ABSTRACT
Background: The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with decreased susceptibility to neutralizing antibodies are of clinical importance. While several demographic and clinical correlates of Coronavirus Disease 2019 (COVID-19) outcome have been identified, their relationship to virological and immunological parameters remain poorly defined. Here, we evaluate viral diversity and the accumulation of intra-host mutations over time in a population of hospitalized adults positive for SARS-CoV-2. Methods: We performed longitudinal collection of nasopharyngeal swabs and blood samples from a small cohort of hospitalized adults with COVID-19. Clinical information regarding study subject's immunocompromised status was collected. Samples were assessed for SARS-CoV-2 viral load, viral genotype, viral diversity, and antibody titer. Results: Intra-host viral genetic diversity remained constant through disease course in study subjects that were non-immunocompromised and resulted in changes in viral genotype in some participants. We report the de novo emergence of Spike mutations that have been previously associated with circulating variants of concern in two immunosuppressed patients with persistent SARS-CoV-2 infection. Conclusion: Constant rates of viral evolution suggest the emergence of variants as a function of time, emphasizing the need for effective antivirals to control viral load over long disease courses.
ABSTRACT
Background: The greater Chicagoland area has recorded over 10,000 COVID-related deaths and nearly 600,000 cases since the start of the COVID-19 pandemic in March of 2020. SARS-CoV-2, the causative agent of COVID-19, has continually changed over that time, with some variants evolving to become more transmissible or more resistant to neutralizing antibody responses. Methods: To better understand how viral genetic variation has contributed to differences in COVID-19 pathogenesis and patient outcome, we established a biobank of residual diagnostic samples from adult patients who tested positive for COVID-19 in a PCR-based test at Northwestern Memorial Hospital. Thus far, we have collected samples from 6448 out-patients and 632 in-patients. Of these, we have performed whole genome sequencing and viral load calculations on 1373 samples. Clinical and demographic information, including composite measures of disease severity, were extracted from available electronic health records. These data were assessed for longitudinal patterns and for specific association with viral lineage. Results: We found that the early epidemic in March of 2020 was defined by three distinct lineages reflecting the outbreaks in China (19B/A), Washington state (19B/A.1), and New York state (19A/B.1). By November of 2020, we saw a large increase in the number of confirmed cases, dominated by the 20G clade. This lineage remained predominant until March of 2021, when the Alpha and Gamma variants of concern became more established. These were recently supplanted by the Delta variant, which now accounts for over 90% of Chicago cases. At the height of the pandemic in November of 2020, case counts peaked at over 5000 cases per day, but hospitalizations, ICU admissions, and deaths over this period remained flat. Statistical testing revealed that the predominant clade at that time, 20G, was associated with better outcomes and less severe disease as measured by clinical measures of patient deterioration, even when controlling for patient demographics. These results suggest that a viral variant associated with less severe disease was predominant in late 2020 before the emergence of the more transmissible variants of concern. Conclusion: Current work is being done to determine if the less severe outcomes associated with this clade also contributed to more asymptomatic transmission, potentially contributing to the high case counts recorded over this period. These data emphasize the need for continued genomic surveillance of SARS-CoV-2 to character.
ABSTRACT
Background: The rapid spread of SARS-CoV-2, the causative agent of Coronavirus disease 2019 (COVID-19), has been accompanied by the emergence of viral mutations, some of which may have distinct virological and clinical consequences. While whole genome sequencing efforts have worked to map this viral diversity at the population level, little is known about how SARS-CoV-2 may diversify within a host over time. This is particularly important for understanding the emergence of viral resistance to therapeutic interventions and immune pressure. The goal of this study was to assess the change in viral load and viral genome sequence within patients over time and determine if these changes correlate with clinical and/or demographic parameters. Methods: Hospitalized patients admitted to Northwestern Memorial Hospital with a positive SARS-CoV-2 test were enrolled in a longitudinal study for the serial collection of nasopharyngeal specimens. Swabs were administered to patients by hospital staff every 4 ± 1 days for up to 32 days or until the patients were discharged. RNA was extracted from each specimen and viral loads were calculated by quantitative reverse transcriptase PCR (qRT-PCR). Specimens with qRT-PCR cycle threshold values less than or equal to 30 were subject to whole viral genome sequencing by reverse transcription, multiplex PCR, and deep sequencing. Variant populations sizes were estimated and subject to phylogenetic analysis relative to publicly available SARS-CoV-2 sequences. Sequence and viral load data were subsequently correlated to available demographic and clinical data. Results: 60 patients were enrolled from March 26th to June 20th, 2020. We observed an overall decrease in nasopharyngeal viral load over time across all patients. However, the temporal dynamics of viral load differed on a patient-by-patient basis. Several mutations were also observed to have emerged within patients over time. Distribution of SARS-CoV-2 viral loads in serially collected nasopharyngeal swabs in hospitalized adults as determined by qRT-PCR. Samples were collected every 4 ± 1 days (T#1-8) and viral load is displayed by log(copy number). Conclusion: These data indicate that SARS-CoV-2 viral loads in the nasopharynx decrease over time and that the virus can accumulate mutations during replication within individual patients. Future studies will examine if some of these mutations may provide fitness advantages in the presence of therapeutic and/or immune selective pressures.
ABSTRACT
Background: The rapid spread of SARS-CoV-2, the causative agent of Coronavirus disease 2019 (COVID-19), has been accompanied by the emergence of distinct viral clades, although their clinical significance has yet to be fully elucidated. While whole genome sequencing efforts have identified viral diversity over time, less is known about the clinical significance of this diversity. This study assessed the nasopharyngeal viral loads within patients over time to determine if these changes affect clinical parameters. Methods: Samples were collected from patients presenting to Northwestern Memorial Hospital in Chicago, IL with a positive SARS-CoV-2 RT-PCR from nasopharyngeal swabs. Cycle threshold (Ct) values less than 35 were considered positive, and whole genome sequencing was performed by reverse transcription, multiplex PCR, and Nanopore sequencing. Phylogenetic analysis was conducted on sequenced isolates and compared with publicly available global sequences. Sequence characteristics and viral loads were correlated with each clade. Results: 177 samples were analyzed from March 14, 2020, through May 1, 2020. Most of the sequences (92.6%) clustered in three main clades [Figure 1]. Clade IDs were ordered by relative abundance as Clades 1 (n=122, 68.9%), 2 (n=34, 19.2%), and 3 (n=8, 4.5%). Over this time, Clade 1 viruses have been increasing in incidence across the USA and globally while Clade 2 viruses were uniquely predominant in Illinois with limited global distribution. Ct values were compared across clades [Figure 2]. Significantly lower average Ct values (higher viral loads) were observed in Clade 1 relative to both Clade 2 (p=0.0002) and Clade 3 (p=0.0011). These findings were independent of time from symptom onset to specimen collection. Phylogenetic Analysis of SARS-CoV-2 Isolates with Number of Clades and Clade Distribution Conclusion: These data suggest that SARS-CoV-2 genotype may impact viral load in the upper airways. It remains to be determined whether this difference in clades may impact transmission potential and overall viral spread. Further longitudinal studies with more specimens and associated clinical data are needed.