Hepatitis C virus (HCV) infects millions of people worldwide and is one of the leading causes of liver damage. Infection with HCV is strongly correlated with an increased risk of steatosis, or fatty liver disease, which is caused by a build-up of fat deposits in hepatocytes. All genotypes of HCV appear to cause some degree of steatosis in approximately 50% of infected individuals, especially in the presence of contributing host factors such as diabetes, obesity and alcoholism. However, approximately 70% of genotype 3a infections exhibit steatosis. Furthermore, successful clearance of the genotype 3a virus results in eradication of the steatosis, suggesting the genotype 3a virus may be able to directly cause steatosis.<p> Research suggests a role for the core protein of HCV, which forms the capsid of the virus, in the alteration of lipid metabolism pathways during infection. As such, I hypothesized that: 1) HCV alters lipid metabolism pathways and causes the build up of lipid in hepatocytes and the development of steatosis; 2) HCV-3a core protein has a differential or increased effect on these pathways in comparison to 1b core protein; and 3) other HCV proteins could also play a role in the altering of lipid metabolism. My research characterized the subcellular localization on lipid droplets of the HCV-3a core protein in comparison to HCV-1b core protein. It was found that HCV-3a core causes increased transcriptional activity from the Fatty Acid Synthase (FAS) promoter, an important enzyme involved in the synthesis of triglycerides in hepatocytes. In addition, one specific amino acid of HCV-3a core was determined to be partially responsible for this effect. Further research determined that the effect of HCV-3a core on FAS was dependent on the transcription factor Sterol Response Element Binding Protein-1 (SREBP-1) and the presence of HCV-3a core increased the processing and activity of SREBP-1. HCV core was also able to increase activity of Akt 1 and Akt2; inhibition of Akt activity resulted in decreased SREBP-1 activity thereby indicating that HCV core partially mediates SREBP-1 via Akt. Further experiments examined the role of another HCV protein, NS2, in these same lipid metabolism pathways. NS2 was also able to increase transcription from the FAS promoter via SREBP-1, suggesting that this HCV protein may also be important in the development of steatosis during HCV infection.<p> The evidence provided in these studies shows a very important role for HCV in altering lipid metabolism during infection that may lead to the development of steatosis. Current research suggests that the SREBP-1 pathway may be critical in the life cycle of the virus and these studies have provided important information on how lipid metabolism pathways are being changed by the virus. Hopefully this work can help identify potential treatment options for HCV that can slow down disease progression by preventing the development of steatosis or by decreasing viral replication.

El virus de l’hepatitis A (HAV), el prototip del gènere Hepatovirus, té una sèrie de característiques que el diferencien de la resta de membres de la família Picornaviridae, entre aquestes l’existència d’un únic serotip del virus, a pesar de que replica seguint una dinàmica de quasiespècies. La falta de correlació entre la variabilitat a nivell de nucleòtids i a nivell d’aminoàcids fa pensar que l’HAV presenta unes fortes constriccions estructurals i biològiques a nivell de la càpsida. Cal tenir en compte que el virus ha de superar el pH àcid de l’estómac en la fase d’entrada, l’eliminació de sang en la fase virèmica i l’acció de proteases i detergents, especialment sals biliars, en la fase de sortida. Per fer front al pH àcid, les proteases i les sals biliars, l’HAV disposa d’una càpsida altament estable i cohesiva. Respecte l’eliminació de sang, l’HAV s’uneix a la glicoforina A dels eritròcits, la qual s’ha descrit que pot actuar com a receptor esquer interaccionant amb els patògens i impedint que arribin al seu teixit o òrgan diana, però la interacció és ineficient a pH fisiològic. Com que aquesta interacció és òptima a pH àcid, es creu que l’HAV ha adoptat una conformació de la càpsida que li permet evadir la interacció amb l’eritròcit i evitar així ser eliminat de sang. Per tant, aquest seria un factor que contribuiria a la baixa variabilitat antigènica de la càpsida, ja que els mutants amb canvis d’aminoàcids en la regió d’unió a la glicoforina A podrien ser més fàcilment eliminats de sang. De fet, en aquest treball s’ha observat com una única mutació en aquesta regió genera un mutant amb una capacitat de replicació in vitro idèntica a la del virus parental però amb una reducció del fitness in vivo pel fet de presentar una cinètica d’eliminació en sang més ràpida. Una altra característica única de l’HAV és que presenta un ús de codons altament deoptimitzat i en certa manera antagònic al de la cèl•lula. Com a conseqüència, el virus utilitza molts codons rars, que s’aparellen amb tRNAs poc abundants, ja que utilitza com a codons rars els codons que són rars per la cèl•lula però també els que la cèl•lula utilitza freqüentment, i que, per tant, no estan disponibles per al virus. Aquesta estratègia li permet minimitzar la competència que ha d’establir amb la cèl•lula hoste pels tRNAs, ja que no posseeix d’un mecanisme específic per induir shut-off de la síntesi proteïca cel•lular. S’ha demostrat que els codons rars tenen un paper important en la regulació de la cinètica de traducció, ja que causen pauses del ribosoma degudes a la dificultat de trobar el tRNA correcte, que es troba en concentracions baixes. Aquestes pauses permeten el correcte plegament de les proteïnes, que té lloc de forma co-traduccional. En el cas de l’HAV, la importància de mantenir les pauses de la traducció per assegurar el correcte plegament de les proteïnes de la càpsida és tal que quan es feia replicar el virus en condicions de shut-off cel•lular artificial, el virus re-deoptimitzava l’ús de codons, al contrari del que s’esperaria en aquestes condicions de major disponibilitat de tRNAs. En aquest treball s’ha estudiat la relació entre les pèrdues i recuperacions del fitness del virus en les diferents condicions de shut-off cel•lular i els canvis en la conformació de la càpsida, els quals afectaven l’estabilitat i la eficiència amb que aquesta interaccionava amb el receptor i/o desencapsidava. Durant l’adaptació a aquestes condicions, els canvis en l’ús de codons que experimentava el virus li permetien recobrar un plegament de la càpsida per a recuperar la interacció amb el receptor o augmentar la eficiència de desencapsidació. A més, les poblacions adaptades a les condicions de shut-off cel•lular presentaven un increment de la infectivitat específica i una replicació més ràpida que el virus parental. Tot plegat ens dona una idea de com la càpsida de l’HAV s’adapta a colls d’ampolla imposats pel seu cicle biològic. / Hepatitis A virus (HAV) is a hepatotropic member of the Picornaviridae family. A single serotype exists, in spite of similar nucleotide diversity to that of the other picornaviruses. This discrepancy between nucleotide and amino acid variability suggests capsid structural and biological constraints. For the development of an infection cycle, HAV has to overcome the challenges posed by the acid pH of the stomach and the action of intestinal proteases and detergents (particularly biliary salts) during the entry phase, the decoy factors during the viremic phase, and again the action of proteases and detergents during the exit phase. HAV binds erythrocytes through the interaction with glycophorin A, however hemagglutination at physiological pH is highly inefficient. As erythrocyte glycoproteins may function as decoy receptors, attracting pathogens and keeping them away from target tissues, the actual virion conformation could represent an escape mechanism from blood clearance due to inefficient erythrocyte binding at physiological pH. The G1217D mutant, with a single mutation in the glycophorin A binding site, binds more efficiently to erythrocytes than the parental strain. In a rat model, it is eliminated from serum more rapidly and consequently reaches the liver with a certain delay compared to the parental strain, suggesting a low fitness phenotype which could explain the lack of natural antigenic variants of the glycophorin A binding site. Another unique characteristic of HAV is the codon usage. HAV don’t induce cellular shut-off and has adopted a deoptimazed codon usage, in order to reduce the competence for the cellular tRNAs. A consequence of this deoptimazed codon usage is an increase of the number of rare codons used by HAV. Adaptation of HAV to replicate in changing conditions of artificially induced cellular shut-off, results in dynamic adjustments of codon usage and in alternate capsid conformations which in turn influence the effectiveness of the initiation of the replicative cycle and the capsid stability. The adaptation processes also involve significant increases in virus specific infectivity and enable the selection of fast growing populations. Fine-tuning translation kinetics selection, i.e., the proper combination of abundant and rare codons in order to get a controlled translation speed and a proper capsid folding plays a critical role in HAV evolution during adaptation in conditions of cellular shut-off. These results suggest and adaptation of the capsid to bottlenecks during the replication cycle.

Dendritic cells (DCs) function mainly as antigen presenting cells (APCs) and as such they play a significant role in activating the adaptive immune system. Dendritic cells express toll-like receptors (TLR), and when these receptors are engaged by their cognate agonists, they promote DC maturation, which is critical in the induction of potent T helper (Th) cell -1 responses. Due to the multifunctional abilities of DCs, they have been explored as vaccine carriers, largely in cancer immunotherapy and some infectious diseases including hepatitis C. Previous studies showed that DCs loaded with mRNA of hepatitis C virus (HCV) antigen(s) induced strong immune responses but immune protection was not complete. Therefore, I expected that adoptive transfer of DCs transfected with HCV NS3/4A and/or NS5A mRNA and further treated with TLR agonist(s) ex vivo would induce HCV-specific immunity in vivo. Bone marrow-derived DCs generated with Flt3L (FL-DCs) or GM-CSF (GM-DCs), and loaded with HCV NS3/4A and/or NS5A mRNA showed maturation characteristics and produced substantial amounts of IL-12 after ex vivo activation with CpG ODN or CpG ODN plus Poly I:C, when compared to their untreated counterparts. Treatment with a combination of CpG ODN and Poly I:C synergized to augment IL-12 production in comparison with stimulation with CpG ODN alone. IL-12 secretion by DCs is pivotal in directing immune responses towards a Th1-bias response, which is needed to eliminate HCV. However, the ex vivo responses of stimulated DCs bearing HCV antigen(s) were not efficiently translated in mice to potentiate vigorous antigen-specific T cell responses. This resulted in a lack of protection after challenge with recombinant vaccinia virus expressing HCV NS3/NS4/NS5 in immunized mice. In contrast, both antigen-specific humoral and cell-mediated immune responses were induced in mice vaccinated with HCV recombinant NS3 or NS5A protein co-formulated with CpG ODN, host defense peptide and polyphosphazene. These responses, however, did not mediate viral clearance, as vaccinated mice remained unprotected from infection with recombinant vaccinia virus expressing HCV antigens. Taken together, these results suggest HCV recombinant protein co-formulated with triple adjuvant to be a better vaccine candidate than the DC-based vaccine.

Hepatitis C virus (HCV) infects millions of people worldwide and is one of the leading causes of liver damage. Infection with HCV is strongly correlated with an increased risk of steatosis, or fatty liver disease, which is caused by a build-up of fat deposits in hepatocytes. All genotypes of HCV appear to cause some degree of steatosis in approximately 50% of infected individuals, especially in the presence of contributing host factors such as diabetes, obesity and alcoholism. However, approximately 70% of genotype 3a infections exhibit steatosis. Furthermore, successful clearance of the genotype 3a virus results in eradication of the steatosis, suggesting the genotype 3a virus may be able to directly cause steatosis.<p> Research suggests a role for the core protein of HCV, which forms the capsid of the virus, in the alteration of lipid metabolism pathways during infection. As such, I hypothesized that: 1) HCV alters lipid metabolism pathways and causes the build up of lipid in hepatocytes and the development of steatosis; 2) HCV-3a core protein has a differential or increased effect on these pathways in comparison to 1b core protein; and 3) other HCV proteins could also play a role in the altering of lipid metabolism. My research characterized the subcellular localization on lipid droplets of the HCV-3a core protein in comparison to HCV-1b core protein. It was found that HCV-3a core causes increased transcriptional activity from the Fatty Acid Synthase (FAS) promoter, an important enzyme involved in the synthesis of triglycerides in hepatocytes. In addition, one specific amino acid of HCV-3a core was determined to be partially responsible for this effect. Further research determined that the effect of HCV-3a core on FAS was dependent on the transcription factor Sterol Response Element Binding Protein-1 (SREBP-1) and the presence of HCV-3a core increased the processing and activity of SREBP-1. HCV core was also able to increase activity of Akt 1 and Akt2; inhibition of Akt activity resulted in decreased SREBP-1 activity thereby indicating that HCV core partially mediates SREBP-1 via Akt. Further experiments examined the role of another HCV protein, NS2, in these same lipid metabolism pathways. NS2 was also able to increase transcription from the FAS promoter via SREBP-1, suggesting that this HCV protein may also be important in the development of steatosis during HCV infection.<p> The evidence provided in these studies shows a very important role for HCV in altering lipid metabolism during infection that may lead to the development of steatosis. Current research suggests that the SREBP-1 pathway may be critical in the life cycle of the virus and these studies have provided important information on how lipid metabolism pathways are being changed by the virus. Hopefully this work can help identify potential treatment options for HCV that can slow down disease progression by preventing the development of steatosis or by decreasing viral replication.

<p>HDV antigenomic RNA is processed in two distinct pathways; it can be cleaved at the polyA site and polyadenylated to become mRNA for the delta antigens, or the RNA can be cleaved by the antigenomic ribozyme to become full-length antigenomic RNA that is used for synthesis of genomic HDV RNA. The polyA site is located just 33 nucleotides upstream of the ribozyme cleavage site. If processing occurs primarily at the upstream polyA site, there may not be enough full-length antigenomic RNA to support replication. On the other hand, ribozyme cleavage downstream of the polyA site could inhibit polyadenylation by interfering with polyadenylation complex assembly. Thus, it appears that HDV may need a mechanism to control RNA processing so that both products can be generated in the proper amounts during the infection cycle. </p><p>A model has been proposed in which the choice between ribozyme cleavage and polyadenylation is determined by alternative RNA secondary structures formed by the polyA sequence (Wadkins and Been 2002). One of the hypothetical structures, AltP2, is a pairing between part of the upstream polyA sequence and the 3' end of the ribozyme sequence. For this model, the same upstream sequence that forms AltP2 could also form a stem loop, P(-1), within the leader, by pairing with sequences located farther upstream. A processing choice is possible because AltP2 is predicted to inhibit ribozyme cleavage and favor polyadenylation resulting in mRNA production, whereas P(-1) would inhibit polyadenylation and favor ribozyme cleavage resulting in full-length replication product. </p><p>The P(-1) vs. AltP2 model was tested using an antigenomic HDV ribozyme construct with the 60-nucleotide sequence upstream of the ribozyme cleavage site. This leader sequence contains the proposed polyA sequence elements. In vitro analysis of this construct revealed that the kinetic profile of ribozyme self-cleavage was altered in two ways. Relative to the ribozyme without upstream sequences, the fraction of precursor RNA that cleaved decreased to about 50%, but the active ribozyme fraction cleaved faster. Native gel electrophoresis revealed that the active and inactive precursor RNAs adopted persistent alternative structures, and structure mapping with Ribonuclease T1 and RNase H provided evidence for structures resembling P(-1) and AltP2.</p><p>Sequence changes in the 5' leader designed to alter the relative stability of P(-1) and AltP2 increased or decreased the extent of ribozyme cleavage in a predictable way, but disrupting AltP2 did not completely restore ribozyme activity. The analysis of deletion and base change variants supported a second alternative pairing, AltP4, formed by the pyrimidine-rich sequence immediately 5' of the ribozyme cleavage site and a purine-rich sequence from the 5' side of P4. A similar approach was used to test if the effect of disrupting both AltP2 and AltP4 might be additive, and the results suggested that ribozyme precursors with 5' leader sequences could fold into multiple inactive conformations, which can include, but may not be limited to, AltP2, AltP4, or a combination of both.</p><p>Luciferase expression constructs with HDV polyA and ribozyme sequences were used to investigate the effects of RNA structure and ribozyme cleavage on polyadenylation in cells. One hypothesis was that P(-1) could inhibit polyadenylation by making the polyA sequence elements less accessible to polyA factors, but sequence changes designed to alter the stability of the stem loop had no effect on polyadenylation. The model also predicts that the ribozyme sequence downstream of the polyA site could affect polyadenylation, possibly in two different ways. Ribozyme cleavage could interfere with polyadenylation by uncoupling transcription from processing, however, the ribozyme sequence might also influence polyadenylation in a manner independent of the ribozyme cleavage activity. As such, the AltP2 structure could potentially have a positive effect on polyadenylation either by inhibiting ribozyme cleavage or by making the polyA signal sequences more accessible to the polyA factors. To distinguish between the effects of ribozyme cleavage and alternative RNA structures, luciferase expression levels from constructs with an HDV polyA sequence followed by the active wild-type ribozyme or the inactive C76u version of the ribozyme were compared. For the wild-type HDV polyA sequence, the active ribozyme reduced expression, whereas the inactive ribozyme control had no effect on expression. However, for the modified leader sequences, which were efficiently polyadenylated in the absence of ribozyme, there were changes in expression that appeared to be independent of ribozyme cleavage. Based on these findings, two alternative models are proposed. One model predicts that protein factors might affect antigenomic RNA processing, and the other model suggests that additional alternative structures, such as AltP4, might influence the choice between ribozyme cleavage and polyadenylation.</p> / Dissertation

This dissertation, studies two specific topics related to the research of computer-aided drug design(CADD) by employing the molecular simulations approach, that of protein drugs and that of small agents. These results can help drug designers to improve their products for treating special diseases. This work is divided into two parts: Protein drugs: Potential of mean force of the hepatitis C virus core protein¡Vmonoclonal 19D9D6 antibody interaction: Antigen-antibody interactions are critical for understanding antigen-antibody associations in immunology. To shed further light on this question, we studied a dissociation of the 19D9D6-HCV core protein antibody complex structure. However, forced separations in single molecule experiments are difficult, and therefore molecular simulation techniques were applied in our study. The stretching, that is, the distance between the centre of mass of the HCV core protein and the 19D9D6 antibody, has been studied using the potential of mean force calculations based on molecular dynamics and the explicit water model. Our simulations indicate that the 7 residues Gly70, Gly72, Gly134, Gly158, Glu219, Gln221 and Tyr314, the interaction region (antibody), and the 14 interprotein molecular hydrogen bonds might play important roles in the antigen-antibody interaction, and this finding may be useful for protein engineering of this antigen-antibody structure. In addition, the 3 residues Gly134, Gly158 and Tyr314 might be more important in the development of bioactive antibody analogues. Potential of mean force for syrian hamster prion epitope protein - monoclonal fab 3f4 antibody interaction studies: Simulating antigen-antibody interactions is crucial for understanding antigen-antibody associations in immunology. To shed further light into this question, we study a dissociation of syrian hamster prion epitope protein-fab3f4 antibody complex structure. The stretching (the distance between the center of mass of the prion epitope protein and the fab3f4 antibody) have been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) and implicit water model. For the complex structure, there are four important intermediates and two inter protein molecular hydrogen bonds in the stretching process. Inclusion of our simulations may help to understand the binding mechanics of the complex structure and will be an important consideration in design of antibodies against the prion disease. Potential of mean force for human lysozyme - camelid vhh hl6 antibody interaction studies: Calculating antigen-antibody interaction energies is crucial for understanding antigen-antibody associations in immunology. To shed further light into this equation, we study a separation of human lysozyme-camelid vhh hl6 antibody (cAb-HuL6) complex. The c-terminal end-to-end stretching of the lysozyme-antibody complex structures have been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) and explicit water model. For the lysozyme-antibody complex, there are six important intermediates in the c-terminal extensions process. Inclusion of our simulations may help to understand the binding mechanics of lysozym- cAb-HuL6 antibody complex. Small agents: Predictions of binding for dopamine D2 receptor antagonists by the SIE method: The control of tetralindiol derivative antagonists released through the inhibition of dopamine D2 receptors has been identified as a potential target for the treatment of schizophrenia. We employed molecular dynamics simulation techniques to identify the predicted D2 receptor structure. Homology models of the protein were developed on the basis of crystal structures of four receptor crystals. Compound docking revealed the possible binding mode. In addition, the docking analyses results indicate that five residues (Asp72, Val73, Cys76, Leu183, and Phe187) were responsible for the selectivity of the tetralindiol derivatives. Our molecular dynamics simulations were applied in combination with the solvated interaction energies (SIE) technique to predict the compounds' docking modes in the binding pocket of the D2 receptor. The simulations revealed satisfactory correlations between the calculated and experimental binding affinities of all seven tetralindiol derivative antagonists, as indicated by the obtained R2 value of 0.815. Combining homology modeling, docking, and molecular dynamics to predict the binding modes of oseltamivir, zanamivir, and Chinese natural herb products with the neuramindase of the H1N1 influenza A virus: The neuraminidase of the influenza virus is the target of the anti-flu drugs oseltamivir and zanamivir. Clinical practices show that zanamivir and oseltamivir are effective to treat the 2009 H1N1 influenza virus. Herein, we report the findings of molecular simulations for zanamivir, oseltamivir, and Chinese natural herb products with the neuramindase of the 2009 H1N1 influenza. Our approach theoretically suggests that the Glu278 residue is responsible for the neuramindase of the 2009 influenza drug selectivity.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

Generally Hepatitis C Virus tropism is limited to hepatocytes. This limited tropism is a result of the receptors HCV requires for cellular entry and other host cellular factors including, uniquely, a liver specific miRNA, miR-122. The relationship between HCV and miR-122 is interesting, as commonly, miRNA are associated with suppression of function, but in the case of HCV, miR-122 actively promotes HCV proliferation. In-depth studies have demonstrated that miR-122 along with the RNA induced silencing complex (RISC) protein Argonaute 2 (Ago2) binds directly to two seed sequences separated by 8-9 nucleotides on HCV 5’UTR. Binding to the 5’UTR results in an increase in viral replication and translation. The method by which miR-122 promotes HCV translation and replication is not fully understood but evidence suggests that part of the function of miR-122 is to stabilize the HCV genome and protect it from exonuclease degradation by Xrn1, but other mechanisms remain to be identified. The reliance of HCV on miR-122 is best exemplified by the fact that removal of miR-122 by a miR-122 antagonist drastically impedes HCV viral titers in Chimpanzees and humans with no indication of escape mutants. The observation that HCV augmentation of the HCV life cycle by miR-122 requires Ago2 suggests that other components downstream in the miRNA suppression pathway may also be part of the mechanism of action. Our studies focused specifically on the processing body (p-body) associated DEAD-box helicase DDX6. DDX6 is essential for p-body assembly, required for robust miRNA suppression activity and elevated in HCV associated hepatocellular carcinomas. As such we hypothesized that DDX6 and p-bodies were directly or in-directly associated with the mechanism of action of miR-122. Knocking down DDX6 with siRNA indicated that DDX6 augments both HCV replication and translation. To examine whether DDX6 augmentation of HCV replication was related to the effects of miR-122 on the HCV life cycle, HCV replication and translation were assessed in the presence or absence of miR-122 when DDX6 was knocked down. Our data indicated that HCV replication and translation were augmented equally by miR-122 whether DDX6 was present or not. Our data also demonstrated that HCV replication and translation that was occurring independent of miR-122 was also still affected by DDX6 knockdown. Taken together our observations strongly suggest that the role DDX6 has on HCV is independent of HCV and miR-122’s relationship. In order to better understand miR-122’s relationship with HCV, we hypothesized that targeting the miR-122 binding region with siRNA would inhibit HCV replication initially, but that over the course of several rounds of treatment with the same siRNA, HCV would mutate to escape the siRNA, producing escape mutants that replicate without a dependency on miR-122. These escape mutants could be evaluated on how they replicate without using miR-122, shedding light on miR-122 and HCV’s relationship. Conversely if no escape mutants arose the siRNA could be further studied as a potential therapeutic for HCV. siRNA designed to target the miR-122 binding region inhibited HCV replication, confirming that the designed siRNAs could access the miR-122 binding region and function as an siRNA. Interestingly, when the siRNAs were used against a replication competent HCV RNA having a single nucleotide mutation in the first miR-122 binding site, instead of abolishing siRNA knockdown, two of the siRNA showed enhanced inhibition activity. The target sequences of these siRNAs spanned both miR-122 binding sites and we speculate that their inhibitory activity was due to competition for miR-122 binding to site 2. This observation indicates that siRNA targeting the miR-122 binding region have dual activity, by siRNA induced cleavage, and as a competitive inhibitor of miR-122 binding. Selection for viral escape mutants of the miR-122-binding site targeting siRNAs revealed viral RNAs having mutations within the miR-122 binding sites, in the surrounding region, and to other areas within the HCV IRES. The mutant viruses will be used to assess the influence of miR-122 binding site mutations on HCV replicative fitness, and to determine if the virus can evolve to replicate independent from augmentation by miR-122.

Hepatitis C virus (HCV) is a small single stranded RNA virus, that belongs to the Flaviviridae family. HCV can be classified into six major genotypes and more than 50 subtypes.