Adeno-associated virus type 2 in US children with acute severe hepatitis

Abstract

As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA^1,2. Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA^3-7, although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls (P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology (P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases (P < 0.001). Co-infections by Epstein-Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls (P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.

Fig. 1. Epidemiology of cases and controls.

Geographic distribution of the 16 acute severe hepatitis cases and 113 controls in the study, showing the hospital or public health laboratory sites providing samples and associated clinical data from cases and/or controls. WB, whole blood; NP, nasopharyngeal.

Fig. 2. Sequencing and molecular-based testing for viruses carried out on cases and controls.

a, Analyses carried out on the different sample groups. The specific assays carried out for each group are shown in green whereas assays that were not carried out are shaded in black. WGS, whole-genome sequencing. mNGS, metagenomic next-generation sequencing. b, Graphical chart showing results of sequencing or PCR-based assays for virus detection. Each circle represents a sequenced sample with at least three non-overlapping reads aligning to a viral reference sequence. Circles are colour-coded on the basis of sample type and scaled according to normalized viral read counts. Viral PCR positive and negative results are denoted by plus and minus symbols, respectively. For the HAdV-F PCR, the HAdV subtype identified by hexon sequencing is shown to the left of the viral PCR result. PBV, picobirnavirus; bx, biopsy; ND, not determined. c, Associations between viruses detected in blood and cases of acute severe hepatitis of unknown aetiology in children. Red and blue shading indicate positive and negative detection, respectively. Uncorrected P values were calculated using two-tailed Fisher’s exact test. A Bonferroni-corrected significance level of P < 0.002 was considered significant (n = 24 comparisons). Exact P values are provided in Supplementary Table 2. Ca, cases; Co, controls; AL, Alabama; CA, California; FL, Florida; GA, Georgia; IL, Illinois; NC, North Carolina; OH, Ohio; SD, South Dakota; TX, Texas; WA, Washington; NS, not significant; ***P < 0.001; ****P < 0.0001.

Fig. 3. Phylogenetic and substitution analysis of AAV2 genomes.

a, Phylogenetic tree of 119 AAV2 genomes available from GenBank as of 18 August 2022. The 12 recovered genomes from this study with >25% coverage are denoted in red. The location of the AAV2 reference genome (NC_001401.2) is marked with a black asterisk. The phylogenetic tree was constructed by multiple sequence alignment of the AAV genomes or amino acid sequences using the MAFFT algorithm, followed by maximum-likelihood-based tree construction using IQ-TREE. b, Multiple sequence alignment of 13 AAV2 genomes recovered from cases. Nucleotide mismatches are represented by black-coloured vertical lines, whereas areas of missing coverage are represented by dark grey rectangles. Amino acid variants with respect to the reference genome (NC_001401.2) are denoted in blue and red arrows; red arrows indicate shared substitutions that were reported in another study from the UK and Scotland. Substitution sites that were identified in 100% of cases with sufficient coverage in both studies are highlighted in bold red text. ITR, inverted terminal repeat.

Extended Data Fig. 1. Amino acid phylogenetic analysis of AAV2 genomes from cases, related to Fig. 3.

(A) Phylogenetic tree corresponding to the Assembly Activating Protein (AAP) protein. (B) Phylogenetic tree corresponding to the VP1 protein. Phylogenetic trees was constructed by multiple sequence alignment of the AAV genomes or amino acid sequences using the MAFFT algorithm, followed by maximum likelihood based tree construction using IQ-TREE.