usefulness of bioarchives is unknown. In this study, we assessed the stability of SARS-CoV-2, influenza viruses, noroviruses and the faecal indicator virus, crAssphage, in raw wastewater and purified nucleic aacid extracts stored at -80 °C for 6-24 months. We found that the isolated viral RNA and DNA showed little signs of degradation in storage over 8-24 months, whereas extensive decay viral and loss of qPCR signal was observed during the storage of raw unprocessed wastewater. The most stable viruses were noroviruses and crAssphage, followed by SARS-CoV-2 and influenza A virus. Based on our findings, we conclude that bioarchives comprised of nucleic acid extracts derived from concentrated wastewater samples may be archived long-term, for at least two years, whereas raw wastewater samples may be discarded after one year.

Environmental Stability of Enveloped Viruses Is Impacted by Initial Volume and Evaporation Kinetics of Droplets.

Efficient spread of respiratory viruses requires the virus to maintain infectivity in the environment. Environmental stability of viruses can be influenced by many factors, including temperature and humidity. Our study measured the impact of initial droplet volume (50, 5, and 1 µL) and relative humidity (RH; 40%, 65%, and 85%) on the stability of influenza A virus, bacteriophage Phi6 (a common surrogate for enveloped viruses), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under a limited set of conditions. Our data suggest that the drying time required for the droplets to reach quasi-equilibrium (i.e., a plateau in mass) varied with RH and initial droplet volume. The macroscale physical characteristics of the droplets at quasi-equilibrium varied with RH but not with the initial droplet volume. Virus decay rates differed between the wet phase, while the droplets were still evaporating, and the dry phase. For Phi6, decay was faster in the wet phase than in the dry phase under most conditions. For H1N1pdm09, decay rates between the two phases were distinct and initial droplet volume had an effect on virus viability within 2 h. Importantly, we observed differences in virus decay characteristics by droplet size and virus. In general, influenza virus and SARS-CoV-2 decayed similarly, whereas Phi6 decayed more rapidly under certain conditions. Overall, this study suggests that virus decay in media is related to the extent of droplet evaporation, which is controlled by RH. Importantly, accurate assessment of transmission risk requires the use of physiologically relevant droplet volumes and careful consideration of the use of surrogates. IMPORTANCE During the COVID-19 pandemic, policy decisions were being driven by virus stability experiments with SARS-CoV-2 in different droplet volumes under various humidity conditions. Our study, the first of its kind, provides a model for the decay of multiple enveloped RNA viruses in cell culture medium deposited in 50-, 5-, and 1-µL droplets at 40%, 65%, and 85% RH over time. The results of our study indicate that determination of half-lives for emerging pathogens in large droplets may overestimate transmission risk for contaminated surfaces, as observed during the COVID-19 pandemic. Our study implicates the need for the use of physiologically relevant droplet sizes with use of relevant surrogates in addition to what is already known about the importance of physiologically relevant media for risk assessment of future emerging pathogens.

Interface behavior and removal mechanisms of human pathogenic viruses in anaerobic membrane bioreactor (AnMBR).

Effective removal of human pathogenic viruses is an indispensable yet rarely studied aspect for sustainable treatment of domestic wastewater by anaerobic membrane bioreactor (AnMBR). In this study, the interface behaviors and removal mechanisms of norovirus genogroup I (GI), genogroup II (GII), and rotavirus A from domestic wastewater was systematically investigated in a one-stage AnMBR. On average, norovirus GI, GII and rotavirus were reduced by 4.64, 5.00, and 2.31 logs, respectively. Viruses tended to be transferred to larger-sized suspended solids from sewage influent to the mixed liquor, and the weight-specific concentration of the virus in >100 µm particles of the mixed liquor was significantly higher than that of sewage, indicating a particle scale-dependent affinity with the virus. In-series membrane filtration test showed the main contribution of the membrane retention, which was dominated by the bio-cake layer and the pristine membrane, while the membrane and associated pore foulants can retain viruses in a filtration resistance-efficient way. An unsteady-state mass balance model revealed that free viruses in the bulk liquid of AnMBR were minimally attached to the cake layer but mainly retained by the membrane and pore foulants (>99%). In addition, despite the small virus decay rates in the mixed liquor, the associated contribution increased with run time due to the prolonged sludge retention time. These insights into virus behaviors and removal mechanisms may provide novel regulation strategies for enhanced virus removal by AnMBR.

Effects of UV Radiation on the Chlorophyte Micromonas polaris Host-Virus Interactions and MpoV-45T Virus Infectivity.

Polar seas are under threat of enhanced UV-radiation as well as increasing shipping activities. Considering the ecological importance of marine viruses, it is timely to study the impact of UV-AB on Arctic phytoplankton host-virus interactions and also test the efficacy of ballast water (BW) UV-C treatment on virus infectivity. This study examined the effects of: (i) ecologically relevant doses of UV-AB radiation on Micromonas polaris RCC2258 and its virus MpoV-45T, and (ii) UV-C radiation (doses 25-800 mJ cm-2) on MpoV-45T and other temperate algal viruses. Total UV-AB exposure was 6, 12, 28 and 48 h (during the light periods, over 72 h total). Strongest reduction in algal growth and photosynthetic efficiency occurred for 28 and 48 h UV-AB treatments, and consequently the virus production rates and burst sizes were reduced by more than half (compared with PAR-only controls). For the shorter UV-AB exposed cultures, negative effects by UV (especially Fv/Fm) were overcome without impacting virus proliferation. To obtain the BW desired log-4 reduction in virus infectivity, a UV-C dose of at least 400 mJ cm-2 was needed for MpoV-45T and the temperate algal viruses. This is higher than the commonly used dose of 300 mJ cm-2 in BW treatment.

Environmental Stability of SARS-CoV-2 on Different Types of Surfaces under Indoor and Seasonal Climate Conditions.

Transmission of severe acute respiratory coronavirus 2 (SARS-CoV-2) mainly occurs through direct contact with an infected person via droplets. A potential role of contaminated surfaces in SARS-CoV-2 transmission has been suggested since the virus has been extensively detected on environmental surfaces. These findings have driven the investigation of virus stability on surfaces under several conditions. However, it remains unclear how long the infectious virus survives on surfaces under different climate conditions, which could play a role in predicting the seasonality of SARS-CoV-2. Therefore, the aim of this study was to estimate the virus stability and its biological half-life on various types of surfaces under indoor and seasonal climate conditions. This study revealed that SARS-CoV-2 survived the longest on surfaces under winter conditions, with a survival post-contamination on most surfaces up to 21 days, followed by spring/fall conditions, with a survival up to 7 days. Infectious virus was isolated up to 4 days post-contamination under indoor conditions, whereas no infectious virus was found at 3 days post-contamination under summer conditions. Our study demonstrates the remarkable persistence of SARS-CoV-2 on many different common surfaces, especially under winter conditions, and raises awareness to the potential risk of contaminated surfaces to spread the virus.