High-density amplicon sequencing identifies community spread and ongoing evolution of SARS-CoV-2 in the Southern United States

McNamara, Ryan P.; Caro-Vegas, Carolina; Landis, Justin T.; Moorad, Razia; Pluta, Linda; Eason, Anthony; Thompson, Cecilia M.; Bailey, Aubrey; Villamor, Femi Cleola S.; Lange, Philip T.; Wong, Jason P.; Seltzer, Tischan; Seltzer, Jedediah; Zhou, Yijun; Vahrson, Wolfgang; Juarez, Angelica; Meyo, James O.; Calabre, Tiphaine; Broussard, Grant; Rivera-Soto, Ricardo; Chappell, Danielle L.; Baric, Ralph S.; Damania, Blossom; Miller, Melissa B.; Dittmer, Dirk P.

doi:10.1101/2020.06.19.161141

Cited by 23 publications

(29 citation statements)

References 57 publications

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“…We tested for differences in viral loads between patients infected with either the 614D or the 614G viral variants by comparing cycle threshold (Ct) values. Ct values are inversely correlated with viral load, and differences in Ct values between these two variants have been reported previously ( 4 , 7 ). To test this, we analyzed 1770 SARS-CoV-2 sequenced samples from Washington State for which we had access to Ct values.…”

Section: Introductionsupporting

confidence: 80%

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State

Mueller

Wagner

Frazar

et al. 2020

Preprint

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The rapid spread of SARS-CoV-2 has gravely impacted societies around the world. Outbreaks in different parts of the globe are shaped by repeated introductions of new lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State to characterize how the spread of SARS-CoV-2 in Washington State (USA) was shaped by differences in timing of mitigation strategies across counties, as well as by repeated introductions of viral lineages into the state. Additionally, we show that the increase in frequency of a potentially more transmissible viral variant (614G) over time can potentially be explained by regional mobility differences and multiple introductions of 614G, but not the other variant (614D) into the state. At an individual level, we see evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we do not find any evidence that the 614G variant impacts clinical severity or patient outcomes. Overall, this suggests that at least to date, the behavior of individuals has been more important in shaping the course of the pandemic than changes in the virus.

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Section: Introductionsupporting

confidence: 80%

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State

Mueller

Wagner

Frazar

et al. 2020

Preprint

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“…Answering this question will require correlation with rates of transmission through populations, or with disease outcome, in combination with detailed assessment of its effects on viral biochemistry and replication, some of which are examined in experiments described below. To date, more severe disease outcome with D614G has not been reported, but several studies suggest that it is associated with a modest increase in viral load (Korber et al, 2020;McNamara et al, 2020;Wagner et al). Frequencies are interpolated for dates without published sequence.…”

Section: Resultsmentioning

confidence: 99%

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

Yurkovetskiy

Wang

Pascal

et al. 2020

Preprint

153

160

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AbstractVirus genome sequence variants that appear over the course of an outbreak can be exploited to map the trajectory of the virus from one susceptible host to another. While such variants are usually of no functional significance, in some cases they may allow the virus to transmit faster, change disease severity, or confer resistance to antiviral therapies. Since the discovery of SARS-CoV-2 as the cause of COVID-19, the virus has spread around the globe, and thousands of SARS-CoV-2 genomes have been sequenced. The rate of sequence variation among SARS-CoV-2 isolates is modest for an RNA virus but the enormous number of human-to-human transmission events has provided abundant opportunity for selection of sequence variants. Among these, the SARS-CoV-2 Spike protein variant, D614G, was not present in the presumptive common ancestor of this zoonotic virus but was first detected in late January in Germany and China. The D614G variant steadily increased in frequency and now constitutes >97% of isolates world-wide, raising the question whether D614G confers a replication advantage to SARS-CoV-2. Structural models predict that D614G would disrupt contacts between the S1 and S2 domains of the Spike protein and cause significant shifts in conformation. Using single-cycle vectors we showed that D614G is three to nine-fold more infectious than the ancestral form on human lung and colon cell lines, as well as on other human cell lines rendered permissive by ectopic expression of human ACE2 and TMPRSS2, or by ACE2 orthologues from pangolin, pig, dog, or cat. Nonetheless, monoclonal antibodies targeting the receptor binding domain of the SARS-CoV-2 Spike protein retain full neutralization potency. These results suggest that D614G was selected for increased human-to-human transmission, that it contributed to the rapidity of SARS-CoV-2 spread around the world, and that it does not confer resistance to antiviral therapies targeting the receptor binding domain.

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“…Monitoring of global and local disease spread during the SARS-CoV-2 pandemic requires fast, sensitive and precise methods for pathogen genotyping. Amplicon-based approaches are currently the most widely used method in this context (16-21), yet their limits of performance have not been systematically evaluated. Consequently, the quality of the genomes obtained cannot be easily assessed, resulting in errors that are difficult to trace and submission of viral genome sequences of poor quality to public repositories (11-14).…”

Section: Discussionmentioning

confidence: 99%

Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

Kubik

Marques

Xing

et al. 2020

Preprint

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Background: SARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. Results: We used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. Conclusions: We present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

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High-density amplicon sequencing identifies community spread and ongoing evolution of SARS-CoV-2 in the Southern United States

Cited by 23 publications

References 57 publications

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

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