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Molecular and phylogenetic analysis of HIV-1 variants circulating among injecting drug users in Mashhad-Iran

Molecular and phylogenetic analysis of HIV-1 variants circulating among injecting drug users in... Genetic and phylogenetic information on the HIV-1 epidemic in Middle-East Countries, and in particular in Iran, are extremely limited. By March 2004, the Iranian Ministry of Health officially reported a cumulative number of 6'532 HIV positive individuals and 214 AIDS cases in the Iranian HIV-1 epidemic. The intra-venous drug users (IDUs) represent the group at highest risk for HIV-1 infection in Iran, accounting for almost 63% of all HIV-infected population. In this regards, a molecular phylogenetic study has been performed on a sentinel cohort of HIV-1 seropositive IDUs enrolled at the end of 2005 at the University of Mashhad, the largest city North East of Tehran. The study has been performed on both gag and env subgenomic regions amplified by Polymerase Chain Reaction (PCR) from peripheral blood mononuclear cells (PBMCs) and characterized by direct DNA sequence analysis. The results reported here show that the HIV-1 subtype A is circulating in this IDUs sentinel cohort. Moreover, the single phylogenetic cluster as well as the intra-group low nucleotide divergence is indicative of a recent outbreak. Unexpectedly, the Iranian samples appear to be phylogenetically derived from African Sub-Saharan subtype A viruses, raising stirring speculations on HIV-1 introduction into the IDUs epidemic in Mashhad. This sentinel study could represent the starting point for a wider molecular survey of the HIV-1 epidemics in Iran to evaluate in detail the distribution of genetic subtypes and possible natural drug-resistant variants, which are extremely helpful information to design diagnostic and therapeutic strategies. Background spread among hemophiliacs has dramatically dropped The first case of HIV/AIDS in Iran was reported in 1987 in and the intra-venous drug users (IDUs) have progressively a 6-year-old child with hemophilia and since then, the become the group at highest risk for HIV-1 infection number of HIV infections in Iran has increased exponen- (62.8% of all reported infections) [1]. In particular, the tially within the hemophiliacs group [1]. After the intro- needle sharing among inmate IDUs represents a specific duction in 1989 of the mandatory blood (and its high risk behavior for HIV-1 transmission in Iran [2,3]. derivatives) screening for HIV-1 positivity, the HIV-1 Furthermore, in the last years the HIV infection is increas- Page 1 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:4 http://www.infectagentscancer.com/content/1/1/4 ingly spread in the Iranian population through sexual ual behavior. Some of them have spent few years in route (7.3% of all reported cases), although a considera- prison, representing an IDU specific high risk group ble number of infections (26.1%) are reported to be trans- within the Iranian HIV-1 epidemic [2,3]. The full designa- mitted through unknown routes [4]. By March 2004, the tion of samples, according to WHO-proposed nomencla- Iranian Ministry of Health officially reported a cumulative ture, is MSH05.00XE or MSH05.00XG, where 05 stands number of 6'532 HIV positive individuals and 214 AIDS for the year of study and E (or G) stands for env (or gag). cases [5,6]. However, the latest report from UNAIDS esti- For the sake of simplicity, however, in this paper the sam- mates a number of HIV/AIDS cases in Iran which could be ples have been indicated only with the isolate's number as much as four times higher than those officially regis- (e.g., 001) (Table 1). tered [7]. DNA was extracted at the Imam Reza General Hospital Limited data are available on HIV-1 subtype distribution from 5 × 10 peripheral blood mononuclear cells in the Middle East region where the B and C subtypes are (PBMCs) by the QIAamp DNA blood kit (Qiagen, prevalent, with the exception of Lebanon where the A sub- Alameda, CA – USA), according to the manufacturer's type is predominant [8]. Molecular epidemiology studies instructions. The quality of target DNA was verified by are extremely important to know the HIV-1 subtype distri- PCR amplification of p53 housekeeping cellular gene as bution in a specific population/region which may signifi- routinely performed at the INT in Naples, prior to viral cantly influence the diagnostic and therapeutic strategies. genes amplification [14,15]. In fact, a correlation between HIV-1 genetic subtypes and natural resistance to antiretroviral drugs, as well as effi- The hypervariable C2-V5 region of the HIV-1 env gene ciency of diagnostic serological and molecular tests, has (667 bp) and the fragment spanning the p24 and p7 been observed [9-12]. Moreover, the degree of cross-pro- region of the gag gene (460 bp) have been amplified from tection induced by vaccines, based on subtypes not pre- approximately 1 μg of purified DNA (corresponding to dominant in the target population, is still a debated 1.5 × 10 cells) by nested PCR, as previously described matter [13]. [14,15]. The DNA nucleotide sequence analysis has been performed on uncloned PCR products to identify the In this regards, a molecular phylogenetic study has been prevalent viral quasispecie. Nucleotide sequences (appr. performed on a sentinel cohort of Iranian HIV-1 seropos- 300 nucleotides) were aligned using CLUSTAL W [16], itive IDUs enrolled at the end of 2005 at the University of with minor manual adjustments, and pairwise compared Mashhad, the largest city northeast of Tehran. Blood sam- to HIV-1 reference standards of different subtypes availa- ples were obtained from 12 HIV-1 positive patients ble through the Los Alamos Database [17]. Sites with gaps attending the Department of Infectious Diseases, Imam in any of the sequences, as well as areas of uncertain align- Reza General Hospital where the HIV-1 infection was ment, were excluded from all sequence comparisons. Phy- diagnosed by immunological methods (ELISA, Western logenetic trees were constructed with the neighbor joining blot) and the CD4+ T-cell population counted. At the method [18] and the Tree View software application was enrollment, all of them declared to live in Mashhad and used to draw dendrograms. Genetic distances were calcu- to be IDUs, except one patient who reported a homosex- lated with Kimura's two-parameter method [19]. Table 1: Epidemiological and Clinical Characteristics of Mashhad samples. Sample Risk exposure Status CD4 (cells/μl) ARV PCR gag PCR env MSH01 IDU Alive 428 Yes Pos Neg MSH02 IDU Alive 444 No Pos Pos MSH03 IDU Alive 111 No Pos Pos MSH04 IDU Alive 191 No Pos Pos MSH05 Homo Dead n.a. No Neg Pos MSH06 IDU Alive 400 No Pos Pos MSH07 IDU Alive 450 Yes Pos Pos MSH08 IDU Alive 408 No Pos Pos MSH09 IDU Alive 446 No Pos Neg MSH10 IDU Dead n.a. No Pos Pos MSH11 IDU Alive 133 Yes Pos Neg MSH12 IDU Alive 129 No Pos Pos n.a, not available; homo, homosexual; IDU, injecting drug user. Page 2 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:4 http://www.infectagentscancer.com/content/1/1/4 All Mashhad samples were positive for the highly con- subgenomic regions (approx. 300 bp), which have been served p24 gag subgenomic region, with the exception of repeatedly shown to be informative for an accurate sub- the 005 sample; on the contrary, only 7 out of 12 samples type classification [14,15]. All Mashhad HIV-1 samples (58.3%) were positive for the C2-V5 env subgenomic phylogenetically cluster with reference sequences of A region. An alternative primer pair (ED5-ED12) in the first subtype in gag as well as env subgenomic regions, showing amplification round and less stringent annealing condi- no close phylogenetic relations with reference sequences tions (45°C vs 55°C) yielded the amplification of the env of neither A sub-subtypes (A2, A3, A4) nor A-based circu- subgenomic region from 2 additional samples, from the 5 lating recombinant forms (Fig. 1A and 1B). Considering previously negative ones (Table 1). The negative amplifi- that most of the CRFs show a discordant phylogenetic cation results in gag (MSH05) or env (MSH01, 09, 11) classification in the gag and env subgenomic regions [20], subgenomic regions could be explained by a significant the consistent clustering in the A subtype observed in the number of nucleotide substitutions or deletions in the present study suggests the absence of intra-genomic primers' target sequence, resulting in an inefficient recombination events which, however, need to be con- primer-to-target annealing. firmed by near full-length sequence analyses. The Mash- had samples group in a single cluster indicating a strong The HIV-1 samples identified in Mashhad were analyzed phylogenetic correlation and a recent introduction of the by phylogenetic analysis performed on both gag and env HIV-1 infection in this community with a limited genetic IR-MSH-04 IR-MSH-04 92 80 IR-MSH-06 IR-MSH-12 A IR-MSH-11 93 IR-MSH-07 IR-MSH-12 IR-MSH-10 Mashhad IR-MSH-09 IR-MSH-05 Mashhad IR-MSH-02 IR-MSH-06 10 91 880 IR-MSH-08 IR-MSH-08 IR-MSH-03 92 90 IR-MSH-02 IR-MSH-01 IR-MSH-03 IR-MSH-07 83 UG037 IR-MSH-10 UG029 UG037 PVPI UG031 80 SF1703 UG029 UG273A KE-K112 UG275A KE-K88 KE-Q168 RW-VI415 TZ03 CM4041 KE-Q259 CRF11-cpx CM0186 DJ263 VI310 92 DJ264 CRF02-AG BY1044 CM5288 CRF03-AB 94 KAL153 MP807 UG266 KE1144 UG455 TZ02 01.DDI579 DDI579 96.DDJ360 A3 A3 DDJ360 01.DDJ369 DDJ369 CM243 CRF01-AE CM181 CM238 CRF11-cpx CM449 CY032 CRF04-cpx CM184 PVCH CRF13-cpx CM416 87 DJ263 CRF02-AG 97CD KCC2 DJ264 97CD KTB13 92 A4 97CD KCC2 88 93 02CD KTB035 92 02CD KTB035 A4 CDKS10 97CD KTB13 A2 CY017 97CDKTB CF057 M389 A2 91 92 H VI991 94CY017 SE7022 DRCBL 94 J SE7887 NG083 MP255 BFP90 F2 CRF06-cpx MP257 ML127 BR020 VI557 98 95 F1 VI850 VI1991 DRCBL SE7887 90 97 86 G SE7022 NG083 VI174 BR025 89 C F1 87 DJ259A VI69 MP255 BZ167 87 93 F2 B MP257 SF2 BY1044 EQTB 85 90 CRF03-AB MP535 KAL153 ZAM18 EQTB11C C 95 BR025 MP535 JRFL ELI 100 90 D JH31 NDK NDK 97 FIN168 D 87 CRF01-AE ELI JPNH1 0.1 0.1 Phylog Figure 1 enetic trees based on HIV-1 p24 gag (A) and C2-V3 env (B) regions Phylogenetic trees based on HIV-1 p24 gag (A) and C2-V3 env (B) regions. Nucleotide sequences of Mashhad iso- lates are compared with HIV-1 reference strains of subtypes A – J of Group M. Trees have been constructed by the neighbor- joining method on 300 unambiguously aligned positions; the reliability has been estimated from 1'000 bootstrap replicates and values above 65% are indicated. The Mashhad sequences are indicated in bold characters. Page 3 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:4 http://www.infectagentscancer.com/content/1/1/4 evolution of the circulating virus. This observation is fur- A-subtype reference sequences from Sub-Saharan African ther confirmed by the overall low nucleotide divergence Countries along with sequences from Eastern European in env (10.8%, ± 2.39%) and gag (3.56%, ± 1.52%) sub- and Middle-East Countries. Unexpectedly, the Mashhad genomic regions of virus samples identified in the present cluster is closely related to the African Sub-Saharan cohort study. In particular, within the Mashhad cluster, a viruses, with any phylogenetic correlation to the Eastern single variant pair (007 – 010) is observed in both gag and European and Middle-East variants. In particular, the env phylogenetic trees. The genetic relationship between Ugandan UG037 isolate is strongly correlated to the these two viral variants is further supported by the founder of the epidemic (Fig. 2A and 2B). This is further extremely limited nucleotide divergence (10.1% in env supported by the 100% homology in the amino acid com- and 2.16% in gag), which strongly suggest a possible inter- position of the V3 Loop tip between the consensus of personal direct transmission of the HIV-1 virus, although Mashhad and sub-Saharan sequences (GPGQAFYAT), this is not confirmed by anamnestic data. while the East European consensus sequence shows an A- to-T amino acid substitution (GPGQTFYAT) [21]. In order to identify the possible geographic origin of the founder virus of HIV-1 epidemic in Mashhad, a phyloge- These findings, although obtained in a small number of netic analysis was performed including a larger number of samples and, for each sample, on limited sub-genomic IR-MSH-06 CM181 91 91 CRF11-cpx IR-MSH-04 CM449 IR-MSH-11 CM184 89 B 92 CRF13-cpx IR-MSH-12 CM416 IR-MSH-01 DDI579 IR-MSH-02 DDJ360 A3 IR-MSH-08 Mashhad DDJ369 IR-MSH-03 UG029 92 IR-MSH-09 DJ263 85 97 IR-MSH-07 DJ264 CRF02-AG IR-MSH-10 MP807 UG037 90 CM5288 UG031 TZ02 UG029 95 KE1144 KE-K112 IR-MSH-06 KE-K88 89 IR-MSH-08 TZ-HS507 Sub-Saharan IR-MSH-02 RW-VI415 92 IR-MSH-03 Africa SE7253 98 IR-MSH-04 Mashhad SE7535 94 IR-MSH-12 86 89 Q2317 A IR-MSH-07 TZ-HS123 IR-MSH-10 CM0186 89 IR-MSH-05 CRF11-cpx CM4041 UG037 RW-VI310 UG273A UZ740 UG275A 100 Sub-Saharan UZ0659 PVPI Former Russian UZ0667 95 SF1703 Africa UZ0663 Republics Q259 BY-006 Q168 93 GE-006 TZ03 KAL153 98 KZ-812 CRF03-AB BY1044 97KZ-813 UA0116 KZ-827 UG266 RU-20 UG455 UA-K127 87 88 SN.01.DDI579 UA-20 SN.96.DDJ360 A3 BL-006 Former Russian 89 SN.01.DDJ369 KZ-836 100 Republics CM243 UZ-77 CRF01-AE CM238 94 UZ-78 CY032 92 UZ-79 CRF04-cpx PVCH UZ-80 DJ263 GE-011 89 CRF02-AG DJ264 CDKS10 A2 97CD KCC2 96 CY017 02CD KTB035 A4 02CD KTB035 97CD KTB13 97CD KCC2 A4 94CY017 97 97CD KTB13 M389 CF057 A2 92 97CDKTB VI991 ZAM18 89 BR020 C F1 100 BR025 VI850 NG083 MP255 97 89 G F2 97 87 DRCBL MP257 BFP90 DRCBL CRF06-cpx 83 G ML127 NG083 VI557 100 FIN168 H CRF01-AE VI1991 JPNH1 SE7887 BR025 J 95 SE7022 DJ259A EQTB 91 91 ELI K 95 D MP535 NDK VI174 BY1044 F1 CRF03-AB VI69 KAL153 MP255 94 BZ167 F2 MP257 SF2 JRFL 89 EQTB11C B K JH31 92 MP535 ELI SE7022 93 92 NDK SE7887 0.1 0.1 Phylog dle-East Coun Figure 2 enetic trees based on HIV-1 p24 ga tries g (A) and C2-V3 env (B) regions including sequences from Eastern European and Mid- Phylogenetic trees based on HIV-1 p24 gag (A) and C2-V3 env (B) regions including sequences from Eastern European and Middle-East Countries. Nucleotide sequences of Mashhad isolates are compared with HIV-1 reference strains of Group M subtypes including a larger number of A-subtype reference sequences from Sub-Saharan African Countries along with sequences from Eastern European and Middle-East Countries. Trees have been constructed as in Fig. 1. The Mash- had sequences are indicated in bold characters. Sequence groups from different geographical regions are indicated. Page 4 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:4 http://www.infectagentscancer.com/content/1/1/4 nity-based Injecting Drug Users in Tehran, Iran. J Acquir regions, suggest that the HIV-1 epidemic currently affect- Immune Defic Syndr 2006, 42:342-346. ing the risk groups for HIV-1 infection in Iran (predomi- 4. Gheiratmand R, Navipour R, Mohebbi MR, Mallik AK: Uncertainty nantly IDUs) is driven by viral variants of the A subtype. on the number of HIV/AIDS patients: our experience in Iran. Sex Transm Infect 2005, 81:279-280. Furthermore, the strong phylogenetic correlation between 5. Montazeri A: AIDS knowledge and attitudes in Iran: results the Mashhad and the sub-Saharan isolates (in particular from a population-based survey in Tehran. Patient Educ Couns 2005, 57:199-203. Ugandan) supports the possibility that the founder virus 6. Ministry of Health: HIV/AIDS statistics; update March 2004. has been introduced from African Countries more than Tehran, Iran: Ministry of Health, Center for Disease Manage- from the neighboring Countries, which instead represent ment. 2004. 7. UNAIDS: AIDS epidemics by country. [http://data.unaids.org/ the main source of A-subtype variants in the whole Euro- pub/GlobalReport/2006/2006_GR_CH02_en.pdf]. (accessed 9 May pean Continent. A similar phylogenetic link to African 2004) isolates of A-subtype variants identified in Tehran has 8. [http://hiv-web.lanl.gov/components/hiv-db/new_geography/geogra phy.comp?region=world&form=all]. been recently reported also by Sarrami-Forooshani et al., 9. Apetrei C, Descamps D, Collin G, et al.: Human immunodefi- although a geographic origin from Former Soviet Union ciency virus type 1subtype F reverse transcriptase sequence and drug susceptibility. J Virol 1998, 72:3534-3538. Countries has been proposed [22]. Considering the Afri- 10. Descamps D, Apetrei C, Collin G, Damond F, Brun-Vezinet F: Natu- can origin of all A-subtype HIV-1 epidemics, this hypoth- rally occurring decreased susceptibility of HIV-1 subtype G esis could be supported by a HIV-1 speciation occurred at to protease inhibitors. AIDS 1998, 12:1109-1111. 11. Loussert-Ajaka I, Ly TD, Chaix ML, et al.: HIV-1/HIV-2 seronega- a different rate in the distinct regional epidemics, which, tivity in HIV-1 subtype O infected patients. Lancet 1994, at this stage, is not supported by solid epidemiological 343:1393-4. 12. Parekh B, Phillips S, Granade TC, Baggs J, Hu DJ, Respess R: Impact data. Therefore, assuming a similar HIV-1 speciation rate of HIV type 1 subtype variation on viral RNA quantitation. in Iran and neighboring Countries, the phylogenetic pat- AIDS Res Hum Retroviruses 1999, 15:133-42. tern described in the present study, together with the 13. Gao F, Korber BT, Weaver E, Liao HX, Hahn BH, Haynes BF: Cen- tralized immunogens as a vaccine strategy to overcome results reported by Sarrami-Forooshani et al., would HIV-1 diversity. Expert Rev Vaccines 2004, 3(4 Suppl):S161-8. strongly suggest an independent and more recent direct Review introduction of African A subtype virus in Iran. This could 14. Buonaguro L, Del Gaudio E, Monaco M, et al.: Heteroduplex mobility assay and phylogenetic analysis of V3 region possibly be correlated to the relevant role played by this sequences of HIV 1 isolates from Gulu – Northern Uganda. J Country, in particular Mashhad, in the Muslim religious Virol 1995, 69:7971-7981. 15. Buonaguro L, Tagliamonte M, Tornesello ML, et al.: Screening of culture. HIV-1 Isolates by Reverse Heteroduplex Mobility Assay and Identification of Non-B Subtypes in Italy. J Acquir Immune Defic These results on a sentinel cohort need to be confirmed by Syndr 2004, 37:1295-1306. 16. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving a nationwide molecular survey to verify the real distribu- the sensitivity of progressive multiple sequence alignment tion of A subtype in the country as well as in other risk through sequence weighting, position-specific gap penalties groups (Homo- and heterosexuals groups). This molecu- and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680. 17. [http://hiv-web.lanl.gov]. lar epidemiological information will be extremely rele- 18. Saitou N, Nei M: The neighbor-joining method: a new method vant to guide the development and implementation of for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-425. diagnostic as well as preventive/therapeutic approaches in 19. Kimura M: A simple method for estimating evolutioanary Iran. rates of base substitution through comparative studies of nucleotide sequence. J Mol Evol 1980, 16:111-120. 20. McCutchan F: Global epidemiology of HIV. J Med Virol 2006, Acknowledgements 78:S7-S12. This study was supported by grants from the Ministero Italiano Università 21. Korber B, MacInnes K, Smith RF, Myers G: Mutational trends in e Ricerca (MIUR, 2004), the Ministero Italiano della Sanità (Ricerca Cor- V3 loop protein sequences observed in different genetic lin- rente and Progetto Finalizzato AIDS 2004). The GeneBank accession num- eages of Human immunodeficiency virus type 1. J Virol 1994, 68:6730-6744. bers of the env sequences are DQ788541-DQ788549. The GeneBank 22. Sarrami-Forooshani R, Ranjan Das S, Sabahi F, et al.: Molecular accession numbers of the gag sequences are DQ788550- DQ788560. Analysis and Phylogenetic Characterization of HIV in Iran. J Med Virol 2006, 78(7):853-863. References 1. Gheiratmand R, Navipour R, Mohebbi MR, Hosseini KM, Motaghian- Monazzam M, Mallik AK, Samarbakhsh GR, Jamili P, de Lindvan Wijn- gaarden JW, Ahmadzadeh N, Goroohi F: A country studyto review existing capacity building and management of the trainingof teachers on preventive education against HIV/ AIDS in the schoolsin IR Iran. Available from the official website of theDeputy of Physical Education and Health, Ministry of Education of the IR Iran 2003 [http://www.neshat.org/papers/files/paper_1251.mht]. 2. Vazirian M, Nassirimanesh B, Zamani S, et al.: Needle and syringe sharing practices of injecting drug users participating in an outreach HIV prevention program in Tehran, Iran: a cross- setional study. Harm Reduct J 2005, 2:19. 3. Zamani S, Kihara M, Gouya MM, et al.: High Prevalence of HIV Infection Associated With Incarceration Among Commu- Page 5 of 5 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

Molecular and phylogenetic analysis of HIV-1 variants circulating among injecting drug users in Mashhad-Iran

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Springer Journals
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Copyright © 2006 by Naderi et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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Abstract

Genetic and phylogenetic information on the HIV-1 epidemic in Middle-East Countries, and in particular in Iran, are extremely limited. By March 2004, the Iranian Ministry of Health officially reported a cumulative number of 6'532 HIV positive individuals and 214 AIDS cases in the Iranian HIV-1 epidemic. The intra-venous drug users (IDUs) represent the group at highest risk for HIV-1 infection in Iran, accounting for almost 63% of all HIV-infected population. In this regards, a molecular phylogenetic study has been performed on a sentinel cohort of HIV-1 seropositive IDUs enrolled at the end of 2005 at the University of Mashhad, the largest city North East of Tehran. The study has been performed on both gag and env subgenomic regions amplified by Polymerase Chain Reaction (PCR) from peripheral blood mononuclear cells (PBMCs) and characterized by direct DNA sequence analysis. The results reported here show that the HIV-1 subtype A is circulating in this IDUs sentinel cohort. Moreover, the single phylogenetic cluster as well as the intra-group low nucleotide divergence is indicative of a recent outbreak. Unexpectedly, the Iranian samples appear to be phylogenetically derived from African Sub-Saharan subtype A viruses, raising stirring speculations on HIV-1 introduction into the IDUs epidemic in Mashhad. This sentinel study could represent the starting point for a wider molecular survey of the HIV-1 epidemics in Iran to evaluate in detail the distribution of genetic subtypes and possible natural drug-resistant variants, which are extremely helpful information to design diagnostic and therapeutic strategies. Background spread among hemophiliacs has dramatically dropped The first case of HIV/AIDS in Iran was reported in 1987 in and the intra-venous drug users (IDUs) have progressively a 6-year-old child with hemophilia and since then, the become the group at highest risk for HIV-1 infection number of HIV infections in Iran has increased exponen- (62.8% of all reported infections) [1]. In particular, the tially within the hemophiliacs group [1]. After the intro- needle sharing among inmate IDUs represents a specific duction in 1989 of the mandatory blood (and its high risk behavior for HIV-1 transmission in Iran [2,3]. derivatives) screening for HIV-1 positivity, the HIV-1 Furthermore, in the last years the HIV infection is increas- Page 1 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:4 http://www.infectagentscancer.com/content/1/1/4 ingly spread in the Iranian population through sexual ual behavior. Some of them have spent few years in route (7.3% of all reported cases), although a considera- prison, representing an IDU specific high risk group ble number of infections (26.1%) are reported to be trans- within the Iranian HIV-1 epidemic [2,3]. The full designa- mitted through unknown routes [4]. By March 2004, the tion of samples, according to WHO-proposed nomencla- Iranian Ministry of Health officially reported a cumulative ture, is MSH05.00XE or MSH05.00XG, where 05 stands number of 6'532 HIV positive individuals and 214 AIDS for the year of study and E (or G) stands for env (or gag). cases [5,6]. However, the latest report from UNAIDS esti- For the sake of simplicity, however, in this paper the sam- mates a number of HIV/AIDS cases in Iran which could be ples have been indicated only with the isolate's number as much as four times higher than those officially regis- (e.g., 001) (Table 1). tered [7]. DNA was extracted at the Imam Reza General Hospital Limited data are available on HIV-1 subtype distribution from 5 × 10 peripheral blood mononuclear cells in the Middle East region where the B and C subtypes are (PBMCs) by the QIAamp DNA blood kit (Qiagen, prevalent, with the exception of Lebanon where the A sub- Alameda, CA – USA), according to the manufacturer's type is predominant [8]. Molecular epidemiology studies instructions. The quality of target DNA was verified by are extremely important to know the HIV-1 subtype distri- PCR amplification of p53 housekeeping cellular gene as bution in a specific population/region which may signifi- routinely performed at the INT in Naples, prior to viral cantly influence the diagnostic and therapeutic strategies. genes amplification [14,15]. In fact, a correlation between HIV-1 genetic subtypes and natural resistance to antiretroviral drugs, as well as effi- The hypervariable C2-V5 region of the HIV-1 env gene ciency of diagnostic serological and molecular tests, has (667 bp) and the fragment spanning the p24 and p7 been observed [9-12]. Moreover, the degree of cross-pro- region of the gag gene (460 bp) have been amplified from tection induced by vaccines, based on subtypes not pre- approximately 1 μg of purified DNA (corresponding to dominant in the target population, is still a debated 1.5 × 10 cells) by nested PCR, as previously described matter [13]. [14,15]. The DNA nucleotide sequence analysis has been performed on uncloned PCR products to identify the In this regards, a molecular phylogenetic study has been prevalent viral quasispecie. Nucleotide sequences (appr. performed on a sentinel cohort of Iranian HIV-1 seropos- 300 nucleotides) were aligned using CLUSTAL W [16], itive IDUs enrolled at the end of 2005 at the University of with minor manual adjustments, and pairwise compared Mashhad, the largest city northeast of Tehran. Blood sam- to HIV-1 reference standards of different subtypes availa- ples were obtained from 12 HIV-1 positive patients ble through the Los Alamos Database [17]. Sites with gaps attending the Department of Infectious Diseases, Imam in any of the sequences, as well as areas of uncertain align- Reza General Hospital where the HIV-1 infection was ment, were excluded from all sequence comparisons. Phy- diagnosed by immunological methods (ELISA, Western logenetic trees were constructed with the neighbor joining blot) and the CD4+ T-cell population counted. At the method [18] and the Tree View software application was enrollment, all of them declared to live in Mashhad and used to draw dendrograms. Genetic distances were calcu- to be IDUs, except one patient who reported a homosex- lated with Kimura's two-parameter method [19]. Table 1: Epidemiological and Clinical Characteristics of Mashhad samples. Sample Risk exposure Status CD4 (cells/μl) ARV PCR gag PCR env MSH01 IDU Alive 428 Yes Pos Neg MSH02 IDU Alive 444 No Pos Pos MSH03 IDU Alive 111 No Pos Pos MSH04 IDU Alive 191 No Pos Pos MSH05 Homo Dead n.a. No Neg Pos MSH06 IDU Alive 400 No Pos Pos MSH07 IDU Alive 450 Yes Pos Pos MSH08 IDU Alive 408 No Pos Pos MSH09 IDU Alive 446 No Pos Neg MSH10 IDU Dead n.a. No Pos Pos MSH11 IDU Alive 133 Yes Pos Neg MSH12 IDU Alive 129 No Pos Pos n.a, not available; homo, homosexual; IDU, injecting drug user. Page 2 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:4 http://www.infectagentscancer.com/content/1/1/4 All Mashhad samples were positive for the highly con- subgenomic regions (approx. 300 bp), which have been served p24 gag subgenomic region, with the exception of repeatedly shown to be informative for an accurate sub- the 005 sample; on the contrary, only 7 out of 12 samples type classification [14,15]. All Mashhad HIV-1 samples (58.3%) were positive for the C2-V5 env subgenomic phylogenetically cluster with reference sequences of A region. An alternative primer pair (ED5-ED12) in the first subtype in gag as well as env subgenomic regions, showing amplification round and less stringent annealing condi- no close phylogenetic relations with reference sequences tions (45°C vs 55°C) yielded the amplification of the env of neither A sub-subtypes (A2, A3, A4) nor A-based circu- subgenomic region from 2 additional samples, from the 5 lating recombinant forms (Fig. 1A and 1B). Considering previously negative ones (Table 1). The negative amplifi- that most of the CRFs show a discordant phylogenetic cation results in gag (MSH05) or env (MSH01, 09, 11) classification in the gag and env subgenomic regions [20], subgenomic regions could be explained by a significant the consistent clustering in the A subtype observed in the number of nucleotide substitutions or deletions in the present study suggests the absence of intra-genomic primers' target sequence, resulting in an inefficient recombination events which, however, need to be con- primer-to-target annealing. firmed by near full-length sequence analyses. The Mash- had samples group in a single cluster indicating a strong The HIV-1 samples identified in Mashhad were analyzed phylogenetic correlation and a recent introduction of the by phylogenetic analysis performed on both gag and env HIV-1 infection in this community with a limited genetic IR-MSH-04 IR-MSH-04 92 80 IR-MSH-06 IR-MSH-12 A IR-MSH-11 93 IR-MSH-07 IR-MSH-12 IR-MSH-10 Mashhad IR-MSH-09 IR-MSH-05 Mashhad IR-MSH-02 IR-MSH-06 10 91 880 IR-MSH-08 IR-MSH-08 IR-MSH-03 92 90 IR-MSH-02 IR-MSH-01 IR-MSH-03 IR-MSH-07 83 UG037 IR-MSH-10 UG029 UG037 PVPI UG031 80 SF1703 UG029 UG273A KE-K112 UG275A KE-K88 KE-Q168 RW-VI415 TZ03 CM4041 KE-Q259 CRF11-cpx CM0186 DJ263 VI310 92 DJ264 CRF02-AG BY1044 CM5288 CRF03-AB 94 KAL153 MP807 UG266 KE1144 UG455 TZ02 01.DDI579 DDI579 96.DDJ360 A3 A3 DDJ360 01.DDJ369 DDJ369 CM243 CRF01-AE CM181 CM238 CRF11-cpx CM449 CY032 CRF04-cpx CM184 PVCH CRF13-cpx CM416 87 DJ263 CRF02-AG 97CD KCC2 DJ264 97CD KTB13 92 A4 97CD KCC2 88 93 02CD KTB035 92 02CD KTB035 A4 CDKS10 97CD KTB13 A2 CY017 97CDKTB CF057 M389 A2 91 92 H VI991 94CY017 SE7022 DRCBL 94 J SE7887 NG083 MP255 BFP90 F2 CRF06-cpx MP257 ML127 BR020 VI557 98 95 F1 VI850 VI1991 DRCBL SE7887 90 97 86 G SE7022 NG083 VI174 BR025 89 C F1 87 DJ259A VI69 MP255 BZ167 87 93 F2 B MP257 SF2 BY1044 EQTB 85 90 CRF03-AB MP535 KAL153 ZAM18 EQTB11C C 95 BR025 MP535 JRFL ELI 100 90 D JH31 NDK NDK 97 FIN168 D 87 CRF01-AE ELI JPNH1 0.1 0.1 Phylog Figure 1 enetic trees based on HIV-1 p24 gag (A) and C2-V3 env (B) regions Phylogenetic trees based on HIV-1 p24 gag (A) and C2-V3 env (B) regions. Nucleotide sequences of Mashhad iso- lates are compared with HIV-1 reference strains of subtypes A – J of Group M. Trees have been constructed by the neighbor- joining method on 300 unambiguously aligned positions; the reliability has been estimated from 1'000 bootstrap replicates and values above 65% are indicated. The Mashhad sequences are indicated in bold characters. Page 3 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:4 http://www.infectagentscancer.com/content/1/1/4 evolution of the circulating virus. This observation is fur- A-subtype reference sequences from Sub-Saharan African ther confirmed by the overall low nucleotide divergence Countries along with sequences from Eastern European in env (10.8%, ± 2.39%) and gag (3.56%, ± 1.52%) sub- and Middle-East Countries. Unexpectedly, the Mashhad genomic regions of virus samples identified in the present cluster is closely related to the African Sub-Saharan cohort study. In particular, within the Mashhad cluster, a viruses, with any phylogenetic correlation to the Eastern single variant pair (007 – 010) is observed in both gag and European and Middle-East variants. In particular, the env phylogenetic trees. The genetic relationship between Ugandan UG037 isolate is strongly correlated to the these two viral variants is further supported by the founder of the epidemic (Fig. 2A and 2B). This is further extremely limited nucleotide divergence (10.1% in env supported by the 100% homology in the amino acid com- and 2.16% in gag), which strongly suggest a possible inter- position of the V3 Loop tip between the consensus of personal direct transmission of the HIV-1 virus, although Mashhad and sub-Saharan sequences (GPGQAFYAT), this is not confirmed by anamnestic data. while the East European consensus sequence shows an A- to-T amino acid substitution (GPGQTFYAT) [21]. In order to identify the possible geographic origin of the founder virus of HIV-1 epidemic in Mashhad, a phyloge- These findings, although obtained in a small number of netic analysis was performed including a larger number of samples and, for each sample, on limited sub-genomic IR-MSH-06 CM181 91 91 CRF11-cpx IR-MSH-04 CM449 IR-MSH-11 CM184 89 B 92 CRF13-cpx IR-MSH-12 CM416 IR-MSH-01 DDI579 IR-MSH-02 DDJ360 A3 IR-MSH-08 Mashhad DDJ369 IR-MSH-03 UG029 92 IR-MSH-09 DJ263 85 97 IR-MSH-07 DJ264 CRF02-AG IR-MSH-10 MP807 UG037 90 CM5288 UG031 TZ02 UG029 95 KE1144 KE-K112 IR-MSH-06 KE-K88 89 IR-MSH-08 TZ-HS507 Sub-Saharan IR-MSH-02 RW-VI415 92 IR-MSH-03 Africa SE7253 98 IR-MSH-04 Mashhad SE7535 94 IR-MSH-12 86 89 Q2317 A IR-MSH-07 TZ-HS123 IR-MSH-10 CM0186 89 IR-MSH-05 CRF11-cpx CM4041 UG037 RW-VI310 UG273A UZ740 UG275A 100 Sub-Saharan UZ0659 PVPI Former Russian UZ0667 95 SF1703 Africa UZ0663 Republics Q259 BY-006 Q168 93 GE-006 TZ03 KAL153 98 KZ-812 CRF03-AB BY1044 97KZ-813 UA0116 KZ-827 UG266 RU-20 UG455 UA-K127 87 88 SN.01.DDI579 UA-20 SN.96.DDJ360 A3 BL-006 Former Russian 89 SN.01.DDJ369 KZ-836 100 Republics CM243 UZ-77 CRF01-AE CM238 94 UZ-78 CY032 92 UZ-79 CRF04-cpx PVCH UZ-80 DJ263 GE-011 89 CRF02-AG DJ264 CDKS10 A2 97CD KCC2 96 CY017 02CD KTB035 A4 02CD KTB035 97CD KTB13 97CD KCC2 A4 94CY017 97 97CD KTB13 M389 CF057 A2 92 97CDKTB VI991 ZAM18 89 BR020 C F1 100 BR025 VI850 NG083 MP255 97 89 G F2 97 87 DRCBL MP257 BFP90 DRCBL CRF06-cpx 83 G ML127 NG083 VI557 100 FIN168 H CRF01-AE VI1991 JPNH1 SE7887 BR025 J 95 SE7022 DJ259A EQTB 91 91 ELI K 95 D MP535 NDK VI174 BY1044 F1 CRF03-AB VI69 KAL153 MP255 94 BZ167 F2 MP257 SF2 JRFL 89 EQTB11C B K JH31 92 MP535 ELI SE7022 93 92 NDK SE7887 0.1 0.1 Phylog dle-East Coun Figure 2 enetic trees based on HIV-1 p24 ga tries g (A) and C2-V3 env (B) regions including sequences from Eastern European and Mid- Phylogenetic trees based on HIV-1 p24 gag (A) and C2-V3 env (B) regions including sequences from Eastern European and Middle-East Countries. Nucleotide sequences of Mashhad isolates are compared with HIV-1 reference strains of Group M subtypes including a larger number of A-subtype reference sequences from Sub-Saharan African Countries along with sequences from Eastern European and Middle-East Countries. Trees have been constructed as in Fig. 1. The Mash- had sequences are indicated in bold characters. Sequence groups from different geographical regions are indicated. Page 4 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:4 http://www.infectagentscancer.com/content/1/1/4 nity-based Injecting Drug Users in Tehran, Iran. J Acquir regions, suggest that the HIV-1 epidemic currently affect- Immune Defic Syndr 2006, 42:342-346. ing the risk groups for HIV-1 infection in Iran (predomi- 4. Gheiratmand R, Navipour R, Mohebbi MR, Mallik AK: Uncertainty nantly IDUs) is driven by viral variants of the A subtype. on the number of HIV/AIDS patients: our experience in Iran. Sex Transm Infect 2005, 81:279-280. Furthermore, the strong phylogenetic correlation between 5. Montazeri A: AIDS knowledge and attitudes in Iran: results the Mashhad and the sub-Saharan isolates (in particular from a population-based survey in Tehran. Patient Educ Couns 2005, 57:199-203. Ugandan) supports the possibility that the founder virus 6. Ministry of Health: HIV/AIDS statistics; update March 2004. has been introduced from African Countries more than Tehran, Iran: Ministry of Health, Center for Disease Manage- from the neighboring Countries, which instead represent ment. 2004. 7. UNAIDS: AIDS epidemics by country. [http://data.unaids.org/ the main source of A-subtype variants in the whole Euro- pub/GlobalReport/2006/2006_GR_CH02_en.pdf]. (accessed 9 May pean Continent. A similar phylogenetic link to African 2004) isolates of A-subtype variants identified in Tehran has 8. [http://hiv-web.lanl.gov/components/hiv-db/new_geography/geogra phy.comp?region=world&form=all]. been recently reported also by Sarrami-Forooshani et al., 9. Apetrei C, Descamps D, Collin G, et al.: Human immunodefi- although a geographic origin from Former Soviet Union ciency virus type 1subtype F reverse transcriptase sequence and drug susceptibility. J Virol 1998, 72:3534-3538. Countries has been proposed [22]. Considering the Afri- 10. Descamps D, Apetrei C, Collin G, Damond F, Brun-Vezinet F: Natu- can origin of all A-subtype HIV-1 epidemics, this hypoth- rally occurring decreased susceptibility of HIV-1 subtype G esis could be supported by a HIV-1 speciation occurred at to protease inhibitors. AIDS 1998, 12:1109-1111. 11. Loussert-Ajaka I, Ly TD, Chaix ML, et al.: HIV-1/HIV-2 seronega- a different rate in the distinct regional epidemics, which, tivity in HIV-1 subtype O infected patients. Lancet 1994, at this stage, is not supported by solid epidemiological 343:1393-4. 12. Parekh B, Phillips S, Granade TC, Baggs J, Hu DJ, Respess R: Impact data. Therefore, assuming a similar HIV-1 speciation rate of HIV type 1 subtype variation on viral RNA quantitation. in Iran and neighboring Countries, the phylogenetic pat- AIDS Res Hum Retroviruses 1999, 15:133-42. tern described in the present study, together with the 13. Gao F, Korber BT, Weaver E, Liao HX, Hahn BH, Haynes BF: Cen- tralized immunogens as a vaccine strategy to overcome results reported by Sarrami-Forooshani et al., would HIV-1 diversity. Expert Rev Vaccines 2004, 3(4 Suppl):S161-8. strongly suggest an independent and more recent direct Review introduction of African A subtype virus in Iran. This could 14. Buonaguro L, Del Gaudio E, Monaco M, et al.: Heteroduplex mobility assay and phylogenetic analysis of V3 region possibly be correlated to the relevant role played by this sequences of HIV 1 isolates from Gulu – Northern Uganda. J Country, in particular Mashhad, in the Muslim religious Virol 1995, 69:7971-7981. 15. Buonaguro L, Tagliamonte M, Tornesello ML, et al.: Screening of culture. HIV-1 Isolates by Reverse Heteroduplex Mobility Assay and Identification of Non-B Subtypes in Italy. J Acquir Immune Defic These results on a sentinel cohort need to be confirmed by Syndr 2004, 37:1295-1306. 16. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving a nationwide molecular survey to verify the real distribu- the sensitivity of progressive multiple sequence alignment tion of A subtype in the country as well as in other risk through sequence weighting, position-specific gap penalties groups (Homo- and heterosexuals groups). This molecu- and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680. 17. [http://hiv-web.lanl.gov]. lar epidemiological information will be extremely rele- 18. Saitou N, Nei M: The neighbor-joining method: a new method vant to guide the development and implementation of for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-425. diagnostic as well as preventive/therapeutic approaches in 19. Kimura M: A simple method for estimating evolutioanary Iran. rates of base substitution through comparative studies of nucleotide sequence. J Mol Evol 1980, 16:111-120. 20. McCutchan F: Global epidemiology of HIV. J Med Virol 2006, Acknowledgements 78:S7-S12. This study was supported by grants from the Ministero Italiano Università 21. Korber B, MacInnes K, Smith RF, Myers G: Mutational trends in e Ricerca (MIUR, 2004), the Ministero Italiano della Sanità (Ricerca Cor- V3 loop protein sequences observed in different genetic lin- rente and Progetto Finalizzato AIDS 2004). The GeneBank accession num- eages of Human immunodeficiency virus type 1. J Virol 1994, 68:6730-6744. bers of the env sequences are DQ788541-DQ788549. The GeneBank 22. Sarrami-Forooshani R, Ranjan Das S, Sabahi F, et al.: Molecular accession numbers of the gag sequences are DQ788550- DQ788560. Analysis and Phylogenetic Characterization of HIV in Iran. J Med Virol 2006, 78(7):853-863. References 1. Gheiratmand R, Navipour R, Mohebbi MR, Hosseini KM, Motaghian- Monazzam M, Mallik AK, Samarbakhsh GR, Jamili P, de Lindvan Wijn- gaarden JW, Ahmadzadeh N, Goroohi F: A country studyto review existing capacity building and management of the trainingof teachers on preventive education against HIV/ AIDS in the schoolsin IR Iran. Available from the official website of theDeputy of Physical Education and Health, Ministry of Education of the IR Iran 2003 [http://www.neshat.org/papers/files/paper_1251.mht]. 2. Vazirian M, Nassirimanesh B, Zamani S, et al.: Needle and syringe sharing practices of injecting drug users participating in an outreach HIV prevention program in Tehran, Iran: a cross- setional study. Harm Reduct J 2005, 2:19. 3. Zamani S, Kihara M, Gouya MM, et al.: High Prevalence of HIV Infection Associated With Incarceration Among Commu- Page 5 of 5 (page number not for citation purposes)

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Infectious Agents and Cancer – Springer Journals

Published: Sep 19, 2006

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