Correlation of Redondovirus and Entamoeba gingivalis Detections in the Human Oral Cavity Suggests That This Amoeba Is Possibly the Redondovirus Host.

The virome of the human oral cavity and the relationships between viruses and diseases such as periodontitis are scarcely deciphered. Redondoviruses were reported in the human oral cavity in 2019, including in periodontitis patients. Here, we aimed at detecting redondoviruses and at searching for a potential viral host in human saliva. Non-stimulated saliva was collected between December 2020 and June 2021. These samples were tested using real-time PCR regarding the presence of redondovirus and Entamoeba gingivalis DNA. Similarity searches were performed using BLAST against eukaryotic and prokaryotic sequences from GenBank. The redondovirus DNA was detected in 46% of the 28 human saliva samples. In addition, short fragments of redondovirus genomes were detected in silico within Entamoeba sequences. Finally, Entamoeba gingivalis DNA was detected in 46% of the 28 saliva samples, with a strong correlation between redondovirus DNA and E. gingivalis DNA detections, in 93% of the cases. Regarded together, these findings and previous ones strongly support the presence of redondoviruses in the human oral cavity and their association to E. gingivalis as their likely host.

Simple Wastewater Preparation Protocol Applied to Monitor the Emergence of the Omicron 21L/BA.2 Variant by Genome Sequencing.

Detecting and monitoring viruses in wastewater samples have been reported as useful ways of tracking SARS-CoV-2 epidemic trends. However, there is currently no unanimously recognised method of processing samples to identify and quantify SARS-CoV-2 variants in wastewater. We aimed to implement a method that was as simple as possible in order to be used universally. In a study performed between January 2022 and June 2022 in the city of Marseille, France, we first evaluated the impact of the sample preservation strategy. We then compared ultracentrifugation to ultrafiltration and several steps of filtration to determine the optimal approach for virus concentration. As a proof-of-concept, the definitive protocol was applied to next-generation sequencing of SARS-CoV-2 in wastewater to monitor the emergence of the Omicron variant in the city. For sewage water to be processed in the week following the sampling, storage at +4 °C is sufficient, with less than 1 Ct loss. Filtration with a 5 µm syringe filter, then with a 0.8 µm filtration unit, followed by ultrafiltration was the optimal protocol, leading to an average increase of 3.24 Ct when the starting Ct was on average 38 in the wastewater. This made it possible to observe the emergence of the Omicron 21L/BA.2 variant after Omicron 21K/BA.1 by genome sequencing over a period ranging from 20 February to 10 April 2022 in agreement with observations based on patient data. To conclude, by using a simple method requiring only basic filters and a centrifuge as equipment, it is possible to accurately track the relative incidence rates and the emergence of SARS-CoV-2 variants based on sewage samples.

Limited permissibility of ENL-R and Mv-1-Lu mink cell lines to SARS-CoV-2.

The SARS-CoV-2 pandemic started in the end of 2019 in Wuhan, China, which highlighted the scenario of frequent cross-species transmission events. From the outbreak possibly initiated by viral spill-over into humans from an animal reservoir, now we face the human host moving globally while interacting with domesticated and peridomestic animals. The emergence of a new virus into the ecosystem leads to selecting forces and species-specific adaptations. The adaptation of SARS-CoV-2 to other animals represents a risk to controlling the dissemination of this coronavirus and the emergence of new variants. Since 2020, several mink farms in Europe and the United States have had SARS-CoV-2 outbreaks with human-mink and mink-human transmission, where the mink-selected variants possibly hold evolutionary concerning advantages. Here we investigated the permissibility of mink lung-derived cells using two cell lines, Mv-1-Lu and ENL-R, against several lineages of SARS-CoV-2, including some classified as variants of concern. The viral release rate and the infectious titers indicate that these cells support infections by different SARS-CoV-2 lineages. The viral production occurs in the first few days after infection with the low viral release by these mink cells, which is often absent for the omicron variant for lung cells. The electron microscopy reveals that during the viral replication cycle, the endomembrane system of the mink-host cell undergoes typical changes while the viral particles are produced, especially in the first days of infection. Therefore, even if limited, mink lung cells may represent a selecting source for SARS-CoV-2 variants, impacting their transmissibility and pathogenicity and making it difficult to control this new coronavirus.

SARS-CoV-2 Testing of Aircraft Wastewater Shows That Mandatory Tests and Vaccination Pass before Boarding Did Not Prevent Massive Importation of Omicron Variant into Europe.

BACKGROUND: Most new SARS-CoV-2 epidemics in France occurred following the importation from abroad of emerging viral variants. Currently, the risk of new variants being imported is controlled based on a negative screening test (PCR or antigenic) and proof of up-to-date vaccine status, such as the International Air Transport Association travel pass. METHODS: The wastewater from two planes arriving in Marseille (France) from Addis Ababa (Ethiopia) in December 2021 was tested by RT-PCR to detect SARS-CoV2 and screen for variants. These tests were carried out between landing and customs clearance and were then sequenced by MiSeq Illumina. Antigenic tests and sequencing by NovaSeq were carried out on respiratory samples collected from the 56 passengers on the second flight. RESULTS: SARS-CoV-2 RNA suspected of being from the Omicron BA.1 variant was detected in the aircraft's wastewater. SARS-CoV2 RNA was detected in 11 [20%) passengers and the Omicron BA.1 variant was identified. CONCLUSION: Our work shows the efficiency of aircraft wastewater testing to detect SARS-CoV-2 cases among travellers and to identify the viral genotype. It also highlights the low efficacy of the current control strategy for flights entering France from outside Europe, which combines a requirement to produce a vaccine pass and proof of a negative test before boarding.

Microscopic Observation of SARS-Like Particles in RT-qPCR SARS-CoV-2 Positive Sewage Samples.

The ongoing outbreak of novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection has spread rapidly worldwide. The major transmission routes of SARS-CoV-2 are recognised as inhalation of aerosol/droplets and person-to-person contact. However, some studies have demonstrated that live SARS-CoV-2 can be isolated from the faeces and urine of infected patients, which can then enter the wastewater system. The currently available evidence indicates that the viral RNA present in wastewater may become a potential source of epidemiological data. However, to investigate whether wastewater may present a risk to humans such as sewage workers, we investigated whether intact particles of SARS-CoV-2 were observable and whether it was possible to isolate the virus in wastewater. Using a correlative strategy of light microscopy and electron microscopy (CLEM), we demonstrated the presence of intact and degraded SARS-like particles in RT-qPCR SARS-CoV-2-positive sewage sample collected in the city of Marseille. However, the viral infectivity assessment of SARS-CoV-2 in the wastewater was inconclusive, due to the presence of other viruses known to be highly resistant in the environment such as enteroviruses, rhinoviruses, and adenoviruses. Although the survival and the infectious risk of SARS-CoV-2 in wastewater cannot be excluded from our study, additional work may be required to investigate the stability, viability, fate, and decay mechanisms of SARS-CoV-2 thoroughly in wastewater.

Culture of SARS-CoV-2 in a panel of laboratory cell lines, permissivity, and differences in growth profile.

The emergence of COVID-19 disease due to SARS-CoV-2 at the end of 2019 was rapidly associated with the isolation of the strain from co-culture onto VERO cells. These isolations quickly made it possible to carry out the first tests for antiviral agents' susceptibility and drug repurposing. However, it seems important to make an inventory of all the cells that can support the growth of this virus and evaluate possible differences between isolates. In the present work, we tested 4 strains of SARS-CoV-2 locally isolated on a panel of 34 cell lines present in our laboratory and commonly used for the isolation of human pathogenic microorganism. After inoculation, cells were observed for cytopathic effects and quantitative real-time polymerase reaction was used to measure the virus replication on the cells. We were able to obtain growth on 7 cell lines, 6 simian, and the human Caco-2. The cytopathogenic effects are variable, ranging from lysis of the cell monolayer in 48-72 h to no cytopathic effect in spite of intense multiplication, as in Caco-2 cells. Interestingly, effect and multiplication varied widely according to the strain tested. In this paper, we explored the species specificity and tissue tropism of SARS-CoV-2 in vitro on a panel of cells available in our laboratory and identified human and animal cell lines susceptible to support SARS-CoV-2 replication. Our work highlights the importance of testing multiple strains when testing antiviral molecules and performing patho-physiological analyzes.