Assessing infectivity of emerging enveloped viruses in wastewater and sewage sludge: Relevance and procedures.

The emergence of SARS-CoV-2 has heightened the need to evaluate the detection of enveloped viruses in the environment, particularly in wastewater, within the context of wastewater-based epidemiology. The studies published over the past 80 years focused primarily on non-enveloped viruses due to their ability to survive longer in environmental matrices such as wastewater or sludge compared to enveloped viruses. However, different enveloped viruses survive in the environment for different lengths of time. Therefore, it is crucial to be prepared to assess the potential infectious risk that may arise from future emerging enveloped viruses. This will require appropriate tools, notably suitable viral concentration methods that do not compromise virus infectivity. This review has a dual purpose: first, to gather all the available literature on the survival of infectious enveloped viruses, specifically at different pH and temperature conditions, and in contact with detergents; second, to select suitable concentration methods for evaluating the infectivity of these viruses in wastewater and sludge. The methodology used in this data collection review followed the systematic approach outlined in the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) guidelines. Concentration methods cited in the data gathered are more tailored towards detecting the enveloped viruses' genome. There is a lack of suitable methods for detecting infectious enveloped viruses in wastewater and sludge. Ultrafiltration, ultracentrifugation, and polyethylene glycol precipitation methods, under specific/defined conditions, appear to be relevant approaches. Further studies are necessary to validate reliable concentration methods for detecting infectious enveloped viruses. The choice of culture system is also crucial for detection sensitivity. The data also show that the survival of infectious enveloped viruses, though lower than that of non-enveloped ones, may enable environmental transmission. Experimental data on a wide range of enveloped viruses is required due to the variability in virus persistence in the environment.

First detection of Hepatitis E virus (Rocahepevirus ratti) in French urban wastewater: Potential implications for human contamination.

Hepatitis E virus (HEV) is considered as an emerging zoonotic pathogen circulating in a wide range of animals. In recent decades, the genus Paslahepevirus frequently isolated in pigs were the most involved in human clinical practice. In addition, the genus Rocahepevirus have been isolated in rodents, and transmission to humans is increasingly reported worldwide, although gaps remain regarding the exposure factors. In this study, the presence of HEV was investigated in urban wastewater, swine slaughterhouse wastewater and river waters, in a geographical area where its circulation had previously been reported. In addition to the expected detection of Paslahepevirus in almost all waters samples collected, Rocahepevirus strains were detected with the same frequencies in urban and river waters, at concentrations up to 40-fold higher. No Rocahepeviruses were detected in swine slaughterhouse wastewater. This is the first study demonstrating the presence of Rocahepevirus in French wastewater. Although no evidence of transmission was reported among patients followed for a suspected HEV infection in the same area between April 2019 and October 2023 (i.e. 135/3078 serological tests positive for anti-HEV IgM detection; 46/822 blood samples positive for Paslahepevirus genome detection but none for Rocahepevirus), the circulation of Rocahepevirus in waters in such concentrations raises the question of the possible zoonotic transmission to human. Indeed, the waterborne transmission of HEV is now well documented in industrialized countries, and the exploration of the growing number of human infections in Europe involving Rocahepevirus has not until now made it possible to clarify the transmission routes.

Definition of a concentration and RNA extraction protocol for optimal whole genome sequencing of SARS-CoV-2 in wastewater (ANRS0160).

Monitoring the presence of RNA from emerging pathogenic viruses, such as SARS-CoV-2, in wastewater (WW) samples requires suitable methods to ensure an effective response. Genome sequencing of WW is one of the crucial methods, but it requires high-quality RNA in sufficient quantities, especially for monitoring emerging variants. Consequently, methods for viral concentration and RNA extraction from WW samples have to be optimized before sequencing. The purpose of this study was to achieve high coverage (≥ 90 %) and sequencing depth (at least ≥200×) even for low initial RNA concentrations (< 105 genome copies (GC)/L) in WW. A further objective was to determine the range of SARS-CoV-2 RNA concentrations that allow high-quality sequencing, and the optimal sample volume for analysis. Ultrafiltration (UF) methods were used to concentrate viral particles from large influent samples (up to 500 mL). An RNA extraction protocol using silica beads, neutral phenol-chloroform treatment, and a PCR inhibitor removal kit was chosen for its effectiveness in extracting RNA and eliminating PCR inhibitors, as well as its adaptability for use with large influent samples. Recovery rates ranged from 24 % to 63 % (N = 17) for SARS-CoV-2 naturally present in WW samples. 200 mL WW samples can be enough for UF concentration, as they showed high quality sequencing analyses with between 5 × 104 GC/L and 6 × 103 GC/L. Below 6 × 103 GC/L, high-quality sequencing was also achieved for ∼40 % of the samples using 500 mL of WW. Sequencing analysis for variant detection was performed on 200 mL WW samples with coverage of >95 % and sequencing depth of >1000×. Analyses revealed the predominance of variant EG.5, known as Eris (66 %-100 %). The use of UF methods in combination with a suitable RNA extraction protocol appear promising for sequencing enveloped viruses in WW in a context of viral emergence.

SARS-CoV-2 Variability in Patients and Wastewaters-Potential Immuno-Modulation during the Shift from Delta to Omicron.

The continuous emergence of SARS-CoV-2 variants favors potential co-infections and/or viral mutation events, leading to possible new biological properties. The aim of this work was to characterize SARS-CoV-2 genetic variability during the Delta-Omicron shift in patients and in a neighboring wastewater treatment plant (WWTP) in the same urban area. The surveillance of SARS-CoV-2 was performed by routine screening of positive samples by single nucleotide polymorphism analysis within the S gene. Moreover, additionally to national systematic whole genome sequencing (WGS) once a week in SARS-CoV-2-positive patients, WGS was also applied when mutational profiles were difficult to interpret by routine screening. Thus, WGS was performed on 414 respiratory samples and on four wastewater samples, northeastern France. This allowed us to report (i) the temporally concordant Delta to Omicron viral shift in patients and wastewaters; (ii) the characterization of 21J (Delta) and 21K (Omicron)/BA.1-21L (Omicron)/BA.2-BA.4 mixtures from humans or environmental samples; (iii) the mapping of composite mutations and the predicted impact on immune properties in the viral Spike protein.

Multiorgan and Vascular Tropism of SARS-CoV-2.

Although the respiratory tract is the main target of SARS-CoV-2, other tissues and organs are permissive to the infection. In this report, we investigated this wide-spectrum tropism by studying the SARS-CoV-2 genetic intra-host variability in multiple tissues. The virological and histological investigation of multiple specimens from a post-mortem COVID-19 patient was performed. SARS-CoV-2 genome was detected in several tissues, including the lower respiratory system, cardio-vascular biopsies, stomach, pancreas, adrenal gland, mediastinal ganglion and testicles. Subgenomic RNA transcripts were also detected, in favor of an active viral replication, especially in testicles. Ultra-deep sequencing allowed us to highlight several SARS-CoV-2 mutations according to tissue distribution. More specifically, mutations of the spike protein, i.e., V341A (18.3%), E654 (44%) and H655R (30.8%), were detected in the inferior vena cava. SARS-CoV-2 variability can contribute to heterogeneous distributions of viral quasispecies, which may affect the COVID-19 pathogeny.

Anti-SARS-CoV-2 Vaccines and Monoclonal Antibodies Facing Viral Variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is genetically variable, allowing it to adapt to various hosts including humans. Indeed, SARS-CoV-2 has accumulated around two mutations per genome each month. The first relevant event in this context was the occurrence of the mutant D614G in the Spike gene. Moreover, several variants have emerged, including the well-characterized 20I/501Y.V1, 20H/501Y.V2, and 20J/501Y.V3 strains, in addition to those that have been detected within clusters, such as 19B/501Y or 20C/655Y in France. Mutants have also emerged in animals, including a variant transmitted to humans, namely, the Mink variant detected in Denmark. The emergence of these variants has affected the transmissibility of the virus (for example, 20I/501Y.V1, which was up to 82% more transmissible than other preexisting variants), its severity, and its ability to escape natural, adaptive, vaccine, and therapeutic immunity. In this respect, we review the literature on variants that have currently emerged, and their effect on vaccines and therapies, and, in particular, monoclonal antibodies (mAbs). The emergence of SARS-CoV-2 variants must be examined to allow effective preventive and curative control strategies to be developed.