Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities

Crystal structure of the sugar acid‐binding protein CxaP from a TRAP transporter in Advenella mimigardefordensis strain DPN7 T

Recently, CxaP, a sugar acid substrate binding protein (SBP) fromAdvenella mimigardefordensis strain DPN7 T, was identified as part of anovel sugar uptake strategy. In the present study, the protein was success-fully crystallized. Although several SBP structures of tripartite ATP-inde-pendent periplasmic transporters have already been solved, this is the firststructure of an SBP accepting multiple sugar acid ligands. Protein crystalswere obtained with bound D -xylonic acid, D-fuconic acid D -galactonic andD-gluconic acid, respectively. The protein shows the typical structure of anSBP of a tripartite ATP-independent periplasmic transporter consisting oftwo domains linked by a hinge and spanned by a long α-helix. By analysisof the structure, the substrate binding site of the protein was identified.The carboxylic group of the sugar acids interacts with Arg175, whereas thecoordination of the hydroxylic groups at positions C2 and C3 is mostprobably realized by Arg154 and Asn151. Furthermore, it was observedthat 2-keto-3-deoxy- D-gluconic acid is bound in protein crystals that werecrystallized without the addition of any ligand, indicating that this mole-cule is prebound to the protein and is displaced by the other ligands if theyare available

Early detection of SARS-CoV-2 Omicron BA.4/5 in German wastewater

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. Omicron sub-variants BA.4/5 have spread globally displacing the predeceasing variants. Due to the severe transmissibility and immune escape potential of BA.4/5, early monitoring was required to asses and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North-Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability to detect BA.4/5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022 and the presence of BA.4/5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V. Finally, the mutant fractions were quantitatively monitored by digital PCR confirming BA.4/5 as the majority variant by 5th June 2022. In conclusions, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variant spreading independent of individual test capacities