Journal of Medical Virology
Phylogenetic Analysis of Hepatitis C Virus Strains and Risk Factors Associated With Infection and Viral Subtypes Among Iranian Patients Faraz Salehi Moghadam,1,2 Seyed Reza Mohebbi,1* Seyed Masoud Hosseini,2 Sara Romani,1 Hanieh Mirtalebi,1 Pedram Azimzadeh,1 Behzad Damavand,1 Hamed Naghoosi,1 Mahsa Khanyaghma,1 Azar Sanati,1 and Mohammad Reza Zali1 1 2
Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran
Hepatitis C virus (HCV) has infected approximately 170 million people worldwide. While the seroprevalence of anti-HCV antibody among Iranian blood donors is 0.13%, HCV infection is prevalent in 59–80% of Iranian injecting drug users. One hundred seventyeight anti-HCV positive patients were referred to the Gastroenterology Department at the Taleghani Hospital (Tehran, Iran) between June 2007 and June 2012. Out of 178 samples, 142 were positive for HCV-RNA. HCV subtypes were determined using phylogenetic analysis of the NS5B or 50 UTR/core regions. Of 142 viremic patients, 71 (50%) were infected with HCV subtype 1a, 43 (30.3%) with subtype 3a, 20 (14.1%) with subtype 1b, 3 (2.1%) with subtype 4d, 2 (1.4%) with subtype 4a, 1 (0.7%) with subtype 2b, and 1 (0.7%) with subtype 6a. Interestingly, genetic analysis of a sub-genomic fragment from one patient identified a non-subtypeable HCV genotype-3 strain. There was a significant association between HCV subtype and a history of injecting drug use (P ¼ 0.003). Subtype 3a was predominant among patients with such a history. Injecting drug use was associated with younger age (P < 0.001). HCV subtype was also significantly associated with a history of upper gastrointestinal endoscopy (P ¼ 0.02). Subtype 1a was more frequent among patients with such a history. In addition, history of upper gastrointestinal endoscopy was significantly associated with older age (P ¼ 0.002). In conclusion, while HCV subtype 1a is predominant among infected Iranian individuals, subtype 3a is predominant among Iranian injecting drug users. J. Med. Virol. # 2014 Wiley Periodicals, Inc. C 2014 WILEY PERIODICALS, INC.
KEY WORDS:
hepatitis C virus; genotype; subtype; phylogenetic analysis; molecular epidemiology; Iran
INTRODUCTION Hepatitis C virus (HCV) is one of the main causes of chronic liver disease and liver transplantation [Alter, 1997]. Twenty to thirty percent of chronic hepatitis C cases will develop cirrhosis and/or hepatocellular carcinoma after 20–30 years [Heintges and Wands, 1997]. According to the World Health Organization (WHO), HCV has infected approximately 170 million people worldwide [Lavanchy, 2009]. According to the Iranian Blood Transfusion Organization, the seroprevalence of HCV among Iranian blood donors is 0.13% [Amini Kafi-abad et al., 2009]. Due to the efficient blood screening program adopted since 1996, HCV transmission through blood or blood products has reduced significantly in Iran [Rezvan et al., 2007]. The prevalence of HCV infection among intravenous drug abusers is as high as 59–80% [Kheirandish et al., 2009; Zamani et al., 2010; Samimi-Rad et al., Grant sponsor: Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Correspondence to: Seyed Reza Mohebbi, Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, 7th floor of Taleghani Hospital, Velenjak, Tehran, Iran. E-mail: [email protected] Accepted 28 March 2014 DOI 10.1002/jmv.23947 Published online in Wiley Online Library (wileyonlinelibrary.com).
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2012]. This indicates that similar to many other countries, intravenous drug abuse has become the major route of HCV transmission in Iran. HCV is an enveloped single stranded positive sense RNA virus which belongs to the Hepacivirus genus of the Flaviviridae family. The viral genome is about 9.6 kb in length and contains a single open reading frame (ORF) encoding a polyprotein precursor of about 3,000 amino acids [Moradpour et al., 2002]. To date, six major genotypes and more than eighty different subtypes have been identified for HCV. Furthermore, one strain has been proposed as a candidate for the seventh genotype. While different genotypes have the similarity of around 60–70% in their nucleotide sequences, the similarity of different subtypes of a genotype is around 75–85% [Nakano et al., 2012]. HCV genotypes have different geographical distribution patterns. In Iran, the predominant subtype is 1a followed by 3a and 1b [Samimi-Rad et al., 2004; Kabir et al., 2006; Keyvani et al., 2007; Jahanbakhsh Sefidi et al., 2013]. Among neighboring countries, genotypes 1b, 3, and 4 are predominant in Turkey, Pakistan, and Arab countries, respectively [Bozdayı et al., 2004; Ramia and Eid-Fares, 2006; Idrees and Riazuddin, 2008; Idrees et al., 2009]. There are few phylogenetic studies that have been carried out on HCV strains obtained from Iranian patients. Most of the previous studies were performed on one particular group of patients (e.g., injecting drug users or patients with thalassemia). Also, there were not sufficient data on the statistical associations between different risk factors and HCV subtype in Iran. In this study, molecular epidemiology of HCV strains obtained from infected individuals with chronic hepatitis or cirrhosis of the liver was investigated using phylogenetic analysis of the NS5B and 50 UTR/ core regions. Furthermore, associations of HCV subtype with age, sex, and the history of exposure to various risk factors were assessed. MATERIALS AND METHODS One hundred seventy-eight Iranian individuals with positive anti-HCV antibody were referred to the Gastroenterology Department at the Taleghani Hospital (Tehran, Iran) between June 2007 and June 2012. Study participants were from various ethnicities (Fars: 52.5%, Turk: 22.8%, Lor: 8.9%, Kurd: 6.9%, Gilaki: 6.9%, Armenian: 1%, and Arab: 1%). Informed consent was provided by each participant. The study protocol was approved by the Ethics Committee at the Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Serological tests for hepatitis B virus (anti-HBc antibody and HBs antigen) and human immunodeficiency virus (anti-HIV antibody) were performed using enzyme-linked immunosorbent assay (DIA PRO Diagnostic Bioprobes, Milan, Italy). J. Med. Virol. DOI 10.1002/jmv
HCV RNA was extracted from plasma samples using QIAamp Viral RNA mini kit (QIAGEN, Hilden, Germany). Complementary DNA synthesis was performed using Random Hexamer primers and RevertAid Reverse Transcriptase enzyme (Fermentas, Vilnius, Lithuania). Polymerase chain reaction (PCR) was used for HCV-RNA detection. A partial segment of the NS5B region was amplified using semi-nested PCR with primers and thermal conditions described elsewhere [Norder et al., 1998; Kalinina et al., 2001]. Briefly, primers hep-101 and hep-120 were used to amplify a 463-bp segment (corresponding to nucleotides 8,260–8,722 in the H77 reference sequence, accession number AF009606) in the first-round PCR, and primers hep-101 and hep105 were used to amplify a 380-bp segment (nucleotides 8,260–8,639) in the second-round PCR. If amplification with the mentioned sets of primers was not successful for a sample, primers hep-106 and hep-105 were used alternatively in the first-round PCR to amplify a 473-bp segment (nucleotides 8,167–8,639). The NS5B region of one strain with subtype 6a could not be amplified using the mentioned primers. For this strain, subtype-specific primers were designed: primers HCV6a-F1 and HCV6a-R1 were used for the first-round PCR and primers HCV6a-F2 and HCV6a-R2 were used for the second-round PCR. The sizes of the first- and second-round PCR amplicons were 535 (nucleotides 8,131–8,665) and 389 bp (nucleotides 8,210–8,598), respectively. Thermal cycling conditions for both first- and second-round PCRs were 5 min at 95˚C, followed by 35 cycles of 1 min at 94˚C, 45 sec at 54˚C and 45 sec at 72˚C. Final elongation step was performed for 7 min at 72˚C. Samples with negative results for the NS5B amplification were subjected to another PCR targeting the 50 UTR/core junction (a 681-bp segment corresponding to nucleotides 51–731) with primers NCR-F and NCR-R. The reaction conditions were described previously [Salehi Moghadam et al., 2013b]. If the result was negative for a sample, 3 ml of the PCR product was used as template and a new PCR was carried out with the same primers and thermal conditions. Table I shows sequences and positions of the primers used in this study. Besides, to assess the suitability of the selected positions of the 50 UTR/core region for accurate subtyping, 23 (16%) samples were randomly selected and amplified in both 50 UTR/core and NS5B regions and the results were compared. PCR products were direct-sequenced bi-directionally, and sequences were edited using BioEdit version 7.0.5.3. The NS5B sequences together with the corresponding regions of 85 other strains obtained from the Los Alamos or GenBank databases were aligned using ClustalX version 2.0.12 [Thompson et al., 1997]. The same approach was adopted for the 50 UTR/core sequences with 37 strains from the databases. Phylogenetic trees for the NS5B and 50 UTR/core regions
Molecular Epidemiology of Hepatitis C Virus in Iran
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TABLE I. Sequences and Positions of the Primers used in this Study Target region NS5B
50 UTR/core a
Primer name hep-101 hep-120 hep-106 hep-105 HCV6a-F1 HCV6a-R1 HCV6a-F2 HCV6a-R2 NCR-F NCR-R
Primer sequence 0
Position 0
5 -ATACCCGCTGCTTTGACTC-3 50 -TGCGCGACBGABACRTTKGAGGA-30 50 -GCTCNTAYGGRTTYCARTACTC-30 50 -ATACCTAGTCATAGCCTCCGTGA-30 50 -RGCATACGGATTCCAGTAC-30 50 -YGAGCAYGATGTTATGAGC-30 50 -GGCTTTTCTTACGACACCA-30 50 -CTGGTCATAGCMTCCGTGA-30 50 -GGAACTACTGTCTTCACGCAGAAAGC-30 50 -GAAGCCGCACGTAAGGGTATCG-30
8,260 8,722 8,167 8,639 8,131 8,665 8,210 8,598 51 731
a
Reference Norder et al. [1998] Kalinina et al. [2001] Kalinina et al. [2001] Norder et al. [1998] This study This study This study This study Salehi Moghadam et al. [2013b] Salehi Moghadam et al. [2013b]
Nucleotide positions are relative to the start of the H77 reference sequence (AF009606).
were constructed using the Kimura two-parameter algorithm [Kimura, 1980] with the neighbor-joining method. Genetic distances were also estimated using the Kimura two-parameter algorithm. MEGA software version 5 was used for the analyses [Tamura et al., 2011]. Associations of sex, age, and exposure to various risk factors with HCV subtype were assessed using the x2 test. The independent Student’s t-test was used to analyze continuous variables. P < 0.05 was considered as the level of significance. Statistical analyses were performed using the Statistical Package for the Social Sciences version 16 (SPSS, Inc., Chicago, IL). RESULTS Of 178 anti-HCV positive individuals, 146 (82%) were male and 32 (18%) were female. Two (1.1%) patients were positive for anti-HBc antibody but not for HBsAg. No patient was anti-HIV-seropositive. One hundred forty-two (79.8%) samples were positive for HCV-RNA. The mean age of the viremic patients was 46.3 13.5 (range 22–82 years). Of 142 viremic patients, 121 (85.2%) were male (mean age 44.9 13.9) and 21 (14.8%) were female (mean age 52.7 9.6). The mean alanine aminotransferase and aspartate aminotransferase levels were 61.08 50.5 and 51.8 35.5 IU/L, respectively. According to their clinical condition, 124 (87.3%) patients were diagnosed with chronic hepatitis. Of these, 106 (85.5%) were male (mean age 43.6 13.9) and 18 (14.5%) were female (mean age 51.9 9.7). Eighteen (12.7%) patients were diagnosed with cirrhosis of the liver. Of these, 15 (83.3%) patients were male (mean age 52.7 10.9) and 3 (16.7%) were female (mean age 57.3 8.5). Of 15 cirrhotic male patients, 6 were infected with subtype 1a, 6 with subtype 1b and 3 with subtype 3a. All three cirrhotic females were infected with HCV subtype 3a. Subtype 1a was predominant among patients over 40 years of age, followed by 3a (53.2% and 26.6%, respectively). Among patients under 40, however, subtypes 1a and 3a had the same frequencies (40.9%).
Of 142 HCV-RNA positive samples, 137 (96.5%) could be amplified in the NS5B region. In the remaining 5 (3.5%) samples, only 50 UTR/core junction could be amplified. Besides, to assess the suitability of the selected 50 UTR/core segment for precise subtype determination, 23 (16%) samples were randomly selected and amplified in both 50 UTR/core and NS5B regions. No inconsistency was observed in these 23 samples. All of the 142 samples were sequenced directly, though due to the short sequencing output, 3 NS5B sequences with subtype 1a could not be included in the dendrogram. All sequences were deposited in the NCBI GenBank under the accession numbers KC285197–KC285362, KF218585, and JQ341409. Based on the phylogenetic analysis of the NS5B and 50 UTR/core sequences, of 142 HCV-RNA positive patients, 71 (50%) were infected with HCV subtype 1a, 43 (30.3%) with subtype 3a, 20 (14.1%) with subtype 1b, 3 (2.1%) with subtype 4d, 2 (1.4%) with subtype 4a, 1 (0.7%) with subtype 2b, and 1 (0.7%) with subtype 6a. The subtype of one strain (0.7%) could not be determined. According to the phylogenetic tree of the 50 UTR/core region, this strain belonged to HCV genotype 3 (Fig. 1). In the phylogenetic tree constructed based on the 50 UTR/core sequences, one strain (FSM16; GenBank accession number KF218585) within the clade 3 was separated from other subtypes with the bootstrap value of 92% over 1,000 replicates (Fig. 1b). For this strain, phylogenetic analysis was limited to HCV genotype 3 (Fig. 2). In the new tree, strain FSM16 was classified in a separate branch between subtype 3h and an unassigned subtype recently reported from Canada (GenBank accession number JF735124) [Lu et al., 2013]. Based on the nucleotide sequences corresponding to positions 83–715 in the H77 reference sequence, FSM16 had the mean intra-genotypic distances of 6.8% and 7.1%, respectively from subtype 3h and the Canadian strain with an unassigned subtype. To include a new subtype of genotype 3 that was previously reported by Amini et al. [2006], in the analysis, the genetic distances were also calculated based on the positions 142–315, as the only sequence of the new subtype which corresponded to the FSM16 J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Phylogenetic trees constructed based on (a) a 254-base segment of the NS5B region corresponding to positions 8330 to 8583 in the H77 reference sequence (AF009606), and (b) a 473-base segment of the 50 UTR/core region corresponding to positions 207 to 679, using the neighbor-joining method. The HCV strains that were obtained in this study are indicated by gray triangles. Reference sequences are shown with their GenBank accession numbers, countries of isolation and subtypes. Numbers at the nodes show the percentages of bootstrap values (1,000 replicates).
J. Med. Virol. DOI 10.1002/jmv
Molecular Epidemiology of Hepatitis C Virus in Iran
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Fig. 2. Phylogenetic analysis of strain FSM16 and the representatives of various subtypes of HCV genotype 3, based on 633 nucleotides of the 50 UTR/core region (corresponding to positions 83–715 in the H77 reference sequence) using the neighbor-joining method. Note that the only available 50 UTR/core sequence of the tentative subtype 3l in the GenBank, corresponded to a short segment (174 nucleotides) of the FSM16 sequence and thus, subtype 3l could not be included in the tree. The subtype of the sequence JF735124 has not yet been assigned. Numbers at the nodes represent the percentages of bootstrap values over 1,000 replicates.
sequence was related to these positions. The FSM16 strain had the same mean distance of 1.15% from subtype 3h, the unassigned subtype reported from Canada and the new subtype reported from Iran (DQ202322). Eleven patients had a history of a single risk factor. The remaining had a history of exposure to more than one risk factor. The most common risk factors among the patients were periodontal procedure and surgery, respectively with 53 and 52 patients in each group, compared with only 2 patients with high-risk sexual behavior. Since the frequencies of patients infected with other subtypes were too
small to be statistically analyzed, analyses were only focused on subtypes 1a and 3a. Table II shows associations of HCV subtype with age, sex, and risk factors that the patients had been exposed to. There was a significant association between HCV subtype and a history of injecting drug use (P ¼ 0.003). Subtype 3a was predominant among patients with such a history. Furthermore, history of injecting drug use was associated with younger age (P < 0.001). HCV subtype was also significantly associated with a history of upper gastrointestinal endoscopy (P ¼ 0.02). Subtype 1a was more frequent among J. Med. Virol. DOI 10.1002/jmv
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TABLE II. Associations of HCV Subtype with Age, Sex, and History of Various Risk Factors HCV subtype
Age 40 >40 Sex Male Female Transfusion No Yes Cupping No Yes Tattooing No Yes Periodontal procedures No Yes Shared shaving razor No Yes UGI endoscopy No Yes Injecting drug use No Yes Surgery No Yes Hemodialysisb No Yes High-risk sexual behaviorb No Yes Needle stickb No Yes Unknown risk factor
1a, No. (%)
3a, No. (%)
18 (50) 42 (66.7)
18 (50) 21 (33.3)
61 (62.2) 10 (62.5)
37 (37.8) 6 (37.5)
37 (59.7) 12 (60)
25 (40.3) 8 (40)
31 (58.5) 18 (62.1)
22 (41.5) 11 (37.9)
36 (58.1) 13 (65)
26 (41.9) 7 (35)
25 (62.5) 24 (57.1)
15 (37.5) 18 (42.9)
37 (57.8) 12 (66.7)
27 (42.2) 6 (33.3)
28 (50.9) 21 (77.8)
27 (49.1) 6 (22.2)
39 (70.9) 10 (37)
16 (29.1) 17 (63)
21 (52.5) 28 (66.7)
19 (47.5) 14 (33.3)
48 (60) 1 (50)
32 (40) 1 (50)
48 (60) 1 (50)
32 (40) 1 (50)
45 (59.2) 4 (66.7) 22 (31)
31 (40.8) 2 (33.3) 10 (23)
P-value 0.103 0.984 0.98 0.752 0.582 0.621 0.499 0.02a 0.003a 0.191 — — — —
a
Statistically significant. The frequency of the risk factor among HCV-1a and -3a infected patients was not adequate to be statistically analyzed. b
patients with such a history. In addition, history of upper gastrointestinal endoscopy was significantly associated with older age (P ¼ 0.002). DISCUSSION The prevalent subtype among the patients was 1a followed by subtype 3a. This is in agreement with the results of previous studies from Iran [Samimi-Rad et al., 2004; Kabir et al., 2006; Keyvani et al., 2007; Amini et al., 2009; Jahanbakhsh Sefidi et al., 2013]. Additionally, in this study two rare HCV subtypes were identified. The first one was HCV subtype 6a. This specific case was previously discussed in detail [Salehi Moghadam et al., 2013b]. The second one, strain FSM16, was genetically more close to subtype J. Med. Virol. DOI 10.1002/jmv
3h and also to a subtype isolated from a Middle Eastern patient residing in Canada. The latter was reported to be a new subtype but has been remained unassigned as of the time of the preparation of this article [Lu et al., 2013]. In recent years, a new subtype of HCV genotype 3 has been isolated from Iranian patients and it has been proposed to provisionally assign this subtype as “3l” [Amini et al., 2006; Salehi Moghadam et al., 2013a]. The relatedness of this new subtype and the strain FSM16 could not be shown in this study, as the only available 50 UTR sequence of the new subtype had the same genetic distance of 1.15% from the strain FSM16, subtype 3h and a new subtype reported recently from Canada. For accurate subtype determination of FSM16, sequences from other parts of the genome were needed. As described previously, nucleotide sequencing of the NS3 region following by phylogenetic analysis can be used for HCV subtype determination [Colson et al., 2011]. With strains that seem to belong to a new subtype, however, phylogenetic analysis of both core/E1 and NS5B regions or preferably the complete genome sequence will be required prior to subtype assignment [Simmonds et al., 2005]. Due to the limited amount of the plasma sample, it was impossible to characterize other parts of the genome for FSM16. Prior to this report, there was no Iranian study that phylogenetically analyzed subtypes of genotype 4. This is mainly due to the fact that few Iranian studies used direct sequencing method and phylogenetic analysis of the nucleotide sequences to investigate HCV epidemiology in Iran. Apart from the HCV4 sequences obtained in this study, there have been 11 HCV-4 sequences deposited in the HCV database. Of these, subtypes of three sequences were not determined. The remaining eight sequences were from a previous study [Samimi-Rad et al., 2004]. Although in the original report, subtypes of these eight strains were not determined, seven sequences are classified as subtype 4d and one is classified as subtype 4a in the Los Alamos HCV database. In the current study, three strains with subtype 4d and two with subtype 4a were identified. It should be mentioned that among the Middle Eastern countries, subtype 4a is predominant in Egypt, whereas 4c/4d are the most frequent subtypes in Saudi Arabia [Kamal and Nasser, 2008]. Although genotype 4 has a low prevalence among infected Iranian individuals, by considering the frequencies of HCV-4 strains mentioned above, two facts reveal: (1) At least two subtypes of genotype 4 including subtypes 4a and 4d are responsible for HCV-4 infections in Iran; (2) Subtype 4d is predominant among Iranian HCV-4 infected patients. This is not only important from the epidemiological point of view but it might be important clinically because according to a retrospective study, HCV-4a infections are associated with higher SVR rates than those of HCV-4d [Kamal and Nasser, 2008].
Molecular Epidemiology of Hepatitis C Virus in Iran
In this study, it was observed that HCV subtype was significantly associated with a history of injecting drug use. Subtype 3a was more frequent among the patients with such a history. Predominance of subtype 3a among Iranian injecting drug users was also reported in a recent study, though no associations between HCV genotype and injecting drug use characteristics (being a current or former, short-term or long-term injecting drug user) were detected in the mentioned study [Samimi-Rad et al., 2012]. Results of the current study showed that in comparison with the older patients, HCV subtype 3a was more frequent among the patients under 40. In fact, in the younger subgroup of patients, subtypes 1a and 3a had the same frequencies. It was also observed that history of injecting drug use was associated with younger age (P < 0.001). These findings are in agreement with the previous reports from Iran [Samimi-Rad et al., 2012; Jahanbakhsh Sefidi et al., 2013], France [Payan et al., 2005], Germany [Ross et al., 2000], Serbia and Montenegro [Svirtlih et al., 2007], and Slovenia [Seme et al., 2009]; and highlight the fact that intravenous drug abusers have become the major source of HCV infection in Iran. On the one hand, due to the successful harm reduction strategies adopted since 1996, HCV transmission through blood transfusion and clinical procedures has reduced. On the other hand, the number of injecting drug users has increased and the needle-sharing habit among them has caused an increase in HCV infections in this group. These facts together with the findings mentioned above reinforce the prediction of a shift in the predominant subtype from 1a to 3a in future in Iran. Nevertheless, 1a and 3a are the most frequent HCV subtypes in Iran. In comparison with genotype 1, infection with HCV genotype 3 is associated with higher SVR rate. The current standard treatment for genotypes 1a and 1b is triple combination therapy with pegylated interferon alpha, ribavirin and one of the approved protease inhibitors, boceprevir or telaprevir. It has been shown, however, that both boceprevir and telaprevir, have poor antiviral activities against genotype 3 [Pearlman, 2012]. This might hamper the treatment of HCV-3 infected patients. In a case–control study on Iranian blood donors, Alavian et al. [2002] reported that history of endoscopy was an independent risk factor of being anti-HCV positive (OR: 4, CI: 1.3–12.5). Results of the current study revealed that there was a significant association between HCV subtype and a history of upper gastrointestinal endoscopy (P ¼ 0.02). There was no information available on when the patients underwent upper gastrointestinal endoscopy and thus, it was not clear whether these patients had undergone endoscopy a long time ago when HCV was more prevalent in Iranian clinical settings or they were exposed to such procedure in recent years. Nevertheless, history of upper gastrointestinal endoscopy was significantly associated with older age (P ¼ 0.002).
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In conclusion, consistent with previous studies, the results of this study showed that HCV subtype 1a is currently predominant in Iran. Results also revealed that HCV-3a is more frequent among younger Iranian patients. There was a significant association between HCV subtype and a history of injecting drug use. There was also a significant association between HCV subtype and a history of upper gastrointestinal endoscopy. According the results of this study and the HCV-4 sequences reported previously, it seems that subtype 4d is predominant among Iranian HCV-4 infected patients. The results of this study also reinforce the prediction of a shift in the predominant subtype from 1a to 3a among Iranian patients in future. ACKNOWLEDGMENTS We wish to thank our colleagues and laboratory personnel, especially Mehdi Tolouei and Parvaneh Mohammadi for their valuable help in the study. REFERENCES Alavian SM, Gholami B, Masarrat S. 2002. Hepatitis C risk factors in Iranian volunteer blood donors: A case-control study. J Gastroenterol Hepatol 17:1092–1097. Alter MJ. 1997. The epidemiology of acute and chronic hepatitis C. Clin Liver Dis 1:559–568. Amini S, Ahmadi pour M-H, Azadmanesh K. 2006. The phylogenetic analysis of hepatitis C virus isolates obtained from two Iranian carriers revealed evidence for a new subtype of HCV genotype 3. Virus Genes 33:271–278. Amini S, Majd Abadi MM, Alavian SM, Joulaie M, Ahmadipour MH. 2009. Distribution of hepatitis C virus genotypes in Iran: A population-based study. Hepat Mon 9:95–102. Amini Kafi-abad S, Rezvan H, Abolghasemi H, Talebian A. 2009. Prevalence and trends of human immunodeficiency virus, hepatitis B virus, and hepatitis C virus among blood donors in Iran 2004 through 2007. Transfusion 49:2214–2220. Bozdayı AM, Aslan N, Bozdayı G, Tu¨rkyılmaz AR, Sengezer T, ¨ , Aydemir F, Zakirhodjaev S, Orucov S¸, Wend U, Erkan O Bozkaya H, Gerlich W, Karayalc¸ın S, Yurdaydın C, Uzunalimo¨ . 2004. Molecular epidemiology of hepatitis B, C and D g˘lu O viruses in Turkish patients. Arch Virol 149:2115–2129. Colson P, Gayet S, Gerolami R. 2011. NS3 protease of genotype 3 subtype h HCV identified in southeastern France. Antivir Ther 16:615–619. Heintges T, Wands JR. 1997. Hepatitis C virus: Epidemiology and transmission. Hepatology 26:521–526. Idrees M, Riazuddin S. 2008. Frequency distribution of hepatitis C virus genotypes in different geographical regions of Pakistan and their possible routes of transmission. BMC Infect Dis 8: 69–77. Idrees M, Butt S, Awan Z, Aftab M, Khubaib B, Rehman I-u, Akram M, Manzoor S, Akbar H, Rafiqe S. 2009. Nucleotide identity and variability among different Pakistani hepatitis C virus isolates. Virol J 6:130–135. Jahanbakhsh Sefidi F, Keyvani H, Monavari SH, Alavian SM, Fakhim S, Bokharaei-Salim F. 2013. Distribution of hepatitis C virus genotypes in Iranian chronic infected patients. Hepat Mon 13:e7991. Kabir A, Alavian S-M, Keyvani H. 2006. Distribution of hepatitis C virus genotypes in patients infected by different sources and its correlation with clinical and virological parameters: A preliminary study. Comp Hepatol 5:4–9. Kalinina O, Norder H, Vetrov T, Zhdanov K, Barzunova M, Plotnikova V, Mukomolov S, Magnius L. 2001. Shift in predominating subtype of HCV from 1b to 3a in St. Petersburg mediated by increase in injecting drug use. J Med Virol 65:517– 524.
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8 Kamal SM, Nasser IA. 2008. Hepatitis C genotype 4: What we know and what we don’t yet know. Hepatology 47:1371–1383. Keyvani H, Alizadeh AHM, Alavian SM, Ranjbar M, Hatami S. 2007. Distribution frequency of hepatitis C virus genotypes in 2231 patients in Iran. Hepatol Res 37:101–103. Kheirandish P, SeyedAlinaghi S, Jahani M, Shirzad H, Seyed Ahmadian M, Majidi A, Sharifi A, Hosseini M, Mohraz M, McFarland W. 2009. Prevalence and correlates of hepatitis C infection among male injection drug users in detention, Tehran, Iran. J Urban Health 86:902–908. Kimura M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. Lavanchy D. 2009. The global burden of hepatitis C. Liver Int 29: 74–81. Lu L, Li C, Yuan J, Lu T, Okamoto H, Murphy DG. 2013. Fulllength genome sequences of five hepatitis C virus isolates representing subtypes 3g, 3h, 3i and 3k, and a unique genotype 3 variant. J Gen Virol 94:543–548. Moradpour D, Brass V, Gosert R, Wo¨lk B, Blum HE. 2002. Hepatitis C: Molecular virology and antiviral targets. Trends Mol Med 8:476–482. Nakano T, Lau GM, Lau GM, Sugiyama M, Mizokami M. 2012. An updated analysis of hepatitis C virus genotypes and subtypes based on the complete coding region. Liver Int 32:339–345. Norder H, Bergstro¨m A, Uhnoo I, Alde´n J, Weiss L, Czajkowski J, Magnius L. 1998. Confirmation of nosocomial transmission of hepatitis C virus by phylogenetic analysis of the NS5-B region. J Clin Microbiol 36:3066–3069. Payan C, Roudot-Thoraval F, Marcellin P, Bled N, Duverlie G, Fouchard-Hubert I, Trimoulet P, Couzigou P, Cointe D, Chaput C. 2005. Changing of hepatitis C virus genotype patterns in France at the beginning of the third millenium: The GEMHEP GenoCII Study. J Viral Hepatitis 12:405–413. Pearlman BL. 2012. Protease inhibitors for the treatment of chronic hepatitis C genotype-1 infection: The new standard of care. Lancet Infect Dis 12:717–728. Ramia S, Eid-Fares J. 2006. Distribution of hepatitis C virus genotypes in the Middle East. Int J Infect Dis 10:272–277. Rezvan H, Abolghassemi H, Amini Kafiabad S. 2007. Transfusiontransmitted infections among multitransfused patients in Iran: A review. Transfus Med 17:425–433. Ross R, Viazov S, Renzing-Ko¨hler K, Roggendorf M. 2000. Changes in the epidemiology of hepatitis C infection in Germany: Shift in
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Salehi Moghadam et al. the predominance of hepatitis C subtypes. J Med Virol 60:122– 125. Salehi Moghadam F, Mohebbi SR, Hosseini SM, Damavand B, Zali MR. 2013a. A new subtype of hepatitis C virus genotype 3: Analysis of available evidence. Hepat Mon 13:e13380. Salehi Moghadam F, Mohebbi SR, Hosseini SM, Mirtalebi H, Romani S, Azimzadeh P, Sanati A, Zali MR. 2013b. Hepatitis C virus subtype 6a infection in an Iranian patient: A case report. Jundishapur J Microbiol 6:e6560. Samimi-Rad K, Nategh R, Malekzadeh R, Norder H, Magnius L. 2004. Molecular epidemiology of hepatitis C virus in Iran as reflected by phylogenetic analysis of the NS5B region. J Med Virol 74:246–252. Samimi-Rad K, Nasiri Toosi M, Masoudi-nejad A, Najafi A, Rahimnia R, Asgari F, Shabestari AN, Hassanpour G, Alavian S-M, Asgari F. 2012. Molecular epidemiology of hepatitis C virus among injection drug users in Iran: A slight change in prevalence of HCV genotypes over time. Arch virol 157:1959–1965. Seme K, Vrhovac M, Mocˇilnik T, Maticˇicˇ M, Lesˇnicˇar G, Baklan Z, Volkar JM, Rajter M, Sˇtepec S, Lunar M. 2009. Hepatitis C virus genotypes in 1,504 patients in Slovenia, 1993–2007. J Med Virol 81:634–639. Simmonds P, Bukh J, Combet C, Dele´age G, Enomoto N, Feinstone S, Halfon P, Inchauspe´ G, Kuiken C, Maertens G, Mizokami M, Murphy DG, Okamoto H, Pawlotsky J-M, Penin F, Sablon E, Shin-I T, Stuyver LJ, Thiel H-J, Viazov S, Weiner AJ, Widell A. 2005. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 42:962–973. Svirtlih N, Delic D, Simonovic J, Jevtovic D, Dokic L, Gvozdenovic E, Boricic I, Terzic D, Pavic S, Neskovic G. 2007. Hepatitis C virus genotypes in Serbia and Montenegro: The prevalence and clinical significance. World J Gastroenterol 13:355–360. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882. Zamani S, Radfar R, Nematollahi P, Fadaie R, Meshkati M, Mortazavi S, Sedaghat A, Ono-Kihara M, Kihara M. 2010. Prevalence of HIV/HCV/HBV infections and drug-related risk behaviours amongst IDUs recruited through peer-driven sampling in Iran. Int J Drug Policy 21:493–500.
Phylogenetic Analysis of Hepatitis C Virus Strains and Risk Factors Associated With Infection and Viral Subtypes Among Iranian Patients Faraz Salehi Moghadam,1,2 Seyed Reza Mohebbi,1* Seyed Masoud Hosseini,2 Sara Romani,1 Hanieh Mirtalebi,1 Pedram Azimzadeh,1 Behzad Damavand,1 Hamed Naghoosi,1 Mahsa Khanyaghma,1 Azar Sanati,1 and Mohammad Reza Zali1 1 2
Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran
Hepatitis C virus (HCV) has infected approximately 170 million people worldwide. While the seroprevalence of anti-HCV antibody among Iranian blood donors is 0.13%, HCV infection is prevalent in 59–80% of Iranian injecting drug users. One hundred seventyeight anti-HCV positive patients were referred to the Gastroenterology Department at the Taleghani Hospital (Tehran, Iran) between June 2007 and June 2012. Out of 178 samples, 142 were positive for HCV-RNA. HCV subtypes were determined using phylogenetic analysis of the NS5B or 50 UTR/core regions. Of 142 viremic patients, 71 (50%) were infected with HCV subtype 1a, 43 (30.3%) with subtype 3a, 20 (14.1%) with subtype 1b, 3 (2.1%) with subtype 4d, 2 (1.4%) with subtype 4a, 1 (0.7%) with subtype 2b, and 1 (0.7%) with subtype 6a. Interestingly, genetic analysis of a sub-genomic fragment from one patient identified a non-subtypeable HCV genotype-3 strain. There was a significant association between HCV subtype and a history of injecting drug use (P ¼ 0.003). Subtype 3a was predominant among patients with such a history. Injecting drug use was associated with younger age (P < 0.001). HCV subtype was also significantly associated with a history of upper gastrointestinal endoscopy (P ¼ 0.02). Subtype 1a was more frequent among patients with such a history. In addition, history of upper gastrointestinal endoscopy was significantly associated with older age (P ¼ 0.002). In conclusion, while HCV subtype 1a is predominant among infected Iranian individuals, subtype 3a is predominant among Iranian injecting drug users. J. Med. Virol. # 2014 Wiley Periodicals, Inc. C 2014 WILEY PERIODICALS, INC.
KEY WORDS:
hepatitis C virus; genotype; subtype; phylogenetic analysis; molecular epidemiology; Iran
INTRODUCTION Hepatitis C virus (HCV) is one of the main causes of chronic liver disease and liver transplantation [Alter, 1997]. Twenty to thirty percent of chronic hepatitis C cases will develop cirrhosis and/or hepatocellular carcinoma after 20–30 years [Heintges and Wands, 1997]. According to the World Health Organization (WHO), HCV has infected approximately 170 million people worldwide [Lavanchy, 2009]. According to the Iranian Blood Transfusion Organization, the seroprevalence of HCV among Iranian blood donors is 0.13% [Amini Kafi-abad et al., 2009]. Due to the efficient blood screening program adopted since 1996, HCV transmission through blood or blood products has reduced significantly in Iran [Rezvan et al., 2007]. The prevalence of HCV infection among intravenous drug abusers is as high as 59–80% [Kheirandish et al., 2009; Zamani et al., 2010; Samimi-Rad et al., Grant sponsor: Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Correspondence to: Seyed Reza Mohebbi, Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, 7th floor of Taleghani Hospital, Velenjak, Tehran, Iran. E-mail: [email protected] Accepted 28 March 2014 DOI 10.1002/jmv.23947 Published online in Wiley Online Library (wileyonlinelibrary.com).
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2012]. This indicates that similar to many other countries, intravenous drug abuse has become the major route of HCV transmission in Iran. HCV is an enveloped single stranded positive sense RNA virus which belongs to the Hepacivirus genus of the Flaviviridae family. The viral genome is about 9.6 kb in length and contains a single open reading frame (ORF) encoding a polyprotein precursor of about 3,000 amino acids [Moradpour et al., 2002]. To date, six major genotypes and more than eighty different subtypes have been identified for HCV. Furthermore, one strain has been proposed as a candidate for the seventh genotype. While different genotypes have the similarity of around 60–70% in their nucleotide sequences, the similarity of different subtypes of a genotype is around 75–85% [Nakano et al., 2012]. HCV genotypes have different geographical distribution patterns. In Iran, the predominant subtype is 1a followed by 3a and 1b [Samimi-Rad et al., 2004; Kabir et al., 2006; Keyvani et al., 2007; Jahanbakhsh Sefidi et al., 2013]. Among neighboring countries, genotypes 1b, 3, and 4 are predominant in Turkey, Pakistan, and Arab countries, respectively [Bozdayı et al., 2004; Ramia and Eid-Fares, 2006; Idrees and Riazuddin, 2008; Idrees et al., 2009]. There are few phylogenetic studies that have been carried out on HCV strains obtained from Iranian patients. Most of the previous studies were performed on one particular group of patients (e.g., injecting drug users or patients with thalassemia). Also, there were not sufficient data on the statistical associations between different risk factors and HCV subtype in Iran. In this study, molecular epidemiology of HCV strains obtained from infected individuals with chronic hepatitis or cirrhosis of the liver was investigated using phylogenetic analysis of the NS5B and 50 UTR/ core regions. Furthermore, associations of HCV subtype with age, sex, and the history of exposure to various risk factors were assessed. MATERIALS AND METHODS One hundred seventy-eight Iranian individuals with positive anti-HCV antibody were referred to the Gastroenterology Department at the Taleghani Hospital (Tehran, Iran) between June 2007 and June 2012. Study participants were from various ethnicities (Fars: 52.5%, Turk: 22.8%, Lor: 8.9%, Kurd: 6.9%, Gilaki: 6.9%, Armenian: 1%, and Arab: 1%). Informed consent was provided by each participant. The study protocol was approved by the Ethics Committee at the Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Serological tests for hepatitis B virus (anti-HBc antibody and HBs antigen) and human immunodeficiency virus (anti-HIV antibody) were performed using enzyme-linked immunosorbent assay (DIA PRO Diagnostic Bioprobes, Milan, Italy). J. Med. Virol. DOI 10.1002/jmv
HCV RNA was extracted from plasma samples using QIAamp Viral RNA mini kit (QIAGEN, Hilden, Germany). Complementary DNA synthesis was performed using Random Hexamer primers and RevertAid Reverse Transcriptase enzyme (Fermentas, Vilnius, Lithuania). Polymerase chain reaction (PCR) was used for HCV-RNA detection. A partial segment of the NS5B region was amplified using semi-nested PCR with primers and thermal conditions described elsewhere [Norder et al., 1998; Kalinina et al., 2001]. Briefly, primers hep-101 and hep-120 were used to amplify a 463-bp segment (corresponding to nucleotides 8,260–8,722 in the H77 reference sequence, accession number AF009606) in the first-round PCR, and primers hep-101 and hep105 were used to amplify a 380-bp segment (nucleotides 8,260–8,639) in the second-round PCR. If amplification with the mentioned sets of primers was not successful for a sample, primers hep-106 and hep-105 were used alternatively in the first-round PCR to amplify a 473-bp segment (nucleotides 8,167–8,639). The NS5B region of one strain with subtype 6a could not be amplified using the mentioned primers. For this strain, subtype-specific primers were designed: primers HCV6a-F1 and HCV6a-R1 were used for the first-round PCR and primers HCV6a-F2 and HCV6a-R2 were used for the second-round PCR. The sizes of the first- and second-round PCR amplicons were 535 (nucleotides 8,131–8,665) and 389 bp (nucleotides 8,210–8,598), respectively. Thermal cycling conditions for both first- and second-round PCRs were 5 min at 95˚C, followed by 35 cycles of 1 min at 94˚C, 45 sec at 54˚C and 45 sec at 72˚C. Final elongation step was performed for 7 min at 72˚C. Samples with negative results for the NS5B amplification were subjected to another PCR targeting the 50 UTR/core junction (a 681-bp segment corresponding to nucleotides 51–731) with primers NCR-F and NCR-R. The reaction conditions were described previously [Salehi Moghadam et al., 2013b]. If the result was negative for a sample, 3 ml of the PCR product was used as template and a new PCR was carried out with the same primers and thermal conditions. Table I shows sequences and positions of the primers used in this study. Besides, to assess the suitability of the selected positions of the 50 UTR/core region for accurate subtyping, 23 (16%) samples were randomly selected and amplified in both 50 UTR/core and NS5B regions and the results were compared. PCR products were direct-sequenced bi-directionally, and sequences were edited using BioEdit version 7.0.5.3. The NS5B sequences together with the corresponding regions of 85 other strains obtained from the Los Alamos or GenBank databases were aligned using ClustalX version 2.0.12 [Thompson et al., 1997]. The same approach was adopted for the 50 UTR/core sequences with 37 strains from the databases. Phylogenetic trees for the NS5B and 50 UTR/core regions
Molecular Epidemiology of Hepatitis C Virus in Iran
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TABLE I. Sequences and Positions of the Primers used in this Study Target region NS5B
50 UTR/core a
Primer name hep-101 hep-120 hep-106 hep-105 HCV6a-F1 HCV6a-R1 HCV6a-F2 HCV6a-R2 NCR-F NCR-R
Primer sequence 0
Position 0
5 -ATACCCGCTGCTTTGACTC-3 50 -TGCGCGACBGABACRTTKGAGGA-30 50 -GCTCNTAYGGRTTYCARTACTC-30 50 -ATACCTAGTCATAGCCTCCGTGA-30 50 -RGCATACGGATTCCAGTAC-30 50 -YGAGCAYGATGTTATGAGC-30 50 -GGCTTTTCTTACGACACCA-30 50 -CTGGTCATAGCMTCCGTGA-30 50 -GGAACTACTGTCTTCACGCAGAAAGC-30 50 -GAAGCCGCACGTAAGGGTATCG-30
8,260 8,722 8,167 8,639 8,131 8,665 8,210 8,598 51 731
a
Reference Norder et al. [1998] Kalinina et al. [2001] Kalinina et al. [2001] Norder et al. [1998] This study This study This study This study Salehi Moghadam et al. [2013b] Salehi Moghadam et al. [2013b]
Nucleotide positions are relative to the start of the H77 reference sequence (AF009606).
were constructed using the Kimura two-parameter algorithm [Kimura, 1980] with the neighbor-joining method. Genetic distances were also estimated using the Kimura two-parameter algorithm. MEGA software version 5 was used for the analyses [Tamura et al., 2011]. Associations of sex, age, and exposure to various risk factors with HCV subtype were assessed using the x2 test. The independent Student’s t-test was used to analyze continuous variables. P < 0.05 was considered as the level of significance. Statistical analyses were performed using the Statistical Package for the Social Sciences version 16 (SPSS, Inc., Chicago, IL). RESULTS Of 178 anti-HCV positive individuals, 146 (82%) were male and 32 (18%) were female. Two (1.1%) patients were positive for anti-HBc antibody but not for HBsAg. No patient was anti-HIV-seropositive. One hundred forty-two (79.8%) samples were positive for HCV-RNA. The mean age of the viremic patients was 46.3 13.5 (range 22–82 years). Of 142 viremic patients, 121 (85.2%) were male (mean age 44.9 13.9) and 21 (14.8%) were female (mean age 52.7 9.6). The mean alanine aminotransferase and aspartate aminotransferase levels were 61.08 50.5 and 51.8 35.5 IU/L, respectively. According to their clinical condition, 124 (87.3%) patients were diagnosed with chronic hepatitis. Of these, 106 (85.5%) were male (mean age 43.6 13.9) and 18 (14.5%) were female (mean age 51.9 9.7). Eighteen (12.7%) patients were diagnosed with cirrhosis of the liver. Of these, 15 (83.3%) patients were male (mean age 52.7 10.9) and 3 (16.7%) were female (mean age 57.3 8.5). Of 15 cirrhotic male patients, 6 were infected with subtype 1a, 6 with subtype 1b and 3 with subtype 3a. All three cirrhotic females were infected with HCV subtype 3a. Subtype 1a was predominant among patients over 40 years of age, followed by 3a (53.2% and 26.6%, respectively). Among patients under 40, however, subtypes 1a and 3a had the same frequencies (40.9%).
Of 142 HCV-RNA positive samples, 137 (96.5%) could be amplified in the NS5B region. In the remaining 5 (3.5%) samples, only 50 UTR/core junction could be amplified. Besides, to assess the suitability of the selected 50 UTR/core segment for precise subtype determination, 23 (16%) samples were randomly selected and amplified in both 50 UTR/core and NS5B regions. No inconsistency was observed in these 23 samples. All of the 142 samples were sequenced directly, though due to the short sequencing output, 3 NS5B sequences with subtype 1a could not be included in the dendrogram. All sequences were deposited in the NCBI GenBank under the accession numbers KC285197–KC285362, KF218585, and JQ341409. Based on the phylogenetic analysis of the NS5B and 50 UTR/core sequences, of 142 HCV-RNA positive patients, 71 (50%) were infected with HCV subtype 1a, 43 (30.3%) with subtype 3a, 20 (14.1%) with subtype 1b, 3 (2.1%) with subtype 4d, 2 (1.4%) with subtype 4a, 1 (0.7%) with subtype 2b, and 1 (0.7%) with subtype 6a. The subtype of one strain (0.7%) could not be determined. According to the phylogenetic tree of the 50 UTR/core region, this strain belonged to HCV genotype 3 (Fig. 1). In the phylogenetic tree constructed based on the 50 UTR/core sequences, one strain (FSM16; GenBank accession number KF218585) within the clade 3 was separated from other subtypes with the bootstrap value of 92% over 1,000 replicates (Fig. 1b). For this strain, phylogenetic analysis was limited to HCV genotype 3 (Fig. 2). In the new tree, strain FSM16 was classified in a separate branch between subtype 3h and an unassigned subtype recently reported from Canada (GenBank accession number JF735124) [Lu et al., 2013]. Based on the nucleotide sequences corresponding to positions 83–715 in the H77 reference sequence, FSM16 had the mean intra-genotypic distances of 6.8% and 7.1%, respectively from subtype 3h and the Canadian strain with an unassigned subtype. To include a new subtype of genotype 3 that was previously reported by Amini et al. [2006], in the analysis, the genetic distances were also calculated based on the positions 142–315, as the only sequence of the new subtype which corresponded to the FSM16 J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Phylogenetic trees constructed based on (a) a 254-base segment of the NS5B region corresponding to positions 8330 to 8583 in the H77 reference sequence (AF009606), and (b) a 473-base segment of the 50 UTR/core region corresponding to positions 207 to 679, using the neighbor-joining method. The HCV strains that were obtained in this study are indicated by gray triangles. Reference sequences are shown with their GenBank accession numbers, countries of isolation and subtypes. Numbers at the nodes show the percentages of bootstrap values (1,000 replicates).
J. Med. Virol. DOI 10.1002/jmv
Molecular Epidemiology of Hepatitis C Virus in Iran
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Fig. 2. Phylogenetic analysis of strain FSM16 and the representatives of various subtypes of HCV genotype 3, based on 633 nucleotides of the 50 UTR/core region (corresponding to positions 83–715 in the H77 reference sequence) using the neighbor-joining method. Note that the only available 50 UTR/core sequence of the tentative subtype 3l in the GenBank, corresponded to a short segment (174 nucleotides) of the FSM16 sequence and thus, subtype 3l could not be included in the tree. The subtype of the sequence JF735124 has not yet been assigned. Numbers at the nodes represent the percentages of bootstrap values over 1,000 replicates.
sequence was related to these positions. The FSM16 strain had the same mean distance of 1.15% from subtype 3h, the unassigned subtype reported from Canada and the new subtype reported from Iran (DQ202322). Eleven patients had a history of a single risk factor. The remaining had a history of exposure to more than one risk factor. The most common risk factors among the patients were periodontal procedure and surgery, respectively with 53 and 52 patients in each group, compared with only 2 patients with high-risk sexual behavior. Since the frequencies of patients infected with other subtypes were too
small to be statistically analyzed, analyses were only focused on subtypes 1a and 3a. Table II shows associations of HCV subtype with age, sex, and risk factors that the patients had been exposed to. There was a significant association between HCV subtype and a history of injecting drug use (P ¼ 0.003). Subtype 3a was predominant among patients with such a history. Furthermore, history of injecting drug use was associated with younger age (P < 0.001). HCV subtype was also significantly associated with a history of upper gastrointestinal endoscopy (P ¼ 0.02). Subtype 1a was more frequent among J. Med. Virol. DOI 10.1002/jmv
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TABLE II. Associations of HCV Subtype with Age, Sex, and History of Various Risk Factors HCV subtype
Age 40 >40 Sex Male Female Transfusion No Yes Cupping No Yes Tattooing No Yes Periodontal procedures No Yes Shared shaving razor No Yes UGI endoscopy No Yes Injecting drug use No Yes Surgery No Yes Hemodialysisb No Yes High-risk sexual behaviorb No Yes Needle stickb No Yes Unknown risk factor
1a, No. (%)
3a, No. (%)
18 (50) 42 (66.7)
18 (50) 21 (33.3)
61 (62.2) 10 (62.5)
37 (37.8) 6 (37.5)
37 (59.7) 12 (60)
25 (40.3) 8 (40)
31 (58.5) 18 (62.1)
22 (41.5) 11 (37.9)
36 (58.1) 13 (65)
26 (41.9) 7 (35)
25 (62.5) 24 (57.1)
15 (37.5) 18 (42.9)
37 (57.8) 12 (66.7)
27 (42.2) 6 (33.3)
28 (50.9) 21 (77.8)
27 (49.1) 6 (22.2)
39 (70.9) 10 (37)
16 (29.1) 17 (63)
21 (52.5) 28 (66.7)
19 (47.5) 14 (33.3)
48 (60) 1 (50)
32 (40) 1 (50)
48 (60) 1 (50)
32 (40) 1 (50)
45 (59.2) 4 (66.7) 22 (31)
31 (40.8) 2 (33.3) 10 (23)
P-value 0.103 0.984 0.98 0.752 0.582 0.621 0.499 0.02a 0.003a 0.191 — — — —
a
Statistically significant. The frequency of the risk factor among HCV-1a and -3a infected patients was not adequate to be statistically analyzed. b
patients with such a history. In addition, history of upper gastrointestinal endoscopy was significantly associated with older age (P ¼ 0.002). DISCUSSION The prevalent subtype among the patients was 1a followed by subtype 3a. This is in agreement with the results of previous studies from Iran [Samimi-Rad et al., 2004; Kabir et al., 2006; Keyvani et al., 2007; Amini et al., 2009; Jahanbakhsh Sefidi et al., 2013]. Additionally, in this study two rare HCV subtypes were identified. The first one was HCV subtype 6a. This specific case was previously discussed in detail [Salehi Moghadam et al., 2013b]. The second one, strain FSM16, was genetically more close to subtype J. Med. Virol. DOI 10.1002/jmv
3h and also to a subtype isolated from a Middle Eastern patient residing in Canada. The latter was reported to be a new subtype but has been remained unassigned as of the time of the preparation of this article [Lu et al., 2013]. In recent years, a new subtype of HCV genotype 3 has been isolated from Iranian patients and it has been proposed to provisionally assign this subtype as “3l” [Amini et al., 2006; Salehi Moghadam et al., 2013a]. The relatedness of this new subtype and the strain FSM16 could not be shown in this study, as the only available 50 UTR sequence of the new subtype had the same genetic distance of 1.15% from the strain FSM16, subtype 3h and a new subtype reported recently from Canada. For accurate subtype determination of FSM16, sequences from other parts of the genome were needed. As described previously, nucleotide sequencing of the NS3 region following by phylogenetic analysis can be used for HCV subtype determination [Colson et al., 2011]. With strains that seem to belong to a new subtype, however, phylogenetic analysis of both core/E1 and NS5B regions or preferably the complete genome sequence will be required prior to subtype assignment [Simmonds et al., 2005]. Due to the limited amount of the plasma sample, it was impossible to characterize other parts of the genome for FSM16. Prior to this report, there was no Iranian study that phylogenetically analyzed subtypes of genotype 4. This is mainly due to the fact that few Iranian studies used direct sequencing method and phylogenetic analysis of the nucleotide sequences to investigate HCV epidemiology in Iran. Apart from the HCV4 sequences obtained in this study, there have been 11 HCV-4 sequences deposited in the HCV database. Of these, subtypes of three sequences were not determined. The remaining eight sequences were from a previous study [Samimi-Rad et al., 2004]. Although in the original report, subtypes of these eight strains were not determined, seven sequences are classified as subtype 4d and one is classified as subtype 4a in the Los Alamos HCV database. In the current study, three strains with subtype 4d and two with subtype 4a were identified. It should be mentioned that among the Middle Eastern countries, subtype 4a is predominant in Egypt, whereas 4c/4d are the most frequent subtypes in Saudi Arabia [Kamal and Nasser, 2008]. Although genotype 4 has a low prevalence among infected Iranian individuals, by considering the frequencies of HCV-4 strains mentioned above, two facts reveal: (1) At least two subtypes of genotype 4 including subtypes 4a and 4d are responsible for HCV-4 infections in Iran; (2) Subtype 4d is predominant among Iranian HCV-4 infected patients. This is not only important from the epidemiological point of view but it might be important clinically because according to a retrospective study, HCV-4a infections are associated with higher SVR rates than those of HCV-4d [Kamal and Nasser, 2008].
Molecular Epidemiology of Hepatitis C Virus in Iran
In this study, it was observed that HCV subtype was significantly associated with a history of injecting drug use. Subtype 3a was more frequent among the patients with such a history. Predominance of subtype 3a among Iranian injecting drug users was also reported in a recent study, though no associations between HCV genotype and injecting drug use characteristics (being a current or former, short-term or long-term injecting drug user) were detected in the mentioned study [Samimi-Rad et al., 2012]. Results of the current study showed that in comparison with the older patients, HCV subtype 3a was more frequent among the patients under 40. In fact, in the younger subgroup of patients, subtypes 1a and 3a had the same frequencies. It was also observed that history of injecting drug use was associated with younger age (P < 0.001). These findings are in agreement with the previous reports from Iran [Samimi-Rad et al., 2012; Jahanbakhsh Sefidi et al., 2013], France [Payan et al., 2005], Germany [Ross et al., 2000], Serbia and Montenegro [Svirtlih et al., 2007], and Slovenia [Seme et al., 2009]; and highlight the fact that intravenous drug abusers have become the major source of HCV infection in Iran. On the one hand, due to the successful harm reduction strategies adopted since 1996, HCV transmission through blood transfusion and clinical procedures has reduced. On the other hand, the number of injecting drug users has increased and the needle-sharing habit among them has caused an increase in HCV infections in this group. These facts together with the findings mentioned above reinforce the prediction of a shift in the predominant subtype from 1a to 3a in future in Iran. Nevertheless, 1a and 3a are the most frequent HCV subtypes in Iran. In comparison with genotype 1, infection with HCV genotype 3 is associated with higher SVR rate. The current standard treatment for genotypes 1a and 1b is triple combination therapy with pegylated interferon alpha, ribavirin and one of the approved protease inhibitors, boceprevir or telaprevir. It has been shown, however, that both boceprevir and telaprevir, have poor antiviral activities against genotype 3 [Pearlman, 2012]. This might hamper the treatment of HCV-3 infected patients. In a case–control study on Iranian blood donors, Alavian et al. [2002] reported that history of endoscopy was an independent risk factor of being anti-HCV positive (OR: 4, CI: 1.3–12.5). Results of the current study revealed that there was a significant association between HCV subtype and a history of upper gastrointestinal endoscopy (P ¼ 0.02). There was no information available on when the patients underwent upper gastrointestinal endoscopy and thus, it was not clear whether these patients had undergone endoscopy a long time ago when HCV was more prevalent in Iranian clinical settings or they were exposed to such procedure in recent years. Nevertheless, history of upper gastrointestinal endoscopy was significantly associated with older age (P ¼ 0.002).
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