Review HBV genotypes: relevance to natural history, pathogenesis and treatment of chronic hepatitis B

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1 Antiviral Therapy 2011; 16: (doi: /IMP1982) Review HBV genotypes: relevance to natural history, pathogenesis and treatment of chronic hepatitis B Beom Kyung Kim 1, Peter A Revill 2, Sang Hoon Ahn 1,3,4,5 * 1 epartment of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea 2 Victorian Infectious iseases Reference Laboratory, North Melbourne, Australia 3 Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea 4 Brain Korea 21 Project for Medical Science, Seoul, South Korea 5 Liver Cirrhosis Clinical Research Center, Seoul, South Korea *Corresponding author ahnsh@yuhs.ac Although chronic HBV infection is the leading cause of chronic liver disease and death worldwide, there are substantial differences in its clinical courses regarding prevalence, mode of transmission, characteristics of each phase, responses to antiviral therapy, and development of cirrhosis and hepatocellular carcinoma, according to geographical areas (Asia versus Western Europe and North America versus Africa). Furthermore, the clinical course in infected individuals depends on a complex interplay among various factors including viral, host, environmental and other factors. Recently, understanding of molecular characteristics of the prevailing HBV genotypes, frequently accompanied mutations and their clinical implications might explain these geographical differences more pertinently. Hence, in this article, we review the global epidemiology and the natural history of HBV infection, with emphasis on summarizing the different HBV genotypes according to regions. Introduction Chronic hepatitis B (CHB) is the leading cause of chronic liver disease (CL) and death worldwide. Of the 400 million people chronically infected with HBV across the globe, over 1 million die annually from primary adverse outcomes such as hepatocellular carcinoma (HCC) and cirrhosis [1 3]. Infected persons can pass through several phases (immune tolerant, immune clearance, inactive, reactivation and immune control phase) during their life time. However, the natural history in individuals is complex and highly variable, depending on the interplay among virus replication, host immune responses and a number of factors, including age, sex, age at infection, duration of infection, route of acquisition, race, infecting HBV genotype plus associated mutations, socioeconomic status and the presence of comorbidities [4]. This article reviews the global epidemiology, virology and the natural history of HBV infection, with emphasis on the different HBV genotypes and treatment response according to region. Prevalence and endemicity CHB, defined as the persistence of hepatitis B surface antigen (HBsAg) for greater than 6 months, has varying epidemiology between regions of the world with high and low endemicity of infection [1,5]. The prevalence of persons chronically infected with HBV ranges from 0.1 2% in low prevalence areas (United States, Canada, Western Europe, Australia and New Zealand) to 3 5% in intermediate prevalence areas (Mediterranean countries, Japan, Korea, Central Asia, Middle East, Latin and South America and Saharan Africa) and to 10 20% in high prevalence areas (Southeast Asia, China and sub- Saharan Africa; Figure 1) [6 8]. This wide range in chronic HBV prevalence in different parts of the world is largely related to differences in age at infection, which is inversely related to the risk of chronicity. The rate of progression from acute to chronic HBV infection is approximately 90% for infections acquired in the perinatal period, 30 50% for infections between the ages of 1 and 5 years, and <5% for infections acquired in adulthood [9]. For chronic infection to develop, the source of transmission is typically hepatitis B e antigen (HBeAg)-positive HBV. When the mother is HBeAgpositive, infants infected at birth have virtually a 100% risk of acquiring HBV and about 90% develop chronic HBV infection unless hepatitis B vaccine and hepatitis B immunoglobulin are administered shortly after birth 2011 International Medical Press (print) (online) 1169

2 BK Kim et al. Figure 1. Global distribution of chronic hepatitis B 8% High 2 7% Intermediate <2% Low Colours represent hepatitis B surface antigen prevalence. Modified from [171]. [10,11]. However, when the mother is HBeAg-negative and antibody to HBeAg (anti-hbe)-positive, the infant has a <25% risk of acquiring HBV and only 10 15% of becoming chronically infected [12]. Recent studies have also demonstrated that maternal serum HBV NA levels correlate better with the risk of transmission. Vertical transmission of HBV occurs in 9 39% of infants of highly viraemic mothers ( 10 8 copies/ml) despite postnatal vaccination [1,13 16]. In regions of Asia and Africa with medium to high HBV endemicity, most HBV infections occur within the first 5 years of life [9,17]. There is little evidence for significant adult HBV transmission in those areas, even among individuals with high-risk sexual behaviors. However, potential risks for adult transmission via sexual transmission and blood transfusions still exist, the latter due, in part, to the absence of screening HBV in blood banks in many low-income countries [5,18]. In East and Southeast Asia, perinatal transmission from HBeAg-positive mothers predominates [19]. By contrast, in Africa, most infections are believed to occur in childhood before the age of 5 years through the so-called inadvertent horizontal route accompanied by open cuts and scratches [5,20]. The risk of developing chronic HBV infection from horizontal transmission in this setting has been estimated between 30% and 50% for children infected between birth and 5 years of age, but only 7 10% thereafter [10,21,22]. Horizontal transmission also occurs either by sexual transmission or by needlestick transmission. The latter includes infections acquired from injecting drug use or inoculations from inadequately sterilized needles and from medical procedures with inadequate infection control precautions [10]. In Africa, cultural practices such as scarification may also be important. As is also the case in Asian countries, a family history is common. However, perinatal transmission plays a less important role in African CHB and the reasons why perinatal transmission is common in some areas of the world, such as Asia, and horizontal transmission in children is the dominant mode in other parts of the world, such as the sub-saharan Africa, are unclear. One hypothesis is that the age at which seroconversion from HBeAg to anti-hbe occurs is a key determinant in whether HBV is transmitted at or after birth [12]. Thus, if HBeAg seroconversion occurs early in a woman s life, the chances of perinatal transmission decreases significantly. It has been shown that >50% of women of child-bearing age in Eastern and Southeast Asia, where perinatal transmission is common, are HBeAg-positive at the time of delivery in comparison to <15% in Africa, where perinatal transmission is uncommon [10,12,23,24]. Likewise, the age at which HBeAg seroconversion occurs is different according to prevalence regions. This may be closely associated with the different distribution of HBV genotypes, which are predominantly restricted to different geographical regions. A detailed explanation on the characteristics of each HBV genotype is provided in HBV genotypes in this review, but in brief the median age at HBeAg seroconversion is <20 years for people infected with HBV genotypes A, B, and F, but approximately 3 decades later for persons infected with HBV genotype C [25] International Medical Press

3 The global epidemiology and natural history of chronic hepatitis Table 1. Prevalence and endemicity of HBV infection according to geographic area and HBV genotypes Clinical Western Europe/ characteristic Asia Africa North America Endemicity High High Low Mode of transmission Perinatal (vertical) Horizontal (frequently through Horizontal (mostly through percutaneous route) sexual or percutaneous route) Age of infection At birth or infant Childhood (<5 years) Adolescent or adult Chronicity Very high (>90%) High (30 50%) Low (1 5%) Family history HBeAg-positive mother HBeAg negative mother or Often none or relatives HBeAg-positive relatives ominant HBV genotype B1, B2, C1, C2 A1, E, A2, HBeAg, hepatitis B e antigen. In regions with low endemicity, most infections occur in adolescents and young adults [4]. For example, in the US and Western Europe (where endemicity is low and HBV genotypes A2 and/or dominate), sexual transmission and percutaneous transmission are the first and second most common routes of transmission [8]. Thus, a family history of HBV infection is uncommon and chronicity is low (1 5%) [9]. In the US, heterosexual and homosexual transmission (mostly among men having sex with men) accounts for approximately 39% and 24% of new HBV infections, respectively [26]. Percutaneous transmission usually occurs among injecting drug users (IUs) who share syringes and needles; in the US, IU accounts for 16% of new HBV infections [26]. The risk of HBV transmission increases with increasing duration of drug use, frequency of injection and sharing of drug preparation equipment [27]. Contact through the sharing of razors or toothbrushes, as well as certain practices, such as acupuncture, tattooing and body piercing, can also transmit HBV. Since most cases of HIV and HBV transmission occur sexually or percutaneously in low endemicity areas, the prevalence of CHB is higher in HIV-infected populations [5]. Prevalence and endemicity according to geographic area are summarized in Table 1 and Figure 1. HBV genotypes Eight HBV genotypes (HBV genotypes A H) have been identified by a sequence divergence greater than 8% in the entire HBV genome [28]. Thereafter, HBV isolated in Vietnam and Laos has been suggested to form a ninth HBV genotype I and, more recently, an HBV strain isolated from a Japanese patient has been designated as HBV genotype J [29,30]. HBV genotypes have further been separated into several subgenotypes if the divergence in whole nucleotide (nt) sequence is between 4% and 8% [31]. The geographic and ethnic distributions of HBV genotypes and subtypes are well-characterized, and increasing evidence suggests that the HBV genotype is a factor in determining disease progression [32]. However, existing information is still incomplete, as data is not available from many parts of the world or is based upon very small numbers of patients [28]. The geographic distribution of HBV genotypes are summarized in Figure 2 and Table 2. HBV genotype A HBV genotype A is found in North America, Southern and East Africa, and Western Europe; however, the HBV genotype A viruses in these regions are of differing subgenotypes and accumulating evidence suggests that their respective natural histories differ significantly [33]. For example, the African HBV subgenotype (A1 or Aa) of HBV genotype A is associated with HCC in young men who are usually HBeAg-negative and anti-hbe-positive, have low levels of HBV NA and rarely have cirrhosis [34]. Exposure to aflatoxin may be an important cofactor in the occurrence of this outcome [35,36]. HBV subgenotype A1 is associated with lower levels of HBV NA and earlier HBeAg seroconversion than other HBV genotypes [37], including the HBV subgenotype A2 or Ae (European HBV subgenotype), which is associated with HCC in older European persons [38]. Compared with HBV genotype (which also prevails in Western Europe), HBV subgenotype A2-infected patients tend to be HBeAg-positive and is associated with a lower risk of HCC, a greater likelihood of resolution of active hepatitis and clearance of HBV NA and HBsAg [39]. HBV subgenotype A3 is found in West Africa; however, little is known about its clinical significance [40]. HBV genotype B One of the two most prevalent HBV genotypes in Asia, HBV genotype B, is divided into two major groups: Bj, which is found in Japan, and Ba, which is found in the rest of Asia. Bj (for B Japan [B1 and B6]) is a pure strain of HBV genotype B, whereas Ba (for B Asia [B2-5]) contains a portion of the genome of HBV genotype C recombined Antiviral Therapy

4 BK Kim et al. Figure 2. Geographic distribution of HBV genotypes and subgenotypes A2 A2 A2 G A A2 2 A1 C2 B2 1 C1 B1 C2 C1 J B2 B6 F1 A2 B6 B6 G F1 A2 B6 E A3 E E A1 A1 C1 B4 C5 I B5 B3 C1 C3 C4 4 A2 H F2 F2 H A1 F1 F2 F3 A1 F4 The size of letter reflects the prevalence of the genotype in each region, with larger fonts representing increased prevalence. with the core region of HBV genotype B [28]. HBV subgenotype B1 is the original Bj subgenotype, found in Japan, whereas HBV subgenotype B6 is a new genotype recently found in the Arctic region in indigenous populations of Alaska, Canada and Greenland [41]. Although HBV subgenotype B6 is phylogenetically related to HBV subgenotype B1 and may have originated from Japan, it is distant enough that this HBV subgenotype is likely to have been removed from Japan for many centuries [41]. HBV subgenotype Ba consists of four distinct HBV subgenotypes, B2 (China and Taiwan), B3 (Indonesia), B4 (Vietnam) and B5 (Philippines) [10]. HBV subgenotype Ba is associated with older age at the time of HBeAg seroconversion, a higher risk for HCC, more severe liver disease and a higher frequency of basal core promoter (BCP) mutations as compared with HBV subgenotype Bj [42,43]. In fact, in persons older than 30 years, 32% versus 9% of those infected with HBV subgenotype Ba were found to be HBeAg-positive compared with those individuals infected with Bj [44]. A cross-sectional study from Japan found that cases of acute and fulminant hepatitis were associated with infection with HBV subgenotype B1, absence of HBeAg and high levels of replication due to the presence of precore mutants [45]. HBV genotype C HBV genotype C, which may be the most insidious of the HBV genotypes, is another prevalent HBV genotype in Asia [33]. There is compelling data from multiple population-based and clinic-based prospective trials that show that HBV genotype C is independently associated with a higher risk of HCC than HBV genotypes A2, Ba and Bj or [46]. The median age at HBeAg seroconversion is <20 years for people infected with HBV genotypes A, B, and F [25]. By contrast, the average age of HBeAg seroconversion in persons infected with HBV genotype C is 47 years [25]. To date, five subclassifications of HBV genotype C have been reported. In 2004, two major subgroups were identified within HBV genotype C from five Asian countries: one for strains from Southeast Asia including Vietnam, Myanmar and Thailand, named Cs and the other for strains from East Asia including Japan, Korea and China, named Ce [47,48]. The former was designated as C1(s), while the latter as C2(e). Thereafter, HBV subgenotype C3 confined to New Caledonia and Polynesia, C4 confined to Australian aborigines in Northeast Australia, and C5 from the Philippines were additionally identified [10,49,50]. A recent prospective study from Hong Kong showed that HBV subgenotype C2(e) is associated with a higher risk of HCC as compared with HBV subgenotype C1(s) [51]. Certain mutations at sr24k (HBsAg), SI126T (HBsAg) and pca1846t (precore gene) may contribute to the higher risk of disease progression in HBV genotype C2(e) [52]. However, whether CHB patients with HBV International Medical Press

5 The global epidemiology and natural history of chronic hepatitis Table 2. Geographic distribution of HBV genotypes/subgenotypes Genotype Geographic region isease association A1 Sub-Saharan Africa, India, Brazil, Argentina HCC develops in young males often without cirrhosis. HBeAg seroconversion occurs earlier. A2 Europe, United States, Arctic, Australia HCC and cirrhosis develop in older persons. A3 West Africa Not studied. B1 Japan HCC and cirrhosis develop in older persons. B2-5 East Asia HBeAg seroconversion occurs at relatively older age. HCC and cirrhosis develop in younger persons. Risks for HCC and cirrhosis are higher than HBV genotype B1. BCP mutations are more frequently observed than HBV genotype B1. B6 Alaska, Northern Canada, Greenland Not studied. C1-5 China, Korea, Southeast Asia, Japan, Risks for HCC and cirrhosis are higher than HBV genotypes B. South Pacific Islands, Australia HBeAg seroconversion occurs 1 3 decades later than in HBV genotypes A, B, and F. 1-4 Russia, Middle East, Mediterranean, HBeAg-negative CHB, HCC and cirrhosis develop in older individuals. Mongolia, North Africa, Europe, Indian It frequently harbours precore mutations. Subcontinent, Arctic, North and South America, Australia E West and Central Africa Both precore and BCP mutations are common. F1 Alaska, Central America, South America, Bolivia Associated with HCC, which developed in children and young adults in Alaska only. F2,3 Central America, Amazon region Associated with fulminant hepatitis with HV coinfection. F4 Argentina Not studied. G Europe, United States Almost exclusively found in persons coinfected with other HBV genotypes, mainly A. H Central America, Amazon region Not studied. I a Vietnam and Laos Not studied. J a Ryukyu Islands (Japan) Not studied. a The classification of these genotypes is yet to be ratified by the International Committee on the Taxonomy of viruses. BCP, basal core promoter; CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen; HCC, hepatocellular carcinoma. genotype C2(e) have the higher risk of progression to liver fibrosis/cirrhosis is uncertain [53]. According to the study conducted in 139 Chinese HBeAg-negative CHB patients, there was no difference between two HBV subgenotypes C in the severity of histological fibrosis [54]. HBV genotype HBV genotype, which is distributed in Northern Africa, Southern and Eastern Europe, and the Middle East, is associated with early HBeAg seroconversion, which is probably due in part to the predilection to develop mutations in the precore gene that abrogate HBeAg production [55,56]. In Mediterranean countries, this has led to high rates of HBeAg-negative CHB [57]. However, persons infected with HBV genotype in the inactive hepatitis B phase are likely to remain in this phase without developing complications of liver disease or HCC. In one study, 97% of those patients with minimal or no fibrosis or inflammation on liver biopsy had no histological progression upon repeat liver biopsy after 4 years of follow-up [58]. A possible reason for these conflicting findings could be that persons infected with HBV genotype either go into the inactive hepatitis B phase and stay there or develop active chronic hepatitis at the time of or shortly after HBV seroconversion [4]. Other HBV genotypes Infections with HBV genotypes E, G and H are uncommon, and their epidemiology and effects on disease outcome are not well-characterized. HBV genotype E (a variant from sub-saharan Africa that has also been reported in Spain [59,60]) is found in Western and Central Africa [61]. In the limited studies done with HBV genotype E, both precore and BCP mutations were commonly identified [60]. Other than that, little is known about its influence on disease outcome. HBV genotype F is found in native populations in North and South America [4,62]. HBV subgenotype F1 has recently been shown to be associated with a high risk of HCC in Alaska, as compared with HBV genotypes A, B and, particularly in children and young adults <30 years of age [10,25]. Besides, outbreaks of fulminant hepatitis in coinfection with HBV genotype F (F2 and F3) and HV were observed, but it is not Antiviral Therapy

6 BK Kim et al. clear whether both viruses together play a role or if this severe outcome is due primarily to HV [10]. HBV genotype G is the most uncommon of all HBV genotypes, which has been found in France and the US in only a few patients [30]. Since HBV genotype G has a defective precore open reading frame and therefore is not able to process HBeAg, it is almost exclusively found in persons coinfected with another HBV genotype, most commonly HBV genotype A, which provides transcription factors necessary for replication [63]. This HBV genotype is characterized by a 36 nt insertion within the core region and therefore is not able to process HBeAg. HBV genotype H is found in Central America and the Amazon region [64]. It is most closely related to HBV genotype F and likely evolved from this HBV genotype after it established itself in the so-called New World, although its complete genome differs by >8% and thus is a separate HBV genotype [62]. There are no reports of the relationship of HBV genotype H to disease outcome in HBV. Recently, HBV genotype I, a novel inter-genotypic recombination among HBV genotypes A, C and G, was isolated in Vietnam and Laos [31,65]. This strain HBV- VH24 showed high similarity ( %) to the aberrant strains among Vietnamese isolates reported by Hannoun et al. [66] and had similar breakpoints of recombination. However, classification of HBV genotype I is controversial, with Kurbanov et al. [67] questioning its validity based on their finding that the four Vietnamese isolates only varied from HBV subgenotypes C1 C5 by 7.0% ±0.4, which is less than the 8% difference required for establishment of a separate HBV genotype. The most recently identified HBV genotype is HBV genotype J, identified in the Ryukyu Islands of Japan. This HBV genotype was divergent from eight human (A H) and four ape (chimpanzee, gorilla, gibbon and orangutan) HBV genotypes, as well as from the recently proposed ninth human HBV genotype I, by % of the entire genomic sequence [68]. HBV genotype J has a close relationship with gibbon/orangutan HBV genotype and human HBV genotype C [31]. HBV natural history After establishment of chronic HBV infection, the course of CHB can be divided into five phases: immune tolerant, immune clearance, inactive, reactivation and immune control phase. These phases differ according to geographical locations and HBV genotypes, as described below. Based on knowledge of the natural history of chronic HBV infection, patients can be classified by serological and virological status (Table 3 and Figure 3). Table 4 summarizes the differences in the natural history of HBV infection according to geographic area and HBV genotypes. Immune tolerant phase HBV-infected persons in the immune tolerant phase are HBeAg-positive, they have normal alanine aminotransferase (ALT) levels and high levels of HBV NA that typically exceed 20,000 IU/ml (10 5 copies/ml) and frequently well above 20,000,000 IU/ml (10 8 copies/ml) [69]. The immune tolerant phase commonly occurs in infants who are infected via perinatal transmission from HBeAg-positive mothers [70]; this period can last for two or three or even more decades. In childhood-acquired HBV (for example, HBV subgenotype A1 and HBV genotypes E and in Africa) and adult-acquired HBV (for example, HBV subgenotypes A2 and 1 in Western Europe), this phase is much shorter or even absent [71]. These phenomena are most likely due to the different timing of HBeAg seroconversion according to HBV genotype. HBeAg may act as an immune tolerant protein that renders the virus undetectable by the host s immune system [72]. Under these circumstances, HBV is regarded as non-autopathic to hepatocytes and is the main reason for the absence of liver disease despite high levels of HBV replication [73]. In fact, during this immune tolerant phase, there appears to be either no or minimal liver inflammation or fibrosis. A study from Taiwan showed there was a low risk for the development of cirrhosis or HCC during this phase (<0.5% per year) [74]. However, because HBV is a NA virus that may integrate randomly into the host s hepatocyte NA, persistently high levels of HBV NA over many years could lead to an accumulation of integrated sites, which has been associated with increased risk of HCC over time [75]. Immune clearance phase The immune clearance phase, sometimes referred to as the HBeAg-positive CHB phase or the immune active phase, is characterized by elevated serum ALT and HBV NA levels in patients who are HBeAg-postive. Serum HBV NA levels generally exceed 20,000 IU/ml (10 5 copies/ml) [4,75]. Active liver inflammation is usually present with or without liver fibrosis. An important outcome of the immune clearance phase is seroconversion from HBeAg to anti-hbe, control of HBV NA to undetectable levels and the eventual normalization of ALT. Persons infected after birth (either in childhood or adulthood) may advance to the immune clearance phase shortly after the time of infection (in other words, they have a short or absent immune tolerant period). By contrast, those infected via the perinatal route may transition into this phase several years after experiencing a long immune tolerant phase. Thus, transition from the immune tolerant to the immune clearance phase in patients with perinatally acquired HBV infection International Medical Press

7 The global epidemiology and natural history of chronic hepatitis usually occurs during the second and third decades [16]. The triggers for transition from immune tolerant to clearance phase are unknown, but presumably the host s immune system recognizes HBV as foreign and initiates an immune response that results in hepatocyte damage. It is unclear why this does not occur during the immune tolerant phase, although there is increasing evidence that the HBeAg is a suppressor of the innate immune response, which may promote persistence [76,77]. uring successful HBeAg seroconversion, the HBV NA levels progressively fall. Seroconversion to anti-hbe can be preceded by a flare of hepatitis (Figure 3). This phenomenon is believed to be due to a sudden increase in immune-mediated lysis of infected hepatocytes and is often preceded by an increase in serum HBV NA and a shift of hepatitis B core antigen from nuclear to cytoplasmic sites within hepatocytes, suggesting that immune clearance may be triggered by an increase in viral load or change in the presentation of viral antigens [78,79]. How these changes occur is not known. Clearance of HBeAg occurring spontaneously or as a result of antiviral therapy usually reduces the risk of disease progression including hepatic decompensation and improves survival, although very early immune clearance in young children (<3 5 years) has been associated with increased rates of HCC [80,81]. HBeAg seroconversion, the most important event during this period, may vary according to region and HBV genotypes [16]. In general, Asian people infected with HBV genotypes B or C experience HBeAg seroconversion later in life, primarily due to a long immune tolerant phase. Several longitudinal follow-up studies have shown that the cumulative rate of spontaneous HBeAg seroconversion is significantly higher in patients with HBV genotype B, as compared with those with HBV genotype C, the two main HBV genotypes of HBV in Asia. These studies revealed that spontaneous HBeAg seroconversion occurred about 10 years earlier and possibly at a higher rate in patients infected with the HBV genotype B, as compared with those with HBV genotype C [82,83]. A longer duration of high levels of HBV replication may contribute to more active liver disease and, in turn, a Table 3. Phases of chronic hepatitis B Immune tolerant phase Occurs primarily after perinatal infection from HBsAg/HBeAg-positive mother. Normal ALT levels, HBeAg-positive and HBV NA>20,000 IU/ml, often above 20,000,000 IU/ml. Normal or minimal grade of necroinflammation with no or minimal fibrosis on liver biopsy. This phase occurs most frequently in Asian patients (HBV genotypes B or C) where spontaneous HBeAg seroconversion occurs in the later life. HBsAg seroconverion does not occur. Precore and core promoter are wild type. Immune clearance phase Referred as HBeAg-positive CHB. HBeAg seroconversion occurs during this phase, but HBsAg seroconversion does not occur. Elevated ALT levels and HBV NA>20,000 IU/ml. Hepatic inflammation with or without fibrosis on biopsy often present. Transition from immune tolerant to clearance phase occurs in the earlier life in HBV subgenotypes A1, A2 and. Inactive phase HBeAg-negative and anti-hbe-positive. Normal ALT levels and undetectable or low levels of HBV NA. Hepatic inflammation minimal or absent. Hepatic fibrosis may improve over time. May convert into immune clearance phase, reactivation phase or immune control phase. Reactivation phase Referred as HBeAg-negative CHB. HBeAg-negative and anti-hbe-positive due to mutations at precore and BCP region. Elevated ALT level and detectable HBV NA (>2,000 IU/ml). Moderate or severe necroinflammation with variable amounts of fibrosis on liver biopsy. HBV precore mutations occur most frequently in HBV genotype infection, so the proportion of HBeAg-negative CHB is higher in regions where HBV genotype infection dominates (that is, Euro-Mediterranean and African areas). Immune control phase Loss of HBsAg and development of anti-hbs. Patients infected with HBV genotypes A or B have a higher chance of HBsAg seroclearance compared with other HBV genotypes in Western European and Chinese population, while no difference in HBsAg seroclearance by HBV genotypes was observed in a population-based study from Alaska. The prognosis is generally good after clearance of HBsAg; however, it does not always signify HBV eradication. ALT, alanine aminotransferase; anti-hbe, antibody to hepatitis B e antigen; anti-hbs, antibody to hepatitis B surface antigen; BCP, basal core promoter; CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma. Antiviral Therapy

8 BK Kim et al. higher rate of progression to cirrhosis among patients with HBV genotype C. What drives relatively early seroconversion in HBV genotype B patients is unclear. A possible hypothesis is that HBV genotype B develops the G1896A precore mutation (which abolishes HBeAg expression) at a higher efficiency, whereas HBV genotype C develops BCP mutations more efficiently (which reduces but does not abolish HBeAg expression). The reasons for the predominance of G1896A precore mutation in some HBV genotypes and not others is explained in HBV virology and clinical significance. It remains to be determined whether the emergence of these two types of mutations or an additional intrinsic HBV genotypic difference in HBeAg expression level solely accounts for the differing rates of seroconversion. In Africa, the great majority of individuals seroconvert from HBeAg to anti-hbe before reaching early adulthood [16,84]. In patients with CHB in South Africa, HBeAg expression is lost very early in the course of infection; only 5% are HBeAg-positive in adulthood, as compared with approximately 40% found in other hyperendemic areas of the world, such as Southeast Asia [85]. This may at Figure 3. Schematic representation of the natural history of HBV infection: immune tolerant phase, immune clearance phase, inactive phase, reactivation phase and immune control phase Reversion to HBeAg (+) CHB HBsAg (+) HBeAg (+) Anti-HBe (-) Immune tolerant phase HBsAg (+) HBeAg (+) Anti-HBe (-) Immune clearance phase HBsAg (+) HBeAg (+) (-) Anti-HBe (-) (+) Inactive phase HBsAg (+) HBeAg (-) Anti-HBe (+) Reactivation phase HBsAg (+) HBeAg (-) Anti-HBe (+) HBV NA level ALT level Immune control phase HBsAg (+) (-) HBeAg (-) Anti-HBe (+) The immune tolerant phase is characterized by high levels of HBV replication, as manifested by the presence of hepatitis B e antigen (HBeAg) and high levels of HBV NA in serum, but no evidence of active liver disease as manifested by normal serum alanine aminotransferase (ALT) concentrations. uring the immune clearance phase, HBeAg seroconversion is frequently, but not always, accompanied by biochemical exacerbations (abrupt increases in serum ALT). The inactive phase is usually characterized by the absence of HBV NA and normalization of serum ALT. Some patients with inactive phase may experience viral exacerbations of either HBeAgnegative chronic hepatitis B (CHB; reactivation phase) or reversion to HBeAg-positive CHB. In reactivation phase, elevated serum ALT and positive HBV NA are observed with negative HBeAg and positive antibody to HBeAg (anti-hbe). In a small portion of patients with inactive phase, hepatitis B surface antigen (HBsAg) loss with development of neutralizing antibody to HBsAg may eventually occur, which is so called immune control phase. +, positive; -, negative International Medical Press

9 The global epidemiology and natural history of chronic hepatitis least partially explain the reason why perinatal transmission has the less important role in Africa, compared with Asia. Early seroconversion is most likely due to the fact that the majority of south African HBV patients harbour double or triple point mutations at nt 1809, 1811 and 1812 at the precore translation initiation codon, which may have a greater effect in accelerating HBeAg loss in this population [86]. These phenomena were studied primarily in African subjects with HBV genotype A (subgenotype A1). Another common HBV genotype in Africa is and these patients also seroconvert from HBeAg to anti-hbe in adolescence or early childhood. The median age of HBeAg seroconversion in those with HBV genotype was 18.0 years [25]. Further studies are warranted in investigating differences among common HBV genotypes in Africa, such as HBV genotypes A, E and. In Western Europe, HBeAg seroconversion also occurs earlier in life, primarily due to a shorter duration of the immune tolerant and clearance phase [16]. However, compared with Africans, seroconversion occurs relatively later in Europeans, given that Europeans are primarily infected in their adulthood (rather than in childhood) and that Africans have mutations facilitating HBeAg loss, accelerating seroconversion in many cases [4]. Although studies comparing HBV genotypes A2 and (which are prevalent in Europe) are scarce, patients with HBV genotype tend to undergo HBeAg seroconversion at a slightly earlier age [25,55,56,87 89]. The ages when 50% of subjects with HBV genotypes and A2 experience HBeAg seroconversion were 18.0 and 19.4 years respectively, whereas the ages when 75% HBeAg seroconverted were 27.3 and 32.1 years, respectively [25]. Inactive phase The important outcome of the immune active phase is HBeAg seroconversion that marks the transition from active phase to inactive phase. This phase is characterized by HBeAg negativity and anti-hbe positivity, undetectable or low levels of HBV NA, persistently normal ALT levels and inactive liver histology with what is usually a minimal amount of fibrosis [75]. The likelihood of converting spontaneously from active to inactive infection is approximately 10 15% per year. However, this phase is not always associated with a permanent termination of viral replication and of HBVinduced chronic liver damage. Overall, 10 20% of persons with inactive phase may have viral replication and exacerbation of hepatitis after years of quiescence again [1]. Approximately 4 20% of persons with inactive phase have one or more reversions back to positive HBeAg, high levels of HBV NA and elevated ALT levels, which means reversion back to immune clearance phase (HBeAg-positive CHB). Among those who still remain anti-hbe positive, 10 30% experience elevated ALT and high HBV NA levels after HBeAg seroconversion, so called reactivation phase (HBeAg-negative CHB) [1]. In a small portion of those with inactive phase, HBsAg loss with development of neutralizing antibody to HBsAg (anti-hbs) may eventually occur, which is the so called immune control phase. The annual rate of spontaneous clearance of HBsAg has been estimated at 0.5 2% per year in Western patients who acquire CHB during adolescence and adulthood and 0.1 1% per year in those who acquire CHB early in life (that is, Asian, African and some Mediterranean patients) [4,90 92], depending on whether the HBV was acquired horizontally or vertically. Reactivation phase Although the majority of patients remain in the inactive phase throughout their lifetime, others retain or redevelop significant HBV replication, active inflammation Table 4. ifferences in natural history of HBV infection according to geographic area and HBV genotypes Clinical phases Western Europe/ of CHB Asia Africa North America ominant HBV genotype B1, B2, C1, C2 A1, E, A2, Immune tolerant phase uration Long (2 3 decades) Short Usually absent Immune clearance phase Timing at HBeAg seroconversion elayed Very early Relatively early Early HBeAg seroconversion Relatively more frequent Common in HBV genotype Common in HBV genotype in HBV genotype B than C A1, A2, Transition from inactive phase Low Low High to immune control phase Reactivation phase (a proportion Relatively low, but increasing High High, of HBeAg-negative CHB among very high in active CHB) Mediterranean area CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen. Antiviral Therapy

10 BK Kim et al. and progressive liver damage over time [16]. Since HBV covalently closed circular (ccc) NA persists in the liver, a number of individuals in the inactive phase may eventually develop HBV reactivation with recrudescence of liver disease, either spontaneously or triggered by active immunosuppression. Although viral replication may occur due to reactivation with the wild-type virus (that is, reversion back to the HBeAg-positive state) much more frequently, it occurs due to replication-competent HBV variants that prevent HBeAg expression. Therefore, the reactivation phase of CHB is characterized by HBeAg negativity usually with anti-hbe positivity, detectable serum HBV NA levels (200 20,000,000 IU/ml [ copies/ml]), ALT elevation and moderate or severe necroinflammation with variable amounts of fibrosis on liver biopsy, referred to as HBeAg-negative CHB. This was first recognized in 1981 and is typically associated with HBV that has lost the ability to express HBeAg due to mutations at precore and BCP regions [16,93]. The incidence of HBeAg-negative CHB is on the rise worldwide, with several clinical implications. First, patients with HBeAg-negative CHB have, in general, lower levels of serum HBV NA (2,000 20,000,000 IU/ml [ copies/ml]) than patients with HBeAgpositive CHB (200,000 2,000,000,000 IU/ml [ copies/ml]). However, many patients with HBeAgnegative CHB have wide fluctuations in both HBV NA and serum ALT levels, and HCC is more likely to develop even in those with relatively low HBV NA level (>2,000 IU/ml) [94]. Second, the treatment of HBeAg-negative CHB with interferon is more difficult because of the high rate of relapse after cessation of treatment [95]. Third, long-term prognosis is poorer among individuals with HBeAg-negative CHB in terms of more rapid progression to cirrhosis and frequent development of HCC, compared with those with HBeAg-positive CHB [96]. Furthermore, considering that those who undergo HBeAg seroconversion following antiviral therapy are likely to have HBeAg-negative CHB when reactivations occur, the incidence and prevalence of HBeAg-negative CHB are expected to rise continuously with the increasing number of patients given long-term suppressive antiviral therapy [4,96,97]. The increasing number of patients with HBeAg-negative CHB in the world may also reflect successful universal vaccination programmes for infant or child, which prevents HBV infection and thereby decreases the pool of HBeAg-positive persons. These reasons explain why HBeAg-negative hepatitis has recently received greater emphasis in the clinical field. Although HBeAg-negative CHB can be now found worldwide, prevalence varies widely by region. This is in part due to variability in study design and sample selection, but geographical variation in HBV genotypes is of notable importance [93,98,99]. In the Euro-Mediterranean and African area the great majority (>85%) of patients with biochemically and histologically active disease are HBeAg-negative, whereas in Asia, North Europe and the USA, HBeAg-positive CHB still predominates [16]. Such geographical distribution might be also explained by differences in HBV genotypes. Precore mutations occur most frequently in HBV genotype infection, so the proportion of HBeAg- negative CHB is higher in regions where HBV genotype infection dominates (that is, Euro-Mediterranean and African areas) [100]. Immune control phase After clearance of HBsAg (that is, in the immune control phase ), the clinical outcome is generally better than in persons who continue to be HBsAg-positive and liver inflammation and fibrosis may improve over time [75,101]. Predictors of HBsAg clearance are older age and sustained presence of the inactive phase [102,103]. Besides, patients infected with HBV genotype A or B have a higher chance of HBsAg seroclearance compared with other HBV genotypes in Western European and the Chinese population [104,105], while a recent population-based study from Alaska had shown no difference in HBsAg seroclearance by HBV genotypes. In this regard, further studies are required [106]. espite a generally favourable prognosis, HBsAg loss and development of anti-hbs do not signify HBV eradication or cure. Almost all patients who experience HBsAg seroconversion still harbour HBV NA in the form of the cccna minichromosome complex, in the nuclei of infected hepatocytes [92,101, ]. This phenomenon is the so-called occult HBV infection, defined as HBsAg negativity and the presence of HBV NA in the liver (with detectable or undetectable HBV NA in the serum) [110]. Such patients can be subclassified as having seropositive or seronegative HBV depending upon whether they are positive or negative for other HBV markers, most commonly antibody to hepatitis B core antigen [111,112]. The long-lasting persistence of the virus in the liver may provoke a very mild but continuing necroinflammation that, in the presence of other causes of liver damage, may contribute over time to the progression from chronic liver damage to cirrhosis [110]. Moreover, occult infection seems to maintain the pro-oncogenic properties typical of overt infection, and, in fact, is an important risk factor for HCC development [92,101,110]. HBV virology and clinical significance Because of the spontaneous error rate of viral reverse transcriptase, HBV mutants often emerge during the course of infection. One of the most common naturally occurring HBV mutations is the precore G to A International Medical Press

11 The global epidemiology and natural history of chronic hepatitis mutation at nt 1896 (G1896A), creating a stop codon at codon 28 and abrogating expression of HBeAg. This mutation is often accompanied by another point mutation (G to A) at nt 1899 in patients. Nt 1896 is located opposite to nt 1858 in the stem loop epsilon structure of the encapsidation signal, and nt 1858 is a C in HBV genotype A and a T in most of the other HBV genotypes. The G1896A mutation pairs with T1858, increasing the stability of the stem loop, a critical factor for viral replication. Therefore, non-a HBV genotypes such as, E, G and some HBV subgenotypes of B and C favour the emergence and selection of G1896A precore mutants [113]. The other common variation in the HBV genome is the dual A1762T/G1764A BCP mutations, which result in reduced expression of HBeAg [86]. ue to the aforementioned requirement for epsilon stability, BCP mutations are more prevalent than the G1896A precore mutation in HBV genotype A and some subgenotypes of HBV genotypes B, C and F. From a regional perspective, the precore variant is most prevalent in the Mediterranean area, while the BCP variant is more prevalent in Asia. In several studies conducted in Asia, HBV genotype C tends to have a higher proportion of the A1762T/ G1764A BCP mutations than HBV genotype B, suggesting a role in the association of HBV genotype C with poor histology and elevated risk of HCC [114,115]. Cross-sectional studies have reported that BCP mutations are associated with more severe liver damage. This may be due to changes in the overlapping X gene and, therefore, in the X protein at codon positions 131 and 132. The HBx protein is recognized as a transactivator of cellular genes (including oncogenes) [116]. Furthermore, the frequency of pre-s deletion, which was an independent risk factor associated with disease progression (odds ratio 3.91, 95% CI ; P=0.003) as well as HCC development (odds ratio 3.72, 95% CI ; P=0.007) was significantly higher in patients with HBV genotype C than those with HBV genotype B [117,118]. A case-control study in Asia confirmed a higher prevalence of such mutations including pre-s deletion, A1762T/G1764A BCP mutations, and G1896A precore mutation in patients with progressive liver disease (including patients with HCC), as compared with age-matched chronic HBV-infected individuals with persistently normal ALT [119,120]. In population-based studies in South Africa, 80% of HBV subgenotype A1 sequences harbour double or triple point mutations at nt 1809, 1811 and 1812 of the HBV genome, corresponding to the -5, -3 and -2 positions of the precore translation initiation codon. These mutations partly contribute to the very early HBeAg loss observed in African people. In Western Europe, HBV genotype A (70%) was more prevalent than HBV genotype (30%) in the HBeAg-positive population, whereas the HBeAgnegative population was dominated by HBV genotype infection. These data suggest that HBV genotype infected patients are more likely to be associated with HBeAg-negative CHB than HBV genotype A. This is presumably reflective of the greater frequency of the G1896A precore mutation in HBV genotype and is consistent with previous observations of the restriction of the G1896A precore mutation in HBV genotype A [55,121]. Table 5 summarizes virological characteristics according to geographic area and HBV genotypes. Progression to cirrhosis, HCC and death The ultimate complications of HBV infection are progression to cirrhosis and the development of HCC. Since the natural courses of HBV infection differ somewhat by geographic area, the pattern of development of these complications also vary. This is summarized in Table 6. Progression to cirrhosis Asian and Caucasian patients in the inactive phase of CHB have a very low risk of cirrhosis development of below 0.1 per 100 person-years [ ]. In patients with HBeAg-positive CHB, the cirrhosis incidence rates are 1.6 and 3.8 per 100 person-years in East Asian countries and European countries, respectively; the corresponding 5-year cumulative incidences of cirrhosis were 8% and 17% [ ]. An incidence rate ratio (IRR) of 0.41 (95% CI ; P=0.004) was found for studies performed in East Asia, as compared with those from Europe; the corresponding IRR when adjusting for age and gender was 0.17 ( ; P<0.003) [74,126,127,129,130]. By contrast, in patients with HBeAg-negative CHB, the cirrhosis incidence rates were 2.8 and 9.7 per 100 person-years in East Asian and European countries, respectively; the corresponding 5-year cumulative incidences of cirrhosis were 13% and 38%, respectively [122,131,132]. Overall, Europeans tend to have a higher morbidity of cirrhosis due in part to the higher incidence of HBV genotype encoding the G1896A mutation and consequent HBeAg-negative HBV in the region. Since each study had different inclusion criteria, further comprehensive and prospective studies that include adjustment for age, sex, HBV genotype, age at infection, duration of infection and viral replication status should be performed in the future. Progression to HCC Incidence rates of HCC among people with chronic HBV infection vary depending on demographic characteristics of not only the study population (including age, sex, ethnicity and underlying conditions), duration Antiviral Therapy

12 BK Kim et al. Table 5. Patterns of some HBV mutations according to geographic area and HBV genotypes Western Europe/ Asia Africa North America ominant HBV genotype B1, B2, C1, C2 A1, E, A2, Naturally occurring HBV G1896A T1809-T1812 in HBV subgenotype A1 G1896A in HBV genotype mutations A1762T/G1764A and Pre-S deletion G1896A in HBV genotype and E in HBV genotype C Response to interferon- Poor (relatively better in HBV Unknown Good (much better in HBV based therapy genotype B than C) genotype A than ) Table 6. ifferences in disease progression, regarding progression to cirrhosis, HCC and liver-related death according to geographic area and HBV genotypes Western Europe/ Asia Africa North America ominant HBV genotype B1, B2, C1, C2 A1, E, A2, Risk of cirrhosis a [122, ,131,132] 1.6 in HBeAg-positive CHB Unknown 3.8 in HBeAg-positive CHB 2.8 in HBeAg-negative CHB 9.7 in HBeAg-negative CHB Risk of HCC High High Relatively low Age at risk of HCC Older persons Young males often without cirrhosis Older persons (male>female) (especially in HBV subgenotype A1) (male>female) Mortality from HBV High High Relatively low a Expressed as incidence per 100 person-years. HBeAg, hepatitis B e antigen; HCC, hepatocellular carcinoma. of infection, the level of HBV replication, but also HBV genotypes [ ]. In Asia, the relative risk of HCC in these HBsAgpositive individuals was 223 times higher than that of non HBsAg-positive [137]. In another series, the relative risk of HCC was 6.9 in a sample of 917 Japanese patients with cirrhosis or chronic hepatitis [133,134]. A similar relationship was demonstrated in a prospective study of patients with CHB in North America, 71% of whom were Asian [135]. The majority of HCCs occur in patients with CL or cirrhosis, suggesting that older patients with long-standing liver disease are more likely to develop HCC. This finding is consistent with a study conducted in Korea [138]. HBV genotype and specific mutations are also associated with HCC development [105,115]. A study involving 2,762 Taiwanese patients with HBsAg positivity showed that the presence of the A1762T/G1764A BCP mutation and HBV genotype C were independent factors with an adjusted hazard ratio of 1.73 and 1.76, respectively [139]. Several large prospective studies conducted in both Asia and Western Europe have noted a mean age at presentation between 50 and 60 years [133,134,140,141]. Male predominance is pronounced ( times, mean 3.7:1) in high-incidence regions (that is, Asia and Africa) [4]. In Africa, increased HCC risk was also strongly associated with chronic HBV infection (odds ratio 16.7, 95% CI ) [142]. However, in contrast to Western Europe and Asia, young African males who often do not have cirrhosis have a high morbidity of HCC, with a mean age of 33 years at presentation [133,134,140,141]. This finding is consistent with that of another study conducted in Gambia showing a mean age of 40 years at diagnosis of HBV-related HCC [142]. Some of the heterogeneity in age-specific HCC rates among regions is not fully understood. However, this finding may be due in part to the high prevalence of HBV genotype A1 in Africa, which is associated with 4.5-fold increase in the relative risk of developing HCC in HBsAg-positive Black sub-saharan Africans, compared with other genotypes [34]. In Western Europe, the risk of HCC among asymptomatic HBsAg-positive patients in inactive phase may be lower in Caucasians [ ]. In a series of 317 HBsAg-positive patients in Canada, most of whom were French-Canadian, 16-year follow-up revealed no cases of HCC. Similar results were noted in a report from Austria: only one case of HCC occurred in 242 asymptomatic HBsAg-positive patients after a followup period of 3.5 years [136,143]. The age at presentation was years, similar to that in Asians [144]. A male predominance still existed, but decreased to a mean of 2.4:1 in intermediate incidence areas and was lower in low incidence regions. Although not fully understood, these differences in sex distribution are thought to be due to variations in chronic carriage rates, host genetics, exposure to environmental toxins and the trophic effect of androgens International Medical Press