Zhong-Liao Fang

HBV core promoter mutations prevail in patients with hepatocellular carcinoma from Guangxi, China

The development of primary liver cancer frequently is associated with persistent HBV infection, and tumours may arise in individuals who are anti‐HBe positive. However, it is unlear whether viruses with an HBeAg‐negative phenotype are associated with tumour development or are selected, during seroconversion, after chromosomal integration of wild‐type viral DNA. In order to investigate the temporal evolution of the HBV genome in such individuals, the polymerase chain reaction was used to amplify HBV DNA from tumour tissue and serum of 14 patients from Guangxi, China with hepatocellular carcinoma. Comparison of nucleotide and amino acid sequences of the precore and proximal core region of HBV from the two sites in each patient produced evidence of divergence following integration in the tumour, but in most cases, HBeAg‐negativity could not be explained by precore mutations. Sequences from the core promoter region were therefore examined and mutations were found in the majority, which are believed to upregulate transcription of the core (and pregenomic) RNA but to downregulate precore mRNA. To determine whether this finding merely reflected sequence variation among geographical isolates of HBV, the same region of HBV DNA from asymptomatic controls was sequenced and these mutations were found to be rare. We hypothesise that HBV with the core promoter mutations replicates at higher levels than the wild type, with the consequence that more integrations occur into the hepatocyte chromosomes during the early stages of infection. These hepatocytes may expand clonally and be targets for further mutagenic events leading to tumour development. J. Med. Virol. 56:18–24, 1998.

Prevalence of hepatitis B virus infection in a highly endemic area of southern China after catch-up immunization†

The Chinese national goals for control of hepatitis B virus (HBV) infection were to achieve a prevalence of HBsAg below 7% for the entire population, and 1% for children under 5‐year old, by 2010. To determine whether Guangxi, a multi‐minority province with a low socio‐economic status and a very high prevalence of HBV, achieved this goal, a seroepidemiological survey of HBV infection was carried out using stratified, random cluster sampling. The results show that the overall prevalence of HBsAg is 9.16% [95% confidence interval (CI) = 8.32–10%]. The prevalence in males (10.96%, 95% CI = 9.64–12.28%) is significantly higher than in females (7.71%, 95% CI = 6.64–8.78%; χ2 = 10.5923, P < 0.05). The prevalence in children under 5‐year old is 3.62% (95% CI = 0.60–6.64%) and increases with age. The prevalence of HBsAg in non‐immunized individuals is significantly higher than in those immunized completely, although not within 24 hr of birth (χ2 = 31.426, P < 0.05); a significant difference was found in those below the age of 20 years but not in older persons. Gender, age, immunization history, and familial HBsAg carriers are risk factors for infection. In conclusion, this study indicates that Guangxi has not reached the goal for the control of HBV infection. Catch‐up HBV immunization may not protect adults effectively against infection in highly endemic regions. J. Med. Virol. 84:878–884, 2012.

Prevalence of transmitted HIV-1 drug resistance remains low in Guangxi, China, eight years after scale-up of highly-active antiretroviral therapy.

Objectives: Highly-active antiretroviral therapy (HAART) was scaled up in Guangxi, China in 2005. The number of individuals receiving free HAART increased dramatically from June 2010 under the Guangxi Governments anti-HIV programme. We aimed to determine the prevalence of HIV-transmitted drug resistance (TDR) of Guangxi. Methods: HIV-positive, antiretroviral-naive individuals were recruited from the east (Hezhou), south (Qinzhou), west (Baise), north (Guilin) and centre (Laibin) of Guangxi. The pol gene of the virus from the individuals was analysed. Results: The overall prevalence of HIV TDR was 3.2% (7/216, 95% CI 0.9-5.5). The prevalence rates in Baise, Guilin, Hezhou, Qinzhou and Laibin are 4.9% (2/41, 95% CI -1.7 to 11.5), 2.3% (1/44, 95% CI -2.1 to 5.7), 4.7% (2/43, 95% CI -1.6 to 11.0), 2.6% (1/38, 95% CI -2.5 to 7.7) and 2.0% (1/50, 95% CI -1.9 to 5.9), respectively. No significant difference in the prevalence was found among them. No factors were found to be associated with TDR, including CD4 cell counts, viral loads and genotypes. The subtypes CRF01_AE, CRF07_BC, CRF08_BC and B were found. Subtype CRF08_BC is the predominant subtype in Baise while CRF01_AE is the predominant subtype elsewhere in Guangxi. Conclusions: The prevalence of TDR in antiretroviral-naive patients in Guangxi remains low 8 years after scale-up of HAART.

Hepatitis B virus candidate subgenotype I1 varies in distribution throughout Guangxi, China and may have originated in Long An county, Guangxi†

Sequencing of the complete hepatitis B virus (HBV) genomes from Vietnam, China and Laos led to the identification of a complex recombinant, referred to initially as an aberrant genotype and later proposed to be a new genotype, I. However, epidemiological data regarding this new genotype are lacking. A cross‐sectional study was carried out to investigate the epidemiology of HBV candidate genotype I in Guangxi, China using stratified, random cluster sampling. Four thousand five hundred thirteen subjects were recruited from five counties within Guangxi. Three genotypes, B, C, and I, were identified with a prevalence of 32.6% (114/350), 64% (224/350), and 3.4% (12/350), respectively. All the genotype I isolates belong to candidate subgenotype I1 and were found in Bing Yang (15.3%, 9/59) and Na Po (5.0%, 3/60) counties only. The prevalence of this subgenotype is significantly higher in males (5.1%, 10/195) than in females (1.3%, 2/155; X2 = 3.959, P < 0.05) but does not differ significantly with age. It was found in the Han (4.5%, 9/201) and Zhuang (3.1%, 3/97) ethnic populations only. There is no significant difference from other genotypes in the prevalence of HBV serological markers. Phylogeographic analysis revealed that genotype I1 likely arose in Long An county, then spread later to Bing Yang, Na Po counties and elsewhere in southeast Asia. In conclusion, the distribution of candidate genotype I within Guangxi is not even and it is highly endemic in some counties. Its prevalence is associated with gender and ethnicity. Subgenotype I1 likely originated in Long An county. J. Med. Virol. 85:799–807, 2013.

The Effect of HBV Genotype C on the Development of HCC Differs Between Wild-Type Viruses and Those With BCP Double Mutations (T 1762 A 1764 )

Background: Association of hepatitis B virus (HBV) genotype C with hepatocellular carcinoma (HCC) development remains controversial. HBV basal core promoter (BCP) double mutations (T1762A1764) are very strong confounding factors of genotypes B and C in HCC development. Objectives: To investigate the association of HBV genotype C with HCC development after controlling for BCP double mutations. Materials and methods: Four hundred and two serum samples from patients with HCC, liver cirrhosis (LC) and chronic hepatitis (CH) and also from asymptomatic HBsAg carriers were analyzed. Results: Genotypes B (31.1%), C (62.8%), and I (6.1%) were detected. With the severity of liver disease the prevalence of genotype B decreased, but genotype C increased. No trend was found for genotype I. The prevalence of BCP double mutations in genotypes C and I viruses was significantly higher than genotype B. BCP double mutations are risk factors for CH, LC and HCC. Genotype C was not identified as a particular risk factor for HCC prior to the stratification analysis but after that genotype C viruses with BCP double mutations were found to be a particular risk factor for HCC (P = 0.008, OR = 17.19 [95% CI: 2.10 - 140.41]), but those with the wild-type BCP were not. In the interaction analysis, genotype C and BCP double mutations were found to have a synergistic effect on HCC development (P < 0.0001, OR = 52.56 [95% CI: 11.49-240.52]). Conclusions: The effect of HBV genotype C on the development of HCC differs between wild-type viruses and those with BCP double mutations, suggesting that not all individuals infected with genotype C HBV are at increased risk of HCC.

Occult HBV Infection May Be Transmitted through Close Contact and Manifest as an Overt Infection.

The importance of transmission of occult HBV infection (OBI) via transfusion, organ transplantation and hemodialysis has been widely recognized. However, data regarding the transmission of OBI through close contact remain limited. In this study, serum samples were obtained from a child and his parents. The child had received the standard vaccination regimen at birth and produced protective antibody. Sera were tested for HBV serological markers. Nested PCR assays were used to detect HBV DNA and the amplicons were cloned and their sequences subjected to phylogenetic analysis. The results showed that both parents had occult infections while the child had an overt infection. Twelve, eleven and nine clones, from the father, mother and son, respectively, were sequenced. Serotypes adrq+, ayw1, ayw and ayr were found in the father and ayw1, adw2 and adwq+ in the mother; adrq+ was the only serotype in son. Genotype B, subgenotype C2 and a recombinant were identified in the father and genotype B, subgenotype C5 and three recombinants were found in the mother. Subgenotype C2 was the only genotype identified in the child. A phylogenetic tree showed that all of the child’s sequences and most of the father’s sequences clustered together. However, none of mother’s sequences clustered with those of the child. The surface gene from the child and his father had the same amino acid substitution pattern (T118K, T123N and G145A). We concluded that the father was the source of the son’s HBV infection, suggesting that occult HBV infection may be transmitted through close contact and manifest as an overt infection.

Short Communication: Low False Recent Rate of Limiting-Antigen Avidity Assay Among Long-Term Infected Subjects from Guangxi, China

Assays used for HIV cross-sectional incidence testing can misclassify some individuals with nonrecent HIV infection as recently infected, overestimating HIV incidence. We analyzed the frequency and factors associated with false-recent misclassification on subjects from Quangxi, China known to have long-term infection using the limited antigen-avidity assay (LAg-Avidity). Stored samples from treatment-naive individuals from Guangxi, China were tested using the LAg-Avidity. A total of 362 samples from individuals known to be infected 2 to 13.5 years were tested and the false-recent rate (FRR), the frequency of samples with a positive result, was determined at different cutoff values of the assay. Additionally, factors associated with misclassification were determined. The FRR of the LAg-Avidity was 1.1% (4/362) using a cutoff of 1.5 normalized optical density units (OD-n). All four samples had viral loads >1,000 copies/ml. Using a cutoff of 3.0 OD-n the FRR was 5.5% (20/362), with four samples having viral loads <1,000 copies/ml. Factors associated with a higher odds of misclassification were female gender (OR 7.7, 95% CI 1.0-56.4) and being a female sex worker (OR 31.3, 95% CI 4.0-242). At the higher cutoff, being of Zhuang decent, relative to Han, had higher odds of misclassification (OR 6.2, 95% CI 1.99-19.0). The LAg-Avidity assay had a low FRR in this Chinese population. Further investigations of the higher frequency of low LAg-Avidity results seen in female sex workers and individuals of Zhuang descent should be explored in a larger study.

Combined core promoter mutations and pre-S deletion of HBV may not increase the risk of HCC: a geographical epidemiological study in Guangxi, China

Although persistent hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), the mechanisms of oncogenesis remain obscure.

Des-γ carboxyprothrombin may not be a good biomarker for hepatocellular carcinoma in those chronically infected with hepatitis B virus with basal core promoter double mutations (T^{1762}, A^{1764}), a prospective study

BACKGROUND The accuracy of des-γ -carboxyprothrombin (DCP) in the detection of hepatocellular carcinoma (HCC) in those infected hepatitis B virus (HBV) from cross-sectional or case-control studies is contradictory. OBJECTIVE To resolve this contradiction using a prospective study. METHODS Three hundred male individuals persistently infected with HBV were recruited from the Chinese cohort and followed up once per year from 2012 to 2015. Each subject was screened for HCC by measurements of serum alpha-fetoprotein (AFP), lectin-bound α -fetoprotein (AFP-L3), DCP concentrations and ultrasonographic examinations. RESULTS Nineteen HCC cases were identified. The area under receiver operating characteristic (AUROC) at first, second and third visit for AFP, AFP-L3 and DCP ranges from 0.710-0.897, 0.566-0.637 and 0.520-0.595, respectively. The rate of elevated DCP is not significantly different between the HCC cases and controls (52.6% vs. 47.4%) (P > 0.05). The incidence of HCC in subjects with elevated DCP is not significantly higher than that of those with normal DCP (9.5% vs. 4.6%) (P > 0.05). The AUROC of combinations of these biomarkers was higher than that of AFP alone at the first visit. However, it was reduced at the second visit. At the third visit, the AUROCs of AFP + DCP and AFP + AFP-L3 + DCP, but not that of AFP + AFP-L3, were higher than that of AFP alone. CONCLUSIONS AFP but DCP or AFP-L3 remains a valuable biomarker for HCC in those chronically infected with HBV. The combination with AFP-L3 and DCP may not increase the accuracy of AFP in differentiating HCC cases from controls, among those infected with HBV.

Hepatitis B Virus Core Promoter Double Mutations (A1762T, G1764A) Are Associated with Lower Levels of Serum Dihydrolipoyl Dehydrogenase.

Objectives: The aim of this study was to identify serum proteins with differential concentrations between hepatocellular carcinoma (HCC) patients and HBsAg asymptomatic carriers among individuals infected with hepatitis B virus (HBV) with basal core promoter (BCP) double mutations (A1762T, G1764A). Methods: iTRAQ and liquid chromatography-tandem mass spectrometry were used to identify differentially expressed protein, and an ELISA test was used for the validation test. Results: The total number of proteins identified was 1,125, of which 239 showed statistically significant differences in their expression. The relative concentrations of serum dihydrolipoyl dehydrogenase (DLD), which showed the most significant correlation with liver diseases and infection, were significantly lower in HCC patients than asymptomatic HBsAg carriers and individuals negative for HBsAg. However, only the difference between HCC patients with BCP double mutations and HBsAg-negative individuals could be confirmed by ELISA. Meanwhile, we found that the concentrations of serum DLD in those infected with HBV with BCP double mutations were significantly lower than in individuals with the wild-type BCP. However, the difference in the concentrations of serum DLD between individuals with wild-type BCP and those negative for HBsAg was not significant. Conclusions: HBV with BCP double mutations are associated with lower concentrations of serum DLD.