Global prevalence and molecular characteristics of three clades within hepatitis B virus subgenotype C2: Predominance of the C2(3) clade in South Korea

**Background:** Hepatitis B virus (HBV) genotype C is the most prevalent genotype in Asia and is associated with higher replication capacity, chronic infection, and progression to liver cirrhosis and hepatocellular carcinoma compared to genotype B. Subgenotype C2 is endemic in East Asia (China, Japan, South Korea) and has been further divided into three phylogenetically distinct clades: C2(1), C2(2), and C2(3). However, the global distribution and molecular characteristics of these clades had not been systematically analyzed. South Korea has a notable HBV burden (HBsAg prevalence 3.4% in males, 2.6% in females) and reports higher rates of naturally occurring variants, occult infection, and antiviral drug resistance.

**Methods:** The authors retrieved 1,315 HBV genotype C full-genome sequences from public databases (1,096 from a prior dataset plus 219 newly downloaded from NCBI). Phylogenetic analysis was performed using the approximate maximum likelihood method (FastTree) and confirmed with MrBayes (MCMC, GTR substitution model) and maximum likelihood (Tamura-Nei model). A total of 683 subgenotype C2 sequences (261 C2(1), 270 C2(2), 152 C2(3)) were used for genetic distance calculations and mutation analysis. Mutation frequencies for preC/C (G1896A), basal core promoter (BCP) double mutations (A1762T/G1764A), rtL269I polymorphism, and 42 potential nucleos(t)ide analog resistance (NAr) amino acid positions were compared across clades using chi-square tests. Signature sequences (≥70% frequency in one clade) were identified. A validation cohort of 127 Korean chronic HBV patients from Konkuk University Hospital was used to assess the frequency of two C2(3)-specific signature mutations: sV184A in HBsAg and xT36P in HBx.

**Key Results:** Of 1,315 genotype C sequences, 893 belonged to subgenotype C2, separated into C2(1) (n=299), C2(2) (n=321), and C2(3) (n=273). Subgenotype C2 accounted for 88.4% of genotype C strains in China (457/517), 97.4% in Japan (111/114), and 100% in South Korea (108/108). Strikingly, C2(3) comprised 96.3% (104/108) of South Korean strains but only 5.4% (28/457) of Chinese and 35.1% (40/114) of Japanese strains. Genetic diversity was lowest in C2(2) (mean substitution/site 0.013±0.004) compared to C2(1) (0.011±0.004, p<0.0001) and C2(3) (0.012±0.004, p<0.01). No significant difference in preC/C G1896A mutation frequency was found among clades. BCP double mutations (A1762T/G1764A) were significantly higher in C2(2) and C2(3) versus C2(1) (p<0.05). The rtL269I polymorphism was significantly higher in C2(2) than in C2(1) and C2(3). For NAr mutations, C2(3) showed a significantly higher total mutation rate across 42 positions (3.3%) compared to C2(1) (2.8%) and C2(2) (2.5%) (p=0.0312). Specifically, rtM204I (primary resistance) was detected in 11.84% of C2(3) versus 5.75% of C2(1) and 7.43% of C2(2) (p=0.0003). rtL180M (secondary resistance) was found in 10.5% of C2(3) versus 4.6% of C2(1) and 4.4% of C2(2) (p=0.0209). In treatment-naïve patients (n=183), C2(3) still showed significantly higher total NAr mutation frequency (p<0.0001). Twenty-one signature sequences were identified: 1 nonsynonymous (NS) for C2(1) (L116V in HBx, 81.6%), 3 NS for C2(2) (S314P in Pol, 93.7%; F321L in Pol spacer, 91.1%; A90V in preS1, 89.3%), and 4 NS for C2(3) (sV184A in HBsAg, 78.9%; T/S/A36P in HBx, 82.9%; H81N in Pol terminal protein, 97.4%; T272A in Pol spacer, 76.3%). In the Korean validation cohort, phylogenetic analysis of 131 RT sequences showed 89.3% (117/131) belonged to C2(3). Among these, 65.8% carried sV184A and 79.5% carried xT36P; 61.5% of 65 patients with both regions sequenced carried both mutations.

**Clinical Implications:** The near-exclusive dominance of C2(3) in South Korea (96.3%) versus its low prevalence in China (5.4%) and moderate prevalence in Japan (35.1%) represents a striking epidemiological difference among three geographically proximate East Asian nations. This clonal expansion may explain previously reported higher rates of NAr mutations, BCP mutations, and HBeAg-negative HCC in Korean cohorts. The higher frequency of primary (rtM204I) and secondary (rtL180M) drug resistance mutations in C2(3) suggests that Korean patients may be at increased risk of NA treatment failure, warranting closer monitoring and potentially different treatment strategies. The identified signature mutations (sV184A, xT36P) could serve as genetic markers for rapid clade identification. Limitations include reliance on public database sequences with incomplete treatment history data and potential selection bias. Future studies should investigate the functional impact of C2(3)-specific mutations on viral replication, drug susceptibility, and clinical outcomes.