Michael C. Kew

Epidemiology of hepatitis B virus in Africa, its genotypes and clinical associations of genotypes

Of approximately 360 million people in the world chronically infected with hepatitis B virus (HBV), 65 million reside in Africa. Thus, Africa, with 12% of the worlds population, carries approximately 18% of the global burden of HBV infection, with hepatocellular carcinoma and cirrhosis accounting for 2% of the continents annual deaths. Despite HBV being endemic or hyperendemic in Africa, there is a paucity of data on the genotypes and their distribution. Genotype A is found mainly in southern, eastern and central Africa. Most African genotype A strains belong to subgenotype A1, with subgenotype A3 found in western Africa. Genotype D prevails in northern countries and genotype E in western and central Africa. Ithas become increasingly evident that heterogeneity in the global distribution of HBV genotypes may be responsible for differences in the clinical outcomes of HBV infections and the response to antiviral treatment and vaccination. A limited number of studies have been published relating genotypes to clinical outcomes in African countries. Because observations from other regions of the world can not be extrapolated from one locale to another, the HBV strains circulating in Africa should be studied and related to clinical outcomes.

A unique segment of the hepatitis B virus group A genotype identified in isolates from South Africa.

The preS2/S genes of hepatitis B virus isolated from 29 acutely or chronically infected individuals in the Gauteng province of South Africa were sequenced. Phylogenetic analysis of these sequences in comparison with global isolates from the GenBank database showed that 24 sequences clustered with genotypic group A, three with genotypic group D and one each with genotypic groups B and C. Group A isolates had greater identity with groups D (variation of 6.6%) and E (6.8%) than with the Eastern groups B (7.4%) and C (8.1%) and were most different from group F (11.0%). Of the South African group A specimens, 59.1% clustered with two global sequences to form a discrete segment which we have called subgroup A. The amino acid differences that set these isolates apart from the rest of group A tended to cluster in the preS2 region (amino acids 7, 10, 32, 35, 47, 48, 53 and 54), with a few changes occurring in the major surface antigen (amino acid sites 207 and 209). Analysis of isolates showed that there was a 9-fold higher prevalence of the ay determinant in South Africa than previously reported.

Hepatitis B virus x protein in the pathogenesis of hepatitis B virus-induced hepatocellular carcinoma.

Currently available evidence supports a role for the hepatitis B virus (HBV) x gene and protein in the pathogenesis of HBV‐induced hepatocellular carcinoma (HCC). HBx gene is often included, and remains functionally active, in the HBV DNA that is frequently integrated into cellular DNA during hepatocellular carcinogenesis. HBx protein promotes cell cycle progression, inactivates negative growth regulators, and binds to and inhibits the expression of p53 tumour suppressor gene and other tumour suppressor genes and senescence‐related factors. However, the molecular mechanisms responsible for HBx protein‐induced HCC remain uncertain. Only some of the more fully documented or more recently recognised mechanisms are reviewed. During recent years evidence has accumulated that HBx protein modulates transcription of methyl transferases, causing regional hypermethylation of DNA that results in silencing of tumour suppressor genes, or global hypomethylation that results in chromosomal instability, thereby playing a role in hepatocarcinogenesis. HBx protein has both anti‐apoptotic and pro‐apoptotic actions, apparently contradictory effects that have yet to be explained. Particularly important among the anti‐apoptotic properties is inhibition of p53. Recent experimental observations suggest that HBx protein may increase the expression of TERT and telomerase activity, prolonging the life‐span of hepatocytes and contributing to malignant transformation. The protein also interferes with nucleotide excision repair through both p53‐dependent and p53‐ independent mechanisms. Carboxy‐terminal truncated HBx protein loses its inhibitory effects on cell proliferation and pro‐apoptotic properties, and it may enhance the proteins ability to transform oncogenes. Dysregulation of IGF‐II enhances proliferation and anti‐apoptotic effects of oncogenes, resulting in uncontrolled cell growth.

Cytokeratin expression in hepatocellular carcinoma: an immunohistochemical study

Normal human hepatocytes express cytokeratins no. 8 and 18, whereas bile duct cells contain the same cytokeratins and, in addition, cytokeratins no. 7 and 19. This cytokeratin pattern is believed to be preserved during neoplastic transformation. Thirty-four cases of hepatocellular carcinoma (11 well differentiated, 16 moderately differentiated, 7 poorly differentiated) were studied on frozen sections using monoclonal antisera directed against individual cytokeratins no. 7, 8, 18, and 19 in an immunoperoxidase procedure. In 17 of 34 cases, tumor cells showed only reactivity with monoclonals anticytokeratin no. 8 and 18. However, 17 of 34 cases showed an aberrant pattern in that a variable number of tumor cells were stained with anticytokeratins no. 7 and/or 19 in addition to no. 8 and 18. Only three of 11 well-differentiated cases displayed an unexpected cytokeratin pattern, whereas an aberrant pattern was present in all seven of seven poorly differentiated cases. These results are in conflict with previously published data obtained by two-dimensional gel electrophoresis and immunohistochemistry. They indicate that the cytokeratin pattern might not always be preserved during neoplastic transformation. The implication of this finding for the differential diagnosis of metastatic gastrointestinal carcinomas is discussed.

A case‐control study for differences among hepatitis B virus infections of genotypes A (subtypes Aa and Ae) and D

There are two subtypes of hepatitis B virus genotype A (HBV/A) and they are provisionally designated Aa (“a” standing for Africa/Asia) and Ae (“e” for Europe). In a case‐control study, 78 HBV/Aa, 78HBV/Ae, and 78HBV/D carriers from several countries were compared. The prevalence of HBe antigen (HBeAg) in serum was significantly lower in carriers of HBV/Aa than in carriers of HBV/Ae (31% vs. 49%; P = .033), with a difference more obvious in the carriers aged 30 years or younger (34% vs. 67%; P = .029). HBV DNA levels in the carriers of HBV/Aa (median, 3.46 log copies/mL; 95% CI, 2.93–3.95) were significantly lower than those of carriers of HBV/Ae (6.09 log copies/mL; 95% CI, 4.24–7.64) or of carriers of HBV/D (5.48 log copies/mL; 95% CI, 4.06–7.02), regardless of the HBeAg status (P < .001). The most specific and frequent substitutions in 54 HBV/Aa isolates were double substitutions for T1809 (100%) and T1812 (96%) immediately upstream of the precore initiation codon, which would interfere with the translation of HBeAg in HBV/Aa infections. They were not detected in 57 HBV/Ae or 61 HBV/D isolates examined. The double mutation in the core promoter (T1762/A1764) was more frequent in both HBV/Aa (50%) and HBV/Ae (44%) than in HBV/D isolates (25%; P < .01), whereas the precore mutation (A1896) occurred in HBV/D isolates only (48%; P < .0001). In conclusion, the clearance of HBeAg from serum may occur by different mechanisms in HBV/Aa, HBV/Ae, and HBV/D infections, which may influence clinical manifestations in the Western countries where both genotypes A and D are prevalent. (HEPATOLOGY 2004;40:747–755.)

Hepatitis B virus and human immunodeficiency virus co-infection in sub-Saharan Africa: a call for further investigation.

Abstract: A growing body of evidence indicates that human immunodeficiency virus (HIV)‐positive individuals are more likely to be infected with hepatitis B virus (HBV) than HIV‐negative individuals, possibly as a result of shared risk factors. There is also evidence that HIV‐positive individuals who are subsequently infected with HBV are more likely to become HBV chronic carriers, have a high HBV replication rate, and remain hepatitis Be antigen positive for a much longer period. In addition, it is evident that immunosuppression brought about by HIV infection may cause reactivation or reinfection in those previously exposed to HBV. Furthermore, HIV infection exacerbates liver disease in HBV co‐infected individuals, and there is an even greater risk of liver disease when HIV and HBV co‐infected patients are treated with highly active anti‐retroviral therapy (HAART). Complicating matters further, there have been several reports linking HIV infection to ‘sero‐silent’ HBV infections, which presents serious problems for diagnosis, prevention, and control. In sub‐Saharan Africa, where both HIV and HBV are endemic, little is known about the burden of co‐infection and the interaction between these two viruses. This paper reviews studies that have investigated HIV and HBV co‐infection in sub‐Saharan Africa, against a backdrop of what is currently known about the interactions between these two viruses.

Hepatitis B virus precore mutants in serum and liver of Southern African Blacks with hepatocellular carcinoma

BACKGROUND/AIM The aim of this study was to sequence the precore region of HBV isolated from serum and tumorous and non-tumorous liver tissue from patients with hepatocellular carcinoma to identify mutations that might play a role in malignant transformation. METHODS HBV DNA was extracted from 62 sera, 14 tumorous and 12 non-tumorous liver tissue samples of patients with hepatocellular carcinoma, amplified by the polymerase chain reaction and sequenced directly. RESULTS Thirty-nine patients were HBeAg-negative and 23 HBeAg-positive. Missense mutations were present predominantly in HBeAg-negative sera. The most common missense mutation, a guanine to thymine transversion, occurred at nucleotide 1862 in the bulge of the encapsidation signal; it was more prevalent in HBeAg-negative (10/39) than in HBeAg-positive patients (1/23) (p = 0.03). Mutations known to prevent HBeAg synthesis were detected in seven sera; five with an 1896 stop-codon mutation, one with an 1817 nonsense mutation, and one with a frameshift mutation caused by an insertion between 1838 and 1839. Missense mutations and deletions were present more often in tumorous tissue derived from HBsAg-negative patients. In the tumours missense mutations occurred at position 1862 and 1899, and the deletions affected direct repeat 1 and/or the encapsidation signal and included the x gene stop-codon. CONCLUSIONS The 1862 mutation, and other missense mutations and deletions detected in the precore gene, may disrupt HBV DNA replication and/or signal peptide cleavage leading to HBeAg-negativity. Disruption of viral replication may promote integration of unencapsidated replicative intermediates and hence contribute to hepatocarcinogenesis.

Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma

To search for events underlying reduction of peripheral viremia and integration of hepatitis B virus (HBV) DNA into the liver cell genome in long-term virus carriers with hepatocellular carcinoma, paired samples of liver and tumor tissue were analyzed by molecular hybridization and immunological methods. Most tumor tissues contained integrated viral DNA; in none was extrachromosomal HBV DNA detected. Integrated HBV DNS was also found in peritumor liver tissue in the majority of patients. However, liver of patients either with or without peripheral viremia also contained free HBV DNA and replicative intermediates. In three nonviremic patients with replicative HBV DNA in liver, viral core antigen expression was markedly reduced or absent, whereas viral envelope protein (surface antigen) expression was normal. In one case, replicative intermediates in liver were sensitive to DNase I digestion, indicating that viral DNA was not encapsidated in normal viral core particles. These results suggest that decreased or defective core antigen production can lead to reduced viremia associated with blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell. Accumulation of such HBV DNA molecular forms in the liver may lead to an increased propensity for HBV DNA to integrate into the host genome, which has been found with high frequency in hepatic neoplasms from patients infected with hepatitis B virus.

Effect of age on the etiologic role of the hepatitis B virus in hepatocellular carcinoma in blacks

Hepatocellular carcinoma often affects blacks at an early age. The purpose of this study was to ascertain if the association between chronic hepatitis B virus infection and hepatocellular carcinoma is the same in young and older black patients. Serum markers of hepatitis B infection were measured by radioimmunoassay in 391 blacks with hepatocellular carcinoma, 173 of whom were less than or equal to 30 yr old and 218 of whom were greater than or equal to 50 yr old. Only 2 of the young patients showed no markers of current or past hepatitis B infection compared with 31 (14.3%) of the older patients (p less than 0.001). Hepatitis B surface antigen was present in 81.5% of the young patients and of these 34.5% were e antigen-positive. The corresponding figures in the older patients were 29.8% and 10.9% (p less than 0.001 in each instance). It is concluded that whereas the association between hepatocellular carcinoma and hepatitis B infection is almost universal in young blacks, a subgroup of older blacks shows no evidence of ever having been infected with this virus.

Molecular characterization of subgenotype A1 (subgroup Aa) of hepatitis B virus

Subgenotypes of hepatitis B virus (HBV) were first recognized after a unique segment of genotype A was identified when sequencing the preS2/S region of southern African HBV isolates. Originally named subgroup A′, subsequently called subgroup Aa (for Africa) or subgenotype A1, this subgenotype is found in South Africa, Malawi, Uganda, Tanzania, Somalia, Yemen, India, Nepal, the Philippines and Brazil. The relatively higher mean nucleotide divergence of subgenotype A1 suggests that it has been endemic and has a long evolutionary history in the populations where it prevails. Distinctive sequence characteristics could account for the high hepatitis B e‐antigen (HBeAg) negativity and low HBV DNA levels in carriers of this subgenotype. Substitutions or mutations can reduce HBeAg expression at three levels: (i) 1762T1764A atthe transcriptional level; (ii) substitutions at nt 1809–1812 at the translational level; and (iii) 1862T at the post‐translational level. Co‐existence of 1762T1764A and nt 1809–1812 mutations reduces HBeAg expression in an additive manner. In addition, subgenotype A1 has unique sequence alterations in the transcriptional regulatory elements and the polymerase coding region. The distinct sequence characteristics of subgenotype A1 may contribute to the 4.5‐fold increased risk of heptocellular carcinoma in HBV carriers infected with genotype A, which is entirely attributable to subgenotype A1.