Emergence of New Forms of Human Immunodeficiency Virus Type 1 Intersubtype Recombinants in Central Myanmar

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1 AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 16, Number 17, 2000, pp Mary Ann Liebert, Inc. Emergence of New Forms of Human Immunodeficiency Virus Type 1 Intersubtype Recombinants in Central Myanmar KAZUSHI MOTOMURA, 1 SHIGERU KUSAGAWA, 1 KAYOKO KATO, 1 KYOKO NOHTOMI, 1 HLA HTUT LWIN, 3 KHIN MAUNG TUN, 3 MIN THWE, 3 KHIN YI OO, 3 SOE LWIN, 3 OHN KYAW, 3 MYINT ZAW, 3 YOSHIYUKI NAGAI, 2 and YUTAKA TAKEBE 1 ABSTRACT We have previously shown that HIV-1 env subtypes B9 (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B9 (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B9/env subtype C, one gag subtype B9/env subtype E, one gag subtype C/env subtype B9, and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B9 and C/B9/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B9, C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region. INTRODUCTION MYANMAR (FORMER BURMA) IS SURROUNDED by three nations where HIV has spread rapidly. It is bordered by Thailand to the east, Yunnan Province of China to the northeast, and the states of Manipur and Nagaland of India to the west. The geographical location of Myanmar has influenced the characteristics of the HIV epidemic in this country. Since national sentinel surveillance first detected high HIV prevalence rates (23.7%) among injecting drug users (IDUs) in 1989, HIV has spread to various risk populations. A total of 2854 AIDS cases had been reported by the end of 1998, and among them 60% of cases were due to heterosexual transmission and 29% were due to injecting drug use. The national HIV prevalence rates (average) in 1998 were 61.9% (range, %) for IDUs, 29.8% (range, %) for female commercial sex workers (fcsws), 6.2% (range, %) for male sexually transmitted disease (STD) patients, 7.3% (range, %) for female STD patients, 1.8% (range, %) for pregnant women, 0.8% (range, %) for blood donors, and 1.8% (range, %) for military recruits. 1 The highest HIV prevalence rates among IDUs and CSWs were observed in Mandalay, the second largest city located in central Myanmar: Sentinel surveillance rates in 1998 were 85.0% for IDUs and 39.8% for fcsws. The infection rates are among the highest in Asia. To date, however, the HIV strains from Mandalay have not yet been genetically characterized. Genetic subtyping has been a powerful molecular tool for tracking the global spread of HIV. 2 In contrast to the extensive knowledge about HIV-1 causing the epidemic in neighboring countries, especially Thailand, only a few studies have been conducted in Myanmar. Using dried blood spots from Myanmar, Cassol et al. identified six subtype B9 (Thai-B cluster within subtype B, also referred to as B B ) 3,4 and one subtype E (HIV-1 intersubtype A/E recombinant, CRF01_AE 5 ) among the specimens collected in Myanmar in According to the genetic and serologic study by Kusagawa et al. 7 the epidemiological patterns of HIV spread in Myanmar are classified into 1 Laboratory of Molecular Virology and Epidemiology, 2 AIDS Research Center, National Institute of Infectious Diseases, Tokyo , Japan. 3 AIDS Prevention and Control Programme, Department of Health, Yangon, Myanmar. 1831

2 1832 the following three categories by different geographic regions: (1) In Yangon, the capital city of Myanmar, HIV-1 subtype B9 predominated both in IDUs and heterosexuals, whereas a few HIV-1 subtype E were detected only among persons presumably infected sexually; (2) in the cities near the border with Thailand, where heterosexual transmission is a major pathway of HIV spread, CRF01_AE predominated; in contrast, (3) in central and northeast Myanmar, both HIV-1 subtype B9 and CRF01_AE occurred in a mixed distribution, without showing any significant segregation by risk group. Moreover, a serologic study based on V3 peptide enzyme immunoassay suggested the occurrence of subtype(s) other than subtypes B9 and E in central and northeast Myanmar. 7 However, HIV strains circulating outside Yangon were not genetically characterized, largely because of the difficulty in collecting appropriate specimens of sufficient quality from remote areas in this country. In the present study, we investigated the HIV-1 genetic subtypes found in Mandalay, which has one of the highest HIV infection rates observed among IDUs and CSWs in Southeast Asian countries. We have identified, for the first time, HIV-1 subtype C in nearly one-third of the specimens from Mandalay and provide evidence of the presence of new forms of HIV-1 recombinants between multiple HIV-1 subtypes, B9, C, and CRF01_AE. On the basis of the data in the present study, we discuss the mechanism and interrelationship of the epidemic in Myanmar with those in surrounding countries. MATERIALS AND METHODS Study subjects and specimens Serum specimens were collected from 34 HIV-positive asymptomatic patients from various risk populations in Mandalay, central Myanmar from November 1997 through May 1999, including 17 specimens from male IDUs, 9 from fcsws, 4 from male STD patients, and 4 from women presumably infected heterosexually (Table 1). Other background, clinical, and laboratory information, including the time of seroconversion, was not available on these patients. All 34 specimens were serologically determined as HIV-1 infections. No HIV-2 infections were detected. Determination of nucleotide sequence The nucleotide sequences of 432-bp gag (p17) and 336-bp env (C2/V3) regions were determined from plasma viral RNA by a method described previously, 8 except that some of the primer sets for polymerase chain reaction (PCR) amplifications were slightly modified. For the nested PCR amplification of the gag (p17) region, the first (outer) primer pair, PBS-172A (sense, 59-ATCTCTAGCAGTGGCGCCCGAACAG-3 9) and p24-173b (antisense, 59-CTGATAATGCTGAAAACATGGGTAT-39), and the second (inner) primer pair, PSI-174A (sense, 59-CTCTC- GACGCAGGACTCGGCTTGCT-3 9) and p17-175b (antisense, 59-CCCATGCATTCAAAGTTCTAGGTGA-3 9), were used. For the nested PCR amplification of env (C2/V3) regions, the first (outer) primer pair, ED-5 (sense, 59-ATGGGATCAA- AGCCTAAAGCCATGTG-3 9) and ED-12 (antisense, 59-AGT- GCTTCCTGCTGCTCCCAAGAACCCAAG-3 9), and the second (inner) primer pair, ED-31 (sense, 59-CCTCAGCCATTA- CACAGGCCTGTCCAAAG-3 9) and ED-33 (antisense, 59-TT- ACAGTAGAAAAATTCCCCTC-3 9), 9 were used. The lengths of the RNA nested-pcr products of gag (p17) and env (C2/V3) regions are 520 and 353 bp, respectively. The PCR products were purified with a Centricon-100 (Amicon, Danvers, MA) and then used as templates for direct sequencing on an automated ABI PRISM310 DNA sequencer (Perkin-Elmer, Foster City, CA). The primers used in the second round of PCR were used to determine the nucleotide sequences of the respective strands. To search for possible recombination breakpoints in the specimens that showed discordance between gag (p17) and env (C2/V3) subtypes, the nucleotide sequences of an additional two regions, including a 671-bp segment encompassing the 39 part of vif through vpr (vif-vpr) and a 796-bp segment encompassing the 39 part of vpr through the 59 part of env (vpr-env) of HIV-1, were determined from plasma viral RNA by the same method. For the nested PCR amplification of the vif-vpr region, the first (outer) primer pair, VIF-362A (sense, 59-GGCAGGT- GATGATTGTGTGGC-39) and VIF-364B (antisense, 59- GGCTGACTTCCTGGATGGTTCCAGGGC-3 9), and the second (inner) primer set, VIF-363A (sense, 59-GGATGAGGA- TTAGAACATGG-39) and VIF-365B (antisense, 59-GCCT- ATTCTGCTATGTTGACACCC-3 9), were used. For the nested PCR amplification of the vpr-env region, the first (outer) primer pair, VIF-368A (sense, 59-GCCCCAGAAGACCAAGGGCC- 39) and ENV-360B (antisense, 59-GGGGTCTGTGGGTA- CACAGGCATGTGT-39), and the second (inner) primer pair, VIF-369A (sense, 59-GACATTTTCCTAGGCCATGGC-3 9) and ENV-370B (antisense, 59-GTGGGTACACAGGCATGT- GTGGC-39), were used. The lengths of the RNA nested-pcr products of the vif-vpr and vpr-env regions are 714 and 839 bp, respectively. The nucleotide sequences of both segments overlap by 106 bp. Data analysis MOTOMURA ET AL. All the nucleotide sequences obtained in the present study were screened by the BLAST 2.0 program (National Center for Biotechnology Information, Bethesda, MD) to search for sequence similarities to previously reported sequences in the databases, and to rule out potential laboratory errors. The highest scores (ranging from 92 and 97% in the present data sets) were all from HIV-1 gag (p17) or env (C2/V3) sequences from Southeast Asia. Nucleotide sequences were aligned with the reference strains 10 and with HIV-1 subtypes B9, 3,11 C, 12,13 and CRF01_AE 7,14 16 previously found in Southeast Asia, using CLUSTAL W version 1.4, 17 and were corrected manually to ensure that gaps did not alter the reading frame. Frequencies of nucleotide substitutions were estimated by the Kimura twoparameter method. 18 Phylogenetic trees were constructed by the neighbor-joining method 19 with 100 bootstrap replicates, 20 based on the aligned data sets of the 432-bp sequences starting from the coding frame of gag (p17) and the 286-bp sequences in the env (C2/V3) region, which overlap with those available in databases. Analyses were implemented by MEGA, version Phylogenetic trees based on the maximum-likelihood method were also constructed by the DNAML program of PHYLIP version Since the trees constructed by the two algorithms resulted in the same topology, only the neighborjoining trees are presented.

3 NEW FORMS OF HIV-1 RECOMBINANTS IN CENTRAL MYANMAR 1833 To identify the recombination breakpoints, Simplot 2.5 software 23 was used to calculate similarity plots and bootstrap plots. For similarity plots, the percent similarity between the selected pairs was determined by moving a window of 200 bp along the alignment in increments of 10 bp. The similarity values for each pairwise comparison were plotted at the midpoint of the 200- bp segment. For bootstrap plots, the bootscanning was performed on neighbor-joining trees for a window of 200 bp, moving along the alignment in 10-bp increments, evaluating 100 replicates for each phylogeny. The data were also analyzed by the Recombinant Identification Program (RIP). 24 Since the results obtained by both programs were identical, only the plots generated by Simplot software are presented. Intrasubtype nucleotide diversity of the gag (p17) and env (C2/V3) regions was estimated from the Kimura two-parameter substitution matrix. 8,18 Differences in variation between groups were tested by nonparametric statistics (Mann Whitney U test). RESULTS Occurrence of HIV-1 subtype C and the discordance of gag and env subtypes Genetic subtypes for both gag (p17) and env (C2/V3) regions were determined in 25 of 34 (73.5%) serum specimens (Table 1). The genetic subtype for one region, either gag (p17) or env (C2/V3), was determined for the remaining nine specimens (26.5%) (Table 1). TABLE 1. LIST OF STUDY SPECIMENS FROM MANDALAY AND SUMMARY OF THEIR GENETIC CHARACTERISTICS a HIV-1 subtype b Risk Age Clinical Collection Specimen group Sex (years) status date gag (p17) vif/vpr vpr/env env (C2/V3) midu 1 IDU M 21 AC 18 Nov. 97 B9 B9 midu 2 IDU M 20 AC 18 Nov. 97 B9 C C/B9 C midu 3 IDU M 30 AC 18 Nov. 97 n/a C midu 4 IDU M 23 AC 18 Nov. 97 n/a E midu 5 IDU M 29 AC 18 Nov. 97 B9 B9 midu 7 IDU M 37 AC 11 June 98 n/a B9 midu 8 IDU M 39 AC 11 June 98 B9 B9 midu 9 IDU M 27 AC 11 June 98 A E midu 10 IDU M 35 AC 11 June 98 C C C/B9 B9 midu 11 IDU M 27 AC 11 June 98 C C n/a C midu 12 IDU M 25 AC 11 June 98 C C C C midu 13 IDU M 22 AC 11 June 98 B9 n/a midu 14 IDU M 20 AC 11 June 98 n/a C midu 15 IDU M 19 AC 19 Nov. 98 B9 C C C midu 16 IDU M 22 AC 19 Nov. 98 C C midu 17 IDU M 28 AC 19 Nov. 98 A E midu 18 IDU M 25 AC 15 May 99 C C C/B9/E E mcsw 1 CSW F 21 AC 11 June 98 A E mcsw 2 CSW F 28 AC 11 June 98 B9 B9 B9 C mcsw 5 CSW F 26 AC 11 June 98 B9 B9 mcsw 6 CSW F 19 AC 11 June 98 A E mcsw 8 CSW F 33 AC 11 June 98 A E mcsw 9 CSW F 20 AC 11 June 98 A E mcsw 10 CSW F 22 AC 11 June 98 C n/a mcsw 11 CSW F 22 AC 19 Nov. 98 A E mcsw 12 CSW F 23 AC 19 Nov. 98 B9 B9 B9 E mstd 1 STD M 30 AC 11 June 98 n/a E mstd 3 STD M 39 AC 19 Nov. 98 A E mstd 5 STD M 23 AC 15 May 99 A E mstd 6 STD M 24 AC 15 May 99 n/a E mhetero 1 Hetero F 34 AC 20 Nov. 97 A n/a mhetero 2 Hetero F 18 AC 20 Nov. 97 A E mhetero 4 Hetero F 21 AC 21 Nov. 97 C C mhetero 5 Hetero F 20 AC 21 Nov. 97 C C Abbreviations: IDU, injecting drug user; CSW, commercial sex worker; STD, sexually transmitted disease clinic patient; Hetero, person infected via heterosexual contact; M, male; F, female; AC, asymptomatic carrier; n/a, not amplified. a Samples that show discordance between HIV-1 gag (p17) and env (C2/V3) subtypes are in boldface (HIV-1 intersubtype A/E recombinants [CRF01_AE] are not included). b HIV-1 subtypes: A, gag subtype A; B9, subtype B9 within subtype B; C, subtype C; E, env subtype E; blank, not tested.

4 1834 Neighbor-joining analysis placed 10 of 25 sequences (40%) in CRF01_AE with high bootstrap values: 90 of 100 and 99 of 100 for gag (p17) and env (C2/V3) regions, respectively (Fig. 1A and B). In gag (p17) trees, all 10 sequences belonged to the MOTOMURA ET AL. cluster of HIV-1 CRF01_AE (gag A/env E, A/E) within subtype A, distinct from the nonrecombinant form of subtype A (gag A/env A, pure A in Fig. 1), and CRF02_AG 25 and CRF04_cpx 5 found in Africa (Fig. 1A). The CRF01_AE was FIG. 1. Neighbor-joining tree based on the gag (p17) (A) and env (C2/V3) (B) nucleotide sequences from central Myanmar (Mandalay) with HIV-1 group M (subtypes A J) reference sequences. 10 SIV CPZ GAB is used as an outgroup. Subtypes and CRF (circulating recombinant form) codes are indicated on the right side of the tree. Bootstrap values greater than 50 are shown at corresponding nodes. The specimens from Mandalay in the present study are shown against the gray background: IDUs (rectangular boxes); persons with sexual exposure (oval-shaped boxes). Symbols for the specimens with discordance in gag and env subtypes are indicated in the inset in (A).

5 NEW FORMS OF HIV-1 RECOMBINANTS IN CENTRAL MYANMAR 1835 FIG. 1. Continued. predominantly distributed among the persons presumably infected sexually (8 of 13, 61.5%), whereas it was less frequently detected among IDUs (2 of 12, 16.7%) (Table 2). All six HIV-1 env subtype B9 sequences formed a monophyletic cluster (designated gag B9) within gag subtype B, except for midu10, which is placed in gag subtype C, despite its belonging to the env subtype B9 cluster (see below) (Fig. 1A and B). HIV-1 subtype B9 (gag B9/env B9) was found more often among IDUs (3 of 12, 25%) than in persons with sexual exposure (1 of 13, 7.7%) (Table 2). In addition to HIV-1 env subtypes B9 and E, which were identified in previous studies in Myanmar, 6,7 HIV-1 subtype C (gag C/env C, C/C in Fig. 2) was detected in 5 of 25 (20%) specimens: 3 specimens from IDUs (3 of 12, 25%) and 2 specimens (2 of 13, 15.4%) from persons with sexual exposure (Table 2). The HIV-1 subtype C sequences in either the gag

6 ì í î ì í î ì í î 1836 MOTOMURA ET AL. TABLE 2. DISTRIBUTION OF HIV-1 SUBTYPES AMONG DIFFERENT RISK POPULATIONS IN CENTRAL MYANMAR (MANDALAY) a Risk group n A/E B9 C B9/C B9/E C/B9 C/E IDUs (16.7) (25.0) (25.0) (16.7) (8.3) (8.3) (33.3%) Persons with sexual exposure (61.5) (7.7) (15.4) (7.7) (7.7) (15.4%) CSW STD 2 2 Hetero Total (40.0) (16.0) (20.0) (12.0) (4.0) (4.0) (4.0) (p17) or env (C2/V3) region were detected in 13 of 34 specimens (38.2%) from Mandalay (Table 1). The prevalence of HIV-1 subtype C in either the gag (p17) or env (C2/V3) region was slightly higher among IDUs (9 of 17, 52.9%) than in persons infected sexually (4 of 17, 23.5%) (p ) (Table 1). Interestingly, 4 of 12 specimens (33.3%) from IDUs and 2 of 13 specimens (15.4%) from persons with sexual exposure showed a discrepancy between gag (p17) and env (C2/V3) subtypes: Three specimens (3 of 25, 12.0%) exhibited gag subtype B9 and env subtype C (B9/C); the remaining three discordant specimens were assigned as either gag subtype B9 and env subtype E (B9/E), gag subtype C and env subtype B9 (C/B9), or gag subtype C and env subtype E (C/E) (1 of 25, 4.0%, respectively) (Tables 1 and 2, and Fig. 1). Closeness of HIV-1 subtype C sequences from Mandalay to those found in China and India The phylogenetic analysis of gag (p17) and env (C2/V3) regions showed a tendency of the majority of HIV-1 subtype C sequences from central Myanmar to be phylogenetically more closely related to those found in southern China, whereas they were weakly related to Indian subtype C strains and distantly related to the HIV-1 subtype C distributed in African countries and Brazil (Figs. 1 and 2). (24.0%) a A/E, HIV-1 intersubtype A/E recombinant; B9, subtype B9; C, subtype C. Specimens exhibiting discordance between gag (p17) and env (C2/V3) subtypes are designated B9/C (gag B9/env C), B9/E (gag B9/env E), C/B9 (gag C/env B9), and C/E (gag C/env E). To search for the possible amino acid sequence signature shared among the HIV-1 subtype C circulating in Mandalay and the surrounding countries, the amino acid sequences of env (C2/V3) regions from various geographical locales were aligned with HIV-1 subtype C consensus sequence 26 and compared extensively with those distributed in Mandalay (Fig. 3). As seen in Fig. 3, the combination of glutamine (Q) substitution at position 37 (37Q) and serine (S) substitution at position 108 (108S) (37Q/108S) was found almost exclusively in HIV-1 env subtype C sequences from Mandalay (7 of 10, 70%) and southern China (Yunnan Province) (17 of 17, 100%) (Figs. 2 and 3). This combination of the signature (37Q/108S) was observed only in a few env subtype C sequences from India (IN3.D1024 from Bombay- Goa and IN3.IND2 from New Delhi) (2 of 31, 6.5%) and was not found among subtype C sequences from Africa (0 of 30) and Brazil (0 of 7), according to the database entries as of 1998 (Fig. 3). Similarly, the 37Q/108S substitution was not found among the latest subtype C sequence data entries from other countries, including France 27 (0 of 5), The Netherlands 28 (0 of 3), the Russian Federation 29 (0 of 4), Nepal 30 (0 of 19), Tanzania 31,32 (0 of 11), Uganda 33 (0 of 2), Senegal 34 (0 of 2), and Ethiopia 35 (0 of 3). In addition, the single-amino acid 37Q substitutions was found almost exclusively among subtype C specimens from India (19 of 31, 61.3%): the only exception was one env subtype C sequence from Mandalay (midu3) (1 of 10, 10%) (Fig. 3). FIG. 2. Phylogenetic relationship of HIV-1 subtype C from central Myanmar (Mandalay) with that from surrounding countries based on neighbor-joining analysis of env (C2/V3) regions. HIV-1 subtype B strain JRFL is used as an outgroup. The specimens from Mandalay are shown in boxes against a gray background: IDUs (rectangular boxes); persons with sexual exposure (ovalshaped boxes). Bootstrap values are shown at the corresponding nodes. The name of the country where the specimen originated is indicated at the right. The open and gray arrowheads indicate the HIV-1 subtype C specimens with glutamine substitution at position 37 (37Q) and serine substitution at position 108 (108S) in the env (C2/V3) region, respectively (see Fig. 3). The closed arrowheads indicate the HIV-1 subtype C specimens with the combination of 37Q and 108S substitutions, which are almost exclusively observed in the subtype C specimens from southern China (Yunnan Province) and central Myanmar. A small square after the specimen code indicates a specimen with discordance in gag (p17) and env (C2/V3) subtypes: subtype B9 in the gag (p17) region and subtype C in the env (C2/V3) region. The gag (p17) and env (C2/V3) subtype of each specimen from central Myanmar (Mandalay) is indicated in parentheses [gag (p17) subtype/env (C2/V3) subtype] after the specimen code: B9, HIV-1 subtype B9; C, HIV-1 subtype C;?, subtype not determined because the corresponding region was not PCR amplified.

7 NEW FORMS OF HIV-1 RECOMBINANTS IN CENTRAL MYANMAR 1837

8 1838 MOTOMURA ET AL.

9 NEW FORMS OF HIV-1 RECOMBINANTS IN CENTRAL MYANMAR 1839 In summary, the possible amino acid signature 37Q/108S appeared to characterize the subtype C strains from southern China and central Myanmar, whereas 37Q was relatively specific to some Indian subtype C strains (Figs. 2 and 3). Identification of recombination breakpoints To discern whether the apparent discrepancy of subtype assignments in the gag (p17) and env (C2/V3) regions of six specimens (midu2, 10, 15, and 18 and mcsw2 and 12) from Mandalay (Tables 1 and 2, and Fig. 2) was due to coinfections or recombinations of multiple HIV-1 subtypes, we attempted to amplify and determine the sequence of a 671-bp region from vif to vpr (vif/vpr) and a 796-bp region containing the 39 half of vpr and the 59 part of env sequences (vpr/env) from plasma RNA. Both sequences overlap by 106 bp and altogether constituted a 1361-bp nucleotide sequence encompassing the 39 part of vif through the 59 part of the env gene (Fig. 4). We succeeded in amplifying and sequencing both regions in all six specimens. Since the 106-bp overlapped nucleotide sequences of the above two segments were completely identical in all six cases, the combined nucleotide sequences of the 1361-bp segment encompassing the 39 part of vif through the 59 part of the env gene (vif/env) were subjected to analysis by recombinant identification programs, including Simplot 23 and RIP. 24 Since both programs gave similar results, only the plots created by the Simplot program are presented (Fig. 4). As seen in Fig. 4, we were able to identify the recombination breakpoints between HIV-1 subtype C and B9 in the contiguous 796-bp vpr/env regions of midu2 and midu10, whereas the locations of the recombination breakpoints in these specimens were obviously not identical (left and middle panels of Fig. 4). Similarly, multiple recombination breakpoints between HIV-1 subtypes C, B9, and E (CRF01_AE) were identified in the vpr/env region of midu18 (the right panel in Fig. 4). Recombination breakpoints are not identified in the vif/env region of the remaining three specimens (midu15 and mcsw2 and 12) (Table 1). Intrasubtype nucleotide diversity of HIV-1 subtypes in Mandalay To assess the genetic diversity of HIV-1 subtypes circulating in Mandalay, we determined the intrasubtype nucleotide diversity in env (C2/V3) regions based on the Kimura two-parameter data matrix (Table 3). As shown in Table 3, the CRF01_AE distributed in Mandalay IDUs ( %) showed significantly lower genetic divergence than that of HIV- 1 subtypes B9 ( %) (p ) and C ( %) (p ) in the same risk population, respectively. Moreover, the intrasubtype nucleotide diversity of CRF01_AE among IDUs was significantly lower than that among the persons infected sexually ( %) (p ). In contrast, among the persons infected sexually, the intrasubtype genetic divergence of CRF01_AE was essentially the same as that of subtype C ( %; n 5 3; range, %) (p ) (Table 3). Similarly, the genetic divergence of HIV-1 subtype C among IDUs ( %) was slightly higher than among persons infected sexually ( %), whereas the difference was not significant (p ) (Table 3). DISCUSSION Two HIV-1 subtypes, B9 and E (CRF01_AE), were previously identified in studies performed in Myanmar. 6,7 However, the occurrence of subtype(s) other than subtypes B9 and CRF01_AE in central and northeast Myanmar was suggested by unusual serological reactivity in the V3 loop peptide enzyme immunoassay, 37 using V3 loop peptides of subtypes E and B9 from a previous study. 7 Approximately one-third of the serum specimens from central and upper Myanmar were untypable (nonreactive or dual reactive) by this method. 7 In the present study we have, for the first time, genetically identified HIV-1 subtype C (gag C/env C) in 20% (5 of 25) of the specimens from Mandalay in which both gag (p17) and env (C2/V3) subtypes were determined (Table 2, and Fig. 1). Moreover, HIV- 1 subtype C sequences in either the gag (p17) or env (C2/V3) region were identified in more than two-thirds (13 of 34, 38.2%) of the specimens from Mandalay, whereas HIV-1 subtype B9 and CFR01_AE sequences occurred in 32.4% (13 of 34) and 47.1% (16 of 34), respectively (Table 1). HIV-1 subtype C is thus indeed one of most prevalent subtypes in central Myanmar. Interestingly, phylogenetic tree analyses revealed the discordance between the env (C2/V3) and gag (p17) subtypes in 6 of 25 (24.0%) specimens, including 3 gag B9/env C (midu2, midu15, and mcsw2), 1 gag C/env E (midu18), 1 gag C/env B9 (midu10), and 1 gag B9/env E (mcsw12) (Tables 1 and 2, and Fig. 1). Such discordance was detected frequently among IDUs (4 of 12, 33.3%) and CSWs (2 of 8, 25%) in Mandalay FIG. 3. Alignment of the deduced amino acid sequences of the env (C2/V3) region of HIV-1 subtype C sequences from central Myanmar (Mandalay) with those from surrounding countries. The deduced amino acid sequences of the env (C2/V3) regions were aligned with the HIV-1 subtype C consensus sequence (CON.C) 26 (top). A period (.) indicates identity with the subtype C consensus sequence. A dash ( ) indicates that the corresponding amino acid sequence is not available in the data sets from the databases in this alignment. The deduced amino acid sequence of the env (C2/V3) region is numbered every 10 residues from the first amino acid, which was determined by direct sequencing in the present study and is shown at the top of the alignment. Boxed amino acids at positions 37 (37Q) and 108 (108S) are possible amino acid sequence signatures specific to the HIV-1 subtype C sequences from southern China and central Myanmar. The name of the country and the region/city where each HIV-1 subtype C strain originated are indicated at the left of the alignment: MMR (MND), Myanmar (Mandalay, central Myanmar); CHN, China; GX, Guangxi Province; YU, Yunnan Province; IND, India; BG, Bombay-Goa; ND, New Delhi; VL, Vellore; BRA, Brazil; AFR, Africa; ET, Ethiopia; UG, Uganda; ZM, Zambia; ZA, South Africa; MW, Malawi. The single nucleotide changes (underlined) shown at the top are required for 37Q and 108S substitutions, respectively. The hept-amino acid motif of the V3 loop crowns of subtype C strains (consensus sequence, RIGPGQT) is boxed.

10 1840 MOTOMURA ET AL. TABLE 3. (Tables 1 and 2). Although we were not able to discern in every case, except for midu2, 10, and 18 (see below), whether the discordance in gag and env subtypes was caused by coinfections or recombinations of different subtypes, this may strongly imply the cocirculation of multiple subtypes among persons engaging in extremely high-risk behaviors, including IDUs and CSWs, in Mandalay. Indeed, the seroprevalence rates were 85% among IDUs and 39.8% among CSWs in Mandalay in These values are among the highest in Myanmar as well as in Southeast Asian countries. Such extremely high infection rates would explain the high incidence of coinfection and/or recombination among these particular high-risk populations in Mandalay. The analyses for intersubtype mosaicism detected the possible recombination breakpoints in at least three specimens (midu2, 10, and 18) from central Myanmar (Table 1 and Fig. 4). The former two specimens (midu2 and 10) showed the intersubtype recombination between HIV-1 subtypes C and B9, whereas midu18 appeared to have multiple breakpoints between subtypes C, B9, and E, in the region between the 39 part of vpr and the 59 part of the env gene (Fig. 4). Although the analyses were not done in contiguous segments, additional breakpoints between subtypes B9 and C are suggested in midu2 and midu15 (Table 1). The recombination breakpoints that were identified in the present study appeared to be unique in each case. Although the exact recombination breakpoints were not fully described, Shao et al. previously reported that two COMPARISON OF INTRASUBTYPE NUCLEOTIDE DIVERSITY IN env (C2/V3) REGION OF HIV-1 SUBTYPES B9, C, AND CRF01_AE CIRCULATING IN DIFFERENT RISK POPULATION S IN MANDALAY OF CENTRAL MYANMAR % Nucleotide diversity a Persons with HIV-1 subtype IDUs sexual exposure B % Not available (n 5 5; range, %) C % % (n 5 7; range, %) (n 5 3; range, %) CRF01_AE % % (n 5 4; range, %) (n 5 11; range, %) a Intrasubtype nucleotide diversity was calculated on the basis of the Kimura two-parameter data matrix. 18 Significant differences in intrasubtype nucleotide diversity are observed in the following pairs of data sets (Mann Whitney U Test): CRF01_AE in IDUs versus subtypes B9, (p ) and C (p ) in IDUs, respectively; CRF01_AE in IDUs versus CRF01_AE (p ) and subtype C (p ) among persons infected sexually, respectively. forms of intersubtype B9/C recombinants are circulating in China and that the prevalence of recombinants showed the gradient along the presumed drug trafficking pathway from Yunnan (4.5%) via central provinces (14.3 to 50%) to the northwestern province (100%). 38,39 It is thus tempting to speculate that there is an interrelationship between the epidemics in central Myanmar and China. Similar to Thailand, CRF01_AE was found more frequently among persons infected sexually (8 of 13, 61.5%) and was less frequently distributed among IDUs (2 of 12, 16.7%) (Tables 1 and 2). In contrast, subtype B9 was found more frequently among IDUs (3 of 12, 25%) and less frequently among persons with sexual exposure (1 of 13, 7.7%) (Tables 1 and 2). This weak but similar trend of segregation of subtypes in different risk groups in central Myanmar appears to suggest an interrelationship with the epidemic in Thailand. 3 Moreover, the apparent closeness of HIV-1 subtype C sequences found in central Myanmar with those distributed in southern China, in terms of phylogenetic relationship (Figs. 1 and 2) and the common amino acid signatures characterized by 37Q/108S substitutions (Figs. 2 and 3), suggested that HIV-1 subtype C circulating in central Myanmar were close in origin to those found in southern China. The nucleotide sequence determination of longer HIV-1 sequences is now in progress to further confirm these results. The higher intrasubtype diversity of HIV-1 subtypes B9 and C among IDUs (Table 3) could be due to the introduction of FIG. 4. Recombination breakpoint analyses of 1285-bp region encompassing vif through the 59 terminal of the env region for midu10, mcsw12, and midu18. Similarity plots (A) and bootstrap plots (B) depicting the relationship to reference sequences of HIV-1 subtype A through H and RL42, which belongs to the HIV-1 subtype B9 cluster within subtype B. 36 At the top of each panel, the strategy of nucleotide sequence determination in relation to reading frames of corresponding HIV-1 genes is illustrated. The percent similarity (A) and bootstrap values (B) were plotted for a window of 200 bp moving in increments of 10 bp along the alignment. Analyses were implemented by Simplot 2.5 software. 23 The regions for subtype B9, C, or E are identified by high bootstrap values (.90%). Point of cross-over of the two curves indicates recombination breakpoint. The black triangles represent the locations where the sequences were broken to construct phylogenetic trees. The phylogenetic trees were computed by the neighbor-joining method with parsimony bootstrap values at the nodes containing the specimen of interest. The deduced subtype structure is illustrated above the similarity plot. Region in white could not be assigned to any known subtype.

11 NEW FORMS OF HIV-1 RECOMBINANTS IN CENTRAL MYANMAR 1841

12 1842 multiple strains of respective subtypes from surrounding areas (as suggested also in Figs. 2 and 3). Alternatively, it might reflect that HIV-1 subtypes B9 and C have resided longer than CRF01_AE among IDUs in Mandalay. Further sampling and characterization of HIVs are needed to confirm or test these interpretations. Mandalay functions as a crucial intersection connecting the cities throughout this country as well as the intercountry traffic to and from Thailand, southern China (Yunnan Province), and eastern India (Manipur and Nagaland). Reflecting this geographical location of Mandalay, the data in the present study elucidated the unique distribution and occurrence of HIV-1 subtypes and their intersubtype recombinants. In summary, the cocirculations of multiple subtypes among persons engaging in extremely high-risk behaviors in Mandalay provide the unique opportunity of coinfection of different HIV-1 subtypes and of creating the various forms of HIV-1 intersubtype recombinants. Obviously, more structural information on the HIV-1 strains prevalent in these geographically intertangled regions is critically important to elucidate the dynamics of HIV spread and to formulate vaccine strategies in this particular area in Asia. ACKNOWLEDGMENTS The first two authors contributed equally to this work. We thank Edward Zan, Khin Ohmar San, and the staff of the Department of Health, Union of Myanmar for their logistical help, and Shudo Yamazaki, Kazunori Oishi, Tsuyoshi Nagatake, Bruce Weniger, Chantapong Wasi, Yasushi Sawazaki, Yoshiki Sakurai, Tamami Umeda, Takashi Kitamura, Akira Oya, and the late Souroku Yamagata for their generous support for our study. 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