Detecting SARS-CoV-2 lineages and mutational load in municipal wastewater and a use-case in the metropolitan area of Thessaloniki, Greece.

The COVID-19 pandemic represents an unprecedented global crisis necessitating novel approaches for, amongst others, early detection of emerging variants relating to the evolution and spread of the virus. Recently, the detection of SARS-CoV-2 RNA in wastewater has emerged as a useful tool to monitor the prevalence of the virus in the community. Here, we propose a novel methodology, called lineagespot, for the monitoring of mutations and the detection of SARS-CoV-2 lineages in wastewater samples using next-generation sequencing (NGS). Our proposed method was tested and evaluated using NGS data produced by the sequencing of 14 wastewater samples from the municipality of Thessaloniki, Greece, covering a 6-month period. The results showed the presence of SARS-CoV-2 variants in wastewater data. lineagespot was able to record the evolution and rapid domination of the Alpha variant (B.1.1.7) in the community, and allowed the correlation between the mutations evident through our approach and the mutations observed in patients from the same area and time periods. lineagespot is an open-source tool, implemented in R, and is freely available on GitHub and registered on bio.tools.

A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain.

Mutations resulting in amino-acid substitutions of the SARS-CoV-2 spike protein receptor-binding domain (RBD) have been associated with enhanced transmissibility and immune escape of the respective variants, namely Alpha, Beta, Gamma or Delta. Rapid identification of the aforementioned variants of concern and their discrimination of other variants is thus of importance for public health interventions. For this reason, a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed, to target signature mutations associated with amino-acid substitutions at positions 478, 484 and 501 present in the receptor-binding motif (RBM) of the spike protein RBD. This region contains most contacting residues of SARS-CoV-2 that bind to ACE2. A novel strategy employing the use of non-extendable LNA oligonucleotide blockers that can reduce non-specific hybridization of probes increased the number of different mutated sites examined in a multiplex PCR. The combinatory analysis of the different fluorescence signals obtained enabled the preliminary differentiation of SARS-CoV-2 variants of concern. The assay is sensitive with a LOD of 263 copies/reaction for the Delta variant, 170 copies/reaction for the Beta variant, amplification efficiencies > 91% and a linear range of >5 log10 copies/reaction against all targets. Validation of the assay using known SARS-CoV-2-positive and negative samples from humans and animals revealed its ability to correctly identify the targeted mutations and preliminary characterize the SARS-CoV-2 variants. The novel approach for mutation typing using LNA oligonucleotide blockers can be modified to target signature mutations at four different sites in the RBM and further expand the range of variants detected.

Outbreaks of SARS-CoV-2 in naturally infected mink farms: Impact, transmission dynamics, genetic patterns, and environmental contamination.

SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3-1.3 log10 RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R0 of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions.

The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94 % and a linear range of over 5 log10 copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.