Topics:
pediatric inflammatory multisystem syndrome, sars-cov-2, polymorphism

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China, in December 2019. It rapidly spread across the world, and on March 11, 2020, the World Health Organization declared COVID-19 a global pandemic.1 

In adults, SARS-CoV-2 can manifest as severe interstitial pneumonia and hyperinflammation, with∼3% to 5% of infections requiring admission to critical care.2,3  In contrast, severe illness and death due to SARS-CoV-2 infection in children is rare.4  However, recently, a small number of cases of shock and multisystem inflammation have been reported in children who have either tested positive for SARS-CoV-2 (by polymerase chain reaction or serology) or have had epidemiological links to it. This new syndrome, which has overlapping features of Kawasaki disease, is called multisystem inflammatory syndrome in children (MIS-C).5 

The exact pathogenesis of MIS-C is as yet unknown. However, it has been suggested that part of the SARS-CoV-2 viral spike protein may resemble a superantigen that could drive the development of MIS-C and trigger cytokine storms in adults.6  Specifically, polymorphic residues in the viral spike protein, including A831V and D839Y/N/E, which are predicted to enhance binding affinity to the T-cell receptor, have been observed in lineages circulating in Europe and North America, where most MIS-C cases have been described. In addition, the 614G spike protein polymorphism may be associated with increased transmission and altered SARS-CoV-2 biology.7  Phylogenetic comparisons of SARS-CoV-2 viral sequences from patients with MIS-C and patients without MIS-C are necessary to identify the significance of viral polymorphisms in the etiology of MIS-C.

To this end, we compare SARS-CoV-2 viral sequences found in 5 children diagnosed with MIS-C with sequences from 8 children not diagnosed with MIS-C and 130 community case patients in North London.

We sequenced SARS-CoV-2 from children hospitalized for COVID-19 in London between late-March and mid-May 2020. Of 61 children hospitalized with COVID-19, confirmed by polymerase chain reaction for SARS-CoV-2 RNA in nasopharyngeal aspirates and/or by the presence of SARS-CoV-2 antibodies (Meso Scale Discovery Chemiluminescent binding assay; Meso Scale Diagnostics, LLC, Rockville, MD), 36 were diagnosed with MIS-C on the basis of the case definition of Royal College of Paediatrics and Child Health.8,9  The patients classified as not having MIS-C were asymptomatically infected or had predominantly respiratory symptoms with no evidence of multiorgan involvement, widespread inflammation, shock, or Kawasaki-like symptoms. Eleven of the 36 patients with MIS-C were positive for SARS-CoV-2 viral RNA, and the remainder were positive for antibodies to SARS-CoV-2 (Meso Scale Discovery Chemiluminescent binding assay; Meso Scale Diagnostics, LLC). All 25 patients without MIS-C were positive for SARS-CoV-2 viral RNA. Full-length SARS-CoV-2 genome sequences were obtained from 5 children classified as having MIS-C and 8 children classified as not having MIS-C (Table 1) by using SureSelectXT target enrichment and Illumina sequencing. Reads generated were quality checked and mapped to the SARS-COV-2 reference genome (NC_045512) by using Burrows-Wheeler Aligner software version 0.7.17. Sequences are available on GISAID (Accession identifier: EPI_ISL_479777 to EPI_ISL_479789). Demographics of the 5 children with MIS-C and the 8 children without MIS-C are summarized in Table 1.

TABLE 1

Clinical Characteristics of Patients With and Without MIS-C

Patient12345678910111213
Age12 y, 1 mo5 y 8 mo14 y 8 mo15 y 0 mo8 y 9 mo5 y 11 mo8 y 11 mo1 y 8 mo2 y 11 mo10 mo10 y 3 mo1 y 4 mo1 mo
Days in hospital 10 15 18 82 8 44 14 19 284 268 22 87 23 
Days in ICU 3 0 10 16 2 — 11 19 — — — — 22 
Sex Male Female Male Female Male Female Female Male Male Male Male Female Male 
Ethnicity Black  White Mixed Asian Asian Asian Asian Black  Other Other Black  Other White 
Comorbidities Seizure disorder Seizure disorder, right upper lobectomy Previously well Previously well Previously well Metabolic disease Bronchiectasis, lobectomy Hepatoblastoma, chronic lung disease, BiPAP Metastatic neuroblastoma  Prematurely born infant 23 wk, short gut; chronic lung disease T-cell lymphoma Primitive neuroectodermal tumor Extremely premature infant 32 + 6/7 wk gestation, cleft palate 
Clinical presentation Borderline MIS-C MIS-C MIS-C MIS-C MIS-C Asymptomatic Respiratory deterioration Respiratory deterioration URT URT Enterobacter line infection Asymptomatic Respiratory deterioration 
Fever Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No No 
Fatigue Yes Yes Yes Yes Yes No Yes No Yes No No No No 
Cough No Yes Yes Yes Yes No Yes Yes Yes Yes No No Yes 
URT symptoms No No Yes Yes Yes No No Yes Yes Yes No No Yes 
Dyspnea Yes Yes Yes Yes Yes No Yes No Yes No No No Yes 
Abdominal pain No No Yes No No No Yes Yes No No Yes No No 
Vomiting No Yes Yes Yes Yes No Yes Yes No Yes Yes No No 
Diarrhea No No Yes Yes Yes No Yes Yes No No No No No 
Headache No No No No Yes No Yes No No No Yes No No 
Seizure Yes Yes No No No No No No No No No No No 
Meningitis No No No No No No No No No No No No No 
Anosmia No No Yes No No No No No No No No No No 
Rash No No No Yes Yes No No No No No No No No 
Edema No No Yes Yes No No No No No No No No No 
Shock requiring resuscitation Mild Yes Yes Yes Yes No No No No No No No No 
Mechanical ventilation Yes No Yes Yes Yes No Yes No No No No No Yes 
Highest neutrophil count, × 109/L 3.98 11.58 15.46 30.34 13.91 0.94 5.67 0.13 2.3 3.52 2.74 2.39 8.49 
Lowest lymphocyte count, × 109/L 0.57 3.98 1.26 0.35 0.47 1.47 0.19 0.66 0.43 3.58 0.19 0.51 1.18 
Highest CRP level, mg/L 158 323 449 290 311 <5 284 253 49 <5 28 <5 63 
Highest ferritin level, μg/L — — 1452 63 626 990 — 366 2496 754 9.5 786 — 789 
Highest D-dimer level, μg/L — — 6742 14 798 7085 212 1737 2332 — 415 265 — 1160 
Highest fibrinogen level, g/L 3.4 7.49 12.8 5 6.6 2.4 6.5 5.7 — 2.6 5.7 3.3 3.9 
Lowest albumin level, g/L 34 34 29 <20 27 40 25 29 29 27 34 36 20 
Highest LDH level, U/L — — 1905 5764 1018 — 1022 685 602 — 423 868 — 
Highest ALT level, U/L 108 6 121 482 101 104 41 44 85 89 25 147 59 
Patient12345678910111213
Age12 y, 1 mo5 y 8 mo14 y 8 mo15 y 0 mo8 y 9 mo5 y 11 mo8 y 11 mo1 y 8 mo2 y 11 mo10 mo10 y 3 mo1 y 4 mo1 mo
Days in hospital 10 15 18 82 8 44 14 19 284 268 22 87 23 
Days in ICU 3 0 10 16 2 — 11 19 — — — — 22 
Sex Male Female Male Female Male Female Female Male Male Male Male Female Male 
Ethnicity Black  White Mixed Asian Asian Asian Asian Black  Other Other Black  Other White 
Comorbidities Seizure disorder Seizure disorder, right upper lobectomy Previously well Previously well Previously well Metabolic disease Bronchiectasis, lobectomy Hepatoblastoma, chronic lung disease, BiPAP Metastatic neuroblastoma  Prematurely born infant 23 wk, short gut; chronic lung disease T-cell lymphoma Primitive neuroectodermal tumor Extremely premature infant 32 + 6/7 wk gestation, cleft palate 
Clinical presentation Borderline MIS-C MIS-C MIS-C MIS-C MIS-C Asymptomatic Respiratory deterioration Respiratory deterioration URT URT Enterobacter line infection Asymptomatic Respiratory deterioration 
Fever Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No No 
Fatigue Yes Yes Yes Yes Yes No Yes No Yes No No No No 
Cough No Yes Yes Yes Yes No Yes Yes Yes Yes No No Yes 
URT symptoms No No Yes Yes Yes No No Yes Yes Yes No No Yes 
Dyspnea Yes Yes Yes Yes Yes No Yes No Yes No No No Yes 
Abdominal pain No No Yes No No No Yes Yes No No Yes No No 
Vomiting No Yes Yes Yes Yes No Yes Yes No Yes Yes No No 
Diarrhea No No Yes Yes Yes No Yes Yes No No No No No 
Headache No No No No Yes No Yes No No No Yes No No 
Seizure Yes Yes No No No No No No No No No No No 
Meningitis No No No No No No No No No No No No No 
Anosmia No No Yes No No No No No No No No No No 
Rash No No No Yes Yes No No No No No No No No 
Edema No No Yes Yes No No No No No No No No No 
Shock requiring resuscitation Mild Yes Yes Yes Yes No No No No No No No No 
Mechanical ventilation Yes No Yes Yes Yes No Yes No No No No No Yes 
Highest neutrophil count, × 109/L 3.98 11.58 15.46 30.34 13.91 0.94 5.67 0.13 2.3 3.52 2.74 2.39 8.49 
Lowest lymphocyte count, × 109/L 0.57 3.98 1.26 0.35 0.47 1.47 0.19 0.66 0.43 3.58 0.19 0.51 1.18 
Highest CRP level, mg/L 158 323 449 290 311 <5 284 253 49 <5 28 <5 63 
Highest ferritin level, μg/L — — 1452 63 626 990 — 366 2496 754 9.5 786 — 789 
Highest D-dimer level, μg/L — — 6742 14 798 7085 212 1737 2332 — 415 265 — 1160 
Highest fibrinogen level, g/L 3.4 7.49 12.8 5 6.6 2.4 6.5 5.7 — 2.6 5.7 3.3 3.9 
Lowest albumin level, g/L 34 34 29 <20 27 40 25 29 29 27 34 36 20 
Highest LDH level, U/L — — 1905 5764 1018 — 1022 685 602 — 423 868 — 
Highest ALT level, U/L 108 6 121 482 101 104 41 44 85 89 25 147 59 

ALT, alanine transaminase; BiPAP, bilevel positive airway pressure; CRP, C-reactive protein; LDH, lactose dehydrogenase; URT, upper respiratory tract infection; —, not applicable.

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We reconstructed a maximum likelihood phylogenetic tree of all sequences using RAxML version 8.0.0 using model GTRGAMMA and 1000 bootstrap replicates.10  In addition, single-nucleotide polymorphisms (SNPs) were identified by comparison with the SARS-CoV-2 reference genome (NC_045512) and classified as synonymous or nonsynonymous.

Categorical data are presented as proportions. Comparison of categorical data between groups (MIS-C, non-MIS-C, and community cases) was made by using a χ2 test in R.11 

The maximum likelihood phylogeny of 13 pediatric SARS-CoV-2 cases and 130 SARS-CoV-2 sequences generated from community cases across North London is shown in Fig 1A. We found no clustering of viral sequences from patients with MIS-C (red) or patients without MIS-C (blue) in relation to other local sequences (black).

FIGURE 1
FIGURE 1. Characteristics of SARS-CoV-2 sequences from children with and without MIS-C. A, Phylogenetic tree of sequences analyzed in this study. Tips colored in red represent children with MIS-C; blue, children without MIS-C; and black, other sequences from London with no association to MIS-C. B, Frequency of occurrence of SNPs. In the top graph, 130 London samples are represented in yellow. In the bottom graph, 13 pediatric samples from Great Ormand Street Hospital are presented: red represents 5 MIS-C samples, and blue represents 8 non–MIS-C samples. The x-axis is annotated with a map of the reading frames in the viral genome. E, envelope protein; M, membrane protein; N, nuclearprotein; S, spike protein; UTR, untranslated region.
View largeDownload slide

Characteristics of SARS-CoV-2 sequences from children with and without MIS-C. A, Phylogenetic tree of sequences analyzed in this study. Tips colored in red represent children with MIS-C; blue, children without MIS-C; and black, other sequences from London with no association to MIS-C. B, Frequency of occurrence of SNPs. In the top graph, 130 London samples are represented in yellow. In the bottom graph, 13 pediatric samples from Great Ormand Street Hospital are presented: red represents 5 MIS-C samples, and blue represents 8 non–MIS-C samples. The x-axis is annotated with a map of the reading frames in the viral genome. E, envelope protein; M, membrane protein; N, nuclearprotein; S, spike protein; UTR, untranslated region.

FIGURE 1
FIGURE 1. Characteristics of SARS-CoV-2 sequences from children with and without MIS-C. A, Phylogenetic tree of sequences analyzed in this study. Tips colored in red represent children with MIS-C; blue, children without MIS-C; and black, other sequences from London with no association to MIS-C. B, Frequency of occurrence of SNPs. In the top graph, 130 London samples are represented in yellow. In the bottom graph, 13 pediatric samples from Great Ormand Street Hospital are presented: red represents 5 MIS-C samples, and blue represents 8 non–MIS-C samples. The x-axis is annotated with a map of the reading frames in the viral genome. E, envelope protein; M, membrane protein; N, nuclearprotein; S, spike protein; UTR, untranslated region.
View largeDownload slide

Characteristics of SARS-CoV-2 sequences from children with and without MIS-C. A, Phylogenetic tree of sequences analyzed in this study. Tips colored in red represent children with MIS-C; blue, children without MIS-C; and black, other sequences from London with no association to MIS-C. B, Frequency of occurrence of SNPs. In the top graph, 130 London samples are represented in yellow. In the bottom graph, 13 pediatric samples from Great Ormand Street Hospital are presented: red represents 5 MIS-C samples, and blue represents 8 non–MIS-C samples. The x-axis is annotated with a map of the reading frames in the viral genome. E, envelope protein; M, membrane protein; N, nuclearprotein; S, spike protein; UTR, untranslated region.

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We observed no SNPs unique to the MIS-C or to the other childhood cases and no difference in the distribution of SNPs between MIS-C, non-MIS-C, and community cases, as depicted in Fig 1B. The proportion of nonsynonymous SNPs did not differ in the MIS-C, non-MIS-C, and community cases (χ2 test, degrees of freedom = 2, P =.64).

All childhood cases were D839 and A831, as were all the locally circulating samples. The majority of pediatric inflammatory multisystem syndrome (3 of 5), non–pediatric inflammatory multisystem syndrome (6 of 8), and community cases (118 of 130) were 614G-positive.

Understanding the immunopathological etiology of MIS-C is useful in effective management and treatment of the disease. In this report, we compare viral sequences from children diagnosed with MIS-C with viral sequences from children without MIS-C as well as the wider London community. Overall, the data suggest that the viruses causing MIS-C in our patients are representative of locally circulating SARS-CoV-2. We found no evidence of an association of MIS-C with the presence of new or unusual sequence polymorphisms. This suggests that alternative factors, such as host genetics, may trigger MIS-C. Further studies are required to address this.

This study was approved by Great Ormond Street Hospital (clinical audit number 2857) and Public Health England Research Ethics and Governance Group (R&D NR0195).

This work was supported by the Covid-19 Genomics UK Consortium, the John Black Charitable Foundation, and the University College London Hospitals and Great Ormond Street Hospital National Institute for Health Research biomedical research centers. J.P. is supported by the Rosetrees Foundation, and F.A.T.B is supported by a Wellcome Trust Collaborative Award to J.B. We thank Richard Goldstein, Kathryn Harris, Julianne Brown, Jack Lee, Rachel Williams, Helena Tutill, and Sunando Roy for their contribution to this work. We also acknowledge the contributions of the University College London Pathogen Genomics Unit, University College London Genomics, and the Great Ormond Street Hospital Departments of Infectious Disease and Microbiology, Virology and Infection Control.

Ms Pang and Dr Boshier completed the data analysis and literature search, drafted the initial manuscript, and reviewed and revised the manuscript; Drs Alders and Dixon collected the data and reviewed the manuscript; Dr Breuer conceived the study and completed the data interpretation and literature search; and all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

FUNDING: No external funding.

     
  • COVID-19

    coronavirus disease 2019

    •  
  • MIS-C

    multisystem inflammatory syndrome in children

    •  
  • SARS-CoV-2

    severe acute respiratory syndrome coronavirus 2

    •  
  • SNP

    single-nucleotide polymorphism

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Competing Interests

POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.

Copyright © 2020 by the American Academy of Pediatrics
2020