Investigating the Cause of the 2022 Severe Acute Hepatitis Outbreak in Children
AAV2 virus detected with metagenomics, TE sequencing, and PCR in non-A–E hepatitis cases from two cohorts in Scotland/the UK and the US.
More than a thousand children across 35 countries were reported to have severe acute hepatitis as of July 8, 2022, resulting in 22 deaths.1 In viral hepatitis, inflammation of the liver is caused by one of the main hepatitis viruses (A–E), but the cause of this 2022 outbreak remained unknown. While human adenoviruses (HAdV) were suspected to be underlying in these cases of non-A–E hepatitis, two recent and simultaneous Nature publications present evidence for the role of adeno-associated virus type 2 (AAV2) in cases from Scotland and the UK reported by Ho et al.,2 as well as from the US by Servellita et al.3
What Is AAV2?
AAV2 is a small, non-enveloped virus with a single-stranded 4,675-nucleotide DNA genome. It typically infects people at a young age and is present in up to 80 percent of the adult population, so it is considered nonpathogenic in humans and even used as a gene therapy vector.4 AAV2 is known to require coinfection with a helper virus for replication, most commonly HAdV or human herpesvirus (HHV).
What Is Non-A–E Hepatitis?
Both studies obtained blood, liver tissue, fecal, and various types of swab samples from children hospitalized with non-A–E hepatitis after presenting with gastroenteritis-like symptoms such as nausea, vomiting, jaundice, generalized weakness, and abdominal pain. In the US cohort, acute elevation in the liver enzymes alanine transaminase (ALT) or aspartate transaminase (AST) was confirmed for all cases. In the Scotland and UK cohorts, liver biopsies and samples were reported to show the following:
- Lobular hepatitis
- Presence of immune cellular markers, including CD3, CD4, CD8, PD-L1, CD107a, CD20, CD31, CD44, CD68, MX1, and panCK—revealed through co-detection by indexing (CODEX)
- Disordered proliferation of epithelial cells
- Increased numbers of CD68+ macrophages, activated CD4+ and CD8+ T cells, and CD20+ B cells
- High expression of interferon-induced GTP-binding protein MX1
Scotland and UK Cohort Studied for Non-A–E Hepatitis
Ho et al. recruited 32 children affected by non-A–E hepatitis and compared samples from these children to unaffected control samples obtained from existing study cohorts. The usual causes of hepatitis were ruled out: most of the affected children had no known medical conditions nor did they test positive in routine blood tests for viral hepatitis (types A, B, C, and E), acute Epstein–Barr virus (EBV), cytomegalovirus (CMV), HHV-6 and HHV-7, herpes simplex virus (HSV), or autoimmunity markers.
Therefore, to understand the etiology underlying these pediatric cases of acute non-A–E hepatitis, the Scotland and UK-based researchers performed metagenomics and target enrichment (TE) next-generation sequencing (NGS) on plasma, liver biopsy, throat and rectal swabs, and fecal samples. The samples were obtained between seven and 80 days after initial symptom onset, with an average of 14 million sequence reads per sample.
Presence of AAV2 in the Scotland and UK Study Samples
Metagenomics and TE sequencing on samples from the first nine recruited patients—confirmed with PCR testing—most frequently detected the viral genome of AAV2 in affected patients and did not detect any AAV2 in the two comparison groups—healthy controls (group 1) and children with HAdV infection and normal liver function (group 2). The study found seven distinct sequences of AAV2, alongside four previously detected AAV2 genomes. HAdVs and human herpesvirus 6B (HHV-6B) were also detected in affected and comparison samples.
Next, as part of a case-control study, samples from all 32 cases of hepatitis were compared with samples from comparison groups 1 and 2, as well as two additional comparison groups—children with increased transaminase levels that were HAdV-negative (group 3) and those attending the hospital at the same time as the affected children (group 4). Again, AAV2 was shown to be significantly higher in the children affected by acute hepatitis, with AAV2 infection being detected in 81 percent of the hepatitis cases.
US Cohort Studied for Non-A–E Hepatitis
Servellita et al. analyzed a combination of whole blood, plasma, liver tissue, nasopharyngeal swab, and fecal samples from 16 children with severe, acute non-A–E hepatitis who also all tested positive for HAdV. The 113 controls included those:
- without hepatitis,
- with acute hepatitis of defined etiology,
- with acute gastroenteritis, and
- blood donors.
The California-based researchers and their collaborators detected AAV2 in whole blood or plasma samples for 93 percent of affected cases but not in most of the control cases. The methods they used were metagenomic sequencing, including probe capture viral enrichment, tiling multiplex PCR amplicon sequencing for AAV2 and HAdV-41, and virus-specific PCR testing for HAdV with genotyping by hexon gene sequencing. Some of the cases were also positive for other viruses (EBV, CMV, enterovirus A71 (EV-A71), HAdV-1, HAdV2, and HHV-6).
Phylogenetic Analysis of AAV2 Recovered from Non-A–E Hepatitis Cases
Multiple sequence alignment and phylogenetic analysis of 13 recovered AAV2 genomes from 12 cases by Servellita et al. revealed that the 13 genomes were located within a distinct subgroup of a large human-infecting AAV2 clade. They found 35 substitutions, of which 25 were the same as those independently identified by Ho et al. These substitutions are thought to impact the binding of AAV2 with its heparan sulfate proteoglycan receptor, improving the virus’ ability to infect its host and cause severe illness.
Genetic Susceptibility to Non-A–E Hepatitis in Scotland and the UK
To investigate the children’s genetic susceptibility to non-A–E hepatitis, Ho et al. also performed high-resolution genotyping for all human leukocyte antigen (HLA) loci. The HLA-DRB1*04:01 allele had a significantly higher frequency (93 percent) in hepatitis cases. Along with the DRB1 allele, there were also associations with the DQA1 and DRB4 alleles, so it is unclear if there is a single causal allele underlying the observed susceptibility.
Conclusions
AAV2 was detected in most of the affected children, particularly in whole blood or plasma samples. HAdV, HHV-6, and other viruses were also detected. Presence of SARS-CoV-2 was ruled out. Phylogenetic analysis of the AAV2 genomes from the affected children revealed several substitutions mapping to a distinct subclade, while genotyping the children shed light on specific HLA alleles in disease susceptibility. Together, these genetic changes provide researchers hints regarding AAV2-dependent hepatitis mechanisms.
The studies by Ho et al. and Servellita et al. implicate AAV2 coinfection in the 2022 outbreak of pediatric non-A–E hepatitis and suggest that coinfection exacerbates liver disease more than infection by an adenovirus or herpesvirus alone. Further investigation will be necessary to help establish a causal link and disease mechanism for AAV2 coinfection underlying severe acute non-A–E hepatitis in children.
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