This document summarizes genetic analysis of HIV-1 strains circulating in Russia. The key points are: 1. Subtype A1 is the most prevalent, accounting for 75% of strains, but subtypes B, G, and CRF03_AB also contribute significantly to the epidemic in Russia. 2. The distribution of subtypes varies by geographical region within Russia. For example, subtype G strains were highly concentrated in two southern cities. 3. Many strains showed natural polymorphisms in protease and reverse transcriptase genes that could influence response to antiretroviral treatment, highlighting the need for genotype testing to guide therapy.

1. Conclusions
l 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 Russia
Natalia 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
1
Celera, Alameda, USA; 2
Abbott Diagnostics, Abbott Park, USA; 3
Abbott Molecular, Germany; 4
Russian HIV-1 Study Group
Abstract
Background: 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 OBJECTIVEs
l 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 & METHODS
l 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)
RESULTS
Phylogenetic Analysis
Genetic 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 YekaterinburgKazan
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
93
99
97
100
98
83
76
66
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)
Yekaterinburg
Vologda
A1-RU00051
Ust-Izhora
Ust-Izhora
Rostov
Khabarovsk
St Petersburg Pasteur
St Petersburg AIDS
St Petersburg Pasteur
Krasnoyarsk
St Petersburg AIDS
Noyabrsk
Noyabrsk
Noyabrsk
Yekaterinburg
Rostov
Kazan
Vologda
Krasnoyarsk
A1-RUPok
St Petersburg AIDS
Ust-Izhora
A1-RU20061
Khabarovsk
Krasnoyarsk
Khabarovsk
Rostov
Kazan
Yekaterinburg
Kazan
Vologda
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
JAPAN
N. KOREA
KAZAKHSTAN
UZBEKISTAN
KIRGHIZIA
TURKMENISTAN
TAJIKISTAN
AZE
RB
AIJAN
ARMENIA
LITHUANI
ESTONIA
R U S S I A
GEORGIA
Kola
Peninsula
Novaya Zemlya
Yamal
Pen.
Gyda
Pen.
Taymyr
Peninsula
Severnaya
Zemlya
FranzJosef Land
New
Siberian
Islands
Chukchi
Pen.
Kamchatka
Peninsula
Sakhalin
MOSCOW
Novosibirsk
Kazan
Rostov
Chelyabinsk
Omsk
Perm
St. Petersburg
Nizhniy-Novgorod
Ust-Izhora
Vologda
Yekaterinburg Khanty-Mansiysk
Krasnoyarsk Khabarovsk
Noyabrsk
Rostov
Ust-Izhora
Saint Petersburg AIDS
Saint Petersburg
Pasteur Institute
YekaterinburgKazan
Vologda
N O R W A Y
E N
LITHLITII
EESTONININIE AAA
OSCO
Rostov
N
UKRAINE
MO
I R A N
AZE
RB
ARMENIAAAGEORGIA
ssk
KK
k
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 (natalia.marlowe@celera.com)
Celera, 1401 Harbor Bay Parkway, Alameda, CA 94502