Fig 1.
Family trees of family members with their status of HBsAg and HBV DNA.
A) Family 1, B) Family 2, C) Family 3, D) Family 4. The index persons are indicated with bold rim. Their age at the study conducted are shown. We could detect HBV DNA in 13 cases with nested PCR for sequence.
Table 1.
Demographic characteristics of residents and family members.
Table 2.
Primers for PCR and sequencing.
Fig 2.
Phylogenetic tree of mitochondrial hyper variable region (HVR) sequences of family members.
NJ method was used for construction of the phylogenetic tree, by bootstrap method with one thousand-time resampling. Outgroup is EF556173 (haplogroup L5). Every child was identified having the same sequence as his or her mother, meaning they are in a blood relation.
Fig 3.
Phylogenetic tree constructed with sequences of HBV polymerase region of obtained isolates.
Out of analyzed 48 isolates, forty-four residents and family participants were determined genotype B4 and also four residents were as genotype C1. Obtained residents’isolates are shown by their isolate names with arrow heads. Family member isolates are shown by their family names with arrow. Family 2, 3 and 4 member’s isolates accumulated in respective family’s clusters, except F3-5 marked with an asterisk that was the mother of family 3. Some residents’ isolates were confirmed as neighborhood of family members in each family cluster. Among residents’ isolates, some pair were so close composing a cluster, for example, 4445-Viet12 (28 years old female) and 3770-Viet12 (39 years old male), 4061-Viet12 (30 years old female) and 4454-Viet12 (58 years old female), 736-Viet (27 years old female) and 3968-Viet (25 years old male).
Fig 4.
Phylogenetic tree constructed with sequences of HBV complete genome.
HBV genotype FX75663 was used for outgroup. An arrow head is indicated for obtained residents isolate and an arrow for family member isolate. (A): Phylogenetic tree of full HBV genome of twenty-one isolates. Obtained all family members isolates were reconfirmed genotype B4 with whole HBV genome analysis. (B): Phylogenetic tree based on the core region sequences of twenty-one whole genome isolates, showed every genotype B4 isolate had a similarity as genotype C at the core region. Consequently, isolates were recombinants, genotype B4/C.
Fig 5.
Full genome mapping image with a standard strain D00329, genotype B1.
Full HBV genome mapping of obtained twenty-one isolates and known HBV genomes of genotype B and C. Besides the isolate name, age, sex, and nucleotide length are described. At the core region, all genotype B4 isolates have different pattern from genotype B1 or B6 (square in broken line), and almost the same pattern as genotype B2, B3, B5, B7, and C, suggesting they were recombinant at core region strains. Pre-S deletions as shaded squares were recognized in five isolates.
Fig 6.
Partial nucleotide sequences from nt1601 to nt1790 including core promoter of 48 obtained isolates.
Each genotype is shown besides each isolates name. The upper line sequence is AB073835, genotype B4, as a standard. HBeAg and HBeAb status are described at the right side of figure. In forty-four genotype B4 isolates, mutation rate at nt1613 was 13.6%, at nt1753 was 4.5%, at nt1762/64 was 11.4%. Insertions were detected in three isolates with 24 base starting from nt1673, just middle in the promoter, in X region.