HIV-1 genetic subtype A/B recombinant strain causing an... : AIDS (original) (raw)
Introduction
In Western Europe, the HIV/AIDS epidemic has been driven to a large extent by injecting drug use [1]. As much as 43% of all cumulative AIDS diagnoses in Europe to the end of 1995 were directly or indirectly associated with drug use [1]. Injecting drug use has served a major role in spreading HIV heterosexually by introduction of the virus into the general population by means of sexual partners of injecting drug users (IDU) [1]. Epidemics amongst IDU have in the past been characterized frequently by the rapid spread of the virus in the IDU population and subsequent spread to other population groups. Examples of such IDU-associated epidemics have been described in Edinburgh, Scotland, where large numbers of IDU became infected by sharing contaminated needles and syringes in the late 1980s [2], and in Italy and Spain [3–7]. All these epidemics have been caused by strains belonging to the HIV-1 genetic subtype B.
In tracing the geographical spread of HIV-1, studies on the molecular epidemiology of different strains of HIV-1 have proved useful. Such studies rely on the variability of the virus, which has resulted in the formation of distinct genetic subtypes of HIV-1 (subtypes A–J). These subtypes can be identified by phylogenetic analysis and strains from different areas and populations can be compared to find related viruses and therefore identify epidemiological links that otherwise may have gone unnoticed. Since the distribution of the subtypes varies in different geographical regions, links between distant geographical regions may be identified. Previous examples of such studies have shown that the strain causing a sexually transmitted epidemic in Thailand was caused by a virus belonging to subtype E that probably originated in Africa, whereas another simultaneous epidemic amongst IDU was found to be caused by a virus of sub-type B, prevalent in North America and Europe. Molecular epidemiology studies have also been applied for estimating the proportion of imported versus domestic cases in low prevalence areas [8–10], and to identify transmission links [11,12]. Furthermore, molecular epidemiology has been used to track the global distribution of HIV-1 variants for the purpose of vaccine development [13–17]. Although there has been only limited evidence for correlation between subtype and specificity of immune response, the tracking of genetic subtypes is still seen as a useful tool for the selection of geographically representative viral strains to be used for vaccine development.
In Russia, and other countries of the former Soviet Union, the number of HIV infections has until recently remained low. There have been many indications of the potential for rapid spread of HIV-1, such as the increased incidence of sexually transmitted disease (STD) and a poor knowledge of risk factors predisposing to HIV-infection, especially among young people [18–21]. When the Soviet Union still existed, HIV/AIDS was perceived as a taboo issue, and as a disease only affecting the West, especially among healthcare professionals. This also applied to injecting drug use [22,23]. These factors, combined with the recent economic difficulties and social uncertainty have created a situation favourable for the spread of many infectious diseases, and has prompted several warnings of the potential for the explosive spread of HIV in the region [24–26]. Previous studies on the molecular epidemiology of HIV-1 in the countries of the former Soviet Union have shown that practically all genetic subtypes can be found [10,14,27–33]. However, the number of strains of a given subtype has been fairly limited, and epidemic spread has been limited to nosocomial outbreaks [30,34]. The number of different subtypes present in the countries of the former Soviet Union suggests that there have been frequent introductions of the virus into the region, not only from the West, but also from other parts of the world. However, except for the few nosocomial epidemics previously described [30,34], there has been little evidence of significant epidemic spread in the region. This picture is now changing, and IDU-associated HIV-1 epidemics have been reported in the Ukraine, Belarus and southern parts of the Russian Federation [1,35–40]. In the Ukraine and Southern Russia, the IDU epidemic has been caused by a subtype A virus. An even more recent example of an explosive epidemic has occurred in the Russian Kaliningrad region.
In July 1996, the monthly number of newly diagnosed HIV-1 infections in the Russian Kaliningrad oblast (district) situated at the Baltic sea between Poland and Lithuania (Fig. 1) began to rise dramatically, reaching numbers of over 100 new cases per month before the end of the year. Preliminary information from the area indicated an association with IDU [39,40]. In September 1996 investigators of the St Petersburg Pasteur Institute (Russia), the National Public Health Institute in Helsinki (Finland) and the Kaliningrad AIDS Centre initiated a collaborative effort for a molecular epidemiological characterization of the outbreak and its relation to some other HIV-1 epidemics in the Commonwealth of Independent States region. This report describes the results of the study.
. Map of the Baltic region. Est, Estonia; Lat, Latvia; Lit, Lithuania.
Materials and methods
HIV testing program in Kaliningrad
The study serological surveillance period covers the time period from 1988 to June 1997. Surveillance of HIV-1 infections in the Kaliningrad oblast (population, 932 000; main city Kaliningrad, 421 000; 1996 Kaliningrad regional census data) was performed by a population-based screening program, and the results of the tests were centrally registered. Through this program, all patients of STD clinics, drug rehabilitation centres, blood donors, maternity care receivers, military recruits on entry into service, prisoners, individuals held in custody before trial, and registered commercial sex workers were tested for HIV antibodies. In addition, an HIV-1 test may have been conducted for patients visiting hospitals or health-care centres for an HIV-unrelated medical problem. These tests were based on the treating physicians judgement of the likelihood that the patient might be HIV-1-infected (i.e., if the patient presented with HIV/AIDS-related symptoms). Drop-in testing (including anonymous testing) was provided at the AIDS centre in Kaliningrad city. In 1996, more than 190 000 HIV-1 antibody enzyme immunoassay (EIA) tests were performed in the region. Similar or higher numbers of annual tests have been performed since the inception of testing in 1988. Active but voluntary contact tracing was employed for those individuals who tested positive, and their sexual partners were contacted and separately advised to have their HIV antibody status tested. All HIV-1-infected patients who could be reached after test verification were asked about their epidemiological risk factors using a standardized questionnaire distributed in association with psychological counselling sessions.
Patient samples
A total of 67 blood samples from Kaliningrad (n = 61), neighbouring Lithuania (n = 4), and the Ukraine (n = 2) were collected in 1996 and 1997 for genetic characterization of HIV-1. All samples were donated with informed consent by previously identified HIV-1-positive individuals. The donors were outpatients of the AIDS treatment centre in Kaliningrad city and individuals in police custody pending trial for drug-related or other crimes (Kaliningrad only). Samples from Kaliningrad were collected in October 1996 (n = 22) and April 1997 (n = 39). Samples from Lithuania and the Ukraine were collected at AIDS treatment centres in Vilnius in April 1997 and in Kiev in July 1997. The sample collection was obtained as a convenience sample, with the only common denominator being the HIV-1-positive status of the patients. However, age, transmission risk factors and sex ratio of the individuals sampled in Kaliningrad did not significantly differ from those of the total infected population in Kaliningrad. The majority of the donors indicated injecting drug use as their probable transmission risk factor, except for those from Lithuania, where only one individual reported this risk factor. Possible source of bias in the samples collected for genetic characterization may have been introduced by inclusion of samples from patients held in police custody, for whom the risk of injecting drug use transmission may have differed from the HIV-1 infected AIDS centre outpatient group. Samples from Lithuania and the Ukraine were not necessarily representative of the epidemics in these countries, but were randomly chosen. In addition to genetic characterization of HIV-1, the samples from Kaliningrad and Lithuania were tested for the presence of anti-hepatitis C virus (HCV) antibodies. To better estimate the seroprevalence of HCV, hepatitis B virus (HBV) and syphilis antibodies in the HIV-1-positive IDU population in the Kaliningrad district, 353 serum samples from individuals reporting drug use were randomly chosen for testing among samples collected for the population-based HIV-1 screening program. The overlap between the samples collected for the genetic typing of HIV-1 and the 353 samples chosen for the estimation of other STD was unknown.
Sample preparation
For the primary HIV-1 antibody screening program, serum was separated by centrifugation from clotted venous blood and stored at −20°C prior to testing. For the samples collected for the genetic typing of HIV-1, venous blood was collected in EDTA-anticoagulated tubes, and peripheral blood mononuclear cells (PBMC) and plasma were separated by density gradient centrifugation (CPT tube; Beckton Dickinson, Lincoln Park, California, USA). Plasma was aspirated and stored at −20°C. PBMC were washed once with 15 ml of phosphate-buffered saline and for a second time with 1.5 ml of the same (centrifugation for 10 min at 500 g) and counted by microscopy. Isolated PBMC were processed into lysates suitable for PCR by a modification of the method of Higuchi as previously described [13,41].
Antibody assays
All HIV antibody testing for the primary HIV screening program was performed in Kaliningrad by EIA (Screen-HIV, St Petersburg, Russia). Positive samples were verified by a second EIA (Vironostika HIV UniForm II, Organon Teknika, Boxtel, The Netherlands) and by Western blot (New LAV Blot 1–2, Diagnostics Pasteur, Paris, France) at the Pasteur Institute in St Petersburg. From those samples that were verified as HIV-1-positive, 353 samples from individuals reporting injecting drug use were also tested for the presence of HBV surface antigen, and antibodies against HCV (AquaPast, St Petersburg, Russia) and Syphilis (EIA Lues Screen, Moscow, Russia; Syphilia TPHA, Diagnostics Pasteur). Plasma samples from the sample sets collected for genetic typing of HIV-1 were assayed at the National Public Health institute in Helsinki by EIA for antibodies against HCV (Murex anti-HCV, Wellcome, Temple Hill, UK).
HIV-1 PCR
The _vpu-env-nef_-5′ (∼3.1 kb) and NCp7-p6 gag (∼600 base pairs) regions of HIV-1 were amplified from patient primary PBMC lysates by nested PCR, as previously described [42]. Briefly, the env region was amplified with primers JL86 (5′-CCGTCTAGATGCTGTTTATTCATTTCAGAATTGG-3′), JL89 (5′-TCCAGTCCCCCCTTTTCTTTTAAAAA-3′) and ED3 (5′-TTAGGCATCTCCTATGGCAGGAA GAAGCGG-3′), JL88 (5′-TAAGTCATTGGTCT TAAAGGTACCTG-3′) in the first- and second-round PCR, respectively. Expand Long Template kit (Boehringer Mannheim, Indianapolis, Indiana, USA) was used according to the manufacturer's recommendations. For PCR, a program of 94°C for 10 sec, 55°C for 30 sec and 68°C for 4 min for a total of 30 cycles was used. Hot start was used by separating primer and template from enzyme using DynaWax (Finnzymes, Espoo, Finland). Final reaction volume was 50 ml, and 5 ml of first-round product was transferred to the second round reaction.
For NCp7 amplification primers, BJGAG1 (5′-TAGAAGAAATGATGACAGCATG-3′), BJPOL2 (5′-TGGCTTTAATTTTACTGGTACAG-3′) and MSGAG7 (5′-GATGACAGCATGTCAGGGAG-3′), BJPOL3 (5′-GTTGACAGGTGTAGGTCCTAC-3′) were used in a similar nested fashion [13,43]. For this region, reaction conditions were 0.3 mmol/l for both primers, 62.5 mmol/l each dNTP, 1 µl enzyme (1 : 6 mixture of Taq/Pfu, (Life Technologies, Gaithersburg, Maryland, USA/Stratagene, La Jolla, California, USA) in a buffer comprising 50 mmol/l Tris-HCl, 15 mmol/l (NH4)2SO2, 1.5 mmol/l MgCl2 (pH 9.3). The PCR program was 10 sec denaturation at 95°C, 30 sec annealing and 1 min extension at 68°C for a total of 30 cycles. For the first cycle, the annealing temperature was 65°C, for the second and third it was 60°C, and 55°C thereafter.
Amplified products were purified by Qiaquick purification (Qiagen GmbH, Hilden, Germany) and the NCp7 (∼250 base pairs) and env C2-V3 (∼400 base pairs) coding regions were directly sequenced on both strands using specific primers and dye-terminator cycle sequencing chemistry (ABI PRISM Readymix dye-terminator kit) and an ABI 377-18 automated sequencer (Perkin-Elmer Applied Biosystems, Foster City, California, USA). Direct sequences of both amplified regions were obtained from all Lithuanian and Ukrainian samples and 27 samples from Kaliningrad. In addition, single sequences from either the gag NCp7 or env C2-V3 region were obtained from the majority of the rest of the samples from Kaliningrad. The direct sequences of the NCp7 coding and C2-V3 regions have been submitted to Genbank (accession numbers for NCp7: AF082396–AF082449 and for C2–V3: AF082450–AF082484).
From one of the Kaliningrad patient samples (Kal68) the amplified complete envelope region was cloned and cloning of the same region was attempted from the two Ukrainian samples (ukr970063 and ukr960047). Cloning conditions have been previously described [42]. Briefly, the 3 kb segment covering vpu, env and approximately one half of nef (amplified and Qiaquick-purified as above) was cloned into the commercially available vector pCR2.1 following the manufacturers instruction (TA-cloning kit, Invitrogen, San Diego, California, USA). The Ukrainian envelope sequences proved unclonable due to insert instability upon culture scale-up. Instead, the amplification product from the ukr970063 sample was directly sequenced.
The cloned envelope gene from Kaliningrad (clone kal68.1) and the amplified envelope gene from Ukraine was sequenced as above by primer walking using a previously described panel of oligonucleotide primers [44]. Sequences were assembled into contigs using Sequencher software (Gene Codes Corp., Ann Arbor, Michigan, USA). The _vpu-env-nef_-5′ sequences have been submitted to GenBank (accession numbers AF082485 and AF082486).
Phylogenetic analysis, subtyping and genetic variation
Virus subtype was determined by phylogenetic analysis of the sequences compared with previously published sets of reference sequences [45]. In addition, previously published and unpublished sequences from Finnish, Swedish, Estonian, Latvian, Lithuanian, Russian, Belorussian and Ukrainian strains were included in the phylogenetic analyses [10,13,14,27–30,35,46,47]. Alignments were assembled with GDE2.2a [48] and phylogenetic analysis was performed on gapstripped alignments with ClustalW (neighbour joining with the Kimura two-parameter model [49,50]). Bootstrap values (500 iterations) were estimated by the neighbour joining method as implemented in ClustalW. Maximum parsimony and maximum likelihood methods of the Phylip 3.52c package and using the FastDNAml program resulted in comparable results [51–53] (not shown). Genetic variation among sequences was estimated using the Felsenstein 84 maximum likelihood model of evolution and the program DNADIST [54]. Transition/transversion rates were estimated from the datasets using PUZZLE [55].
Results
Epidemiology of HIV-1 in Kaliningrad
Despite a rigorous seroepidemiological surveillance program, only 31 HIV-1-infected individuals were identified in Kaliningrad in the time period of 1988 to June 1996 (Fig. 2, upper panel). The yearly incidence rate from 1988–1995 in the region was from 0 to 0.09 per 10 000 calculated for the total population. In July 1996, a rapid increase in monthly new HIV-1 infections was detected, which continued through the following year (Fig. 2, lower panel). Between 1 July 1996 and 30 June 1997, 1335 newly diagnosed HIV-1-infected individuals (plus 141 who were tested anonymously, total cumulative number 1507) were detected in Kaliningrad (Fig. 2). This gives a yearly incidence rate of 6.07 per 10 000 in 1996 (an increase of 6744% compared with 1995) and the incidence for the first 6 months of 1997 was 8.79 per 10 000. Most of the infected individuals were young, over 65% were in the 14–25-year age-group, and 20% were aged 14–18 years (Fig. 3). Amongst 1269 infected individuals who were asked about their possible risk factors for HIV infection, the majority (n = 1208) reported injecting drug use. In 47 cases the reported risk factor was sexual contact (homo-/heterosexual) and 14 infected individuals were children born to infected mothers (Fig. 4). Individuals were asked about risk factors using a standardized questionnaire; 335 individuals were asked about their transmission risk before testing, and 934 were asked when positive test results were delivered. In addition to the 141 anonymous tests, risk factors could not be determined for 97 individuals. The drug that the majority of the interviewed individuals with the injecting drug use risk factor reported using (> 95%) was a home-made opiate extracted from locally produced or imported poppy stems and heads.
. Epidemiology of HIV-1 infections in Kaliningrad. Upper panel: annual numbers of newly diagnosed HIV-1 infections reported in Kaliningrad 1988–June 1997 (log scale; total, 1507, including those anonymously reported). Lower panel: monthly numbers of new HIV-1 infections in Kaliningrad from 1 January 1996 to 30 June 1997 (months shown as numbers; total, 1345, anonymous excluded). Arrows show sample collection dates.
. Age distribution of newly infected individuals reported in Kaliningrad from 1 January 1996 to 30 June 1997 (n = 1345, anonymous excluded).
. Proportions of HIV-1 transmission associated risk factors reported (n = 1507). ‘Unknown’ includes those anonymously tested and those who could not be reached after diagnosis.
Evidence of exposure to other parenterally/sexually transmitted diseases
Out of a sample of 353 HIV-1 positive sera from Kaliningrad taken from individuals reporting a history of injecting drug use, 120 (34%) had a positive syphilis serology, 287 (81%) were positive for antibodies against HCV, and 32 (9%) were positive for the HBV surface antigen. Of the 61 samples collected for genetic typing of HIV from Kaliningrad, 59 (97%) also reacted positive for antibodies against HCV (Table 1). For the four samples from Lithuania, only the individual with a history of injecting drug use in Kaliningrad was positive for HCV.
. Epidemiological information and variation.
Genetic variation and genetic typing
Sequence data from one or both of the two regions sequenced was successfully obtained from 50 out of the 61 samples collected from Kaliningrad. From 27 samples, sequence data was obtained from both the gag NCp7 and env C2–V3 regions. In addition, single gag NCp7 sequences were obtained from 21 samples and single env C2–V3 sequences from two samples. Sequencing of both regions was successful for all the samples from Lithuania (n = 4) and the Ukraine (n = 2). All except one (kal168, see below) of the HIV-1 sequences obtained from the individuals in Kaliningrad were very similar to one another (Table 1). Interpatient genetic variation was low [mean, 0.5% (range, 0–2.5%) in gag NCp7; mean, 0.5% (range, 0–1.9%) in env C2-V3], and there was no statistically significant difference between the variation of the November and April sample sets nor between samples collected at the AIDS centre and the pretrial custody prison (P < 0.01, t-test; two-sample assuming unequal variances). Two sequences (kal184, kal190) displayed a G→A mutation pattern in the gag NCp7 region, which is characteristic for hypermutation [56], and these sequences were excluded from analyses of interpatient variation. The low variation between individual samples supports a recent and rapid spread of the virus in the population. Positions in the V3 loop associated with viral phenotype and cell tropism (coreceptor usage), carried amino acids (Gly at amino-acid position 11 and Asp at position 18 of the V3-loop) linked to non-syncytium-inducing phenotype and macrophage/monocyte tropism (usage of CC-chemokine-5 receptor) [57–59].
Phylogenetic analysis of both the gag NCp7 and env C2-V3 regions showed that the sequences form a tight cluster with high bootstrap values (99.5 and 99.7%, respectively), and that the NCp7 sequences grouped together with previously described sequences belonging to subtype A (Fig. 5A). However, even though the sequences formed a similar tight cluster in the env region (Fig. 5B), they grouped together with viruses of subtype B, indicating that the virus may be a recombinant between subtypes A and B.
. Phylogenetic analysis of (A) the gag NCp7 coding region, and (B) the env C2-V3 coding region. Included in the trees are all the sequences produced for this study for which both gag and env sequences were obtained. From Kaliningrad, both regions were sequenced from 27 samples (sample prefix kal), from Lithuania from four samples (prefix lit), and from the Ukraine from two samples (prefix ukr). Included are also a set of subtype reference sequences [45,75,76]. In the env region, additional sequences collected from Lithuania, Belarus and Ukraine are included [10,29,35]. Bootstrap values (500 replicates, neighbour joining with Kimura two-parameter model of evolution) are shown next to the subtype designations. In the trees, the clusters of sequences from Kaliningrad and the Ukraine/Southern Russia are boxed, with corresponding supporting bootstrap values shown next to the box. Samples from Lithuania produced during this study are shown in italics (also sample kal168, which is unrelated to the main epidemic strain; see text for details), and those from the Ukraine produced during this study are shown in larger print. Alignments are available from the authors upon request.
None of the sequences from Kaliningrad collected in this study were closely related (maximum similarity, 93%) to any sequences from strains previously described from the Baltic countries, Russia, Belarus, Sweden, Finland or any of the sequences in the Los Alamos HIV database [8,10,13,27–30,33,35,47,60], with the exception of Ukrainian subtype A sequences (see below). One sequence (kal168; obtained from a sample collected from an individual at the pretrial custody prison) was unrelated to the Kaliningrad major strain (mean difference, 15.4 and 12.5% in gag NCp7 env C2–V3, respectively; Fig. 5). This sequence belonged to genetic subtype B in both regions analysed and was related to sample lit172 (Fig. 5). This individual was among the two reporting sexual transmission (homo-sexual) outside Kaliningrad as his probable transmission risk factor, and was also HCV-negative. The only other individual (kal181) who reported sexual transmission (heterosexual), carried the same NCp7 subtype A/C2–V3 subtype B strain as the majority of the other individuals from Kaliningrad. Two other HCV-negative individuals (kal68 and kal182) also carried the A/B recombinant strain.
The sequences from samples collected from individuals in the neighbouring Lithuania during this study were more heterogeneous. Two of the viruses (lit172 and lit175) were classified as subtype B strains unrelated to the main Kaliningrad strain (mean difference 13% in gag NCp7 and 11.6% in env C2-V3 compared with the main Kaliningrad strains), one (lit174) was classified as subtype A in both gag NCp7 and env C2-V3, but unrelated to the Kaliningrad strain (mean difference, 6.3% in gag NCp7 and 20.4% in env C2-V3 compared with the main Kaliningrad strains), and one (lit173) was similar to the strains from Kaliningrad (mean difference, 0.3% in gag NCp7 and 1.2% in env C2-V3 compared with the main Kaliningrad strain). This patient reported injecting drug use in Kaliningrad as his main risk factor (Table 1) and he was the only one who was HCV-positive. For the other three, the reported risk factors were homo-/heterosexual contact outside Kaliningrad.
Sequences from the two IDU samples from the Ukraine (ukr970063 and ukr960047) both grouped tightly together with those from Kaliningrad in the NCp7 region (mean difference, 0.5% in gag NCp7 compared with the main Kaliningrad strains), classifying them as belonging to subtype A (Fig. 5A). However, in the env C2-V3 region, the viruses formed a cluster within the A subtype rather than in the B subtype (mean difference, 23% in env C2-V3 compared with the main Kaliningrad strains). Furthermore, the Ukrainian strains were extremely similar (cluster bootstrap value of 100%) to previously described sequences from Ukrainian IDU [35], strengthening the results of the analysis (Fig. 5B). It is evident that the subtype A parent of the recombinant is a representative of the strain that has been reported to be prevalent amongst IDU in the Ukraine and some cities of Southern Russia [35].
To differentiate between recombination and coinfection followed by biased PCR amplification, we sequenced the complete 3 kb _vpu-env_-5′ nef regions from one sample each from Kaliningrad and Ukraine (kal68 and ukr970063) in the hope that this longer region would span a recombination point. Indeed, phylogenetic analysis showed that the sequence of sample ukr970063 consisted of completely subtype A-like sequences (Fig. 6). In contrast, the majority of the kal68 sequence belonged to subtype B, but the 5′ nef part was very similar to the Ukrainian subtype A sequence, both by phylogenetic analysis and visual examination (Figs 6 and 7). The results indicate that the Kaliningrad epidemic strain is indeed recombinant, and not a spreading coinfection of two different subtypes. A genetic structure, where the beginning of the nef gene has been acquired from a sub-type A strain is very similar to that previously described for subtype A/E recombinant viruses [61,62].
. Phylogenetic classification of the gag NCp7 and _vpu-env-nef_-5′ regions of the Kaliningrad and Ukrainian HIV-1 strains. Upper panel: phylogenetic classification of the strains. Clusters defining the A and B subtypes are boxed and the corresponding bootstrap value (bootstrapping as in Fig. 5) supporting the cluster is shown next to the box. For the NCp7 coding region, the same tree is shown as for Fig. 5 (although for clarity only the position of the kal68 and ukr970063 samples are shown). For the envelope and nef 5′ region, all available sequences were used [45]. Lower panel: schematic of corresponding regions in HIV-1 provirus (not to scale) showing the subtype classification. The filled and open areas indicate the approximate segments used for the analysis, which are joined to the corresponding tree in the upper panel by a broken line. Broken line box: schematic of the HIV-1 proviral genome. Alignments are available from the authors upon request.
. Alignment of the region covering the recombination breakpoint (in the gp41-nef region) between the Kaliningrad A/B recombinant (kal68.1) and the Ukrainian subtype A strain (ukr970063). The sequences are individually numbered excluding gaps, and only changes relative to ukr970063 are shown in the alignment (dots represent similarity). The putative recombination breakpoint area is shown by asterisks above and below the two sequences. The stop codon of the env gene and the start codon of the nef gene are shown.
Discussion
In this report we have described the outbreak of an IDU-associated HIV-1 epidemic in the Russian Kaliningrad region (Fig. 1). The epidemic started in July-August 1996, and rapidly expanded to 100–200 newly identified cases per month (Fig. 2). HIV-1 surveillance in the area had been ongoing since 1988, and the principles for testing had been largely similar for the complete period. No change in either the volume of yearly tests or the population groups being tested had taken place before the 1996 epidemic started, indicating that the monthly increase in newly diagnosed HIV-1-positive samples was caused by a true increase in the number of monthly transmission events. The majority of those infected reported injecting drug use as their main risk factor for transmission (Fig. 4). Transmission by injecting drug use as the main route was supported by the rapid epidemiology of the epidemic and the fact that the majority of those infected were also infected by HCV. The majority of those infected were young, 65% belonged to the 14–25-year age-group, and 95% of all the infected were aged younger than 35 years (Fig. 3). The very rapid epidemiology (an increase from less than one case per month to > 100 per month in just 3 months) suggests that injecting drug use has been widespread among young people in the area even before the epidemic, and that infected dealers/users may have been responsible for introduction of the virus into the IDU population.
As in previous examples of IDU-associated HIV-1 epidemics [1,2,16,63–70], the main route of transmission is likely to have been syringe and needle-sharing. However, according to local health-care officials, IDU and producers claim one major risk factor for the rapid spread may be found in the method of drug preparation. In the final preparation of the locally produced opiate, kompot (also called maki and sjirevo in Kaliningrad, hymka and kuknar in the Ukraine), human blood was reported to be added to act as a clarifier. Whether the virus could survive in the product is unknown. Although not directly investigated, this was reported to us by several sources, and has also been independently reported [71]. In addition to HIV-1, our results show evidence for IDU spread of HCV and HBV in Kaliningrad. Since coinfection with these viruses and HIV-1 may aggravate disease course [72,73], further difficulties are likely to develop in the treatment of those infected.
Molecular characterization of the HIV-1 strains in Kaliningrad shows similar characteristics to those from other previously described IDU-associated HIV-1 epidemics, with low inter-patient variability and a V3-loop sequence associated primarily with replication in cells of the monocyte/macrophage lineage [58]. We were able to demonstrate a direct molecular epidemiological link to similar IDU-associated HIV-1 epidemics in the Ukraine and the southern parts of the Russian Federation [35] suggesting that these epidemics have a common origin. However, the strain in Kaliningrad is recombinant between subtypes A and B, whereas the Ukrainian and Southern Russian epidemic strains belong to the genetic subtype A of HIV-1 in both the gag and env regions of the proviral genome [35]. Analysis of a long segment covering the complete env gene and approximately half of the nef gene from both viruses showed that the Kaliningrad strain is truly recombinant, with A subtype nef and B subtype env gp160 coding regions. Phylogenetic analysis also showed that the Ukrainian A subtype strain is the direct ancestor of the Kaliningrad recombinant strain. In contrast, we were not able to find a close relationship to previously described subtype B sequences from the Ukraine, Belorussia and Lithuania, or to previously published sequences from the other Baltic and Scandinavian countries. Comparison with sequences in the Los Alamos HIV-1 sequence database also failed to reveal closely related sequences [8,10,13,27–30,33,35,47,60]. Therefore, the subtype B parent of the Kaliningrad A/B recombinant remains unknown.
This is the first report of a subtype A/B recombinant virus, extending the range of possible recombinants and suggesting that most HIV-1 subtypes can exchange genetic material by recombination. On the basis of our data it is not possible to differentiate where and when the actual recombination event occurred. However, since we did not find evidence for the presence of the parental subtype B and A strains in Kaliningrad, it may have occurred before the introduction of the strain to the region. The similarity of the gag and nef regions to Ukrainian and southern Russian subtype A strains [35] suggests that the subtype A parent virus of the Kaliningrad strain may have been introduced from this region, most likely by illegal drug trafficking. The exact time and location of the recombination event are not possible to deduce based on the available data. It may have occurred in the Ukraine, along its way to Kaliningrad, or in Kaliningrad itself.
The finding that the 5′ nef region of the Kaliningrad virus belongs to subtype A may be significant. Comparison of the Kaliningrad virus with another strain (the A/E recombinant strain) that has previously caused an explosive epidemic, spreading through Southeast Asia, reveals unexpected similarity in structure. In both cases, the gag regions of the virus belong to subtype A and the envelope to another subtype, which in the Southeast Asian case is subtype E and in the Kaliningrad case is subtype B. Furthermore, in both viruses there is a putative recombination point in the downstream end of the env gene, resulting in subtype A sequences in the nef gene (Figs 6 and 7) [61,62]. The recombination points are not identical, but the common denominator for both viruses is a subtype A nef gene. It is possible to speculate that some selection force would be favouring the A subtype nef gene in subtype A recombinant viruses. A more likely explanation may be that this region is simply a hotspot for recombination. This would be supported by the fact that the region is bounded by insertion/deletion events in alignments covering all subtypes. Further analysis of recombinant viruses will be needed to answer this question.
Variation between individual HIV-1 strains was extremely low (0.5% on average in both regions analysed) among the samples collected in Kaliningrad, which is consistent with spread from a common point source of the virus. There was no statistically significant difference in variability between the two sample sets, collected almost 6 months apart, clearly suggesting that the same virus was circulating in the population and was probably introduced into new hosts before significant variation had developed. The low variation may suggest that many individuals were infected during the primary viraemic stage of the transmitter. The epidemiological and molecular data both suggest that the virus was introduced into the area by very few, or possibly a single infected individual, who then rapidly transmitted the virus further. It is likely that this source of the virus was an infected drug producer/dealer. The clonal nature of the epidemic and its detection almost immediately after it commenced opens up the possibility for natural history studies, as well as studies of drug efficacy and the relationship of virus evolution and host factors. Since the subtype A parental virus and the A/B recombinant are both available, studies of biological differences between these two subtypes may also be possible.
The explosive epidemiology of HIV-1 in Kaliningrad is not surprising. Previous examples of similar IDU HIV epidemics abound elsewhere in the world [1,2,16,63–70] and for some time there have been signs of a radical change in the epidemiology of HIV infection in the countries of the former Soviet Union [24–26,35,36]. According to health statistics of the Russian Federation, 3513 new HIV infections were diagnosed by 25 October 1997, of which approximately half can be attributed to the Kaliningrad epidemic [74].
The long history of a large military naval base in the region, the breakdown of economic and social structures following the fall of the Soviet system, and the relative isolation of the region have all contributed to conditions that favour the spread of STD and the use of addictive substances, and have paved the way for the explosive HIV epidemic. The Lithuanian case with connections to Kaliningrad shows that the epidemic has a true potential to spread wider along with increasing travel. The fact that the epidemic strain is a recombinant between subtypes A and B may have significance for future prevention and treatment strategies in the wider European context.
The region's health policy is facing major challenges due to the epidemic. A majority of infected people are young, aggravating the societal damage that will come when the infected people will become invalidated by the onset of clinical AIDS. In addition, the simultaneous acquisition of the infection by so many will probably result in a sudden increase of AIDS incidence at some time in the future. This will cause problems in mobilizing sufficient resources for the health services. Another major challenge is to prevent the sexual spread of the current epidemic. This requires effective health education but also innovative new strategies that can take into account the unusual local circumstances. First steps to develop effective health education have already been taken, but international collaboration and help will be needed to solve the problems caused by the present epidemic [40].
Acknowledgements
The authors thank Richard Pebody for critical review of the manuscript, Rachanee Cheinsong-Popov and Aleksei Bobkov for access to information prior to publication, Marja Leena Kantanen for her support of this work, and the International Department of the Kaliningrad City Council for the practical arrangements enabling the successful completion of the study.
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Keywords:
Injecting drug users; outbreak; epidemic; intersubtype recombinant; molecular epidemiology
© 1998 Lippincott Williams & Wilkins, Inc.