High Prevalence of SARS-CoV-2 Genetic Variation and D614G Mutation in Pediatric Patients With COVID-19

Abstract

Background: The full spectrum of the disease phenotype and viral genotype of coronavirus disease 2019 (COVID-19) have yet to be thoroughly explored in children. Here, we analyze the relationships between viral genetic variants and clinical characteristics in children.

Methods: Whole-genome sequencing was performed on respiratory specimens collected for all SARS-CoV-2-positive children (n = 141) between March 13 and June 16, 2020. Viral genetic variations across the SARS-CoV-2 genome were identified and investigated to evaluate genomic correlates of disease severity.

Results: Higher viral load was detected in symptomatic patients (P = .0007) and in children <5 years old (P = .0004). Genomic analysis revealed a mean pairwise difference of 10.8 single nucleotide variants (SNVs), and the majority (55.4%) of SNVs led to an amino acid change in the viral proteins. The D614G mutation in the spike protein was present in 99.3% of the isolates. The calculated viral mutational rate of 22.2 substitutions/year contrasts the 13.5 substitutions/year observed in California isolates without the D614G mutation. Phylogenetic clade 20C was associated with severe cases of COVID-19 (odds ratio, 6.95; P = .0467). Epidemiological investigation revealed major representation of 3 of 5 major Nextstrain clades (20A, 20B, and 20C) consistent with multiple introductions of SARS-CoV-2 in Southern California.

Conclusions: Genomic evaluation demonstrated greater than expected genetic diversity, presence of the D614G mutation, increased mutation rate, and evidence of multiple introductions of SARS-CoV-2 into Southern California. Our findings suggest a possible association of phylogenetic clade 20C with severe disease, but small sample size precludes a definitive conclusion. Our study warrants larger and multi-institutional genomic evaluation and has implications for infection control practices.

Keywords: COVID-19; D614G; SARS-CoV-2; children; clade 20C; genomic epidemiology; viral sequencing.

Figure 1.

Viral genotypes and clinical features of CHLA patients. Maximum likelihood tree showing phylogenetic clades of CHLA isolates in relation to a subsample of global isolates. CHLA isolates are shown in red. Clade-defining mutations are shown with various clinical metadata indicated on the right. Each row represents a single patient. Nextstrain clades: orange = 20B, blue = 20C, green = 20A. Disease severity: pink = severe, orange = moderate, yellow = mild, green = asymptomatic. Race/ethnicity: blue = Hispanic, white = unknown, purple = Black, gray = other, red = Asian, green = White. Location: red = ICU, blue = inpatient, green = ED, pink = outpatient. Age: green = ≥10 years, gray = ≥5 to <10 years, black = ≥1 to <5 years, blue = <1 year. Chronic diseases: red = present, green = absent. Days since symptom onset: purple = 1–1.5 days, black = >2 days, white = unknown. Imaging: red = chest imaging with significant findings, green = clear, white = unknown. Abbreviations: CHLA, Children’s Hospital Los Angeles; ED, emergency department.

Figure 2.

Viral load comparison among patient groups. Increased viral loads were observed in (A) symptomatic individuals, (B) patients tested within 2 days of symptoms, and (C) patients who were <5 years old. The line depicts the median viral load. Statistical analysis was performed using the Mann-Whitney test (^**P < .005; ^***P < .0005). Abbreviation: CHLA, Children’s Hospital Los Angeles.

Figure 3.

Single nucleotide variations in genomes obtained from 88 CHLA patients with available medical records. A, Summary of characteristics of SNVs in open reading frames and noncoding regions. B, Spread of SNVs across the genome in 88 CHLA isolates as compared with the Wuhan isolate (NC_045512.2). Vertical red lines on the genomes indicate presence of an SNV relative to the reference genome. Genomes are grouped based on disease severity seen in patients. The panel on the right shows the Nextstrain clade for each isolate. Upper panel shows the structure of the severe acute respiratory syndrome coronavirus 2 genome. Abbreviations: CHLA, Children’s Hospital Los Angeles; ORF, open reading frame; SNV, single nucleotide variant.