AIDS RESEARCH AND HUMAN RETROVIRUSES SEQUENCE NOTE Volume 25, Number 9, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=aid.2009.0075

Epidemiological Networks and Drug Resistance of HIV Type 1 in Region,

Olga A. Rumyantseva,1,2 Igor A. Olkhovskiy,1 Marina A. Malysheva,1 Ludmila A. Ruzaeva,1 Alexander V. Vasiliev,2 Elena V. Kazennova,2 Marina R. Bobkova,2 and Vladimir V. Lukashov2,3

Abstract

To study the molecular epidemiology of HIV-1 in Krasnoyarsk region, Russia, where HIV-1 has spread rapidly since 2000, we obtained pol sequences from individuals living in this region (n ¼ 67) as well as in the geo- graphically closely related Altay region (n ¼ 13). In both regions, subtype A viruses specific for the former (IDU-A strains) were dominant (92.5%). Virus sequences clustered according to the geographic origin of the infected individuals rather than to their risk group, demonstrating the role of geographically defined epi- demiological networks in the propagation of the HIV-1 epidemic in the region. Six viruses belonged to subtype B. Three of them were phylogenetically (and therefore epidemiologically) closely related to each other, dem- onstrating that even though IDU-A viruses dominate the epidemic, the spread of other virus strains does occur. Most viruses (75%) had an A62V mutation in reverse transcriptase, specific for HIV-1 strains in Russia. Re- markably, 26 of 47 (55%) patients under HAART with detectable virus loads did not have any known drug- resistant mutation, indicating the need to increase compliance to therapy.

he HIV-1 epidemic in Russia and other new indepen- is growing (Fig. 1B). Most HIV-1-infected individuals (75%) Tdent states of the former Soviet Union started later than in are <30 years old. West Europe. The first massive outbreaks and subsequent Because of the relatively late beginning of the HIV-1 epi- explosive spread of HIV-1 in the former Soviet Union have demic in the Krasnoyarsk region, until recently highly active been registered since 1996 with the estimated total number of antiretroviral therapy (HAART) was not widely used to treat HIV-1 infections exceeding one million.1 Molecular epidemi- HIV-1-infected individuals. HAART has become widely used ological studies demonstrated that the HIV-1 epidemic in only since 2004–2006. To study the molecular epidemiology of Russia and other countries of the former Soviet Union is HIV-1 and the effectiveness of HAART, a ViroSeq HIV-1 caused primarily by the spread of subtype A HIV-1 strains Genotyping System (Abbott Laboratories, Abbott Park, IL) is (known as IDU-A strains, up to 90% of all HIV-1 cases in the used in the Krasnoyarsk Regional AIDS Center. Specific at- region).2–14 tention is given to the study of HIV-1-infected individuals The Krasnoyarsk region (Krasnoyarskiy kray, in Russian) is failing their HAART regimens (to identify possible drug- one of the largest federal subjects of Russia, being the second resistant mutations) and to newly infected individuals largest in territory and the thirteenth largest in population (to monitor possible transmissions of drug-resistant HIV-1 among the 83 federal subjects (Fig. 1A). Before 2000, only 217 mutants). HIV-1 infections had been reported in the region. Since 2000, In this study, we analyzed HIV-1 sequences ( pol gene, full the epidemic in Krasnoyarsk region has been rapidly ex- protease, and partial reverse transcriptase regions, sequence panding (Fig. 1B). As of January 1, 2009, 9581 HIV-1-infected length—1302 nucleotides) obtained from 80 infected indi- individuals were registered in the region, which places it viduals from the Krasnoyarsk (n ¼ 67) and Altay regions among the 20 regions most affected by HIV-1 in Russia (331 (Altayskiy kray, in Russian, n ¼ 13). Epidemiological informa- HIV-1-infected individuals per 100,000 inhabitants). Although tion for the study individuals is presented in Table 1. Sequences most of these HIV-1 infections are associated with drug usage, obtained in this study have been deposited in GenBank (ac- the proportion of individuals infected through sexual contact cession numbers FJ822055–FJ822134). Phylogenetic analysis

1Krasnoyarsk Regional AIDS Center, Krasnoyarsk, Russia. 2D.I. Ivanovsky Institute of Virology, Moscow, Russia. 3Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

931 932 RUMYANTSEVA ET AL.

B Number of infections Transmission routes 10000 100%

9000 90% Number of infections 8000 80% New infections 7000 70% Cumulative number 6000 60%

5000 50%

4000 40% Transmission routes 3000 30% IDU 2000 20% Heterosexual

1000 10% Other (homosexual, vertical, hospital infections) 0 0%

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 1989-1997 Year

FIG. 1. Map of Russia showing the geographic origin of study individuals (A) and dynamics of the HIV-1 epidemic in the Krasnoyarsk region (B). Table 1. Epidemiological Data and HIV-1 Subtypes for Study Individuals Study subject Residence Transmission route Gender Year of birth First seropositive Sampling year HIV-1 subtype ( pol) 1 Krasnoyarsk IDU m 1971 2005 2008 A 2 Krasnoyarsk IDU f 1979 2008 2008 A 3 Krasnoyarsk IDU m 1978 2004 2008 A 4 Krasnoyarsk IDU m 1979 2008 2008 A 5 Krasnoyarsk IDU m 1979 2005 2008 A 6 Krasnoyarsk IDU m 1974 2002 2008 A 7 Krasnoyarsk IDU m 1973 2000 2008 A 8 Krasnoyarsk IDU m 1973 1999 2008 A 9 Krasnoyarsk IDU m 1964 2003 2008 A 10 Krasnoyarsk IDU f 1988 2007 2008 A 11 Krasnoyarsk IDU m 1970 2008 2008 A 12 Krasnoyarsk IDU m 1982 2001 2008 A 13 Krasnoyarsk IDU f 1982 2001 2008 A 14 Krasnoyarsk IDU m 1976 2006 2008 A 15 Krasnoyarsk IDU m 1968 1999 2008 A 16 Krasnoyarsk IDU m 1962 2002 2008 A 17 Krasnoyarsk IDU f 1985 2008 2008 A 18 Krasnoyarsk IDU f 1977 2008 2008 A 19 Krasnoyarsk IDU m 1977 2004 2007 A 20 Krasnoyarsk IDU m 1984 2001 2007 A 21 Krasnoyarsk IDU m 1982 2001 2007 A 22 Krasnoyarsk IDU m 1981 2000 2008 A 23 Krasnoyarsk IDU m 1978 2009 2009 A 24 Krasnoyarsk IDU m 1969 2001 2009 A 25 Krasnoyarsk IDU f 1981 2008 2009 A 26 Krasnoyarsk IDU m 1982 2008 2009 A 27 Krasnoyarsk Heterosexual f 1962 2008 2008 B 28 Krasnoyarsk Heterosexual m 1977 2003 2008 A 29 Krasnoyarsk Heterosexual f 1978 2006 2008 A 30 Krasnoyarsk Heterosexual m 1974 2003 2008 A 31 Krasnoyarsk Heterosexual f 1980 2008 2008 A 32 Krasnoyarsk Heterosexual f 1954 2002 2008 A 33 Krasnoyarsk Heterosexual f 1987 2008 2008 A 34 Krasnoyarsk Heterosexual m 1954 2002 2008 B 35 Krasnoyarsk Heterosexual m 1968 2007 2008 B 36 Krasnoyarsk Heterosexual m 1971 2007 2008 A 37 Krasnoyarsk Heterosexual f 1972 2006 2008 A 38 Krasnoyarsk Heterosexual f 1979 2001 2008 A

39 Krasnoyarsk Heterosexual f 1982 2008 2008 A 40 Krasnoyarsk Heterosexual f 1976 2007 2007 A 41 Krasnoyarsk Heterosexual f 1991 2007 2007 A 42 Krasnoyarsk Heterosexual f 1976 2006 2007 A 43 Krasnoyarsk Heterosexual f 1986 2006 2008 A 44 Krasnoyarsk Heterosexual m 1979 2007 2008 A 45 Krasnoyarsk Heterosexual f 1958 2003 2008 B 46 Krasnoyarsk Heterosexual f 1985 2008 2008 A 47 Krasnoyarsk Heterosexual m 1982 2002 2008 A 48 Krasnoyarsk Heterosexual f 1984 2006 2008 A 49 Krasnoyarsk Heterosexual m 1972 2002 2008 A 50 Krasnoyarsk Homosexual m 1965 2006 2008 A 51 Krasnoyarsk Homosexual m 1952 2007 2007 A 52 Krasnoyarsk Vertical m 2000 2002 2008 A 53 Krasnoyarsk Vertical m 2001 2003 2008 A 54 Krasnoyarsk Vertical m 2004 2005 2008 A 55 Krasnoyarsk Vertical f 1999 2002 2008 A 56 Berezovka Heterosexual f 1986 2008 2008 A 57 Divnogorsk Heterosexual m 1977 2000 2008 A 58 Divnogorsk Homosexual m 1952 2008 2008 B 59 Zheleznogorsk Heterosexual f 1979 2001 2008 A 60 Sosnovoborsk Homosexual m 1981 2008 2008 B 61 Borodino Heterosexual m 1978 2007 2008 A 62 Vertical f 2007 2007 2008 A 63 Heterosexual f 1971 2001 2008 A 64 Partizanskoe Heterosexual f 1962 2008 2008 A 65 Uyar IDU m 1973 2008 2008 A 66 Uyar IDU m 1976 2007 2007 A 67 IDU f 1980 2001 2008 A 68 Biysk, Altayskiy kray Unknown m 1963 2007 2008 A 69 Biysk, Altayskiy kray Unknown f 1973 2004 2008 A 70 Biysk, Altayskiy kray Unknown m 1980 2006 2008 A 71 Biysk, Altayskiy kray Unknown m 1971 2001 2008 A 72 Biysk, Altayskiy kray Unknown f 1981 2005 2008 A 73 Biysk, Altayskiy kray Unknown f 1989 2000 2008 A 74 Biysk, Altayskiy kray Unknown m 1975 2000 2008 A 75 Biysk, Altayskiy kray Unknown m 1979 2001 2008 A 76 Biysk, Altayskiy kray Unknown f 1983 2000 2008 A 77 Biysk, Altayskiy kray Unknown m 1987 2002 2008 A 78 Biysk, Altayskiy kray Unknown m 1978 2001 2008 A 79 Biysk, Altayskiy kray Unknown m 1975 2006 2008 A 80 Biysk, Altayskiy kray Unknown m 1979 2000 2008 A 934 RUMYANTSEVA ET AL.

FIG. 2. Phylogenetic analysis of HIV-1 pol sequences from study individuals. Sequences obtained in this study are labeled as in Table 1. Reference sequences of HIV-1 subtypes and sequences of virus strains specific for the epidemic in the former Soviet Union are included in the analysis. For IDU-A viruses, the consensus of viruses from the Krasnoyarsk and Altay regions is included. Bootstrap values 70% are shown. HIV-1 IN KRASNOYARSK REGION, RUSSIA 935 was performed by using the MEGA4 program (Kimura dis- Disclosure Statement tance estimation model, pairwise gap deletion, neighbor- No competing financial interests exist. joining method, bootstrap analysis with 1000 replications). Phylogenetic analysis demonstrated that most of virus References strains (n ¼ 74, 92.5%) belonged to subtype A and clustered together with IDU-A strains from other regions of the former 1. Hamers FF and Downs AM: HIV in central and eastern Soviet Union (Fig. 2). The remaining six virus strains (7.5%) Europe. Lancet 2003;361:1035–1044. sequences belonged to subtype B. Phylogenetic clustering of 2. Lazouskaya NV, Eremin VF, Adema KW, Gasich EL, Baan E, and Lukashov VV: The HIV type 1 epidemic in Belarus: virus strains according to the geographic origin of the infected Predominance of Eastern European subtype A strains and individuals, rather than to their risk group, was observed. The circulation of subtype B viruses. AIDS Res Hum Retro- largest cluster (cluster 1, Fig. 2) contained 25 sequences from viruses 2005;21:830–833. Krasnoyarsk and nearby cities (Zheleznogorsk and Bere- 3. Lukashov VV, Karamov EV, Eremin VF, Titov LP, and zovka). Also, 10 of 13 strains from Biysk clustered together Goudsmit J: Extreme founder effect in an HIV type 1 sub- (although with low bootstrap support, cluster 2). Genetic het- type A epidemic among drug users in Svetlogorsk, Belarus. erogeneity among sequences from Biysk (the mean p-distance AIDS Res Hum Retroviruses 1998;14:1299–1303. of 0.019 0.002) was much lower than that among sequences 4. Lukashov VV, Huismans R, Rakhmanova AG, Lisitsina ZN, from the city of Krasnoyarsk (0.026 0.002). Akhtyrskaya NA, Vlasov NN, Melnick , and Goudsmit J: Five statistically supported smaller phylogenetic clusters, Circulation of subtype A and gagA=envB recombinant HIV containing two or three sequences each (clusters 3–7), were also type 1 strains among injecting drug users in St. Petersburg, formed by virus strains with close geographic origins. For in- Russia, correlates with geographical origin of infections. stance, both sequences from Uyar in our study clustered to- AIDS Res Hum Retroviruses 1999;15:1577–1583. gether (cluster 4). All seven clusters were also observed when 5. Nabatov AA, Kravchenko ON, Lyulchuk MG, Shcherbinskaya synonymous substitutions were analyzed (data not shown), AM, and Lukashov VV: Simultaneous introduction of HIV indicating that clustering was not influenced by convergent type 1 subtype A and B viruses into injecting drug users in evolution (the same drug-resistant mutations). Interestingly, southern Ukraine at the beginning of the epidemic in the former three of six subtype B viruses from our study clustered together Soviet Union. AIDS Res Hum Retroviruses 2002;18:891–895. (cluster 7, bootstrap value 100%). Although individual cases of 6. Bobkov A, Cheingsong-Popov R, Selimova L, Ladnaya N, subtype B infections have been registered in Russia before, no Kazennova E, Kravchenko A, Fedotov E, Saukhat S, Zverev S, Pokrovsky V, and Weber J: An HIV type 1 epidemic outbreaks caused by (closely related) subtype B viruses were among injecting drug users in the former Soviet Union known. The only outbreak of subtype B has been registered in 5 caused by a homogeneous subtype A strain. AIDS Res Hum

Nikolaev, Ukraine, by a virus known as the IDU-B strain, to Retroviruses 1997;13:1195–1201. which these three subtype B viruses were unrelated (Fig. 2). 7. Bobkov A, Kazennova E, Selimova L, Bobkova M, Khanina This cluster included individuals whose only reported risk T, Ladnaya N, Kravchenko A, Pokrovsky V, Cheingsong- factor was sexual contact. Thus our findings indicate that even Popov R, and Weber J: A sudden epidemic of HIV type 1 though IDU-A viruses dominate the epidemic in Russia, the among injecting drug users in the former Soviet Union: introduction and spread of other virus strains do occur. Identification of subtype A, subtype B, and novel gagA= Our analysis revealed that 30 of 33 therapy-naive individ- envB recombinants. AIDS Res Hum Retroviruses 1998; uals in the study did not have drug-resistant mutations. Yet in 14:669–676. the majority of these individuals, mutations that do not cause 8. Bobkov A, Kazennova E, Khanina T, Bobkova M, Selimova resistance themselves but are associated with drug-resistant L, Kravchenko A, Pokrovsky V, and Weber J: An HIV type 1 mutations were identified. In particular, 21 of 33 individuals subtype A strain of low genetic diversity continues to spread (64%), as well as 39 of 47 patients under HAART (83%), had an among injecting drug users in Russia: Study of the new local A62V mutation in reverse transcriptase. HIV-1 strains with outbreaks in Moscow and Irkutsk. AIDS Res Hum Retro- this mutation are dominant in Russia.15 Two therapy-naive viruses 2001;17:257–261. individuals had K101E and V108I mutations (possibly) con- 9. Novitsky VA, Montano MA, and Essex M: Molecular epi- tributing to NNRTI resistance; one therapy-naive individual demiology of an HIV-1 subtype A subcluster among injec- had a K103N mutation causing resistance to NNRTI. tion drug users in the Southern Ukraine. AIDS Res Hum The K103N as well as M184V mutations were the most Retroviruses 1998;14:1079–1085. prevalent drug-resistant mutations among patients receiving 10. Kurbanov F, Kondo M, Tanaka Y, Zalalieva M, Giasova G, Shima T, Jounai N, Yuldasheva N, Ruzibakiev R, Mizokami HAART: each mutation was found in 10 of 47 individuals M, and Imai M: Human immunodeficiency virus in Uzbe- (21%); in two patients (4%) both mutations were found. In kistan: Epidemiological and genetic analyses. AIDS Res general, drug-resistant mutations were identified in 21 of 47 Hum Retroviruses 2003;19:731–738. individuals (45%). However, 26 of 47 patients (55%) under 11. Eyzaguirre LM, Erasilova IB, Nadai Y, Saad MD, Kovtu- HAART with detectable virus loads did not have any known nenko NG, Gomatos PJ, Zeman VV, Botros BA, Sanchez JL, drug-resistant mutation. This observation indicates the need to Birx DL, Earhart KC, and Carr JK: Genetic characterization increase compliance to therapy in individuals under HAART. of HIV-1 strains circulating in Kazakhstan. J Acquir Immune Defic Syndr 2007;46:19–23. Acknowledgments 12. Carr JK, Nadai Y, Eyzaguirre L, Saad MD, Khakimov MM, Yakubov SK, Birx DL, Graham RR, Wolfe ND, Earhart KC, The study was financially supported by grants from INTAS and Sanchez JL: Outbreak of a West African recombinant of (05-1000004-7749) and the Russian Foundation for Basic HIV-1 in Tashkent, Uzbekistan. J Acquir Immune Defic Research (09-04-90702). Syndr 2005;39:570–575. 936 RUMYANTSEVA ET AL.

13. Saad MD, Shcherbinskaya AM, Nadai Y, Kruglov YV, An- Zverev SI, Grishechkin AE, Pokrovskii VV, Bobkova MR, tonenko SV, Lyullchuk MG, Kravchenko ON, Earhart KC, and Bobkov AF: Protease and reverse transcriptase genetic Sanchez JL, Birx DL, and Carr JK: Molecular epidemiology polymorphism in HIV type 1 subtype A variants predomi- of HIV type 1 in Ukraine: Birthplace of an epidemic. AIDS nating in CIS countries. Mol Biol (Mosk) 2005;39:1063–1071. Res Hum Retroviruses 2006;22:709–714. 14. Thomson MM, de Parga EV, Vinogradova A, Sierra M, Ya- Address correspondence to: kovlev A, Rakhmanova A, Delgado E, Casado G, Munoz M, Vladimir V. Lukashov Carmona R, Vega Y, Perez-Alvarez L, Contreras G, Medrano Laboratory of Experimental Virology L, Osmanov S, and Najera R: New insights into the origin of Department of Medical Microbiology the HIV type 1 subtype A epidemic in former Soviet Union’s Academic Medical Center countries derived from sequence analyses of preepide- University of Amsterdam mically transmitted viruses. AIDS Res Hum Retroviruses Meibergdreef 15 2007;23:1599–1604. 1105AZ Amsterdam, The Netherlands 15. Sukhanova AL, Bogoslovskaia EV, Kruglova AI, Bashkirova LI, Tsyganova GM, Shipulin GA, Kazennova EV, Alikina Iu I, E-mail: [email protected] This article has been cited by:

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