National-scale surveillance of emerging SARS-CoV-2 variants in wastewater

SARS-CoV-2 surveillance is crucial to identify variants with altered epidemiological properties. Wastewater-based epidemiology (WBE) provides an unbiased and complementary approach to sequencing individual cases. Yet, national WBE surveillance programs have not been widely implemented and data analyses remain challenging. We deep-sequenced 2,093 wastewater samples representing 95 municipal catchments, covering >57% of Austrias population, from December 2020 to September 2021. Our Variant Quantification in Sewage pipeline designed for Robustness (VaQuERo) enabled us to deduce variant abundance from complex wastewater samples and delineate the spatiotemporal dynamics of the dominant Alpha and Delta variants as well as regional clusters of other variants of concern. These results were cross validated by epidemiological records of >130,000 individual cases. Finally, we provide a framework to predict emerging variants de novo and infer variant-specific reproduction numbers from wastewater. This study demonstrates the power of national-scale WBE to support public health and promises particular value for countries without dense individual monitoring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/21267633v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1044af0org.highwire.dtl.DTLVardef@d3fe2borg.highwire.dtl.DTLVardef@1d3bfa1org.highwire.dtl.DTLVardef@c9fc95_HPS_FORMAT_FIGEXP M_FIG C_FIG

High-throughput Mutational Surveillance of the SARS-CoV-2 Spike Gene

SARS-CoV-2 has evolved rapidly towards higher infectivity and partial immune escape over the course of the pandemic. This evolution is driven by the enormous virus population, that has infected close to 200 million people by now. Therefore, cost effective and scalable methods are needed to monitor viral evolution globally. Mutation-specific PCR approaches have become inadequate to distinguish the variety of circulating SARS-CoV-2 variants and are unable to detect novel ones. Conversely, whole genome sequencing protocols remain too labor- and cost-intensive to monitor SARS-CoV-2 at the required density. By adapting SARSeq we present a simple, fast, and scalable S-gene tiling pipeline for focused sequencing of the S-gene encoding for the spike protein. This method reports on all sequence positions with known importance for infectivity and immunity, yet scales to >20K samples per run. S-gene tiling is used for nationwide surveillance of SARS-CoV-2 at a density of 10% to 50% of all cases of infection in Austria. SARSeq S-tiling uncovered several infection clusters with variants of concern such as the biggest known cluster of Beta/B.1.351 outside Africa and successfully informed public health measures in a timely manner, allowing their successful implementation. Our close monitoring of mutations further highlighted evolutionary constraints and freedom of the spike protein ectodomain and sheds light on foreseeable evolutionary trajectories.

SARS-CoV-2 escapes CD8 T cell surveillance via mutations in MHC-I restricted epitopes

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control, though direct evidence has been lacking so far. Here, we identified non-synonymous mutations in MHC-I restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV- 2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding, which was associated with a loss of recognition and functional responses by CD8+ T cells isolated from HLA-matched COVID-19 patients. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through escape mutations in MHCI-restricted viral epitopes. This provides evolutionary evidence for CD8+ T cell immunity controlling SARS-CoV-2 with consequences for COVID-19 vaccine design.

Mutational dynamics and transmission properties of SARS-CoV-2 superspreading events in Austria

Superspreading events shape the COVID-19 pandemic. Here we provide a national-scale analysis of SARS-CoV-2 outbreaks in Austria, a country that played a major role for virus transmission across Europe and beyond. Capitalizing on a national epidemiological surveillance system, we performed deep whole-genome sequencing of virus isolates from 576 samples to cover major Austrian SARS-CoV-2 clusters. Our data chart a map of early viral spreading in Europe, including the path from low-frequency mutations to fixation. Detailed epidemiological surveys enabled us to calculate the effective SARS-CoV-2 population bottlenecks during transmission and unveil time-resolved intra-patient viral quasispecies dynamics. This study demonstrates the power of integrating deep viral genome sequencing and epidemiological data to better understand how SARS-CoV-2 spreads through populations. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY