Phylogenetic analysis of the 5ʹ untranslated region of HCV from cirrhotic patients in Khyber Pakhtunkhwa, Pakistan

Hepatitis C virus (HCV), a small, single-stranded RNA virus with a 9.6 kb genome, is one of the most common causes of liver diseases. Sequencing of the 5ʹ untranslated region (UTR) is usually used for HCV genotyping, but it is less important in numerous subtypes due to its scarce sequence variations. This study aimed to identify genotypes using the 5ʹ UTR of HCV from cirrhotic patients of Khyber Pakhtunkhwa (KP). Serum RNA samples (44) were screened by real time PCR to determine the HCV viral load. Nested PCR was performed to identify cDNA and the 5ʹ UTR. The HCV 5′ UTR was sequenced using the Sanger method. MEGA-7 software was used to analyze evolutionary relatedness. After 5ʹ UTR sequencing, 26 samples (59%) were identified as genotype 3, and 2 samples (6%) were identified as genotypes 1, 2 and 4. The most predominant genotype was 3a, and genotype 4 was rarely reported in the phylogenetic tree. Analysis of the HCV 5ʹ UTR is an efficient alternative method for confirmation of various genotypes. Phylogenetic analysis showed that genotype 3 was dominant in the area of KP, Pakistan.

www.nature.com/scientificreports/ makes the 5ʹ UTR the region of choice for performing (RT)-PCR detection tests, such as the HCV amplicor test 12 . A number of genotyping schemes have been established and utilized in this region to obtain phylogenetic genotype information 13,14 . Sequencing data of the 5ʹ UTR and other regions, such as the NS-3, NS-4, core and NS-5, have been used in phylogenetic studies and genotyping of HCV [15][16][17] . Geographically, HCV genotype 1 is prevalent in Europe, Japan and the USA 18 , whereas genotype 2 is found in Korea and Taiwan, and genotype 3 is detected in Pakistan, India and Thailand. Genotype 4 is the most frequent genotype in Saudi Arabia, Egypt, Syria, Iraq, Vietnam and Lebanon. Genotype 5 is found in South Africa, and genotype 6 is found in Vietnam [19][20][21] . The current study focused on the analysis of 5′ UTR sequencing and identification of genotypes by comparison with reference genotypes.

Materials and methods
RNA isolation and cDNA preparation. All samples were collected from HCV cirrhotic patients treated at the Hayatabad Medical Complex (HMC) and Khyber Teaching Hospital (KTH), Peshawar, during January 2017-May 2018. The current study was approved by the competent authorities of the institute, and the whole study was carried out according to the ethical guidelines given by the institute. RNA was isolated from serum by a QIAamp viral RNA kit in accordance with the manufacturer's protocol and stored at − 80 °C.
The cDNA was formed using 1 µl outer antisense primer (OAS), 10 µl RNA, 1 µl (200 U) M-MLV reverse transcriptase enzyme (BIORON Life Science cDNA Kit), 4 µl complete RT buffer, 2.5 µl PCR water, 1 µl dNTPs (10 mM) and 0.5 µl RNA inhibitors with a total volume of 20 µl. The following temperature cycle was then applied: 37 °C/60 min, 70 °C/10 s, and 22 °C/∞. The outer antisense primer was the reverse primer used in the first round of nested PCR amplification of the HCV 5ʹ UTR. megas oftwa re. net) was used to sequence the 5′ UTR, sequences were aligned by the maximum likelihood method and associated with the reference sequence of the identified genotype. The neighbor-joining algorithm of MEGA 7 was used to calculate the p-distance and the differences in each nucleotide sequence. The phylogenetic tree analysis utilized 1000 bootstrap resampling to test the robustness of the observed dominant clades.

Statistical analysis. Statistical analysis was performed by IBM SPSS version 25, Windows 7 and Microsoft
Excel version 13.
Ethics approval. This study was approved by the Ethics Committee, Centre of Biotechnology and Microbiology, University of Peshawar, Pakistan. Written informed consent was obtained from all the individuals who participated in the study.

Results
HCV cirrhotic samples (n = 44) were collected on the basis of their demographic variables. The average age and standard deviation (SD) of the patients was 48.69 ± 11.28 yrs. The numbers of male and female patients were n = 25 and 19, respectively. The highest viral load was recorded prior to and during the treatment > 200,0000 IU/ mL, as shown in Table 2. A high number of HCV cirrhotic patients were identified or noted between the ages of 31-50 ( Fig. 1). Out of all the patients (n = 44), sequencing was successful for samples from 32 patients, and the remaining 12 (8 mild, 2 moderate and 2 gross) patients had sequences that showed less similarity during BLAST because of their small size/nucleotide bp and hence were excluded. The sequence alignment of the 5ʹ-UTR isolates (entitled Hepacivirus C isolate AIMZ7 KP 5ʹ-UTR) was performed with reference genotype (1-7) sequences from the database. The well-conserved areas and few nucleotide substitutions in the 5ʹ-UTR of the HCV genome are shown in Fig. 2. The data also revealed that the length of the 5ʹ-UTR of HCV was up to 183 nucleotides. In the phylogenetic tree, most of the isolates were in a clad and clustered perfectly with the reference sequence. The two isolates from the group did not cluster with the reference sequence. The phylogenetic tree indicated that most of the isolates clustered with subtypes 3a and 3b, and few of them clustered with other reference genotype sequences (Fig. 3).
Sequencing of the HCV 5ʹ-UTR was performed with a forward primer (red color), and the nucleotide similarity of the 5ʹ-UTR sequence was checked by using multiple alignment fast Fourier transform methods (MAFFT version 7 software: https:// mafft. cbrc. jp/ align ment/ server/ large. html). All the sequences show significant similarity with sample and sequence no. 22, and sequence 28 shows significant similarity with sequence no. 1, as shown in Fig. 4.

Discussion
Hepatitis C virus (HCV) has a broad range of genotypes and quasispecies due to frequent genetic mutations in its RNA. The identification of HCV genotypes through direct sequencing of the 5ʹ-UTR (Fig. 5) is a great technique because it does not require pattern processing steps and uses amplified products obtained from onestep, nonnested PCR. Additionally, direct sequencing of PCR products provides more comprehensive sequence information than different genotyping analyses. Different studies have reported that the HCV 5ʹ-UTR of 324 to www.nature.com/scientificreports/ 341 nucleotide sequences is the most conserved region 22 and can be used for the sequence-based identification of HCV genotypes [17][18][19][20][21][22][23] .
The purpose of the current research work was to identify different HCV genotypes from the 5ʹ UTR isolated from patients of the Khyber Pakhtunkhwa (KP) region of Pakistan. The results showed that different genotypes were present in the phylogenetic tree, and genotype 3 was noted as the most predominant. Genotype 3 (a, b) was reported in 26 (59%) isolates of the phylogenetic tree and coincided with the position of each genotype 1,  www.nature.com/scientificreports/ 2, and 4. Sequence conservation in the 5ʹ-UTR HCV genome is vital for the detection of genotypes by sequence evaluation from samples that are not typed with more generally used assays. In the geographic area of Pakistan, HCV genotypes have varied distributions. Genotype 3 was detected more than the other genotypes in this area, as clearly stated in the study of Idress and Riazudin 24 . Another finding of the study was that genotypes 1 and 2 are also found in this area. Genotypes 1 and 2 are mostly found in Europe and East Asian countries. The detection of these genotypes in KP revealed the immigration of people from one state to another 25 . The rise of genotypes 1 and 2 also causes major problems during treatment, as these patients show less response to antiviral drugs 26 . The occurrence of genotype 2 was also previously reported but was very rare in the local province and neighboring states of Pakistan 24 .
One of the main important findings of our results was that genotype 4 was found very rarely in our state. Recently, HCV genotype 4 has spread in parts of Europe due to variations in the populace structure, ways of transmission and migration. The characteristics of genotype 4 infection and proper therapeutic programs are not well described 27 . Genotype 4 is mostly found in Northern Africa and the Middle East (Egypt, Iran). As these are neighboring countries of Pakistan, immigration could be a factor [28][29][30] . In general, in this study, the number of samples was small, and available funding was limited. Further study with a larger number of samples is necessary to completely identify the genotypes of this region.

Conclusion
The study concludes that genotype 3a is the predominant genotype of HCV in the Khyber Pakhtunkhwa region, Pakistan. Direct sequencing of the 5ʹ UTR is a valuable method for clinical detection of different HCV genotypes. Genotype 4 was rarely detected in the tribal area of Khyber Pakhtunkhwa. I am very thankful to the gastroenterology laboratories of tertiary hospitals of Peshawar and the diagnostic laboratory of the Centre of Biotechnology and Microbiology University of Peshawar, Pakistan. I am highly thankful to the Institute of Pathology, in particular to the Laboratory of Translational Molecular Pathology (University Hospital of Cologne Germany) and Prof. Dr. Margarete Odenthal for the helpful discussions and in proofreading of the manuscript.  www.nature.com/scientificreports/ Figure 4. Sequencing of the HCV 5ʹ UTR was performed with a forward primer (red color), and the nucleotide similarity of the 5ʹ UTR sequence was checked by using multiple alignment fast Fourier transform (MAFFT version 7 software: https:// mafft. cbrc. jp/ align ment/ server/ large. html). All the sequences show significant similarity with sample 1, and sequence no. 22 shows significant similarity with sequences no. 1 and 28.