Comparison of three pediatric studies investigating acute hepatitis of unknown etiology

Abstract

Between 2021 and 2023, approximately 400 pediatric cases of acute hepatitis of unknown etiology (AHUE) were reported in European countries and the United States. In 2023, three pediatric studies revealed that adeno-associated virus serotype 2 (AAV-2) infection was associated with AHUE. This article presents a summary and comparison of the results of metagenomic sequencing, viral whole-genome sequencing, virus-specific real-time polymerase chain reaction (PCR) and histological analysis of the liver, all of which were among the common investigative methods used in the three pediatric studies. All three pediatric studies revealed 80% or greater rates of positivity for AAV-2 in cases of AHUE according to metagenomic sequencing. Moreover, on the basis of PCR results, two studies revealed high AAV-2 positivity rates (96.4% and 81.2%) among cases of AHUE. These findings suggest that AAV-2 is a pathogen in AHUE. Coinfection with AAV-2 and one or more helper viruses (human adenovirus, human herpesvirus 6B, Epstein–Barr virus, etc.), high viral loads of AAV-2 in blood, anti-AAV-2 IgM and human leukocyte antigen typing could be candidate diagnostic criteria for AHUE. AAV-2 infection should be incorporated into clinical guidelines for the management of acute liver failure. Cidofovir can be administered if coinfection with AAV-2 and HAdV is detected.

Key Words: Adeno-associated virus; Cidofovir; Coinfection; Helper virus; Herpes virus; Human adenovirus; Human leukocyte antigen; Metagenomic sequencing; Phylogenetic tree analysis; Real-time PCR

Core Tip: The rate of positivity for adeno-associated virus serotype 2 (AAV-2) was 80% or greater in cases of acute hepatitis of unknown etiology (AHUE) according to metagenomic sequencing and real-time polymerase chain reaction. AAV-2 RNA was detected in the nuclei and cytoplasm of ballooned hepatocytes through in situ hybridization. No distinct clade was observed in the phylogenetic tree analysis of whole genomes of AAV-2 isolated from cases. Coinfection of AAV-2 with one or more helper viruses (HAdV, HHV-6B, EBV, etc.), a high viral load of AAV-2 in blood, anti-AAV-2 IgM and human leukocyte antigen (HLA) typing (HLA-DRB1*04:01, HLA-DQA1*03:03 and HLA-DRB4*01:03) could be candidate diagnostic criteria for AHUE.

INTRODUCTION

From October to November 2021, five pediatric patients with severe acute hepatitis and human adenovirus (HAdV) viremia were identified in a children’s hospital in Alabama, United States; all five were previously healthy young children[1]. Moreover, in April 2022, the United Kingdom Health Security Agency and Public Health Scotland reported an increase in pediatric cases of severe acute hepatitis of unknown origin[2,3]. In collaboration with the United Kingdom Health Security Agency, the World Health Organization (WHO) published the first Disease Outbreak News on acute hepatitis of unknown etiology (AHUE) in children under 10 years old[4]. Following the first news report, the WHO reported as of 8 July 2022 that 1010 probable cases of AHUE in children, including 22 deaths, were identified in 35 countries, mainly European countries and the United States[5]. In 2024, the United States Centers for Disease Control and Prevention announced that a total of 392 cases of AHUE in children were reported in the United States from October 2021 to June 2023. Among the 392 patients, 8 were fatally ill children who met the reporting criteria[6]. Findings from several investigations suggest that HAdV alone or in combination with other factors could play a crucial role in cases of AHUE[7-10]. In May 2022, the United Kingdom Health Security Agency reported that high quantities of adeno-associated virus serotype 2 (AAV-2) were detected in the blood and liver tissue of children with AHUE through metagenomic sequencing[11]. AAV-2 is a small, nonenveloped DNA virus that requires helper viruses such as adenovirus and herpesviruses for viral replication. AAV infection is common and nonpathogenic in the human population[12-16]. Thus, contamination or reactivation of AAV-2 was suspected at the early stage of investigation[11]. However, in March 2023, three pediatric studies from the United Kingdom (by an English group and a Scottish group) and the United States revealed that high-level replication of AAV-2 in the context of coinfection with helper viruses was related to AHUE in children[17-19]. These studies provide clues to clarify the pathogenesis of AAV-2 infection. To gain further understanding of the association between AAV-2 infection and AHUE, the data from the three pediatric studies were analyzed[17-19]. This study is focused especially on metagenomic sequencing, viral whole-genome sequencing, virus-specific real-time polymerase chain reaction (PCR) and histological analysis of the liver, all of which are common investigative methods used in the three pediatric studies.

DEFINITIONS OF AHUE CASES AND CONTROLS

The characteristics of the cases of AHUE and controls in the United Kingdom studies (English and Scottish)[17,18] and the United States study[19] are shown in Table 1. For the Scottish study, patients were prospectively recruited. The United States study was a retrospective observational study. Although the English study seems to be a prospective study, the design of the study was not described.

Table 1 Characteristics of cases of acute hepatitis of unknown etiology and controls in the United Kingdom and United States studies.

	England	Scotland	United States
Case definition of acute hepatitis of unknown etiology	A person presenting since January 1, 2022 with acute hepatitis which is not due to hepatitis A-E viruses, or an expected presentation of metabolic, inherited or genetic, congenital or mechanical cause with serum transaminase greater than 500 IU/L (AST or ALT), who is 10 years old and under	A person presenting with a serum transaminase greater than 500 IU/L (AST or ALT) without known cause (excluding hepatitis A-E, CMV and EBV), who is 10 years of age and under or a contact of any age of a confirmed case, since 1 January 2022	Children < 10 years of age with elevated (> 500 U/L) AST or ALT since October 1, 2021 who have an unknown etiology for their hepatitis (with or without any adenovirus testing results, irrespective of the results)
Number of cases	n = 38	n = 32	n = 16
	Male: 12, Female: 15, Unknown: 11	Male: 11, Female: 21	Male: 9, Female: 7
	1-9 years of age (median 3)	2.7-5.5 years of age (median 4.1)	Mean age: 3.8 ± 2.0 (median 3)
	Liver transplantation, n = 12	Liver transplantation, n = 1	Liver transplantation, n = 2
	Treatment with cidofovir, n = 14	Treatment with cidofovir, n = 1	Treatment with cidofovir, no data
	Blood HAdV PCR was positive in 22/23 cases	All routine blood tests for A-E hepatitis, EBV, CMV, HHV-6, HHV-7 and HSV were negative. Of 30 cases, 5 were positive for plasma HAdV PCR	All 16 cases had positive testing for HAdV from blood
Controls (comparators)	Immunocompetent control: n = 65	Group 1: Age-matched healthy control = 13	Group 1: Without hepatitis, n = 42 (non-inflammatory, non-hepatitis hospitalized control, n = 27, inflammatory, non-hepatitis hospitalized control, n = 15)
	Healthy control, n = 13	Group 2: HAdV infection (HAdV PCR positive with normal ALT) control, n = 12	Group 2: Acute hepatitis (ALT > 100 U/L, defined etiology), n = 30
	HAdV infection, normal ALT, n = 17	Group 3: Hepatitis control (HAdV PCR negative with increased ALT), if reactivation of AAV-2 in severe hepatitis, n = 33	Group 3: Acute gastroenteritis, n = 23 (HAdV positive stool, n = 12)
	HAdV infection, normal ALT (HAdV in blood), n = 8	Group 4: Contemporaneous control, if circulating widely across Scotland, n = 16	Group 4: Blood donor, n = 18
	HAdV infection, raised ALT, n = 5
	Critical illness, raised ALT, n = 11
	Non-HAdV infection, raised ALT, n = 5
	Other hepatitis, n = 6
	Immunocompromised comparator: n = 17
	HAdV infection, raised ALT > 500 U/L, n = 14
	CMV infection, raised ALT > 500 U/L, n = 3

AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; CMV: Cytomegalovirus; EBV: Epstein-Barr virus; HAdV: Human adenovirus; HHV: Human herpes virus; PCR: Polymerase chain reaction.

Definition of AHUE cases

Among the three studies, the case definitions of AHUE were almost the same[17-19]. The case definitions were as follows: (1) The child was 10 years of age or younger; (2) The child’s level of aspartate aminotransferase (AST) or alanine aminotransferase (ALT) was > 500 U/L; and (3) Hepatitis A-E viruses and metabolic, inherited/genetic, congenital, mechanical and other underlying causes were excluded. Cases from January 1, 2022, were included in the United Kingdom studies, and cases from October 1, 2021, were included in the United States study.

Cases

The number of cases was 38 (male/female = 12/15, sex unknown = 11, median age = 3 years) in the English study[17], 32 (male/female = 11/21, median age = 4.1 years) in the Scottish study[18] and 16 (male/female = 9/7, median age = 3 years) in the United States study[19]. Among these cases, 12 (32%, England), one (3%, Scotland) and two (13%, United States) of the patients underwent liver transplantation. The rate of liver transplantation in England was much higher than those in Scotland and the United States. Antiviral treatment with cidofovir, which is a broad-spectrum antiviral agent that acts as a viral DNA inhibitor[20], was administered to control HAdV infection in both the English study and the Scottish study[17,18,21]. In the English study, 14 patients were treated with cidofovir. Among the 12 patients who underwent transplantation, 11 received cidofovir (before transplantation: n = 1; after transplantation: n = 4; unknown: n = 6). In the Scottish study, one patient underwent transplantation, and that patient was treated with cidofovir afterward. In the English study, for 22/23 (96%) cases, blood samples (mainly whole-blood samples; personal communication with Prof. Judith Breuer) collected at the time of study enrollment were positive for HAdV according to PCR[17]. In the Scottish study, for 5/30 (17%) cases (calculated from the data in supplementary table 1 of the Scottish study), plasma samples collected at the time of study enrollment were positive for HAdV according to PCR[18]. The rate of plasma positivity for HAdV according to PCR in the Scottish study was much lower than the rate of blood positivity for HAdV according to PCR in the English study. In the United States study, blood samples were positive for HAdV in all 16 cases (100%), but the details of the testing methods used were not described[19].

Controls

Various controls were included in the studies as points of comparison for children with AHUE[17-19]. For example, 65 immunocompetent controls (healthy controls: n = 13; patients with HAdV infection and normal ALT levels: n = 17; patients with blood HAdV infection and normal ALT levels: n = 8; patients with HAdV infection and increased ALT levels: n = 5; critically ill patients with increased ALT levels: n = 11; non-HAdV-infected patients with increased ALT levels: n = 5; other hepatitis patients: n = 6) and 17 immunocompromised controls [HAdV-infected patients with increased ALT levels > 500 U/L: n = 14; cytomegalovirus (CMV)-infected patients with increased ALT levels > 500 U/L: n = 3] were included in the English study[17].

Four control groups were included in the Scottish study: Group 1, age-matched healthy controls (n = 13); group 2, HAdV-infected controls (PCR positive with normal liver function; n = 12); group 3, hepatitis controls (HAdV-negative according to PCR with increased ALT levels) for the evaluation of whether reactivation of AAV-2 could be caused by severe hepatitis (n = 33); and group 4, contemporaneous controls for the evaluation of whether AAV-2 was circulating widely across Scotland (n = 16)[18].

Four control groups were also included in the United States study: Group 1, individuals without hepatitis (n = 42; hospitalized controls with non-inflammatory non-hepatitis conditions, n = 27; hospitalized controls with inflammatory non-hepatitis conditions, n = 15); group 2, individuals with acute hepatitis (ALT level > 100 U/L, defined etiology; n = 30); group 3, individuals with acute gastroenteritis (n = 23; HAdV-positive stool, n = 12); and group 4, blood donors (n = 18)[19].

CLINICAL PRESENTATIONS OF AHUE IN CHILDREN

The clinical signs and symptoms of AHUE reported in the three studies are shown in Table 2[17-19]. Jaundice and vomiting were present in 50% or more of cases in all three studies. In 13% to 62.5% of cases, the patients experienced diarrhea, abdominal pain and lethargy. In the English study, clinical presentations were unavailable for approximately 40% of cases. There were no specific clinical presentations of AHUE in children.

Table 2 Clinical presentation of acute hepatitis of unknown etiology in the United Kingdom and United States studies, n (%).

Symptoms		England (n = 38)	Scotland (n = 32)	United States (n = 16)
Jaundice	Yes	21 (55.2)	21 (66)	13 (81.3)
	Uknown	15 (39.5)	0 (0)	1 (6.3)
Vomiting	Yes	20 (52.6)	22 (69)	11 (68.8)
	Uknown	15 (39.5)	0 (0)	5 (31.3)
Diarrhea	Yes	13 (34.2)	4 (13% )	8 (50.0)
	Uknown	15 (39.5)	0 (0)	1 (6.3)
Abdominal pain	Yes	8 (21.0)	10 (31)	6 (37.5)
	Uknown	15 (39.5)	0 (0)	0 (0)
Lethargy	Yes	8 (21.0)	10 (31)	10 (62.5)
	Uknown	15 (39.5)	0 (0)	1 (6.3)
Pale stool	Yes	7 (18.4)	NA	NA
	Uknown	15 (39.5)
Cough or coryza	Yes	5 (13.1)	NA	5 (31.3)
	Uknown	15 (39.5)		3 (18.8)
Fever	Yes	3 (7.9)	NA	6 (37.5)
	Uknown	15 (39.5)		1 (6.3)
Poor appetite	Yes	1 (2.6)	12 (38)	10 (62.5)
	Uknown	15 (39.5)	0 (0)	0 (0)

NA: Not available.

INVESTIGATIVE METHODS AND CLINICAL SAMPLES

A comparison of the investigative methods of the three studies is shown in Table 3[17-19]. Metagenomic sequencing, whole-genome sequencing for viruses and virus-specific real-time PCR were applied in all three studies. In particular, metagenomic sequencing played a central role in identifying viral pathogens associated with AHUE in children. Liver histology was evaluated in all three studies. Although there was no description in the results section of the English study, human leukocyte antigen (HLA) typing analysis was performed in the two United Kingdom studies. Additionally, untargeted nanopore sequencing, transduction of AAV-2 capsid mutations, cytokine transcriptomic analysis and proteomic analysis were carried out in the English study[17]. In the Scottish study, longitudinal analyses of AAV-2 PCR/anti-AAV IgG/IgM and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection evaluated by PCR/IgG antibody were performed[18].

Table 3 Methods of investigation for acute hepatitis of unknown etiology in children.

	England	Scotland	United States
1 Metagenomic sequencing	○	○	○
2 Viral whole-genome sequencing	○	○	○
3 Virus-specific real-time PCR	○	○	○
4 Liver histology	○	○	○
5 Nanopore sequencing	○
6 Transduction of AAV-2 capsid mutation	○
7 Cytokine transcriptomics analysis	○
8 Proteomic analysis	○
9 Longitudinal analysis (AAV-2 PCR and AAV2 IgG/IgM)		○
10 Severe acute respiratory syndrome coronavirus 2 infection		○
11 HLA typing	○	○

AAV: Adeno-associated virus; HLA: Human leukocyte antigen; PCR: Polymerase chain reaction.

In the English study, 24 of 38 patients had one clinical sample each (whole blood: n = 11; liver: n = 6; stool: n = 3; plasma: n = 2; nasopharyngeal aspirate: n = 1; nasopharyngeal swab: n = 1). The label “Serum” in Table 1 of the English study was incorrect; the samples were actually plasma (personal communication)[17]. There were two or three clinical samples for each of the remaining 14 patients. In the Scottish study, plasma, serum, and swab samples (throat/nose, stool and rectal swabs) were collected and evaluated, but whole-blood samples were not. The exact number of samples could not be obtained from the supplementary data of the Scottish study. In the United States study, 9 of 16 patients had one clinical sample each (whole blood: n = 6; nasopharyngeal swab: n = 1; plasma: n = 1; and stool: n = 1). There were several clinical samples for each of the remaining 7 patients, including one or more whole-blood samples each. The type and number of clinical samples used for investigation differed across studies. Because the number of positive cases was calculated on the basis of various clinical samples in the individual studies, understanding the results of the investigations is somewhat complicated.

METAGENOMIC SEQUENCING

In the three studies, there were two types of metagenomic sequencing: Agnostic (untargeted) metagenomic sequencing and target-enrichment metagenomic sequencing. Agnostic sequencing has an advantage in detecting novel viruses whose genome sequences are unknown[22]. In contrast, target-enrichment sequencing is not useful for the detection of novel viruses but has a better ability to detect viruses through the enrichment of viral DNA using probes or PCR. Various samples, such as whole-blood, plasma, serum, liver, stool and pharyngeal swab samples, were used for metagenomic sequencing in the three studies.

In the English study, agnostic metagenome sequencing was performed for 10 cases: Whole-blood samples were analyzed for 5 cases (cases 6-10, but case 8 had both whole-blood and plasma samples), and liver samples were analyzed for another 5 cases (cases 1-5) (Table 4)[17]. In whole-blood samples, AAV-2 [DNA: 4/5 cases (80%) and 1.9-42.1 reads/million, RNA: 2/3 cases (67%); 3.1-7.8 reads/million], HAdV [DNA: 1/5 cases (20%) and 0.1 reads/million; RNA: 0/3 cases (0%)], Epstein-Barr virus (EBV) [DNA: 1/5 cases (20%); RNA: 0/3 cases (0%)] and torque teno virus (TTV) [DNA: 1/5 cases (20%); RNA: 0/3 cases (0%)] were detected. AAV-2 had the highest rate of positivity among the viruses detected in blood. Although the rate of positivity for HAdV according to PCR was 96% in blood samples collected at the time of enrollment in the study, the rate of whole-blood sample positivity for HAdV was only 20% according to metagenomic sequencing.

Table 4 Detected viruses via metagenomic sequencing in the United Kingdom and United States studies, n (%).

England, agnostic (untargeted) metagenomic sequencing (n = 10)			Scotland, target enrichment metagenomic sequencing (n = 9)			United States, agnostic (untargeted) and target enrichment metagenomic sequencing (n = 16)
Whole- blood			Serum or plasma			Whole-blood or plasma
AAV-2 positive	DNA: 4/5 cases (80), RNA: 2/3 cases (66.7) (Two cases were positive for DNA and RNA)	DNA: 1.9-42.1 reads/million, RNA: 3.1-7.8 reads/million	AAV-2 positive	DNA: 9/9 cases (100), RNA: 6/6 cases (100)	DNA: 152.06-24967.81 reads/million, RNA: 113.25-12119.78 reads/million	Agnostic metagenomic sequencing (n = 14)
HAdV positive	DNA: 1/5 cases (20), RNA: 0/3 cases (0)	DNA: 0.1 reads/million	HAdV positive	DNA: 3/9 cases (33.3), RNA: cases1/9 (11.1)	DNA: 0.16-1.66 reads/million, RNA: 577.57 reads/million	AAV-2 positive	8/14 cases (57.1)	0.033-3.87 reads/million
HHV-6B positive	DNA: 0/5 cases (0), RNA: 0/3 cases (0)		HHV-6B positive	DNA: 3/4 cases (75), RNA: 0/4 cases (0)	DNA: 0.41-3.89 reads/million median	HAdV-41 positive	0/14 cases (0)
EBV positive	DNA: 1/5 cases (20), RNA: 0/3 cases (0)	Read count, no data	VZV positive	DNA: 1/4 cases (25), RNA: 0/4 cases (0)	DNA: 2.14 reads/million	EBV positive	2/14 cases (14.3)	0.00057-0.0056 reads/million
TTV positive	DNA: 1/5 cases (20), RNA: 0/3 cases (0)	Read count, no data	EBV positive	DNA: 1/4 cases (25), RNA: 0/4 cases (0)	DNA: 0.53 reads/million	CMV positive	1/14 cases (7.1)	0.0068 reads/million
			CMV positive	DNA: 1/4 cases (25), RNA: 1/4 cases (25)	DNA: 9.85 reads/million, RNA: 0.21 reads/million	HAdV-2 positive	1/14 cases (7.1)	0.74 reads/million
			HHV-7 positive	DNA: 0/4 cases (0), RNA: 1/4 cases (25)	RNA: 0.31 reads/million	EV-A71 positive	1/14 cases (7.1)	4.10 reads/million
						Tilling multiplex PCR amplicon for
						AAV-1- AAV-8 and HAdV-41 (n = 10)
						AAV-2 positive	9/10 cases (90)	0.513-388.147 reads/million
						HAdV-41 positive	7/10 cases (70)	0.012-0.97 reads/million
						Probe capture viral enrichment (n = 4)
						AAV-2 positive	4/4 cases (100)	0.054-183.976 reads/million
						HAdV-41 positive	3/4 cases (75)	0.126-0.803 reads/million
						Total (target enhancement)
						AAV-2 positive	13/14 cases (92.9)
						HAdV-41 positive	10/14 cases (71.4)
Liver			Liver			Liver
AAV-2 positive	DNA: 5/5 cases (100), RNA: 4/4 cases (100)	DNA: 7.3-42 reads/million, RNA: 0.6-9.9 reads/million	AAV-2 positive	DNA: 4/4 cases (100), RNA: 1/1 case (100)	DNA: 637.16-39653.48 reads/million, RNA: 3089.09 reads/million	Agnostic metagenomic sequencing (n = 1)
HAdV positive	DNA: 0/5 cases (0), RNA: 0/4 cases (0)		HAdV positive	DNA: 3/4 cases (75), RNA: 0/4 cases (0)	DNA: 419.33-1349.78 reads/million	AAV-2 positive	1/1 case (100)	1.42 reads/million
HHV-6B positive	DNA: 5/5 cases (100), RNA: 0/4 cases (0)	DNA: 0.09-4.1 reads/million	Feces
TTV positive	DNA: 2/5 cases (40), RNA: 0/4 cases (0)	Read count, no data	AAV2 positive	DNA: 1/7 cases (14.2), RNA: 1/7 cases (14.2)	DNA: 3156.64 reads/million, RNA: 554.03 reads/million	Tilling multiplex PCR amplicon for AAV1-AAV8 and HAdV-41 (n = 1)
Astrovirus VA3 positive	DNA: 1/5 cases (20), RNA: 0/4 cases (0)	Read count, no data	HAdV positive	DNA: 4/7 cases (57.1), RNA: 0/7 cases (0)	DNA: 0.72-10512 reads/million	AAV-2 positive	1/1 case (100)	9210.435 reads/million
			Rectal swab			HAdV-41 positive	1/1 case (100)	0.012 reads/million
			AAV2 positive	DNA: 1/1 case (100), RNA: 0/1 case (0)	DNA: 108.68 reads/million	Nasopharyngeal swab
			HAdV positive	DNA: 1/1 case (100), RNA: 0/1 case (0)	DNA: 2.93 reads/million	Agnostic metagenomic sequencing (n = 1)
			Throat swab			HAdV-1 positive	1/1 case (100)	23.58 reads/million
			AAV2 positive	DNA: 1/6 cases (16.7), RNA: 0/6 case (0)	DNA: 670.26 reads/million	Feces
			HAdV positive	DNA: 2/6 cases (33.3), RNA: 0/6 case (0)	DNA: 152.96-14453.17 reads/million	Agnostic metagenomic sequencing (n = 2)
						Picobirnavirus positive	1/2 cases (50)	0.32 reads/million
Total			Total			Total
AAV-2 positive	9/10 cases (90)		AAV-2 positive	9/9 cases (100)		AAV-2 positive	13/16 cases (81.3)
HAdV positive	1/10 cases (10)		HAdV positive	6/9 cases (66.7)		HAdV (serotypes 1, 2 and 41) positive	12/16 cases (75)
HHV-6B positive	5/10 cases (50)		HHV-6B positive	3/4 cases (75)		EBV positive	2/16 cases (12.5)
EBV positive	1/10 cases (10)		VZV positive	1/4 cases (25)		CMV positive	1/16 cases (6.3)
TTV positive	3/10 cases (30)		EBV positive	1/4 cases (25)		EV-A71 positive	1/16 cases (6.3)
Astrovirus VA3 positive	1/10 cases (20)		CMV positive	2/4 cases (50)		Picobirnavirus positive	1/16 cases (6.3)
			HHV-7 positive	1/4 cases (25)
Control			Control			Control
No data			Plasma or serum			Whole blood or plasma
			AAV2 positive			Agnostic metagenomic sequencing (n = 113)
			G1	DNA: 0/13 (0), RNA: 0/13 (0)		AAV-2 positive	1/113 (0.9)	97.65 reads/million
			G2	DNA: 0/12 (0), RNA: 0/12 (0)		HAdV-41 positive	4/113 (3.5)	0.07-6.14 reads/million
			HAdV positive			HAdV-2 positive	2/113 (1.8)	0.015-0.035 reads/million
			G1	DNA: 0/13 (0), RNA: 0/13 (0)
			G2	DNA: 6/12 (50), RNA: 2/12 (16.7)	DNA: 1.63-29950.78 reads/million, RNA: 2.36-13.74 reads/million	Tilling multiplex PCR amplicon for AAV-1-AAV-8 and HAdV-41 (n = 113)
			HSV1			AAV-2 positive	4/113 (3.5)	2.178-44265.146 reads/million
			G1	DNA: 0/13 (0), RNA: 0/13 (0)		G1	0/42
			G2	DNA: 1/12 (8.3), RNA: 1/12 (8.3)	DNA: 3.5 reads/million, RNA: 11.38 reads/million	G2	0/30
			HSV2			G3	3/23
			G1	DNA: 0/13 (0), RNA: 0/13 (0)		G4	1/18
			G2	DNA: 1/12 (8.3), RNA: 1/12 (8.3)	DNA: 10.37 reads/million, RNA: 22.92 reads/million	HAdV-41 positive	9/113 (8.0)	0.412-7857.512 reads/million
			VZV			G1	0/42
			G1	DNA: 0/13 (0), RNA: 0/13 (0)		G2	0/30
			G2	DNA: 1/12 (8.3), RNA: 1/12 (8.3)	DNA: 2.68 reads/million, RNA: 4.02 reads/million	G3	9/23 (Of HAdV positive 9 cases, 3 were positive for AAV-2)
			EBV			G4	0/18
			G1	DNA: 3/13 (23.1), RNA: 3/13 (23.1)	DNA: 1.23-114.38 reads/million, RNA: 0.1-1.0 reads/million
			G2	DNA: 3/12 (25), RNA: 3/12 (25)	DNA: 1.64-272.4 reads/million, RNA: 1.38-17.58 reads/million
			CMV
			G1	DNA: 1/13 (7.7), RNA: 0/13 (0)	DNA: 17.06 reads/million
			G2	DNA: 5/12 (41.7), RNA: 1/12 (8.3)	DNA: 8.26-3634.02 reads/million, RNA: 3185.33 reads/million
			HHV6A
			G1	DNA: 0/13 (0), RNA: 3/13 (23.1)	RNA: 0.08-5.1 reads/million
			G2	DNA: 1/12 (8.3), RNA: 1/12 (8.3)	DNA: 1.67 reads/million, RNA: 12.38 reads/million
			HHV6B
			G1	DNA: 0/13 (0), RNA: 0/13 (0)
			G2	DNA: 3/12 (25), RNA: 2/12 (16.7)	DNA: 0.18-5062.23 reads/million, RNA: 206.17-5819.09 reads/million
			HHV7
			G1	DNA: 0/13 (0), RNA: 1/13 (7.7)	RNA: 0.1 reads/million
			G2	DNA: 0/12 (0), RNA: 2/12 (16.7)	RNA: 2.0-10.54 reads/million
			HHV8
			G1	0/13 (0)
			G2	DNA: 2/12 (16.7), RNA: 1/12 (8.3)	DNA: 2.01-75.91 reads/million, RNA: 1.34 reads/million

AAV: Adeno-associated virus; CMV: Cytomegalovirus; EBV: Epstein-Barr virus; EV: Enterovirus; HAdV: Human adenovirus; HHV: Human herpes virus; TTV: Torque teno virus; VZV: Varicella-zoster virus; PCR: Polymerase chain reaction.

In five liver samples, AAV-2 [DNA: 5/5 cases (100%), 7.3-42 reads/million; RNA: 4/4 cases (100%), 0.6-9.9 reads/million], HHV-6B [DNA: 5/5 cases (100%), 0.09-4.1 reads/million; RNA: 0/4 cases (0%)], TTV [DNA: 2/5 cases (40%); RNA: 0/4 cases (0%)] and astrovirus VA3 [DNA: 1/5 cases (20%); RNA: 0/4 cases (0%)] were detected. HAdV was not detected in liver samples. AAV-2 had the highest positivity rate among the viruses detected in liver and whole-blood samples. The presence of AAV-2 RNA reads in both blood and liver samples suggests viral replication of AAV-2[16,23,24]. Although human herpes virus (HHV)-6B was not detected in any of the blood samples from the five patients, HHV-6B was detected in the liver samples from all five patients. This result indicates that HHV-6B is an important helper virus for AAV-2 replication in the liver. In a total of 10 cases, the rates of positivity for AAV-2, HAdV, HHV-6B, EBV, TTV and astrovirus were 90% (9/10), 10% (1/10), 50% (5/10), 10% (1/10), 30% (3/10) and 10% (1/10), respectively. There were no metagenomic sequencing data for the control samples. As a result, in the English study, metagenomic sequencing findings suggest that AAV-2 infection could be a cause of AHUE in children.

In the Scottish study, agnostic and target-enrichment metagenomic sequencing (VirCapSeq-VERT) was performed on DNA and RNA extracted from plasma, liver, feces, rectal swab and throat swab samples[18]. VirCapSeq-VERT is a system for the detection, typing and strain differentiation of both RNA and DNA viruses with sensitivity to quantitative PCR. This system uses 2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans, and its use can result in a 100- to 10000-fold increase in the number of viral reads[25,26]. The agnostic metagenomic sequencing data were not available. The results of the target-enrichment metagenomic sequencing of the clinical samples from nine cases (cases 1-9) are shown in Table 4. All DNA and RNA extracted from the plasma and liver samples tested positive for AAV-2 (plasma DNA: 152.06-24967.81 reads/million; plasma RNA: 113.25-12119.78 reads/million; liver DNA: 637.16-39653.48 reads/million; and liver RNA: 3,089.09 reads/million). The read count for AAV-2 was much greater than that reported in the English study. This finding might be due to the difference in sensitivity between agnostic and target sequencing. HAdV DNA and RNA were detected in plasma [DNA: 3/9 cases (33.3%); RNA: 1/9 cases (11.1%)] and liver samples [DNA: 3/4 cases (75%); RNA: 0/4 cases (0%)]. AAV-2 and HAdV were also detected in feces, rectal swab and throat swab samples. The target sequencing data of human herpes viruses [herpes simplex virus (HSV)-1, HSV-2, varicella-zoster virus (VZV), EBV, CMV, HHV-6A, HHV-6B, HHV-7 and HHV-8] were available for four cases (cases 6-9). HHV-6B was detected in 3/4 (75%) of the DNA samples extracted from plasma. VZV (DNA: 1/4 cases), EBV (DNA: 1/4 cases), CMV (DNA: 1/4 cases; RNA: 1/4 cases) and HHV-7 (RNA: 1/4 cases) were detected in plasma samples. In the plasma, feces, rectal swab and throat swab samples collected from 9 patients, the overall rates of positivity for AAV-2, HAdV, HHV-6B, VZV, EBV, CMV and HHV-7 were 100% (9/9), 66.7% (6/9), 75% (3/4), 25% (1/4), 25% (1/4), 50% (2/4) and 25% (1/4), respectively. Additionally, in the Scottish study, plasma or serum samples from two control groups (group 1: Age-matched healthy controls; group 2: PCR-positive HAdV-infected patients with normal liver function) were evaluated via targeted-enrichment metagenomic sequencing. Neither AAV-2 nor HAV was detected in any of the group 1 samples. HAdV DNA and RNA were detected in 50% (6/12) and 16.7% (2/12) of the group 2 samples, respectively. However, AAV-2 was not detected in any of the group 2 samples. Taken together, these findings suggest that AAV-2 could be a pathogen of AHUE.

In the United States study, both agnostic and target-enrichment metagenomic sequencing were carried out for both DNA and RNA extracted from whole-blood or plasma, liver, nasopharyngeal swab and fecal samples. For target-enrichment metagenomic sequencing, two methods of viral genome enrichment were used[19]: Multiplex PCR of tiled amplicons (tiling multiplex PCR) and the commercially available viral probe capture method (VirCapSeq-VERT and Twist Respiratory Virus Panel)[19,27]. The Twist Respiratory Virus Panel targets 29 human respiratory viruses[27]. The tiling multiplex PCR method combines multiplex PCR for targeted enrichment of the viral genome with next-generation sequencing[28,29]. The detection limit of the tiling multiplex PCR method is approximately 10 copies per mL[29]. In the United States study, custom primers for multiplex PCR were designed to target HAdV-41 or AAV-1 of the AAV-8 genome[19]. In the United States study, DNA and RNA were not differentiated according to the results of the metagenomic sequencing. The agnostic metagenomic sequencing of whole-blood (n = 12), plasma (n = 1, 14_NC) and whole-blood and plasma (n = 1, 9_CA) samples revealed that AAV-2 was detected in 8/14 cases (57.1%) (0.033-3.87 reads/million), but HAdV-41 was not detected. EBV, CMV, HAdV-2 and enterovirus (EV)-71 were detected in 2 (14.3%), 1 (7.1%), 1 (7.1%) and 1 (7.1%) of 14 cases, respectively (Table 4). The multiplex PCR method yielded higher rates of positivity for AAV-2 and HAdV [AAV-2: 9/10 cases (90%); HAdV-41: 7/10 cases (70%)] than did agnostic metagenomic sequencing of whole-blood or plasma samples. The probe capture method targeted two viruses, AAV-2 and HAdV-41, in whole-blood or plasma samples. Like multiplex PCR, the probe capture method detected AAV-2 and HAdV-41 at high rates [AAV-2: 4/4 cases (100%); HAdV-41: 3/4 cases (75%)]. In total, AAV-2 and HAdV-41 were detected in whole-blood or plasma samples from 13 (92.9%) and 10 (71.4%) of 14 patients, respectively. Moreover, one liver sample, one nasopharyngeal swab sample and two fecal samples were examined via metagenomic sequencing in the United States study. AAV-2 was detected in the liver sample [agnostic sequencing (1.42 reads/million) and target enrichment (9210.435 reads/million)]. HAdV-41 [target enrichment (0.012 reads/million)] and HAdV-1 [agnostic sequencing (23.58 reads/million)] were detected in the liver and nasopharyngeal swab samples, respectively. In the blood, plasma, liver, nasopharyngeal and fecal samples from 16 patients, the rates of positivity for AAV-2, HAdV (serotypes 1, 2 and 41), EBV, CMV, EV-A71 and picobirnavirus were 81.3% (13/16 patients), 75% (12/16 patients), 12.5% (2/16 patients), 6.3% (1/16 patients), 6.3% (1/16 patients) and 6.3% (1/16 patients), respectively. In the United States study, samples from 113 controls (whole blood: n = 78; serum: n = 1; plasma: n = 34) were examined via agnostic metagenomic sequencing and the tiling multiplex PCR method. In contrast to the United Kingdom studies, more than 100 controls were evaluated via metagenomic sequencing in the United States study. Agnostic metagenomic sequencing detected AAV-2, HAdV-41 and HAdV-2 in one (0.9%), four (3.5%) and two (1.8%) of the 113 controls, respectively. The rates of positivity for AAV-2 and HAdV-41 via the multiplex PCR method were 3.5% (4/113 controls) and 8.0% (9/113 controls), respectively. All four controls [group 3: n = 3 (AAV-positive stool: n = 2); group 4: n = 1] in whom AAV-2 was detected via multiplex PCR were coinfected with HAdV (HAdV-41: n = 3, HAdV-2: n = 1), which was detected via agnostic sequencing. The rates of positivity for AAV-2 and HAdV-41 in the cases were significantly greater (P < 0.001) than those in the controls. These findings suggest that AAV-2 and HAdV-41 infection are associated with AHUE. In cases of AAV-2 coinfection with HAdV, the mean normalized viral count for AAV-2 was approximately 12.7 times greater than that for HAdV-41 according to target sequencing. Previous studies have revealed that AAV-2 inhibits the replication of helper viruses, such as HAdV, without preventing helper virus function[16].

VIRUS-SPECIFIC REAL-TIME PCR

To confirm the results of the metagenomic sequencing, virus-specific real-time PCR was carried out. Qualitative and quantitative evaluations of AAV-2, HAdV and HHV-6 were performed using real-time PCR. AAV-2 real-time PCR was not performed in the United States study.

In the English study[17], AAV-2 DNA was detected using Aurnhammer’s PCR protocol, which targets the inverted terminal repeat sequence[30]. In addition, AAV-2 RNA was detected by PCR with reverse transcription targeting the cap open reading frame sequence[17]. These PCR protocols can be used to amplify AAV-2 and AAV-6. It is not clear whether the applied HHV-6 PCR method can differentiate HHV-6B from HHV-6A[31]. HAdV DNA was detected via the pan-HAdV detection PCR method[32]. The detection limit of AAV-2, HAdV and HHV-6 was a cycle threshold (Ct) value of 45. A Ct value between 38 and 45 was defined as a low positive value. The term “blood” referred to whole blood in the English study (personal communication)[17]. The sample type obtained from the controls was whole blood.

Among the 38 patients, 28, 32 and 23 underwent AAV-2, HAdV and HHV-6 PCR tests, respectively (Table 5). Among the 28 patients who underwent AAV-2 PCR, 22 had one sample [whole-blood samples: 11 cases; formalin-fixed paraffin-embedded (FFPE) liver samples: Five cases; liver samples: Four cases; and plasma samples: Two cases], and the remaining 6 had two different types of samples (FFPE liver and plasma samples: Four cases; liver and respiratory samples: One case; and stool and respiratory samples: One case). A total of 34 samples were examined via AAV-2 PCR. Thirty-three samples were positive for AAV-2 according to PCR, while one blood sample was not. Therefore, AAV-2 was detected in 27 (96.4%) of the 28 patients. Moreover, the Ct values of AAV-2 PCR were low in whole-blood (Ct: 19-24) and liver (Ct: 17-21) samples. These findings indicate that the viral load of AAV-2 is high in the blood and liver. Among the 65 immunocompetent pediatric controls, 6 (9.2%) had whole-blood samples that tested positive for AAV-2 according to PCR. Although the rate of AAV-2 positivity according to PCR was 50% (4/8 controls) in children with normal ALT levels and HAdV viremia, the Ct values of AAV-2 ranged from 27 to 38 in the controls. The 1/Ct values of AAV-2 were significantly greater (P < 0.05) in whole-blood samples from the cases than in those from the controls[17]. These findings suggest that AAV-2 is related to AHUE. However, consideration should be given to immunocompromised patients with HAdV infection. Among the 14 immunocompromised controls with HAdV infection and increased ALT levels, blood samples from 6 (42.9%) were positive for AAV-2 according to PCR. Although the 1/Ct values of AAV-2 were significantly greater (P = 0.00169) in blood samples from the cases than in those from the immunocompromised controls, the detection of AAV-2 does not always indicate AHUE in children with HAdV infection.

Table 5 Rate of positivity for adeno-associated virus, human adenovirus, human herpes virus-6, Epstein-Barr virus and cytomegalovirus via real-time polymerase chain reaction, n (%).

England					Scotland				United States
Virus	Cases of acute hepatitis of unknown etiology		Controls		Cases of acute hepatitis of unknown etiology		Controls		Cases of acute hepatitis of unknown etiology		Controls
	Sample	Positivity rate	Sample and group	Positivity rate	Sample	Positivity rate	Sample and group	Positivity rate	Sample	Positivity rate	Sample and group	Positivity rate
AAV-2	Whole-blood	10/11 cases (91) (Ct: 19-24)	Whole-blood, immunocompetent control	6/65 controls (9.2)	Plasma or serum	26/32 cases (81.2), viral load, median: 66100 copies/mL (IQR; 13461-300277 copies/mm3)	Plasma or serum	5/74 controls (6.7)	No data		No data
			Healthy control	1/13 (7.7) (Ct: 39)			Group 1	0/13 (0)
			HAdV infection, normal ALT	0/17 (0)			Group 2	0/12 (0)
			HAdV infection, normal ALT (HAdV in blood)	4/8 (50) (Ct: 27-38)			Group 3	0/33 (0)
			HAdV infection, raised ALT	0/5 (0)			Group 4	5/16 (31.2), viral load, median: 3267 copies/mL
			Critical illness, raised ALT	1/11 (9.1) (Ct: 37)	Liver	5 cases, viral load, median: 3721497 copies/mm3 (IQR; 3308243-6717616 copies/mm3)	Liver (controls under 18 years old)	19 controls, viral load, median: 64 copies/mm3 (IQR; 20-83 copies/mm3)
			Non-HAdV infection, raised ALT	0/5 (0)
			Other	0/6 (0)
			Whole-blood, immunocompromised control	6/17 (35.3)
			HAdV infection, raised ALT > 500 U/L	6/14 (42.9) (Ct 24-37)
			CMV infection, raised ALT > 500 U/L	0/3 (0)
	Liver	5/5 cases (100) (Ct: 17-21)	Liver, immunocompromised	1/4 (25) (Ct: 39)
	Throat swab	2/2 cases (100) (Ct: 25, 30)
	Stool	1/1 case (100) (Ct: 30)
	Plasma	6/6 cases (100) (Ct: 27-32)
	Formalin-fixed, paraffin-embedded liver	9/9 (cases 100) (Ct: 23-36)
		Total: 27/28 cases (96.4%)				Total: 26/32 cases (81.2%)
HAdV	Whole-blood	13/15 cases (86.7) (Ct: 31-37)	Whole-blood, immunocompromised, HAdV infection, raised ALT > 500 U/L	14/14 controls (100) (Ct: 29-42)	Plasma or serum	5/27 cases (18.5) (Ct: 37-38)	Plasma or serum	6/58 controls (10.3)	Whole-blood	21/21 samples (100) (Ct: 30-37)		2/113 controls (1.8)
	Liver	5/5 cases (100) (Ct: 37-42)	Whole-blood, immunocompromised, CMV infection, raised ALT > 500 U/L	0/2 (0) (Ct: 35)			Group 1	0/13 (0)	Plasma	1/2 cases (50) (Ct: 39)	G1 (whole blood)	0/42 (0)
	Throat swab	2/3 cases (66.7) (Ct: 35-39)	Liver, immunocompromised	0/4 (0)			Group 2	5/12 (41.7)		(Total: Whole blood or plasma, 14/14 cases 100)	G2 (whole blood)	0/30 (0)
	Stool	0/1 case (0)					Group 3	1/33 (3.0)	Liver	1/1 case (100) (Ct: 37)	G3 (plasma: n = 22, serum = 1)	2/23 (8.7) (Ct: 35, 36)
	Plasma	2/6 cases (33.3) (Ct: 39-42)							Nasopharyngeal swab	1/1 case (100) (Ct: 23)	G4 (whole blood: n = 6, plasma: n = 12)	0/18 (0)
	Formalin-fixed, paraffin-embedded liver	4/9 cases (44.4) (39-44)							Stool	1/2 cases (50) (Ct: 35)
		Total: 24/32 cases (75%)								Total: 16/16 cases (100%)
HHV-6	Whole-blood	4/6 cases (66.7) (Ct: 37)	Whole-blood, immunocompromised, HAdV infection, raised ALT > 500 U/L	4/12 controls (33.3) (Ct: 30-36)	Plasma or serum	1/27 cases (3.7) (Ct: 37)	Plasma or serum	4/58 controls (6.9)	Whole-blood	10/21 samples (47.6) (Ct: 37-40)		1/113 controls (0.9)
			Whole-blood, immunocompromised, CMV infection, raised ALT > 500 U/L	1/3 (33.3) (Ct: 35)			Group 1	0/13 (0)	Plasma	0/2 cases (0)	G1 (whole- blood)	0/42 (0)
	Liver	5/5 cases (100) (Ct: 27-32)	Liver, immunocompromised	1/4 (25) (Ct: 34)			Group 2	2/12 (16.7)		(Total: Whole- blood and plasma, 7/14 cases, 50)	G2 (whole- blood)	0/30 (0)
	Throat swab	1/2 cases (50) (Ct: 45)					Group 3	2/33 (6.1)	Liver	1/1 case (100) (Ct: 30)	G3 (plasma: n = 22, serum=1)	0/23 (0)
	Stool	0/1 case (0)			Liver	2/4 cases (50) (Ct: 33, 36)			Nasopharyngeal swab	No data	G4 (whole- blood: n = 6, plasma: n = 12)	1/18 (5.6) (Ct: 39)
	Plasma	0/6 cases (0)							Stool	0/2 case (0)
	Formalin-fixed, paraffin-embedded liver	6/9 cases (66.7) (Ct: 30-37)
		Total: 16/23 cases (69.6%)				Total: 3/27 cases (11.1%)				Total: 7/16 cases (43.8%)
EBV	No data		No data		Plasma	0/9 cases (0)	No data		Whole-blood	17/21 samples (80.9) (Ct: 31-43)		1/113 controls (0.9)
									Plasma	1/2 cases (50) (Ct: No data)	G1 (whole- blood)	1/42 (2.4) (Ct: 44)
										(Total: Whole- blood and plasma, 11/14 cases, 78.6)	G2 (whole- blood)	0/30 (0)
									Liver	0/1 case (0)	G3 (plasma: n = 22, serum = 1)	0/23 (0)
									Nasopharyngeal swab	0/1 case (0)	G4 (whole- blood: n = 6, plasma: n = 12)	0/18 (0)
									Stool	1/2 cases (50) (Ct: No data)
										Total: 11/16 cases (68.8%)
CMV	No data		No data		Plasma	0/9 cases (0)	No data		Whole-blood	0/21 samples (0)		2/113 controls (1.8)
									Plasma	0/2 cases (0)	G1 (whole- blood)	1/42 (2.4) (Ct: 38)
										(Total: Whole- blood and plasma, 0/14 cases, 0)	G2 (whole- blood)	0/30 (0)
									Liver	0/1 case (0)	G3 (plasma: n = 22, serum: n = 1)	1/23 (4.3) (Ct: 39)
									Nasopharyngeal swab	0/1 case (0)	G4 (whole- blood: n = 6, plasma: n = 12)	0/18 (0)
									Stool	0/2 cases (0)
										Total: 0/16 cases (0%)

AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; AAV: Adeno-associated virus; CMV: Cytomegalovirus; HAdV: Human adenovirus; HHV: Human herpes virus.

HAdV PCR tests were performed in 32 cases. Among the 32 patients, 25 had one sample (whole-blood samples: 14 cases; FFPE liver samples: Five cases; liver samples: Three cases; plasma samples: Two cases; respiratory sample: One case), and the remaining 7 had two different types of samples (FFPE liver and plasma samples: Four cases; liver and respiratory samples: One case; liver and whole-blood samples: One case; and stool and respiratory samples: One case). A total of 39 samples from 32 cases were evaluated. HAdV was detected in 86.7% (13/15) of the whole-blood samples, 100% (5/5) of the liver samples, 66.7% (2/3) of the respiratory samples, 0% (0/1) of the plasma samples, 33.3% (2/6) of the plasma samples and 44.4% (4/9) of the FFPE liver samples from the patients according to HAdV PCR. Thus, 24 (75%) of the 32 patients were positive for HAdV according to PCR. The Ct values of the whole-blood samples ranged from 31 to 37. The 1/Ct values of HAdV were significantly greater (P = 0.03651) in the blood of the patients (n = 14) than in that of the immunocompromised controls (HAdV-infected patients with elevated ALT levels > 500 U/L, n = 14)[17]. These findings suggest that AAV-2 infection with a high viral load of HAdV might be a clue for the diagnosis of AHUE.

Among the 23 cases in which HHV-6 PCR was performed, 17 had one sample (whole-blood samples: Six cases; FFPE liver samples: Five cases; liver samples: Four cases; and plasma samples: Two cases), and the remaining 6 had two different types of samples (FFPE liver and plasma samples: Four cases; liver and respiratory samples: One case; and stool and respiratory samples: One case). A total of 29 samples from 23 patients were examined by HHV-6 PCR. HHV-6 was detected by PCR in 66.7% (4/6) of the whole-blood samples, 100% (5/5) of the liver samples, 50% (1/2) of the respiratory samples, 0% (0/1) of the stool samples, 0% (0/6) of the plasma samples and 66.7% (6/9) of the FFPE liver samples. Thus, 16 (69.6%) of the 23 cases were positive for HHV-6 according to PCR. Among the immunocompromised controls, four (33.3%) of the 12 HAdV-infected patients whose ALT levels were > 500 U/L and one (33.3%) of the three CMV-infected patients whose ALT levels were > 500 U/L were positive for HHV-6 according to PCR. However, there was no significant difference in the 1/Ct values of HHV-6 in blood samples between the cases and immunocompromised controls[17]. These findings suggest that the viral load of HHV-6 in the blood is not useful for the diagnosis of AHUE in children. The 1/Ct values of AAV-2, HAdV and HHV-6 were significantly greater (HAdV: P = 0.01508; AAV-2: P = 0.01794; HHV-6: P = 0.01794) in liver samples from patients than in those from immunocompromised controls[17]. Although liver tissue is not a routinely evaluated type of clinical sample, the viral loads of AAV-2, HAdV and HHV-6 in the liver could be helpful for the diagnosis of AHUE.

In the Scottish study[18], extracted RNA from plasma or serum samples was used for viral PCR (Table 5). AAV-2 was detected by real-time quantitative PCR (Aurnhammer’s PCR method) with reverse transcription[30]. The plasmid was used to generate a standard curve. A Ct value ≤ 31 cycles was considered positive. The detection limit of AAV-2 quantitative PCR was 3200 copies/mL[33]. HAdV-41 and HHV-6 were also evaluated by quantitative PCR. Among the 32 patients, plasma or serum samples from 26 (81.2%) were positive for AAV-2 according to PCR. Plasma or serum samples from 74 controls were also examined via AAV-2 PCR. AAV-2 was not detected in plasma or serum samples from control group 1 (age-matched healthy controls), control group 2 (HAdV-infected patients with normal liver function) or control group 3 (patients negative for HAdV according to PCR and having increased ALT levels). Because AAV-2 was undetectable in control group 3, AAV-2 reactivation was unlikely to have occurred in severe hepatitis. Among the 16 contemporaneous controls (group 4), however, plasma or serum samples from 5 (31.2%) were positive for AAV-2 according to PCR. This finding suggested that AAV-2 was circulating widely at a low level in children across Scotland between March and April 2022. The viral loads of AAV-2 were significantly greater (P < 0.001) in plasma or serum samples from patients (median: 66100 copies/mL) than in those from any of the control groups[18]. The high rate of positivity for AAV-2 and the high viral load of AAV-2 in patients were consistent with the results of the English study. The viral loads of AAV-2 in liver samples from 5 patients (median: 3721497 copies/mm³) were significantly greater (P = 0.000997) than those in liver samples from 19 controls (median: 64 copies/mm³)[18]. Taken together with the findings of the English study, these findings suggest that AAV-2 is associated with AHUE in children.

Among 27 cases, plasma or serum samples from 5 (18.5%) were positive for HAdV according to PCR. The Ct values ranged from 37 to 38, which indicated that the viral load was not high. None of the 13 age-matched healthy controls (group 1) was positive for HAdV. Plasma or serum samples from 5 (41.7%) of the 12 controls infected with HAdV (group 2) and one (3.0%) of the 33 non-HAdV-infected hepatitis controls (group 3) were positive for HAdV according to PCR. Among the 27 cases, plasma or serum samples from 1 (3.7%) were positive for HHV-6 according to PCR. HHV-6 was detected in liver samples from 2 (50%) of 4 cases. Among 58 controls (groups 1, 2 and 3), 4 (8.9%) plasma or serum samples were positive for HHV-6 according to PCR. In contrast to the results of the English study, the rates of positivity for HAdV and HHV-6 viremia were low. The authors speculated that plasma and serum samples were less suitable for the detection of HAdV than were whole-blood samples[18]. In the technical briefing published by the United Kingdom Health Security Agency, it was reported that whole blood was a better matrix for the detection of HAdV than were plasma and serum[34]. Neither EBV nor CMV was detected in plasma samples from the cases in the Scottish study.

In the United States study, HAdV, HHV-6, EBV and CMV infections, but not AAV-2 infection, were confirmed via virus-specific real-time PCR (Table 5)[19]. Real-time PCR can detect both HHV-6A and HHV-6B simultaneously[35]. A pan-adenovirus PCR method targeting all serotypes of HAdVs was used[36]. For the HAdV PCR, a Ct value of 40 or less was considered a positive result. Additionally, conventional PCR following Sanger sequencing was performed to identify species F HAdV (serotypes 40 and 41)[37]. Among 16 patients, 8 had one whole-blood sample each, and 5 had two or more whole-blood samples each. The remaining 3 patients had one clinical sample each (10_CA: Nasopharyngeal swab; 11_CA: Stool; and 14_CA: Plasma). Therefore, whole-blood or plasma samples were available for 14 patients. HAdV, HHV-6, EBV and CMV were detected in 16/16 patients (100%), 7/16 patients (43.8%), 11/16 patients (68.8%) and 0/16 patients (0%), respectively. In contrast to the United Kingdom studies, EBV was frequently detected in cases of AHUE in the United States study. HAdV-41 and HAdV-40 sequences were detected in 10 (62.5%) and 1 (6.3%) of the 16 cases, respectively. In blood samples from the 113 controls, the rates of HAdV, HHV-6, EBV and CMV positivity were 1.8% (2/113), 0.9% (1/113), 0.9% (1/113) and 1.8% (2/113), respectively.

COMPARISON OF RATES OF VIRUS POSITIVITY ACCORDING TO METAGENOMIC SEQUENCING AND REAL-TIME PCR

The rates of AAV-2, HAdV and HHV positivity in cases of AHUE according to metagenomic sequencing and real-time PCR are shown in Table 6. In all three studies[17-19], the rates of AAV-2 positivity were 80% or greater according to both metagenomic sequencing and PCR. However, there was a discrepancy in the rates of positivity for other viruses between metagenomic sequencing and PCR. For example, the English study revealed that the HAdV detection rates were 10% and 75% according to metagenomic sequencing and PCR, respectively. Conversely, the Scottish study revealed that the rates of HAdV positivity were 66.7% and 18.5% according to metagenomic sequencing and PCR, respectively. A discrepancy was also observed in the rates of HHV-6 positivity. The Scottish study revealed that the rates of HHV-6 positivity were 75% and 11.1% according to metagenomic sequencing and PCR, respectively. On the other hand, the United States study revealed that the rates of HHV-6 positivity were 0% and 43.8% according to metagenomic sequencing and PCR, respectively. Although the timing of sampling and type of sample can influence the rates of positivity for viruses, it is necessary to keep in mind that there is some degree of discrepancy in the rates of positivity for viruses between metagenomic sequencing and real-time PCR.

Table 6 Rates of positivity for adeno-associated virus, human adenovirus and human herpes viruses in cases of acute hepatitis of unknown etiology according to metagenomic sequencing and real-time polymerase chain reaction.

Viruses	England		Scotland		United States
	Metagenomic sequencing	PCR	Metagenomic sequencing	PCR	Metagenomic sequencing	PCR
AAV-2	90% (9/10)	96.4% (27/28)	100% (9/9)	81.2% (26/32)	81.3% (13/16)	ND
HAdV	10% (1/10)	75% (24/32)	66.7% (6/9)	18.5% (5/27)	75% (12/16)	100% (16/16)
HHV-6	50% (5/10)	69.6% (16/23)	75% (3/4)	11.1% (3/27)	0% (0/16)	43.8% (7/16)
VZV	0% (0/10)	ND	25% (1/4)	ND	0% (0/16)	ND
EBV	10% (1/10)	ND	25% (1/4)	ND	12.5% (2/16)	68.8% (11/16)
CMV	30% (3/10)	ND	50% (2/4)	ND	6.3% (1/16)	0% (0/16)
HHV-7	20% (2/10)	ND	25% (1/4)	ND	0% (0/16)	ND

AAV: Adeno-associated virus; CMV: Cytomegalovirus; EBV: Epstein-Barr virus; EV: Enterovirus; HAdV: Human adenovirus; HHV: Human herpes virus; VZV: Varicella-zoster virus; ND: No data.

AAV-2 COINFECTION WITH HADV AND/OR HHVS

The rates of AAV-2 coinfection with HAdV and/or HHVs detected via metagenomic sequencing are shown in Table 7. The English, Scottish and United States studies revealed that AAV-2 was detected in 9, 9 and 13 cases, respectively, via metagenomic sequencing[17-19]. AAV coinfection with HAdV and/or HHVs was detected in 78% (7/9) of the AAV-2-infected patients in the English study, 78% (7/9) of the AAV-2-infected patients in the Scottish study and 77% (10/13) of the AAV-2-infected patients in the United States study according to metagenomic sequencing. At the early stage of the outbreak of AHUE in children, HAdV was considered a candidate pathogen of AHUE. Metagenomic sequencing detected HAdV in all 10 patients with AAV-2 coinfection in the United States study and in six (86%) of the seven patients with AAV-2 coinfection in the Scottish study. However, in the English study, metagenomic sequencing detected HAdV in only one (14%) of seven patients with AAV-2 coinfection. Instead of HAdV, HHV-6B was detected in five (71%) of seven patients with AAV-2 coinfection according to metagenomic sequencing. In the English study, in all five cases of AAV-2 coinfection with HHV-6B, the patients underwent liver transplantation[17]. In the United States study, AAV-2 was detected in four (3.5%) of the 113 controls via metagenomic sequencing (Table 4). Although the rate of AAV-2 positivity was low in the controls, three (75%) of the four controls infected with AAV-2 were coinfected with HAdV.

Table 7 Rate of positivity for human adenovirus and/or human herpes viruses in adeno-associated virus-2-infected cases of acute hepatitis of unknown etiology according to metagenomic sequencing.

Detected viruses	England	Scotland	United States
	n = 9	n = 9	Case, n = 13	Control, n = 4
AAV-2 + HAdV	11% (1/9)	33% (3/9)	46% (6/13)	75% (3/4)
AAV-2 + HAdV + HHV-6B + VZV		11% (1/9)
AAV-2 + HAdV + HHV-7 + CMV		11% (1/9)
AAV-2 + HAdV + HHV-6B + CMV		11% (1/9)
AAV-2 + HAdV + EBV			15% (2/13)
AAV-2 + HAdV + CMV			8% (1/13)
AAV-2 + HAdV + EV-71			8% (1/13)
AAV-2 + HHV-6B	56% (5/9)
AAV-2 + EBV	11% (1/9)
AAV-2 + HHV-6B + EBV		11% (1/9)
Total	78% (7/9)	78% (7/9)	77% (10/13)	75% (3/4)

AAV: Adeno-associated virus; CMV: Cytomegalovirus; EBV: Epstein-Barr virus; EV: Enterovirus; HAdV: Human adenovirus; HHV: Human herpes virus; VZV: Varicella-zoster virus.

The rates of AAV-2 coinfection with HAdV and/or HHV-6 detected via real-time PCR are shown in Table 8. Twenty-three cases and 27 cases positive for AAV-2 according to PCR were also examined for HAdV and HHV-6 infection via real-time PCR in the English and Scottish studies, respectively[17,18]. In the English study, among 23 patients in whom AAV-2 was detected (29 samples; FFPE liver samples: n = 8; whole-blood samples: n = 7, serum samples: n = 6, liver samples: n = 5, respiratory samples: n = 2, and stool samples: n = 1), 18 (78.3%) were coinfected with HAdV and/or HHV-6. AAV-2 coinfection with HAdV and HHV-6 (triple infection) was detected in 13 of 23 cases (56.5%). Among the 12 cases in which the patient underwent transplantation in the English study, nine involved triple infections (AAV-2, HAdV, and HHV-6), two involved double infections (AAV-2 and HHV-6), and one involved infection with only AAV-2[17]. Although the rate of AAV-2 positivity was low (9.2%) in the controls in the English study (Table 5), the coinfection rate was high (85.7%) in the AAV-2-positive controls. Although the rates of AAV-2 coinfection with HAdV and/or HHV-6 detected via real-time PCR were almost the same, approximately 80%, as those of HAdV and/or HHV detection via metagenomic sequencing, the details of the viruses detected via real-time PCR were not consistent with those detected via metagenomic sequencing. In the Scottish study, AAV-2 coinfection involved only HAdV in 5 (18.5%) of 27 cases. In the Scottish study, no AAV-2 coinfection with either HHV-6 or HAdV+HHV-6 was detected via real-time PCR. As mentioned above, the use of plasma samples might cause a low detection rate of coinfection[18].

Table 8 Rate of positivity for human adenovirus and/or human herpes virus-6 infection in adeno-associated virus-2-infected cases of acute hepatitis of unknown etiology according to real-time polymerase chain reaction, n (%).

	England				Scotland
Detected viruses	Sample	Case, n = 23	Control, n = 7		Sample	Case, n = 27
AAV-2 + HAdV + HHV-6 (triple infection)	Whole-blood	3/7 (42.9)	Whole-blood, immunocompromised, HAdV infection, raised ALT > 500 U/L	1/6 (16.7)	Plasma or serum	0/27 (0)
	Liver	5/5 (100)	Liver, immunocompromised	0/1 (0)
	Respiratory sample	1/2 (50)
	Stool	0/1 (0)
	Plasma	0/6 (0)
	Formalin-fixed, paraffin-embedded liver	4/8 (50)
	Total	13/23 (56.5)		1/7 (14.2)
AAV-2 + HAdV	Whole-blood	1/7 (14.3)	Whole-blood, immunocompromised, HAdV infection, raised ALT > 500 U/L	5/6 (83.3)	Plasma or serum	5/27 (18.5)
	Liver	0/5 (0)	Liver, immunocompromised	0/1(0)
	Respiratory sample	0/2 (0)
	Stool	0/1 (0)
	Plasma	2/6 (33.3)
	Formalin-fixed, paraffin-embedded liver	0/8 (50)
	Total	3/23 (13.0)		5/7 (71.4)
AAV-2 + HHV-6	Whole-blood	0/7 (0)	Whole-blood, immunocompromised, HAdV infection, raised ALT > 500 U/L	0/6 (0)	Plasma or serum	0/27 (0)
	Liver	0/5 (0)	Liver, immunocompromised	0/1 (0)
	Respiratory sample	0/2 (0)
	Stool	0/1 case (0)
	Plasma	0/6 (0)
	Formalin-fixed, paraffin-embedded liver	2/8 (25)
	Total	2/23 (8.7)		0/7 (0)
Total		18/23 (78.3)		6/7 (85.7)		5/27 (18.5)

AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; AAV: Adeno-associated virus; HAdV: Human adenovirus; HHV: Human herpes virus.

Because real-time PCR for AAV-2 was not performed in the United States study, the frequency of AAV-2 coinfection detected via real-time PCR could not be evaluated. However, in the United States study, AAV-2 coinfection detected via metagenomic sequencing combined with herpes virus-specific PCR was evaluated[19]. In the United States study, 14 cases in which whole-blood or plasma samples were available were compared with 113 controls in terms of the rates of positivity for AAV, HHV-6, EBV and CMV. Because samples obtained at enrollment from all 14 patients were positive for HAdV according to PCR, HAdV was excluded from the statistical analysis. The rate of positivity was calculated on the basis of the total number of positive metagenomic sequences (positive results among cases: AAV-2, n = 13; HHV-6, n = 0; EBV, n = 2; and CMV, n = 1; positive results among controls: AAV-2, n = 4) and virus-specific PCRs (positive result among cases: HHV-6, n = 7; EBV, n = 11, and CMV, n = 0; positive result among controls: HHV-6, n = 1; EBV, n = 1; and CMV, n = 2). There was a significant difference (P < 0.001) in the rates of positivity for AAV-2, HHV-6 and EBV between the cases and each of the four control groups, except for HHV-6 between the cases and control group 4. These findings suggest that HHV-6, EBV, and AAV-2 contribute to the development of AHUE in children.

PHYLOGENETIC TREE ANALYSIS OF WHOLE AAV-2 GENOMES

A total of 37 isolates of AAV-2, including fifteen isolates from the English study (GenBank accession number OP161114-OP161128)[17], nine isolates from the Scottish study (GenBank accession number OP019741-OP019749)[18] and 13 isolates from the United States study (United States supplementary data)[19], were used for phylogenetic tree analysis of the AAV genomes. Phylogenetic tree analysis including 97 reference sequences revealed that all isolates from children with AHUE except one (OP161117.1; liver case 2, England 2022) belonged to a subgroup of AAV-2 (Figure 1). However, analysis of contemporaneous non-AHUE isolates from England (GenBank accession number OP113129-OP113134) and other reference sequences revealed high similarity of viral genome sequences to isolates from cases of AHUE and that these sequences belonged to the same cluster of AAV-2. These findings suggest that there are no specific viral genotypes that cause severe pediatric hepatitis. The AAV-2 genome encodes structural protein/capsid proteins (VP1, VP2 and VP3), large nonstructural/replication-related proteins (Rep78/68, Rep52/40), and small nonstructural/viral accessory proteins (assembly activating protein [AAP], membrane-associated accessory protein and X protein)[38]. The frequencies of amino acid mutations are shown in Table 9. GenBank accession number NC_001401.2 was used as a reference whole-genome sequence. Amino acid mutations were distributed throughout the whole genome of AAV-2. The frequencies of AAV-2 amino acid mutations in 37 isolates from AHUE cases were compared with those in isolates from two control groups—one with 30 published isolates, including contemporaneous data, and one with 30 isolates published before 2016. The three studies revealed six, 17 and 20 amino acid mutations in the Rep 78/52, AAP and VP1-VP3 regions, respectively. Among these mutations, three amino acid mutations in the Rep 78/52 region (T183A, V508A and F619S), 10 amino acid mutations in the AAP region (Q6P, E70A, P73 L, T80I, S81C, N83S, K84R, I95T, L103P, and D132G), and seven amino acid mutations in the VP1-VP3 region (V151A, Q457M, S492A, E499D, F533Y, R585S, and R588T) were detected in 50% or more of the cases in all three studies. However, these mutations were also detected in 50% or more of the controls, except for three mutations (Q457M, E499D, and F533Y) in the VP3 region. The VP3 region is associated with the characteristics of viral structure. Thirteen single-amino-acid mutations, including Q457M, E499D and F533Y, lead to the ability to evade adaptive host immunity[39]. Therefore, these mutations might be related to AHUE in children. Additionally, the English study revealed that the majority of cases and contemporaneous isolates contained stop codons in the X region. Among the 37 AAV isolates from patients with AHUE, all but one (97%) had a stop codon in the X region. On the other hand, the frequency of the X region containing a stop codon was 30% (9/30) in the control group including contemporaneous control isolates and 30% (9/30) in the control group from before 2016. The X protein functions in the replication of AAV-2 DNA[38]. Mutating the X region in an AAV-2 packaging plasmid resulted in an approximately 33% reduction in recombinant AAV vector DNA replication and virion production[40]. Although the reduction in DNA replication seems to reduce the severity of hepatitis caused by AAV-2, the influence of the stop codon in the X region must be clarified.

Figure 1 A phylogenetic tree of the whole adeno-associated virus serotype 2 genomes, including 37 isolates from three studies (England: n = 15, Scotland: n = 9, United States: n = 13) and 97 reference strains from GenBank, was constructed via the maximum likelihood method. The reliability of the phylogenetic tree was assessed with 500 bootstrap replicates. The evolutionary distances were computed using the maximum likelihood method. These analyses were performed with the MEGA software program (version 11.0.13). Bootstrapped values less than 80 are not shown.

Table 9 Frequencies of amino acid mutations in adeno-associated virus-2 genomes from cases of acute hepatitis of unknow etiology.

Proteins	Amino acid mutation	England	Scotland	United States	Controls
					Including recent published data	Published data before 2016
Rep 78, 52	T183A	100 (15/15)	100 (9/9)	100 (12/12)	97 (29/30)	93 (28/30)
	K474Q	33 (5/15)	66 (6/9)	67 (4/6)	20 (6/30)	13 (4/30)
	V508A	100 (15/15)	100 (9/9)	100 (4/4)	97 (29/30)	93 (28/30)
	T605S	27 (4/15)	0 (0/9)	60 (3/5)	17 (5/30)	17 (5/30)
	L609V/M	73 (11/15)	89 (8/9)	25 (1/5)	33 (10/30)	20 (6/30)
	F619S	100 (15/15)	100 (9/9)	67 (2/3)	97 (29/30)	97 (29/30)
AAP	Q6P	100 (15/15)	100 (9/9)	60 (3/5)	93 (28/30)	93 (28/30)
	E70A	93 (14/15)	89 (8/9)	100 (13/13)	57 (17/30)	30 (9/30)
	P73 L	93 (14/15)	89 (8/9)	100 (13/13)	73 (22/30)	50 (15/30)
	T80I	87 (13/15)	100 (9/9)	100 (11/11)	67 (20/30)	47 (14/30)
	S81C	87 (13/15)	100 (9/9)	100 (11/11)	67 (20/30)	43 (13/30)
	N83S	73 (11/15)	89 (8/9)	100 (11/11)	80 (24/30)	93 (28/30)
	K84R	93 (14/15)	67 (6/9)	100 (11/11)	73 (22/30)	57 (17/30)
	N88S	27 (4/15)	0 (0/9)	56 (5/9)	20 (6/30)	33 (10/30)
	Q89 L	27 (4/15)	0 (0/9)	56 (5/9)	20 (6/30)	33 (10/30)
	R92A	53 (8/15)	0 (0/9)	83 (5/6)	40 (12/30)	37 (11/30)
	I95T	100 (15/15)	100 (9/9)	86 (6/7)	93 (28/30)	97 (29/30)
	L103P	87 (13/15)	56 (5/9)	100 (6/6)	57 (17/30)	73 (22/30)
	F116S	20 (3/15)	67 (6/9)	50 (2/4)	23 (7/30)	23 (7/30)
	H117P	13 (2/15)	67 (6/9)	67 (2/3)	23 (7/30)	20 (6/30)
	D132G	73 (11/15)	67 (6/9)	57 (4/7)	60 (18/30)	53 (16/30)
	T137A	20 (3/15)	11 (1/9)	67 (4/6)	10 (3/30)	17 (5/30)
	K146R	47 (7/15)	22 (2/9)	100 (6/6)	47 (14/30)	43 (13/30)
VP1-3	R81Q	0 (0/15)	0 (0/9)	50 (6/12)	0 (0/30)	0 (0/30)
	V125I	0 (0/15)	0 (0/9)	60 (6/10)	3 (1/30)	10 (3/30)
	V151A	87 (13/15)	89 (8/9)	82 (9/11)	60 (18/30)	0 (0/30)
	A162S	33 (5/15)	44 (4/9)	56 (5/9)	23 (7/30)	10 (3/30)
	Q164N	47 (7/15)	67 (6/9)	11 (1/9)	37 (11/30)	27 (8/30)
	T205S	60 (9/15)	22 (2/9)	25 (1/4)	23 (7/30)	43 (13/30)
	N312S	20 (3/15)	11 (1/9)	60 (3/5)	10 (3/30)	17 (5/30)
	R447K	20 (3/15)	56 (5/9)	18 (2/11)	30 (9/30)	33 (10/30)
	T450A	14 (2/14)	56 (5/9)	10 (1/10)	10 (3/30)	0 (0/30)
	Q457M1	93 (13/14)	100 (9/9)	86 (6/7)	40 (12/30)	23 (7/30)
	S492A	100 (14/14)	100 (9/9)	50 (1/2)	87 (26/30)	80 (24/30)
	S492D	0 (0/14)	0 (0/9)	50 (1/2)	0 (0/30)	0 (0/30)
	E499D1	93 (13/14)	100 (9/9)	100 (3/3)	47 (14/30)	23 (7/30)
	P521T	67 (10/15)	89 (8/9)	33 (1/3)	20 (6/30)	0 (0/30)
	F533Y1	87 (13/15)	100 (9/9)	50 (1/2)	43 (13/30)	20 (6/30)
	E548G	20 (3/15)	44 (4/9)	50 (1/2)	37 (11/30)	33 (10/30)
	D553E	7 (1/15)	0 (0/9)	50 (1/2)	0 (0/30)	7 (2/30)
	R585S	93 (14/15)	100 (9/9)	100 (10/10)	73 (22/30)	50 (15/30)
	R588T	100 (15/15)	100 (9/9)	100 (10/10)	97 (29/30)	93 (28/30)
	A593S	47 (7/15)	44 (4/9)	70 (7/10)	53 (16/30)	37 (11/30)

¹Frequency of amino acid mutations is 50% or more in all three studies, but under 50% in the recent data and published data before 2016. Gray background: Frequency of amino acid mutation is 50% or more.

HISTOLOGICAL ANALYSIS OF THE LIVER

All three studies performed histological analysis of the liver (Table 10). In the English study[17], 12 explanted liver samples and two liver biopsy samples were examined. Liver histological analysis revealed ballooning hepatocytes and perivenular, bridging or pan-acinar necrosis. Although the lobular structure of the liver was disrupted, there was no evidence of fibrosis. These findings were consistent with nonspecific acute hepatitis. There were no features of autoimmune hepatitis. Viral inclusions were not observed. The infiltrating lymphocytes were predominantly CD8+ T cells but included CD20+ B cells. Staining for the CD79a pan-B-cell lineage was also performed. Although macrophage-derived cells were positive for antibodies against HHV-6B in explanted livers, immunochemical staining for adenovirus and AAV-2 was negative. Electron microscopy did not identify any viral particles in hepatocytes, blood vessel endothelial cells or Kupffer cells.

Table 10 Histological analysis of liver from cases of acute hepatitis of unknown etiology.

	England	Scotland	United States
Histology	Ballooning hepatocytes	Destruction of the sinus structures and irregularly arranged fibers were detected	Acute hepatitis with portal and lobular inflammation, ranging from mild to severe activity
	Disrupted liver architecture with varying degrees of perivenular, bridging or pan-acinar necrosis	Lobular hepatitis with periportal and interface inflammation	Increased sinusoidal macrophage with occasional forms demonstrating hemophagocytosis
	No evidence of fibrosis	Intracellular inclusions, bile duct proliferation and ballooning of hepatocyte of varying severity were observed	Marked fibrosis is rare
		Mild-to-moderate fibrotic changes were noted, but no evidence of confluent fibrosis
		Modified hepatic activity index for scoring of necroinflammatory ranged from 6 to 11 (maximum score, 18)
Immunohistochemistry	Infiltrating cells were predominantly stained with CD8+ T cells, but also included CD20+ B cells	Accumulation of MHC class II + cells was observed	Negative for adenovirus, HSV1, HSV2, CMV or VZV
	Widespread staining with the CD79a pan-B cell lineage, which identified plasma cells, was observed	Negative for complement
	Staining for Adenovirus and AAV-2 was negative
	MHC class I and II staining were increased but not specific
	C4d staining showed very weak
Macrophage-derived cells were positive for staining with HHV-6B antibody in all five explanted livers
In situ hybridization	No data	AAV-2 RNA was detected in the nuclei and cytoplasm of ballooned hepatocytes and arterial endothelial cells	No data
		AAV-2 RNA-positive cells were quantified at a high level
		HAdV RNA and HHV6 RNA were detectable, but at negligible levels
Co-detection by indexing analysis	No data	Prominent proliferation of epithelial cells was detected with increased CD68+ macrophages and activated CD4+ and CD8+ T cells and CD20+ B cells	No data
		High expression of the interferon-induced GTP-binding protein MX1 was observed

AAV: Adeno-associated virus; CMV: Cytomegalovirus; HAdV: Human adenovirus; HHV: Human herpes virus; MHC: Major histocompatibility complex; VZV: Varicella-zoster virus.

In the Scottish study[18], five liver biopsy samples and one explant liver sample were evaluated. In addition to conventional staining, in situ hybridization and immune typing were carried out. Liver histological analysis revealed lobular hepatitis. Moreover, periportal and interface inflammation, bile duct proliferation, ballooning and vacuolated hepatocytes were observed. The inclusion bodies were detected in hepatocytes. Mild to moderate fibrotic changes were noted, but there was no evidence of confluent fibrosis. The modified hepatic activity index for scoring necroinflammatory activity, in which the maximum score is 18, ranged from 6 to 11. Immunohistochemistry revealed an increase in MHC class II+ cells. In situ hybridization revealed the presence of AAV-2 RNA in the nuclei and cytoplasm of ballooned hepatocytes and arterial endothelial cells in the samples of all patients. AAV-2 RNA was detected in 1.2% to 4.7% of cells, which was as high as the frequency of HCV-infected cells in the liver of patients with chronic hepatitis C[41]. These findings suggest that AAV-2 replicates in the liver. HAdV RNA and HHV6 RNA were also detectable but at negligible levels. Codetection by indexing (CODEX), a multiplexed single-cell imaging technology, is used for the detection of various immune markers[42]. CD68-positive macrophages and activated CD4+ and CD8+ T cells were increased in liver samples from patients. High interferon expression induced the expression of the GTP-binding protein MX1, which indicated the activation of the innate immune response in the explant liver. Considering the significantly high frequency of the MHC class II HLA-DRB1*04:01 allele in patients, the authors speculated that a CD4+ T-cell-mediated immunopathological response was triggered by exposure to AAV-2 infection.

The United States study reported the findings of the histological analysis in the supplementary table of the United States study[19]. Liver histological analysis data were obtained from eight patients. Portal and lobular inflammation were observed. The degree of inflammation ranged from mild to severe. Bile duct damage was also observed. These findings were described as interface hepatitis. Marked fibrosis was rare, and there was no evidence of chronicity. No adenovirus was detected by electron microscopy. Immunohistochemistry was negative for adenovirus, HSV1, HSV2, CMV and VZV. Immunohistochemistry and in situ hybridization for AAV-2 were not performed.

All three histological investigations revealed nonspecific acute hepatitis. Although the degree of fibrosis was inconsistent across these studies, there was no advanced fibrosis. The features of autoimmune hepatitis were not observed. Immunohistochemistry revealed an increase in activated T cells and B cells and the accumulation of MHC class II+ cells. These findings indicate that the immune-mediated response may be the cause of AHUE. Although immunohistochemistry could not detect AAV-2 or HAdV proteins, in situ hybridization detected AAV-2 RNA in ballooned hepatocytes. However, further studies are needed to confirm the replication of AAV-2 in the liver.

HLA TYPING

To explore the disease susceptibility of patients, the two United Kingdom studies investigated the relationship between HLA and AHUE. The results of HLA typing are shown in Table 11. In the Scottish study, 27 samples from cases and 64 controls (apheresis donors in Scotland) were genotyped for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 by high-resolution typing[18]. Among the 27 samples, 25 (92.6%), 23 (85.2%) and 26 (96.3%) were positive for at least one copy of HLA-DRB1*04:01, HLA-DQA1*03:03 and HLA-DRB4*01:03, respectively. Among the 64 controls, 10 (15.6%), 11 (17.2%) and 21 (32.8%) were positive for at least one copy of HLA-DRB1*04:01, HLA-DQA1*03:03 and HLA-DRB4*01:03, respectively. The allele frequencies of HLA-DRB1*04:01, HLA-DQA1*03:03 and HLA-DRB4*01:03 in cases were 0.54 (controls: 0.08, odds ratio of 13.7, P = 5.49 × 10^-12), 0.54 (controls: 0.09, odds ratio of 12.3, P = 1.9 × 10^-11) and 0.67 (controls: 0.17, odds ratio of 9.4, P = 1.8 × 10^-10), respectively. Although the authors mentioned that it was not possible to be certain which was the causal susceptibility allele, these findings suggest that the HLA class II alleles were significantly related to disease susceptibility. Although comparisons with controls were not available, 13 cases were genotyped in the English study[17]. Among the 13 patients, 12 (92.3%) and 11 (84.6%) were positive for at least one copy of HLA-DRB1*04:01 and HLA-DQA1*03:03, respectively. These genetic predispositions are consistent with the findings of the Scottish study. Therefore, these studies suggest that strong linkage disequilibrium is present in these regions.

Table 11 Frequencies of human leukocyte antigen.

	Scotland			England
	Case	Control		Case
Positive for at least one copy of the HLA-DRB1*04:01	92.6% (25/27)	15.6% (10/64)		92.3% (12/13)
Allele frequency of the HLA-DRB1*04:01	0.54	0.08	Odds ratio 13.7 (95%CI: 5.5-35.1), P = 5.49 × 10-12	0.54
Positive for at least one copy of the HLA-DQA1*03:03	85.2% (23/27)	17.2% (11/64)		84.6% (11/13)
Allele frequency of the HLA-DQA1*03:03	0.54	0.09	Odds ratio 12.3 (95%CI: 5.1-30.7), P = 1.9 × 10-11	0.5
Positive for at least one copy of the HLA-DRB4*01:03	96.3% (26/27)	32.8% (21/64)		No data
Allele frequency of the HLA-DRB4*01:03	0.67	0.17	Odds ratio 9.4 (95%CI: 4.4-21.3), P = 1.8 × 10-10

HLA: Human leukocyte antigen.

CONCLUSION

Jaundice and vomiting are common clinical symptoms in patients with AHUE. Although one-third of children with AHUE underwent liver transplantation in England, only a few children underwent liver transplantation in Scotland and the US. Targeted and untargeted metagenomic sequencing detected AAV-2 in 80% or more of the patients. The high frequency of AAV-2 was confirmed by PCR. Findings from three studies suggest that AAV-2 is a pathogen for AHUE. Although there were discrepancies in the rates of positivity for viruses between metagenomic sequencing and PCR, HAdV, HHV-6B and EBV were frequently detected. As helper viruses, not only HAdV but also HHV-6B and EBV were indispensable for the development of severe hepatitis caused by AAV-2. No distinct clade was observed in the phylogenetic tree analysis of the whole AAV-2 genomes from the patients. However, three mutations (Q457M, E499D and F533Y) in the VP3 region might be associated with the virulence of AAV-2. Most liver samples from cases presented evidence of interface hepatitis without marked fibrosis. Although the samples were negative for AAV-2 and adenovirus according to the immunohistochemical analysis, in the Scottish study, AAV-2 RNA was detected in the nuclei and cytoplasm of ballooned hepatocytes by in situ hybridization. The presence of viral RNA indicates that AAV-2 can replicate in the liver. As host factors, HLA-DRB1*04:01, HLA-DQA1*03:03 and HLA-DRB4*01:03 were significantly associated with disease susceptibility. Coinfection with AAV-2 and one or more helper viruses, high viral loads of AAV-2 in the blood, anti-AAV-2 IgM and human leukocyte antigen typing could be candidate diagnostic criteria for AHUE. AAV-2 infection should be incorporated into clinical guidelines for the management of acute liver failure. Cidofovir can be administered if coinfection with AAV-2 and HAdV is detected.

ACKNOWLEDGEMENTS

I thank Professor Emma Thomson and Professor Judith Breuer for providing me with the details of their published research.