Long-read deep sequencing analysis of hepatitis B virus quasispecies in two elderly cases of interspousal transmission

Hepatitis B virus (HBV) can be transmitted within a family, but an interspousal transmission in elderly cases is rare and the change of viral quasispecies during the event is unclear. We experienced two acute hepatitis B males (AH1 and AH2, 67 and 71 years old, respectively) whose HBV was transmitted from their wives with chronic HBV infection (CH1 and CH2, 67 and 66 years old, respectively). To clarify the characteristics of HBV quasispecies in such cases, we performed long-read deep sequencing of HBV preS1/preS2/S domain using samples from the 2 couples. HBV full-genome sequences determined with direct sequencing showed that the HBV sequences belonged to subgenotype B1. AH1 was 98.0–99.2 % identical to CH1, and AH2 was 98.5–99.5 % identical to CH2, whereas the identity between AH1 and AH2 was 96.9 %. The long-read deep sequencing of amplicons including preS1/preS2/S domains with PacBio Sequel IIe showed the numbers of nucleotides with >5 % substitution frequencies in AH1, AH2, CH1 and CH2 were 0 (0 %), 4 (0.31 %), 39 (3.06 %) and 28 (2.20 %), respectively, indicating that CH1 and CH2 were more heterogeneous than AH1 and AH2. From a phylogenetic analysis based on the deep sequencing, minor CH1/CH2 clones that were close to AH1/AH2 clones were considered to be substantially distinct from the major populations in CH1/CH2. The major population formed during chronic infection under the immune pressure might not be suitable to establish new infection and this might be one of the reasons why the transmission had not occurred for a long time after marriage.