Within-host genetic diversity of SARS-CoV-2 in the context of large-scale hospital-associated genomic surveillance (preprint)

The COVID-19 pandemic has resulted in extensive surveillance of the genomic diversity of SARS-CoV-2. Sequencing data generated as part of these efforts can also capture the diversity of the SARS-CoV-2 virus populations replicating within infected individuals. To assess this within-host diversity of SARS-CoV-2 we quantified low frequency (minor) variants from deep sequence data of thousands of clinical samples collected by a large urban hospital system over the course of a year. Using a robust analytical pipeline to control for technical artefacts, we observe that at comparable viral loads, specimens from patients hospitalized due to COVID-19 had a greater number of minor variants than samples from outpatients. Since individuals with highly diverse viral populations could be disproportionate drivers of new viral lineages in the patient population, these results suggest that transmission control should pay special attention to patients with severe or protracted disease to prevent the spread of novel variants.

Analysis of the ARTIC version 3 and version 4 SARS-CoV-2 primers and their impact on the detection of the G142D amino acid substitution in the spike protein (preprint)

The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses. ImportanceARTIC Network primers are commonly used by laboratories worldwide to amplify and sequence SARS-CoV-2 present in clinical samples. As new variants have evolved and spread, it was found that the V3 primer set poorly amplified several key mutations. In this report, we compare the results of sequencing a matched set of samples with the V3 and V4 primer sets. We find that adoption of the ARTIC V4 primer set is critical for accurate sequencing of the SARS-CoV-2 spike region. The absence of metadata describing the primer scheme used will negatively impact the downstream use of publicly available SARS-Cov-2 sequencing reads and assembled genomes.

Delta variants of SARS-CoV-2 cause significantly increased vaccine breakthrough COVID-19 cases in Houston, Texas (preprint)

Genetic variants of SARS-CoV-2 have repeatedly altered the course of the COVID-19 pandemic, and disease in individual patients. Delta variants (B.1.617.2, AY.2, and AY.3) are now the focus of international concern because they are causing widespread COVID-19 disease globally. Vaccine breakthrough cases caused by SARS-CoV-2 variants also are of considerable public health and medical concern worldwide. As part of a comprehensive project, we sequenced the genomes of 3,913 SARS-CoV-2 from patient samples acquired March 15, 2021 through July 3, 2021 in the Houston Methodist hospital system and studied vaccine breakthrough cases. During the study period Delta variants increased to cause 58% of all COVID-19 cases and spread throughout the metropolitan Houston area. In addition, Delta variants caused a significantly higher rate of vaccine breakthrough cases (19.7% compared to 5.8% for all other variants). Importantly, only 6.5% of all COVID-19 cases occurred in fully immunized individuals, and relatively few of these patients required hospitalization. Our genomic and epidemiologic data emphasize that vaccines used in the United States are highly effective in decreasing severe COVID-19 disease, hospitalizations, and deaths.

The rapid reemergence of seasonal respiratory viruses in Houston, Texas, after relaxing COVID-19 restrictions (preprint)

Brief abstract Implementation of measures to limit the spread of the SARS-CoV-2 virus at the start of the COVID-19 pandemic resulted in a rapid decrease in all other respiratory pathogens. As COVID-19 containment measures were relaxed, the first non-COVID respiratory viruses to return to prepandemic levels were members of the rhinovirus/enterovirus, followed by the rapid return of seasonal coronaviruses, parainfluenza, and respiratory syncytial virus after the complete removal of COVID-19 precautions at the state level, including an end to mask mandates. Inasmuch as COVID-19 has dominated the landscape of respiratory infections since early 2020, it is important for clinicians to recognize the return of non-COVID respiratory pathogens may be rapid and significant when COVID-19 containment measures are removed.

Identification and trajectory of growth of concerning SARS-CoV-2 variants in Houston, Texas, January through April 2021 based on 11,568 genome sequences (preprint)

Genetic variants of the SARS-CoV-2 virus are of substantial concern because they can detrimentally alter the pandemic course and disease features in individual patients. Here we report SARS-CoV-2 genome sequences from 12,476 patients in the Houston Methodist healthcare system diagnosed from January 1, 2021 through May 31, 2021. The SARS-CoV-2 variant designated U.K. B.1.1.7 increased rapidly and caused 63%-90% of all new cases in the Houston area in the latter half of May. Eleven of the 3,276 B.1.1.7 genomes had an E484K change in spike protein. Compared with non-B.1.1.7 patients, individuals with B.1.1.7 had a significantly lower cycle threshold value (a proxy for higher virus load) and significantly higher rate of hospitalization. Other variants (e.g., B.1.429, B.1.427, P.1, P.2, and R.1) also increased rapidly, although the magnitude was less than for B.1.1.7. We identified 22 patients infected with B.1.617 "India" variants; these patients had a high rate of hospitalization. Vaccine breakthrough cases (n=207) were caused by a heterogeneous array of virus genotypes, including many that are not variants of interest or concern. In the aggregate, our study delineates the trajectory of concerning SARS-CoV-2 variants circulating in a major metropolitan area, documents B.1.1.7 as the major cause of new cases in Houston, and heralds the arrival and spread of B.1.617 variants in the metroplex.

Rapid, widespread, and preferential increase of SARS-CoV-2 B.1.1.7 variant in Houston, TX, revealed by 8,857 genome sequences (preprint)

Genetic variants of the SARS-CoV-2 virus have become of great interest worldwide because they have the potential to detrimentally alter the course of the SARS-CoV-2 pandemic, and disease in individual patients. We recently sequenced 20,453 SARS- CoV-2 genomes from patients with COVID-19 disease in metropolitan Houston (population 7 million), dating from March 2020 to early February 2021. We discovered that all major variants of concern or interest are circulating in the region. To follow up on this discovery, we analyzed 8,857 genome sequences from patients in eight Houston Methodist hospitals dispersed throughout the metroplex diagnosed from January 1, 2021 to March 7, 2021. This sample represents 94% of Houston Methodist cases and 4.8% of all reported cases in metropolitan Houston during this period. We discovered rapid, widespread, and preferential increase of the SARS-CoV-2 UK B.1.1.7 throughout the region. The estimated case doubling time in the Houston area is 6.9 days. None of the 648 UK B.1.1.7 samples identified had the E484K change in spike protein that can cause decreased recognition by antibodies.

Sequence Analysis of 20,453 SARS-CoV-2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern (preprint)

[Abstract]Since the beginning of the SARS-CoV-2 pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several "variants of concern" emerged globally, including the UK variant (B.1.1.7), South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California "variants of interest" (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility capacity. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. Based on our extensive genome sequencing program involving 20,453 virus specimens from COVID-19 patients dating from March 2020, we report identification of all important SARS-CoV-2 variants among Houston Methodist Hospital patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency in the population, they are geographically widespread. Houston is the first city in the United States to have all variants documented by genome sequencing. As vaccine deployment accelerates worldwide, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence and frequency of consequential variants and their patterns and trajectory of dissemination. This information is critical for medical and public health efforts to effectively address and mitigate this global crisis.

Molecular Architecture of Early Disseminationand Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area (preprint)

We sequenced the genomes of 5,085 SARS-CoV-2 strains causing two COVID-19 disease waves in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston, and 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 genotypes 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 CR30022. Our study is 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. IMPORTANCEThere 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, Texas, 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, evolution, and relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our study provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.

Treatment of COVID-19 Patients with Convalescent Plasma in Houston, Texas (preprint)

Background: COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for more than 100 years. Methods: Patients (n=25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28 to April 14, 2020. Patients were transfused with convalescent plasma obtained from donors with confirmed SARS-CoV-2 infection and had been symptom free for 14 days. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 post-transfusion. Clinical improvement was assessed based on a modified World Health Organization 6-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. Results: At baseline, all patients were receiving supportive care, including anti-inflammatory and anti-viral treatments, and all patients were on oxygen support. At day 7 post-transfusion with convalescent plasma, nine patients had at least a 1-point improvement in clinical scale, and seven of those were discharged. By day 14 post-transfusion, 19 (76%) patients had at least a 1-point improvement in clinical status and 11 were discharged. No adverse events as a result of plasma transfusion were observed. The whole genome sequencing data did not identify a strain genotype-disease severity correlation. Conclusions: The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease. Randomized, controlled trials are needed to determine its efficacy.

Molecular Architecture of Early Dissemination and Evolution of the SARS-CoV-2 Virus in Metropolitan Houston, Texas (preprint)

We sequenced the genomes of 320 SARS-CoV-2 strains from COVID-19 patients in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. These genomes were from the viruses causing infections in the earliest recognized phase of the pandemic affecting Houston. Substantial viral genomic diversity was identified, which we interpret to mean that the virus was introduced into Houston many times independently by individuals who had traveled from different parts of the country and the world. The majority of viruses are apparent progeny of strains derived from Europe and Asia. We found no significant evidence of more virulent viral types, stressing the linkage between severe disease, underlying medical conditions, and perhaps host genetics. We discovered a signal of selection acting on the spike protein, the primary target of massive vaccine efforts worldwide. The data provide a critical resource for assessing virus evolution, the origin of new outbreaks, and the effect of host immune response. SignificanceCOVID-19, the disease caused by the SARS-CoV-2 virus, is a global pandemic. To better understand the first phase of virus spread in metropolitan Houston, Texas, we sequenced the genomes of 320 SARS-CoV-2 strains recovered from COVID-19 patients early in the Houston viral arc. We identified no evidence that a particular strain or its progeny causes more severe disease, underscoring the connection between severe disease, underlying health conditions, and host genetics. Some amino acid replacements in the spike protein suggest positive immune selection is at work in shaping variation in this protein. Our analysis traces the early molecular architecture of SARS-CoV-2 in Houston, and will help us to understand the origin and trajectory of future infection spikes.