IN SILICO ANALYSIS OF NS5B GENE OF HCV GENOTYPE 4 IN NORTH EGYPT

Background: Hepatitis C is a liver disease caused by the hepatitis C virus (HCV). HCV genotypes and subtypes are related to antiviral therapy response. Our purpose was to identify genotypes and subtypes of HCV isolates collected from Menoufia, Egypt and to analyze the effect of the known drug resistance mutations on the RdRp catalytic domain of HCV-NS5B protein. Methods: Serum of 5 HCV samples


Introduction:-
Hepatitis C virus (HCV) is a major cause of liver diseases in the world (Mohd et al., 2013 andMosaad et al., 2010). HCV genotypes are geographically distributed worldwide, Genotype 4 and subtype 4a are the most dominant in Egypt (Fakhr et al., 2013, Ray et al., 2000and Youssef et al., 2009. HCV belongs to the Flaviviridae family and consists of at least 10 distinct protein products (C, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Lyra et al., 2004 andMoradpour et al., 2007). HCV genotyping is important for prediction of the efficiency of antiviral therapy and for determining treatment duration (Cavalcante and Lyra, 2015). NS5B is a nonstructural protein encoded on the HCV genome, it is an RNA-dependent RNA polymerase (RdRp) responsible for HCV genome replication. Castilho et al. (2011) studied the genetic variability and the genotype's mutations of the RdRp catalytic domain that are typically associated with interferon/ribavirin (IFN/RIB) combination therapy and new non-nucleoside drugs.
In this study, we determined the genotypes and subtypes of HCV isolates, collected from North Egypt (Menoufia) and we detected4 known mutations related to drug resistance as this will influence the expected therapeutic response and could be used as a marker for clinical decisions.

Viral RNA extraction and partial NS5B gene amplification:
The method of Demetriou et al., (2009) was used to extract RNA, primer design and partial NS5B gene amplification. Viral RNA was extracted from plasma samples of HCV-infected patients collected in 2018 from Menufia governorate-Egypt using the QIAmp1 UltraSens1 Virus kit (Qiagen, Venlo, The Netherlands), 15 µl of the RNA was used in a one-step RT-PCR HiSenScript RH (-) RT PreMix kit (intron Biotechnology, Korea), following a heat-shock step at 70°C for 20 sec to denature the RNA secondary structure. RT-PCR was done in a 50 µl reaction with 20 pmol each of the outer sense and antisense degenerate primers correspond to the NS5B region of HCV genome. A nested PCR was performed using 3 µl of the RT-PCR product with 40 pmol each of the inner PCR primers, using 2x PCR master mix (I-Taq) (intron Biotechnology, Korea) in a 50 µl reaction. Amplified product was confirmed using a 2% agarose gel electrophoresis. DNA sequencing was carried out with the ABI 310 capillarity sequencer, sequences were edited using FinchTV version 1.4.0.

Sequence analysis:
Sequence similarity search was done using BLAST programs from National Center for Biotechnology Information (NCBI), USA, (http://www.ncbi.nlm.nih.gov/Blast). HCV isolates correspond to different genotypes and subtypes were obtained from GenBank database (NCBI). Sequence comparison between our HCV isolates and those of GenBank database was used to detect drug resistance mutations and to construct Neighbor-Joining phylogenetic tree.

GenBank Submission:
NS5B sequences were submitted to GenBank under the accession numbers MN068031-MN068035.

Phylogenetic analysis:
Molecular Evolutionary Genetics Analysis (MEGA) software version MEGA6 was used to construct the phylogenetic tree (Tamura et al., 2013).

Analysis of RdRp catalytic domain of NS5B protein:
PROSITE is a protein database for identifying protein domains, families and functional sites as well as associated patterns and profiles (Sigrist et al., 2012). ScanProsite tool was used to predict the effect of the drug resistance mutations on the RdRp catalytic domain of NS5B protein.

Amplification of partial HCV NS5B gene and sequence analysis:
A total of 5 serum samples of HCV-infected patients collected in 2018 from Menufia governorate-Egypt were used to amplify HCV NS5B gene. PCR amplification of partial sequence of NS5B gene resulted in a product of 500bp, then PCR product of the 5 isolates was sequenced and the sequences were edited using FinchTV. Using BLAST,our isolates showed 88.59%-95.11% identity and 91.80%-99.18% similarity to NS5B gene of HCV isolates in NCBI database for nucleotide and amino acid sequence, respectively.

Effect of the drug resistance mutationson RdRp catalytic domain of NS5B protein:
PROSITE database was used to analyze the effect of the detected drug resistance mutations on the NS5B-RdRp catalytic domain (Fig 2). ScanProsite tool recognized the full length of RdRp catalytic domain (P 214 -D 332 ) of the reference isolate 430(accession number AUF37153.1) (Fig. 2a). Similarly, the prediction of the partial NS5B-RdRp catalytic domain of our isolates and those representHCV genotypes (represented in Fig. 1) confirm that no effect of the detected drug resistance mutations on the NS5B-RdRp catalytic domain (data not shown).The predicted partial RdRp catalytic domain (C 243 -D 332 ) of NS5B protein of isolate Menoufia-1 is shown in Fig. 2b.

Phylogenetic classification of HCV isolates based on NS5B protein sequence:
To identify genotypes and subtypes of HCV isolates,our HCV isolates and those represent genotypes and subtypes of HCV-NS5B, retrieved from GenBank database were used to construct the phylogenetic tree (Fig. 3). Phylogentically, our isolates were grouped with HCV isolates of genotype 4 (Fig. 3a). In Fig. 3b, isolates Menoufia-1, 2, 4 and 5 were clustered with subtype 4a and showed the lowest divergence (0.00%-0.044) with 6 isolates from Egypt and one isolate from France. However isolate Menoufia-3 was clustered with subtype 4c where divergence was 0.00% with 2 isolates from Gabon, and one isolate from Canda.

Discussion:-
It is important to classify HCV genotypes, since HCV genotypes respond differently to several treatment regimens (Kumar et al., 2018 andOmata et al., 2012). The objective of this study was to classify HCV isolates collected from HCV-infected patients from Egypt based on HCV NS5B gene and to predictthe presence and the effect of the known drug resistance mutations on the RdRp catalytic domain of NS5B protein.
As reported previously, genotype 4a is the most predominant HCV genotype in Egypt (Fakhr et al., 2013, Rost et al., 2004and Youssef et al., 2009). This was confirmed in the current study, in which genotype 4a was observed in 4/5 of our isolates (Menoufia-1, 2, 4 and 5). Isolate Menoufia-3 was clustered with subtype 4c from Gabon; similarly, Mahmoud and Hashem (2012) found a close relationship between an Egyptian isolate of HCV from Sohag and an isolate from Gabon. It is possible that subtype 4c circulates in Egypt and originated from Gabon (Central Africa). Iles et al. (2014) reported that genotype 4 originated in central Africa before disseminating elsewhere.
Four known drug-resistance mutations (D244N, Q309R, D310N and C316N) were detected in the analyzed HCV genotypes. D244N, Q309R and D310N were highly represented in genotype 3 in comparison to the other genotypes (80% for D244N and 100% for both Q309R and D310N). It was noticed that D244N was detected only in the isolates of genotype 3; in a study by Asahina et al., (2005), D244N was mainly represented in subtype 3a. Similarly, castilho et al., (2011) analyzed mutations related to non-response to IFN/RIB observed in subtypes 1a, 1b, 2b, 3a and 4 and found that D244N was detected only in subtype 3a. We detected Q309R with frequencies 40%, 100%, 10% and 50% for genotype 1, 3, 4 and 5, respectively); Q309R mutation was the most common mutation related to non-response to IFN/RIB in genotypes 1a, 1b, 3a and 4, except subtype 2b with frequency above 20% (castilho et al., 2011). Also, D310N was found to be associated with IFN/RIB resistance (Asahina et al., 2005). In our study, C316N was detected only in genotype 2 (10%), in castilho et al., (2011) study, it was observed in only 24% of isolates subtype 1b. The codon 316 implicated with re¬sistance to new NS5B inhibitors; C316N increased the replication fitness by 1.6-fold using AG-021541 inhibitor (Shi et al., 2008, Dutartre et al., 2006and McCown et al., 2009). In our study, the S282T resistance substitution was not detected in any of the analyzed genotypes which was previously confirmed by castilho et al., (2011). Although S282T has been associated with NS5B polymerase inhibitor Sofosbuvir (SOF) resistance in vitro, it has very rarely been detected, both before and after SOF-based treatment failure, mainly because it seems to be a highly unfit substitution that is usually only detectable for very short periods of time (Dietz et al., 2018). Detection of drug-resistance mutations in HCV infected-patients before treatment indicates that the detection of these mutations could be used as a marker for clinical decisions. When a patient has a confirmed increase in HCV RNA levels while adhering to therapy, implying development of resistance, antiviral drug should be quickly discontinued to prevent evolution of viral population (Strahotin and Babich 2012).
Functional analysis of NS5B protein indicated that the detected drugresistance mutations did not alter the RdRpcatalytic domain of NS5B protein in all genotypes and in our isolates. Nachappa et al., (2018) did not find change in the motifs identified in the RpoB and KatG wild-type protein sequences compared with the RpoB Ser531Leu and KatG Ser315Thr mutants.However, single nucleotide polymorphism detected in the human proteome resulted in domain alteration led to loss of connectivity of cell signaling pathways (Liu and Tozeren 2010).
In conclusion, the data suggest that genotype 4a is still the predominant genotype in Egypt. Furthermore, the study indicates that isolate Menoufia-3, classified ad subtype 4c, could be introduced from Central Africa as new subtype of genotype 4 in Egypt. Also, the detection of NS5B drug-resistance mutations in HCV infected-patients before treatment could be used as a marker to determine the suitable treatment.