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• W3124451844 abstract "Background: Hepatitis C virus (HCV) infection is associated with increased morbidity and mortality worldwide. In Cameroon, HCV infection is characterized by a greater genetic diversity that could represent significant threat and challenges for successful treatment and response to potential vaccine. Effective therapeutics have been developed against HCV isolates circulating in the western countries; however, it is under-described whether these therapies are equally efficient on strains circulating in sub-Saharan Africa. Furthermore, whether African isolates replicate in cell culture to produce infectious virions like the Japanese fulminant hepatitis (JFH) is unknown. This study aimed at developing infectious HCV clones from Cameroon clinical isolates, and testing infectivity of the given strains in cell culture. Methods & Materials: Clinical HCV isolates were transferred from Cameroon to Yale University, US, for molecular characterization, cloning and cell culture. To confirm the genotypes, the HCV capsid and NS5B genes were amplified by RT-PCR, nested and semi-nested PCR respectively. PCR products were resolved in 1.5% agarose gel electrophoresis, purified, cloned into a TOPO vector. Clones were used to transform chemically competent DH5-a cells. Plasmid DNA obtained was used for DNA sequencing, and the nucleotide sequences were analyzed using the Geneious 12.2.3 software. Consensus/FASTA sequences were used for genotyping and phylogenetic analysis. Clinical isolates/viral RNA were used to transfect Huh7.5 cells, and the HCV infectivity assay/anti-NS5A staining was performed. The median tissue culture infectious dose (TCID50/ml) was determined and the growth curves plotted. Results: Successfully analyzed HCV isolates were shown as belonging to genotypes 1 and 4 (subgenotypes 1b & 4a); these are virulent HCV genotypes and sub-genotypes. The anti-NS5A antibodies’ staining performed using the infected Huh7.5 cells was slightly pronounced compared to the positive control, Jc1 wild type – GLuciferase (WT-GLuc). The growth curves show that HCV replicate very fast between 24 and 48 hours’ time points, so that the TCID50/ml reaches the maximum, but there is a decrease after 48 h. Conclusion: Virulent HCV genotypes circulate in Cameroon confirming the chronic infection noticed in the country. The determination of the optimal time points for HCV replication (24–48 h) is critical for vaccine development, preclinical drug testing, and basic virology research." @default.
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• W3124451844 date "2020-12-01" @default.
• W3124451844 modified "2023-09-27" @default.
• W3124451844 title "Enabling HCV replication in cell culture: An approach for vaccine and drug development" @default.
• W3124451844 doi "https://doi.org/10.1016/j.ijid.2020.09.1325" @default.
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