E2 GLYCOPROTEIN OF GENOTYPE Ⅲ CHINESE ISOLATES OF HEPATITIS C VIRUS EXPRESSED IN MAMMALIAN CELL AS ANTIGEN FOR ANTI-E2 ANTIBODY DETECTION

Expression vector inserted with E2/NS1 gane derived from ganotype Ⅲ Chinese isolates of HCV was transfected into mammalian cells to express E2 glycoprotein. Expressed protein was used as antigen for anti-E2 antibody detection in 19 cases of hepatitis C patients by Western blot. It was first to express E2 glycoprotein of genotype Ⅲ Chinese hepatitis C virus isolates. For anti-E2 detection, 14 cases of patients were positive of antibodies against E2(73.7%). These results indicated that E2 glycoprotein expressed in mammalian cells had good immunoganicity and cross reactivity to serum infected with genotype Ⅱ Chinese hepatitis C virus isolates.     

Clinical application and analysis of hepatitis C virus NS3 antigen detection by ELISA in human serum

Background Hepatitis C virus （HCV） core antigen assays have been produced to exclude infectious donations collected during the preseroconversion window phase （PWP）. For the same purpose, we evaluated the specificity and sensitivity of a novel hepatitis C virus NS3 antigen detection immunoassay and the application of this assay in clinical diagnosis. Methods Samples from 77 healthy subjects, 173 anti-HCV positive patients and 3708 hepatitis patients other than HCV positive were tested with the HCV NS3 antigen assay. Some HCV NS3 antigen positive samples were further validated with HCV-RNA, neutralization and immunodot assays. Twenty-five sequential samples from 11 HCV NS3 antigen positive patients were subjected to kinetic study. Results Only 48 （1.3%） of 3708 anti-HCV negative samples were positive for HCV NS3 antigen. Among them, 44 of 3030 samples from patients only infected with HBV were HCV NS3 antigen positive, 4 of the 445 samples from patients infected with other type hepatitis were HCV NS3 antigen positive. In addition, 42 （24.3%） of 173 anti-HCV positive samples were HCV NS3 antigen positive and all 77 samples from healthy subjects were negative to HCV NS3 antigen assay. Of the 15 HCV NS3 antigen positive samples, 9 （60%） were HCV-RNA positive. The neutralization and positive percentage of immunodot assay for 23 HCV NS3 antigen positive sera were 87.0% （20/23） and 69.6% （16/23） respectively. Of the 25 sequential samples from 11 HCV NS3 antigen positive patients, there was a negative correlation between the OD values and the duration of test （r=-0.989, P〈0.05）, and there were correlations among their HCV NS3 antigen, HCV-RNA and anti-HCV Utres. The anti-HCV antibodies of two sera were detected while their OD values of HCV NS3 antigen decreased gradually. Conclusions The HCV NS3 antigen detection assay showed perfect specificity and high sensitivity. Thus, it would be useful and economical as a routine test in laboratories for early diagnosis of HCV infection and prevention.     

Efficacy of hepatitis B immunoglobulin in relation to the gene polymorphisms of human leukocyte Fcγ receptor III (CD16) in Chinese liver transplant patients

Background Although the use of hepatitis B immunoglobulin (HBIG) may lead to a significant reduction in recurrent hepatitis B virus (HBV) infection and improve the survival of patients who have undergone liver transplantation (LT) for hepatitis B-related diseases, the recurrence of the disease still remains at a lower level. Different clinical curative effects were observed in patients with the same HBV-related diseases and the same therapy. This study was undertaken to investigate whether the efficacy of HBIG is associated with FCGR3A gene polymorphisms in Chinese liver transplant patients.Methods Altogether 77 patients who had received liver transplantation for hepatitis B-related diseases with more than one-year survival after surgery were studied. The recurrence of HBV was characterized by the appearance of HBsAg in serum after the operation. The FCGR3A genotyping was performed using genomic DNA sequencing (ABI 3037). Single nucleotide polymorphism at nucleotide 559 was detected by Polyphred. Results Of the 77 patients, 14 were complicated with HBV recurrence post-transplant. The FCGR3A at nucleotide 559 TT was observed in 35 (45.5％) subjects, whereas TG in 31(40.3％) and GG in 11(14.3％). In the 559G carrier group (n=42, 54.5%), the risk of HBV recurrence was 9.5%, and 1- and 2-year recurrence-free survival rates were 95.2% and 88.7%, respectively. In the 559G noncarrier group (n=35, 45.5%), the risk of HBV recurrence was 28.6%, and 1- and 2-year recurrence-free survival rates were 74.3% and 69.3%, respectively. The risk of HBV recurrence and the recurrence-free survival rate were both statistically different between the 559G carrier and noncarrier groups (P<0.05).Conclusions A single nucleotide polymorphism（T/G）at position 559 of the FCGR3A gene was found in Chinese patients. The efficacy of HBIG in prophylaxis of HBV recurrence after LT is associated with the gene polymorphism, so detecting FCGR3A genotypes can be a clinical reference of the HBIG administration.     

EFFECTS OF HBV preS AS A HUMORAL ENHANCER ON THE ABILITIES OF HCV E2 PROTEIN TO INDUCE IMMUNE RESPONSESIN THE DNA-IMMUNIZED MICE

Objective. To study whether the abilities of hepatitis C virus (HCV) E2 gene immunization to induce humoral and cellular immune responses to E2 protein were affected by hepatitis B virus (HBV) preS gene when they were fused in DNA-immunized mice.Methods. Mice were immunized with E2, preS-E2(preS gene was upstream of E2 gene), and E2 - preS (preS gene was downstream of E2 gene)gene by their eukaryotic expression vectors, respectively. The anti - E2 or anti - preS antibodies were detected using the E2 and preS antigens. The cellular immune response to E2 protein in immunized mice was presented by its survival time after injecting SP2/O myeloma cells expressing HCV E2 protein into the abdominal cavity.Results. Chimeric E2 and preS gene immunization can induce mice to develop anti-preS and anti-E2 antibodies. The number of the mice developing anti-E2 antibody and the antibody titers in preS-E2 gene-injected group were higher than those in E2-preS gene-immunized group. However, the mice injected with E2 gene     

Detection of HBV and HCV Coinfection by TEM with Au Nanoparticle Gene Probes

Gold(Au) nanoparticle HBV DNA or HCV cDNA gene probes were prepared and were used to detect HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection directly by transmission electron microscopy(TEM).PCR identifying HBV and HCV in serum of patients with HBV and HCV coinfection was established.Alkanethiol-modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA or HCV cDNA gene probes through covalent binding of Au-S.HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection was added to the detection system composed of nanoparticle HBV DNA and(or) HCV cDNA gene probes.The results showed that HBV DNA and HCV RNA could be specifically amplified by PCR.The zones of DNA amplification appeared in 431 bp and 323 bp respectively.When HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection were added to the detection system,TEM displayed the nanoparticles self-assembled into large network aggregates.It was concluded that the detection of HBV and HCV coinfection by TEM was convenient and efficient with high specificity and sensitivity.     

Relationship of hepatitis C virus infection with primary hepatic carcinoma: an investigation of serum HCV RNA in different population groups

A preliminary study on the serum hepatitis C virus(HCV)RNA in 182 pa-tients with different chronic liver diseases and 35 blood donors was carried out with“nested”polymerase chain reaction.It was found that serum HCV RNA was detected in36.6%(34/93)cases of primary hepatic carcinoma(PHC),31.7%(20/63)liver cirrhosis,15.4%(4/26)chronic hepatitis and 2.9%(1/35)blood donors;most of the HCV RNA pos-itive cases(41/58)were complicated with HBV replicating markers;the rate of single posi-tive for HCV RNA was 15.1%(14/93)in PHC,3.2%(2/63)in liver cirrhosis,and 3.8%(1/26)in chronic hepatitis.These findings imply that about 20% of PHC cases appear tobe related to the coinfection of HCV and HBV and 15% of PHC cases are related to sin-gle HCV infection.     

The preliminary efficacy of interferon-α and ribavirin combination treatment of chronic hepatitis C in HIV-infected patients

Background It is internationally accepted that in drug-naYve individuals with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co-infection, chronic hepatitis C should be treated first if the CD4 cell count does not require the initiation of anti-retroviral therapy. Present paper evaluated the clinical effect and side-effect of interferon-α (IFN-α) and ribavirin (RBV) combination therapy for Chinese patients with HCVHIV co-infection, and compared with them for HIV infection alone. Methods Ten patients with HCV-HIV and 17 patients with HCV received 5 million unit IFNα-2b every other day intramuscularly, and 300 mg RBV triple daily by oral. Dynamic observations were made for HCV RNA and HIV RNA loads, CD4^ and CD8^ T lymphocyte counts, liver function and blood cell measurement, and the medicine side-effects.Results After 12-week and 24-week treatments of IFN-α and RBV combination therapy, mean HCV RNA levels reduced 1. 14 logs and 1.56 logs from the baseline at week 0 in HCV-HIV co-infection, and reduced 1.48 logs and 1.75 logs in HCV infection, respectively. The HIV RNA levels decreased 1.22 logs and 1.32 logs from the base line; however, there were no obvious different changes at T lymphocyte counts of HCV-HIV and HCV patients through 24-week treatments. Whole 27 patients showed satisfactory biochemical response to therapy. There were some mild or mediate influence-like symptoms, intestinal uncomfortable and depressed blood cell counts in early stage of the treatments. No neuropsychiatric and auto-immune disorders were found. Conclusions IFN-α and RBV combination therapy had similar anti-HCV effects during 24-week treatment for HCV-HIV and HCV infected Chinese patients, and some anti-HIV effect. There were no obvious different biochemical responses and side-effects between two groups above.     

Single-strand conformation polymorphism for analysis of genomic variability of hepatitis C virus nonstructure 5A region

OBJECTIVE To establish a convenient method to detect the genomic population with hepatitis C virus (HCV) at nonstructure 5A (NS5A) region and to determine the correlation between the genomic population complexity at NS5A region and disease stage.

METHODS The sera from 52 patients with chronic hepatitis C virus infections were analysed using single strand conformation polymorphism (SSCP). In the SSCP, an asymmetric polymerase chain reaction (PCR) was carried out on the 455 bp products of the first round PCR at the NS5A region and the number of band of single strand deoxyribonucleic acid (DNA) which reacted with complemental DNA probe specific for the NS5A region after gel electrophoresis was analyzed.

RESULTS In 90% patients with chronic persistent hepatitis, the bands of single strand DNA was limited to one, and in those with chronic active hepatitis or liver cirrhosis, two or more bands of DNA were frequently detected. In about half of patients with hepatocellular-carcinoma, three or more bands were found. The number of bands increased with the progression of liver disease. The multivariate analysis showed that the progression of liver disease was the independent factor of viral diversity (P < 0.025) and was not related to the age, sex, the route of infection and the titer of hepatitis C virus ribonucleic acid (HCV RNA).

CONCLUSIONS These results suggest that the genomic variability of HCV at NS5A region increases with the progression of liver disease, and this may be closely related to the clinical features of type C liver disease.

     

Preliminary research on the co-infection of human immunodeficiency virus and hepatitis virus in intravenous drug users

Objective To confirm the close relationship of high co-infection rate between HIV and hepatitis virus in intravenous drug users (IVDUs).Methods Anti-HIV, HBV and HCV were detected by ELISA in the serum from 35 scattered and 15massed IVDUs. PCR and RT-PCR were performed to confirm the infection of HIV, HBV, HCV, HGVand TTV among the 15 massed intravenous drug abusers.Results Among the 50 IVDUs, the positive rates of anti-HCVl HBsAg, anti-HBe and anti-HBc were 92% (46/50), 12% (6/50), 10% (5/50) and 66% (33/50), respectively. In the samples of HBsAg positive, their HBeAg was also positive. Although the positive rate of serum markers was different in the massed IVDUs compared to the scattered IVDUs, no significant difference was shown.In the cases of massed IVDUs, the positive rates of HIV DNA, HBV-DNA, HCV-RNA, HGV-RNA,and TTV-DNA were 100% (15/15), 26. 6% (4/15), 53. 3% (8/15), 33. 3% (5/15) and 26. 6%(4/15), respectively. Among the 15 massed intravenous drug users, one was infected with HIV,HBV, HCV, HGV and TTV; two were infected with HIV, HBV, HCV and HGV; three were infected only with HIV; and the remaining had other forms of co-infection.Conclusion The co-infection rate of HIV, HBV, HCV, HGV and TTV in intravenous drug users is very high.     

Effects of the Ganning Formula (肝宁方) on Liver Fibrosis in Patients With Chronic Hepatitis B

Objective: To investigate the clinical efficacy and safety of the Ganning formula (肝宁方) for the treatment of liver fibrosis in patients with chronic hepatitis B. Methods: In a multicenter, randomized, controlled clinical trial, 150 patients with liver fibrosis secondary to hepatitis B virus (HBV) infection were randomly assigned in equal numbers to receive either the Ganning formula (a Chinese herbal decoction; active treatment group) or oral entecavir (control group) for two 3-month courses. Patients were ...     

RANTES gene single nucleotide polymorphisms and expression in patients with chronic hepatitis B virus infection

Background Regulated on activation, normal T-cell expressed and secreted (RANTES) plays a critical role in T-lymphocyte activation and proliferation. The process is involved in both acute and chronic phases of inflammation. The present study was to ascertain the possible correlations between chronic hepatitis B virus (HBV) infection and the RANTES gene polymorphisms and their expression. Methods The study included 130 HBV negative healthy donors and 152 patients with chronic hepatitis B (CHB) virus infection. The polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLPs) were used to detect RANTES gene single nucleotide polymorphisms (SNPs). RANTES levels in the platelet depleted plasma were detected by enzyme linked immunosorbent assay (ELISA). Results RANTES alleles -403G, -28C and In1.1T were the predominant alleles in the subjects studied. No significant correlation was found between CHB infection and the RANTES alleles, while a significant correlation was found between CHB infection and increased RANTES expression in platelet depleted plasma (P&lt;0.05). Conclusions SNPs in RANTES gene do not affect chronic HBV infection or the outcome of interferon-α treatment in patients positive for HBV “e” antigen (HBeAg+). However, patients with CHB infection express the higher levels of plasma RANTES, which is thus associated with CHB infection.     

Cloning and sequencing of core gene cDNA of Chinese hepatitis C virus

A hepatitis C virus(HCV)cDNA fragment,534bp in length and designated asQ534,was obtained by PCR amplification with self-designed primers.Q534 was cloned in-to Hinc Ⅱ site of pUC18 and the recombinant plasmid pQ534 was then selected from thebacterial transformants.The sequence analysis indicated that Q534 was a cDNA fragmentof HCV core gene,and located in HCV genome from positions 320 to 853 incorrespondence with Chiron's prototype sequence.The homologies between Q534 and theprototype at the levels of nucleotides and amino acids were 90.0% and 97.6%,respectively.The homologies of Q534 with Japanese HCV-J and HCV-BK strains were 96.6% and97.0% at the nucleotide level,and 98.2% and 98.8% at the amino acid level.In terms ofthe sequence,this Chinese HCV isolate should belong to HCV group Ⅱ.     

Bosentan and antiretroviral therapy in an HIV/HBV/HCV coinfected Chinese patient with HIV-related pulmonary arterial hypertension

To the editor:Pulmonary arterial hypertension (PAH) is an infrequent but nevertheless,serious life threatening complication of human immunodeficiency virus (HIV) infection.People infected with HIV have a high prevalence of chronic hepatitis B virus (HBV) and/or hepatitis C virus (HCV).Bosentan is effective in patients with HIV-related PAH (HRPH);1 but hardly used on Chinese patients so far.In this rare case,we present successful,long-term coadministration of bosentan and antiretroviral therapy in an HIV/HBV/HCV coinfected Chinese patient with HRPH.     

Prevalence of antibodies to hepatitis C virus in Chinese patients with viral hepatitis and hepatic failure.

Anti-HCV assay with ORTHO kits was done in 100 blood donors and recipients and 374 cases of viral hepatitis, including 65 cases of fulminant, subacute and chronic hepatic failure. None of the 100 blood donors and recipients showed positive anti-HCV response. Anti-HCV was positive in 7.6% of the patients with chronic persistent hepatitis, 9.7% of the patients with chronic active hepatitis and 23.1% of the patients with liver cirrhosis. High prevalence of anti-HCV was observed in subacute hepatic failure (60.8%) and chronic hepatic failure (53.9%). Fifty-two (83.9%) of 62 anti-HCV positive cases were infected concurrently with HBV. The incidence of HBV replicating marker in patients with HCV or co-infected with HBV was lower than that of those with HBV alone. It is suggested that HCV might inhibit the replication of hepatitis B virus. The mortality rate of patients with anti-HCV positive hepatic failure was higher than that of those with HBV infection. Therefore, anti-viral therapy for anti-HCV positive hepatic failure should be considered.

     

Extrahepatic manifestations of chronic hepatitis C virus infection: 297 cases from a tertiary medical center in Beijing, China

Background Chronic hepatitis C virus （HCV） infection can affect multiple organ systems and cause a variety of extrahepatic manifestations （EMs）.We sought to assess the constituent ratio of EMs in Chinese patients with chronic HCV infection and identify the clinical and biological factors associated with EM.Methods The medical records of 297 patients with chronic HCV infection were analyzed and demographic and epidemiological information was collected.The diagnosis of chronic HCV infection was based on positive anti-HCV combined with a positive HCV-RNA or at least two times of elevated aminotransferases attributable to HCV infection.Patients with HBV and/or HIV coinfection,autoimmune hepatitis,and history of alcohol abuse were excluded.Results Sixty-two percent （184/297） of the patients had at least one EM,including fatigue （29.4％）,type 2 diabetes mellitus （28.2％）,renal involvement （12.5％）,lymphadenopathy （9.6％）,fever （9.4％）,thyroid dysfunction （8.1％）,and arthralgia （7.4％）.Neuropathy,sicca syndrome,B-cell lymphoma,Raynaud＇s phenomenon,and lichen planus were rare.The mean age of patients with EM was older compared with those without EM.Conclusions EMs were common in Chinese patients with chronic HCV infection,particularly fatigue,type 2 diabetes,renal impairment,lymphadenophy,fever,and thyroid dysfunction.Older age was associated with EMs.     

Expression of toll-like receptor 9 in peripheral blood mononuclear cells from patients with different hepatitis B and C viral loads

The aim of the present study was to investigate the expression of toll-like receptors （TLR） 9 in peripheral blood mononuclear cells （PBMC） of patients with chronic hepatitis B and C with different virus copies. The study group included 90 patients （60 with chronic hepatitis B, and 30 with chronic hepatitis C）, and 20 healthy people served as control group. The protein and mRNA levels of TLR9 were detected by using flow cytometry and real-time PCR. The serum viral copies of HBV and HCV were measured in all patients, and the correlation between HBV-DNA copies or HCV-RNA copies and the TLR9 expression was analyzed. Our results demonstrated that HBV or HCV infection led to a decreased expression of TLR9 mRNA and protein compared to the control group （P〈0.05）. The TLR9 protein and mRNA levels were negatively correlated with serum viral copies of HBV and HCV （r=-0.632, r=-0.909, P〈0.01）. It was concluded that TLR9 mRNA and protein are down-regulated in PBMC of HBV-infected or HCV-infected patients, and they are negatively correlated with serum viral copies and play an important role in detecting viral replication of HBV and HCV.     

Allele polymorphisms of interleukin-lO and hepatitis B,C virus infection

Background Interleukin 10 （IL-10） is an important cytokine with anti-inflammatory, anti-immune and anti-fibrotic functions. This study aimed at evaluating the relationship between allele polymorphisms in the IL-10 promoter region and hepatitis B virus （HBV） or hepatitis C virus （HCV） infection. Methods The odds ratios （ORs） of IL-10 allele distributions in patients with HBV or HCV infection were analyzed against healthy controls. All the relevant studies in PubMed were identified, and poor qualified studies were excluded. The meta-analysis software REVMAN 4.2 was applied for investigating heterogeneity among individual studies and summarizing all the studies. The publication bias was also evaluated. Results This study demonstrated a significant association between the IL-10-592 A/C polymorphism and HBV infection in the Asian population under the overall effect size of allele A versus C. In our subgroup meta-analysis, we found a significant association of IL-10-592 A/C polymorphism to HCV infection susceptibility in Asian populations, although sensitivity analysis showed that the combined result was not associated with the worldwide population. Other IL-10 allele polymorphisms were not associated with HBV or HCV infection. Conclusion IL-10-592 A/C allele might be a risk factor for HBV or HCV in Asians but not in Europeans.