Developing a genotyping assay for HCV resistance for new direct acting antivirals

(1)

1

Developing a

genotyping assay

for HCV resistance

for new direct

acting antivirals

Division molecular diagnostics,

Department of Viroscience

Erasmus MC

Author: Iris Kluijfhout 2061171

I.Kluijfhout@student.avans.nl /

I.Kluijfhout@erasmusmc.nl

Biology and medical Research Avans

University of applied sciences, Breda

Supervised by: Jolanda Maaskant, Bsc

Dr. Suzan Pas

Version 2.1 06-06-2016

Bachelor thesis

(2)

2

Summary

Hepatitis C is a liver disease caused by hepatitis C virus that affects millions of people worldwide. This year, new medicines (direct acting-antivirals) have been approved. Until now, no HCV resistance analysis assay was available. Therefore, the primary objective of this project was to develop a genotyping assay for HCV resistance for new direct acting antivirals for the NS3, NS5a and NS5b gene regions and to determine the lower limit of detection of the assay. This project had several side questions: one of the side questions of this project was if there was any difference in sequence quality if samples were isolated with QiaSymphony or MagNA Pure LC. Another one of the side questions of this project was whether it mattered for quality of sequencing results if samples were diluted with RPMI-culture medium or negative human plasma. For the last side question the effectiveness of cDNA synthesis was compared with random hexamer primers or specific forward or reverse primers.

The primers for the project were developed according to gene region and genotype. The genotypes tested were genotype 1a, 1b, 2, 3a and 4, as they are the most commonly occurring genotypes. RNA from patient plasma samples was isolated with the QiaSymphony and/ or MagNA Pure LC. cDNA was synthesized from the isolated RNA and an outer PCR and nested PCR were performed. The PCR results were analyzed on a agarose gel and positive samples were analyzed by Sanger sequencing. For analyzing the results, software tools were utilized for the clinical interpretation of found drug resistance

associated mutations (DRMs).

The assay was successfully developed and can be used for diagnostic purposes. The lower limit of detection was determined for all genotypes and gene regions, only the NS5a and NS5b gene regions of genotype 2 did not meet the criteria for a solid assay. The side questions were also answered, it can be concluded that the QiaSymphony and MagNA Pure LC isolation methods can be used interchangeably. Comparison of dilution of samples with RPMI-culture medium or negative human plasma showed no significant difference. However it was discovered that using random hexamer primers for cDNA synthesis gives better results than using specific primers.

(3)

3

Samenvatting

Hepatitis C is een ontsteking van de lever die door het hepatitis C virus is veroorzaakt en treft miljoenen mensen wereldwijd. Dit jaar zijn er nieuwe medicijnen (direct-acting antivirals) goedgekeurd. Voor deze direct-acting antivirals is het nodig om een genotypisch assay voor HCV resistentie op te zetten. Om deze reden was het hoofddoel van dit onderzoek het ontwikkelen van een genotypisch assay voor HCV resistentie voor nieuwe direct acting antivirals, voor de NS3, NS5a en NS5b gen regio’s en het bepalen van het laagste detectie limiet van de assay. Er waren ook enkele andere onderzoeksvragen; maakt het uit voor het sequensen of samples met de QiaSymphony of MagNA Pure LC werden geïsoleerd. Een andere onderzoeksvraag was of de sequenties beter waren als de samples met RPMI medium of negatief humaan plasma werden verdund. De laatste onderzoeksvraag was of het uitmaakte voor sequensen of cDNA synthese met random hexameer of specifieke forward of reverse primers werd uitgevoerd.

De gebruikte primers zijn ontwikkeld afhankelijk van genotype en gen regio. De genotypen 1a, 1b, 2, 3a en 4 zijn getest, aangezien dat de meest voorkomende genotypen zijn. RNA van patiënt plasma samples zijn geïsoleerd met de QiaSymphony en/of MagNA Pure LC. cDNA werd gesynthetiseerd uit het

geïsoleerde RNA en een conventionele en nested PCR zijn daarop uitgevoerd. Deze PCR resultaten zijn geanalyseerd op een agarose gel en de positieve samples zijn doormiddel van sequensen geïsoleerd. Het analyseren van de resultaten voor klinische interpretatie is uitgevoerd met verschillende software voor het vinden van resistentie geassocieerde mutaties.

De assay is succesvol ontwikkeld en kan in de diagnostiek gebruikt worden. Het laagste detectie limiet van de assay is bepaald voor alle genotypen en gen regio’s, alleen de NS5a en NS5b gen regio’s van genotype 2 voldoen niet aan de criteria voor een goed assay. Alle overige onderzoeksvragen zijn ook beantwoord, er kan geconcludeerd worden dat de QiaSymphony en MagNA Pure LC isolatie methoden beide gebruikt kunnen worden. Het vergelijken van verdunningen van samples met RPMI medium of negatief humaan plasma maakte geen significant verschil voor de sequentie uitslagen. Er is echter wel ontdekt dat het gebruik van random hexameer primers leidt tot beter sequenties dan het gebruik van specifieke primers.

(4)

4

1. Introduction

Hepatitis C is a liver disease caused by the Hepatitis C virus that affects millions of people worldwide. There is no vaccine available for the Hepatitis C virus, as it has a high degree of genetic variability and mutates easily. Infection with the virus can result in death, though the death rate has decreased since more medicines have become available. This year, new medicines (direct acting-antivirals) have been approved. However, for these direct acting-antivirals, a genotyping assay for HCV resistance had to be developed. Therefore, the primary objective of this project was to develop a genotyping assay for HCV resistance for new direct acting antivirals for the NS3, NS5a and NS5b gene regions and to determine the lower limit of detection of the assay. The project itself had several side questions: one of the side

questions of this project was whether it mattered for sequencing if samples were isolated with

QiaSymphony or MagNA Pure LC. Another of the side questions of this project was whether sequences were better if samples were diluted with RPMI or negative plasma. The last side question was whether it mattered for sequencing if cDNA synthesis was performed with random hexamer primers or specific forward and reverse primers.

In-house developed primers (kindly provided by Dr. R. Molenkamp , Amsterdam Medical Centre ) were used for this project to accomplish the aforementioned objective and answer the side questions. The primers were developed according to gene region and genotype. The genotypes tested were genotype 1a, 1b, 2, 3a and 4, as they are the more commonly occurring genotypes. RNA from patient plasma samples was isolated with the QiaSymphony and/ or MagNA Pure LC. cDNA was synthesized from the isolated RNA and an outer PCR and nested PCR were performed. The PCR results were analyzed on a agarose gel and positive samples were analyzed by sequencing. For analyzing the results,(online) software tools were utilized for the clinical interpretation of found drug resistance associated mutations (DRMs).

Chapter two describes the theoretical background of topics important to the research, chapter three describes the materials and methods used to accomplish the primary objective and answer the side questions. In chapter four the results are presented and interpreted. Chapter five contains the

discussion and conclusion of the research. Chapter six contains recommendations for follow-up research and clinical use of the assay. The literature list follows chapter six. Finally, the appendix contains

information on the current state of HCV medicine, a DRM table, a list of primers and miscellaneous information.

(6)

6

2. Theoretic background

2.1 Hepatitis C

Hepatitis C is an inflammation of the liver that is caused by infection of the hepatitis C virus (HCV). Hepatitis C virus can cause a chronic and an acute hepatitis. An acute hepatitis C virus infection is defined as an infection which is cleared within six months; about 15-45% of the people infected with hepatitis C virus clear the infection within these six months. The acute infection is usually without symptoms and is cleared by the immune system. This acute infection becomes a chronic infection if it is not cleared within six months, the chronic infection can last a lifetime. 55-85% of people infected with hepatitis C virus develop a chronic infection; they have a 15-30% chance of developing liver cirrhosis within 20 years. Recent antiviral medication can cure around 90% of the infected people and so reduce the risk of death from liver cirrhosis and liver cancer. (1)

Less than 50% of the people infected with HCV develop symptoms. Most of the people that show symptoms, report tiredness, having to throw up, nausea, dark urine, jaundice and pain on the upper right side of the belly during an acute infection. People with a chronic infection usually go up to 25-30 years without any symptoms. Some people report nonspecific symptoms, such as tiredness and a general feeling of being unwell. Only when the infection has reached an advanced stage, such as severe liver cirrhosis, symptoms will occur. The symptoms will be typical of liver failure. (2)

As mentioned before, hepatitis C virus infection has several different stages. The initiation of infection comes first; the virus enters through the bloodstream. This can occur through blood to blood contact with an infected person or by having unprotected sex with an infected person. The moment the virus has entered the bloodstream, the acute phase begins. During the acute phase, HCV RNA can be detected in serum in the first two weeks after exposure to HCV, in 70% of the infected people. Subsequently, the serum alanine aminotransferase (ALT) levels begin rising 2 to 8 weeks after HCV infection. Rising ALT levels signify necrosis of the hepatocytes, the ALT levels can rise up to ten times as high as the normal levels. After the initial infection of the host the body begins to develop antibodies against HCV, which occurs 1 to 3 months after exposure. This process is depicted in figure 1. (3)

Figure 1: Course of a Hepatitis C virus infection: the ALT, anti-HCV and HCV RNA levels are shown.. In blue the stages of fibrosis and HCC are shown. Source: Natural history of chronic HCV infection (Seeff, 2002).

(7)

7 When chronic hepatitis C develops, the HCV RNA persist in the peripheral blood for at least six months after the start of infection. The rate of chronic HCV infections is affected by several factors, like age, gender, ethnicity and immune response. The infection progresses from chronic hepatitis C to fibrosis, cirrhosis, hepatocellular carcinoma (HCC) and death, this process can take decennia. (the progression of fibrosis can be seen in figure 1). During fibrosis, excessive amounts of extracellular matrix proteins (ECM), like collagen, accumulate in the liver. The number of mononuclear inflammatory cells and dead hepatocytes rises in the afflicted areas of the liver. At the end of the fibrosis stage, ECM proteins distort the liver tissue and form scar tissue, causing cirrhosis. Cirrhosis is only caused in 15% of people with a chronic HCV infection. The progression of cirrhosis is often without symptoms, until the end stage of cirrhosis. The scarring of the liver causes the liver to lose function, the more cirrhosis proceeds, the more hepatocytes are replaced with scar tissue. In the end the liver stops working altogether and can cause death. HCC can develop during cirrhosis and can lead to loss of liver function, like cirrhosis, and death. (3) (4)

2.2 Hepatitis C virus

Hepatitis C virus is an enveloped RNA virus, which belongs to the Hepacivirus genus of the Flaviviridae family. This virus has a high degree of genetic variability, but mutation rates vary per region. The genome of the HCV consists of a single stranded positive-sense RNA strand, of approximately 9.6 kb. This genome contains an open reading frame (ORF), coding for a polyprotein precursor of 3000 residues and is flanked by untranslated regions (UTRs) on both C- and N-terminal ends. This precursor protein gets cleaved into about 10 different proteins: the structural proteins core, E1, E2 and p7 and also the non-structural proteins NS2, NS3, NS4A, NS4B and NS5A and NS5B, as shown in figure 2. The structural proteins are the proteins that are expressed on the virion particle, the envelope around the viral RNA/DNA. The non-structural proteins are expressed inside the infected cells. (5)

Figure 2: Genomic organization of HCV; translation of the 3000bp region to proteins. The three structural proteins are shown in grey, the non-structural proteins in white. (5)

(8)

8

2.2.1 Genomic organization of HCV

All proteins have a different function in the hepatitis C virus, the E1 and E2 structural proteins make up the viral envelope and are the most variable proteins of HCV. The glycosylated E1 and E2 proteins are essential for viral entry of the host. E1 has a molecular weight of 30-35 kDa and E2 has a molecular weight of 70-72 kDa. (5)

It has been suggested that the C-terminal transmembrane domains of E1 and E2, along the precursor polyproteins, form hairpin structures that pass twice through the membrane. Doing this would allow processing by a signal peptide in the Endoplasmic Reticulum (ER) lumen. It is thought that the C-termini are translocated into the cytoplasm during signal peptidase cleavage, to allow for the transmembrane membrane topology of mature E1 and E2 proteins. These mature proteins are bound together in a noncovalent way, they interact through their C-terminal transmembrane domains. These domains also mediate the attachment of the E1-E2 complex in the ER. Recently, it has been demonstrated that the E1 proteins also adopts a topology in which the ER membrane is spanned twice by the E1 protein with a cytoplasmic loop. (5), (6)

The other structural protein is the HCV core protein, it is highly likely that this protein forms the viral nucleocapsid. The core protein is also essential for viral replication, maturation and pathogenesis. It also has a few regulatory functions, such as gene expression, cell transformation, lipid metabolism,

regulation of signaling pathways and apoptosis. The core protein has a very conserved amino acid sequence among different HCV strains. This core protein is mostly a 191 residue-long precursor protein of 23 kDa, and after further processing 21 kDa protein. The C-terminus of this protein is hydrophobic and the N-terminal domain very basic. The core protein can already be detected two weeks after HCV has entered a host, and is primarily detected in the cytoplasm of the cells. (5), (7)

The non-structural proteins also have some important functions, the p7 protein is the smallest HCV protein. It is only 63 residues long, and is essential for the production of infectious virus particles. It is a hydrophobic polypeptide, which has a double membrane-spanning topology. It belongs most likely to a protein family of viroporins that are known for enhancing membrane permeability. (5)

The next protein is the NS2 protein, which is a transmembrane protein of 21 to 23 kDa. It has 96

hydrophobic N-terminal residues that form three of four transmembrane helices into the ER membrane of a cell. NS2’s C-terminal part resides in the cytoplasm, where it facilitates zinc-stimulated NS2/NS3 auto protease activity, together with the N-terminal domain of NS3. The NS2 protein is not essential for viral replication, but it is essential for completion of the replication cycle, as it plays an important role in HCV particle assembly, not much else is known about NS2. (5), (8)

The NS3 protein (69 kDa) is hydrophobic. It has a serine protease encoded by the N-terminal region, which non-covalently binds to the NS4A co-factor. By binding to the NS4A co-factor, it forms the NS3-4A complex, which is a bifunctional molecule that is essential for polyprotein processing and RNA

replication. The NS4A co-factor is a 54 residue long polypeptide, of which the central region is important for processing the nonstructural proteins by NS3. The N-terminus of NS4A forms a transmembrane helix, which keeps the NS3-4A complex attached to the cellular membrane. The N-terminal region of NS3 codes for the helicase-NTPase domain, which unwinds RNA-RNA substrates. (5), (9)

The NS4B protein is very much different than the NS4A co-factor. The NS4B protein is much bigger, about 27 kDa and is an integral membrane protein. It has at least four transmembrane regions and a N-terminal amphipathic helix that is responsible for membrane association. The NS4B protein can induce the formation of a sort of membranous web, in which viral RNA replication takes place. (5)

(9)

9 The NS5 is the last gene encoded the HCV genome, which is translated into NS5A and NS5B proteins. NS5A is a phosphoprotein that is anchored into the membrane and has two forms. It has a basally phosphorylated form of 56 kDa and a hyper phosphorylated form of 58 kDa. The function of NS5A is not fully known, but it is expected to interact with viral and cellular proteins, membranes and also RNA. It is also believed to be important for viral replication, in the regulation of RNA replication. (10)

NS5B protein, is somewhat bigger than NS5A protein (68 kDa) and has a conserved motif that is characteristic for viral RNA-dependent RNA polymerase (RdRp). It is a protein that is tail-anchored and can form a transmembrane domain that is responsible for translational targeting on the cytoplasmic side of the ER. The NS5B protein is a RNA polymerase that initiates the RNA synthesis properly and inhibits extension from a primed template. (5)

2.2.2 HCV genotypes

As mentioned before, HCV is a virus with a high degree of genetic variability. Because of this genetic variability, HCV is classified into seven genotypes, with 30-35% genomic variation between the

genotypes. These genotypes have several subtypes that are based on phylogeny and sequence analyses of the whole HCV genome, having less than 15% differing nucleotide sequences. (11)

The most common HCV genotype worldwide(except for Africa) is genotype 1 and has two major subtypes: 1a and 1b. Genotype 1 has a wide geographical distribution, as it has been located in Europe, North and South America, Asia and also Australia (see figure 3). Genotype 1 is estimated to cause 46.2% of the HCV cases world-wide. Genotype 2 is estimated to cause only 9.1% of the cases and is mostly present nearby its origin: West and Central Africa. HCV genotype 3 is also one of the major genotypes; it is estimated to account for 30.1% of the cases. It is mostly prevalent in South Asia. Genotype 4 is a genotype that causes only 8.3% of the HCV cases and is prevalent in the Middle East, and especially Egypt. Genotype 5 only occurs in South Africa and no prevalence percentages are known. Genotype 6 is estimated to cause 5.4% of the HCV cases and is mostly found in South-East Asia, especially in Hong Kong and Southern China. To this day, only a few genotype 7 cases reported. The strains were all isolated from people from Central Africa. (11)

Figure 3: Relative prevalence of each HCV genotype by GBD region. source: Messina et al. 2015 Global distribution and prevalence of hepatitis C virus genotypes.

(10)

10

2.3 HCV replication cycle

The replication cycle of HCV starts with the entry of HCV into a cell, the next step in the replication cycle is translation of the HCV RNA and polyprotein processing. Hereafter the HCV genomic RNA is replicated, followed by assembly of the virus and the release of the virus out of the cell. This process can be seen in figure 4.

Many cellular molecules are involved in HCV entry into liver cells, and more are being discovered. The HCV particles attach themselves initially to the cell surface through LDL receptors and

glycosaminoglycans. After attaching themselves to the cell’s surface, the E1 and E2 proteins bind with the SR-BI48 an CD81 co-receptors. The virus particles are then relocalized to the tight junction proteins; Claudin-1 (CLDN1) and occludin (OCLN). The virus particles then become internalized via endocytosis, which is followed by pH-dependent viral fusion to the endosome and uncoating, which occurs inside endosomes after the fusion. Afterwards, the viral genome is released into the cytoplasm of the cells. (12), (13)

After the HCV particle has released its genome into the cytosol, translation and polyprotein processing occurs. The internal ribosomal entry site (IRES) sequence that is located at the 5’ end of the UTR region is recognized by ribosomes located in the ER of the cell. These ribosomes mediate viral polyproteins translation, the seven non-structural proteins make up the RNA replication machinery within ER-derived structures that are known as the membranous web. In replication sites at the membranous web lipid droplets can be found, which play an essential role in RNA synthesis. The RNA is replicated and the assembly of the HCV particles starts. The coordinated action of the ER-resident E1 and E2 glycoprotein complex, the recruitment of lipid droplet associated core protein and several viral and host factors, package the viral RNA. The HCV particles are further assembled and released from the cells with the help of the lipoprotein production pathway through the Golgi system. (13), (14)

(11)

11

Figure 4: HCV replication Cycle: The replication cycle of HCV starts with the entry of HCV into a cell, then translation of the HCV RNA and polyprotein processing occurs. The next step is RNA replication, followed by assembly of the virus and the release of the virus out of the cell. source: www.tibotec.com

2.4 Antiviral drugs against HCV: past and present

The first medicine developed for HCV was interferon-α (IFN-α) (13). IFN-α is a component of the innate immune system and possesses antiviral activity (15). IFN-α induces a general antiviral response in cells. The second HCV therapy was a combination of pegylated IFN-α and ribavirin for up to 48 weeks, this treatment cures about 50% of the patients. Pegylated IFN-α induces a more potent antiviral response in the cells than non-pegylated IFN-α. Ribavirin is a guanoside analog, it induces IFN-stimulated genes, favors the T helper type 1 immune response, inhibits factors leading to GTP depletion, and directly inhibits the HCV polymerase and causes mutagenesis of newly synthesized viral RNA. This treatment can, however, have severe side effects, leading to discontinuing of the treatment. The side effects are caused by IFN, some side effects are flu-like and neuropsychiatric symptoms, hemolytic anemia and autoimmune disease. Years later , in 2013 direct acting-antivirals (DAAs) were developed, these DAAs are directed against specific proteins involved in HCV replication. Currently there are four classes of DAAs, as shown in figure 5. There are four classes of DAAs, which are defined by their mode of action and therapeutic target. The four classes are (1) NS3 and NS4A protease inhibitors, (2) NS5A inhibitors, (3) NS5B nucleoside polymerase inhibitors and, (4) NS5B non-nucleoside polymerase inhibitors. Each

(12)

12 class has different advantages and disadvantages. The NS3 and NS4A protease inhibitors, have a high potency, but cannot cover multiple HCV genotypes and have a low barrier for resistance. The NS5A inhibitors are also highly potent, and cover multiple HCV genotypes, but have an intermediate barrier for resistance. The NS5B nucleoside polymerase inhibitors have an intermediate potency, but can cover all HCV genotypes and have generally a high barrier for resistance. The NS5B non-nucleoside polymerase inhibitors have an intermediate potency, cover a limited amount of genotypes and have a low barrier for resistance. (13), (16)

Figure 5: DAAs for HCV treatment; the four classes of DAAs for HCV treatment, some of the DAAs are shown per class. DAAs in clinical use or development are listed in black. Those approved by the FDA(when the figure was made) are highlighted in bold. NB: see appendix I for the current state of HCV medicine. (17)

The first DAAs that were developed and approved for use, were the NS3 and NS4A protease inhibitors telaprevir and boceprevir. These protease inhibitors can be used in combination with pegylated IFN and Ribavirin for treatment of HCV genotype 1. Nowadays, they are not regularly used anymore because they are less potent and cause more adverse effects than newer protease inhibitors. The first NS5B nucleoside polymerase inhibitors, NS5B non-nucleoside polymerase inhibitors, and NS5A inhibitors were approved a few years later, along with the second generation of NS3 and NS4 protease inhibitors. (13) The most recent approved DAAs are Daclatasvir (brand name: Daklinza) and the

Ombitasvir/paritaprevir/ritonavir combination (brand name: Technivie) (18). To detect antiviral resistance against protease and polymerase (NS5A) inhibitors, an antiviral genotyping resistance assay had to be developed, which is exactly what has been accomplished in this research. Daclatasvir is a strong NS5A protein inhibitor and covers all HCV genotypes. It has a low barrier to resistance, but can be used for treatment with other DAAs without addition of IFN (19). The Ombitasvir/paritaprevir/ritonavir combination is for treatment of patients with a genotype 4 infection. It contains a NS3 and NS4A protease inhibitor (Paritaprevir), a NS5A inhibitor (Ombitasvir) and a CyP3A inhibitor (Ritonavir), which mediates the metabolism of paritaprevir and so increases its concentration in plasma (20). Many more DAAs are being developed or in clinical trials in the hope of finding a 100% cure for HCV. (13)

(13)

13 The current approved HCV medication and the HCV genotypes they are effective on can be seen in appendix I.

2.4.1 HCV and antiviral drug resistance

As mentioned before, HCV is a virus with a high degree of genetic variability and it mutates easily. Some of those mutations can lead to resistance against HCV medicines and some genotypes are naturally resistant for certain HCV drugs. Drug resistance is therefore a big problem in treating HCV, the high mutation rate of HCV stems from the HCV polymerase. This polymerase generally causes 10-3 to 10-5 misincorporations per copied nucleotide and the high viral production rates in vivo (1012 viruses per patient per day) only add to the fast mutation rate of HCV. If these mutations occur in the wrong places, they can lead to drug resistance (see appendix II for mutations in genotype1, 2, 3 and 4 that can lead to drug resistance).

External pressure to the HCV genome can lead to a faster rise of drug resistant mutations. The HCV strains that are resistant to a certain drug, will survive the treatment and continue to replicate, to combat this, multiple HCV drugs are usually given to patients infected with HCV. Some drugs are more susceptible to HCV resistance, they only need a single mutation in the right place to not have any effect anymore. Some drugs, like the NS5B nucleoside polymerase inhibitors have a high barrier for resistance. (21)

(14)

14

3. Methods

3.1 RNA isolation

RNA was isolated from plasma samples with the QIAsymphony® SP (QS ) (Qiagen, Venlo, the

Netherlands) or MagNA Pure LC (MPLC) (Roche, Almere, the Netherlands). The MPLC and QS isolation robots both operate upon the same principle. The isolation starts with lysing the cells, releasing the nucleic acids and denaturing the nucleases. Proteinase K is used by the isolation robot to digest the proteins. The MPLC method works with magnetic particles, these magnetic particles bind nucleic acids to their silica-coated surface, due to the chaotropic salt conditions, isopropanol and high ionic strength of the lysis buffer. Magnetic particles with bound nucleic acids can then be magnetically separated from the residue of the sample. These beads then undergo washing steps, to remove unbound substances like proteins and to lower the chaotropic salt concentration. The purified particles are then treated with elution buffer at +70⁰C to remove the nucleic acids from the magnetic particles.

For Qiasymphony, extraction was done according to the Complex 200_V6_DSP default IC protocol for QS and using the Virus/Bacteria Kit. An input volume of 300µl and an elution volume of 110µl was used. For the MagNA Pure LC, the Total Nucleic Acid Isolation Kit (Roche) was used for isolation with an input volume of 200µl and an elution volume of 100µl. The QS and MPLC methods were compared, to find out which method led to better results in terms of purity and sensitivity. To determine the lower limit of detection for all regions and genotypes, the samples were diluted from undiluted to 100.000 times dilution in 1log dilution steps.

3.2 cDNA synthesis

A cDNA synthesis was performed to create copy DNA from the isolated RNA. The cDNA synthesis used 5x first strand buffer (Invitrogen, Paisley, United Kingdom), dNTP’s 10mM (Roche), RNAsin 40U/µl (Promega, Leiden, the Netherlands), 0.1 M DTT (Invitrogen) and superscript III 200U/µl (Invitrogen) in a 50µl reaction. The primers used were either random hexamer 50µM (Invitrogen) or specific forward or reverse 20µM (the outer PCR primers, see appendix IV) (Eurogentec, Maastricht, the Netherlands). The cDNA synthesis was performed in a SimpliAmp ThermalCycler cDNA System (Life technologies, Oslo, Norway ), using a 10 minute 25⁰C denaturation step, a 48⁰C synthesis step of an hour and a 5 minute termination step at 95⁰C.

3.3 PCR reactions

Outer PCR amplification was performed on all cDNA sequences, nested PCR amplification was

performed on all products from the outer PCR. This was done according to genotype and gene region. Different primers were ordered (Eurogentec) per gene region (kindly provided by Dr R. Molenkamp, AMC), and genotype. The primers also split the NS5a gene region into two fragments, as the fragment would otherwise be longer than 800 bp and the sequencer cannot sequence bigger fragments. See appendix IV for the list of primers used in this research. All PCR amplifications used the HotStar Taq enzyme 5U/µl (Qiagen), MgCl2+ (Qiagen), 10x PCR buffer (Qiagen), dNTP’s in a 50µl reaction. All PCR reactions used a 15 minutes pre-heating step of 95⁰C, and a 10 minute final extension step of 72⁰C. All PCR reactions also used a 30 seconds denaturation step of 95⁰C, a 1 minute extension of 72⁰C for 40

(15)

15 cycles, only the annealing step was variable. The primers used all had a concentration of 20µM. For all PCR reactions, negative controls (negative process control (NPC), negative template control (NTC)) were included.

To obtain the NS3, NS5a fragment 1 and 2 and NS5b fragments from genotype 1a, cDNA from samples with genotype 1a was amplified by PCR. The outer PCR reaction used a 30 seconds annealing step of 52⁰C for NS3, using the outer NS3 primers. For NS5b and the NS5a fragments an annealing temperature of 55⁰C was used, using the outer genotype 1-4 NS5b primers and the outer fragment 1 and 2 gt1a NS5a primers. The nested PCR used a 55⁰C annealing temperature for the NS3 and NS5b fragments, for the NS5a fragments an annealing temperature of 58⁰C was used. The NS3 nested primers, NS5b nested 1-4 primers and the nested gt1a fragment 1 and 2 NS5a primers were used. See appendix IV for the list of primers.

To obtain the NS3, NS5a fragment 1 and 2 and NS5b fragments from genotype 1b, cDNA from samples with genotype 1b was amplified by PCR. The outer PCR reaction used a 52⁰C annealing step for NS3 and the NS5a fragments, using the outer NS3 primers and the outer fragment 1 and 2 gt1b NS5a primers. For NS5b fragment an annealing temperature of 55⁰C was used, using the outer genotype 1-4 NS5b primers. The nested PCR used a 55⁰C annealing temperature for the NS3 and NS5b fragments, for the NS5a fragments an annealing temperature of 58⁰C was used. The NS3 nested primers, NS5b nested 1-4 primers and the nested gt1b fragment 1 and 2 NS5a primers were used.

To obtain the NS3, NS5a fragment 1 and 2 and NS5b fragments from genotype 2, cDNA from samples with genotype 2 was amplified by PCR. The outer PCR reaction used a 52⁰C annealing step for NS3, using the outer NS3 primers. For NS5b and the NS5a fragments an annealing temperature of 55⁰C was used, using the outer genotype 1-4 NS5b primers and the outer fragment 1 and 2 gt2 NS5a primers. The nested PCR used a 55⁰C annealing temperature for the NS3 and NS5b fragments, for the NS5a fragments an annealing temperature of 58⁰C was used. The NS3 nested primers, NS5b nested 1-4 primers and the nested gt2 fragment 1 and 2 NS5a primers were used.

To obtain the NS3, NS5a fragment 1 and 2 and NS5b fragments from genotype 3, cDNA from genotype 3a samples was amplified by PCR. The outer PCR reaction used a 52˚C annealing step for NS3, using the outer NS3 primers. For NS5b and the NS5a fragments an annealing temperature of 55⁰C was used, using the outer genotype 3a NS5b primers and the outer fragment 1 and 2 gt3a NS5a primers. The nested PCR used a 55⁰C annealing temperature for the NS3 and NS5b fragments, for the NS5a fragments an

annealing temperature of 58⁰C was used. The NS3 nested primers, NS5b nested gt3a primers and the nested gt3a fragment 1 and 2 NS5a primers were used.

To obtain the NS3, NS5a fragment 1 and 2 and NS5b fragments from genotype 4, cDNA from genotype 4 was amplified by PCR. The outer PCR reaction used a 52⁰C annealing step for NS3 and the NS5a

fragments, using the outer NS3 primers and the outer NS5a gt4 1 and 2 fragments. For the NS5b fragment an annealing temperature of 55⁰C was used, using the outer genotype 1-4 NS5b primers. The nested PCR used a 55⁰C annealing temperature for the NS3 and NS5b fragments, for the NS5a fragments an annealing temperature of 56⁰C was used. The NS3 nested primers, NS5b nested 1-4 primers and the nested gt4 fragment 1 and 2 NS5a primers were used.

(16)

16 The PCR products were assayed on a 2% pre-cast agarose gel (Invitrogen), the FastRuler Low Range DNA Ladder (Thermo scientific) was used as marker.

3.4 Sanger sequencing

To analyze the PCR products, the Sanger sequencing method was used. The Sanger sequencing method is based on the dideoxynucleotide chain termination principle and was developed by Fred Sanger. A dideoxynucleotide is a molecule that lacks a hydroxyl-group at the 2´and 3´ carbons of the sugar part of the nucleotide. During normal DNA-replication, a nucleoside triphosphate is linked by its 5´α-phosphate group to the 3´ hydroxyl group of the last nucleotide of the growing chain. However, with Sanger

sequencing, a dideoxynucleotide is incorporated at the end of the growing chain. The DNA-synthesis will then stop because the phosphodiester bond cannot be formed with the nucleotide that is to be

incorporated next. This termination is crucial in Sanger sequencing method.

The first step of the Sanger sequencing method is annealing a synthetic oligonucleotide of 17-24

basepairs to an already known part of the DNA strand that is to be sequenced. This oligonucleotide then acts as a primer, it provides a 3´hydroxyl group for initiation of DNA synthesis. Thereafter, a normal PCR takes place. The DNA-synthesis is stopped when the polymerase chain reaction ends, the product being a unique mix of single stranded DNA-molecules of all possible lengths.

The single stranded DNA strands are then analyzed with a laser that detects the fluorescent labels and assigns the right nucleotide to it, as each nucleotide (thymine, adenine, cytosine and guanine) is coupled to a different fluorescent label. (22)

For this end the in-house sequencing method was used. All sequencing reactions used the Big Dye terminator V3.1 reaction mix(Applied Biosystems), the 5x sequencing buffer (Applied Biosystems) and the primers also used for the PCR reaction plus an extra sequencing primer for nested NS5a fragment 1 (see appendix IV), with an concentration of 5 pmol/µl.

A sequencing PCR reaction was performed on all positive PCR products with the program Big dye terminator V3.1 short: a pre-heating step of 1 minute at 96⁰C. Then 10 seconds at 96⁰C, 5 seconds at 50⁰C, 1 minute at 60⁰C for 25 cycles.

The sequencing PCR products were purified by centrifuging the products at 1000 x g for 5 minutes through a DyeEx 96 plate (Qiagen). After purifying, the PCR products were analyzed by the ABI Prism 3130xl genetic analyzer (Applied Biosystems, Oslo, Norway).

Sequencing results were analyzed with the Seqman Pro software (Lasergene DNA star). Subsequently, the DRMs we interpreted using the website http://hcv.geno2pheno.org/ and the self-assembled DRM table (appendix II).

The DRM table was assembled using literature mentioning resistance mutations for HCV. The search for literature was conducted in the NCBI database, using the search terms: “HCV resistance and genotype”, or “HCV resistance” and “specific drug”. References from the geno2pheno site were also used.

(17)

17

4. Results

4.1 Comparison of the QiaSymphony and MagNa Pure LC isolation methods

HCV positive plasma samples were diluted 100x with RPMI and isolated with the QS and MPLC, a NPC (RPMI-culture medium) was also included. cDNA was synthesised from these samples with random hexamer primers and an outer and nested PCR were performed. The PCR products were sequenced and analyzed.

The goal was to compare the nucleic acid extraction of the QS and MPLC in term of purity and sensitivity using the PCRs of the protocols decribed.

Below, figure 9 and figure 10 show the outer and nested PCR results of a genotype 1b, genotype 2 and genotype 4 NS3 fragment. Successful PCR amplification would result in band of c.a. 750-800 bp for the outer PCR and a band of c.a. 650 bp for the nested PCR. Only the genotype 4 sample shows no bands for the outer and nested PCR, as its concentration was lower than the concentration of the other samples. The other two genotypes show comparable bands between the 400 and 850 bp bands of the marker. The outer PCR products of QS and MPLC isolation barely differ, only the QS genotype 1b band is slightly darker than the MPLC band. The same goes for the nested PCR, only the QS isolated products show less discernable background bands than the MPLC isolated products. The same results were observed for the other samples on which the QS and MPLC method were compared (data not shown). The QS method seemed to generate less discernable background bands, no other effects were observed.

Figure 11 shows the sequencing results of the reverse primers of the nested PCR of genotype 2 for QS (lower sequence) and MPLC (upper sequence) isolation. The same peaks and background can be observed for both the QS and the MPLC method. This was also the case for most other compared sequences. Sometimes the sequences between the QS and MPLC isolation method differed slightly, but never a consistent difference (data not shown).

Figure 9: Outer PCR of genotype 1b, 2 and 4 for NS3 QS and MPLC isolation methods are compared.

Figure10: Nested PCR of genotype 1b, 2 and 4 for NS3 QS and MPLC isolation methods are compared.

-400 bp

-850 bp

-400 bp

(18)

18

Figure 12: Outer and nested PCR for NS5a F1 genotype 1b. The difference between random hexamer primers and specific forward (F1o) and reverse primers (R2o) is being compared. Samples are diluted either with negative plasma (PLE) or RPMI. Figure 11: Nested PCR of genotype 2 NS3 region, reverse primers. The RNA of the upper sequence was isolated with

the MPLC, the RNA of the lower sequence with the QS. Both sequences show the same peaks and background.

4.2 Optimization of cDNA synthesis

HCV positive plasma samples were diluted 100x with RPMI or negative plasma and isolated with the QS and MPLC, a NPC (-QS/-MPLC)(RPMI/negative plasma) was also included. cDNA was made from these samples with random hexamer primers and specific forward and reverse primers and an outer and nested PCR were performed. The PCR products were sequenced and analyzed.

One of the side questions of this project was whether it mattered for sequencing if samples were diluted with RPMI or negative plasma. Another side question was whether it mattered for sequencing if cDNA synthesis was performed with random hexamer primers or specific forward or reverse primers. Figure 12 shows the outer and nested PCR results of a

genotype 1b NS5a fragment 1 product. All NTC’s and NPC’s from the outer PCR are negative, but not all NTC’s and NPC’s from the nested PCR are negative. The NTC from the random hexamers gives a positive band (c.a. 650 bp) and the MPLC NPC from the same section give a vague band just below the 400 bp. The NTC and QS NPC from the F1o section also show a few vague bands below the 800 bp. This indicates contamination of the PCR reagents. Contamination in the NPC’s and NTC’s was also found in other samples (data not shown). The outer PCR shows stronger and more clear bands for the cDNA made with random hexamers than the specific primers. There is not any difference in the nested PCR samples. This was also true for other samples (data not shown).

For both outer and nested PCR, samples diluted with negative plasma seem to give clearer PCR bands. But the difference is barely visible.

(19)

19 The sequencing results for all used samples varied, some sequences were cleaner than others, but no clear difference between the samples diluted with either RPMI or negative plasma was discovered (data not shown).

As for the difference between random hexamer and specific primers; the sequences from the cDNA made with random hexamer primers had less background and longer sequences than those derived from cDNA made with the specific primers. (data not shown).

4.3 Lower limit of detection determined for the HCV resistance assay

HCV positive plasma samples were diluted from undiluted to 100.000 times dilution in 1log dilution steps, and isolated with the QS and MPLC, a NPC (negative plasma) was also included. cDNA was made from these samples with random hexamer primers and an outer and nested PCR were performed. The PCR products were sequenced and analyzed.

The goal of these experiments was to develop a genotyping assay for HCV resistance for new direct acting antivirals and to determine its lower limit of detection.

Table 1 shows the dilution series made for genotype 1b, the dilution series for genotype 1a, 2, 3a and 4 were similar, only with slightly different concentrations.

Figure 6 shows the dilution series of the outer PCR of genotype 1b for the NS3, NS5a fragment 1 and fragment 2 and NS5b gene regions. Successful PCR amplification would result in band of c.a. 750-800 bp for the outer PCR. For all gene regions a band of c.a. 800 bp is visible up to and including the 10.000x dilution (2.25E2 IU/ml). For the other genotypes results were similar, though the concentrations at which the PCR failed varied per genotype and gene region (data not shown).

Dilution series gt1b Concentration

In IU/ml

undiluted (ov) 2.25E6

10x 2.25E5

100x 2.25E4

1000x 2.25E3

10.000x 2.25E2

100.000x 2.25E1

Table 1: Dilution series of genotype 1b. the left column shows the dilution, the right column shows the concentration of the plasma sample.

(20)

20 Figure 6: Determination of LLOD for outer PCR of genotype 1b, all gene regions.

Figure 7 shows the dilution series of the nested PCR of genotype 1b for the NS3, NS5a fragment 1 and fragment 2 and NS5b gene regions. Successful PCR amplification would result in band of c.a. 650 bp for the nested PCR. For all gene regions a band of c.a. 650 bp is visible up to and including the 10.000x dilution (2.25E2 IU/ml) and for NS3 and NS5b up to and including the 100.000x dilution (2.25E1 IU/ml ). All gene regions seem to have a weak background band above 850 bp and NS3 has a stronger

background band just below 400 bp. For the other genotypes results were similar, though the concentrations at which the PCR failed varied per genotype and gene region (see table 2).

As for the NTC’s and NPC’s, the NTC and the NPC of NS5a fragment 1 show weak bands below (NTC, NPC)and above (NTC) the expected height. This indicates contamination of the PCR reagents.

Contamination in the NPC’s and NTC’s was also found in other samples regardless of genotype and gene region (data not shown). After making new primer dilutions and using new, clean reagents, the problem with contamination was solved.

850 bp 400 bp

(21)

21 Figure 7: Determination of LLOD for nested PCR of genotype 1b, all gene regions.

All positive bands of the dilution series of all genotypes were sequenced. The sequences were analyzed with the Seqman Pro software (data not shown), and the lower limit of detection was determined for all genotypes, as can be seen in table 2. It can be seen that the PCRs for genotype 2 are rather weak in comparison with the other genotypes, and for NS5a fragment 1 a very high viral load (1.82E6 IU/ml) is necessary, and even then only the nested PCR will give a band that can be sequenced.

(22)

22 Table 2: Lower limit of detection determined for genotype 1a, 1b, 2, 3a and 4, for all gene regions.

genotype fragment Outer PCR (IU/ml) Nested PCR (IU/ml)

1a NS3 from 1.52E4 from 1.52E3

NS5a fragment 1 from 1.52E2 from 1.52E2

NS5b from 1.52E3 from 1.52E2

1b NS3 from 2.25E3 from 2.25E1

2 NS3 from 1.82E5 from 1.82E2

NS5a fragment 1 no band from 1.82E6

NS5a fragment 2 no band from 1.82E4

NS5b no band from 1.82E5

3a NS3 from 1.45E3 from 1.45E3

4 NS3 from 1.95E5 from 1.95E3

The sequences that were analyzed with the Seqman Pro software were compiled into consensus sequences per genotype and per gene region. These consensus sequences were analyzed using the geno2pheno website. Geno2pheno confirms genotype, determines (DRM) mutations in sequence and interprets the clinical resistance of the uploaded sequence. An example of a geno2pheno result (genotype 1b, NS5a) can be viewed in figure 8. The result confirms that this is a genotype 1b NS5a sequence and shows all the mutations that occur in the sequence. The geno2pheno software then determines which mutations can cause resistance to certain DAAs (none in this case).

(23)

23 Figure 8: geno2pheno result resistance analysis for genotype 1b NS5a gene region. Geno2pheno confirms genotype, determines mutations in sequence and makes a resistance analysis.

Because it is important for diagnostic use of the HCV assay, the sequences resulting from the project were compared with reference sequences (from NCBI) and a table showing the amino acids (aa) that can be covered by this assay was compiled (table 3).

. Gene region aa covered

outer PCR nested PCR total

NS3 1-210 1-199 1-210

NS5A-fragment 1 7-280 12-269

7-436 NS5A-fragment 2 200-436 233-426

NS5B 102-369 116-345 102-369

(24)

24

5. Discussion/conclusion

During this project, the primary objective was to develop a genotyping assay for HCV resistance for new direct acting antivirals for the NS3, NS5a and NS5b gene regions and to determine the lower limit of detection of the assay.

The results for determining the Lower limit of detection are displayed in chapter 4.3. These results show that the Lower limit of detection was determined for all the assayed genotypes (genotype 1a, 1b, 2, 3a and 4) and that the developed assay for determining HCV resistance for DAAs works for all genotypes and gene regions. Furthermore, for diagnostic purposes the assay has to be capable of generating a good sequence for a minimum load of 5000 IU/ml. This criterion was met for genotype 1a, 1b, 3 and 4. For genotype 2, the criterion was only met for the NS3 gene region. Gene region NS5a and NS5b do not yet meet the specified criterion, so further research need to be done for a more solid assay. The genotype 2 assay can be done in the diagnostic routine, but only if the sample has a very high viral load (> 1.82E5 IU/ml).

All of the subsequent sequences were successfully analyzed with the geno2pheno database for HCV, and the given mutations were cross-checked with the self-assembled DRM table. The geno2pheno site compares the sequences with a database and determines the genotype, subtype, gene region (aa covered) and reports all found mutations. The resulting report also shows the mutations that can lead to resistance and shows the HCV drugs the virus is still susceptible for an the drugs the virus is resistant for. The self-assembled DRM table only contains known resistance causing mutations for genotype 1, 2, 3 and 4. The DRM table is more extensive than the geno2pheno drug resistance database for all genotypes except genotype 1, due to the fact that the geno2pheno database was updated in October 2015 and new DRMs were reported. It was also discovered that not all literature references from geno2pheno were fully sound. A few references were completely left out, a few referenced articles do not show the genotype or mutation that is claimed by geno2pheno. This only occurred sparsely and only for the genotypes that are not genotype 1. The geno2pheno site is still trustworthy, especially for genotype 1, but the DRM table is more trustworthy for checking mutations for genotypes other than genotype 1. This project also had several side questions: Whether sequences were better if samples were isolated with QiaSymphony or MagNA Pure LC. Another side question was whether it mattered for sequencing if samples were diluted with RPMI or negative plasma. The last side question was whether sequencing results would be better if cDNA synthesis was performed with random hexamer primers or specific forward and reverse primers.

The results of comparison of the QS and MPLC isolation methods are displayed in chapter 4.1 and the results concerning the other side questions are displayed in chapter 4.2. These results show that there is no significant difference between the QS and MPLC isolation methods. The QS might give slightly more pure sequences (Mogens Kruhøffer et al. 2010 showed the QS to be capable of very pure RNA isolation (23) ) but both methods can be used interchangeably for diagnostic purposes.

As for diluting samples with either negative plasma or RPMI, diluting with negative plasma seems to give barely noticeable better results. So in this case the dilution agent can also be used interchangeably for diagnostic purposes.

(25)

25 The results also show that random hexamer primers (chapter 4.2) give better results than the specific forward and reverse primers, also mentioned in the research of Michael Stangegaard et al. (2006) (24). The random hexamer primers are therefore the primers that will be used for diagnostic use of the assay. All result sections briefly touched upon the subject of positive NPCs and NTCs. During this project there were several instances of positive NPCs and NTCs, with either band at the expected height for a HCV PCR product, but also bands lower and/or higher than that. This meant that the reagents had been

contaminated, all instances of contamination were easily solved by replacing the reagents and making a new 20uM dilution from the primer stock. The positive NPCs and NTCs did not make the assay invalid, but showed that the assay is very sensitive. So use of proper controls and molecular work-up for diagnostic purposes is extremely important. One time the contamination found resulted in a sequence from Propionibacterium acnes strain PA_15_1_R1, 87% similarity, 30% of the genome covered (see appendix IV for NCBI result).

This means that the developed primers are not entirely specific for HCV. The developed primers were also blasted on NCBI, which resulted in blast results showing HCV but also other organisms, such as

Propionibacterium acnes, Macaca fascicularis and homo sapiens. See appendix IV for the NCBI blast

result. Many of the HCV PCR products also had vague background bands. These background bands luckily did not have any effect on the resulting sequences, but do confirm that the primers are not entirely specific. This is not a problem, as the primers do work on HCV. Just as the uncertainty that the developed primers will pick up all HCV variants they were designed for, as HCV is a virus that mutates easily and has many variants. It is a problem noted often in HCV research, for example in the research of Shantanu Prakash et al. (2016) (25) and the research of Verónica Saludesa et al. (2016) (26).

(26)

26

6. Recommendations

A few recommendations can be made for this project. The most important recommendations can be made for genotype 2, as genotype 2 did not meet the criterion mentioned in chapter 5 for the NS5a and NS5b regions. Further research needs to be done, one of the first steps that should be taken is to try different annealing temperatures. This was already tested, but only for a limited set of temperatures and only for the NS5a fragments. For the outer PCR the temperatures tested ranged from 48°C up to and including 57°C. For the nested PCR however, the only two temperatures tested were 55°C and 58°C. Another action that can be taken is designing a new genotype 2 NS5a primer set and also designing a genotype 2 primer set for the NS5b region. This could lead to better results, but also easily to worse, as the HCV genome still differs widely per genotype. The primer concentration for the PCR primers could be raised above the 20uM. The ratio primer:DNA would then be different and it could lead to an increase in PCR product.

Then there are some recommendations of a more diagnostic nature. As came forth from chapter 5, it is extremely important to always take the proper negative controls when performing the HCV resistance assay. When synthesizing cDNA it is important to use random hexamer primers and when sequencing the outer PCR fragments, it is important to sequence with both the outer and nested primers. This, because the sequences from only one primer set are not always long enough or clear enough on deletions and double peaks, so having the double amount of sequences will make the analysis easier and helps providing better results. The sequences from the nested PCR are usually a lot more clear and complete than the outer PCR sequences.

For analyzing the sequences, geno2pheno should be used for checking genotype, subtype and region, for giving out all present mutations, and aa coverage. For the subsequent interpretation of the mutations, the found mutations should be checked with the DRM table.

For the assay no positive control has yet been assigned, the EMC virology unit usually takes a high positive control and a low positive control (mostly for real-time PCR assays). In this case just a high positive control will do, as this assay consists of a conventional PCR and a sequencing analysis, and is not a real time PCR assay. This high positive control should have a concentration of c.a. 1.5E6 IU/ml. As the assay has different primer sets, of which some only work on a specified genotype, there should be a positive control for each genotype. For the genotype 1a samples a positive control of a HCV genotype 1a should be used, the same goes for genotype 1b, 2, 3a and 4. Another option is to make a single positive control from pooling all genotypes together, as this will be easier for diagnostic use. However, there is no guarantee that this will lead to a clear positive control. This control has to be tested first, if it is to be incorporated into the diagnostic routine. The positive control can be previously tested patient samples or commercially available stocks.

(27)

27

Literature list

1. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a

systematic analysis for the Global Burden of Disease Study . Lozano R, Naghavi M, Foreman K, Lim S,

Shibuya K, Aboyans V, Abraham J, et al. 2012, Lancet, pp. 2095-2128.

2. Initial presentation of acute HCV infection among HIV-negative and HIV-positive individuals.

Experience from 2 large German Networks on the study of acute HCV infection. Vogel M, Deterding K,

Wiegand J, et al. 2009, clinical infectious diseases, Vol. 49, pp. 317-319.

3. The natural history of hepatitis C virus. Stephen L. Chen, Timothy R. Morgan. 3, 2006, Int J medical science, Vol. 2, pp. 47-52.

4. Liver fibrosis. Bataller R, Brenner A. D. 115, 2005, J clinical invest., Vol. 2, pp. 209-218.

5. Hepatitis C Viral Life Cycle. Tetsuro Suzuki, Koji Ishii, Hideki Aizaki, Takaji Wakita. 12, 2007, Advanced drug delivery reviews, Vol. 59, pp. 1200-1212.

6. Oligomerization of hepatitis C virus core protein is crucial for interaction with the cytoplasmic domain

of E1 envelope protein. K. Nakai, T. Okamoto, T. Kimura-Someya, K. Ishii, C.K. Lim, H. Tani, E. Matsuo,

T. Abe, Y. Mori, T. Suzuki, T. Miyamura, J.H. Nunberg, K. Moriishi, Y. Matsuura. 22, 2006, journal of virology, Vol. 80, pp. 11265-11273.

7. Maturation and assembly of hepatitis C virus core protein. T.Suzuki, R. Suzuki, M.Kalitzky, P.Borowski. 2006, Horizon bioscience, pp. 295-311.

8. NS2 protein of hepatitis C virus interacts with structural and non-structural proteins toward virus

assembly. Cl Popesu, N. Callens, D. Trinel, P. Roingeard, D. Moradpour. V. Deschamps, G. Duverlie,

F.Penin et al. 2, s.l. : PLoS Pathology, 2011, Vol. 7. e1001278.

9. structure of the catalytic domain of the hepatitis C virus NS2-3 protease. C. Lorenz, J. Marcotrigiano, T.G. Dentzer, C.M Rice. 7104, 2006, Nature, Vol. 442, pp. 831-835.

10. The NS5A protein of hepatitis C virus is a zinc metalloprotein. T.L. Tellinghuisen, J. Marcotrigiano, A.E. Gorbalenya, C.M. Rice. 47, 2004, journal of biological chemistry, Vol. 297, pp. 48576-48587. 11. Hepatitis C virus: A global view. A.A. Mohamed, T.A. Elbedewy, M. El-Serafy, N. El-Toukhy, W. Ahmed, Z.A. El Din. 26, 2015, world journal of hepatology, Vol. 7, pp. 2676-2680.

12. Tight junction proteins claudin-1 and Occludin control hepatitis C virus entry and are downregulated

during infection to prevent superinfection. Liu Shufeng, Yang Wei, Shen Le, Jerrold.R. Turner, Carolyn.B.

Coyne, Tianyi Wang. 83, 2009, Journal of virology, Vol. 4, pp. 2011-2014.

13. understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Troels.K.H. Scheel, Charles.M. Rice. 19, 2013, nature medicine, Vol. 7, pp. 837-849.

14. Hepatitis C virus and antiviral innate immunity: who wins at tug-of-war? Da-Rong Yang, Hai-Zhen-Zhu. 21, 2015, World journal of Gastroentology, Vol. 13, pp. 3786-3800.

15. negative regulation of the interferon response by an interferon-induced long non-coding RNA. Hiroto Kambara, Farshad Niazi, Lenche Kostadinova, Dilip.K. Moonka, Christopher T. Siegel, Anthony B. Post, Elena Carnero, Marina Barriocanal, Prri Fortes, Donal D. Anthony, Saba Valadkhan. 42, 2014, nucleic acids res., Vol. 16, pp. 10668-10680.

(28)

28 16. new era for management of chronic hepatitis C virus using direct antiviral agents: A review. Tamer Elbaz, Mohamed El-Kassas, Gamal Esmat. 6, 2015, journal of Adv Res., Vol. 3, pp. 301-310.

17. antiviral treatment of hepatitis C. Feeney E.R., Chung R.T. 3, 2014, BMJ.

18. FDA. FDA approves new treatment for chronic hepatitis C genotype 3 infections. Silver Spring : FDA, 2015.

19. Daclatasvir for the treatment of chronic hepatitis C. Elisabetta Degasperia, Alessio Aghemoa, Massimo Colomboa. 17, 2015, expert opinion on pharmacotherapy, Vol. 16, pp. 2679-2688.

20. Pharmaceutical approval update. Gohil, Kunj. 40, 2015, Pharmacy and Therapheutics, Vol. 10, pp. 649-650.

21. Hepatitis C virus resistance to protease inhibitors. Philippe Halfon, Stephen Locarnini. 1, s.l. : journal of hepatology, 2011, journal of hepatology, Vol. 55, pp. 192-206.

22. ATP-dependent chromatin remodeling in the DNA-damage response. H. Lans, J.AMarteijn, W. Vermeulen. 4, 2012, Epigenetics and Chromatin, Vol. 5, pp. 1756-8935.

23. Evaluation of the QIAsymphony SP Workstation for Magnetic Particle—Based Nucleic Acid

Purification from Different Sample Types for Demanding Downstream Applications. Mogens Kruhøffer,

Thorsten Voss, Katharina Beller, Mario Scherer, Janina Cramer, Thomas Deutschmann, Cordula Homberg, Martin Schlumpberger, Christian Lenz. 1, s.l. : journal of laboratory automation, 2010, Vol. 15. 41-51.

24. Reverse transcription using random pentadecamer primers increases yield and quality of resulting

cDNA . Michael Stangegaard, Inge Hogh Dufva, Martin Dufva. 5, s.l. : Bio techniques, 2006, Vol. 40.

649-657.

25. Development od novel triples single-step real time PCR assay for detection of HBV and HCV

simultaneously. Shantanu Prakash, Amita Jain, Bhawana Jain. s.l. : Virology, 2016, Vol. 492. 101-107.

26. Identification of HCV genotype 3 by a commercial assay challenged by natural polymorphisms

detected in Spain from patietns with diverse origins. Verónica Saludesa, Josep Querc, Josep Gregoric,

Elisabet Bascuñanaa, Damir García-Cehicc,, Juan Ignacio Estebanc, Vicente Ausinaa, Elisa Martró. s.l. : Journal of clinical virology, 2016, Vol. 78. 14-19.

(29)

29

Appendix

I: Current state of HCV medicine

Figure 13: The current state of HCV medicine. Everything in grey has not yet been approved by the FDA. Green colored blocks under mutations in region means that our HCV resistance assay covers those mutations. Table was made by Dr. Suzan Pas.

(30)

30

II: DRM

Table 4: Drug Resistance Mutations table for genotype 1. The table shows mutations that can cause resistance against antiviral drugs.

Mutation Amino acid position

Region drug resistance sequence in vivo/ vitro references:

V36A/L/G/M 36 NS3 Paritaprevir increase vivo L.L Vidal et al. JVH. 2016

V36A/M 36 NS3 telaprevir(3-25 fold increase), Paritaprevir vivo and vitro S. De Meyer, A. Ghys et al 2013, Lontok E et al. Hepatology. 2015

V36A/M/G/I/L 36 NS3 Boceprevir increase vivo and vitro

Lontok E et al. Hepatology. 2015

V36L/M/G/A 36 NS3 Asunaprevir increase vivo and vitro

Lontok E et al. Hepatology. 2015, L.L.Vidal et al. JVH. 2016

V36M 36 NS3 Faldaprevir vivo and vitro

L.L Vidal et al. JVH. 2016

43I/S/V 43 NS3 Simeprevir increase vivo and vitro

F43L 43 NS3 Paritaprevir increase Vivo L.L Vidal et al. JVH. 2016

F43L/S 43 NS3 Asunaprevir increase vivo and vitro L.L Vidal et al. JVH. 2016 F43S 43 NS3 Telaprevir and Boceprevir increase vivo and vitro L.L Vidal et al. JVH. 2016

T54A/S 54 NS3 telaprevir(3-25 fold increase)

vitro S. De Meyer, A. Ghys et al 2013

T54A/S/C/G 54 NS3 Boceprevir increase vivo and vitro

V55A 55 NS3 Asunaprevir increase vivo and vitro

V55A/I 55 NS3 Boceprevir increase vivo and vitro

V55I 55 NS3 Paritaprevir increase vivo and vitro

Y56H 56 NS3 Paritaprevir increase vivo and vitro

Y56H/L 56 NS3 Asunaprevir increase vivo and vitro

Q80H/K/R 80 NS3 Simeprevir increase vivo and vitro

Q80K 80 NS3 simeprevir (11 fold increase)

vitro Christophe Sarrazin, Erkki Lathouwers et al 2015 Q80K/L/R 80 NS3 Faldaprevir and paritaprevir increase vivo and vitro L.L Vidal et al. JVH. 2016 Q80K/R 80 NS3 Simeprevir and Asunaprevir increase vivo and vitro

(31)

31

V107I 107 NS3 Boceprevir increase vivo and vitro

S122D/G/I/N/T/R 122 NS3 Asunaprevir increase vivo and vitro

S122G/R/A/I/T 122 NS3 Simeprevir increase vivo and vitro

I132V 132 NS3 Telaprevir increase vivo and vitro

S138T 138 NS3 Simeprevir increase vivo and vitro

R155K/C 155 NS3 Boceprevir increase vivo and vitro

R155K/G/M/T/S/L/I 155 NS3 Telaprevir increase vivo and vitro

R155K/G/Q 155 NS3 Asunaprevir increase vivo and vitro

Lontok E et al. Hepatology. 2015, L.L.Vidal et al. JVH. 2016 R155K/Q 155 NS3 Faldaprevir and Simeprevir increase vivo and vitro

R155K/S/T/W 155 NS3 Paritaprevir increase vivo and vitro

R155K/T 155 NS3 telaprevir(3-25 fold increase)

vitro S. De Meyer, A. Ghys et al 2013, Jensen et al.2015

A156S 156 NS3 telaprevir(3-25 fold increase)

vitro S. De Meyer, A. Ghys et al 2013

A156S/F/T/V/N 156 NS3 Telaprevir increase vivo and vitro

A156S/T/V 156 NS3 Boceprevir increase vivo and vitro

Lontok E et al. Hepatology. 2015, L.L.Vidal et al. JVH. 2016 A156T/V 156 NS3 telaprevir(>25 foldincrease),Faldaprevir, Paritaprevir and Asunaprevir

vitro S. De Meyer, A. Ghys et al 2013, L.L Vidal et al. JVH. 2016

V158I 158 NS3 Boceprevir increase vivo and vitro

D168A/G/V 168 NS3 Faldaprevir increase vivo and vitro

D168E/V/A/H/F/T/G/Y/C 168 NS3 Asunaprevir increase vivo and vitro

D168E/V/A/H/F/T/I/N/Y 168 NS3 Simeprevir increase vivo and vitro

Lontok E et al. Hepatology. 2015, L.L.Vidal et al. JVH.

(32)

32 2016

D168E/V/Y/A/K/G/H/N/T 168 NS3 Paritaprevir increase vivo and vitro

Lontok E et al. Hepatology. 2015, L.L.Vidal et al. JVH. 2016 D168N 168 NS3 Telaprevir and Boceprevir increase vivo and vitro

I/V170F/A/T/V/L 170 NS3 Boceprevir increase vivo and vitro

I/V170T/A 170 NS3 Simeprevir, Telaprevir and Asunaprevir increase

vivo and vitro

M175L 175 NS3 Boceprevir increase vivo and vitro

S176G 176 NS3 Boceprevir increase vivo and vitro

L28T/M 28 NS5A Daclatasvir increase vivo and vitro

M28T 28 NS5A Ledipasvir increase vivo and vitro

M28T/A/S/V 28 NS5A Daclatasvir increase vivo and vitro

M28T/V 28 NS5A Ombitasvir increase vivo and vitro

P29/S/X 29 NS5A Daclatasvir increase vivo and vitro

Q30E/H/R/D/G/K/T 30 NS5A Daclatasvir increase vivo and vitro

Q30E/R/H 30 NS5A Ledipasvir increase vivo and vitro

Q30R 30 NS5A Ombitasvir increase vivo and vitro

R30G/H/P/Q 30 NS5A Daclatasvir increase vivo and vitro

L31M 31 NS5A Ledipasvir increase vivo and vitro

L31M/I/V 31 NS5A Daclatasvir increase vivo and vitro

L31M/V/F/I 31 NS5A daclatasvir increase vivo and vitro

P32L/X 32 NS5A Daclatasvir increase vivo and vitro

H58D 58 NS5A Ledipasvir and Ombitasvir increase