Analysis of SARS-CoV-2 variants from patient specimens in Nevada from October 2020 to August 2021.

In early 2020, the emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population quickly developed into a global pandemic. SARS-CoV-2 is the etiological agent of coronavirus disease 2019 (COVID-19) which has a broad range of respiratory illnesses. As the virus circulates, it acquires nucleotide changes. These mutations are potentially due to the inherent differences in the selection pressures within the human population compared to the original zoonotic reservoir of SARS-CoV-2 and formerly naïve humans. The acquired mutations will most likely be neutral, but some may have implications for viral transmission, disease severity, and resistance to therapies or vaccines. This is a follow-up study from our early report (Hartley et al. J Genet Genomics. 01202021;48(1):40-51) which detected a rare variant (nsp12, RdRp P323F) circulating within Nevada in mid 2020 at high frequency. The primary goals of the current study were to determine the phylogenetic relationship of the SARS-CoV-2 genomes within Nevada and to determine if there are any unusual variants within Nevada compared to the current database of SARS-CoV-2 sequences. Whole genome sequencing and analysis of SARS-CoV-2 from 425 positively identified nasopharyngeal/nasal swab specimens were performed from October 2020 to August 2021 to determine any variants that could result in potential escape from current therapeutics. Our analysis focused on nucleotide mutations that generated amino acid variations in the viral Spike (S) protein, Receptor binding domain (RBD), and the RNA-dependent RNA-polymerase (RdRp) complex. The data indicate that SARS-CoV-2 sequences from Nevada did not contain any unusual variants that had not been previously reported. Additionally, we did not detect the previously identified the RdRp P323F variant in any of the samples. This suggests that the rare variant we detected before was only able to circulate because of the stay-at-home orders and semi-isolation experience during the early months of the pandemic. IMPORTANCE: SARS-COV-2 continues to circulate in the human population. In this study, SARS-CoV-2 positive nasopharyngeal/nasal swab samples were used for whole genome sequencing to determine the phylogenetic relationship of SARS-CoV-2 sequences within Nevada from October 2020 to August 2021. The resulting data is being added to a continually growing database of SARS-CoV-2 sequences that will be important for understanding the transmission and evolution of the virus as it spreads around the globe.

Rapid Lineage Assignment of Severe Acute Respiratory Syndrome Coronavirus 2 Cases through Automated Library Preparation, Sequencing, and Bioinformatic Analysis.

The coronavirus disease 2019 (COVID-19) pandemic has provided a stage to illustrate that there is considerable value in obtaining rapid, whole-genome-based information about pathogens. This article describes the utility of a commercially available, automated severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) library preparation, genome sequencing, and a bioinformatics analysis pipeline to provide rapid, near-real-time SARS-CoV-2 variant description. This study evaluated the turnaround time, accuracy, and other quality-related parameters obtained from commercially available automated sequencing instrumentation, from analysis of continuous clinical samples obtained from January 1, 2021, to October 6, 2021. This analysis included a base-by-base assessment of sequencing accuracy at every position in the SARS-CoV-2 chromosome using two commercially available methods. Mean turnaround time, from the receipt of a specimen for SARS-CoV-2 testing to the availability of the results, with lineage assignment, was <3 days. Accuracy of sequencing by one method was 100%, although certain sites on the genome were found repeatedly to have been sequenced with varying degrees of read error rate.

Use of wastewater surveillance for early detection of Alpha and Epsilon SARS-CoV-2 variants of concern and estimation of overall COVID-19 infection burden.

A decline in diagnostic testing for SARS-CoV-2 is expected to delay the tracking of COVID-19 variants of concern and interest in the United States. We hypothesize that wastewater surveillance programs provide an effective alternative for detecting emerging variants and assessing COVID-19 incidence, particularly when clinical surveillance is limited. Here, we analyzed SARS-CoV-2 RNA in wastewater from eight locations across Southern Nevada between March 2020 and April 2021. Trends in SARS-CoV-2 RNA concentrations (ranging from 4.3 log10 gc/L to 8.7 log10 gc/L) matched trends in confirmed COVID-19 incidence, but wastewater surveillance also highlighted several limitations with the clinical data. Amplicon-based whole genome sequencing (WGS) of 86 wastewater samples identified the B.1.1.7 (Alpha) and B.1.429 (Epsilon) lineages in December 2020, but clinical sequencing failed to identify the variants until January 2021, thereby demonstrating that 'pooled' wastewater samples can sometimes expedite variant detection. Also, by calibrating fecal shedding (11.4 log10 gc/infection) and wastewater surveillance data to reported seroprevalence, we estimate that ~38% of individuals in Southern Nevada had been infected by SARS-CoV-2 as of April 2021, which is significantly higher than the 10% of individuals confirmed through clinical testing. Sewershed-specific ascertainment ratios (i.e., X-fold infection undercounts) ranged from 1.0 to 7.7, potentially due to demographic differences. Our data underscore the growing application of wastewater surveillance in not only the identification and quantification of infectious agents, but also the detection of variants of concern that may be missed when diagnostic testing is limited or unavailable.