Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area

Abstract

We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein-the primary target of global vaccine efforts-are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.IMPORTANCE There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.

Keywords: COVID-19; COVID-19 disease; SARS-CoV-2; evolution; genome sequencing; molecular population genomics.

FIG 1

(A) Confirmed COVID-19 cases in the Greater Houston Metropolitan region. Data represent cumulative number of COVID-19 patients over time through 7 July 2020. Counties include Austin, Brazoria, Chambers, Fort Bend, Galveston, Harris, Liberty, Montgomery, and Waller. The shaded area represents the time period (indicated as month/day along the x axis) during which virus genomes characterized in this study were recovered from COVID-19 patients. The red line represents the number of COVID-19 patients diagnosed in the Houston Methodist Hospital Molecular Diagnostic Laboratory. (B) Distribution of strains with either the Asp614 or Gly614 amino acid variant in spike protein among the two waves of COVID-19 patients diagnosed in the Houston Methodist Hospital Molecular Diagnostic Laboratory. The large inset shows major clade frequency for the time frame studied (indicated as month-day to month-day along the x axis).

FIG 2

Sequential time-series heat maps for all COVID-19 Houston Methodist Hospital patients during the study period. The geospatial distribution of COVID-19 patients is based on Zip code. Panel A (left) shows the geospatial distribution of sequenced SARS-CoV-2 strains in wave 1, and panel B (right) shows the wave 2 distribution. The collection dates are shown at the bottom of each panel. The insets refer to numbers of strains in the color spectrum used. Note the differences in the numbers of strains presented in the panel A and panel B insets.

FIG 3

Location of amino acid replacements in RNA-dependent RNA polymerase (RdRp/Nsp12) among the 5,085 genomes of SARS-CoV-2 sequenced. The various RdRp domains are color coded. The numbers refer to amino acid sites. Note that several amino acid sites have multiple variants identified. The dates shown at the bottom of the figure panels represent month/day/year.

FIG 4

Amino acid changes identified in Nsp12 (RdRp) in this study that may influence interactions with remdesivir. The schematic at the top shows the domain architecture of Nsp12. (Left) Ribbon representation of the crystal structure of Nsp12-remdesivir monophosphate-RNA complex (PDB code: 7BV2). (Right) Magnified view of the boxed area in the left panel. The Nsp12 domains are colored as indicated in the schematic at the top. The catalytic site in Nsp12 is marked by a black circle at lower right in the right panel. The side chains of amino acids comprising the catalytic site of RdRp (Ser758, Asp759, and Asp760) are shown as balls and stick and colored yellow. The nucleotide binding site is boxed in the right panel. The side chains of amino acids participating in nucleotide binding (Lys544, Arg552, and Arg554) are shown as balls and sticks and colored light blue. A remdesivir molecule incorporated into the nascent RNA is shown as balls and sticks and colored light pink. The RNA is shown as a blue cartoon, and bases are shown as sticks. The positions of Cα atoms of amino acids identified in this study are shown as red and green spheres and labeled. The amino acids that are shown as red spheres are located above the nucleotide binding site, whereas Cys812 located at the catalytic site is shown as a green sphere. The side chain of active site residue Ser758 is shown as ball and sticks and colored yellow. The location of Cα atoms of remdesivir resistance-conferring amino acid Val556 is shown as a blue sphere and labeled.

FIG 5

Locations of amino acid replacements in spike protein among the 5,085 genomes of SARS-CoV-2 sequenced. The various spike protein domains are color coded. The numbers refer to amino acid sites. Note that many amino acid sites have multiple variants identified.

FIG 6

Location of amino acid substitutions mapped on the SARS-CoV-2 spike protein. The figure presents a model of the SARS-CoV-2 spike protein with one protomer shown as ribbons and the other two protomers shown as a molecular surface. The Cα atom of residues found to be substituted in one or more virus isolates identified in this study is shown as a sphere on the ribbon representation. Residues found to be substituted in 1 to 9 isolates are colored tan, those substituted in 10 to 99 isolates yellow, those substituted in 100 to 999 isolates red (H49Y and F1052L), and those substituted in >1,000 isolates purple (D614G). The surface of the amino-terminal domain (NTD) that is distal to the trimeric axis has a high density of substituted residues. RBD, receptor binding domain.

FIG 7

Cycle threshold (C_T) data for every SARS-CoV-2 patient sample tested using the Hologic Panther assay. Data are presented as means ± standard errors of the means for strains with an aspartate (D614, n = 102 strains, blue) or glycine (G614, n = 812 strains, red) at amino acid 614 of the spike protein. Mann-Whitney test; *, P < 0.0001.

FIG 8

Biochemical characterization of spike RBD variants. (A) Size exclusion chromatography (SEC) traces of the indicated spike-RBD variants. The dashed line indicates the elution peak of spike-6P. mAU, milli-absorbance units. (B) Relative expression levels of all RBD variants as determined by the area under the SEC traces. All expression levels are normalized relative to spike-6P. (C) Thermostability analysis of RBD variants by differential scanning fluorimetry. Each sample had three replicates, and only mean values were plotted. The black vertical dashed line indicates the first melting temperature of 6P-D614G, and the orange vertical dashed line indicates the first melting temperature of the least stable variant (spike-G446V). (D) First apparent melting temperatures of all RBD variants. (E and F) ELISA-based binding affinities for ACE2 receptor (E) and the neutralizing antibody CR3022 (F) to the indicated RBD variants. (G) Summary of EC₅₀ values for all measured RBD variants.