HIV-1 Epidemiology, Genetic Diversity, and Primary Drug Resistance in the Tyumen Oblast, Russia

Introduction. Specific molecular epidemic features of HIV infection in Tyumen Oblast (TO), Russia, were studied. Methods. The genome sequences encoding HIV-1 protease-reverse transcriptase, integrase, and major envelope protein were examined for 72 HIV-1 specimens isolated from the TO resident infected in 2000–2015. Results. The recorded prevalence of HIV-1 subtype A (A1) is 93.1%; HIV-1 subtype B continues to circulate in MSM risk group (1.4%). Solitary instances of HIV-1 recombinant forms, CRF63_02A1 (1.4%) and CRF03_AB (1.4%), were detected as well as two cases of HIV-1 URF63_A1 (2.8%). Phylogenetic analysis showed no HIV-1 clustering according to the duration of infection and risk groups but revealed different epidemic networks confirming that HIV infection spread within local epidemic foci. A high incidence of CXCR4-tropic HIV-1 variants and a higher rate of secondary mutations influencing the virus fitness (K20R, L10V, and I) are observed among the virus specimens isolated from newly infected individuals. Conclusions. The current HIV-1 epidemic in TO develops within the local epidemic networks. Similar to the previous period, HIV-1 subtype A is predominant in TO with sporadic cases of importation of HIV-1 recombinant forms circulating in adjacent areas.


Introduction
The Russian Federation is now in the list of regions with a high rate of HIV epidemic spread [1]. In 2016, the total number of officially registered persons with diagnosed HIV infection in Russia exceeds one million. The HIV prevalence among Russian citizens in 2015 continued to grow. A high HIV prevalence (over 0.5% of total population) is observed in 26 regions, housing 41.5% of the population of this country [2].
The cases of first mass detection of HIV-1 infection in the territories of Russia and other newly independent states of the former Soviet Union date back to 1996. In 2000-2001, local dramatic outbreaks of HIV-1 infection were recorded in many regions of Russia; these outbreaks stemmed from an active spread of HIV-1 among injection drug users (IDUs). HIV-1 subtype A was included into the risk group for IDUs and continued its spread in many territories of this country [3][4][5][6][7][8]. The Tyumen Oblast (TO) is a federal subject of Russia with the administrative center in the city of Tyumen ( Figure 1). This territory with an area of 1435 200 km 2 has 3615 485 residents, with the urban population accounting for 80.12%. TO was among the first Russian territories actively   Since the very beginning of mass HIV spread in TO, several different programs were implemented there that focused on HIV prevention and treatment (information and education of various population cohorts and organization of laboratory facilities allowing for timely detection, prescription, and efficiently control of the relevant therapies) and on the control of drug abuse. This allowed for coping with the drastic growth in the epidemic: its rate in 2003-2008 considerably slowed down (Figure 2). During this period, the minimal growth in HIV morbidity was observed (below 2% or even negative); however, the share of women among the newly diagnosed HIV infection cases increased as well as the contribution of sexual transmission (50% in 2004), while the share of teenagers among HIV-infected individuals reduced. During the overall epidemic, sexual transmission was to a larger degree characteristic of women: up to 70% are annually infected via heterosexual contacts and only 22-27% via drug versus 73-78% of men infected when using drugs and only 29-33%, via sexual contacts.
Since 2008, numerous new drugs and homemade drug variants have appeared in the drug market. Emergence of new psychoactive substances offered as a "legal" alternative for the known drugs [9] controlled at an international level had a significant effect on the further development of HIV epidemic in many regions of this country. The new psychoactive substances (the so-called "synthetics" and "salts") are marketed under harmless names as "salts," such as, bath salts, legal highs, speed euphoria, Barnyi, Cico, Cristalius SEX, Cristalius LOVE, Miff, Ivory Wave, Liquid Snow Berry, Snow Blow, and Spice. These substances were sold almost legally and were considerably less expensive as compared with classical drugs, making them easily accessible.
Synthetic psychoactive substances differ from the opioid drugs not only by their price and accessibility. These substances have a lower toxic threshold, which decreases the fear of death caused by overdosing, and have no effect of withdrawal; that is, they are used not to eliminate the withdrawal symptoms, but to reach an altered state of consciousness (euphoria or almightiness). Specific features of synthetics consist not only in their effect on the nervous system (disturbance of locomotion, memory, and sensitivity to pain as well as an increase in sexual sensations), but also in the injury of the immune system. The dependence on synthetics develops very rapidly. Ever new synthetics with changed structures and different administration methods (injection, smoking mixtures, etc.) constantly appear on the marker with addition of such substances to the lists of illegal drugs [10].
Emergence of such psychoactive substances involved many new persons into practicing risk behavior. The individuals intravenously injecting synthetics lose control over their behavior and start to share syringes. The practice of mindless unprotected sex becomes common when using some synthetics, including smoking mixtures. Consequently, the number of newly diagnosed HIV cases started to increase in TO in the second half of 2009. The epidemic history of HIV + TO residents ever more frequently contains the use of synthetic psychostimulants.
Characteristic of the subsequent years is a more active aggressive spread of psychoactive substances in many Russian regions. It is possible to purchase synthetics just calling by phone or via the internet. The situation continued to deteriorate because of an increased accessibility of these BioMed Research International 3 synthetic psychostimulants and spread of the drugs effective in sexual disinhibition; in addition, many drug addicts commenced practicing combined (multiple) drug use. For 2011-2014, men were prevalent among the newly detected HIV + individuals, also suggesting activation of the drug-associated HIV infection route. Nonetheless, a stable decrease in the annual rate of HIV morbidity growth has been observed starting from 2011 on the background of an absolute increase in the HIV incidence.
As of May 01, 2016, 18 423 cases of HIV infection were recorded in TO. According to the HIV prevalence, TO is the eighth in the list of Russian regions (1107.0 HIV + individuals per 100 000 population) and thirteenth according to the morbidity rate (106.6 per 100 000 population).
Active HIV-1 outbreaks in the regions adjacent to TO (Novosibirsk, Tomsk, and Kemerovo Oblasts; Figure 1) recorded in 2008-2014 were associated with a drastic change in genetic characteristics of the circulating viruses, namely, replacement of the earlier circulating HIV-1 subtype A by CRF63 02A1 [11][12][13]. The studies of the HIV-1 variants circulating in TO have not been conducted so far. A high HIV-1 prevalence in TO and a long history of antiretroviral therapy (ART) there justify the interest to assess the specific molecular genetic features of the virus population circulating there and analyze HIV-1 primary resistance.

Materials and Methods
In total, 74 TO residents with diagnosed HIV infection were involved in this study. Peripheral blood of the HIVinfected individuals was sampled by medical staff of the Tyumen Regional Center for the Prevention and Control of AIDS and Infectious Diseases from September to December 2015 with pretest and posttest psychological consultations; epidemiologists obtained the written informed content from the participants and questioned them. The study of clinical blood samples complied with the ethical standards of the Helsinki Declaration of 1975 (http://www.wma.net/en/ 30publications/10policies/b3/), revised in 2008. The study projects were approved by the local ethical committee (protocols nos. 7 and 15 of March 20, 2013, andMarch 4, 2015). The blood samples were linked with demographic and clinical data via coded ID numbers according to the requirements of medical ethics in Russia. All participants were ART-naïve before the tests. The recorded characteristics for patients included their gender, age, transmission route, estimated time of infection, dates of the last negative and first positive tests for HIV, drug use, viral load, and CD4 cell count at the moment of diagnosis. The group of individuals HIV-infected when using drugs included those infected during nonmedical drug injection of drugs and psychoactive substances.
Virus RNA was isolated from 200 L of blood plasma using RealBest DeltaMag kits according to the manufacturer's protocol (Vector-Best, Russia). Amplification was performed using a lyophilized ready-to-use Reverse Transcription Master Mix containing all the components for a single-tube reverse transcription and PCR (Vector-Best, Russia) and an in-house set of primers. This allowed for amplifying three HIV-1 specific fragments for each clinical sample, namely, two fragments of the pol gene encoding the protease-reverse transcriptase (PR-RT, 1400 nt), integrase (IN, 960 nt), and a fragment encoding a part of the major envelope protein, env (732 nt). Sequencing was conducted in a 3130xl (Applied Biosystems, United States) automated sequencer. All original sequence fragments of the pol and env gene regions were assembled in whole sequences in Sequencher 4.1 software (Gene Codes Corporation, Ann Arbor, MI, United States). The assembled sequences of pol fragments (PR-RT and IN) and env gene fragments were compared to the corresponding reference sequences of various HIV-1 subtypes and recombinant forms extracted from the Los Alamos HIV-1 database using CLUSTALW Multiple Alignment and BioEdit software 7.2.5 [14]. Phylogenetic analysis was performed with MEGA 6.0.6 by neighbor-joining method with 1000 bootstrap replicates based on Kimura's two-parameter model [15]. Statistical significance of phylogenetic tree topologies was estimated using bootstrap analysis. New intersubtype or inter-CRF sequences were analyzed by recombination identification programs (RIP, https://www.hiv.lanl.gov/) and SimPlot 3.5.1 software using a 200 nt window with tree construction by the neighbor-joining method applying Kimura's two-parameter substitution model. The possible intersubtype mosaicisms of URFs were screened using the online jpHMM program (http://jphmm.gobics.de/submission hiv.html) [16]. The V3 loop sequences were analyzed to estimate the genotypic virus tropism and to confirm the phenotypic coreceptor specificity using the online tools Position-Specific Scoring Matrix (PSSM) (http://indra.mullins.microbiol.washington.edu/web -pssm/) and Geno2pheno [coreceptor] 2.5 (g2p) (http://coreceptor.bioinf.mpi-inf.mpg.de/index.php), additionally checking whether the sequence codes for positively charged amino acid residues at 11 and/or 25 codons of the V3 loop [17]. The analyzed pol gene sequences were assayed for the presence of mutations determining resistance to protease, reverse transcriptase, and integrase inhibitors (DR mutations) with the specialized online service (http://sierra2.stanford.edu/ sierra/). The transmitted DR mutations were determined based on the WHO-recommended list of mutations for DR surveillance [18].

Clinical Epidemiological Data on Patients.
In order to assess the current molecular epidemic situation on HIV-1 spread, 74 individuals diagnosed with HIV were involved in this study. Table 1 lists the clinical epidemiological data for the examined patients. The HIV-specific fragments of necessary lengths were not obtained for two HIV-1 samples, so they were discarded. Correspondingly, the examined sample comprised 72 individuals, namely, 33 (45.8%) men and 39 (54.2%) women, with an average age of 35 years (range, 23-60); 33 persons reported injection drug use (IDU); five of them were sex workers; 41 individuals (56.9%) were infected via heterosexual contacts; three of them were IDUs; and one      Summing up the genotyping results, we conclude that HIV-1 subtype A still remains predominant and determines development of the current epidemic in TO, its prevalence being 93.1%. HIV-1 subtype B continues its circulation in MSM risk group and was detected in one case (1.4%). The only cases of HIV-1 recombinant forms CRF63 02A1 (1.4%) and CRF03 AB (1.4%) were detected as well as two instances (Tyumen 31 and Tyumen 33) of HIV-1 unique recombinant forms, URF63 A1. The URF genome is mosaic, partially identical to subtype A and partially to CRF63 02A1, as was confirmed by phylogenetic analysis: some studied loci of the same isolate belong to subtype A1 and the others to subtype 63 02A1 (Figures 3 and 4).

Analysis of Primary Resistance and Tropism of the Studied
HIV-1 Variants. All HIV-1 nucleotide sequences encoding protease ( = 70), reverse transcriptase ( = 70), and integrase ( = 68) of the isolated virus variants were assayed for the presence of mutations influencing the resistance to the corresponding virus reproduction inhibitors, namely, the inhibitors of protease (PIs), reverse transcriptase (nonnucleoside and nucleoside inhibitors, NNRTIs, and NRTIs, resp.), and integrase (IIs) ( Table 2).
The conducted analysis demonstrated a low prevalence of the primary drug resistance (DR) among the studied variants of HIV-1. Nonpolymorphic mutations determining HIV-1 resistance to NNRTIs, G190E, and Y181C were detected only in two HIV-1 specimens circulating in TO. The former mutation causes a high-level resistance to NVP, EFV, RPV, and ETR, while the latter is associated with a decreased response to the therapy with EFV. Likewise, two cases of the resistance to IIs were found. The corresponding mutations are T66I and E157Q. The former is a major mutation selected in the individuals treated with EVG, decreasing 15-fold the sensitivity to the drug, and the latter is a secondary mutation, reducing fivefold the sensitivity to RAL and halving the sensitivity to EVG.
The polymorphic mutations associated with drug resistance had different prevalence. The mutations able to improve HIV-1 reproductive characteristics in the presence of PIs or decrease the PI sensitivity (K20R, T74S, L10V/I, and K20I) were most frequently recordable among the examined HIV-1 variants, mainly in the individuals infected in 2010-2015.    Oblast. Genetic distances were estimated using Kimura's two-parameter model; clustering of strains was tested with 1000 bootstrap replicates; and statistical significance of the phylogenetic tree topology was estimated using bootstrap analysis.  Genetic distances were estimated using Kimura's two-parameter model; clustering of strains was tested with 1000 bootstrap replicates; and statistical significance of the phylogenetic tree topology was estimated using bootstrap analysis.  Solitary cases of the mutations influencing HIV resistance to PIs in combination with other DR mutations (L33F and A71I) were also recorded.
Of the mutations influencing NRTI therapy, only the V118I mutation (of the TAM set, efficient only in combination with other resistance mutations) was found. The mutations with a low effect on the resistance to NNRTIs (E138A, V108I, and V90I) also had a low prevalence. The sequences encoding HIV-1 IN carried the L74I in 94.1% of the examined subtype A viruses. This allows L74I to be regarded as a characteristic mutation for the HIV-1 subtype A population circulating in this area. On the other hand, only the A62VRT mutation of the DR mutations in the pol gene specific of HIV-1 subtype IDUA (V77I PR and A62V RT ) was detected at a rate of 8.3%. The mutation A62V is an accessory mutation that often occurs in combination with the multinucleoside resistance mutations K65R or Q151M. Alone it does not reduce NRTI susceptibility.
The tropism of examined HIV-1 variants, that is, the use of CCR5/CXCR4 coreceptors, was predicted by analyzing the deduced amino acid sequences of HIV-1 env gene V3 loop. The prevalence of CXCR4-tropic variants among the examined HIV-1 specimens was 20%; these variants were isolated from the individuals infected over 5 years ago in 25% of the cases versus 13.6% of the individuals infected less than 3 years ago.

Discussion
The circulation of resistant strains in the population of people living with HIV/AIDS (PLHIV) is a serious factor influencing the selection of first-line antivirals and adoption of the strategies for efficient ART schemes. Unlike the HIV IIs, which in this country are used only recently, the PIs and RTIs were and are still widely used for the specific ART. In Russia and the CIS countries, the HIV variants carrying DR mutations in pol gene are most frequently detectable in the individuals with inefficient ART. Among them, the mutations G190S, K103N, M184V, Y181C, K101E, M41L, and T215F/Y, influencing the HIV resistance to NRTIs and NNRTIs, are observed at a high rate [19][20][21][22][23][24]. Mutations K103N, M184I, T215Y, G190S, Y181C, K65R, and V108I with prevalences of 0.3 to 7% depending on the region are most frequently detected in the naïve HIV-infected individuals [21][22][23][24][25][26][27][28]. Only two HIV-1 variants carrying solitary G190E and Y181C mutations (causing resistance to RTI) and two HIV-1 specimens with resistance to IIs were found in the examined sample of HIV-1 specimens. Detection of the mutations associated with the resistance to IIs on the background of a very limited use of these drugs in clinical practice is explainable by a random selection of such substitutions. Despite a long-term practice of treatment with NRTIs, NNRTIs, and PIs, the transmission of resistant virus variants in the HIV population isolated from infected ART-naïve residents in TO was low. A low prevalence of the HIV-1 primary DR may be a favorable prognostic factor in the epidemic development in TO. However, note that over 10% of the HIV-1 variants circulating in TO had the mutations potentially associated with a decreased sensitivity to PIs and improvement of HIV reproductive characteristics in the presence of PIs.
The mutations A62V RT and V77I PR are certain marker mutations for the subtype A HIV-1 variants circulating in Russia. The circulating subtype A HIV-1 variants characteristic of Russia are those carrying the V77I PR and/or A62V RT mutations and wild-type (WT) HIV-1. The prevalence of such viruses among both the naïve HIV-infected individuals and the patients undergoing ARV therapy considerably varies ranging from solitary cases to 80-100% [27][28][29]. Presumably, this is associated with the importation history of the different subtype A HIV-1 variants to certain regions of the country and with the specific features in subsequent progression of the epidemic. Wild-type HIV-1 viruses were detected in TO as early as the late 1990s [30]. The observed low abundances of A62V RT (8.3%) and V77I PR (11.4%) among the HIV-1 variants in TO may result from importation of WT HIV-1 to this area followed by prevalent development of the "own" focus of HIV-infection epidemic.
The use of CCR5 antagonists in the modern therapeutic schemes makes it necessary to study the tropism of the HIV-1 variants isolated from the ART-naïve patients in order to assess the potential of such drugs [31][32][33]. According to several studies, the onset of HIV-1 infection with the spread of CCR5-tropic viruses in the body is characteristic of HIV-1 subtype A as well as most virus genetic variants except for subtype D. Different studies on the use of CCR5 or CXCR4 coreceptors by HIV-1 belonging to non-B subtypes (including subtype A) involving naïve HIV + patients demonstrate that the abundance of CXCR4-tropic viruses varies from 2 to 40%. This high variability may be influenced by different compositions of the studied populations and by differences in methodology. According to the data of most researchers, the detection rate of CXCR4-tropic HIV-1 variants in the patients at early disease state amounts to only several percent. Typically, the abundance of CCR5-tropic HIV variants decreases with the disease progression so that the CXCR4tropic viruses emerge only at later HIV infection stages [34][35][36][37][38][39][40][41][42][43].
The results of our work demonstrate that the number of HIV-1 variants using CXCR4 coreceptor also increases with the duration of the disease among the studied subtype A viruses isolated from the patients with confirmed date of infection: the CXCR4-tropic viruses were isolated from 13.6% of the subjects infected several months to 3 years ago and 25% of the subjects infected over 5 years ago, which agrees with the data of other researchers. Nonetheless, the recorded levels of CXCR4-tropic HIV-1 variants among the patients infected for less than 3 years are rather high. Our results demonstrate that it is necessary to study the tropism of a virus before planning the use of CCR5 antagonists in the therapeutic schemes for the individuals infected with HIV-1 subtype A and to additionally study the prevalence of CXCR4-tropic HIV-1 variants among the naïve patients infected with other genetic variants specific of different regions of this country.
Comparison of the HIV-1 genetic variants isolated from the infected TO residents in the 2000s (2000-2009) and later (2010-2015) did not reveal any significant differences in the circulating virus variants. HIV-1 subtype A was and still remains the predominant variant. Phylogenetic analysis did not find any clustering of HIV-1 subtype A according to the time of infection and risk groups. Different epidemic networks revealed by phylogenetic analysis of the HIV-1 variants, on the one hand, confirm that HIV infection spreads within local epidemic foci formed in TO during the previous years. On the other hand, the existence of such epidemic chains that include the HIV-1 variants with a high degree of genetic identity should be taken into account in a correct epidemiological investigation. Genetic identities of HIV-1 variants should be obligatorily assessed according to several genome regions. The revealed epidemic networks, which suggest that HIV is spread within an established focus of HIV infection, are also confirmed by analysis of the epidemic progression.
After 16 years of the epidemic in TO, the age cohort of 30-39 years has emerged to be maximally affected. The share of this age cohort is constantly growing, from 6.9% in 2000 to 48-50% in 2014-2015. The share of the individuals over 50 years old is also constantly growing (0.8% in 2000 to 4.6% in 2014). Presumably, part of the newly diagnosed patients has long been infected and, possibly, some "former" IDUs returned to using drugs and were thus involved into the epidemic. Epidemiological investigations recently performed when newly diagnosing a HIV infection demonstrates that HIV reinfection in the local foci of territorial epidemic remains rather high. The cause in the cohort free of drug addiction is a low sexual culture; as for the IDUs, especially using psychostimulants, they almost or completely neglect any of protection tools. Presumably, all of these factors also enhance HIV reinfection.
The detected HIV-1 URF63 A1, the so-called secondary recombinant forms, could have emerged only as a result of reinfection by different HIV-1 genetic variants (subtype A and CRF63 02A1). In the examined group, one patient (Tyumen 31) infected by URF63 A1 practiced intravenous heroin use in the early 2000s and then had accidental unprotected sexual contacts and another patient (Tyumen 33) intravenously uses synthetic psychostimulants since 2012, having a drug-associated contact with a HIV + partner and accidental unprotected sexual contacts in his history. They have never left the TO area, suggesting that distinct HIV-1 URF63 A1 genetic variants circulate in TO. A case of isolation of an ancestral HIV-1 variant, CRF63 02A1, of a TO resident who is a long-term drug user confirms the possibility that new URFs emerge in TO. Presumably, the HIV-1 CRF63 02A1 originated from adjacent Siberian territories (Novosibirsk, Tomsk, and Kemerovo Oblasts; Figure 1), where this genetic variant is actively spreading in recent years [11][12][13].
Despite that the territorial epidemics in the Siberian region and TO display similar characteristics (distribution of HIV-infected individuals according to infection routes, social parameters, gender, age, spread of drug abuse, and types of drugs), the epidemic in Siberia started with a delay of several years. Unlike Novosibirsk, Tomsk, and Kemerovo Oblasts, where the epidemic outbreaks with severalfold increase in the number of newly infected individuals were recorded in 2008-2014 (these territories are still at the top of the Russian rating for increase in newly infected persons), the last wave of HIV spread activation in TO was recorded in 2008-2011. Moreover, the epidemic growth rate was significantly lower: the maximal values recorded in 2009-2011 did not reach 20%; moreover, a slow but stable trend of a decrease in the HIV spread is observed after 2011. Presumably, a likely reason underlying such a "controlled" epidemic in TO is its early onset and, as a consequence, timely development of the medical facilities able to constantly provide preventive activities, professional pretest and posttest services for HIVinfected individuals, and psychological consulting as well as maintenance of the patients from diagnosing and registration to observation and timely therapy. Note that the therapy coverage in TO in 2015 was 98% and its efficiency according to the approved criteria was 79.6% [44].
Despite an apparent positive dynamic of this epidemic, the number of HIV-infected individuals in TO continues to grow. Currently, TO is the region attractive for migrant workers from both the CIS countries and other Siberian regions, where the current epidemics are determined by HIV-1 recombinant forms, CRF02 AG and CRF63 02A1. Therefore, these HIV-1 genetic variants can be imported to TO and spread there, demonstrating the importance of monitoring of the spreading HIV-1 variants on a regular basis, especially, in remote areas with constant presence of seasonal migrant workers.