Sophisticated viral quasispecies with a genotype-related pattern of mutations in the hepatitis B X gene of HBeAg-ve chronically infected patients

Patients with HBeAg-negative chronic infection (CI) have not been extensively studied because of low viremia. The HBx protein, encoded by HBX, has a key role in viral replication. Here, we analyzed the viral quasispecies at the 5′ end of HBX in CI patients and compared it with that of patients in other clinical stages. Fifty-eight HBeAg-negative patients were included: 16 CI, 19 chronic hepatitis B, 16 hepatocellular carcinoma and 6 liver cirrhosis. Quasispecies complexity and conservation were determined in the region between nucleotides 1255 and 1611. Amino acid changes detected were tested in vitro. CI patients showed higher complexity in terms of mutation frequency and nucleotide diversity and higher quasispecies conservation (p < 0.05). A genotype D-specific pattern of mutations (A12S/P33S/P46S/T36D-G) was identified in CI (median frequency, 81.7%), which determined a reduction in HBV DNA release of up to 1.5 log in vitro. CI patients showed a more complex and conserved viral quasispecies than the other groups. The genotype-specific pattern of mutations could partially explain the low viremia observed in these patients.

Compartmentalized evolution of hepatitis B virus contributes differently to the prognosis of hepatocellular carcinoma

Serum hepatitis B virus (HBV) mutations can predict hepatocellular carcinoma (HCC) occurrence. We aimed to clarify if HBV evolves synchronously in the sera, adjacent liver, and tumors and predict HCC prognosis equally. A total of 203 HBV-positive HCC patients with radical hepatectomy in Shanghai, China during 2011-2015 were enrolled in this prospective study. Quasispecies complexity (QC) in HBV core promoter region was assessed using clone-based sequencing. We performed RNA-sequencing on tumors and paired adjacent tissues of another 15 HCC patients and analyzed it with 3 public datasets containing 127 samples. HBV QC was positively correlated to APOBEC3s’ expression level (r=0.28, p&lt;0.001), higher in the adjacent tissues than in the tumors (p=6.50e-3), and higher in early tumors than in advanced tumors (p=0.039). The evolutionary distance between the sera-derived HBV strains and the tumor-derived ones was significantly longer than that between the sera-derived ones and the adjacent tissue-derived ones (p&lt;0.001). Multivariate Cox regression analyses indicated that high HBV QC in the sera predicted an unfavorable overall survival (OS) (p=0.002) and recurrence-free survival (RFS) (p=0.004) in HCC; whereas it in the tumors predicted a favorable RFS (p&lt;0.001), respectively. APOBECs-related HBV mutations including G1764A were more frequent in the sera than in the adjacent tissues. High-frequent A1762T/G1764A in the sera predicted an unfavorable RFS (p&lt;0.001); whereas it in the tumors predicted a favorable RFS (p=0.035). In conclusion, HBV evolves more advanced in the sera than in the tumors. HBV QC and A1762T/G1764A in the sera and tumors have contrary prognostic effects in HCC.