The lack of detectable HBsAg in spite of the presence of episomal, free HBV genomes at intrahepatic level is attributable in some cases to the HBV genetic variability. In fact, a fairly large number of studies have linked OBI occurrence to specific HBV variants. Indeed, it has been reported that OBI individuals are infected with HBV variants showing (a) mutations clustering in major hydrophilic region (MHR) of the small (S) protein, (b) mutations in the pre-S1/S2 genomic region, (c) specific structural alterations in virus regulatory elements, (d) mutations affecting posttranslational production of virus envelope proteins, and (e) mutations selected under antiviral treatment with nucleos(t)ide analogs (NUCs) that may cause amino acid changes both in viral polymerase and S protein [5, 10–12]. A high frequency of mutations has been found particularly within the MHR of HBsAg in HBV strains isolated from OBI individuals [4, 11, 13–24]. These mutations have been functionally associated with S protein structural changes that may lead to an impaired detection by commercially available HBsAg assays. In addition, there is evidence that occult HBV of specific genotypes may show not only the mutations in the MHR, but also a very high frequency of mutations in the T-cell epitopes, thus further supporting the hypothesis that the selection of these HBV variants may represent a mechanism of immune escape, as also suggested by the inability of anti-HBs antibodies from individual patients to recognize their own circulating viruses [11, 19–21, 25, 26]. Some recent studies have strengthened these data by applying ultra-deep pyro-sequencing. Indeed, a higher degree of genetic variability was found in the S gene of occult HBV compared to viruses from HBsAg-positive patients, and it has been postulated that the complex HBV quasi-species with mutations in HBsAg immune-active regions may help HBV to escape both from neutralizing and diagnostic antibodies [24, 27]. However, this evidence has been challenged by a very recent study showing that the genetic heterogeneity of reactivated HBV is significantly lower in patients with reactivation from OBI carrier status than that from HBsAg-positive carriers, suggesting that OBI individuals are infected with HBV populations of low genomic heterogeneity in their liver [28]. The very low or absent viral load characterizing OBI carriers has suggested that viral genomic mutations could also negatively impact any step of HBV life cycle [21, 23, 29, 30]. Indeed, there are data showing that occult HBV with specific amino acid substitutions in the MHR displays an impaired virion and/or S protein secretion when transfected in hepatoma cells or hydrodynamically injected in mice [21, 23, 30]. In this context, it is worth mentioning that also mutations at the level of the pre-S2/S splice donor site have been detected in occult HBV strains. Pre-S2/S splicing occurs during HBV replication, and mutations that interfere with pre-S2/S mRNA splicing may cause a marked reduction of functional unspliced pre-S2/S transcripts and of HBsAg synthesis, thus leading to OBI development. There is evidence that RNA secondary structure at the 5′ splice site can regulate the splicing efficiency of transcripts and modulate the binding of RNA-splicing factors as well as the recognition of splice site consensus elements [31]. Thus, it has been postulated that mutations at the pre-S/S 5′ splice donor site may affect the interaction of RNA with components of the spliceosome, hence impairing posttranscriptional RNA processing and/or nuclear export via the posttranscriptional regulatory element [25, 32, 33]. Pre-S mutations have also been associated with OBI occurrence. In particular, it has been shown that deletions in the pre-S1/S2 genomic region correlate with an impaired expression of envelope proteins, and that some of these deletions may contribute to persistence of the virus in the occult state by implying the elimination of HLA-restricted B-cell and T-cell epitopes [34–36]. The association of mutations and deletions in the pre-S gene with a lack of secreted HBsAg and low levels of HBeAg and HBV DNA was demonstrated using functional analysis by transfection into hepatocyte cell lines [36].

Despite all these lines of evidence, however, it is proved that the great majority of OBI individuals are not infected with specific HBV mutants. Moreover, important data have demonstrated that pre-S/S variants can frequently be found also in patients with overt HBV infection, including subjects with high viral loads [4, 12, 22, 35, 37]. Furthermore, strong evidence from different studies indicates that “occult” HBV genomes are usually replication-competent and that their genetic heterogeneity is comparable with those from HBsAg-positive individuals [4, 28, 37]. In vitro functional analysis showed that occult viral isolates “re-acquire” normal replication, transcription, and protein synthesis abilities once taken out from the host’s liver microenvironment. These viruses appear to normally replicate when transfected in hepatoma cells and to be competent in HBsAg production [4]. Therefore, according to these findings genomic variability does not usually appear to play a fundamental role in inducing the OBI status, which rather seems to be dependent on a strong suppression of the virus replication and transcriptional capabilities in the majority of the cases.

Several studies have shown that HBV replication is frequently impaired in individuals coinfected with other infectious agents. In particular, it has been shown that hepatitis C virus (HCV) infection can strongly suppress HBV replication, and this has led to hypothesize that the inhibitory activity exerted by HCV on HBV might ultimately result in OBI occurrence. This assumption is supported by the large body of evidence showing that OBI has the highest prevalence precisely in HCV-infected patients [5, 39], and by the in vitro data demonstrating that the HCV “core” protein can strongly inhibit HBV replication and gene expression [77–80]. However, more recent evidence has challenged the existence of interplay between HCV and HBV. Indeed, studies performed in animal models coinfected with HCV and HBV or in hepatoma cells transfected with HCV replicon (instead of single viral proteins) and full-length HBV genome have found no interference between the two viruses [81–85]. Thus, the available data cannot allow any definitive conclusion to be drawn for a role of HCV in the induction of the OBI status. It is known that also individuals positive for human immunodeficiency virus (HIV) frequently show either overt or occult HBV coinfection, but there is no evidence of possible direct effects of HIV on HBV activity or of the existence of any peculiar specific mechanisms leading to OBI occurrence in HIV-infected individuals [86–88]. Other infectious agents potentially capable of interfering with HBV activity include Schistosoma mansoni, a parasite that affects more than 200 million people worldwide [89]. Coinfection with HBV and Schistosoma occurs frequently in geographic areas where both agents are endemic [90, 91], and it has been demonstrated that infection with Schistosoma mansoni in HBV transgenic mice induces a strong suppression of HBV replication [92].

The peculiar life cycle of the HBV with its long-term persistence at intrahepatic level regardless of the HBsAg status represents scientific support of the large body of evidence indicating that OBI is a common, worldwide occurrence. Nevertheless, a reliable evaluation of the general prevalence of OBI is at present a very difficult objective to achieve mainly because of the lack of standardized, valid and commercially available assays for its detection, and because the present gold standard for OBI identification (i.e., to test liver DNA extracts by highly sensitive and specific molecular biology approaches such as nested-PCR or real-time PCR) is of course applicable only in the little minority of cases in which a liver specimen is available [1]. In addition, the positivity of circulating anti-HBc antibody—often used as a surrogate marker for OBI identification in HBsAg negative subjects—may be misleading since anti-HBc tests may provide false positive results [1, 93, 94], and also because about 20 % of OBI cases are negative for all HBV serum markers (namely, OBI seronegative individuals) [1, 5, 39]. Despite the above-mentioned limitations and some discrepancies in the available epidemiological data mainly due to the differences in sensitivity and/or specificity of the methods used in the various studies (reviewed in refs. [5, 95]), there is more than one solid piece of evidence that OBI is a largely world-wide diffused entity with a distribution that may reflect the diffusion of the HBV in the various geographic areas and in the various populations [96–99], and thus with a prevalence that appears to be higher in countries where HBV is endemic and among subjects at high risk of parenterally transmitted infections such as drug addicts and hemophiliacs [100, 101]. Of importance, OBI appears to be highly prevalent in chronically HCV infected individuals, and generally in patients with chronic liver diseases (i.e., alcoholic, cryptogenic, etc.) or with hepatocellular carcinoma [5, 39, 46, 78, 102–105]. In fact, HBV DNA is detectable in about one third of HBsAg-negative HCV carriers in the Mediterranean area, in more than 50 % in Far East Asian countries and in 50 % of US patients of Caucasian origin undergoing liver transplantation for end-stage HCV-related liver disease [39, 95, 106]. This last observation is particularly important also considering that the HBV general prevalence in the Caucasian American population is one of the lowest in the world [107].

The vast majority of individuals with OBI will never suffer from any clinical event related to the small amounts of viral genomes segregated in the liver cells. Nevertheless, in some particular circumstances and contexts OBI may acquire a pathogenic role and may become a (co)factor implicated in different clinical conditions that may also have severe sequels (Fig. 13.2). Indeed, since the suppression of viral replication and gene expression typical of the OBI status is a reversible condition, there is no doubt about the possibility that OBI, once transmitted by blood transfusion or liver transplantation from an “occult carrier,” may induce a typical, overt hepatitis B in a recipient naive for HBV infection. In analogy, an occult HBV infection may be reverted in an overt infection and reactivated with development of hepatitis B—often acute and severe—in patients undergoing therapeutic immunosuppression. Moreover, growing evidence exists on the possible contribution of OBI to the progression of liver fibrosis and establishment of cirrhosis as well as to the development of hepatocellular carcinoma, this last effect being related to the maintenance in the OBI phase of the mechanisms responsible for the pro-oncogenic properties of the overt, active HBV infection. In this context, however, it has to be taken into account that OBI appears to shape up as a complex scenario, which includes several different clinical/virological conditions quite different from one another. In fact, it is possible to distinguish seropositive (anti-HBc and/or anti-HBs positive) and seronegative (both anti-HBc and anti-HBs negative) OBI individuals (Fig. 13.3). In seropositive OBI, the HBsAg may have disappeared either very early after the resolution of an acute hepatitis event or after many years of overt carriage, whereas the seronegative OBI cases might have either progressively lost all HBV serum markers or might have been HBV negative since the beginning of the infection. Indeed, one cannot rule out the possibility that each of these conditions may have different roles and/or impacts on the occurrence or outcome of the liver disease.