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ConClusionsl The genetic diversity of HiV-1 circulating in Russia is complex & subtype distri-
bution is related to the geographical region of sample collection.
l A1, the most prevalent subtype, accounts for 75% of strains evaluated.
l A1 strains in Russia (AFsu) appear to cluster independently from A1 reference strains collected in other parts of the world.
l subtypes B (6.8%), G (9.3%) and CRF03_AB (7%) also contribute to the HiV-1 epidemic in Russia.
l other strains (2%) include subtypes A3, C, F1, CRF01_AE, CR02_AG, CRF06_cpx and CRF32_06A1.
l We detected a high frequency of protease polymorphic mutations in positions associated with drug resistance. The influence of these polymorphisms on the success of protease inhibitor therapy deserves further investigation.
l Availability of commercial genotyping assay facilitates management of patients on HAART regimens, and identifies natural polymorphisms in drug naïve patients that may influence resistance pathways.
Genetic Characterization of Diverse HIV-1 Strains Circulating in RussiaNatalia Marlowe1*, Priscilla Swanson2, Lei Fang1, Vera Holzmayer2, Peter Smith1, Robert Bruce1, Sven Thamm3, Tatiana Kondrashova3, John Hackett, Jr.2 and the Russian HIV-1 Study Group4
1Celera, Alameda, USA; 2Abbott Diagnostics, Abbott Park, USA; 3Abbott Molecular, Germany; 4Russian HIV-1 Study Group
ABsTRACTBackground: Of the European countries, Russia has the largest number of HIV infections. The dominant strain in Russia has been a variant of subtype A1 (AFSU), followed by subtypes B and an intersubtype recombinant CRF03_AB. To examine the molecular epidemiology of circulating virus strains, plasma samples were collected from HIV-1 infected subjects attending ten geographically diverse AIDS centers in Russia.
Methods: Plasma samples were collected in 2007-2009 from 611 HIV-1 infected individuals and processed at the clinical laboratories of Rostov (n=29), Ust-Izhora (n=79), St. Petersburg AIDS & Pasteur Institute (n=70, n=20), Yekaterinburg (n=43), Kazan (n=62), Vologda (n=89), Krasnoyarsk (n=67), Noyabrsk (n=129), and Khabarovsk (n=23). Protease and reverse transcriptase were sequenced using the ViroSeq HIV-1 Genotyping System v2.0. Consensus sequences for each sample were aligned against HIV-1 group M reference strains and Russian genomes from the Los Alamos HIV Sequence Database. Phylogenetic analysis was performed using Phylip software to assign HIV-1 subtype.
Results: Complete protease and 5' reverse transcriptase sequences were obtained for 556 samples. The subtype composition of the panel included 6 subtypes and 5 circulating recombinant forms (CRFs): 416 A1 (74.8%), 52 G (9.3%), 39 CRF03_AB (7.0%), 38 B (6.8%), 3 CRF02_AG (0.5%), 2 CRF01_AE (0.4%), 2 F1 (0.4%) as well as 1 each of A3, C, CRF06_cpx, and CRF32_06A1 (0.2% each). A1 strains were identified in all regions and clustered with the Russian AFSU sequences. Subtype G strains were present in nearly 50% of the samples from Rostov and Ust-Izhora sites, and formed closely related clusters suggesting common ancestors. Subtype B was found at all locations except three southern cities, and CRF03_AB was primarily restricted to the northwest and central regions.
Conclusions: These data demonstrate complex genetic diversity of the HIV-1 strains circulating in Russia. While A1 is still the most prevalent subtype, other strains, especially subtype G and CRF03_AB, are contributing to the increasing genetic diversity of non-B HIV-1 epidemic in this country. The subtype distribution was shown to be closely related to the geographical region where samples were collected.
sTudy oBJECTiVEsl To evaluate performance of the ViroSeq™ HIV-1 Genotyping system v 2.0 (Celera, Alameda, CA) on HIV infected
plasma collected (and processed) by AIDS Centers in Russia
l To determine HIV-1 subtypes and analyze natural polymorphisms in virus strains circulating in Russia
MATERiAls & METHodsl Number of HIV-1 samples and panel composition were determined by each participating center
l Samples were collected and processed at the participating site using the ViroSeq system
l ViroSeq PCR, sequencing results and sample information (Viral load, treatment history) were sent to Abbott/Celera for data analysis
l Sequencing data were used for Drug Resistance analysis with ViroSeq v2.8 software
l ViroSeq Protease/Reverse Transcriptase sequences were aligned with reference sequences (Los Alamos HIV Sequence Database) for HIV-1 group M subtypes and circulating recombinant forms (CRF’s)
l Phylogenetic analysis for subtype determination was performed using the PHYLIP software (v3.5c; J. Felsenstein, Univ of Washington, Seattle, WA)
REsulTsPhylogenetic AnalysisGenetic Diversity by site
Site n A1 A3 B C F1 G CRF01 CRF02 CRF03 CRF06 CRF32
Saint Petersburg Pasteur Institute
20 17 1 2
Saint Petersburg AIDS
70 54 13 2 1
Ust-Izhora 72 16 1 7 39 2 7
Vologda 59 45 1 12 1
Rostov 28 14 13 1
Kazan 51 50 1
Yekaterinburg 38 29 9
Noyabrsk 129 117 2 2 8
Krasnoyarsk 67 61 6
Khabarovsk 22 13 8 1
Total (n) 556 416 1 38 1 2 52 2 3 39 1 1
% 74.8 0.2 6.8 0.2 0.4 9.3 0.4 0.5 7.0 0.2 0.2
Distribution of HIV-1 Subtypes in Russia (n = 556)
A1 B G CRF03_AB A3 C
St. Petersburg (AIDS)
St. Petersburg (Pasteur)
Ust-Izhora
Vologda Rostov Yekaterinburg Kazan
Krasnoyarsk
Noyabrsk
Khabarovsk
F1 CRF01_AG CRF02_AG CRF06_cpx CRF32_06A1
A1 (74.8%)
B (6.8%)
G (9.3%)
CRF03_AB (7%)
All Other (2.1%)
(85%)
(77.1%)
(18.6%)
(22.2%)
(9.7%)
(54.2%)
(9.7%)
(76.3%)
(20.3%)
(46.4%)(50%)
(98%)
(76.3%)
(23.7%)
(90.7%)
(6.2%)
(91%)
(9%)
(36.4%)(59.1%)
(10%)
Neighbor-Joining Tree of HIV-1 Subtypes/CRF's in Russia
90
100
97
9399
97
10098
83
7666
100
100
8574
-
-0
--
- - -1
-
-- -
n
v -1v-
0.02
B
C
CRF02_AG
CRF01_AE
CRF06_cpx
CRF32_06A1
D
F1
F2
CRF03_AB
HG
A1
A3
Afsu
Representative virus strains from each of 10 sites (black triangle) are shown relative to HIV-1 reference strains. Numbers at selected nodes indicate bootstrap support (%). Branches are labeled with the HIV-1 subtype or CRF. Outgroup (SIV-cpz) was removed from the tree. AFSU designates the A1 strains observed in Russia.
Neighbor-Joining Tree of HIV-1 Subtype A1 in Russia (Afsu)
YekaterinburgVologda
A1-RU00051Ust-Izhora
Ust-IzhoraRostov
KhabarovskSt Petersburg Pasteur
St Petersburg AIDSSt Petersburg Pasteur
KrasnoyarskSt Petersburg AIDS
NoyabrskNoyabrskNoyabrskYekaterinburgRostov
KazanVologda
KrasnoyarskA1-RUPok
St Petersburg AIDSUst-Izhora
A1-RU20061KhabarovskKrasnoyarskKhabarovsk
RostovKazan
YekaterinburgKazanVologda
SIV-CPZGAB
0.02
CRF01_AE
Afsu
B
A1
99
100
100
78
63
78
71
St Petersburg Pasteur
A1
Representative A1 virus strains from each of 10 Russian sites (AFSU) are shown relative to HIV-1 reference strains A1, B and CRF01_AE. Numbers at selected nodes indicate bootstrap support (%). A1 strains highlighted in red are full genome Russian reference strains. The branch labeled A1 represents reference strains from Kenya, Somalia & Uganda.
Acknowledgements We thank the following investigators and institutions for their collaboration: St. Petersburg (Pasteur): Tatiana Smolskaya, Ph.D. - Pasteur St. Petersburg
Research Institute for Epidemiology and Microbiology, Northwest Federal AIDS Center Diagnostical Division
St. Petersburg (AIDS): Natalya Dementyeva - St. Petersburg Municipal AIDS Center, Clinical Diagnostics Lab
Ust-Izhora: Galina Korovina Ph.D. - Russian Republican Clinical Hospital for Infectious Diseases, Clinical Diagnostics Lab
Vologda: Lyudmila Chernyshova - Vologda Region AIDS Center, Clinical Immunology
Rostov: Svetlana Poddubskaya - Rostov Research Institute for Microbiology and Parasitology, Division of Health Survey and Clinical Laboratory Monitoring of HIV-infected patients
Kazan: Valery Gerasimov Ph.D. - Tatarstan Republican AIDS Center, Molecular Biology Research Lab
Yekaterinburg: Tatiana Sandyreva - Sverdlovsk Region AIDS Center, Division of Clinical Laboratory Diagnostics
Noyabrsk: Lyudmila Volova, Ph.D. - Yamalo-Nenets Region AIDS Center Clinical Diagnostic Lab
Krasnoyarck: Olga Rumyantseva - Krasnoyarsk Region AIDS Center, Division of Molecular Genetic Research
Khabarovsk: Valeria Kotova - Khabarovsk Research Institute for Epidemiology and Microbiology, Diagnostic Lab
#439
substitutions in Protease/Reverse Transcriptase at drug Resistance-Associated Amino Acid Positions^
HiV-1 strains from drug naïve Russian subjects (n = 139)Protease amino acids 10-93
HXB2 L I G K L E M K I Q D I L I H V V N L I
AA position 10 13 16 20 33 35 36 43 47 58 60 62 63 64 69 77 82 83 89 93
Mutation* I, F, V, C V E R, M, I, T, V I, F, V G I, L, V T V E E V P L, M, V K I A, T, F, I, S, L D V L, M
Subtype (n)
A1 (112) I8, V1 V57 E23, A2 R3 D103 I109 T1 M1 E1 V2 I2, P7, T3, V3 M2, V1 K112 I31 M109 L59
B (10) I2, V1 V2 E1 I2 D2 I1 E1 E1 V5 P4, Q1, S2, T1 L1, M1 Q1 I3 I1 L4
G (2) I1 V2 I2 D2 I2 K2 I2 M2
CRF03_AB (15) I3, V1 V8 D15 I15 T1 K15 Y1 M15 L6
TOTAL (139) I14, V3 V69 E24 R3, I2 I2 I127 T1 E1 E2 V7 P11 L1, M3, V1 K129 I34 I3 L69
% of Total 10.0, 2.2 49.6 17.2 2.2, 1.4 1.4 91.4 0.7 0.7 1.4 5.0 7.9 0.7, 2.2, 0.7 92.8 24.4 2.2 49.6
Amino acid (letter) followed by number indicates the number of strains observed with a selected drug-associated substitution. ^Based on guidelines of IAS, CID 2008:47, 266-285
Reverse Transcriptase amino acids 62-210
HXB2 A D V A K F Q V Y G L
AA position 62 67 90 98 101 116 151 179 188 190 210
Mutation* V N I G E, P Y M D, F, T L A, S W
Subtype (n)
A1 (112) V47 N1 I7 S1 R2, Q1 Y1 M1 I1, G1 M1 A1, S1 F1, M1, S1
B (10) S2 I2 F1
G (2)
CRF03_AB (15) N1 S1 D1
TOTAL (139) V47 N2 I7 S4 R2, Q1 Y1 M1 D1, I3, G1 M1 A1, S1 F2, M1, S1
% of Total 33.8 1.4 5.0 0.7 0.7 0.7 0.7, 0.7
HiV-1 strains from drug Treated Russian subjects (n = 119)Protease amino acids 10-93
HXB2 L I G K L E M K I Q D I L I H V V N L I
AA position 10 13 16 20 33 35 36 43 47 58 60 62 63 64 69 77 82 83 89 93
Mutation* I, F, V, C V E R, M, I, T, V I, F, V G I, L, V T V E E V P L, M, V K I A, T, F, I, S, L D V L, M
Subtype (n)
A1 (83) I6, V5 V35 A1, E14 R4, T2 D73 I82 T2 E1 V1 C1, I1, P6, T2, V2 K82 I22 F1 I1, M79, T1 L28
B (9) I6, F1 V1 E3 I1 D7 I5 V1 V1 P4, S1, T3 M1, V2 K1 I2 A1 L5
F1 (2) V1 V2 R2 D1 I2 I1 M2
G (21) I6 V21 E4 I17, T1 I2 D17, N2 I21 R3 E1 P1, R1, T3 M1 K20, R1 F1, I19, S1 I4, M14, V3 M2
CRF02_AG (1) V1 E1 I1 D1 I1 R1 L1 K1 M1 V1
CRF03_AB (2) V2 D2 I2 V1 K2 M2 L1
CRF32_06A1 (1) I1 V1 I1 D1 I1 H1 M1 K1 M1
TOTAL (119) I19, F1, V1 V63 E22 R6, I19, T3 I3 I114 T2 V1 E2 V2 P11 L1, M3, V3 K108 I24 A1, F2, I20, S1 V3 L34, M2
% of Total 16, 0.8, 0.8 52.9 18.5 5.0, 16, 2.5 2.5 95.8 1.7 0.8 1.7 1.7 9.2 0.8, 2.5, 2.5 90.8 20.2 0.8, 1.7, 16.8, 0.8 2.5 28.6, 1.7
Resistance mutation analysis on ViroSeq protease/Reverse Transcriptase sequences from 139 drug naive and 119 drug-treated Russian patients showed that mutations of >10% were observed for these populations at protease positions L10I, I13V, G16E, M36I, H69K, V77I, I93L and reverse transcriptase A62V. Protease K20I and V82I mutations were present in ~16% of only the drug-treated subjects.
Reverse Transcriptase amino acids 62-210
HXB2 A D V A K F Q V Y G L
AA position 62 67 90 98 101 116 151 179 188 190 210
Mutation* V N I G E, P Y M D, F, T L A, S W
Subtype (n)
A1 (83) V24 N1 I7 S1 Q1, R2 Y1 M1 G1, I1 M1 A1, S1 F1, M1, S1
B (9) N2 G1 I4 A1 W1
F1 (2) V1
G (21) N3 G2, S1 E1, Q1 W3
CRF02_AG (1)
CRF03_AB (2) L1
CRF32_06A1 (1)
TOTAL (119) V24 N6 I7 G3 E1 Y1 M1 L1 A2, S1 W3
% of Total 20.2 5.0 5.9 2.5 0.8 0.8 0.8 0.8 1.7, 0.8 2.5
Participating AIDS Centers in Russia
N O R W A Y
E N
F I N L A N D
UKRAINE
I R A N
M O N G O L I A
J A P
A N
N. KOREA
KAZAKHSTAN
UZBEKISTAN
KIRGHIZIA
TURKMENISTAN
TAJIKISTAN
AZERBAIJAN
ARMENIA
LITHUANI
ESTONIA
R U S S I A
GEORGIA
Kola
Peninsula
Novaya Zemlya
Yamal Pen.
Gyda Pen.
TaymyrPeninsula
SevernayaZemlya
FranzJosef Land
New Siberian
Islands
Chukchi Pen.
Kamchatka Peninsula
Sakhalin
MOSCOW
Novosibirsk
Kazan
Rostov
Chelyabinsk
Omsk
Perm
St. Petersburg
Nizhniy-Novgorod
Ust-IzhoraVologda
Yekaterinburg Khanty-Mansiysk
Krasnoyarsk Khabarovsk
Noyabrsk
Rostov
Ust-Izhora
Saint Petersburg AIDS
Saint PetersburgPasteur Institute
YekaterinburgKazan
Vologda
N O R W A Y
E N
LITHLITII
EESTONININIE AAA
OSCO
Rostov
NUKRAINE
MO
I R A N
AZERB
ARMENIAAA
GEORGIA
ssk KKk
NN
Proposed sites
SS
Noyabrsk Krasnoyarsk Khabarovsk
Poster presented at 17th Conference on Retroviruses and Opportunistic Infections — February 16–19, 2010 — San Francisco, CA
Contact*Dr. Natalia Marlowe ([email protected])
Celera, 1401 Harbor Bay Parkway, Alameda, CA 94502
