Comparison of three pediatric studies investigating acute hepatitis of unknown etiology

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.