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Objective: To explore a new method for determining hepatitis B virus (HBV) genotypes B-D with real-time fluorescence-based PCR., Methods: The PCR primers and probes were designed according to the analysis of 143 complete HBV genomes from GenBank and on the basis of a search for genotype-specific nucleotide sequences which were conserved in the 3 HBV genotypes. Real-time fluorescence-based PCR was performed for HBV genotyping of 128 samples collected from Lanzhou. Three samples of each genotype of B, C and D were randomly selected and their S gene was sequenced for confirmation of the results of PCR-based method., Results: Real-time PCR identified genotype B in 26 (20.3%), genotype C in 92 (71.9%), and genotype D in 10 (7.8%) cases. The sequencing results of the S gene of 18 PCR-produced clones were in complete consistence with those of fluorescence-based PCR., Conclusion: The real-time PCR method is convenient, highly sensitive, rapid and accurate, especially suitable in clinical and large-scale epidemiological studies.

Objective: To study the effect of bicyclol tablets on the levels of interleukin (IL)-4, IL-10 and interferon (IFN)-gamma in culture supernatants of peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis B (CHB)., Methods: Whole blood samples were obtained from 30 patients with CHB before and at 1 and 3 months during bicyclol tablet therapy and also from 7 healthy donors for isolation of the PBMCs. The product of IL-4, IL-10 and IFN-gamma in the culture supernatant of PBMCs were determined by enzyme-linked immunosorbent assay (ELISA) after 72 h of cultivation., Results: At 3 months during the therapy, the level of IFN-gamma was increased significantly in the patients from 36.25+/-19.92 pg/ml to 53.19+/-7.28 pg/ml (P<0.05) and the level of IL-4 significantly decreased from 17.18+/-7.43 pg/ml to 9.74+/-7.75 pg/ml (P<0.01). The changes in the levels of IL-4 and IFN-gamma at 3 months during therapy were more obvious in patients with HBeAg positivity than in HBeAg-negative patients (8.74+/-6.12 pg/ml vs 20.51+/-9.16 pg/ml, 50.71+/-30.76 pg/ml vs 26.03+/-10.48 pg/ml, respectively, P<0.05)., Conclusion: Bicyclol tablets not only promote Th1 type cytokine-mediated immune responses but also down-regulates Th2 type cytokine-mediated immune responses.

Objective: To investigate the distribution of hepatitis B virus (HBV) genotypes in Guangdong and explore its clinical significance., Methods: Fifty-five patients with chronic active hepatitis (CAH) from Guangdong province were included in this study. HBV surface gene amplified by PCR was analyzed by restriction fragments length polymorphism (RFLP) for HBV genotyping, and the relationship of HBV genotype with clinical, serological and histological data of the patients was analyzed., Results: Twenty-eight out of 55 patients were infected with HBV strains of genotype B (51.0%), 18 with genotype C (32.7%), 4 with genotype D (7.3%), 4 with HBV classified as genotype B+C (7.3%), and 1 (1.8%) with HBV that did not conform to any of the genotypes from A to G. No significant differences in clinical, histological, or serological data of HBV DNA loading were detected between genotypes B and C. But in patients older than 30 years, the genotype C was accompanied by significantly higher HBV DNA loading and serum HBeAg level than genotype B (Fisher's exact test, P=0.002)., Conclusions: HBV genotype B and C are the major genotypes prevalent in Guangdong Province. Genotype C is associated with the longer duration of HBeAg and higher HBV DNA level in patients older than 30 years, suggesting the higher risk of HBV genotype C infection to progress into chronic liver disease.

Objective: To assess the correlation of routine blood and serum biochemical indices with liver histology, and identify the sensitive non-invasive ones indicative of liver inflammation and fibrosis in patients with chronic hepatitis B., Methods: A total of 252 patients were enrolled in this retrospective analysis. The indices including the patients' age, gender, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, globulin, total bilirubin, prothrombin time (PT) and its activity (PTA), WBC, and platelet count were analyzed statistically., Results: The status of liver inflammation was correlated to patients' routine blood indices (P<0.05). Serum AST, total bilirubin, WBC, and platelet count was related significantly to the degree of liver inflammation, but serum ALT and AST alone did not describe exactly the degree of liver inflammation. The status of liver fibrosis was independent of patients' serum ALT, but correlated to other routine blood indices (P<0.05). The patients' age, serum AST, total bilirubin and platelet count had significant relation to the stage of liver fibrosis, and the age, blood PT, total bilirubin and platelet count were significant relevant indices for early liver cirrhosis, and their sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 31.5%, 94.4%, 60.7%, 83.3% and 80.7%, respectively., Conclusions: Single examination of serum ALT and AST does not help much to predict the status of liver test in patients with chronic hepatitis B. Serum AST, total bilirubin, WBC and platelet count are associated with the degree of liver inflammation. The patients' age, serum total bilirubin, AST and platelet count are relevant indices for liver fibrosis, and patients' age, serum total bilirubin, PT, and platelet count provide valuable assistance in confirmation of early liver cirrhosis with high specificity, but not so for screening purpose.

Objective: To examine the immunological reactions between the peptides of SARS coronavirus (SARS-Cov) S-protein and the serum of SARS patients for identifying the SARS-Cov epitope., Methods: The peptides from S1 domain of SARS-Cov S-protein were synthesized by peptide synthesizer, and the immunological reaction of the peptides with the serum of SARS patients were examined by means of ELISA., Results: The optical density value of the immunological reaction of synthesized 8 peptides with SARS patient serum was 1.5-2.6 times higher than that with normal serum., Conclusion: The 8 peptides from S1 domain of S protein appear to have low immunogenicity to the serum of SARS patients, indicating that these peptides may not be the epitope of the SARS-Cov.

Objective: To study the effects of lidocaine on the balance between cerebral oxygen supply-consumption and on the hemodynamics during anesthesia induction in patients with supratentorial tumor., Methods: Twenty-four patients with supratentorial tumor were randomly divided into lidocaine group (n=12) and control group (n=12). Blood gas analysis and determinations of plasma lactic acid and glucose in the radial artery and internal jugular venous bulb were performed. Oxygen extraction ratio (OER) and blood oxygen content in the artery and internal jugular venous bulb were calculated during anesthesia induction., Results: OER and difference declined in plasma lactic acid level between the internal jugular venous bulb and the artery, and blood oxygen saturation as well as blood oxygen pressure in the internal jugular venous bulb and the artery increased along with blood oxygen content in the internal jugular venous bulb in both groups during anesthesia induction. Comparison between the groups showed that only the changes in blood oxygen pressure in the internal jugular venous bulb were statistically significant. Changes in the hemodynamics in lidocaine group were less obvious than those in the control group during anesthesia induction., Conclusion: Lidocaine does not significantly influence cerebral oxygen balance and may effectively inhibit hemodynamic response during anesthesia induction in patients with supratentorial tumor.

Objective: To investigate the generation of neutralizing anti-interferon-alpha antibodies (NA) in patients with chronic hepatitis B in the course of interferon-alpha treatment, and analyze the factors influencing the production of the antibodies., Methods: A total of 181 patients with histologically confirmed chronic hepatitis B were enrolled in this study. Recombinant interferon-alpha 1b (rIFN-alpha 1b) was given subcutaneously 3 times a week (5 MU once) in a treatment course lasting for 6 to 37 months (median 10.0 months). In each case, serum NA was detected with antiviral neutralization bioassay, HBeAg with enzyme-linked immunoassay (EIA) and hepatitis B virus DNA with fluorescent quantitative PCR every 1 to 3 months starting from 3 months after the initiation of the treatment., Results: Of the 181 patients, 61 were positive for NA during the treatment course, resulting in an overall NA occurrence rate of 33.7%. The overall incidence rate, as well as the overall prevalence rate, was significantly higher in male than in female patients (39.1% vs 20.8%, (2)=5.622, P=0.018), and no correlations of NA generation was observed with age, pretreatment serum ALT level, serum HBeAg, serum HBV DNA level, liver histological findings or treatment course. Binary logistic regression analysis showed that the only factor in relation with NA production was gender. The prevalence rates of NA varied significantly with the treatment courses ( (2)=98.051, P=0.000). Bivariate correlation analysis showed that the prevalence rate of NA, but not the incidence of NA, was strongly related with the treatment course (r=0.855, P<0.001)., Conclusions: In chronic hepatitis B patients treated with rIFN-alpha 1b, the prevalence rate of NA, instead of the incidence of NA, is significantly related to the treatment course. NA was more likely to develop in male patients to contribute to their poorer antiviral response in comparison with the female patients.

Objective: To explore the distribution of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) in SARS autopsy tissues at the molecular level., Methods: In situ hybridization was used to detect the expression and location of SARS-CoV RNA polymerase gene in autopsy tissues from SARS-Cov-infected subjects, including the lung, spleen, lymph nodes, pituitary, pancreas, parathyroid, adrenal glands, gastrointestinal tract, skin, brain, liver, kidney, blood vessels, striated muscles of the limbs, bone marrow, heart, ovary, uterus and testicles., Result: SARS-CoV RNA was detected in the cytoplasm of the alveolar epithelia, infiltrating mononuclear phagocytes in the lungs, serous gland epithelium of the trachea/bronchus, monocytes in the spleen and lymph nodes, acinar cells in the pancreas, acidophilic cells in the parathyroid and pituitary, adrenal cortical cells, epithelia of the alimentary tracts, gastric parietal cells, sweat gland cells, brain neurons, hepatocytes near the central vein, epithelia of the distal renal tubules, bone marrow promyelocytes, and endothelia of the small veins., Conclusions: SARS-CoV invades various organs of the body and distributes in a similar fashion to CD13, the receptor of human coronavirus 229E. The detection of SARS-CoV in the sweat glands, alimentary tracts and epithelia of the distal convoluted tubules of the kidney may help identify the transmission routes of SARS-CoV.

Objective: To assess serum antibody responses of patients with severe acute respiratory syndrome (SARS) to nucleocapsid (N) antigen of SARS-associated coronavirus., Methods: The serum levels of IgM and IgG antibodies to N antigen were measured in 200 healthy blood donors and 13 SARS patients at different time points of acute and convalescent phases using indirect enzyme-linked immunosorbent assay (ELISA) with N fusion proteins of SARS-associated coronaviruses., Results: The IgM positive critical value of 0.233 and IgG of 0.239 were selected as the threshold value for positive results that equals the product of 2.1 and the mean IgM and IgG levels of 200 healthy blood donors. In 13 patients with SARS, the antibody responses to N antigen were not detectable in the first week after the onset of symptoms. The IgM and IgG seroprotection rates were 83.3% and 66.7% respectively in the second week, both reaching 100% at the third week. IgM seroprotection rates was 61.5% in the second month, and 38.5% at third month. The IgG peaked one month after the onset and remained at high levels in the following 2 months., Conclusion: The antibody responses suggest that N protein of SARS is immunodominant and plays an important role in viral pathogenesis. This ELISA-based test for detecting anti- N antigen may be of significant value for SARS diagnosis.

Objective: To analyze the heterogeneity of polymerase gene fragment of SARS-associated coronavirus from SARS patients, and establish a RT-PCR method for detecting SARS-associated coronavirus., Methods: RT-PCR was performed using SARS coronavirus-specific primers to amplify the polymerase gene fragment of SARS-associated coronavirus from specimens of suspected and established SARS cases. The amplicons were cloned and sequenced. All the obtained sequences were compared with the sequence of published SARS-associated coronavirus, and alignment was proceeded with other coronavirus sequences., Results: Specific amplicons can be amplified from the sputum samples, throat swab and plasma of most SARS patients, and 8 were random selected and sequenced. All of them possessed 100% homology with the published SARS-associated coronavirus sequence, while all the negative controls were RT-PCR negative. Nucleotide-sequence and amino acid-sequence alignment of the fragment BNI109 with other six known coronavirus show that the fragment BNI109 is more close to bovine coronavirus(BCV) and murine hepatitis virus(MHV). The BNI109 fragment showed 75% homology with BCV and MHV at amino acid level., Conclusion: The polymerase fragment BNI109 of SARS coronavirus is highly conservative and is suitable for detecting SARS-associated coronavirus using RT-PCR method.

Objective: To obtain human beta2-microglobulin (beta2m) gene that is to be efficiently expressed in E.coli., Methods: beta2m cDNA including only the coding region for the protein was amplified from Raji cell line by reverse transcriptase-PCR via the primers 5'-GGTGGTCATATGGCTATCCAGCGTACTCCA-3' and 3'-GGTGGTTGCTCTTCCGACATGTCTCGATCC-3'. The product was subsequently cloned into a modified pBV220 vector after digestion with Nde I/Sap I. The recombinant plasmid pBV220-beta2m was transformed into E.coli BL21 after sequence analysis, and the fusion protein was then expressed via induction at 42 for 5 h at D(600) of 0.55-0.60, followed by purification through chitin beads., Results: The beta2m cDNA was identical with those published in Genbank. The expressed fusion protein was identified in the form of inclusion body at the ratio more than 45 % of the E.coli proteins, and was denatured with 8 mol/L urea, followed by refold and purification to a high purity, displaying a relative molecular mass of 12 000 on 10 % SDS-PAGE gel., Conclusion: The human beta2m gene was cloned successfully and expressed efficiently and constantly in E.coli BL21,which lays the ground for engineering MHC-tetramers.

Objective: To investigate the role of hepatitis B virus (HBV) DNA levels in HBV-carrier mother and selective transmission of HBV variant with S gene mutation to infant in the failure of neonatal HBV immunization., Methods: Serum HBV DNA level was determined by Roche LightCycler quantities in 95 HBsAg-positive pregnant women and later in their newborns. Hepatitis B virus S gene sequences isolated from mother/child pairs were analyzed in 7 cases which did not respond to HBV vaccination and hepatitis B immunoglobulin., Results: HBV infection was identified in 7.4 % of the infants during their first year after birth. The levels of HBV DNA in the mothers of the infected infants were higher than those of the uninfected group, and selective transmission of HBV variant from mother to infant was observed., Conclusion: HBV DNA levels in HBV-carrier mother and selective transmission of HBV variant from mother to infant contribute to the failure of neonatal HBV immunization.

Objective: To screen the differential points related with response to interferon-alpha in HBV post-transcriptional regulatory element (HPRE)., Methods: HPRE sequence of 31 Chinese patients with HBV infection were detected by direct sequencing of PCR products. Differential points in HPRE between Chinese patients and White patients were compared., Results: The T to C differential point at nt 1 504 and C to T(G) at nt 1 508 were found respectively in HPRE of 21 and 19 patients with chronic hepatitis B, the C to T differential point at nt 1 509 were found in 17 cases. These differential points were not found in HPRE of the White patients. The relationship between the HPRE differential points and the levels of HBV DNA was not observed., Conclusion: There were differential points in the HPRE between Chinese and White HBV patients, which might be correlated with response to interferon-alpha.

Objective: To study the characteristics of intrauterine transmission of hepatitis B virus (HBV) of different genotypes from mother to the newborn babies., Methods: One hundred and thirty-five HBsAg-positive pregnant women from 3 hospitals in Guangzhou municipality and their 139 newborn babies were enrolled in this study. Peripheral blood samples were collected from both the mothers before delivery and the neonates within 3 d after birth, and during the follow-up survey, sampling was also performed in some of the newborn babies. Nested-PCR was employed for the amplification of the S region of HBV genome, the product of which was subjected to digestion with restriction endonuclease to compare the restriction fragments length polymorphism (RFLP) to determine the HBV genotype. Direct sequence analysis was conducted and the results compared with the sequences available in Genebank., Results: HBV infection occurred in 7 (5.0%) neonates through intrauterine transmission, among whom 3 were identified as being infected with C genotype, 3 with B genotype and 1 with D genotype, all identical to the genotypes afflicting their mothers. All of the 7 mothers and the babies were asymptomatic., Conclusion: C and B genotypes are predominant in intrauterine transmission of HBV in Guangzhou, and base mutation, rather than mutation at the endonuclease digestion site, may take place during intrauterine transmission due to impact of the immune system. The genotyping results, however, can not help make prognostic assessment of the infected babies.

Objective: To study the effects of 20/21 bp partial deletion mutation (from nt 1 748 or nt 1 747 to nt 1 767) in the core promoter (CP) region of hepatitis B virus (HBV) genome complicated by precore stop condon mutation at nt 1 896 on the expression of the viral antigens., Methods: Eukaryotic expression vector containing full-length HBV genome with the above mutations was constructed. After transfection of the recombinant HBV plasmids into HepG2 cells, the expression of the viral antigens was examined with enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis., Results: As shown by ELISA and Western blotting analysis, the amount of extracellular secretion of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) along with intracellular hepatitis B core antigen (HBcAg) in the cells transfected with vectors containing HBV genomes with partial deletion in the CP region was markedly reduced compared with that produced by wild-type HBV., Conclusion: The mutations in question causes marked reduction in viral antigen production by HBV in comparison that by wild-type HBV.

Objective: To study the association between immune response to hepatitis B vaccine and HLA-DRB1*02, 07, 09 genes in the population of Han nationality in Guangdong Province., Methods: A total of 1 400 healthy college students of Han nationality from Guangdong Province were recruited in this study, who received standard courses of vaccination with recombinant hepatitis B virus vaccine at month 0, 1 and 6. Eight weeks after the final vaccination, the serum levels of anti-HBs antibody was examined with enzyme-linked immunosorbent assay (ELISA). Detection of HLA-DRB1*02, 07, 09 alleles were performed with PCR using sequence-specific primers in 118 individuals who failed to produce or produced insufficient antibodies (nonresponders or weak responders), 100 normal responders and 45 strong responders., Results: The frequency of HLA-DRB1*07 in nonresponders or weak responders was significantly higher than that in normal or strong responders (15.25% vs 3.44%, P<0.05), while the frequency of HLA-DRB1*02 was much lower (5.08% vs 17.9%, P<0.05). The frequency of HLA-DRB1*09 showed no obvious difference between the 2 groups (19.49% vs 17.9%, P>0.05)., Conclusion: Failure or deficiency in the response to HBV vaccine is associated with the HLA-DRB1*07 gene, while HLA-DRB1*02 gene is related to the normal vaccination process or overreaction. Definite relation of HLA-DRB1*09 to the response to HBV vaccine is not found.

OBJECTIVE: To construct an eukaryotic expression vector containing the full-length genome with partially deleted core promoter of hepatitis B virus (HBV). METHODS: A linearized genome containing the entire HBV 3.5 kb mRNA transcriptional units (P3.8 I plasmid) was used, which initiated from the upstream sequences of the basic core promoter. The objective eukaryotic expression vector was constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identifcation was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by cloning and sequencing analysis. RESULTS: The eukaryotic expression vectors containing HBV genomes with 20/21 bp deletion (position 1 748/1 747 to 1 767) in the core promoter or with precore stop mutation at nucleotide 1896 as well were constructed successfully as confirmed by sequence analysis with RFLP. CONCLUSION: The recombinant expression vector may lay the foundation for further studies into the biological significance of the above mentioned mutations in vitro.

OBJECTIVE: To determine the complete nucleotide sequence of a Chinese hepatitis B virus (HBV) strain of genotype D. METHOD: The complete nucleotide sequence of HBV derived from a Chinese chronic asymptomatic carrier was amplified by PCR and cloned to conduct sequence analysis. Homology of the resulted nucleotide sequence with those of published HBV strains was assessed by using DNASIS, and complete sequence analysis of the phylogenetic tree of 30 genotype D HBV strains conducted by the assistance of Clustalw. RESULTS: With the complete nucleotide sequnce of 3 182 bp, this HBV strain belongs to ayw3 subtype and D genotype, with the Genebank accession number AF280817. Its complete nucleotide homology is 98.3% to 94.5% with the published sequences of genotype D HBV strains and less than 89.5% with the other HBV strains of genotype A, B, C, E, F and G published in GenBank. CONCLUSION: The evolutionary relations of the complete nucleotide sequence of this HBV strain is the closest to those of the 4 Swedish strains out of the 30 genotype D strains published in GenBank.

OBJECTIVE: To establish a convenient approach to implement polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detecting of gene mutation. METHODS: Serum samples were collected from l0 patients with chronic hepatitis B who had received oral lamivudine for at least l year, and 3 methods including mismatched PCR-RFLP, direct sequence analysis of the PCR product and cloning prior to secondary sequence analysis were employed respectively to examine the YMDD motif mutation of P gene of hepatitis B virus (HBV). RESULTS: PCR-RFLP revealed wild-type HBV infection in 7 cases mixed infection in 1 case and HBV mutant infection in 2 cases. With direct sequence analysis of the PCR product, 9 cases were found with Wild-type HBV infection and 1 with HBV mutant infection. However, 2 non-wild-type HBVV-infected cases as approved by PCR-RFLP, opposite to the results by direct sequence analysis, were both shown by sequence analysis after cloning to suffer mixed infection. CONCLUSION: Sequence analysis following cloning is the best way to detect YMDD motif mutation in HBV, but in terms of economy and practicability, mismatched PCR-RFLP is of greater significance in mass screening of YMDD motif mutant HBV than the other 2 approaches.

OBJECTIVE: To investigate the association of hot-spot mutations in hepatitis B virus (HBV) pre-C region with the occurrence and outcome of severe hepatitis B. METHODS: A total of 68 patients with severe hepatitis B negative for hepatits B e antigen (HBeAg) were enrolled in this study, including 6 cases of acute, 38 cases of subacute and 24 chronic severe hepatitis B, with another 44 HBeAg-positive patients with chronic hepatitis B serving as control. Mismatch PCR and restriction fragment length polymorphism analysis were employed to examine the mutations of T1862 and A1896 in this 2 groups of patients. RESULTS: The mutation rates at A1896 and T1862 were 66.7% (4/6) and 0 (0/6) respectively in acute severe hepatitis B cases, 42.1% (16/38) and 15.8% (6/38) in subacute severe hepatitis, 25.0% (6/24) and 16.7% (4/24) in chronic severe hepatitis, and 45.5% (20/24) and 2.3% (1/44) in chronic hepatitis cases. There were significant differences in terms of T1862 mutation between patients with severe hepatitis and chronic hepatitis (P<0.01). CONCLUSION: T1862 mutation is closely related to the exacerbation of chronic hepatitis, while the role of A1896 mutation in this process requires further investigation.