Use of immuno-dominant epitope derived from genotype 4 as a diagnostic reagent for detecting the antibodies against Hepatitis E Virus.

BACKGROUND: Despite the genotype 4 has become the dominant cause of hepatitis E disease in China, none antigen derived from genotype 4 of hepatitis E virus (HEV) was used in current commercial anti-HEV immunoassay, and the serological reactivity of antigen derive from genotype 4 is not well-charactered. METHODS: We expressed and purified the 4 main immuno-dominant epitopes derived from genotype 1 and 4 including ORF2 (410-621aa) of genotype 4, ORF3 (47-114aa) of genotype 4, ORF2 (396-606aa) of genotype 1 and ORF3 (56-123aa) of genotype 4. RESULTS: The ORF2 of genotype 4 displayed good diagnostics performance according to ROC analysis using in-house panel, and the immunoassays based the ORF2 of genotype 4 was then developed to detect the anti-HEV IgG antibodies and evaluated further in 530 anti-HEV IgG positive specimens and 380 negative specimens. The sensitivity and the specificity is 98.1% (520/530) and 94.7% (360/380) for immunoassay based on ORF2 of genotype 4, 96.6% (512/530) and 92.6% (352/380) for commercial immunoassay based on genotype 1. It is noted that all of the positive samples will be detected by combing two assays together. The anti-HEV immunoassays based on genotype 4 are in accordance with Chinese anti-HEV national standard,and show an good agreement of 95.8% with commercial assay (kappa=0.913, P=0.014). CONCLUSIONS: The immunoassay based on ORF2G4 displays good performance, and combining assay based on genotype 1 together with genotype 4 will benefit the HEV diagnosis in large scale samples.

Evaluation of cross-reactive antibody response to HVR1 in chronic hepatitis C.

AIM: To evaluate the presence and cross-reactive antibodies against hypervariable region 1 (HVR1) in hepatitis C virus (HCV) infected patients and its relationship with the progression of the disease. METHODS: Sixteen representative HVR1 proteins selected from a unique set of 1600 natural sequences were used to semiquantitate the cross-reactivity of HVR1 antibodies in the sera of HCV patients. Fifty-five chronic HCV patients including 23 with asymptomatic mild hepatitis, 18 with chronic hepatitis and 16 with liver cirrhosis patients were studied. RESULTS: The degree of the cross-reactivity of anti-HVR1 antibodies in 23 patients with mild asymptomatic hepatitis was 3.09 ± 2.68, which was significantly lower than in those with chronic hepatitis (5.44 ± 3.93, P < 0.05) and liver cirrhosis (7.44 ± 3.90, P < 0.01). No correlation was observed between the broadness of the cross-reactivity anti-HVR1 antibodies and patient's age, infection time, serum alanine aminotransferase activity, or serum HCV-RNA concentration. It was the breath of cross-reactivity rather than the presence of anti-HVR1 antibody in HCV sera that was associated with the progression of liver disease. CONCLUSION: The broadly cross-reactive HVR1 antibodies generated in natural HCV patients can not neutralize the virus, which results in persistent infection in patients with chronic hepatitis.

Induction of an oxalate decarboxylase in the filamentous fungus Trametes versicolor by addition of inorganic acids.

In order to improve yields and to reduce the cost of oxalate decarboxylase (OxDC, EC 4.1.1.2), the induction of OxDC in the white-rot fungus Trametes versicolor was studied in this work. OxDC was induced by addition of inorganic acids including hydrochloric acid, sulfuric acid, and phosphoric acid to culture media. The results showed that all the acids could enhance OxDC expression. The activity of the acid-induced OxDC rose continuously. All of the OxDC volumetric activities induced by the inorganic acids were higher than 20.0 U/L and were two times higher than that obtained with oxalic acid. OxDC productivity was around 4.0 U*L(-1)*day(-1). The highest specific activity against total protein was 3.2 U/mg protein at day 8 after induction of sulfuric acid, and the specific activity against mycelial dry weight was 10.6 U/g at day 9 after induction of hydrochloric acid. The growth of mycelia was inhibited slightly when the pH values in culture media was around 2.5-3.0, while the growth was inhibited heavily when the pH was lower than 2.5.

[Preparation of recombinant IL-1 fusion protein as the substrate for HCV NS3 serine protease].

AIM: To prepare recombinant IL-1 fusion protein as the substrate for the HCV NS3 serine protease. METHODS: NS5A-B gene fragment (2,412-2,427aa) synthesized by PCR was subcloned into prokaryotic expression vector pBVIL1 to fuse with IL-1 gene and the recombinant vector pBVIL1/NS5A-B was transformed into E.coli strain HB101. The fused protein was induced to express at 42degreesCelsius and purified by two-step column chromatography. The proteolysis of the purified IL-1 fusion protein catalyzed by NS3 serine protease was analyzed with SDS-PAGE, Western blot and ELISA. RESULTS: NS5A-B fragment gene was correctly subcloned into pBVIL1 vector and the fusion protein was expressed as inclusion body in transformed HB101 cells. The recombinant fusion protein can be cleaved into smaller fragments by NS3 protease. CONCLUSION: The recombinant fusion protein can be cleaved by NS3 serine protease successfully and specifically, suggesting that it can be used as a surrogate substrate of NS3 serine protease in searching for inhibitors of this protease.

Cross-reactivity of hypervariable region 1 chimera of hepatitis C virus.

AIM: To analyze the amino acid sequences of hypervariable region 1 (HVR1) of HCV isolates in China and to construct a combinatorial chimeric HVR1 protein having a very broad high cross-reactivity. METHODS: All of the published HVR1 sequences from China were collected and processed with a computer program. Several representative HVR1's sequences were formulated based on a consensus profile and homology within certain subdivision. A few reported HVR1 mimotope sequences were also included for a broader representation. All of them were cloned and expressed in E.coli. The cross-reactivity of the purified recombinant HVR1 antigens was tested by ELISA with a panel of sera from HCV infected patients in China. Some of them were further ligated together to form a combinatorial HVR1 chimera. RESULTS: Altogether 12 HVR1(s) were selected and expressed in E.coli and purified to homogeneity. All of these purified antigens showed some cross-reactivity with sera in a 27 HCV positive panel. Recombinant HVR1s of No. 1, 2, 4, and 8# showing broad cross-reactivities and complementarity with each other, were selected for the ligation elements. The chimera containing these 4 HVR1s was highly expressed in E.coli. The purified chimeric antigen could react not only with all the HCV antibody positive sera in the panel but also with 90/91 sera of HCV -infected patients. CONCLUSION: The chimeric antigen was shown to have a broad cross-reactivity. It may be helpful for solving the problem caused by high variability of HCV, and in the efforts for a novel vaccine against the virus.