Clustering of Different Subtypes of Hepatitis B Surface Antigen in Families of Patients with Chronic Liver Diseases

Clustering of hepatitis B surface antigen (HBsAg) with both subtypes adr and adw in three families of patients with chronic liver diseases or hepatocellular carcinoma was demonstrated in Taiwan where adw is the main subtype. The subtype in the children was similar to that in their mothers, suggesting maternal transmission. In all the family units clustered with different subtypes, the same pattern occurred, invariably with fathers carrying HBsAg/adr and the children carrying HBsAg/adw. The subtype difference clearly rules out the transmission of hepatitis B virus (HBV) from father. Horizontal infection with the locally dominant adw-subtyped HBV in the children of fathers carrying HBsAg/adr explains the discrepancy of the subtypes in these families. Clustering of two HBsAg-positive brothers with hepatocellular carcinoma in one of the families was found. That both adr-subtyped and adw-subtyped HBV are capable of inducing chronic active hepatitis in another family suggests that host factors are probably more important in determining the clinical course of HBV infection.     

Maternal transmission of hepatitis B surface antigen in patients with hepatocellular carcinoma in Taiwan

To determine the source of the highly prevalent hepatitis B virus (HBV) infection in our patients with hepatocellular carcinoma (HCC), we examined hepatitis B surface antigen (HBsAg) and its subtypes and antibody in 11 patients with HCC and their parents. All the patients were positive for HBsAg. Eight (73%) of the mothers were also HBsAg-positive, whereas only one of the seven fathers was an HBsAg carrier (P = 0.025). The observation is compatible with maternal transmission as a source of HBV infection in most of our patients with HCC. The subtype was identifiable in 10 patients, 9 with HBsAg/adw and one with adr. The subtype was identical in the patient--mother carrier pairs, suggesting that HBV infection in the patient and the mother is intimately related. This is further evidenced by the observation of a relatively uncommon adr subtype in one patient--mother pair. These observations suggest that the HBV infection in our patients results from vertical transmission from their carrier mothers probably long before the development of HCC.     

Genotypes and subtypes of hepatitis B virus in Thailand

Hepatitis B virus exhibits considerable variability evident in its various antigenic subtypes, which complicates the characterization of epidemiological factors, particularly in areas endemic for hepatitis B. Our group investigated the genotypes and subtypes prevalent in Thailand employing nested PCR and sequencing of the a determinant, as well as the sub-determinants located on the S gene. The sera examined originated from a mixed range of HBV-infected individuals. The results were mostly consistent with those reported for Southeast Asia in that genotype C (54.4%) dominates over genotypes A (22.1%) and B1 (23.5%). Regarding the subtypes, we have exclusively found adw2 (45.6%) and adr (54.4%) as expected for this area, with one case of subtype adw representing the exception. While genotype and/or subtype of HBV do not predispose to clinical disease, they nevertheless may account for those few cases reported in which a mutation, particularly within the a determinant of the S gene, causes evasion of routine detection by commercial kits, particularly as long as the respective individuals remain asymptomatic carriers solely expressing anti-HBc.     

Hepatitis B viral genotypes: clinical relevance and molecular characteristics

Hepatitis B virus (HBV) infection is a global health problem and the clinical outcome of chronic HBV infection depends on the frequency and severity of hepatitis flares in the immune clearance phase. Currently, four subtypes and seven genotypes of HBV are identified and most have specific geographic distributions. The impact of HBV genotypes on the clinical outcome of chronic HBV infection has been partially clarified. In Taiwan, genotype C is associated with more severe liver disease and genotype B is associated with the development of hepatocellular carcinoma (HCC) in young non-cirrhotic patients. In contrast, genotype B has a relatively good prognosis in Japan and China and is rarely associated with the development of HCC. Similarly, genotype D is associated with more severe liver disease than genotype A in India and may predict occurrence of HCC in young patients. Although superinfection of HBV on top of hepatitis B carriers occurs in Taiwan, it is rarely associated with acute exacerbations. As to the response to antiviral treatment, genotypes C and D are associated with a lower response rate to interferon therapy compared with genotypes B and A. In addition, the subtype adw is reported to be associated with a higher risk of lamivudine resistance than ayw. In HBV subtype adw-infected HCC patients, genotype B responds better to embolization therapy and has a lower rate of HCC recurrence than genotype C. In summary, pathogenic and therapeutic differences do exist among HBV genotypes and determining the genotype in patients with chronic HBV infection would help gain further information for etiologic, clinical, virologic and anthropologic investigations. Further studies to clarify the molecular virological factors that contribute to these differences are awaited.     

Molecular epidemiological study of hepatitis B virus in Thailand based on the analysis of pre-S and S genes.

Aims: This study was undertaken to determine the prevalence and characteristics of hepatitis B virus (HBV) genotypes, antigen subtypes, "a" determinant variants and pre-S gene mutations circulating on a large scale in Thailand. Methods: The sequences of the Pre-S1, Pre-S2 and S regions were determined in serum samples of 147 HBsAg and HBV DNA-positive subjects who had been enrolled from the nationwide seroepidemiological survey conducted on 6213 individuals in 2004. Results: The results showed that genotypes C, B and A accounted for 87.1%, 11.6% and 1.3%, respectively. The distribution of the HBV antigen subtypes was: adr (84.4%), adw (14.2%) and ayw (1.4%). Regarding the "a" determinant, 2/43 (4.65%) and 2/104 (1.92%) samples of vaccinated and non-vaccinated subjects, respectively, displayed mutations, all ofwhich were Thr126Asn. Sequencing analysis showed the pre-S mutations in 14 (9.5%) samples, with pre-S2 deletion as the most common mutant (4.1%) followed by pre-S2 start codon mutation (2.9%), both pre-S2 deletion and start codon mutation (2.0%), and pre-S1 deletion (0.7%). The pre-S mutations were associated with older age and higher mean serum HBsAg level. Conclusion: This study demonstrated that HBV genotype/subtype C/adr and B/adw were the predominant strains circulating in Thailand. The "a" determinant variants seemed to be uncommon, and might not be attributed to vaccine-induced mutation.     

Subtypes of HBsAg in Eastern and South-Eastern Europe

Abstract. A total of 481 serum samples was collected from asymptomatic carriers of H B,Ag as well as some patients with acute and chronic hepatitis, type B. The sera were obtained mainly from blood banks in the following countries: Poland, Hungary, Romania, Bulgaria, Yugoslavia, and the European part of the Soviet Union. Asymptomatic carriers of HB_sAg from Poland and Hungary showed a preponderance of adw subtype over uyw subtype (Poland -80.7% adw , Hungary -72.2% adw). Carriers from the remaining countries showed the reverse situation: Bulgaria -71.5% ayw , Yugoslavia -79.0% ayw , Romania -82.8% ayw , Soviet Union -87.5% ayw. No individuals were found among these indigenous populations to have the adr or ayr subtypes but two carriers appeared to have the unusual subtype adyw.     

New Hepatitis B Surface Antigen Subtypes inside the ad Category

In addition to the 10 HBs Ag subtypes already described, 2 new subtypes were defined by using the q determinant. Exceptions to the rule generally accepted were found in that the q determinant was only lacking in HBs Ag/adw4. These exceptions occurred in adw and adr categories. These 2 new subtypes are adw q positive and adr q negative. Out of 98 HBs Ag/adw4 from silent carries and patients from different parts of the world, mainly from France (79), 6 were found q positive. 3 out of these 6 cases came from Montpellier (South of France) and another 3 from Germany. The 92 other cases were found q negative. Further studies will be necessary to better know the location of this new subtype adw4 q positive, but it seems to be present only in certain parts of Europe. Out of 86 HBs Ag/adr from silent carriers from Asia (58), Oceania (17), France (10, most of them contaminated in Asia) and Réunion (1), 10 were found q negative. All these 10 cases were detected in Oceania, 2 out of 2 in carriers from New Caledonia and 8 out of 13 from French Polynesia. The new subtype adr q negative seems localized in Melanesia and Polynesia and absent from Asia. These 2 new markers of hepatitis B virus will allow better epidemiological and geographical studies.     

贵州地区慢性乙型肝炎病毒感染者病毒基因亚型研究

目的调查贵州省B、C基因型慢性HBV感染者的病毒基因亚型。方法PCR扩增HBV P区长309 bp的基因片段,扩增产物分别经限制性内切酶NciⅠ、VspⅠ、BstEⅡ酶切,琼脂糖凝胶电泳,根据酶切图谱多态性,用限制性片段长度多态性分析（PCR-RFLP）检测HBV C基因亚型。直接测序确定B基因亚型、对178例用S基因限制性片段长度多态性鉴定为B、C基因型的不同临床类型慢性HBV感染者进行亚型分析。结果84例C基因型HBV感染者中,27例（32.14%）为C1亚型、56例（66.67%）为C2亚型,1例为C1、C2亚型混合感染。94例B基因型HBV感染者中,93例（98.94%）为Ba、1例为Bj亚型感染。从无症状乙型肝炎表面抗原携带者、CHB到肝硬化/肝癌,C1亚型在各组中的比例逐渐降低,分别为60.00%、30.65%和16.67%;而C2亚型在各对应组中的分布逐渐增高,分别为40.00%、67.74%和83.33%。结论贵州地区B、C基因型慢性HBV感染者中,以Ba、C2亚型为主。C1、C2亚型在疾病中的分布有一定差异。PCR-RFLP分析HBV C1、C2亚型,方法简便、特异性强,适合较大样本分析,可用于流行病学调查。     

Geographical distribution of the subtype of hepatitis B surface antigen in Chinese.

Using the much more sensitive hemagglutination inhibition assay for subtyping of hepatitis B surface antigen (HBsAg), we examined the determinants a, d, w and r in 192 from 228 HBsAg positive adults who had been found after screening with reversed passive hemagglutination method. Sixty-four subtypable cases were asymptomatic carriers and the remaining 128 were liver disease patients. Among them there was no significant difference of the subtypes, invariably with adw as the main subtype. Geographical difference was evident: adr was the main subtype (78 per cent) among the northern Chinese; while adw was dominant (76 per cent) among the southern Chinese with the Yangtze River as a boundary. Eight of the 18 adr-subtyped northern Chinese were born and live in Taiwan where 91 per cent of HBsAg positive Taiwanese were adw-subtyped. This was an indirect evidence that intra-familial spreading from parents played an important role in hepatitis B virus infection.     

Frequency of concurrence of hepatitis B surface antigen and antibody in a large number of carriers in Okinawa,Japan

A large number of chronic hepatitis B surface antigen (HBsAg) carriers in Okinawa, Japan were tested for antibody to HBsAg (anti-HBs), by both radioimmunoassay and enzyme immunoassay methods. Concurrence of HBsAg and anti-HBs was found in 166 (26.1% ). We found no clear predominance of either liver damage or hepatitis B e antigen (HBeAg) in the concurrent carriers studied. Antibody to pre-82 antigen (anti-pre-S2) was detected in 16 (9.6%) of 166 subjects with concurrent markers, 15 of these 16 carriers were positive for antibody to HBeAg (anti-HBe). Anti-pre-S2 was correlated wit anti-HBe rather than with anti-HBs. The distribution of HBsAg subtypes among carriers determined to have subtypes was 76.7% adw, 22.0% adr, 0.2% ayr, 0.9% adwr, and 0.2% adyr. The distribution of anti-HBs subtypes among concurrent carriers was 51.5% anti-r, 21.4% anti-w, 15.5% anti-d, and 10.7% anti-y. Concurrent carriers had HBsAg of one subtype and heterotypic anti-HBs. Because the HBsAg subtype ay is rare in this area, it is hard to believe that the concurrent carriers with anti-y were infected with hepatitis B virus of which the HBsAg subtype was ay. A dual infection was highly unlikely. It seems that some of the concurrent carriers correlate with compound subtypes adwr and adyr.     

Hepatitis B surface antigen disappearance and hepatitis B surface antigen subtype: a prospective, long-term, follow-up study of Japanese residents of Okinawa, Japan with chronic hepatitis B virus infection.

To determine the natural course of hepatitis B surface antigen (HBsAg) disappearance in chronic hepatitis B virus (HBV) infection and the factors related to its disappearance, 946 HBsAg carriers in Okinawa, Japan were prospectively followed for up to 19 years (mean = 9.2 years). The disappearance of HBsAg, as determined by radioimmunoassay (RIA), was observed in 62 (6.6%) and the overall annual disappearance rate was 0.79%/year. Its disappearance was more frequent in 60 (7.4%) of 815 serum samples negative for hepatitis B e antigen (HBeAg) by RIA at entry compared with only two (1.5%) of 131 serum samples that were HBeAg positive by RIA at entry (P < 0.05). Stepwise logistic regression analysis showed that age and HBsAg subtype were significantly associated with HBsAg disappearance (both P < 0.05), and that carriers with subtype adr (odds ratio = 2.87) had an increased probability of clearing HBsAg compared with carriers with subtype adw. Conversely, HBeAg disappearance was earlier in those with the adw subtype than in those with adr. Hepatitis B virus DNA was not detected by the polymerase chain reaction after HBsAg disappearance in any of the 62 from whom it had disappeared. The HBsAg titer, as measured by reverse passive hemagglutination, was related to the time to its disappearance; the higher the titer, the longer the time to disappearance. These findings suggest that HBeAg negativity, a more advanced age, and low titers of HBsAg are favorable factors for HBsAg disappearance in the natural course of chronic HBV infection. Moreover, HBsAg subtype adr was a predictive factor for HBsAg disappearance, whereas subtype adw was predictive of early HBeAg disappearance.     

Relationship of HBsAg Subtypes with HBeAg/anti-HBe Status and Chronic Liver Disease. Part I: Analysis of 1744 HBsAg Carriers

A total of 1744 HBsAg carriers were investigated to determine whether there are clinical differences among HBsAg subtypes or not. Although adr was more predominant than adw in 1078 asymptomatic carriers as well as in 666 carriers with liver dysfunction, the adr carriers had liver dysfunction more frequently than the adw carriers (p = 0.005). In addition, the adr carriers were more often positive for HBeAg and less often positive for anti-HBe than the adw carriers (p less than 0.001). Multivariate analyses indicated that the HBsAg subtypes were associated with liver dysfunction not directly but through the relationship between the HBsAg subtypes and HBeAg/anti-HBe status. HBeAg/anti-HBe status of each age bracket in the adr carriers and in the adw carriers suggested that adr carriers are seroconverted later than adw carriers. In conclusion, HBsAg subtypes may affect the development of chronic liver disease, through their association with HBeAg/anti-HBe status.     

Hepatitis B virus genotypes and clinical manifestation among hepatitis B carriers in Thailand

BACKGROUND: Hepatitis B virus (HBV) genotypes have distinct geographic distributions. The aim of the present study was to evaluate the distribution of HBV genotypes and their clinical relevance in Thailand. METHODS: Hepatitis B virus genotypes among 107 hepatitis B carriers residing in Thailand were evaluated using serologic and genetic methods. They were clinically classified into asymptomatic carriers with normal serum alanine transaminase (ALT) levels and patients with chronic liver disease, such as those with chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). RESULTS: Hepatitis B virus genotype distribution among the 107 patients was 25.2% for genotype B, 72.0% for genotype C and 2.8% for genotype D. The serum ALT levels, HBV-DNA and hepatitis B e antigen positivity were significantly higher in carriers infected with genotype C HBV than in those infected with genotype B (P < 0.05). The proportion of genotype B HBV was higher in asymptomatic carriers than in patients with CH and those who developed liver disease, such as LC and HCC (45.5, 16.9 and 25.0%, respectively; P < 0.05). In contrast, the proportion of genotype C HBV was higher in patients who developed liver disease and CH than in asymptomatic carriers (68.7, 83.0 and 50.0%, respectively; P < 0.05). Phylogenetic analysis based on entire genome sequences revealed three HBV isolates, which were classified into a subgroup of genotype C in isolates from South-East Asian countries. CONCLUSIONS: Genotypes B and C are the predominant types among hepatitis B carriers residing in Thailand and those genotypes influence the clinical manifestation in carriers with chronic hepatitis B infection.     

乙型肝炎病毒感染者病毒基因型和亚型分布及其临床意义

目的研究不同类型乙型肝炎病毒（HBV）感染者中HBV基因型和亚型分布情况及其临床意义。方法应用基因型和亚型特异性引物聚合酶链反应法（PCR），对北京、长春、汉川，深圳，清远和南京等6个城市445份HBV感染者血清进行基因型及亚型分型，其中急性肝炎7例，无症状携带者36例，慢性乙型肝炎352例，肝硬化28例，肝细胞癌22例。通过对PCR产物测序确定其基因型，以验证该法的准确性。结果445份血清中，239例（53．7％）为C型，其中13例（5．4％）为C1亚型，135例（56．5％）为C2亚型，91例（38．1％）为非C1／C2亚型；145例（32．6％）B型，其中100例（69．0％）为Ba亚型，25例（17．2％）为Bj亚型，20例（13．8％）为非Ba／Bj亚型；61例（13．7％）为B型与C型混合感染，其中6例（9．8％）为Ba与C1亚型混合感染，3例（4．9％）为Bj与C1亚型混合感染，15例（24．6％）为Ba与C2亚型混合感染，8例（13．1％）为Bj与C2型混合感染，11例（18．0％）为Ba与非C1／C2亚型混合感染，7例（11．5％）为Bj与非C1／C2亚型混合感染，2例（3．3％）为非Ba／Bj亚型与C1亚型混合感染，3例（4．9％）为非Ba／Bj亚型与C2亚型混合感染，6例（9．8％）为非Ba／BJ亚型与非C1／C2亚型混合感染。未检测到其他基因型及亚型。在不同类型乙型肝炎病毒感染者中，B型，C型、B与C型混合感染及亚型分布差异有统计学意义，在肝硬化和肝细胞癌患者中C型所占比例较高（分别为78．6％和86．4％），无症状携带者中B型所占比例较高（72．2％）。但不同性别患者中，HBV基因型和亚型分布无差异。在HBeAg阳性和阴性感染者中，HBV基因型和亚型分布差异也有统计学意义。结论该6个城市HBV感染者中，以B2和C2亚型占优势，C基因型可能与肝硬化和肝细胞癌的发生有关。     

135例乙型肝炎病毒感染者基因分型及其临床分布

目的研究乙型肝炎病毒（HBV）感染者中HBV基因分型及其临床分布情况。方法应用基因型和亚型特异性引物聚合酶链反应（PCR）法，对鲁西地区135例HBV感染者血清进行HBV基因型及亚型分型。结果未分型11例，已分型124例。其中C型111例（C2基因亚型87例、非C1／C2亚型24例）；B型11例，其中Ba型8例、Bj型3例；B／C混合型2例均为BaC2亚型混和感染。在肝硬化和重度慢性乙型肝炎中C型所占比例较高，分别为100％、88％；无症状携带者中B型所占比例较高为8．51％；HBV基因型分型与性别无关。结论鲁西地区HBV感染者以C基因型为主，其中C2亚型占优势。     

Association of pre-S deletion mutant of hepatitis B virus with risk of hepatocellular carcinoma

BACKGROUND: Pre-S deletion mutant of hepatitis B virus (HBV) affects the expression of middle and small surface proteins, resulting in intracellular accumulation of large surface protein. The correlation between pre-S deletion mutant and risk of hepatocellular carcinoma (HCC) in hepatitis B virus carriers remains unclear. METHODS: Using molecular assays, pre-S deletion mutant of HBV were determined in 266 patients with chronic HBV genotype B or C infection. They included 202 asymptomatic carriers and 64 HCC patients. RESULTS: The overall prevalence of pre-S deletion mutant was 16.5%. Hepatocellular carcinoma (odds ratio [OR], 3.23; 95% confidence interval [CI], 1.23-8.48, P = 0.02) and genotype C (OR, 3.19; 95%CI, 1.54-6.62, P = 0.002) were independently associated with the presence of pre-S deletion mutant. The prevalence of pre-S deletion mutant was comparable between HCC patients with genotype B and C infection. Nevertheless, in asymptomatic carriers, patients with genotype C infection were significantly associated with the presence of pre-S deletion mutant compared to those with genotype B infection (20.8% vs 7.2%, P = 0.007). Compared with age- and genotype B-matched asymptomatic carriers, young HCC patients (<50 years of age) had a significantly higher frequency of pre-S deletion (3.4% vs 20%, P = 0.04). CONCLUSIONS: Pre-S deletion mutant is more frequent in HBV carriers with genotype C infection, and those with pre-S deletion mutant may be associated with the development of HCC, irrespective of HBV genotype.     

20-fold increase in risk of lamivudine resistance in hepatitis B virus subtype adw

We investigated subtype-dependent development of lamivudine resistance in hepatitis B virus (HBV) longitudinally in 26 consecutive patients (13 adw and 13 ayw carriers) during antiviral treatment of chronic hepatitis B. Lamivudine resistance developed in seven adw carriers and one ayw carrier. Risk of lamivudine resistance was significantly higher for adw carriers than for ayw carriers (p=0.03). We believe that the adw subtype of HBV is associated with a high risk of lamivudine resistance, which might be linked to simultaneous changes of the HBsAg that occurs with the emergence of resistance.     

HBs Antigen Subtypes among Acute Hepatitis Patients in Japan: Evidence of Imported Hepatitis

HBs antigen subtypes were determined among 137 acute hepatitis patients in Japan. The distribution among the 99 male patients was 40 adr, 37 adw, one ayw, and 21 undetermined while that among the 38 female patients was 19 adr, 11 adw, and eight undetermined. The distribution among the male patients was different from that among HBs antigen carriers in Japan, suggesting that they were exposed not only to domestic HBs antigen carriers, but also to other sources. Thirty-one (29 males and two females) of the 137 patients (22.6%) were found to have been abroad at some time during the incubation period. the distribution among them was seven adr, 21 adw, and three undetermined. the majority bad the same subtypes that are prevalent in the countries they visited, supporting the hypothesis that most of them bad contracted the disease overseas. The remaining 106 patients bad a distribution of the subtypes similar to that of domestic HBs antigen carriers. "Imported hepatitis" would be an important cause of acute type B hepatitis in Japan.     

Some epidemiological and clinical aspects of hepatitis B antigen and its subtypes.

The close association between hepatitis B antigen (HBAg) and the infectious agent of hepatitis B is clear. Many investigations have shown HBAg to be a useful tool for epidemiological studies of hepatitis B. The relation between HBAg and the postulated hepatitis B virus (HBV) is as yet not clear. In light of recent results a possible candidate could be the so-called Dane particle, which has HBAg reactivity on the surface, but possesses an antigenically distinct core. The core has been shown to have associated DNA polymerase activity. The particles which carry HBAg reactivity have surfaces which are antigenically complex. One common specificity a and 2 pairs of mutually exclusive determinants have been recognized namely d and y and w and r but further possible specificities are under investigation. Four different phenotypes have been described, adw, adr, ayw and ayr. Present evidence indicates that adw, adr and ayw are the phenotypic expression of 3 different transmissible strains of HBV. Studies on the epidemiology of these subtypes have shown 3 different geographic patterns. In the USA and Northern Europe both Dw (adw) and YW (ayw) are common, but in the Eastern Mediterranean and Middle East Yw is practically the only type found. In the Far East DR (adr) is the dominating subtype. Investigations have been done to determine whether there are any clinical differences in hepatitis caused by the different tubtypes. No certain differences have been shown.     

Subtypes of hepatitis B surface antigen in Southeast Asia.

The prevalences of hepatitis B surface antigen (HBs Ag) subtypes in Thais, Cambodians, and Vietnamese were compared with the prevalences in Americans residing in Southeast Asia. HBs Ag was found with approximately equal frequency in Thai (43 percent) and American (39 percent) patients with hepatitis. However, higher prevalences of HBs Ag were found in asympotomatic Thais (9.5 percent), Cambodians (11.9 percent), and Vietnamese (14.3 percent) than in asymptomatic Americans (0.7 percent). Among asymptomatic Thais, the ratio of HBs Ag/adr to HBs Ag/adw was approximately 10:1, with one exception: adw was not detected in a rural population of northern Thailand. The y determinant was not found in Thais. In contrast, both d and y determinants were found in Americans. These observations conform to a geographic pattern, with ad as the predominant combination in the Far East. In Southeast Asia determinants w and r are more useful epidemiologic markers than y and d.