Genetic variability of CYP2B6 polymorphisms in four southern Chinese populations.

AIM: To investigate the genotype and allelic frequencies of Cytochrome P450 2B6 polymorphisms in four southern Chinese populations. METHODS: DNA was obtained from blood samples from Han Chinese from Hong Kong and three minority groups, the Wa, Bulang and Lahu from Yunnan in southern China. Genotyping was performed using real-time PCR and confirmed by direct sequencing. RESULTS: A total of 507 subjects from southern China were studied. Results showed there is a high prevalence of 516G > T (34.5%) in ethnic Chinese compared to literature reports on other Asian populations and Caucasians. The frequency of the 516TT genotype is higher in the Han majority (23.1%) than in three other ethnic minority groups (i.e., 7.4%, 9.1% and 15.8%) in southern China. CONCLUSION: This was the first study to document the spectrum of CYP2B6 allelic variants and genotypes in a southern Chinese population. The 516G > T allele is associated with a defective metabolism of efavirenz (EFV), which therefore may predispose to drug toxicity. Treatment regimens for human immunodeficiency virus (HIV) and heroin addiction may need to be optimized in different populations because of the marked variability of the key metabolizing enzyme.

[Inhibition of HBsAg and HBeAg secretion by RNA interference of the polymerase gene sequence of hepatitis B virus: an experimental study].

OBJECTIVE: To investigate the effects of small interfering RNA (siRNA) targeting the polymerase (P) gene sequence of hepatitis B virus (HBV) on the replication and antigen secretion of HBV. METHODS: From the 29 base sequences of the HBV in the HepG2.2.15 cells that accord with the demands of siRNA designing five sequences targeting the P gene of HBV were selected and cloned into the siRNA expressing vector pGE-1. Then the plasmid pGE-HBVP was transfected into the cultured HepG2.2.15 cells. Chemiluminescent immunoassay was used to determine the levels of HBsAg and HBeAg in the supernatant of culture medium 24, 48, 72, and 96 hours after the transfection and the expression of HBsAg in the 2.2.15 cells 24 hours after the transfection so as to observe the inhibitory effects. Untransfected cells and cells transfected with blank pGE-1 vector were used as controls. RESULTS: Five vectors expressing the siRNAs targeting the HBV P region, pGE-HBVP1-pGE-HBV5 were successfully constructed. The efficiency of transfection of the vectors into the 2.2.15 cells were 30% to 40%. 24, 48, 72, and 96 hours after the transfection of pGE-HBVP2, the strongest inhibitor among the five, the inhibitory rates of HBsAg secretion in the supernatant were 28.88%, 32.28%, 29.10%, and 18.42% respectively; and the inhibitory rates of HBeAg secretion were 38.33%, 27.50%, 33.41%, and 12.60% respectively. In view of the transfection efficiency of 30%-40%, the actual inhibitory rate of HBV antigen secretion might reach 80% and over. 24 hours after the transfection the expression rate of HBsAg in the 2.2.15 cells transfected with pGE-HBVP2 was 50%, significantly lower than that in the cells transfected with the blank vector pGE-1 (82%). CONCLUSION: siRNAs targeting the HBV P gene effectively prevent the HBV gene expression and replication and may play an important role in the clinical anti-viral treatment.

[Relationship between B virus hepatitis genotypes and therapeutic efficacy in early treatment for chronic hepatitis B by using lamivudine].

OBJECTIVE: To investigate the relationship between hepatitis B virus (HBV) genotype and therapeutic efficacy during the early phase of lamivudine treatment. METHODS: Totally 595 patients with chronic hepatitis B were treated with lamivudine 100 mg/day for 12 months. HBV genotypes, contents of HBV DNA, HBeAg/anti-HBe and YMDD mutation after lamivudine treatment for 12 months were determined. The data were analyzed with SPSS software. RESULTS: In 595 patients, 8 (1.4%) were genotype A; 53 (8.9%) genotype B; 360 (60.5%) genotype C; 112 (18.8%) were coinfection of genotype B and C; 14 (2.4%) of A and C; 15 (2.5%) A and B; 6 (1.0%) of A, B, and C, and remaining 27 (4.5%) were unspecified. Patients were treated with lamivudine 100 mg/day for 12 months. Genotype B with HBV DNA levels turned to be negative (HBV DNA < 0.1 ng/L) was 87.2%, genotype C was 89.51%, coinfection of genotype B and C was 93.04% (P > 0.05). HBeAg seroconversion of genotype B was 11.65%, of genotype C was 20.64%, and of coinfection of genotype B and C was 18.57% (P > 0.05). All 69 strains of YMDD mutation were detected after lamivudine treatment for 12 months, in which genotype B was in 16.98%, genotype C in 15.38%, and coinfection of genotype B and C was in 13.86% (P > 0.05). CONCLUSION: There was no difference in HBV genotypes and the rate of development of YMDD mutations, HBeAg seroconversion, descending of HBV DNA level in Chinese patients with chronic hepatitis B.

[Distribution and clinical significance of hepatitis B virus (HBV) genotypes and subtypes in HBV-infected patients].

OBJECTIVE: To study hepatitis B virus (HBV) genotype and subtype distribution and its clinical significance in HBV-infected patients. METHODS: We used type/subtype-specific primers and PCR to detect HBV genotype and subtype of 445 HBV-infected patients from Beijing, Changchun, Hanchuan Shenzhen, Qingyuan and Nanjing, including 7 acute hepatitis (AH), 36 asymptomatic HBV carriers (ASC), 352 chronic hepatitis (CH), 28 liver cirrhosis (LC), and 22 hepatocellular carcinoma (HCC) cases. Genotyping results were confirmed by PCR product sequencing. RESULTS: Among 445 HBV-infected patients, the proportions of genotype B, C, and B/C were 32.6% (145/445), 53.7% (239/445), and 13.7% (61/445), respectively. In genotype C, 13 (5.4%) were subtype C1, 135 (56.5%) were subtype C2, and the remaining 91 (38.1%) were neither C1 nor C2. In genotype B, 100 (69.0%) were subtype Ba, 25 (17.2%) subtype Bj, and the other 20 (13.8%) were neither Ba nor Bj. In genotype B/C, 15 (24.6%) were Ba/C2, 8 (13.1%) Bj/C2, 6 (9.8%) Ba/C1, 3 (4.9%) Bj/C1, 11 (18.0%) Ba/neither C1 nor C2, 7 (11.5%) Bj/neither C1 nor C2, and 6 (9.8%) neither Ba nor Bj/neither C1 nor C2, 2 (3.3%) neither Ba nor Bj/C1, 3 (4.9%) neither Ba nor Bj/C2. The HBV genotype and subtype distribution we found exhibited significant differences in the various clinical types of HBV infection tested, and showed that genotype C was predominant among patients with liver cirrhosis (78.6%) and hepatocellular carcinoma (86.4%) while genotype B was predominant in asymptomatic carriers (72.2%). In addition, genotype and subtype distribution showed no significant differences between male and female patients, but genotype and subtype distribution showed significant differences in patients positive or negative with HBeAg. CONCLUSION: Subtypes Ba and C2 are predominant in patients with hepatitis B from these 6 cities, and genotype C may be associated with the development of liver cirrhosis and hepatocellular carcinoma.

[Detection for severe acute respiratory syndrome (SARS) coronavirus RNA in stool of SARS patients].

OBJECTIVE: To investigate excretion of severe acute respiratory syndrome coronavirus RNA (SARS-CoV) in stool of SARS patients. METHODS: SARS-CoV RNA was detected in stool specimens with fluorescent quantitative polymerase chain reactions (FQ-PCR) in 101 SARS patients on the 10 to 55 days after onset, 27 non-SARS patients and 400 individuals with health check-up. RESULTS: SARS-CoV RNA was positive in stool specimens by FQ-PCR in 58 of 101 SARS patients (57.4%), and all negative in 27 non-SARS patients and 400 healthy individuals. Positive rate of SARS-CoV RNA was 100% (8/8), 67.7% (21/31), 47.4% (27/57) and 40.0% (2/5) on the 10 - 19, 20 - 29, 30 - 39 and 40 - 55 days after onset of fever, respectively, with values of logarithm of SARS-CoV RNA load of 6.06 +/- 2.05, 4.51 +/- 1.23, 3.82 +/- 1.44 and 3.57 +/- 1.25, respectively. CONCLUSION: Positive rate and load of SARS-CoV RNA in stool of SARS patients was the highest at their acute phase, and decreased with the extension of its course.

[Detection of anti-HEV IgG and analysis of partial HEV RNA sequence].

OBJECTIVES: To investigate HEV infection in swine and the genotype relationship between swine and human HEV. METHODS: Anti-HEV IgG antibody was detected in the sera of swine using enzyme linked immunoassay (EIA), and HEV RNA was amplified by reverse transcription nested polymerase chain reaction (RT-nPCR). The Vector NTI Suite 7 and TreeView softwares were used for nucleotide sequences phylogenetic analysis of HEV isolated from human and swine. RESULTS: The anti-HEV IgG positive rate was 16.67% (18/108). Among the 18 anti-HEV IgG positive sera, 2 sequences (11.11%, called S18 and S43, respectively) of HEV ORF1 (102-387bp) were amplified, with the identity of 99% between them. They had 76% to 77%, 78%, 76% to 79%, 85% to 86%, 77%, 80%, 79% and 75% - 79% homology at the nucleotide level with human HEV genotypes 1 to 8, respectively. One (S18) of them was also amplified out in ORF2 region (5,994-6 297bp) and showed 76% to 78%, 74%, 74% to 77%, and 85% to 94% identity with human HEV genotypes 1 to 4 at the nucleotide level, respectively. CONCLUSION: HEV sequences isolated from swine belong to human HEV genotype 4.

Inhibition of HBV replication and gene expression in vitro and in vivo with a single AAV vector delivering two shRNA molecules.

Hepatitis B virus (HBV) infection is highly prevalent worldwide. The major challenge for current antiviral treatment is the elevated drug resistance that occurs via rapid viral mutagenesis. In this study, we developed AAV vectors to simultaneously deliver two or three shRNAs targeting different HBV-related genes. These vectors showed markedly better antiviral effects than ones that delivered a single shRNA in vitro. A dual shRNA expression vector (AAV-157i/1694i), which simultaneously expressed two shRNAs targeted the S and X genes of HBV, reduced HBsAg, HBeAg and HBV DNA levels by 87+/-4, 80.3+/-2.6 and 86.2+/- 7% respectively, eight days post-transduction. In a mouse model of prophylactic treatment, HBsAg and HBeAg were reduced to undetectable levels and the serum HBV DNA level was reduced by at least 100 fold. These results indicate that AAV-157i/1694i generates potent anti-HBV effects and that the strategy of constructing multi-shRNA expression vectors may lead to enhanced anti-HBV efficacy and overcome the evading mechanism of the virus and thus the development of drug resistance. [BMB reports 2009; 42(1): 59-64].

Correlation of hepatitis B virus (HBV) genotypes and mutations in basal core promoter/precore with clinical features of chronic HBV infection.

BACKGROUND/AIMS: To investigate the correlation of hepatitis B virus (HBV) genotypes and basal core promoter (BCP) and precore (PC) mutations in patients with chronic hepatitis B. METHODS: HBV genotyping, nucleotide mutation, serum HBV DNA level and serological markers were analyzed in 121 patients with chronic HBV infection using INNO-LiPA HBV genotyping, polymerase chain reaction (PCR) product-based sequencing, fluorescence quantitative PCR and enzyme-linked immunosorbent assays respectively. RESULTS: Forty (33.0%), 77 (63.6%), two (1.7%) and two (1.7%) patients had genotypes B, C, B/C and D infections respectively. Significant differences were found in serum HBV DNA levels (log10 copies/ml: 6.18 vs. 5.61, P=0.042) and mutations at nucleotide (nt) 1762/1764 (71.4% vs. 42.5%, P=0.002) between genotypes C- and B-infected patients. There were significant differences in the mean age, serum biochemical parameter levels and mutation rates in BCP/PC among hepatitis e antigen (HBeAg)-positive and -negative chronic hepatitis B (CHB) and liver cirrhosis (LC) groups. CONCLUSION: Genotypes C and B are predominant in China, and the frequent nt 1762/1764 mutation, which occurs commonly in HBeAg-negative CHB, especially in genotype C patients, may be associated with the progress of chronic HBV infection.

[Distribution of hepatitis B virus genotypes in patients with chronic hepatitis B virus infection among 12 cities in China].

OBJECTIVE: To investigate the distribution of hepatitis B virus (HBV) genotypes in patients with chronic HBV infection among 11 cities of China. METHODS: A total of 1214 serum samples from patients with chronic HBV infection were collected in 11 cities of China, including Beijing, Qingyuan, Shenzhen, Shijiazhuang, Hanchuan, Nanjing, Changchun, Liaocheng, Jinan, Ningbo and Wenzhou. Genotypes of the 1214 HBV strains were identified by PCR method with type specific primers. Parts of the results were confirmed by direct sequencing analysis of PCR products. RESULTS: Among the 1214 patients with chronic HBV infection, 0.7% (9/1214)were genotype A, 28.4% (345/1214)genotype B, 58.4% (709/1214) genotype C, and 12.4% (151/1214) genotype B and genotype C mixed infection. No other genotypes were found. Genotype C was predominant in the northern part of China, such as Changchun, Beijing, Shijiazhuang,while genotype B was more commonly seen in south of China. 71.4% (20/28) for patients from Qingyuan and 63.6% (70/110) from Shenzhen were infected with genotype B. CONCLUSION: HBV genotypes had distinct geographic distribution. Genotype B and C the predominant strains in patients with chronic HBV infection in China. Genotype C was predominantly identified in the northern part of China versus genotype B the south.

[Detection of SEN virus in sera of patients with chronic hepatitis B and general population in 5 cities of China].

OBJECTIVE: To study the prevalence of SEN virus (SENV) infection in CHB patients in five cities of China. METHODS: A nest-polymerase chain reaction (nPCR) was used for detection of SENV-D and SENV-H in sera of 595 CHB patients from 5 cities of China and 96 normal individuals from Beijing. A total of 7 SENV strains were analyzed by direct sequencing. RESULTS: The prevalence rates of SENV in CHB patients and normal individuals were 61.3% and 62.5%, respectively (chi(2) = 0.047, P = 0.829). The prevalence rates of CHB patients between 5 cities were different. Nucleotide sequence analysis showed that the homology between 4 SENV-D strains was 91% - 98% and 95% - 98% between 3 SENV-H strains isolated from 5 cities in China. CONCLUSION: SENV-D/H were prevalent in CHB patients of China and their prevalence rates were similar to that in normal individuals.

[Rapid detection of genotypes of TT virus using a heteroduplex mobility assay].

OBJECTIVE: To establish a simple, sensitive, specific and less-costly method for detecting genotypes of TT virus (TTV). METHODS: TTV DNA was tested by nested polymerase chain reaction (nPCR) in sera from 180 patients with different types of viral hepatitis and 96 normal individuals in Beijing. TTV genotypes were determined in 40 sera collected from TTV DNA positive patients by heteroduplex mobility assay (HMA) and through sequencing. RESULTS: The positive rates of TTV DNA in viral hepatitis patients and normal individuals were 22.2% (40/180) and 19.8% (19/96), respectively (chi(2) = 0.220, P = 0.639). TTV DNA positive rates of patients with hepatitis A, B, C, E and non-A to E were 20.0% (6/30), 16.7% (5/30), 23.3% (7/30), 36.7% (11/30) and 18.3% (11/60), respectively. Of 40 TTV DNA positive patients, 20 (50.0%) were TTV G1, 7 (17.5%) TTV G2, 10 (25.0%) coinfected with different genotypes of TTV, and 3 untyped by HMA. Twenty G1 and 7 G2 detected by HMA were confirmed by sequence analysis. Of 10 patients coinfected with different genotypes of TTV, 5 were G1 and G2, 2 G1 and G3, 1 G1 and G4, 1 G1 and G3, and 1 with G1, G2 and G3 coinfections. CONCLUSION: HMA was recognized as simple, sensitive, specific and less-costly, thus could be used for genotyping of TTV.

[Dynamics of peripheral blood lymphocytes and their subpopulations in patients with severe acute respiratory syndrome].

OBJECTIVE: To study on the dynamics of peripheral blood lymphocytes and their subpopulations in patients with severe acute respiratory syndrome. METHODS: Using flow cytometry, the absolute numbers of peripheral blood lymphocytes and their subpopulations in 240 SARS patients (696 specimens) and 51 individuals as controls, were counted and compared. RESULTS: The absolute numbers of peripheral blood lymphocytes and their subpopulations (CD45, CD3, CD4, CD8) were 1298 +/- 785, 897 +/- 606, 510 +/- 372, 362 +/- 263/mm(3), respectively, significantly lower in SARS patients as compared to the normal controls (2024 +/- 423, 1391 +/- 289, 795 +/- 129, 551 +/- 183/mm(3)). Of SARS patients, severe group (1095 +/- 740, 740 +/- 562, 419 +/- 346, 304 +/- 244/mm(3)) had lower counts than that of mild group (1404 +/- 788, 991 +/- 612, 564 +/- 378, 396 +/- 267/mm(3)), and in group with deaths (587 +/- 493, 369 +/- 371, 204 +/- 191, 150 +/- 130/mm(3)) was lower than that of recovery group (1355 +/- 776, 948 +/- 603, 539 +/- 375, 382 +/- 263/mm(3)). There were significant differences (P < 0.01) for CD45, CD3, CD4, CD8, but with no significant difference (P > 0.05) for CD4/CD8 ratio between severe and mild, recovery and death groups. The lymphocytes and their subpopulations (CD45, CD3, CD4, CD8) declined in the 1st week and to the lowest level (977 +/- 579, 641 +/- 466, 360 +/- 275, 270 +/- 216/mm(3)) in the 2nd week. Then the lymphocytes and their subpopulations gradually increased during the recovery of the disease. CONCLUSION: The absolute numbers of peripheral blood lymphocytes and their subpopulations in SARS patients might be used as one of the methods for diagnosis on the severity and prognosis of the disease.

[Dynamics of peripheral blood B lymphocytes and natural killer cells in patients with severe acute respiratory syndrome].

OBJECTIVE: To study the dynamics of peripheral blood B lymphocytes and natural killer (NK) cells in patients with severe acute respiratory syndrome (SARS). METHODS: The absolute numbers of peripheral blood B lymphocytes and NK cells in 602 serial samples from 240 patients with SARS were counted, using flow cytometry, and compared with that of normal population. RESULTS: The absolute numbers of peripheral blood B lymphocytes and NK cells in SARS patients were significantly lower than that of the normal population (P < 0.001) and were much lower in SARS patients with severe or extremely severe types, as compared with that of moderate or mild type cases (P < 0.001). The amount of B lymphocytes in recovery SARS patients increased at the 2nd week after onset, and gradually becoming normal at the 5th week of the disease onset. The number of NK cells was in the low level at onset, and keep decreasing at the 2nd week. However, it was increasing with the recovery of the disease, but did not reach to normal level at the 5th week after onset. CONCLUSION: The absolute numbers of peripheral blood B lymphocytes and NK cells were associated with the severity of the disease, and detection of these two kinds of cells was useful for predicting the prognosis of SARS.