Dynamic changes in drug-resistant duck hepatitis B virus DNA in single hepatocyte nucleus after hemihepatectomy using a liver regeneration model

Objective To investigate the dynamic changes in drug-resistant duck hepatitis B virus (DHBV) DNA in single hepatocyte nucleus after hemihepatectomy using a liver regeneration model.Methods Ducks aged 45 days with chronic DHBV infection were inoculated with mutant DHBV after hemihepatectomy and then fed with lamivudine.Blood samples and liver tissue samples were collected at regular intervals.Viral DNA level in peripheral blood and viral strains were measured.Flow cytometry was used to isolate single hepatocyte nuclei and nuclei with or without proliferating cell nuclear antigen (PCNA) .Nested-PCR was used for the multiplication of intranuclear DHBV YMDD region.Direct sequencing was used to determine the absence or presence of mutation.The chi-square test and the Fisher's exact text were used for comparison of categorical data between groups.The Welch method was used for comparison of PCNA before and after hemihepatectomy.Results There were wild strains in single hepatocyte nuclei and serum before hemihepatectomy.After inoculation with mutant strains, the percentage of hepatocyte nuclei infected with mutant strains decreased significantly from 1 week to 12 weeks after hemihepatectomy (χ2=7.225, P<0.01) .The percentage of PCNA-positive hepatocyte nuclei was 0.84%±0.36% before surgery, significantly increased to42.26%±6.48% at 1 week after surgery, and was 11.83%±3.97% at 12 weeks after surgery, and there was a significant difference between different time points (F=190.832, P<0.001) .The percentage of PCNA-positive nuclei infected with mutant strains decreased significantly from 1 week to 12 weeks after surgery (χ2=7.063, P<0.01) , while the percentage of PCNA-negative nuclei infected with mutant stains was extremely low after surgery.Conclusion Hemihepatectomy may provide a replication space for drug-resistant virus, but drug-resistant virus cannot become dominant strains in the peripheral blood and the liver.