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Background Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide, with chronic hepatitis B virus (HBV) infection serving as the primary etiology globally. Although HBV transcripts possess polyA tails and can thus be captured by sequencing platforms, the expression profiling of HBV genes within host cells at single-cell resolution remains a significant knowledge gap.Methods We integrated single-cell transcriptomic data from 8 cases and bulk transcriptomic data from 94 cases, encompassing HBV-positive and HBV-negative samples across both HCC and non-tumor liver tissues, to perform quantitative expression analysis of both human host and HBV genes ( IDDF2026-ABS-0110 Figure 1. Experimental workflow for characterizing HBV gene expression in liver tissues).Results HBV specifically infects and transcribes within NTCP-expressing hepatocyte-like cells, which can be classified into five distinct phenotypes via consensus non-negative matrix factorization (cNMF) analysis. In the NMF2 phenotype (hepatocyte-like metabolic), pgRNA, P, S, and X genes are all actively expressed; in NMF1 (stress/proliferative) and NMF3 (protein synthesis/basal metabolic) phenotypes, HBV selectively expresses only the X gene and pgRNA; conversely, HBV gene expression is not captured in NMF4 (extracellular matrix/pro-metastatic) and NMF5 (cholangiocyte-like) phenotypes ( IDDF2026-ABS-0110 Figure 2. Single-cell expression landscape of HBV genes and functional phenotyping of hepatocyte-like cells). HBV gene expression levels are positively correlated with the degree of copy number variation (CNV) in hepatocyte-like cells, suggesting that HBV activity may promote HCC tumor progression. CNV-based clonal evolution analysis indicates that NMF4 evolved from NMF3 clones, a process during which the loss of NTCP led to the abrogation of HBV activity, while other phenotypes belong to independent clonal evolutionary lineages. The NMF2 phenotype is associated with a favorable prognosis, whereas NMF3/4 phenotypes predict a poor prognosis, potentially due to the distinct gene regulatory modes of HBx on host cells between NMF2 and NMF3 phenotypes (IDDF2026-ABS-0110 Figure 3. CNV alterations clonal evolution and prognostic disparities across different hepatocyte-like cell phenotypes). Furthermore, NMF3 hepatocyte-like cells are capable of recruiting M2 macrophages and Tregs, thereby fostering an immunosuppressive microenvironment that inhibits the cytotoxic functions of CD8+ T cells and NK cells (IDDF2026-ABS-0110 Figure 4. Characterization of the immune microenvironment in HBV-infected liver tissues).Conclusions HBV exhibits distinct gene expression patterns upon infecting host hepatocyte-like cells, which promotes the occurrence and development of HCC with diverse phenotypes and clinical outcomes through differentiated gene regulatory modes.Abstract IDDF2026-ABS-0110 Figure 1Abstract IDDF2026-ABS-0110 Figure 2Abstract IDDF2026-ABS-0110 Figure 3Abstract IDDF2026-ABS-0110 Figure 4