Spanish wastewater reveals the current spread of Monkeypox virus.

Besides nasopharyngeal swabs, monkeypox virus (MPXV) DNA has been detected in a variety of samples such as saliva, semen, urine and fecal samples. Using the environmental surveillance network previously developed in Spain for the routine wastewater surveillance of SARS-CoV-2 (VATar COVID-19), we have analyzed the presence of MPXV DNA in wastewater from different areas of Spain. Samples (n = 312) from 24 different wastewater treatment plants were obtained between May 9 (week 19 of 2022) and August 4 (week 31 of 2022). Following concentration of viral particles by a validated aluminum adsorption-precipitation method, a qPCR procedure allowed us to detect MPXV DNA in 56 wastewater samples collected from May 16 to August 4, 2022, with values ranging between 2.2 × 103 to 8.7 × 104 genome copies (gc)/L. This study shows that MPXV DNA can be reproducibly detected by qPCR in longitudinal samples collected from different Spanish wastewater treatment plants. According to data from the National Epidemiological Surveillance Network (RENAVE) in Spain a total of 6,119 cases have been confirmed as of August 19, 2022. However, and based on the wastewater data, the reported clinical cases seem to be underestimated and asymptomatic infections may be more frequent than expected.

Evaluation of two different concentration methods for surveillance of human viruses in sewage and their effects on SARS-CoV-2 sequencing.

During the current COVID-19 pandemic, wastewater-based epidemiology (WBE) emerged as a reliable strategy both as a surveillance method and a way to provide an overview of the SARS-CoV-2 variants circulating among the population. Our objective was to compare two different concentration methods, a well-established aluminum-based procedure (AP) and the commercially available Maxwell® RSC Enviro Wastewater TNA Kit (TNA) for human enteric virus, viral indicators and SARS-CoV-2 surveillance. Additionally, both concentration methods were analyzed for their impact on viral infectivity, and nucleic acids obtained from each method were also evaluated by massive sequencing for SARS-CoV-2. The percentage of SARS-CoV-2 positive samples using the AP method accounted to 100 %, 83.3 %, and 33.3 % depending on the target region while 100 % positivity for these same three target regions was reported using the TNA procedure. The concentrations of norovirus GI, norovirus GII and HEV using the TNA method were significantly greater than for the AP method while no differences were reported for rotavirus, astrovirus, crAssphage and PMMoV. Furthermore, TNA kit in combination with the Artic v4 primer scheme yields the best SARS-CoV-2 sequencing results. Regarding impact on infectivity, the concentration method used by the TNA kit showed near-complete lysis of viruses. Our results suggest that although the performance of the TNA kit was higher than that of the aluminum procedure, both methods are suitable for the analysis of enveloped and non-enveloped viruses in wastewater by molecular methods.

Monitoring the evolution of SARS-CoV-2 on a Spanish university campus through wastewater analysis: A pilot project for the reopening strategy.

Wastewater surveillance is a fast and cost-effective tool that enables tracing of both symptomatic and asymptomatic transmission of SARS-CoV-2. In this paper, a pilot program carried out at the University Jaume I for monitoring the trends of the presence of SARS-CoV-2 in wastewater. To the best of our knowledge, this is the first such project conducted on a university campus in Spain. Wastewater samples (n = 838) were collected when students returned to campus, from October 2020 until August 2021, at a confluence sewer point and at the building level including different academic departments and services, the library, administration offices and the university student residence. It has been observed that the probability of SARS-CoV-2 RNA detection in wastewater depended on COVID-19 incidence on campus and visitors/occupants of the buildings i.e., high-, or low-traffic buildings with high or low frequency of potential contacts. Moreover, the third wave in Spain (after Christmas 2020) and an outbreak that occurred at the university student's residence could be carefully followed, allowing confirmation of the end of the outbreak. In addition, viral variants (i.e., mutations and linages) from selected time points were detected by sequencing and gave an indication of the evolution of the virus over time. The results illustrate the potential of wastewater-based epidemiology to provide an early warning for SARS-CoV-2 within the university, especially in buildings with low traffic and more defined populations, like the student residence. The strategy and experience gathered in this study will allow for implementation of improvements for reliable monitoring in the future.

The International Virus Bioinformatics Meeting 2022.

The International Virus Bioinformatics Meeting 2022 took place online, on 23-25 March 2022, and has attracted about 380 participants from all over the world. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The participants created a highly interactive scientific environment even without physical face-to-face interactions. This meeting is a focal point to gain an insight into the state-of-the-art of the virus bioinformatics research landscape and to interact with researchers in the forefront as well as aspiring young scientists. The meeting featured eight invited and 18 contributed talks in eight sessions on three days, as well as 52 posters, which were presented during three virtual poster sessions. The main topics were: SARS-CoV-2, viral emergence and surveillance, virus-host interactions, viral sequence analysis, virus identification and annotation, phages, and viral diversity. This report summarizes the main research findings and highlights presented at the meeting.

Monitoring Human Viral Pathogens Reveals Potential Hazard for Treated Wastewater Discharge or Reuse

Wastewater discharge to the environment or its reuse after sanitization poses a concern for public health given the risk of transmission of human viral diseases. However, estimating the viral infectivity along the wastewater cycle presents technical challenges and still remains underexplored. Recently, human-associated crAssphage has been investigated to serve as viral pathogen indicator to monitor fecal impacted water bodies, even though its assessment as biomarker for infectious enteric viruses has not been explored yet. To this end, the occurrence of potentially infectious norovirus genogroup I (GI), norovirus GII, hepatitis A virus (HAV), rotavirus A (RV), and human astrovirus (HAstV) along with crAssphage was investigated in influent and effluent water sampled in four wastewater treatment plants (WWTPs) over 1 year by a PMAxx-based capsid integrity RT-qPCR assay. Moreover, influent and effluent samples of a selected WWTP were additionally assayed by an in situ capture RT-qPCR assay (ISC-RT-qPCR) as estimate for viral infectivity in alternative to PMAxx-RT-qPCR. Overall, our results showed lower viral occurrence and concentration assessed by ISC-RT-qPCR than PMAxx-RT-qPCR. Occurrence of potentially infectious enteric virus was estimated by PMAxx-RT-qPCR as 88–94% in influent and 46–67% in effluent wastewaters with mean titers ranging from 4.77 to 5.89, and from 3.86 to 4.97 log10 GC/L, with the exception of HAV that was sporadically detected. All samples tested positive for crAssphage at concentration ranging from 7.41 to 9.99 log10 GC/L in influent and from 4.56 to 6.96 log10 GC/L in effluent wastewater, showing higher mean concentration than targeted enteric viruses. Data obtained by PMAxx-RT-qPCR showed that crAssphage strongly correlated with norovirus GII (ρ = 0.67, p < 0.05) and weakly with HAstV and RV (ρ = 0.25–0.30, p < 0.05) in influent samples. In effluent wastewater, weak (ρ = 0.27–0.38, p < 0.05) to moderate (ρ = 0.47–0.48, p < 0.05) correlations between crAssphage and targeted viruses were observed. Overall, these results corroborate crAssphage as an indicator for fecal contamination in wastewater but a poor marker for either viral occurrence and viral integrity/infectivity. Despite the viral load reductions detected in effluent compared to influent wastewaters, the estimates of viral infectivity based on viability molecular methods might pose a concern for (re)-using of treated water.

Spatial and temporal distribution of SARS-CoV-2 diversity circulating in wastewater.

Wastewater-based epidemiology (WBE) has proven to be an effective tool for epidemiological surveillance of SARS-CoV-2 during the current COVID-19 pandemic. Furthermore, combining WBE together with high-throughput sequencing techniques can be useful for the analysis of SARS-CoV-2 viral diversity present in a given sample. The present study focuses on the genomic analysis of SARS-CoV-2 in 76 sewage samples collected during the three epidemiological waves that occurred in Spain from 14 wastewater treatment plants distributed throughout the country. The results obtained demonstrate that the metagenomic analysis of SARS-CoV-2 in wastewater allows the detection of mutations that define the B.1.1.7 lineage and the ability of the technique to anticipate the detection of certain mutations before they are detected in clinical samples. The study proves the usefulness of sewage sequencing to track Variants of Concern that can complement clinical testing to help in decision-making and in the analysis of the evolution of the pandemic.

Platinum chloride-based viability RT-qPCR for SARS-CoV-2 detection in complex samples.

Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum.

Monitoring Emergence of the SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19).

Since its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2 has become dominant in several countries raising great concern. We developed a duplex real-time RT-qPCR assay to detect, discriminate, and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the ΔHV69/70 deletion, and used it to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). The B.1.1.7 variant was detected earlier than clinical epidemiological reporting by the local authorities, first in the southern city of Málaga (Andalucía) in week 20_52 (year_week), and multiple introductions during Christmas holidays were inferred in different parts of the country. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from wastewater treatment plants, which reached B.1.1.7 prevalences higher than 90% for ≥2 consecutive weeks showed that 8.1 ± 2.0 weeks were required for B.1.1.7 to become dominant. The study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern as soon as they are identified by clinical sequencing and their integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic.

Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR.

The ongoing coronavirus 2019 (COVID-19) pandemic constitutes a concerning global threat to public health and economy. In the midst of this pandemic scenario, the role of environment-to-human COVID-19 spread is still a matter of debate because mixed results have been reported concerning SARS-CoV-2 stability on high-touch surfaces in real-life scenarios. Up to now, no alternative and accessible procedures for cell culture have been applied to evaluate SARS-CoV-2 infectivity on fomites. Several strategies based on viral capsid integrity have latterly been developed using viability markers to selectively remove false-positive qPCR signals resulting from free nucleic acids and damaged viruses. These have finally allowed an estimation of viral infectivity. The present study aims to provide a rapid molecular-based protocol for detection and quantification of viable SARS-CoV-2 from fomites based on the discrimination of non-infectious SARS-CoV-2 particles by platinum chloride (IV) (PtCl4) viability RT-qPCR. An initial assessment compared two different swabbing procedures to recover inactivated SARS-CoV-2 particles from fomites coupled with two RNA extraction methods. Procedures were validated with human (E229) and porcine (PEDV) coronavirus surrogates, and compared with inactivated SARS-CoV-2 suspensions on glass, steel and plastic surfaces. The viability RT-qPCR efficiently removed the PCR amplification signals from heat and gamma-irradiated inactivated SARS-CoV-2 suspensions that had been collected from specified surfaces. This study proposes a rapid viability RT-qPCR that discriminates non-infectious SARS-CoV-2 particles on surfaces thus helping researchers to better understand the risk of contracting COVID-19 through contact with fomites and to develop more efficient epidemiological measures.

Comparing analytical methods to detect SARS-CoV-2 in wastewater.

Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene glycol (PEG) precipitation protocol. Moreover, two different RNA extraction methods were compared in this study: a commercial manual spin column centrifugation kit and an automated protocol based on magnetic silica beads. Overall, the evaluated concentration methods did not impact the recovery of gamma-irradiated SARS-CoV-2 nor MgV, while extraction methods showed significant differences for PEDV. Mean recovery rates of 42.9 ± 9.5%, 27.5 ± 14.3% and 9.0 ± 2.2% were obtained for gamma-irradiated SARS-CoV-2, PEDV and MgV, respectively. Limits of detection (LoD95%) for five genomic SARS-CoV-2 targets (N1, N2, gene E, IP2 and IP4) ranged from 1.56 log genome equivalents (ge)/mL (N1) to 2.22 log ge/mL (IP4) when automated system was used; while values ranging between 2.08 (N1) and 2.34 (E) log ge/mL were observed when using column-based extraction method. Different targets were also evaluated in naturally contaminated wastewater samples with 91.2%, 85.3%, 70.6%, 79.4% and 73.5% positivity, for N1, N2, E, IP2 and IP4, respectively. Our benchmarked comparison study suggests that the aluminum precipitation method coupled with the automated nucleic extraction represents a method of acceptable sensitivity to provide readily results of interest for SARS-CoV-2 WBE surveillance.

Recovering coronavirus from large volumes of water.

The need for monitoring tools to better control the ongoing coronavirus disease (COVID-19) pandemic is extremely urgent and the contamination of water resources by excreted viral particles poses alarming questions to be answered. As a first step to overcome technical limitations in monitoring SARS-CoV-2 along the water cycle, we assessed the analytical performance of a dead end hollow fiber ultrafiltration coupled to different options for secondary concentrations to concentrate viral particles from large volume of spiked tap water, seawater and surface water together with two quantitative RT-qPCR detection kits. Spiking the porcine epidemic diarrhea virus (PEDV), an enveloped virus surrogate for SARS-CoV-2, together with the mengovirus, we demonstrated that PEG-precipitation and SENS-kit better recovered PEDV (13.10 ± 0.66%) from tap water, while centrifugal filtration resulted the best option to recover mengovirus regardless of the detection kit used. No statistical significant differences were found when comparing high (10,000 ×g) and low (3500 ×g) centrifugation speeds for the secondary PEG- based concentration of spiked seawater, while considerable inhibition was observed for both viruses detected by NoInh-kit assay. Similarly, the co-concentration of PCR inhibitors and viral particles was observed in surface waters detected with either SENS-kit or NoInh-kit and RNA dilution was needed to achieve acceptable recoveries at the expenses of the overall sensitivity of the method. These methodologies represent suitable options to investigate SARS-CoV-2 occurrence in different water resources and allow to conduct on site sampling of large volume of water.