Currently, over 290 million people worldwide suffer from chronic hepatitis B virus (HBV) infection (WHO 2021). In China, approximately 7.18% of the population are HBV carriers who are at risk of developing end stages of liver disease, including liver cirrhosis and hepatocellular carcinoma(Chen et al. 2006; Wang et al. 2014).
HBV contains a partially double-stranded DNA within a nucleocapsid surrounded by envelope proteins(Delius et al. 1983). HBV genome replication relies on a viral polymerase that harbors RNA-dependent reverse transcriptase (Summers and Mason 1982; Hu and Seeger 2015). However, the high error rate of RT coupled with a substantial amount of daily production (approximately 1011 copies/day), as well as selected immune pressures, frequently results in substantial genetic diversity that may evolve into several HBV genotypes(Lau and Wright 1993; Pourkarim et al. 2014; Lin and Kao 2015).
Recently, HBV has been classified into nine genotypes (A–I) and one tentative genotype J based on more than 7.5% differences in the full-length HBV genome sequences(Kramvis and Kew 2007; McNaughton et al. 2020). In addition, each genotype is further divided into subgenotypes based on more than 4.5% and less than 7.5% nucleotide differences(Kramvis and Kew 2007).
It has been shown that the different HBV genotypes display distinct tendencies of chronic infection, HBeAg seroconversion, and responses to interferon-based therapy(Orito et al. 2001; Kao 2002; Kao et al. 2004; Livingston et al. 2007; Ito et al. 2014; McMahon et al. 2021). Although HBV genotyping has not been widely used in clinical settings, the recent European Association for the Study of the Liver guidelines for HBV treatment recommend different timelines for treatment cessation due to a lack of response for genotypes A-D(European Association for the Study of the Liver. Electronic address and European Association for the Study of the 2017). This indicates that the HBV genotype is beginning to be considered in the context of patient management.
Several molecular-based methods have been developed for HBV genotyping, including polymerase chain reaction (PCR) with genotype-specific primers, PCR-restriction fragment length polymorphism, PCR-invader assay, oligonucleotide microarray chip, and sequencing analysis of the whole or portions of the HBV genome(Naito et al. 2001; Kirschberg et al. 2004; Chen et al. 2007). Among them, the sequencing analysis of whole HBV genome is considered to be the “gold standard” method for genotyping; however, it is relatively time-consuming and expensive, especially with respect to the large number of samples(Kramvis and Kew 2007; McNaughton et al. 2020). By contrast, since PCR-based genotyping methods are rapid and inexpensive, they are widely used in HBV genotyping; however, any nucleotide substitution in the PCR primer binding site may alter the HBV genotyping results.
In the present study, we found discrepancies in the results between PCR-based methods and the whole HBV genome sequencing analysis. The results and the possible reason for the discrepancy is described.