Antiviral effect of indigofera geradiana on core protein of HCV

Abstract

Background Hepatitis C virus (HCV) disease is a progressive and fatal infection that can lead to fibrosis and Hepatocellular Carcinoma (HCC). It is a global health challenge. The Core gene of HCV is a well preserved region of the HCV genome and has the capability of interacting with an extensive range of viral proteins and cellular proteins. A close association has been noted with intra-hepatic Core gene expression levels in individuals with chronic HCV disease. There is a need to evaluate the response of new antivirals like plant-derived natural compounds for the treatment of this infection. Objectives The objectives of this study are set as to:- 1. Isolate Core gene from HCV infected patients through conventional PCR. 2. Sequence and clone Core gene. 3. Construct a mammalian expression system. 4. Evaluate the possible antiviral effect of medicinal plant (Indigofera gerardiana) extract against the Core protein of HCV in Huh-7 cell line2 Study Design/Settings This experimental study was conducted at the Institute of Basic Medical Science (IBMS), Khyber Medical University (KMU) Peshawar, Pakistan, and the Institute of Biotechnology & Genetic Engineering (IBGE), Agriculture University Peshawar (AUP) Peshawar, Pakistan. For sequencing and cloning samples were sent to the Center for Applied Molecular Biology (CAMB) Lahore. The work was approved by the ethics board of KMU at the 18th meeting via registration No.DIR/KMU EB/E1/000319. Methodology PCR confirmed HCV (genotype 3a) individuals, belonging to different parts of Peshawar, having the chronic HCV disease were included in this study. Viral load was determined by quantitative PCR. The complete Core region of HCV was amplified, sequenced, and then cloned. The attained sequencing results were submitted to Genbank in the National Center of Biotechnology Information (NCBI) and were applied for construction of phylogenetic tree using CLUSTAL-W and MEGA 6 software. For assessment of the antiviral effect of medicinal plant Indigofera gerardiana (IG) against the Core region of HCV, the extract of IG was screened on the Core gene of HCV (3a genotype) in Huh-7 cell line.3 Results Among the 421, PCR positive samples, 363 (86%) were confirmed HCV genotype 3a. Phylogenetic analysis revealed a distinct cluster of eight viral isolates in the phylogenetic tree. They were found genetically related to HCV 3a strains previously reported from Pakistan. Isolate PK/69 was found closely related to HCV 3a isolate of India. The study of translated amino acids revealed substitutions in functional domains (i.e.; D1, D2, D3) of the Core region. Among studied isolates, Core mutations R70Q (arginine to glutamine) and L/C91M (leucine/cysteine to methionine) were not detected. In the sequence PK/59 at location72, glutamic acid was substituted by aspartic acid. Position 182, in all sequences, was replaced by leucine in place of phenylalanine. Amino acid alanine and serine at positions189 and 191 were replaced with threonine and cysteine respectively. In eight of our reported isolates (Pk/59- PK/68) glutamic acid and serine were found mutated to aspartic acid and cysteine respectively. Quantitative PCR results revealed that the extract of IG by methanol has a 57% inhibition of cells infected with HCV. Conclusions HCV (3a genotype) is prevalent in the region of Peshawar, (Pakistan). In the genome of HCV, the mutation may affect protein interactions and also response to therapeutic agents. We evaluated that an extract of IG has 57% inhibitory activity against the Core protein of HCV. Further studies are strongly suggested for obtaining the importance of this plant as having antiviral activity, against HCV. Keywords Antiviral therapy, Core gene, HCV, Indigofera gerardiana (IG)