ABSTRACT
This study evaluated the stability of differing viral loads of SARS-CoV-2 over 28 days stored at room temperature, 4 degrees Celsius, -20 Celsius, or -80 Celsius. For the high concentration of SARS-CoV-2, regardless of storage conditions, 100% of samples were detected by qRT-PCR through day 28. At room temperature, median cycle threshold (CT) values for lower titers for both N1 and N2 targets remained consistent through day 28, fluctuating less than 1 median CT. For lower concentrations of virus, storage at room temperature was associated with reductions of positivity beginning at day 7, and by day 28, 0% of samples were detected for N1. Storage at room temperature was the least stable of all environmental conditions tested, with 54.2% of negative PCR results. At 4 degrees Celsius, there was minimal change in CTs over time at the higher viral concentration. For lower titers, CTs increased by 2.1 CTs for N1 and 2.6 CTs for N2 over the 28 days. At -20 degrees Celsius, lower titers of virus fluctuated slightly more, increasing by 3 CTs. Storage of SARS-CoV-2 in PBS at -20 degrees Celsius was the second least stable condition, accounting for 37.5% of negative PCR results. Storage at -80 degrees Celsius showed the greatest stability, with all samples detected throughout the 28 days and 1.5 median CTs for both N1 and N2 targets. Here, this study shows that the stability of SARS-CoV-2 can be maintained at 4 degrees Celsius for up to a month when -80 degrees Celsius storage is not available. At viral loads of >5,000 copies/ml corresponding to >75% of positive samples recovered in the clinical lab to date-different storage temperatures did not have a substantial impact on the ability to detect SARS-CoV-2 when stored in PBS.
ABSTRACT
Rapid dissemination of SARS-CoV-2 sequencing data to public repositories has enabled widespread study of viral genomes, but studies of longitudinal specimens from infected persons are relatively limited. Analysis of longitudinal specimens enables understanding of how host immune pressures drive viral evolution in vivo. Here we performed sequencing of 49 longitudinal SARS-CoV-2-positive samples from 20 patients in Washington State collected between March and September of 2020. Viral loads declined over time with an average increase in RT-PCR cycle threshold (Ct) of 0.87 per day. We found that there was negligible change in SARS-CoV-2 consensus sequences over time, but identified a number of nonsynonymous variants at low frequencies across the genome. We observed enrichment for a relatively small number of these variants, all of which are now seen in consensus genomes across the globe at low prevalence. In one patient, we saw rapid emergence of various low-level deletion variants at the N-terminal domain of the spike glycoprotein, some of which have previously been shown to be associated with reduced neutralization potency from sera. In a subset of samples that were sequenced using metagenomic methods, differential gene expression analysis showed a downregulation of cytoskeletal genes that was consistent with a loss of ciliated epithelium during infection and recovery. We also identified co-occurrence of bacterial species in samples from multiple hospitalized individuals. These results demonstrate that the intrahost genetic composition of SARS-CoV-2 is dynamic during the course of COVID-19, and highlight the need for continued surveillance and deep sequencing of minor variants.
Subject(s)
COVID-19 , Severe Acute Respiratory SyndromeABSTRACT
Despite limited genomic diversity, SARS-CoV-2 has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA-sequencing profiles of nasopharyngeal swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with upregulation of antiviral factors such as OAS1-3 and IFIT1-3, and Th1 chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial cultures replicated the in vivo antiviral host response. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of Th1 chemokines CXCL9/10/11 and their cognate receptor, CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B and NK cell-specific transcripts and an increase in inhibitors of NF-{kappa}B signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.