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Occult hepatitis C infection: viruses with infectious potential in Huh7.5 and MDBK cell lines suggest HCV/OCI transmission


Pharmacy & Pharmacology International Journal
Eliane Silva,1,2 Sara Marques,1 Hugo Osorio,3-5 Bernardo Canhao,6,7 Joao Madaleno,6,7 Adelia Simao,6,7 Armando Carvalho6,7

Abstract

Background: Occult hepatitis C infection (OCI) is characterized by the detection of HCVRNA in patient’s peripheral blood mononuclear cells (PBMCs) without detection in serum. We aim to evaluate the capacity of viruses presented in OCI patients to infect and replicate in Huh7.5 (human hepatoma–derived Huh7 cell line) and in naïve Mardin-Darby Bovine Kidney (MDBK) cell lines questioning HCV/OCI transmission.
Methods: Huh7.5 cells were infected with serum and PBMCs samples of 4 OCI patients. Naïve MDBK cells were infected with the same serum samples. Huh7.5 and naïve MDBK cells were also infected and reinfected, with an inoculum of naïve MDBK cell cultures previously infected with the same 4 serum samples, respectively. All infected/reinfected Huh7.5 and MDBK cell cultures were screened for HCV/OCI-RNA detection by droplet digital-PCR (ddPCR). MDBK cell cultures infected with the serum samples were evaluated for HCV/OCI specific proteins by proteomic liquid chromatography-mass spectrometry (LC-MS) analysis. Huh7.5/infected and MDBK/reinfected cell cultures with the inoculum serum samples were also evaluated for the E2, NS3 and NS4a HCV/OCI specific regions by immunogold electron microscopy (IEM) analysis.
Results: HCV/OCI-RNA was detected in Huh7.5 cell cultures infected with serum and PBMCs samples of the OCI patients 4 and 3, respectively, and in Huh7.5/infected and MDBK/reinfected cell cultures with the inoculum serum samples of the 4 OCI patients by ddPCR. HCV specific proteins peptides were detected in MDBK cell cultures (first infection) infected with 1 serum sample by LC-MS. HCV specific immunostaining was shown in Huh7.5 cell cultures infected with PBMCs samples of the OCI patients’ 3- NS3 and 4-NS4a, and in MDBK cell cultures reinfected with inoculum serum sample of the OCI patient 4-E2 and -NS4a by IEM analysis.
Conclusions: Infectious potential of HCV/OCI viruses was demonstrated in Huh7.5 cell cultures infected with serum and PBMCs samples of the 4 OCI patients. Infectious potential of HCV/OCI viruses was also demonstrated in MDBK cell cultures infected with the serum samples, as well as, in Huh7.5/infected and MDBK/reinfected cell cultures with the inoculum serum samples. Overall results suggest HCV/OCI transmission, and could contribute for a better understanding of the HCV/OCI life cycle, infection and transmission.

Keywords

HCV, OCI, Huh7.5 cell line, MDBK cell line, HCV/OCI transmission, Droplet digital PCR, LC-MS, IEM

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