OBJECTIVETo construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells.

METHODSUsing pSG5/M-H05-5/4A as the template (A1-1) and primers designed according to the typing criteria, 4 single point mutation plasmids, namely pSG5/M-H05-5(A1-2)/4A(A1-2) (Y56F), pSG5/M-H05-5(B1-1)/4A(B1-1) (L80Q), pSG5/M-H05-5(B2-1)/4A(B2-1) (V51A), and pSG5/M-H05-5(B2-2)/4A(B2-2) (S61A), were constructed. With A1-2, B2-1, and B2-2 as the templates, the leucine to glutamine mutation at position 80 (L80Q) was induced to construct another 3 double point mutation plasmids pSG5/M-H05-5(B1-2)/4A(B1-2), pSG5/M-H05-5(A2-1)/4A(A2-1), and pSG5/M-H05-5(A2-2)/4A(A2-2), respectively. DNA sequencing was performed for confirmation of the mutations. Huh 7 cells were transfected with the constructs using FuGene 6 transfection reagents. Indirect immunofluorescence staining and Western blotting were used to detect the expression of the constructs.

RESULTSIndirect immunofluorescence assay revealed 4 subcellular localization patterns of NS3 protein, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blotting also demonstrated successful expression of the constructs and weak in cis and in trans NS3 serine protease activities of subtypes A2-1 and B2-1 in comparison with other subtypes.

CONCLUSIONThe point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal are constructed successfully, which provides the basis for further study of different subtypes of HCV.

Amino Acid Sequence ; Cell Line ; Gene Expression ; Genetic Engineering ; methods ; Hepacivirus ; enzymology ; Immunohistochemistry ; Intracellular Space ; metabolism ; Molecular Sequence Data ; Plasmids ; genetics ; Point Mutation ; Protein Structure, Secondary ; Protein Transport ; Viral Nonstructural Proteins ; chemistry ; genetics ; metabolism

Objective: To determine anti-viral activities of three Artocarpus species: Artocarpus altilis, Artocarpus camansi, and Artocarpus heterophyllus (A. heterophyllus) against Hepatitis C Virus (HCV). Methods: Antiviral activities of the crude extracts were examined by cell culture method using Huh7it-1 cells and HCV genotype 2a strain JFH1. The mode of action for anti-HCV activities was determined by time-of-addition experiments. The effect on HCV RNA replication and HCV accumulation in cells were analyzed by quantitative reverse transcription-PCR and western blotting, respectively. Results: The dichloromethane (DCM) extract of A. heterophyllus exhibited strong anti-HCV activity with an inhibitory concentration (IC50) value of (1.5 ± 0.6)μg/mL without obvious toxicity. The DCM extracts from Artocarpus altilis and Artocarpus camansi showed moderate anti-HCV activities with IC50 values being (6.5 ± 0.3) μg/mL and (9.7 ± 1.1) μg/mL, respectively. A time-of-addition studies showed that DCM extract from A. heterophyllus inhibited viral entry process though a direct virucidal activity and targeting host cells. HCV RNA replication and HCV protein expression were slightly reduced by the DCM treatment at high concentration. Conclusions: The DCM extract from A. heterophyllus is a good candidate to develop an antiviral agent to prevent HCV grant reinfection following liver transplantation.

Objective To determine anti-viral activities of three Artocarpus species: Artocarpus altilis, Artocarpus camansi, and Artocarpus heterophyllus (A. heterophyllus) against Hepatitis C Virus (HCV). Methods Antiviral activities of the crude extracts were examined by cell culture method using Huh7it-1 cells and HCV genotype 2a strain JFH1. The mode of action for anti-HCV activities was determined by time-of-addition experiments. The effect on HCV RNA replication and HCV accumulation in cells were analyzed by quantitative reverse transcription-PCR and western blotting, respectively. Results The dichloromethane (DCM) extract of A. heterophyllus exhibited strong anti-HCV activity with an inhibitory concentration (IC

To examine the potential risk of hepatitis B virus (HBV) spread in Indonesia by migrant workers, based on the molecular characteristics of HBV strains. Methods: Sera collected from migrant workers traveling to their destination countries (pre-migrant workers) and those returning to Indonesia (post-migrant workers) were screened for HBsAg by ELISA, followed by HBV DNA detection by PCR and (sub) genotype/subtype determination according to surface region and whole genome sequencing. Results: Of 87 pre-migrant workers, 15 (17.24%) were HBsAgpositive, whereas 15 (12.10%) of 124 post-migrant workers were HBsAg seropositive. HBV genotype analysis based on the S region showed that HBV-B3/adw2 was predominant (96.15%, 25/26) whereas 3.85% (1/26) of isolates were HBV-C3/adrq+. Whole genome sequencing of selected strains and phylogenetic tree analysis identified subgenotype B7 in three samples previously categorized as subgenotype B3 based on S region analysis, supporting a recent argument that subgenotypes B5/B7/B8/B9 could be considered as a quasi-subgenotype of B3. Conclusions: A high prevalence of HBsAg carriers was detected among migrant workers from Lombok Island, with no significant difference in prevalence between before and after returning to Indonesia. All strains were classified into genotypes common in Indonesia, and the results suggested that migrant workers are not a risk factor for HBV transmission into Indonesia.