Of 2023 mothers and 2518 children aged 5-7 years, HBsAg positive rate was 4.7% (95/2023) in mothers and 0.52% (13/2518) in their children and all HBsAg positive samples were included in this study. The mean age of mothers was 32.36±6.01 years. 69.2% of children were 5 years old and 30.8% were 6 years old. Among 95 HBsAg positive mothers, nine of their children were positive for HBsAg giving MTCT rate of 9.5% (9/95). The detail of background demography were already discussed by Vichit et al. In this study, we present the outcomes from genome sequences analysis.
Nucleic acid extraction and HBV genomes amplification
HBV DNA amplified by WA region primer set was detected in 52 samples (41 mothers and 11 children) from which the full genome sequences having 3 kilo base pairs (3kbp) could perform in 78.1% (32/41) of mothers and 90.9% (n= 10/11) of children. After another trial of amplification to those samples undetected by WA primers, the partial sequencing using s-region primers was achieved in the HBV DNA positive samples of 29 mothers and 1 child. Therefore, HBV DNA was extracted from 73.7% (70/95) of mothers and 92.3% (12/13) of children who were positive for HBsAg and all these 82 samples were able to classify HBV genotypes in Cambodia. (Table 1)
Nationwide HBV genotype distribution and phylogenetic tree
HBV genotype was determined by the s-region of each detected strain using the neighbor-joining method. HBV genotype C was abundantly found in 84.3% (59/70) of mothers and 58.3% (7/12) of children. HBV genotype B was found in 15.7% of mothers (11/70) and 36.3% of children (5/12). As the phylogenetic tree was constructed by the strains having 823 base pairs from nt111-nt933, 53 out of 82 HBV DNA positive samples could assign. Almost all HBV genotype C were sub-grouped to C1 and were gathered in the same cluster of China, Hong Kong, Thailand, Laos, Malaysia, Myanmar and India except one (C173433) which is sub-genotype C 8 and is much closed to Indonesian strain (Figure 1). Only a small portion of HBV genotype B was circulated in Cambodia and is in the same cluster to Vietnam in phylogenetic tree except one (C170329) which is adjacent to Taiwanese strain (Figure 1).
Homology of genome sequences in 7 mother-child pairs
Of 9 HBsAg positive mother-child pairs, 2 pairs were excluded for mothers’ refusal to participate. Among them, only one pair could amplify 2630 bp. The rate of base sequences match (homology) in six mother-child pairs ranged from 99.62 to 100%. The analysis of 2630bp (nt1929-nt1343) detected from one mother-child pair (C171408m and C171407c) showed a 99.96% homology in their nucleotide sequence. (Table 2)
Occurrence of S gene mutant strains of HBV in Cambodia
The determination of mutation was confined to the area specific for “a determinant region of hepatitis B surface antigen (nt121- nt149). S gene mutant strains of HBV were detected in 17 mothers and 2 children and were recognized at P120S, T/I126N, P127S/T/A, T131I/N, M133T, F134 and G145R/A. In children, one for each aa120 (P120S) and aa127 (P127S) were found (Table 3). But in mothers, the predominant mutation was found at aa127 (P127S/T/A: 5/17) and aa131 (T131I/N/P: 4/17) and aa145 (G145R/A: 4/17), aa126 (T/I126N: 3/17) and aa133 (M133T: 3/17), aa134 (F134L: 1/17) (Figure 2). The overall S gene mutation rate among HBV DNA positive sera was 24.3% (17/70) in mothers and 16.7% (2/12) in children (p=0.5657). The S gene mutation rate of HBV by the entire participants was 0.8% (17/2023) in mothers and 0.08% (2/2518) in children (p<0.001). Among 17 mothers infected with S gene mutant of HBV, two children (11.7%) were found to be HBsAg positive. Among 53 mothers infected with wild type HBV, 7 children (13.2%) were positive to HBsAg (Table 4, Figure 2).
HBsAg prevalence and S gene mutation rate of HBV among immunized children were 0.4% and 0.08% and that among non-immunized children were 4.8% and 0% respectively. The a determinant mutation rate among children infected from mother with mutant variant is higher than those infected from mother with wild type (5.9% Vs 1.9%). If the child received hepatitis B vaccination birth-dose (HepB-BD) within 24 hours after birth, the infection rate among children with mutant variants is (2% Vs 4.5%). By each genotype, the mutation rate in genotype C was 24.2% (16/66) and that of genotype B was 18.8% (3/16).
Characteristics of S gene mutant strains of HBV found among 13 mother-child pairs
After excluding children with undetectable HBV DNA (n=1), whose mothers’ HBsAg negative (n=2) and whose mothers refused to participate (n=2), 8 mother-child pairs were then analyzed for S gene mutation of HBV. One mother-child pair has mutation at nt127 (P127S) in both mother and her child, one mother-child pair had mutation at nt120 (P120S) only in child and another one pair has mutation at nt145 (G145R) only in mother. Seven out of 13 children had completed at least 2 doses of pentavalent vaccine with or without HepB-BD. (Figure 2)
Double and combination mutant strains among children and their mothers in Cambodia
The double mutation at A1762T/G1764A was found only in HBV genotype C1 strains (12 mothers and 3 children) with the mutation rate of 48.39%. The combination mutation at C1653T and A1762T/G1764A or T1753C and A1762T/G1764A was also only found in HBV genotype C1 strains (10 mothers) with the mutation rate of 32.26%. (Table 5)
Full-length genome sequences and evolutionary analysis of Texa
We could do the full-genome sequences in 42 samples (32 mothers and 10 children) with the nucleotide length from 3161-3239 base pairs amongst which 31 strains were belong to genotype C and the rest (11 strains) were genotype B. All HBV genotype C belongs to sub-genotype C1 which were assumed to be originated from Indonesia, Thailand, India, China and Vietnam. For HBV genotype B, almost all detected strains (n=10) are found to be recombinant genotype B4/C2. Only one strain (C170329) showed recombinant B2/C2. All these recombinant B/C strains build up with circular DNA mixing up of sequences resembling genotype B and a short portion of genotype C in core region (Figure 3) with various breaking points for recombination. By mean of evolutionary relationship of Texa, all recombinant genotype B4/C2 strains are near to Vietnamese strains but B2/C2 is very near to Taiwanese strain.