Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

SARS-CoV-2 and other respiratory pathogens are detected in continuous air samples from congregate settings.

Two years after the emergence of SARS-CoV-2, there is still a need for better ways to assess the risk of transmission in congregate spaces. We deployed active air samplers to monitor the presence of SARS-CoV-2 in real-world settings across communities in the Upper Midwestern states of Wisconsin and Minnesota. Over 29 weeks, we collected 527 air samples from 15 congregate settings. We detected 106 samples that were positive for SARS-CoV-2 viral RNA, demonstrating that SARS-CoV-2 can be detected in continuous air samples collected from a variety of real-world settings. We expanded the utility of air surveillance to test for 40 other respiratory pathogens. Surveillance data revealed differences in timing and location of SARS-CoV-2 and influenza A virus detection. In addition, we obtained SARS-CoV-2 genome sequences from air samples to identify variant lineages. Collectively, this shows air sampling is a scalable, high throughput surveillance tool that could be used in conjunction with other methods for detecting respiratory pathogens in congregate settings.

Construction of a new chromosome-scale, long-read reference genome assembly for the Syrian hamster, Mesocricetus auratus.

BACKGROUND: The Syrian hamster (Mesocricetus auratus) has been suggested as a useful mammalian model for a variety of diseases and infections, including infection with respiratory viruses such as SARS-CoV-2. The MesAur1.0 genome assembly was generated in 2013 using whole-genome shotgun sequencing with short-read sequence data. Current more advanced sequencing technologies and assembly methods now permit the generation of near-complete genome assemblies with higher quality and greater continuity. FINDINGS: Here, we report an improved assembly of the M. auratus genome (BCM_Maur_2.0) using Oxford Nanopore Technologies long-read sequencing to produce a chromosome-scale assembly. The total length of the new assembly is 2.46 Gb, similar to the 2.50-Gb length of a previous assembly of this genome, MesAur1.0. BCM_Maur_2.0 exhibits significantly improved continuity, with a scaffold N50 that is 6.7 times greater than MesAur1.0. Furthermore, 21,616 protein-coding genes and 10,459 noncoding genes are annotated in BCM_Maur_2.0 compared to 20,495 protein-coding genes and 4,168 noncoding genes in MesAur1.0. This new assembly also improves the unresolved regions as measured by nucleotide ambiguities, where ∼17.11% of bases in MesAur1.0 were unresolved compared to BCM_Maur_2.0, in which the number of unresolved bases is reduced to 3.00%. CONCLUSIONS: Access to a more complete reference genome with improved accuracy and continuity will facilitate more detailed, comprehensive, and meaningful research results for a wide variety of future studies using Syrian hamsters as models.

Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16, 2020, to November 19, 2020, surveillance samples (n = 4704) were collected from volunteers and tested for SARS-CoV-2 at 5 sites. Twenty-one samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, whereas 8 tested negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the false-negative rate of the RT-LAMP assay only from July 16, 2020, to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or fewer and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP-negative pools (2493 total samples) testing positive in the more-sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and that can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.