الفهرس 
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Abstract
Hepatitis c virus (HCV) belongs to the Flaviviridea family with its genome consisting of positive sense single strand RNA. Globally 110 million people are infected with HCV out of which 80 million are carriers. Peg interferon /ribavirin are burdened with adverse reactions in at least 10% of patients moreover a sustained virological response is achieved in only 50% of patients.
Aiming at better sustained virus response rate, improved tolerability and shorter treatment duration an increasing number of small molecules inhibitors have been developed which specifically target key regions of the HCV genome known as directly acting antiviral agents (DAAs).
The RNA dependent polymerase protein NS5B , responsible for viral genome replication, the serine protease protein NS3 with protease and RNA helicase activities and the NS5A including the Interferon /Ribavirin Sensitivity determining Region (IRSDR) are the most promising targets.
However, the high genetic diversity, mutation rate and turnover of HCV lead to in vivo development of viral quasispecies with high sequence diversity among various genotypes and subtypes with the potential accumulation of virus variants showing mutations with varying degrees of resistance to DAAs, even in the absence of preexisting drug exposure .therefore a clear understanding will be needed of how the presence of resistance associated variants at baseline influences treatment response.
The aim of the present study was to determine the prevalence of possible mutations expected to induce potential drug acting agent’s resistant variants in HCV NS3, NS5A and NS5B genes from DAAs naïve HCV infected patients.
Twenty HCV naïve infected patients were selected from the outpatient clinic of the Medical Research Institute, Alexandria University. Blood samples were taken from the patients and sera separated, distributed into aliquots stored for further virological investigations:
• For determination of HCV viral load by Taqman probe technique.
• Amplification of NS3 protease, NS5A and NS5B polymerase regions by conventional PCR on Veriti Thermal cycler (Applied Biosystem) using pairs of specifically designed primers.
• The PCR products were analyzed on a 1.7% agarose gel stained with ethidium bromide.
• The PCR product of interest was excised from agarose gel and purified using Thermo Scientific GeneJET Gel Extraction Kit.
• Cycle sequencing of the amplified regions using BigDye Terminator V 3.1 Cycle Sequencing Kit was done in Veriti thermal cycler.
• Removal of excess Dye Terminators was done using CENTRI-SEP Kit before loading them onto the ABI PRISM 310 Genetic Analyzer.
• The genotypes were identified by entering the sequencing data of the 3 amplified regions into HCV BLAST in GeneBank.
• Amino acid sequence variations and signature patterns of the 3 amplified genes were analyzed using the online Viral Epidemiology Signature Pattern Analysis (VESPA) with nearly all HCV subgenotypes other than GT-4:
• BioEdit Sequence alignment was used to undergo alignments of sequenced samples using GT-4 Reference Sequence with accession number (NC009825.1) from NCBI (National Center for Biotechnology Information)as a background reference sequence and HCV GT-4 cases included in this study as the query sequence.
HCV genotyping of our 20 HCV isolates was tried using the full length NS3/4A, NS5B, and the two third of the carboxy terminal region (including ISDR) of NS5A gene sequences.Full concordance was observed in 18 out of the 20 isolates of which 16 were diagnosed as subgenotype 4a while 2 (10%) were of subgenotype 4n.The remaining 2 isolates were successfully sequenced only in the NS5B region and were diagnosed as subgenotype 4a.
NS3 protease gene was successfully sequenced in 18 (90%) out of the 20 HCV isolates.Two types of mutations were observed with different frequencies.Amino acid frequencies at each position in the NS3 protease sequence were determined with the VESPA software program. 24 Genotype 4 -specific amino acid signatures were present in almost all of our sequences, but were absent from all other genotypes.Among the 24 amino acid signatures only one mutation at position 41 (T/S) reported to be associated with resistance to protease inhibitors and was detected in this study.
The second type of mutations was detected by nucleotide and amino acid sequences of the NS3 region aligned with Clustal X and BIOEDIT version 7.2.5 software and analyzed for the presence of previously reported substitutions conferring resistance to NS3 protease inhibitors among our isolates compared to the wild type HCV GT-4.
Mutations at positions 18, 61, 92, 95, 101, 102, 114, 134, and 150 were detected at a high frequency ranging from 27% to 100%; however, none of these mutations were reported to be associated with resistance to protease inhibitors.
On the other hand, 43 mutations compared to the wild type HCV GT-4 were detected among our isolates at a much lower frequency ranging from 5.5% to 16.6%. Only 5out of them were associated with protease inhibitor resistance.
Mutation at T54S detected in 1(5.5%) out of our 18 isolates was associated with Telaprevir and Boceprevir resistance but not with Simeprevir and Grazoprevir.Mutation at I153L detected in 2 (11.1%) of our isolates was associated with resistance to protease inhibitors.Mutation at D168E detected in 1 (5.5%) of our isolates was reported to confer high level resistance to Simeprevir. Mutation at V170I was detected in 2(11.1%) out of our 18 isolates. Naturally occurring mutation M175L in the NS3 protease region was observed by Costantino et al in 100 % of patients infected with genotype 4.16 (88.8%) out of our 18 strains had the naturally occurring mutation M175L while mutation to P or V was detected in only 2(11.1%) out of our isolates.
We amplified domain II and III including interferon sensitivity-determining region and an interferon/ribavirin resistance-determining region of the NS5a region.Out of our 20 isolates 17gave fairly good sequences that have been aligned with Clustal X and BIOEDIT program and analyzed for the presence of mutations compared to the wild type HCV GT-4 .One of our strain has been used for genotyping but has not been successfully aligned with BIOEDIT program ( due to the poor quality of peaks).All our isolates had a number of mutations exceeding 4 and 82.3% of them had from 10-30 mutations.
The primary NPI resistance variant 282T was not detected in our isolates.Two mutations A179P/T and L289M essential for conferring resistance to Sofosbuvir together with S282T were detected in 95% and 15% of our isolates respectively in absence of S282T. 19 mutations were detected in the NS5B region among our 19 isolates ranging from 55% to 100% as well as less frequently detected mutations (26) ranging from 10% to less than 55% using BIOEDIT program.
Amino acid frequencies at each position in the NS5B sequence were determined with the VESPA software program. 32 Genotype 4 -specific amino acid signatures were present in almost all of our sequences, but were absent from all other genotypes.
None of the mutations reported to be associated with resistance were detected among our isolates, however other neighboring mutations such asV410I/L, I420V and V449I were detected in the present study.
Based on our findings , the large number of natural polymorphism in the NS3 and the NS5B regions (as seen by comparison with other genotypes ) among our HCV 4 isolates as well as the common and isolated mutations (compared to an HCV4 reference strain) make the detection of baseline resistance mutations a difficult task.
Moreover, the mutations detected in our study differed from those associated with DAA resistance cited in the literature either in treatment failure or in DAA naïve patients infected with different genotypes.
The sequence heterogeneity of our Hepatitis C genotype 4 isolates (82.3% of them had from 10-30 mutations) was such that comparison with other non HCV 4 genotypes was not possible using the VESPA software program.
It seems more practical to detect resistance associated mutations in DAA treatment failure then to look for these mutations in naïve patients.