Demographic and virological data
Totally 13 7-month-old infants and their mothers were enrolled, and 8 of them were followed up to 3 years old. The levels of serum HBV DNA, HBsAg and HBeAg did not change significantly among mothers, 7-month-old infants and 3-year-old infants. (Table 1) In addition, an average of 16±4.72 full-length HBV genome clones per sample were collected, and no significant differences of the clone numbers were noticed among groups.
Table 1
The demographic, clinical and laboratory data of 13 mother-infant pairs
Subject
|
Age
|
Gender
|
Clone number
|
HBsAg
(log10IU/ml)
|
HBeAg
(log10S/CO)
|
HBV DNA
(log10IU/mL)
|
pair 1
|
M1
|
19Y
|
|
17
|
4.41
|
3.16
|
8.40
|
|
C1-1
|
7M
|
Male
|
24
|
4.01
|
3.00
|
7.65
|
|
C1-2
|
3Y
|
|
25
|
4.05
|
3.07
|
7.87
|
pair 2
|
M2
|
21Y
|
|
19
|
4.52
|
3.04
|
8.23
|
|
C2-1
|
7M
|
Male
|
24
|
2.19
|
2.12
|
5.87
|
|
C2-2
|
3Y
|
|
19
|
3.60
|
3.15
|
7.80
|
pair 3
|
M3
|
19Y
|
|
13
|
4.46
|
3.15
|
8.47
|
|
C3-1
|
7M
|
Male
|
22
|
4.52
|
3.09
|
8.39
|
|
C3-2
|
3Y
|
|
17
|
4.54
|
3.08
|
8.48
|
pair 4
|
M4
|
32Y
|
|
19
|
4.83
|
3.18
|
8.24
|
|
C4-1
|
7M
|
Female
|
10
|
5.14
|
3.24
|
7.97
|
|
C4-2
|
3Y
|
|
20
|
5.01
|
3.18
|
8.16
|
pair 5
|
M5
|
27Y
|
|
12
|
4.81
|
2.93
|
9.22
|
|
C5-1
|
7M
|
Male
|
20
|
4.61
|
2.95
|
9.13
|
|
C5-2
|
3Y
|
|
18
|
4.88
|
2.25
|
8.26
|
pair 6
|
M6
|
23Y
|
|
9
|
4.38
|
3.21
|
8.12
|
|
C6-1
|
7M
|
Female
|
8
|
4.58
|
3.03
|
8.87
|
|
C6-2
|
3Y
|
|
13
|
4.69
|
3.19
|
8.06
|
pair 7
|
M7
|
22Y
|
|
15
|
4.55
|
3.23
|
8.49
|
|
C7-1
|
7M
|
Female
|
12
|
4.96
|
3.07
|
9.37
|
|
C7-2
|
3Y
|
|
12
|
5.07
|
3.07
|
8.89
|
pair 8
|
M8
|
20Y
|
|
14
|
4.43
|
3.11
|
8.08
|
|
C8-1
|
7M
|
Male
|
13
|
4.58
|
3.19
|
8.97
|
|
C8-2
|
3Y
|
|
17
|
4.09
|
3.07
|
8.39
|
pair 9
|
M9
|
20Y
|
|
14
|
4.69
|
3.12
|
8.41
|
|
C9
|
7M
|
Female
|
18
|
4.74
|
3.77
|
8.81
|
pair 10
|
M10
|
29Y
|
|
14
|
4.25
|
2.90
|
8.82
|
|
C10
|
7M
|
Female
|
19
|
4.92
|
2.93
|
8.16
|
pair 11
|
M11
|
22Y
|
|
10
|
4.73
|
2.94
|
8.96
|
|
C11
|
7M
|
Female
|
22
|
4.19
|
0.85
|
8.67
|
pair 12
|
M12
|
25Y
|
|
17
|
4.49
|
2.99
|
8.22
|
|
C12
|
7M
|
Male
|
20
|
4.70
|
2.94
|
8.87
|
pair 13
|
M13
|
20Y
|
|
7
|
3.92
|
3.16
|
7.82
|
|
C13
|
7M
|
Male
|
14
|
3.79
|
3.24
|
8.32
|
M1: mother 1; C1-1: child of mother 1 at 7 months; C1-2: child of mother 1 at 3 years; Y: years; M: months |
The phylogenetic trees were constructed between 13 mothers and their paired 7-month-old infants, 8 infants at 7 months and 3 years old, as well as 8 mothers and their paired infants at 3 years old. (Supplementary Figure S1) All clones clustered together with the reference sequence of genotype C HBV, and the sequences of all clones from the same pair were clustered together, indicating there was no contamination during the acquisition of clones.
Comparative analysis of HBV quasispecies characteristics in mothers and their paired 7-month-old infants
The quasispecies characteristic values, including complexity, mutation frequency and genetic distance, at full-length genome level, 11 coding regions and 7 noncoding regions were analyzed between mothers and 7-month-old infants. As shown in Supplementary Table S1 and Figure 1A-C, the complexity, mutation frequencies and genetic distances of full-length genome and most specific regions at nucleotide level in 7-month-old infants were significantly lower than those in mothers.
For coding regions, the mutation frequencies of Core, PreS2, RT, NTCP-BD, X and PreS1 regions, as well as the complexities and genetic distances of Core, PreS2, P and RT regions at amino acid level in 7-month-old infants were also significantly lower than those in mothers. (Supplementary Table S2 and Figures 1D-F) Further, the synonymous substitution rates of most regions and the non-synonymous substitution rates of Core, PreS2, P and RT regions were dropped significantly after MTCT. (Supplementary Table S3 and Figure1G-H)
The phylogenetic tree and mutation rate of single nucleotide site analyses were performed on each pair of infants and their mothers. Phylogenetic trees of HBV sequences in 10 infants (76.92%, 10/13) segregated from those of mothers, indicating the evolve selection was present during MTCT of HBV. (Supplementary Figure S2) The mutation rates of some nucleotide sites were significantly different in 10 pairs of infants and their mothers (Table 2). In one of the other 3 pairs with sequences mixed in phylogenetic trees, C1826T & A1827C mutations were present in 40% of HBV sequences in infant, which is significantly higher than that of mother. (Table 2)
Table 2
Detailed nucleotide site with mutation rate changed significantly during MTCT
Case
|
Mutants
|
MR
(mother)
|
MR
(7-month-old infants)
|
MR change
|
P
|
Amino acid substitution
|
pair 1
|
G375T
|
0.06
|
0.38
|
0.32
|
0.028
|
sW74L
|
|
C2102T
|
0.12
|
0.46
|
0.34
|
0.039
|
non
|
pair 2
|
C339A
|
0.00
|
0.58
|
0.58
|
<0.001
|
sP62L
|
|
T2555C
|
0.00
|
0.42
|
0.42
|
0.001
|
non
|
|
A2590T
|
0.00
|
0.42
|
0.42
|
0.001
|
pY95F
|
pair 3
|
C105T
|
0.00
|
1.00
|
1.00
|
<0.001
|
PreS2A39V
|
pair 4
|
C1826T&A1827C
|
0.00
|
0.40
|
0.40
|
0.009
|
PrecH5S;xT152P
|
pair 5
|
C2366A
|
0.00
|
0.50
|
0.50
|
0.004
|
cP156T
|
pair 6
|
T3116C
|
0.00
|
0.94
|
0.94
|
<0.001
|
PreS1V90A
|
pair 7
|
T2708G
|
0.00
|
0.95
|
0.95
|
<0.001
|
non
|
pair 8
|
G648C
|
0.00
|
1.00
|
1.00
|
<0.001
|
sW165S
|
|
C732T
|
0.00
|
0.38
|
0.38
|
0.016
|
sS193L
|
pair 9
|
C1T
|
0.50
|
0.00
|
-0.50
|
0.001
|
non
|
|
G20A
|
0.00
|
1.00
|
1.00
|
<0.001
|
PreS2A11T
|
|
A616G
|
0.43
|
0.00
|
-0.43
|
0.003
|
rtI163V
|
|
T999A
|
0.36
|
1.00
|
0.64
|
<0.001
|
non
|
|
C1913A
|
0.50
|
0.00
|
-0.50
|
0.001
|
cP5T
|
|
A2159G
|
0.50
|
0.00
|
-0.50
|
0.001
|
cS87G
|
|
A2189C
|
0.50
|
0.00
|
-0.50
|
0.001
|
cI97L
|
pair 10
|
C26A
|
0.29
|
0.00
|
-0.29
|
0.024
|
PreS2L13I
|
|
T39A
|
0.00
|
0.95
|
0.95
|
<0.001
|
PreS2V17E
|
|
G1386A
|
0.50
|
1.00
|
0.50
|
0.001
|
xV5M
|
|
C2660T
|
0.29
|
1.00
|
0.71
|
<0.001
|
non
|
pair 11
|
T2576C
|
0.00
|
1.00
|
1.00
|
<0.001
|
non
|
MR: Mutation Rate at single nucleotide site |
Dynamics of HBV quasispecies characteristics from 7 months to 3 years old
The quasispecies characteristic values of full-length decreased significantly after MTCT and increased to near maternal level from 7 months to 3 years old, while the levels of serum HBV DNA, HBsAg and HBeAg did not change obviously. (Figure 2A)
At nucleotide acid level, the quasispecies complexities, mutation frequencies and genetic distances of Core, PreS2 and other regions increased to near the maternal level at 3 years of age. (Supplementary Table S4 and Figure 2B-D) For amino acid level, the phenomenon was noticed in Core and PreS2 regions. (Figures 2E-G and Supplementary Table S5) The synonymous substitution rates of Core, P and NTCP-BD regions (Figure 2H and Supplementary Table S6) and the non-synonymous substitution rates of Core and PreS2 regions also significantly increased to near the maternal level at 3 years old. (Figure 2I and Supplementary Table S6)
The phylogenetic tree of each pair revealed that most sequences of infants at 7 months and 3 years old partially mixed. (Figure 2J) The mutation rates of single nucleotide site were analyzed, and 19 mutants with mutation rates significantly changed have been found in 5 infants. (Table 3) Among them, the mutation rates of 3 sites significantly decreased from 7 months to 3 years old, and caused amino acid substitutions; While in 15 nucleotide sites with the mutation rates significantly increased, 13 of them caused amino acid substitution, including 10 located at Core region. Core region is the most diverse region during HBV quasispecies evolution in the early stage of infection. It is worth noticing that these significant changing mutants in Core region were found in 4 of 5 male infants at 3 years old.
Table 3
The detailed nucleotide sites with mutation rate changed significantly from 7 months to 3 years of age
Case
|
Gender
|
Mutant sites
|
MR
(7 months)
|
MR
(3 years)
|
MR change
|
P
|
Amino acid mutation
|
pair 1
|
Male
|
G375T
|
0.38
|
0.04
|
-0.34
|
0.011
|
sW74L
|
|
|
G1613A
|
0.00
|
0.44
|
0.44
|
<0.001
|
pR841K
|
|
|
G1899A
|
0.00
|
0.60
|
0.60
|
<0.001
|
precG29D
|
|
|
T1938C
|
0.00
|
0.28
|
0.28
|
0.01
|
cV13A
|
|
|
T1961G
|
0.00
|
0.24
|
0.24
|
0.022
|
cF21C
|
|
|
C2102T
|
0.46
|
0.04
|
-0.42
|
0.001
|
non
|
|
|
C2288A
|
0.00
|
0.68
|
0.68
|
<0.001
|
cP130T
|
pair 2
|
Male
|
C339A
|
0.58
|
1.00
|
0.42
|
0.004
|
sP62L
|
|
|
T1938C
|
0.00
|
0.84
|
0.84
|
<0.001
|
cV13A
|
|
|
A2119C
|
0.00
|
0.37
|
0.37
|
0.002
|
non
|
|
|
A2159G
|
0.00
|
0.58
|
0.58
|
<0.001
|
cS87G
|
|
|
A2189C
|
0.00
|
1.00
|
1.00
|
<0.001
|
cI97L
|
|
|
C2198A
|
0.00
|
0.42
|
0.42
|
0.001
|
cL100I
|
|
|
C2288A
|
0.42
|
1.00
|
0.58
|
<0.001
|
cP130T
|
|
|
T2555C
|
0.42
|
1.00
|
0.58
|
<0.001
|
non
|
|
|
A2590T
|
0.42
|
1.00
|
0.58
|
<0.001
|
pY95F
|
pair 3
|
Male
|
C2381A
|
0.00
|
0.24
|
0.24
|
0.029
|
cP161T
|
pair 4
|
Female
|
C1826T&A1827C
|
0.40
|
0.00
|
-0.40
|
0.008
|
precH5S;xT152P
|
pair 5
|
Male
|
C2366A
|
0.50
|
0.17
|
-0.33
|
0.031
|
cP156T
|
MR: Mutation Rate at single nucleotide site |
Dynamics of the potential NAs-resistant mutations in RT region and the mutation rates of single amino acid site in Core and PreS2 regions
The potential NAs-resistant mutants defined by previous reports [24, 25] were searched from the clones of 8 mothers (118 clones) and their paired infants at 7 months (133 clones) and 3 years old (141 clones). The deletion rate in RT region was significantly higher in mothers (8.47%, 10/118) than that in 7-month-old infants (0, 0/133) (P<0.001). (Figure 3A) Totally, 21 potential NAs-resistant mutants were found and listed in Supplementary Table S7. The cumulative rate of NAs-resistant mutations (the number of clone with NAs-resistant mutants/total clone number, clones contain multiple NAs-resistant mutants were counted multiple times) was significantly lower in 7-month-old infants (26.32%, 35/133) than that in mothers (49.15%, 58/118) (P=0.003) and 3-year-old infants (39.01%, 55/141) (P=0.031), whereas there was no significant difference between mothers and 3-year-old infants. (Figure 3B) Similarly, the ratio of clones with NAs-resistant mutants (clones contain multiple NAs-resistant mutations were counted only once) was also significantly lower in 7-month-old infants (23.31%, 31/133) than that in mothers (43.22%, 51/118) (P=0.001) and 3-year-old infants (35.46%, 50/141) (P=0.028), whereas there was no significant difference between mothers and 3-year-old infants. (Figure 3C)
Due to the dramatic change of diversity in Core and PreS2 regions during MTCT and early infancy, the substitution rates of single amino acid site were calculated in these regions. More indels were found in mothers (3.40%, 4/118) than that in 7-month-old infants (0, 0/133) (P=0.048), and there were also more indels in 3-year-old infants (5.67%, 8/141) than that in 7-month-old infants (P=0.007), whereas there was no significant difference between mothers and 3-year-old infants. (Figure 3A) As shown in Figure 3D, more substitutions in B cell and CD4+ T cell epitopes in Core and PreS2 regions were found in the clones from mothers and 3-year-old infants than that in 7-month-old infants. For 184 amino acid sites in Core region, the ratio of sites with substitution rate over 1% was lower in 7-month-old infants (2.72%, 5/184) than that in mothers (8.15%, 15/184) (P=0.021) and 3-year-old infants (13.59%, 25/184) (P<0.001). (Figure 3D) For 55 amino acid sites in PreS2 region, the ratio of sites with substitution rate over 0.5% was lower in 7-month-old infants (18.18%, 10/55) than that in mothers (40%, 22/55) (P=0.012) and 3-year-old infants (38.18%, 21/55) (P=0.02). (Figure 3D)