Investigation of a SARS-CoV-2 outbreak in a Texas summer camp resulting from a single introduction

SARS-CoV-2 is the etiological agent responsible for the COVID-19 pandemic. It is estimated that only 10 aerosol-borne virus particles are sufficient to establish a secondary infection with SARS-CoV-2. However, the dispersal pattern of SARS-CoV-2 is highly variable and only 10- 20% of cases are responsible for up 80% of secondary infections. The heterogeneous nature of SARS-CoV-2 transmission suggests that super-spreader events play an important role in viral transmission. Super-spreader events occur when a single person is responsible for an unusually high number of secondary infections due to a combination of biological, environmental, and/or behavioral factors. While super-spreader events have been identified as a significant factor driving SARS-CoV-2 transmission, epidemiologic studies have consistently shown that education settings do not play a major role in community transmission. However, an outbreak of SARS-CoV-2 was recently reported among 186 children (aged 10-17) and adults (aged 18 +) after attending an overnight summer camp in Texas in June 2021. To understand the transmission dynamics of the outbreak, RNA was isolated from 36 nasopharyngeal swabs collected from patients that attended the camp and 19 control patients with no known connection to the outbreak. Genome sequencing on the Oxford Nanopore platform was performed using the ARTIC approaches for library preparation and bioinformatic analysis. SARS-CoV-2 amplicons were produced from all RNA samples and >70% of the viral genome was successfully reconstructed with >10X coverage for 46 samples. Phylogenetic methods were used to estimate the transmission history and suggested that the outbreak was the result of a single introduction. We also found evidence for secondary transmission from campers to the community. Together, these findings demonstrate that super-spreader events may occur during large gatherings of children.

QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing 1,2. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates 3. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif ({Delta}QTQTN). Here we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS, and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated4, and disruption its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site - the FCS, loop length, and glycosylation - are required for efficient SARS-CoV-2 replication and pathogenesis.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called Tiled-ClickSeq, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.