Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation

Table 1 The primers used for amplification of HAV genomic RNA

Cloned fragments	Primers	Sequences
A (0.8 kb)	A1	5'-CGCCGGCGTTCAAGAGGGGTGTCCGGAG-3'
	A2	5'-GAATCTCAATGCCAAATCTTGC-3'
B (0.5 kb)	B1	5'-TCTGAGGTACTCAGGGGC-3'
	B2	5'-CAGTCAATGATGCTATAGAACC-3'
C (1.1 kb)	C1	5'-CCAACAGGGGGGATTGATC-3'
	C2	5'-CGTTAGAAGGAGAGGTCAATC-3'
D (1.0 kb)	D1	5'-CCCTGGATTTCTGACACTCC-3'
	D2	5'-CAGTGGATAACATGGCATTTG-3'
E (1.1 kb)	E1	5'-GTCTGTCACAGAACAATCAGAG-3'
	E2	5'-GATCCCAGAACAGATATCTCTTAA-3'
F (1.2 kb)	F1	5'-GTTAAGAGATATCTGTTCTGGATC-3'
	F2	5'-CCATCCTCCAACGAGCACTCC-3'
G (1.2 kb)	G1	5'-CAGTTCTTTAGTCATGACAGTTG-3'
	G2	5'-GCCATTGGATCAATTTCAGC-3'
H (1.1 kb)	H1	5'-GAGTCCCATTTATCATCACA-3'
	H2	5'-GTCCAATCAGATCAAGATTATC-3'
I (0.5 kb)	I1	5'-GATTCTCTGTTATGGAGATG-3'
	I2	5'-TTTTTTTTTTTTTTTTTTTTTATTT-3'

⁽¹⁾: A-I represent 9 amplified fragments of HAV; ⁽²⁾ 1 and 2 represent positively and negatively oriented primers

Table 2 Titers of different passages of HAVH2K7 on KMB17 for 14 d.

Passages	Antigen titers	Infectious titers (LgCCID50/mL)
1	1:16	5.83
2	1:8	5.5
3	1:16	5.67
4	1:32	6.0
5	1:32	6.5
6	1:64	6.77
7	1:64	6.67
8	1:64	6.5
9	1:64	6.67
10	1:128	6.83
11	1:128	7.0
12	1:256	7.0
13	1:256	7.17
14	1:256	7.0
15	1:256	7.17
16	1:256	7.0
17	1:512	7.17
18	1:512	7.33
19	1:512	7.5
20	1:512	7.67
21	1:1024	7.67
22	1:1024	7.83

Table 3 One-step growth dynamics of HAVH2K7P22 on KMB17 cells

Post inoculation days	Antigen titers	Infectious titers (LgCCID50/mL)
2	1:8	ND*
4	1:16	6.0
6	1:32	6.67
8	1:128	7.17
10	1:512	7.5
12	1:1024	7.8
14	1:1024	7.17
16	1:1024	7.0
18	1:1024	7.0
20	1:1024	6.83
22	1:1024	7.0
24	1:1024	6.8
26	1:2048	6.5

*Not determined

Table 4 Differences in the genome sequence and amino acids of the HAV H2 K7 with HAV H2K7 P6, HAV H2K7 P12, HAV H2K7 P18 and HAV H2 K7 P22

Nucleotide position	Location	Nucleotide					Amino acids
		K7	K7P6	K7P12	K7P18	K7P22	K7	K7P6	K7P12	K7P18	K7P22
33	5'NTR	C	C	C	U	U
263	5'NTR	U	U	U	U	C
378	5'NTR	U	U	U	U	C
591	5'NTR	A	A	G	G	G
646	5'NTR	A	A	A	U	U
858	VP2	G	U	U	U	U	A	S	S	S	S
1178	VP2	C	U	U	U	U	I	I	I	I	I
4022	2B	T	C	C	C	C	I	I	I	I	I
4558	2C	A	A	A	A	C	Q	Q	Q	Q	P
4802	2C	U	U	U	U	C	A	A	A	A	A
4949	2C	A	A	A	A	U	T	T	T	T	T
4968	2C	A	C	C	C	C	N	H	H	H	H
5193	3A	C	U	U	U	U	R	C	C	C	C
5217	3A	G	G	G	G	U	A	A	A	A	S
5336	3C	G	G	G	G	A	L	L	L	L	L
5715	3C	A	A	A	G	G	T	T	T	A	A
6427	3D	U	U	U	G	G	V	V	G	G	G
7256	3D	A	U	U	U	U	A	A	A	A	A

Table 5 Identitiy comparisons of nucleotide sequence of HAVH2K7 with HAVH2K7P6 and HAVH2K7P22

Genomic region	HAVH2K7P6 (%)	HAVH2K7P12 (%)	HAVH2K7P18 (%)	HAVH2K7P22 (%)
GENOME	99.93	99.9	99.87	99.76
5'NTR	100	99.86	99.59	99.32
VP4 ENCODING	100	100	100	100
VP2 ENCODING	99.71	99.71	99.71	99.71
VP3 ENCODING	100	100	100	100
VP1 ENCODING	100	100	100	100
2A ENCODING	100	100	100	100
2B ENCODING	99.74	99.74	99.74	99.74
2C ENCODING	99.9	99.9	99.9	99.6
3A ENCODING	99.42	99.42	99.42	98.84
3B ENCODING	100	100	100	98.53
3C ENCODING	100	100	99.85	99.85
3D ENCODING	99.94	99.86	99.86	99.86
3'UTR	100	100	100	100