Journal of Medical Virology 74:246–252 (2004)
Molecular Epidemiology of Hepatitis C Virus in Iran as Reflected by Phylogenetic Analysis of the NS5B Region Katayoun Samimi-Rad,1,2 Rakhshandeh Nategh,2 Reza Malekzadeh,3* Helene Norder,1 and Lars Magnius1 1
Virological Department, Swedish Institute for Infectious Disease Control, Solna, Sweden Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3 Digestive Disease Research Centre, Tehran University of Medical Sciences, Tehran, Iran 2
Hepatitis C virus (HCV) subtypes were determined in 125 Iranian patients by phylogenetic analysis within the NS5B or 50 -UTR/core regions. Subtypes 1a and 3a were predominant accounting for 47 and 36%, whereas 1b and 4 accounted for 8 and 7%. This subtype distribution differs from that of Turkey and Pakistan, where subtypes 1b and 3a dominate and also from neighbouring Arabic countries where subtype 4 is the prevalent genotype. The Iranian 1a and 3a strains formed subclusters in the dendrogram indicating that these subtypes are indigenous to Iran. In contrast, the 1b strains intermixed with strains derived worldwide. Subtype 1a was frequent in South Iran (70%), while 3a was more prevalent in NorthWest Iran (83%), a region with a high proportion of Turkish inhabitants. Patients infected by blood products had more frequently subtype 1a (57%), while younger drug users had more frequently subtype 3a (54%). Genotype 4 was over-represented among haemodialysis patients in Tehran. One strain, most similar to genotype 5, was highly divergent in the NS5B region and further analysis is needed to assess the systematic status of this strain. In half of the patients with unknown source of infection only the 50 -UTR could be amplified, most of which were from North-West Iran and from patients younger than those with unknown source of infection with typable strains, mean age 29 versus 43 years. In conclusion, the NS5B sequence data revealed population based subtype patterns in Iran, the further study of which may help to understand the molecular epidemiology of HCV in a low-endemic area. J. Med. Virol. 74:246–252, 2004. ß 2004 Wiley-Liss, Inc. KEY WORDS: HCV; genotypes; subtypes INTRODUCTION Hepatitis C virus (HCV) accounts for the majority of post-transfusion non-A, non-B hepatitis cases [Choo ß 2004 WILEY-LISS, INC.
et al., 1989], and worldwide the number of infected individuals is estimated to be about 120 million. Sequence analysis has revealed at least six major types, each type being further divided into subtypes [Simmonds et al., 1993]. The relative frequencies of HCV subtypes have been found to vary for different geographical areas [Mellor et al., 1995]. Studies suggest that infections caused by different HCV subtypes may have different clinical outcome and that some subtypes are associated more frequently with advanced liver disease and hepatocellullar carcinoma [De Mitri et al., 1995]. Also, the route of viral transmission, serum HCV RNA levels and the success of antiviral treatment seem dependent on the subtype [Zein, 2000]. It is, therefore important to determine HCV subtypes in different geographical regions both for epidemiological purposes and for patient management. The limited data available indicate that the general population of Iran has a low prevalence of HCV infections ranging from 0.12 to 0.5% [Ghavanini and Sabri, 2000; Alavian et al., 2002; Ansar and Kooloobandi, 2002]. A preliminary study on HCV subtypes on a limited number of patients shows that subtypes 1a and 3a are the most common [Zali et al., 2000]. However, molecular data on HCV strains in Iran is lacking. The aim of the present study was, therefore, to investigate the prevalence and molecular characteristics of HCV subtypes in Iran. MATERIALS AND METHODS Serum samples from 233 anti-HCV positive subjects were collected during 1999–2003 at two major referral hospitals in Tehran and five other major hospitals throughout the country (Sari in the North, Tabriz and *Correspondence to: Prof. Reza Malekzadeh, Digestive Disease Research Centre, Tehran University of Medical Sciences, Shariati Hospital Kargar Shomali, Avenue, Tehran, Iran. E-mail:
[email protected] Accepted 13 May 2004 DOI 10.1002/jmv.20170 Published online in Wiley InterScience (www.interscience.wiley.com)
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Oromieh in the North-West, Kermanshah in the western part, Mashad in the North-East, and Zahedan in South-East). The sera were tested for HCV RNA by PCR. Anti-HCV was tested by a second generation enzyme immunoassay and confirmed by a recombinant immunoblot assay, RIBA HCV 3.0. The suspected sources of infection together with the age and gender distribution of the patients are given in Table I. RNA Extraction and PCR HCV RNA was isolated from serum by a modified version of Garson et al. [1990] as described [Norder et al., 1998]. The RNA was dissolved in 20 ml distilled water, and 5 ml were used for cDNA synthesis with random hexamer primers (Roche Diagnostics Scandinavia AB, Bromma, Sweden) and 1 U reverse transcriptase Superscript II (Invitrogen, Life Technologies, Sweden). The cDNA was amplified within the NS5B region forming a 371 bp product using primers hep101 and hep120 as outer primers, and hep101 and hep105 for nesting [Kalinina et al., 2001]. Samples for which the NS5B region could not be amplified, the 50 -UTR region was amplified with primers univ1 and univ2 as outer primers, and with univ3 and univ4 for nesting [Stuyver et al., 1995]. Strains, which could not be subtyped by phylogenetic analysis of the NS5B region, and strains, positive only by amplification of the 50 -UTR region, were further amplified in the 50 -UTR/core region using primers univ1 and 185 as described [Kalinina et al., 2002]. Phylogenetic Analysis The NS5B sequences were aligned with the corresponding region of 174 other NS5B sequences from GenBank. Sequences of the 50 -UTR/core region of strains, for which the NS5B region could not be amplified, were aligned with the corresponding region of other Iranian strains from this study and of 155 strains from GenBank. The genetic distances of the aligned sequences were calculated using the Kimura-two parameter model in DNADIST in the Phylip program package version 3.52 [Felsenstein, 1993]. Dendrograms were constructed using the UPGMA algorithm in the program NEIGHBOR of the Phylip program package. Statistical Analysis The 95% confidence intervals for proportion of subtype frequencies were calculated using the binomial distribution.
RESULTS In total, 161 of the 233 samples (69%) were positive for HCV RNA, and 125 could be genotyped by sequencing. The NS5B region could be amplified in 128 samples, although only113 of these generated amplicons that could be sequenced. For 3 of the 113 strains the NS5B fragment allowed subtyping but was too short to be included in dendrograms. In 48 of the 120 sera, with HCV strains untypable by NS5B sequencing, the 50 -UTR region could be amplified, and in 12 of these the viral 50 UTR/core region could also be amplified and sequenced. The 125 Iranian HCV strains were classified into the genotypes 1, 2, 3, and 4 based on analysis of the NS5B and 50 -UTR/core regions (Fig. 1a,b). The most prevalent subtypes, 1a, 59 (47%) strains, and 3a, 45 (36%) strains, were found in most patient groups and regions of Iran (Table II). The mean age of the patients infected by 1a and 3a was 36 years for both groups. 1a was more prevalent in patients receiving blood or blood products (57%) than 3a (29%), and in patients from Zahedan in South Iran (70%) compared to patients from the Oromieh/Tabriz region in North Iran (17%) who more often were infected with 3a. The NS5B region could be sequenced for 53 Iranian 1a strains, 51 of which divided into two clusters. One cluster was formed by 36 strains from Iran and 15 strains from Sweden (Fig. 1a). Twenty-three of the 29 subtype 1a strains from patients receiving blood or blood products were in this cluster. Fifteen other Iranian strains, nine of which from dialysis patients in Tehran formed a second cluster with strains from Japan, the USA, India, and Sweden (Fig. 1a). Six of the 12 strains, for which only the 50 -UTR/core could be amplified, belonged to subtype 1a. Ten strains belonged to subtype 1b, eight of which from Tehran, and two from Mashad. These were intermixed with 68 other strains from Asia and Europe (Fig. 1a). Five Iranian strains derived from haemophiliacs, two from dialysis patients, one from a drug addict, and two from patients with unknown source of infection (Table II). The drug addict strain clustered with strains from drug addicts in St. Petersburg, Russia (Fig. 1a). Three major clusters were formed by the 37 Iranian 3a strains in the NS5B dendrogram (Fig. 1b). One was formed by 16 Iranian strains comprising apart strains from IDUs also strains from dialysis units and multitransfused patients, which indicates its spread into hospital settings. Within the second major cluster 17 subtype 3a strains from Iran were intermixed with
TABLE I. Characteristics and Results on Iranian HCV Infected Patients According to Route of Infection Route of infection IDU Blood or blood products Dialysis Other Unknown Total
n
Males/females
Age: min–max
Mean age
Number PCR positive
Number sequenced
39 67 51 5 71 233
38/1 50/17 24/27 5/0 61/10 178/55
24–67 5–54 28–76 20–52 17–64 5–76
40 29 47 33 34 36
33 (85%) 55 (82%) 36 (71%) 3 (60%) 34 (48%) 161 (69%)
24 (73%) 51 (93%) 30 (83%) 2 (67%) 18 (53%) 125 (78%)
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Fig. 1. UPGMA dendrogram based on 325 nucleotides of the NS5B region of 284 HCV strains. The strains from Iran are shown in bold and other strains in italic. The designations, accession numbers of the strains obtained from GenBank, and origin of the strains are given at the nodes. a: part of the dendrogram showing the clusters formed by the
Samimi-Rad et al.
genotype 1 sequence. The connection of this part of the dendrogram with the other part is indicated with an arrow. b: Part of the dendrogram with the branches formed by genotypes 2–5 and 10. The connection of this part of the dendrogram with the other part is indicated as for a.
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Fig. 1. (Continued )
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Samimi-Rad et al. TABLE II. Distribution of Geno/Subtypes According to Route of Infection and Origin in Iran
Region Tehran IDU Blood or blood products Dialysis Other Unknown source Whole region Zahedan IDU Blood or blood products Whole region Mashad IDU Blood or blood products Dialysis Unknown source Whole region Kermanshah IDU Blood or blood products Unknown source Whole region Sari IDU Blood or blood products Unknown source Whole region Oromieh &Tabriz IDU Blood or blood products Other Unknown source Whole region All regions IDU Blood or blood products Dialysis Other Unknown source Total 95% binomial CI
1a
1b
2b
3a
4
All typable
Only 50 UTR positive
Total
4 16 12 0 3 35
1 4 1 0 2 8
0 1 0 0 0 1
7 8 8 2 1 26
0 1 7 0 0 8
12 30 28 2 6 78
2 1 7a 0 1 11
14 31 35 2 7 89
1 6 7
0 0 0
0 0 0
1 2 3
0 0 0
2 8 10
1 1 2
3 9 12
0 4 0 0 4
0 1 1 0 2
0 0 0 0 0
1 2 0 1 4
0 0 0 1 1
1 7 1 2 11
2 2 0 1 5
3 9 1 3 16
4 2 3 9
0 0 0 0
0 0 0 0
3 0 1 4
0 0 0 0
7 2 4 13
1 0 0 1
8 2 4 14
0 1 2 3
0 0 0 0
0 0 0 0
0 2 1 3
0 0 0 0
0 3 3 6
3 0 7 10
3 3 10 16
1 0 0 0 1
0 0 0 0 0
0 0 0 0 0
1 1 0 3 5
0 0 0 0 0
2 1 0 3 6
0 0 1 7 8
2 1 1 10 14
10 29 12 0 8 59 (47%) 38–56%
1 5 2 0 2 10 (8%) 3.9–14%
0 1 0 0 0 1 (0.8%) 0.02–4.3%
13 15 8 2 7 45 (36%) 27–46%
0 1 7 0 1 9 (7%) 3.3–13%
9 4 7a 1 16 37
33 55 36 3 34 161
24 51 29 2 18 124
a
One strain related to genotype 5.
strains from St. Petersburg, Australia, New Zealand, and Sweden. A third cluster was made up of IDU associated strains from St. Petersburg. For three 3a strains the NS5B fragment was too short to be included in the phylogenetic analysis. Another five Iranian strains were subtyped as 3a by sequencing and analysis of the 50 UTR/core region. Genotype 4 was found in nine subjects (Table I; Fig. 1b). Six of seven strains from dialysis patients formed a cluster with a strain from a patient with haemophilia. In one dialysis strain only the 50 -UTR/core region could be sequenced. The ninth strain was from a patient from Mashad, who had been a prisoner of war in Iraq, and had probably contracted his HCV infection there. A single strain from a patient with haemophilia in Tehran was subtype 2b (Fig. 1b). Another strain (934) from a patient on haemodialysis formed a deep bifurcation from the branch with one genotype 5 strain from
South Africa and one strain from Somalia (Fig. 1b). The 50 -UTR/core-region could not be amplified for this strain. In 36 strains only the 50 -UTR region could be amplified. Eighteen of these were from patients in Sari and the Oromieh/Tabriz region.The source of infection was unknown for 16 patients, seven each from Sari and the Oromieh/Tabriz region (Table II). The mean age of these 14 patients was 29 years compared to 43 years for the patients with unknown source of infection and infected by typable strains. Another nine untypable strains derived from IDUs, and six were from patients on dialysis in Tehran (Table II). Sixty-five percent of the patients were younger than 40 years. The mean age was 37 years for patients infected by typable strains, which did not differ significantly from the mean age of 34 years, of patients with untypable strains. Patients infected by blood or blood products were younger, mean age 29 years, as compared to dialysis patients or injecting drug users (IDUs), mean
Molecular Epidemiology of Hepatitis C Virus in Iran
age 47 and 39 years, and patients with unknown source of infection, mean age 33 years. DISCUSSION The dominant HCV subtypes in Iran were found to be 1a and 3a, which are also the prevalent ones in England, Scotland, and Australia [Mellor et al., 1995; McCaw et al., 1997; Harris et al., 1999]. The subtypes found in Iran, were neither similar to those in neighbouring countries like Turkey or Pakistan, where the dominating subtypes are 1b and 3 [Abacioglu et al., 1995; Shah et al., 1997] nor to those in Middle East countries such as Iraq, Saudi Arabia, Yemen, Kuwait where 4 is the most prevalent HCV genotype [Ohno et al., 1996; Al-Ahdal et al., 1997; Chamberlain et al., 1997; Fakeeh and Zaki, 1999]. The similarities in the distribution of the HCV subtypes in Iran with European countries and the USA might support a common origin through large-scale medical contacts between Iran and these countries. However, although there was some intermixing of Iranian 1a and 3a strains with strains from other parts of the world, they tended to form separate subclusters in the dendrograms, indicating the presence of indigenous 1a and 3a strains. In contrast, for subtype 1b the Iranian strains intermixed with strains derived worldwide. Previously Iran imported blood and other blood products such as commercial coagulation factor concentrates, immunoglobulins from the USA, which may explain the finding of different lineages within 1b suggesting multiple introductions of 1b strains into the country. The subtype distribution within Iran did not differ distinctly according to geographical origin, although subtype 1a strains were significantly more frequent in South Iran, and 3a strains in North Iran. However, regional differences might have been more pronounced in the past with subtype 1a being indigenous to the general Iranian population, and 3a being more characteristic for the population with Turkish background. A later intermixing of strains within the country could have occurred by transmission of infected blood or blood products or nosocomially and during the 8-year Iraqi war in the eighties, in which almost 1 million soldiers from all over Iran participated. The injured soldiers underwent surgery and other treatment often requiring blood transfusions from local donors in different cities of Iran. During the war also major migrations took place with more than 2 million inhabitants living near the Iraqi border moving East to the major cities. These migrations might have disseminated HCV strains into new regions. The spread of subtype 3a in Iran may be associated with an increase in intravenous drug abuse and needle sharing, since 54% of the IDUs were infected by 3a strains. These drug users were younger than drug users infected by 1a, indicating a possible shift from 1a to 3a strains in this sub-community. There was no specific strain of either 1a or 3a spread by intravenous drug abuse. A close relationship has been shown between increase in intravenous drug abusers with needle-
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sharing and subsequent spread of subtype 3a in other countries [Pawlotsky et al., 1995; Kalinina et al., 2001]. The proportion of blood components imported from abroad is declining in Iran, and younger drug users, who will become a major source of HCV infection in the future, were more frequently infected by 3a. Therefore, a shift in prevalent subtype from 1a to 3a is likely to occur in Iran as described from St. Petersburg [Kalinina et al., 2001]. One strain from a patient on dialysis in Tehran was highly divergent from other analysed strains in the NS5B region, and split off from a branch with one genotype 5 strain from South Africa and one strain from Somalia. If this divergent strain represents a genetically drifted type 5 strain or a new subtype will be investigated. Strains of subtype 1b and 4 were rather uncommon in this study. The 1b strains were more divergent from each other than Iranian strains belonging to subtypes 1a and 3a, and were intermixed with 1b strains derived worldwide. A geographical intermixing of 1b strains has been described [Simmonds, 2001], and might be explained by patients receiving blood products being infected by strains from abroad. Half of the Iranian patients with 1b strains had haemophilia or thalassaemia, and had thus received blood products. They were, however, younger than other patients, while in other studies patients infected with 1b are older than those infected with 1a and 3a [Pawlotsky et al., 1995; Feucht et al., 1997; Smith et al., 1997]. This further supports the recent introduction of 1b into Iran while 1a and 3a are the indigenous subtypes of the country. Seven of the eight 4b strains were from patients treated by haemodialysis in Tehran. These strains might have been introduced from one of the neighbouring Arabic countries, where genotype 4 is prevalent [Al-Ahdal et al., 1997; Chamberlain et al., 1997; Fakeeh and Zaki, 1999] and become spread at dialysis units. Imports of foreign HCV strains from other countries have been documented from South Africa [Smuts and Kannemeyer, 1995]. The NS5B or core regions could not be amplified for 36 of the 161 strains positive by PCR. Eighteen of these 36 strains were from young patients with unknown source of infection in North and North-West Iran. These strains might be indigenous strains introduced into this part of Iran from regions not covered in this study. However, since they were more common in younger individuals they may also be associated with intravenous drug abuse and imported, possibly from Afghanistan from where there are few data regarding circulating HCV strains. Since the NS5B or core regions were not amplifiable for the strains from the mentioned part of Iran, they might be divergent from other HCV strains in the country and should be subject to further study. ACKNOWLEDGMENTS The sequences reported herein are deposited in GenBank under accession numbers AY653896-AY6554000.
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