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.

Experimental and CFD evaluation of ozone efficacy against coronavirus and enteric virus contamination on public transport surfaces.

The limited information about the routes of the transmission of SARS-CoV-2 within the ongoing pandemic scenario mobilized the administration, industry and academy to develop sanitation and disinfection systems for public and private spaces. Ozone has been proposed as an effective disinfection method against enveloped and non-enveloped viruses, including viruses with similar morphology to SARS-CoV-2. Due to this efficacy, numerous gaseous and aqueous phase ozone applications have emerged potentially to inhibit virus persistence in aerosols, surfaces, and water. In this work, a numerical model, a RANS CFD model for ozone dispersion inside tram and underground coach has been developed including the chemical self-decomposition and surface reactions of the ozone. The CFD model has been developed for a real tram coach of 28.6 × 2.4 × 2.2 m (L × W × H) using 1.76 million nodes and the Menter's shear stress transport turbulence model. The model predicts the O3 concentration needed to meet disinfection criteria and the fluid dynamics inside the public transport coach. The effectiveness of the system has been validated with laboratory and field tests in real full-scale coach using porcine epidemic diarrhea virus (PEDV) and murine norovirus (MNV-1) as SARS-CoV-2 and human norovirus surrogates, respectively. Lab-scale experiments on plastic surfaces demonstrated O3 disinfection (100 ppm, 95% RH, 25 min) inactivate > 99.8% MNV-1 and PEDV. Additionally, field tests in real full-scale coach demostrate the efficacy of the system as > 98.6% of infectious MNV-1 and > 96.3% PEDV were inactivated.

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.

SARS-CoV-2 RNA and antibody detection in breast milk from a prospective multicentre study in Spain.

OBJECTIVES: To develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies. DESIGN AND PATIENTS: This is a prospective, multicentre longitudinal study (April-December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included. SETTING: Seven health centres from different provinces in Spain. MAIN OUTCOME MEASURES: Presence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)-IgA, IgG and IgM-in breast milk samples from patients with COVID-19. RESULTS: All breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%-87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation). CONCLUSIONS: Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence. Trial registration number NCT04768244.

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.

Rapid Selective Detection of Potentially Infectious Porcine Epidemic Diarrhea Coronavirus Exposed to Heat Treatments Using Viability RT-qPCR.

Coronaviruses (CoVs) cause severe respiratory, enteric, and systemic infections in a wide range of hosts, including humans and animals. Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of porcine epidemic diarrhea (PED), a highly contagious intestinal disease affecting pigs of all ages. In this study, we optimized a viability real-time reverse transcriptase polymerase chain reaction (RT-qPCR) for the selective detection of infectious and heat-inactivated PEDV. PEMAX™, EMA™, and PMAxx™ photoactivable dyes along with PtCl4 and CDDP platinum compounds were screened as viability markers using two RT-qPCR assays: firstly, on PEDV purified RNA, and secondly on infectious and thermally inactivated virus suspensions. Furthermore, PMAxx™ pretreatment matched the thermal inactivation pattern obtained by cell culture better than other viability markers. Finally, we further optimized the pretreatment by coupling viability markers with Triton X-100 in inoculated serum resulting in a better estimation of PEDV infectivity than RT-qPCR alone. Our study has provided a rapid analytical tool based on viability RT-qPCR to infer PEDV infectivity with potential application for feed and feed ingredients monitoring in swine industry. This development would allow for greater accuracy in epidemiological surveys and outbreak investigations.

Metropolitan wastewater analysis for COVID-19 epidemiological surveillance.

The COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a rapidly emerging pandemic which has enforced extreme containment measures worldwide. In the absence of a vaccine or efficient treatment, cost-effective epidemiological surveillance strategies are urgently needed. Here, we have used RT-qPCR for SARS-CoV-2 detection in a series of longitudinal metropolitan wastewaters samples collected from February to April 2020, during the earliest stages of the epidemic in the Region of Valencia, Spain. We were able to consistently detect SARS-CoV-2 RNA in samples taken in late February, when communicated cases in that region were only incipient. We also find that the wastewater viral RNA context increased rapidly and anticipated the subsequent ascent in the number of declared cases. Our results strongly suggest that the virus was undergoing community transmission earlier than previously believed, and suggest that wastewater analysis could be sensitive and cost-effective strategy for COVID-19 epidemiological surveillance. Routine implementation of this surveillance tool would significantly improve our preparedness against new or re-occurring viral outbreaks.

SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200,000 reported COVID-19 cases in Spain resulting in more than 20,800 deaths as of April 21, 2020. Faecal shedding of SARS-CoV-2 RNA from COVID-19 patients has extensively been reported. Therefore, we investigated the occurrence of SARS-CoV-2 RNA in six wastewater treatments plants (WWTPs) serving the major municipalities within the Region of Murcia (Spain), the area with the lowest COVID-19 prevalence within Iberian Peninsula. Firstly, an aluminum hydroxide adsorption-precipitation concentration method was validated using a porcine coronavirus (Porcine Epidemic Diarrhea Virus, PEDV) and mengovirus (MgV). The procedure resulted in average recoveries of 10 ± 3.5% and 10 ± 2.1% in influent water (n = 2) and 3.3 ± 1.6% and 6.2 ± 1.0% in effluent water (n = 2) samples for PEDV and MgV, respectively. Then, the method was used to monitor the occurrence of SARS-CoV-2 from March 12 to April 14, 2020 in influent, secondary and tertiary effluent water samples. By using the real-time RT-PCR (RT-qPCR) Diagnostic Panel validated by US CDC that targets three regions of the virus nucleocapsid (N) gene, we estimated quantification of SARS-CoV-2 RNA titers in untreated wastewater waters of 5.4 ± 0.2 log10 genomic copies/L on average. Two secondary water samples resulted positive (2 out of 18) and all tertiary water samples tested as negative (0 out 12). This environmental surveillance data were compared to declared COVID-19 cases at municipality level, revealing that members of the community were shedding SARS-CoV-2 RNA in their stool even before the first cases were reported by local or national authorities in many of the cities where wastewaters have been sampled. The detection of SARS-CoV-2 in wastewater in early stages of the spread of COVID-19 highlights the relevance of this strategy as an early indicator of the infection within a specific population. At this point, this environmental surveillance could be implemented by municipalities right away as a tool, designed to help authorities to coordinate the exit strategy to gradually lift its coronavirus lockdown.