A novel phenotypic assay of hepatitis B virus polymerase with extensive site-specific mutagenesis

Dear Editor,A common reason for drug failure during long-term treatment of chronic hepatitis B with nucleot(s)ide analogues(NUCs)is the emergence of drug resistance(Das et al.,2001).Most primary NUCs-resistant mutations identified in clinical samples have been limited to a minority of amino acids(usually less

2D-QSAR Studies on Anthranilic Acid Derivatives: A Novel Class of Allosteric Inhibitors of Hepatitis C NS5B Polymerase

Quantitative structure activity relationship （QSAR） studies were performed on 45 anthranilic acid derivatives for their potent allosteric inhibition activities of HCV NSSB polymerase. Genetic algorithm based genetic function approximation （GFA） method of variable selection was used to generate the model. Highly statistically significant model with r^2 = 0.966 and r^2cv = 0.951 was obtained when the number of descriptors in the equation was set to 5. High r^2pred value of 0.884 indicates the good predictive power of the best model. Spatial descriptors of radius of gyration （RadOfGration）, molecular volume （Vm）, length of molecule in the z dimension （Shadow-Zlength）, thermodynamic descriptors of the octanol/water partition coefficient （LogP） and molecular refractivity index （MR） showed enormous contributions to HCV NS5B polymerase inhibition. The validation of the model was done by leave-one-out （LOO） test, randomization tests and external test set prediction. The model gives insight on indispensable structural requirements for the activity and can be used to design more potent analogs against HCV NSSB polymerase.

Efficient detection of pathogen virus in sand dabs,Paralichthys olivaceus using loop-mediated isothermal amplification(LAMP)

Viral hemorrhagic septicemia virus（VHSV） and marine birnavirus（MABV） are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the prevalence of pathogen infection. The loop-mediated isothermal amplification（LAMP） of DNA is currently one of the most commonly used molecular diagnostic tools, as it is simple, quick, and easy to amplify target DNA under isothermal conditions. In the present study, a novel and highly specific LAMP assay for the sensitive and rapid detection of VHSV and MABV infection in fish was developed. Using a set of synthesized primers matching a specific region of the genome, the efficiency and specificity of the LAMP assay were optimized in terms of the reaction temperature and DNA polymerase concentration, as they are the main determinants of the sensitivity and specificity of the LAMP assay. In particular, we demonstrated that our assay could be applied to efficient detection of VHSV and MABV infection in the wild fish, Paralichthys olivaceus. Our results demonstrate the simplicity and convenience of this method for the detection of viral infection in aquatic organisms.

Expression and semi-quantification of hepatitis B virus reverse transcriptase protein in a prokaryotic system

The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus （rt306P） and the polymerase gene from a mutant, with rt306P substituted by serine （rtP306S） were separately fused to the maltose binding protein （MBP） gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Westem blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.

Current progress in the treatment of chronic hepatitis C

Over the last decade, the standard of care for the treat- ment of chronic hepatitis C has been the combination of pegylated-interferon-alfa （PEG-IFN） and ribavirin （RBV） which results in sustained virological response （SVR） rates of 75%-85% in patients with genotypes 2 or 3 but only of 40%-50% in patients with genotype 1. Cur- rently, there are rapid and continuous developments of numerous new agents against hepatitis C virus （HCV）, which are the focus of this review. Boceprevir and tela- previr, two first-generation NS3/4A HCV protease inhibi- tors, have been recently licensed in several countries around the world to be used in combination with PEG- IFN and RBV for the treatment of genotype 1 patients. Boceprevir or telaprevir based triple regimens, com- pared with the PEG-IFN/RBV combination, improve the SVR rates by 25%-31% in treatment-naTve genotype 1 patients, by 40%-64% in prior relapsers, by 33%-45% in prior partial responders and by 24%-28% in prior null responders. At the same time, the application of response-guided treatment algorithms according to the on-treatment virological response results in shortening of the total therapy duration to only 24 wk in 45%-55% of treatment-na＇ive patients. There are, however, several challenges with the use of the new triple combinations in genotype 1 patients, such as the need for immediate results of HCV RNA testing using sensitive quantitative assays, new and more frequent adverse events （anemia and dysgeusia for boceprevir; pruritus, rash and anemia for telaprevir）, new drug interactions and increasing dif- ficulties in compliance. Moreover, the SVR rates are still poor in very difficult to treat subgroups of genotype 1 patients, such as null responders with cirrhosis, while there is no benefit for patients who cannot tolerate PEG- IFN/RBV or who are infected with non-1 HCV genotype. Many newer anti-HCV agents of different classes and numerous combinations are currently under evaluation with encouraging results. Preliminary data suggest that the treatment of chronic HCV patients with well toler- ated combinations of oral agents without PEG-IFN is feasible and may lead to a universal HCV cure over the next 5-10 years.

The Paper Symposium on PLC VIRAL HEPATITIS（HBV AND HCV）BACKGROUND IN CHINESE PATIENTS WITH HEPATOCELLULAR CARCINOMA

Antibody to hepatitis C virus (AntiHCV) was detected using antiHCV EIA (Abbott Kit) in the sera of Chinese patients with hepatocellular carcinoma (HCC), acute hepatitis, chronic hepatitis and blood donors, the positive rates being 10.4% (61/586),11. 8% (10/85),19.2% (44/229), ana 1. 9% (3/160) respectively. HBV DNA was detected by polymerase chain reaction (PCR) in sera from 61 HCC patients with positive antiHCV, the positive rate for HBV DNA being 55.7% (34/61),which was lower than those with negative antiHCV (78.7%) , 413/525). These results indicate that in Chuia the role of HBV infection in the causation of HCC seems to be more important than that of HCV infection.

Detection of herpes simplex virus type 1 in rheumatic valvular tissue

Background Rheumatic heart disease (RHD) is the most important sequela of rheumatic fever (RF): evidence that streptococcal infection is aetiological is prominent, but sometimes contradictory. Acute HSV-1 infection in mouse leads to carditis and valvulitis whereas recurrent infection results in inflammatory granulomatous lesions that resemble Aschoff bodies. Cells containing HSV-1 inclusions or virus infected giant cells appear similar to Anitschkow cells or Aschoff cells respectively. We hypothesized that HSV-1 infection also may be involved in RHD. Methods Formalin-fixed, paraffin-embedded valvular tissue samples from 32 patients with RHD were investigated for evidence of HSV-1 infection. HSV-1 antigen was detected by immunohistochemistry, using HSV-1-specific monoclonal and polyclonal antibodies. HSV-1 glycoprotein D gene sequences were amplified by nPCR, using β-globin gene amplification in the same samples as internal control. Valvular tissue from 5 cases of sudden death and 3 cases died of neisseria meningitis without a history of valvular disease was used for comparison. HSV-1-infected lung tissue was used as positive control. Results HSV-1 antigens were detected in valvular tissues from 21 of 32 (65.6%) patients. Fifteen of these 21 (46.9% of cases), but no antigen-negative sample, were positive also for HSV DNA. Nucleotide sequence of PCR products was homologous to the targeted region of the HSV-1 glycoprotein D gene. HSV-1 antigen was present also in one case of sudden death but viral DNA was not found in any tissue sample from the comparison group. Results from reagent and positive controls were as anticipated.Conclusions This is the first study to show the presence of HSV-1 antigen and genomic DNA in valvular tissues from patients with RHD and provides evidence for an association of HSV-1 infection with some cases of rheumatic valvular disease.