Because genotype C is known to be responsible for the majority of HBV infections in HBV endemic areas such as China, Korea and other Asia-Pacific nations and given that it is reportedly more virulent than genotype B 
, monitoring of mutation types in genotype C by means of nested PCR, particularly in cases associated with occult infections, should be imperative for the appropriate control of hepatitis B in this area. For example, a specific mutation type in the HBV preS/S region in occult cases may cause an alteration of HBsAg antigenicity or the secretion capacity, which may lead to vaccine escape infections and the emergence of high virulence variants in a population. However, mutations related to occult infection from genotype C isolates have rarely been introduced thus far, despite their probable high frequencies due to the mutation-prone features of genotype C.
A large number of unique mutation patterns in S ORF related to occult infections, thus far not introduced, were in fact found in this study. The high frequency of diverse mutation types related to occult infection in Korean subjects may be due to in part the early introduction of the HBV vaccination among the Korean population. HBV vaccinations were initially introduced in 1983 to the Korean population 
, which dramatically reduced the prevalence of HBsAg-positive chronic carriers from the initial level 30 years ago of more than 10% to 3.7% by 2005 
. Selection pressure by this successful vaccination may have led to the generation of diverse mutation patterns capable of causing immune evasion among the vaccinated population 
In this study, we investigated mutation patterns related to occult infections of HBV genotype C, focusing on the S ORF from HBsAg-negative Korean subjects. A total of five major characteristic features are noteworthy in terms of mutation patterns related to occult infections from our HBsAg-negative cohort. First, two deletion types in preS1 and preS2, both of which are reported to be associated with liver disease progression in chronic patients, including HCC 
, were significantly implicated in more than half of the occult subjects (53.7%, 22/41 subjects). This finding strongly supports the previous notion that HBV variants related to occult infection can contribute to the progression of HCC or cirrhosis 
. Furthermore, HBV mutations in occult infection may be due to de novo infection in patients with advanced liver disease rather than the accumulation of independent mutations in individuals. Our data also showed that these two types of deletions existed in an exclusive manner, suggesting that the simultaneous generation of both deletions may have a lethal effect on the viral life cycle. Of the preS1 deletions, the deletions in the preS1 start codon leading to 11 amino acid truncations of LHBs, as shown in genotype D, were significantly related to occult infection in our cohort. This offers strong support of a previous report showing that a mixed infection of both genotype A and genotype D could lead to HBsAg seronegativity 
. The preS2 deletion observed in this study may also contribute to occult infection by shortening the distances between the S promoter and the transcription initiation site of S mRNA, in turn leading to an alteration of the expression ratio of LHBs to HBsAg, which can also result in HCC via the up-regulation of the ER stress pathway, affecting the transactivating capacity 
. The preS2 deletion type is also reported to exist in some occult subjects infected with genotype A or D 
, suggesting its global prevalence in HBV occult infection. Disturbance of the ratios between the three HBV S proteins by LHB overexpression due to these two types of preS deletions may lead to the interruption of virion formation or secretion, thus contributing to HBV occult infection. The questions of whether the two types of preS deletions can lead to the overexpression of LHBs or can contribute to HCC via the ER stress pathway remain to be elucidated in a future study.
Secondly, it is noteworthy that the prevalence of mutations leading to premature termination in LHBs was significantly higher in occult subjects (16 subjects) than in carriers (1 subject) (39.0% vs. 2.5%, p<0.001). The interruption of normal virion formation by this type of mutation may complicate the infection of hepatocytes or the secretion of HBsAg into the blood, leading to occult infection. A phylogenetic analysis of HBV strains showed that three types of premature terminations, PreS1-Q104Stop, PreS2-W3Stop and Pol-W591Stop, all had close relationships within the strains of each type (), suggesting the possibility of horizontal transmission between Korean populations by specific HBV strains related to occult infection.
Thirdly, mutations in the “a” determinant were related to occult infection in our cohort. Mutations in major hydrophilic regions (MHR), particularly the “a” determinant, are known to be associated with occult infection or the generation of vaccine escape variants due to the reduced binding affinity between the HBsAg and the antibody to HBsAg 
. Among the MHR mutations related to vaccine escape, the mutation from glycine to arginine in the 145th
codon of HBsAg, known as G145R, has been most frequently encountered worldwide 
. However, the G145R mutation was found only in one patient in our cohort (KU-II-28) (Table S1
). The 126th
codon of HBsAg was most frequently affected among the MHRs of our occult subjects, thus offering strong support of our previous report which showed that the MHR mutation of the I/T126N/S type prevails in chronic Korean patients with genotype C infection 
. Of the mutation types in the “a” determinant observed in this study, mutations in two codons, G145 and I/T126 may be related to the global pattern in HBV occult infections.
Fourth, notably, including the W182L and sW182* mutations, the 182nd
codon was most frequently affected among the mutations of HBsAg in occult subjects (15/41 subjects). Our previous report showed that HBV variants with sW182* were found at a substantially high frequency in chronic Korean patients of genotype C, particularly in patients with advanced liver disease. Furthermore, a comparison of the clinical data between patients with wild type and sW182* indicated that the HBV DNA levels in patients with sW182* were significantly lower than those with wild types 
, one of the distinctive features of HBV occult infection. This finding provides information about how sW182* can lead to occult infection. However, the exact mechanism of how the sW182* variant leads to occult infection should be addressed in a future study. We also found no W182L mutations in chronic Korean patients of genotype C (data not shown), suggesting that this type of mutation is specific to HBV occult infections. A previous transient transfection study using the HBV full genome construct showed that sW182* interrupts secreted and intracellular virion formation, providing a possible link between sW182* and occult infections 
. However, the mechanism by which W182L leads to occult infection remains to be found.
We also found that all mutation frequencies with the three aforementioned regions (preS1, preS2 and HBsAg) of S ORF, particularly mutations in the “a” determinant, the major target of the host immune response 
, were significantly higher in occult subjects than in carriers (), which leads us to speculate that HBV variants related to occult infection may contribute to liver disease progression via persistent infection. Furthermore, our data showing that the preS deletions and sW182* mutations, reportedly related to disease severity in chronic Korean patients, were also found more frequently in occult subjects than in carriers strongly support the above hypothesis. Compared to the mutation patterns in S ORF from occult subjects of other countries, including other genotypes as well as genotype C, two mutation types, deletions in the preS1 start codon and in sW182*, were found to exist exclusively in Korean occult subjects 
The most important drawback of our study is that the quasispecies analyses of some samples were based on the very few clones. Therefore, given the low number of templates in these samples, it cannot be excluded that they may be subject to PCR artifact in terms of the accuracy of their mutations.
In conclusion, our data suggest that the defect in virion formation induced by the mutations of preS deletions and sW182* in HBsAg, related to the progression of liver disease in chronic patients, a loss of infectivity via the premature termination of LHBs, and a change in HBsAg antigenicity via “a” determinant mutations, all contribute to the occult infection of HBV genotype C among a HBsAg-negative population. The novel mutation patterns related to occult infection introduced in the present study can help to broaden our understanding of not only HBV occult infections, but also HBV immune evasion.