Endothelium-independent vasorelaxant effect of puerarin on rat thoracic aorta.

To investigate the vasorelaxant effect of puerarin, tension was recorded from rat thoracic aortic rings. Puerarin completely relaxed the contractions induced by phenylephrine (PE) in a concentration-dependent manner in endothelium-intact and endothelium-denuded rat aorta, while had no effect on those preconstricted by a high concentration of potassium chloride (60 mM). Also, puerarin had no effects on the transient contraction elicited by PE or caffeine in Ca2+- free medium. The relaxant effect of puerarin was significantly inhibited by pretreatment of endothelium-denuded aorta with potassium channel antagonists tetraethylammonium and 4-aminopyridine, but not glibenclamide. These results indicate that puerarin induces an endothelium-independent relaxation in rat aortic rings. The mechanisms may involve the reduction in Ca2+ influx through the non-voltage-sensitive calcium channels and the activation of the potassium channels (voltage-activated K+ channels and large conductance Ca2+ -activated K+ channels, but not ATP-sensitive K+ channels).

Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing.

AIM: To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome and establish a new sequencing method in China. METHODS: Polymerase chain reaction (PCR) was combined with a DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE) and polyethylene glycol (PEG) respectively. Then, in the presence of a 5' labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC-42 and HC-49) were sequenced. RESULTS: PCR directed sequencing worked best using PCR amplified DNA purified by electrophoresis as a sequencing template. When sequencing a large number of templates, the purification step can be bypassed by using a lower concentration of dNTPs (40 µmol of each dNTP) and primers (10 pmol of each primer) in the first stage of PCR. The aliquot of the first stage of PCR mixture was then directly used for amplification of chain terminated products but the sequencing ladders generated were of low intensity. Polyethylene glycol (PEG) could not remove nonspecific products of PCR, which affected the sequencing result to a certain extent and generated a background in sequencing ladders. Compared with the reported HCVJ and HC-C2, a new three nucleotide deletion was found in HC-42. CONCLUSION: PCR directed sequencing is a rapid, simple and effective method, especially for sequencing large samples. A three nucleotide deletion was first reported.

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins.

AIM: To prepare hybridoma cell lines that secrete monoclonal antibodies against hepatitis C virus (HCV) recombinant proteins NS3 and NS5 and to evaluate their use in the study of HCV NS3 and NS5 antigen distribution in human liver tissue. METHODS: Hybridoma cell lines were generated using spleen cells from BALB/C mice immunized with recombinant NS3 and NS5 proteins, following conventional protocols. Antibody-secreting cells were screened by solid phase ELISA and cloned by limited dilution. The specificity of the monoclonal antibodies was determined by testing hybridoma culture supernatants by Western blots of E. coli expressing the recombinant HCV proteins and ELISA with HCV core and hepatitis B virus (HBV) antigens. The monoclonal antibodies were employed in immunohistochemistry studies to determine the distribution of HCV NS5 and NS3 antigens in 51 paraffin embedded human liver tissue samples. RESULTS: Eight hybridoma cell lines secreting monoclonal antibodies against HCV NS3 and NS5 proteins were generated and named 2B6, 2F3, 3D8, 3D9, 8B2, 6F11, 4C6 and 7D9. Only one of them, 2B6 (secreting antibodies against NS3 protein), cross-reacted with the C7 polypeptide, a different recombinant NS3 polypeptide. The rest of the cell lines showed no cross-reactivity with HCV core or HBV antigens. In addition, monoclonal antibodies against NS3 antigens did not cross-react with NS5 antigens, and vice versa. In immunohistochemistry studies, these monoclonal antibodies did not detect HCV antigens in specimens from patients infected only with HBV (n = 20). In HCV-infected specimens (n = 31), the rates of positive detection of NS3 and NS5 antigens were 51.6% (16/31) and 54.9% (17/31), respectively. Six of these 31 specimens were from patients infected only with HCV and half of them were positive for HCV NS3 and NS5 antigens. In specimens from patients co-infected with HBV and HCV (n = 25), the rates of NS3 and NS5 antigen positive detection were 52% (13/25) and 56% (14/25), respectively, which are similar to those obtained in samples from patients infected only with HCV. In specimens from chronic active cirrhosis patients, the rates of HCV NS3 and NS5 antigen detection were 70.6% (12/17) and 76.5% (13/17), respectively. CONCLUSION: We successfully prepared monoclonal antibodies that are specific against recombinant HCV NS3 and NS5 proteins and could be useful for clinical immunohistochemistry diagnosis.

Hepatitis G virus infection in patients with chronic non-A-E hepatitis.

AIM: To elucidate the role of hepatitis G virus (HGV) infection in chronic non-A-E hepatitis and sequence the partial NS5 genome of HGV isolated from the serum of a Chinese patient with chronic non-A-E hepatitis METHODS: Serum samples of patients with chronic non-A-E hepatitis were collected and total nucleic acids were extracted and subjected to reverse transcriptase-nested-polymerase chain reaction (RT-nested-PCR) using primers from the putative NS5 region of HGV genome. Then, 994bp cDNA was prepared from the positive serum, purified with electrophoresis of polyacrylamide gels, and directly sequenced using the dideoxy-mediated chain-termination method. RESULTS: HGV-RNA was detected in 1 of the 35 patients with chronic non-A-E hepatitis. Compared with the 2 HGV isolates (PNF2161 and R10291) obtained from American patients, the HGV NS5 gene of this Beijing isolate (HG-G) showed homology of 88.0% and 89.2% respectively. On the other hand, in comparison with the West African isolate (GBV-C), the Beijing isolate showed homology of 93.5%. The patient showed persistent increase of alanine transaminase, but normal levels were achieved after interferon therapy with persistent positive HGV RNA. CONCLUSION: HGV is one of the causes of chronic non-A-E hepatitis, but it may not be a very important cause. The nucleotide sequence of partial NS5 gene of HG-G was found to be highly homologous to the West Africa isolate.

[Studies on synthetic peptide. XX: the antigenicity and linear epitope map of synthetic peptide hepatitis C virus].

Hepatitis C virus (HCV), the major causative agent of post transfusion non-A, non-B hepatitis (NANB), had been cloned and expressed. According to the protein sequence of HCV-BK and its epitope profiles which combined the hydrophilicity, accessibility, flexibility, antigenicity, charge distribution and HPLC reserve coefficient of protein using the "Goldkey" computer program, we designed and synthesized the following peptides: P1(475-495), P3(449-468), P4(658-663), P5(645-663), P6(484-489), P7(475-489), P15(655-662), P16(230-237), P17(225-237), P18(1220-1240), P19(1694-1735), P24(1230-1240), P25(1482-1493), P26(384-389), P27(2355-2389). The results of ELISA showed that P6(60% positive results) and P19(63% positive results) testing with PT-HC of Gu An, Hebei province were the major antigens in NS1 and in NS4 region, respectively.

[Studies on peptide. XIX: Hepatitis C virus (HCV) immune selection].

HCV has characteristics of rapid variability. The amino terminus of E2/NS1 of HCV (amino acids sequence 384-414), which is hypervariable with respect to both nucleotide and amino acid sequence, has been termed the E2 HV domain or HVR1. The E2 HV domain appears to be a rapidly evolving region of the HCV genomes which may contain linear neutralizing epitopes and the E2 HV are under immune selection. For further studies of the immunogenicity on E2 HV of HCV, we selected three peptide fragments from the full length of E2 HV region sequence and synthesized them with SPPS method. The amino acid sequences are shown as following: P2: VDGDTHVTGGAQAKTTNR (381-398); P9: STHVTGAVQGHSIRGTTSLFTSGPAQKIQ (384-412); P10: RTYTSGGTAGHTTSGITSLFSPGASQKIQ (384-412). From the results of ELISA and anti-peptide Abs of rabbit sera, we conclude that there are anti-E2 HV Abs in immune host but they could not neutralize HCV, so these Abs were not reactive with all E2 HV epitopes that resulted in immune selection of escape mutants; the anti-E2 HV Abs, probably from the same genotype, contained some common structure in which E2 HV epitopes react with other anti-E2 HV Abs at 30%: the C-terminus of E2 HV region (398-412) caused the immune response to rabbits.

Hepatitis C virus RNA and antibodies among blood donors in Beijing.

Blood units from voluntary as well as commercial donors in Beijing, China, were tested for hepatitis C virus RNA and antibodies, and for serological markers of hepatitis B virus infection. HCV RNA was detected less frequently in 1909 voluntary donors (5 (0.3%)), than in 1017 commercial donors (58 (5.7%)) (p < 0.001). Antibody to hepatitis C virus was detected by the second-generation enzyme immunoassay in 55 (87%) of 63 blood units with viremia. Evidence of present or past infection with hepatitis B virus was common both in voluntary (43.9%) and commercial (46.4%) donors. There were eight (13%) sera with HCV-RNA in which hepatitis C virus antibodies were not detectable by second-generation enzyme immunoassay. Of 63 HCV-RNA samples from donors, 33 (52%) were of genotype II, 18 (29%) of III and one (2%) of II + III. HCV-RNA in the remaining 11 (17%) were not classifiable into any of the genotypes I, II, III, IV and V. Genotype II was more frequent in viremic donors with elevated alanine aminotransferase levels (13/18 or 72%) than in those with normal levels (20/45 or 44%). These results indicate a low prevalence of hepatitis C virus infection in the general population in Beijing, and the limitations of identifying sera with viremia by second-generation enzyme immunoassay.

Prevalence, genotypes, and an isolate (HC-C2) of hepatitis C virus in Chinese patients with liver disease.

China has not been extensively investigated for the prevalence of hepatitis C virus (HCV) infection among people with or without liver disease. We analyzed serum from 2,177 liver disease patients from 7 cities in different areas of China. Of 435 acute hepatitis patients, only 11% were positive for HCV RNA, while hepatitis B surface antigen (HBsAg) was detected in 33%. Of 1,668 patients with chronic liver disease, 14% and 74% were positive for HCV RNA and HBsAg, respectively. Nearly 80% of non-B chronic liver disease were negative for HCV RNA. The frequency of HCV RNA in chronic liver disease was significantly higher in Hami (32%) and Shenyang (30%) than in other cities (6-12%). The HCV genotype distribution varied by region. Genotype III was detected in 46-70% of HCV infections in Hami, Shenyang, and Lanzhou, while more than 90% of patients from southern cities (Nanjing, Nanning, and Chengdu) had genotype II. No evidence for genotype I or IV infections was found. A full-length HCV genome sequence (HC-C2) derived from a Beijing patient with genotype II was closely related to previous isolates from Japanese and Taiwanese patients. These results suggest that HCV prevalence and genotype distribution vary from region to region in China, and that the HCV now predominant in China may have evolved epidemiologically with infections in Japan and Taiwan. The study identified a high frequency of non-B, non-C chronic liver disease in China, suggesting possibly a new agent or infections with extreme variants of HCV.

[Typing on 5'-terminal noncoding region of hepatitis C virus genome with restrict endonuclease].

We reported typing of the 5'-terminal noncoding region of HepCV genome in 219 patients with hepatitis C using restrict endonuclease. These patients came from different areas of China. The infective percentage of HCV type II was 83%, type III 14%, and mixed type II and III (II/III) 2%. It is indicated that type III and II/III. The constituent rate of HCV subgenome type in different areas was not significantly different.

Preliminary report on seroepidemiology of HCV and HBV infection in northern China.

1 141 serum samples from various population groups in north China were examined for C100-3Ab by ELISA. Antibody to C100-3 antigen derived from HCV genome (C100-3A) and HBsAg were measured in 438 normal population in Beijing. The C100-3Ab positive rate was 2.1% and the HBsAg positive rate was 2.5%. There is increased occurrence with age. In 649 cases of chronic liver diseases, the HBsAg positive rate was 87.1% in chronic persistent hepatitis (CPA), 88.8% in chronic active hepatitis (CAH), 64.9% in liver cirrhosis (LC) and 67.3% in hepatocellular carcinoma (HCC). The C100-3Ab positive rate was 10.5% (CPH), 12.1% (CAH), 42.6% (LC) and 38.4% (HCC). It is noteworthy that the C100-3Ab positive rate significantly increased with disease progression from CPH to CAH, LC and HCC. Prevalence of cases positive for both C100-3Ab and HBsAg was 0% in the normal population, 6.7% in CPH, 8.4% in CAH, 31.1% in LC and 28.8% in HCC. Investigation of patients with HCV infection showed that only 36.8% had blood transfusions. HCV and HBV infection may play important pathogenic roles in CPH, CAH, LC and HCC in north China.

[Studies on polypeptides. IX. Synthesis and antigen specificity of pre-S region fragments of hepatitis B surface antigen].

Seven peptide fragments of HBsAg Pre-S region have been synthesized by liquid phase and solid phase methods. All peptide fragments were linked to protein carrier by carbodiimide and glutaraldehyde methods. The antigen specificity of the peptide--protein conjugates was assayed by anti-HBsAg polyclonal antibodies and anti--"a" monoclonal antibody. P1 and P5 were found to have higher antigen specificity.

Similarity and diversity in sequence of HCV genome among Chinese, Japanese and American strains.

To estimate the homology between Chinese HCV strain, Japanese HCV strain and American HCV strain, we isolated and sequenced 8 clones, representing a 277-long fragment of high diversity. Chinese HCV strain was found to be homologous only in 68.6-72.6% for nucleotide sequence and in 69.6-73.9% for amino acid sequence to American HCV strain, but in 83.4-88.4% for nucleotide sequence and in 78.3-88.0% for amino acid sequence to Japanese main strain. The isolated strain may be the main strain in China, which can be divided into several substrains. This result is believed to be of paramount importance for the development of HCV detection method and vaccination as well as for the study on pathogenesis of HCV infection.

Seroepidemiology of HCV and HBV infection in northern China.

The HCVAb positive rate in normal population in Beijing was 2.1% and HBsAg positive rate was 2.5%. There is an increasing tendency in the aged group. Plasmapheresis might have been the major cause of HCV transmission in blood donors in the Hebei area. There was a high prevalence of HCVAb and HBsAg in chronic liver diseases in the Beijing area and the HCVAb-positive rate significantly increased corresponding to disease progression.

Investigation of anti-HCV in 391 serum samples in China.

391 serum samples from healthy blood donors, HBsAg positive blood donors and patients with different kinds of liver diseases in northern China were tested for anti-HCV by ELISA. Of the healthy donors, 4.6% (7/151) were anti-HCV positive; of the HBsAg positive donors, 32.4% (24/74) were positive; and of the liver disease patients, 5.4% (9/166) were positive. Among the 24 cases of HBV-HCV double infection, 10 were assayed for serum ALT level. 40% (4/10) of them were abnormal. The causes of the surprisingly high rate of HCV infection, both in healthy donors and in HBsAg positive donors, were discussed. This is the first report on the distribution of anti-HCV in certain population in China.