ABSTRACT
Transmission clusters of HIV-1 subtype B uniquely associated with the epidemic among men who have sex with men (MSM) in East Asia have recently been identified. Using the Los Alamos HIV sequence database and the UK HIV drug resistance database, we explored possible links between HIV MSM epidemics in East Asia and the rest of the world by using phylogenetic and molecular clock analyses. We found that JP.MSM.B-1, a subtype B MSM variant that accounts for approximately one-third of the infections among Japanese MSM, was detected worldwide, in the United Kingdom (n = 13), mainland China (n = 3), the United States, Germany, Canada, and Taiwan (n = 1 each). Interestingly, 10 United Kingdom samples plus two from Germany and the United States formed a distinct monophyletic subgroup within JP.MSM.B-1. The estimated divergence times of JP.MSM.B-1 and the latter subgroup were ∼1989 and ∼1999, respectively. These dates suggest that JP.MSM.B-1 was circulating for many years in Japan among MSM before disseminating to other countries, most likely through global MSM networks. A significant number of other Asian MSM HIV lineages were also detected in the UK HIV drug resistance database. Our study provides insight into the regional and global dispersal of Asian MSM HIV lineages. Further study of these strains is warranted to elucidate viral migration and the interrelationship of HIV epidemics on a global scale.
IMPORTANCE We previously identified several transmission clusters of HIV-1 subtype B uniquely associated with the epidemic among men who have sex with men (MSM) in East Asia. Using the Los Alamos HIV sequence database and the UK HIV drug resistance database, we explored the possible interplay of HIV MSM epidemics in the different geographic regions and found previously unrecognized interrelationships among the HIV-1 epidemics in East Asia, the United Kingdom, and the rest of the world. Our study provides insight into the regional and global dispersal of Asian MSM HIV lineages and highlights the importance of strengthening HIV monitoring efforts and the need for implementing effective control measures to reduce HIV transmission on a global scale.
INTRODUCTION
HIV-1 transmission and infection among men who have sex with men (MSM) continues worldwide (1, 2). While the HIV-1 incidence in the general adult population is decreasing in most countries, the MSM risk group continues to be disproportionately affected by HIV-1 infection. MSM were approximately 19 times more likely to become infected than average individuals in the global general population in 2000 to 2006 (3). East Asia is no exception to this trend, and HIV-1 epidemics among MSM in China have expanded rapidly (4). According to the Chinese Ministry of Health, the proportion of MSM among newly identified HIV cases in China has risen from 0.3% in 1985 to 2005 to 8.6% in 2009 and to 17.5% as of October 2012 (4, 5). Further, a large cross-sectional study of MSM in 61 Chinese cities in 2008 found that the average HIV prevalence in this group reached 4.9% (6). The highest HIV prevalences among MSM were found in cities in central-western and southwestern China: 8.5% in Chongqing, 9.1% in Chengdu (reviewed in reference 1), and 10.8% in Kunming (7, 8).
While the HIV incidence remains very low in Japan compared with that in many other countries, Japan is also experiencing a similar but smaller increase in HIV-1 infections among MSM (1). By the end of 2012, a cumulative total of 20,036 HIV/AIDS cases had been reported in Japan, of which approximately one-third were associated with MSM (Ministry of Health, Japan; http://api-net.jfap.or.jp/status/). The number of newly reported HIV cases among MSM has more than doubled during the past decade, from 305 in 2002 to 655 in 2012 (9). Despite extensive HIV prevention and education programs targeted at MSM, many MSM in Japan appear to engage in high-risk sexual behaviors, predisposing them to HIV-1 infection and transmission (Ministry of Health, Japan; http://api-net.jfap.or.jp/status/).
In a recent survey of HIV-1 strains circulating among newly diagnosed infected patients in various risk groups in the Tokyo-Kanagawa metropolitan area of Japan in 2004 to 2011, it was found that Japanese MSM were infected predominantly with subtype B HIV-1 strains of U.S.-European origin (96.9%). Detailed phylogenetic analysis revealed that approximately one-third of these subtype B strains formed a single monophyletic cluster (designated JP.MSM.B-1) (10), followed by a number of minor subtype B variants, each accounting for less than 1 to 2% of MSM infections, termed JP.MSM.B-2 through JP.MSM.B-4 (10). While CRF01_AE infections are uncommon among Japanese MSM (1.9%), 30 to 50% of these cases were found to be caused by CRF01_AE variants (11, 12) uniquely associated with transmission among MSM in China (designated CN.MSM.01-1) (10). Virus movement between the two countries appears to be bidirectional, as we also observed that the largest Japanese MSM subtype B variant (JP.MSM.B-1) was detected among MSM in China (10). These findings suggest the presence of previously unknown interactions among the HIV epidemics in China, Japan, and the rest of the world.
Phylogenetic and phylogeographic analyses of HIV genome sequences have revealed the routes by which the virus has been disseminated in many different regions, including the Americas (13), Europe (14), and Africa (15, 16). However, despite the epidemiological importance of HIV-1 subtype B among MSM in Asia, including in Japan and other countries, the global profile of these transmission clusters and their epidemic history remains largely unknown.
In this study, we systematically searched for East Asian subtype B variants by using both the Los Alamos HIV sequence database (Los Alamos HIVDB) and the UK HIV drug resistance database (UKHIVRDB) in order to explore possible linkages between the epidemics in East Asia and those in the rest of the world. We found that HIV-1 subtype B variants associated with transmission among MSM in East Asia have dispersed globally, giving rise to a new chain of infection in the United Kingdom through a founder effect.
MATERIALS AND METHODS
HIV sequence databases.
HIV sequences representing subtype B protease and partial reverse transcriptase coding regions (pro-RT region, ∼1.1 kb long) were obtained from (i) the Los Alamos HIVDB (http://www.hiv.lanl.gov; accessed in June 2012), (ii) the UKHIVRDB (http://www.hivrdb.org; accessed on 20 March 2013, containing sequences up to the end of 2010) of strains from treatment-naive patients (17), and (iii) our in-house HIV database comprising sequences of strains from study participants residing in the Tokyo-Kanagawa metropolitan area of Japan (designated the JP-TK HIVDB) (10). Ethics approval for the use of anonymized data in the UKHIVRDB was given by the London Multicenter Research Ethics Committee. This study project was approved by the Ethics Committee of the National Institute of Infectious Diseases in Japan.
Sample collation and phylogenetic analysis.
We searched the above three HIV databases for the global occurrence of subtype B variants associated with transmission among MSM in East Asia. Figure 1 shows a flow chart of the phylogenetically informed searching procedure undertaken in this study. To identify HIV sequences of interest, we first performed a BLAST (Basic Local Alignment Search Tool) search (18) by using the ∼1.1-kb pro-RT nucleotide sequences of key subtype B MSM variants in Japan and China as queries (this genome region typically corresponds to nucleotide positions 2253 to 3392 of the HXB2 reference strain). The query sequences used were as follows: 06JP.Y306 (accession number AB735881) for the JP.MSM.B-1 lineage, 09JP.Y449 (AB735865) for the JP.MSM.B-2 lineage, 06JP.GM2086 (AB864057) for the JP.MSM.B-3 lineage, 06JP.Y287 (AB864025) for the JP.MSM.B-4 lineage (10), 07CN.BJ07635 (JF906694) for the CN.MSM.B-1 lineage, and 07CN.BJ061217 (JF906568) for the B′/CN.MSM.B-2 lineage (19). The choice of the specific query sequence used to represent each lineage did not significantly influence the search results. The 100 nucleotide sequences with the highest BLAST scores (E values) for each query sequence were retained. When more than one nucleotide sequence appeared to have been reported from the same infected individual, then only one sequence was retained. The top 100 sequences typically showed >94 to 95% sequence homology to the query sequences (see below). To confirm these possible matches to the East Asian HIV lineages of interest, we then verified the phylogenetic relationships of the database sequences. Trees were initially estimated by the neighbor-joining method (20) with the Tamura-Nei 93 (TN93) nucleotide substitution model (21), followed by more rigorous maximum-likelihood estimation based on the Hasegawa-Kishino-Yano nucleotide substitution model (22) and a gamma distribution model for rate variation among sites. Three subtype C strains (95IN21068, 86.ETH2220, and 92.BR025-d) were used as the outgroup. These analyses were undertaken with MEGA 5.05 (23). The geographical origins of the subtype B sequences (n = 272) used in this study were Argentina (AR) (n = 1), Australia (AU) (n = 1), Canada (CA) (n = 1), China (CN) (n = 93), France (FR) (n = 1), Gabon (GA) (n = 1), Germany (DE) (n = 1), Haiti (HT) (n = 5), Japan (JP) (n = 93), Myanmar (MM) (n = 1), Netherlands (NL) (n = 1), Spain (ES) (n = 1), Taiwan (TW) (n = 3), Thailand (TH) (n = 15), Trinidad and Tobago (TT) (n = 5), the United Kingdom (UK) (n = 38), and the United States (US) (n = 11). We also estimated phylogenies after deleting sites linked to drug resistance. The tree topology was very similar to that estimated when these sites were included.
FIG 1.
Flow chart of the procedure used to identify region- and risk factor-specific HIV-1 subtype B variants. To identify HIV sequences of interest, a BLAST search (18) against the Los Alamos HIVDB and the UKHIVRDB was undertaken, by using as queries the ∼1.1-kb pro-RT nucleotide sequences of key subtype B MSM variants in Japan and China (listed in the leftmost box; see text). The 100 nucleotide sequences with the highest BLAST score to each query sequence were retained. We then verified the phylogenetic relationships of the database sequences to confirm possible matches to the East Asian HIV lineages of interest.
Molecular clock analysis.
To determine the time scale of the global dispersal of subtype B, especially in the context of HIV epidemics in East Asia, we undertook a molecular clock phylogenetic analysis by the Bayesian Markov Chain Monte Carlo (MCMC) approach implemented in the BEAST v1.7.4 package (24). To model sequence evolution, we used the generalized time-reversible (GTR) nucleotide substitution model with a gamma distribution model to account for rate variation among sites. We used a relaxed uncorrelated lognormal molecular clock to model rate variation among lineages (25) and a nonparametric Bayesian skyride coalescent model as a prior distribution for phylogeny shape and size (26). The MCMC analyses were run for 50 million generations with sampling every 5,000th state. Multiple chains were executed to ensure that adequate mixing and convergence had been achieved, as assessed with the Tracer software (http://tree.bio.ed.ac.uk/software/tracer/). The posterior distribution of phylogenies was summarized in a maximum clade credibility (MCC) tree, which was calculated with TreeAnnotator (http://tree.bio.ed.ac.uk/software/annotator/). Lineages in the MCC tree were colored according to their countries of origin with FigTree (http://tree.bio.ed.ac.uk/software/figtree/). While there are a number of nucleotide sites in our sequences that were represented by ambiguity codes, this uncertainty is marginalized during maximum-likelihood/Bayesian inference in a statistically correct manner.
Identification of clusters.
The initial identification of transmission clusters was based on the neighbor-joining phylogeny. All clusters of three or more patients with a bootstrap value of >70% were selected. Subsequently, the clusters were verified by Bayesian phylogenetic inference. Only clusters with a posterior probability of 1.0 were considered to be transmission clusters.
RESULTS
Global occurrence of HIV-1 variants associated with MSM in East Asia.
To explore possible epidemiological linkages between the HIV-1 MSM subtype B epidemics in East Asia and those in the rest of the world, we searched three sequence databases for HIV-1 strains that are related to the HIV lineages uniquely associated with transmission among MSM in Japan and China (see Materials and Methods). Our phylogenetically informed search (Fig. 1) identified a total of 48 HIV-1 sequences (“hits”) that were closely related to the transmission clusters identified in East Asia (Table 1). As expected, we found a number of sequences in the Los Alamos HIVDB that belonged to the CN.MSM.B-1, CN.MSM.B-2, JP.MSM.B-1, and JP.MSM.B-2 lineages because many sequences from Chinese and Japanese MSM have already been deposited in that database. About 30% of the Japanese subtype B MSM variants in the JP-TK HIVDB belong to the JP.MSM.B-1 lineage, and <1 to 2% belong to each of the three minor variants (JP.MSM.B-2, JP.MSM.B-3, and JP.MSM.B-4) (10).
TABLE 1.
Summary of the presence of regional HIV-1 subtype B variants from East Asia (Japan and China) in the databases searcheda
| Transmission cluster, country of origin, risk group(s), and database searched | No. of hits (country of origin) |
n | Total | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1997-2003 | 2004 | 2005 | 2006 | 2007 | 2008 | 2009 | 2010 | 2011 | |||
| JP.MSM.B-1, Japan, MSM | |||||||||||
| Los Alamos HIVDB | 1 (DE) | 1 (US) | 2 (CN) | 1 (CN) | 1 (CA) | 1 (TW) | 7 | 20 | |||
| UKHIVRDB | 1 | 4 | 2 | 6 | 13 | ||||||
| JP-TK HIVDB | 0 | ||||||||||
| JP.MSM.B-2, Japan, MSM | |||||||||||
| Los Alamos HIVDB | 0 | 3 | |||||||||
| UKHIVRDB | 1 | 1 | |||||||||
| JP-TK HIVDB | 2 (CN) | 2 | |||||||||
| JP.MSM.B-3, Japan, MSM | |||||||||||
| Los Alamos HIVDB | 0 | 2 | |||||||||
| UKHIVRDB | 1 | 1 | |||||||||
| JP-TK HIVDB | 1 (UK) | 1 | |||||||||
| JP.MSM.B-4, Japan, MSM | |||||||||||
| Los Alamos HIVDB | 1 (TW) | 1 | 2 | ||||||||
| UKHIVRDB | 1 | 1 | |||||||||
| JP-TK HIVDB | 0 | ||||||||||
| CN.MSM.B-1, China, MSM | |||||||||||
| Los Alamos HIVDB | NAb | 1 | |||||||||
| UKHIVRDB | 1 | 1 | |||||||||
| JP-TK HIVDB | 0 | ||||||||||
| B′/CN.MSM.B-2, China, IDU/FPD/heterosexual/MSM | |||||||||||
| Los Alamos HIVDB | NA | 20 | |||||||||
| UKHIVRDB | 1 | 3 | 6 | 4 | 4 | 1 | 1 | 20 | |||
| JP-TK HIVDB | 0 | ||||||||||
| Total | 0 | 1 | 5 | 7 | 11 | 5 | 10 | 7 | 2 | 48 | |
| No. of HIV-1 subtype B sequences in UKHIVRDB | 11,299 | 3,168 | 4,033 | 4,522 | 4,725 | 5,020 | 4,569 | 4,470 | 1,644 | 43,450 | |
| Total no. of HIV-1 sequences in UKHIVRDB | 17,609 | 5,592 | 7,911 | 9,453 | 10,019 | 11,340 | 10,925 | 9,985 | 4,023 | 86,857 | |
| % of HIV-1 subtype B sequences in UKHIVRDB | 64.2 | 56.7 | 51.0 | 47.8 | 47.2 | 44.3 | 41.8 | 44.8 | 40.9 | 50.0 | |
For the Los Alamos HIVDB, we tabulated only the number of hits found outside the country of origin. For the JP-TK HIVDB, we tabulated only the number of hits among foreigners resident in Japan. For the Los Alamos HIVDB and the JP-TK HIVDB, the country of origin is indicated in parentheses as follows: Brazil, BR; Canada, CA; China, CN; France, FR; Germany, DE; Japan, JP; Taiwan, TW; United Kingdom, UK; United States, US.
NA, not applicable.
As shown in Table 1, in the Los Alamos HIVDB, we identified a total of seven sequences sampled outside Japan that matched the JP.MSM.B-1 lineage. These comprised four strains from Asia (06CN.LN107, 06CN.LN126, and 07CN.LN159 from MSM in Liaoning Province, China [27], and 09TW.36497 from Taiwan [28]), two strains from North America (05US.ARC_A221 from the United States [29] and 08CA.PHAC09_054 from Canada), and one strain from Europe (04DE.fd5gjGvO1zxQvv0 from Germany [30]).
Interestingly, a total of 13 JP.MSM.B-1-related variants were identified among the sequences present in the UKHIVRDB that were sampled from 2006 to 2010 (Table 1). This represents approximately 0.04% of the subtype B strains (13/∼32,000) and 0.016% of all of the HIV-1 strains registered in the UKHIVRB (13/∼80,000) from 1997 to 2011 (Table 1). Intriguingly, 10 out of the 13 JP.MSM.B-1 variants identified in the United Kingdom (Table 2) were part of a distinct monophyletic cluster within JP.MSM.B-1 with good statistical support (bootstrap value of 70%) (Fig. 2). Further, this cluster contained a 2004 sample from Germany (04DE.fd5gjGvO1zxQvv0) (30) and a 2005 U.S. sample (05US.ARC_A221) (29) (Fig. 2). We thus designated this the Global (UK)-JP.MSM.B-1 cluster (Fig. 2). We also noted that one of three United Kingdom sequences placed outside this cluster (a 2010 sample, 10UK.a10) was closely related to two 2008 Japanese MSM samples (08JP.Y440 and 06JP.Y308) with a bootstrap value of 95% (Fig. 2; Table 2). In addition, three infections belonging to the JP.MSM.B-1 lineage were identified among MSM in Liaoning Province in northeastern China (06CN.LN107, 06CN.LN126, and 07CN.LN159). These grouped together within the JP.MSM.B-1 lineages with a bootstrap value of 67% and are designated the CN-JP.MSM.B-1 cluster (Fig. 2) (10). These three Chinese MSM had no history of travel outside China (X. Han, personal communication).
TABLE 2.
Summary of epidemiologic and demographic information about subjects identified in this study who harbored HIV-1 subtype B variants associated with transmission among MSM in Japan
| Japanese MSM subtype B variant and strain name or sample ID | Country of origin or birtha | Ethnicity or nationality | Sampling yr | Age (yr) | Sex | Risk factor or origind | Source | Accession no. | Transmission subcluster | Reference or remark |
|---|---|---|---|---|---|---|---|---|---|---|
| JP.MSM.B-1 | ||||||||||
| 04DE.fd5gjGvO1zxQvv0 | Germany | NAb | 2004 | NA | NA | NA | Los Alamos HIVDB | GQ400617 | Global (UK)-JP.MSM.B-1 | 30 |
| 05US.ARC_A221 | US | NA | 2005 | NA | NA | BD | Los Alamos HIVDB | JN697357 | Global (UK)-JP.MSM.B-1 | 29 |
| 06CN.LN107 | China (Liaoning) | Chinese | 2006 | 28 | Mc | MSM | Los Alamos HIVDB | FJ531410 | CN-JP.MSM.B-1 | 27 |
| 06CN.LN126 | China (Liaoning) | Chinese | 2006 | 17 | M | MSM | Los Alamos HIVDB | FJ531423 | CN-JP.MSM.B-1 | 27 |
| 07CN.LN159 | China (Liaoning) | Chinese | 2007 | 22 | M | MSM | Los Alamos HIVDB | FJ531457 | CN-JP.MSM.B-1 | 27 |
| 08CA.PHAC09_054 | Canada | NA | 2008 | NA | NA | NA | Los Alamos HIVDB | JQ675213 | - | Data submission only |
| 09TW.36497 | Taiwan | Taiwanese | 2009 | NA | NA | NA | Los Alamos HIVDB | HQ657934 | - | 28 |
| 06UK.a01 | UK | Caucasian | 2006 | NA | M | MSM/bi | UKHIVRDB | AB906905 | Global (UK)-JP.MSM.B-1 | |
| 07UK.a02 | UK | Caucasian | 2007 | NA | M | MSM/bi | UKHIVRDB | AB906906 | Global (UK)-JP.MSM.B-1 | |
| 07UK.a03 | UK | Caucasian | 2007 | NA | M | MSM/bi | UKHIVRDB | AB906907 | Global (UK)-JP.MSM.B-1 | |
| 07UK.a04 | US | Caucasian | 2007 | NA | NA | NA | UKHIVRDB | AB906908 | Global (UK)-JP.MSM.B-1 | |
| 07UK.a05 | SE Asia | Other Asian | 2007 | NA | M | MSM/bi | UKHIVRDB | AB906909 | - | |
| 09UK.a06 | SE Asia | Other Asian | 2009 | NA | M | NA | UKHIVRDB | AB906910 | - | |
| 09UK.a07 | US | Caucasian | 2009 | NA | M | MSM/bi | UKHIVRDB | AB906911 | Global (UK)-JP.MSM.B-1 | |
| 10UK.a08 | E Europe | Caucasian | 2010 | NA | M | MSM/bi | UKHIVRDB | AB906912 | Global (UK)-JP.MSM.B-1 | |
| 10UK.a09 | UK | Caucasian | 2010 | NA | M | MSM/bi | UKHIVRDB | AB906913 | Global (UK)-JP.MSM.B-1 | |
| 10UK.a10 | NA | NA | 2010 | NA | NA | NA | UKHIVRDB | AB906914 | - | Closely related to 2008 Japanese MSM samples 08JP.Y440 and 06JP.Y308 |
| 10UK.a11 | NA | NA | 2010 | NA | NA | NA | UKHIVRDB | AB906915 | Global (UK)-JP.MSM.B-1 | |
| 10UK.a12 | NA | NA | 2010 | NA | NA | NA | UKHIVRDB | AB906916 | Global (UK)-JP.MSM.B-1 | |
| 10UK.a13 | NA | NA | 2010 | NA | NA | NA | UKHIVRDB | AB906917 | Global (UK)-JP.MSM.B-1 | |
| JP.MSM.B-2 | ||||||||||
| 09JP.CN.GM3061 | China | Chinese | 2009 | 27 | M | MSM | JP-TK HIVDB | AB735867 | Chinese MSM resident in Japan | |
| 09JP.CN.Y519 | China | Chinese | 2009 | 26 | M | MSM | JP-TK HIVDB | AB735896 | Chinese MSM resident in Japan for last 20 years | |
| 05UK.b01 | NA | Other Asian | 2005 | NA | M | MSM/bi | UKHIVRDB | AB906918 | ||
| JP.MSM.B-3 | ||||||||||
| 05JP.UK.GM1736 | UK | Caucasian | 2005 | 39 | M | MSM | JP-TK HIVDB | AB863991 | British MSM resident in Japan, likely infected by Japanese partner | |
| 11UK.c01 | NA | Not known | 2011 | NA | NA | NA | UKHIVRDB | AB906919 | Closely related phylogenetically to 05JP.UK.GM1736 | |
| JP.MSM.B-4 | ||||||||||
| 09TW.36025 | Taiwan | NA | 2009 | NA | NA | NA | Los Alamos HIVDB | HQ657878 | ||
| 06UK.d01 | UK | Caucasian | 2006 | NA | M | MSM/bi | UKHIVRDB | AB906920 |
US, United States; SE, Southeast; UK, United Kingdom; E, Eastern.
NA, not available.
M, male.
BD, blood donation; bi, bisexuality.
FIG 2.
Phylogenetic analysis of HIV-1 subtype B variants identified outside East Asia. Neighbor-joining phylogenies were estimated from nucleotide sequences of the 1.1-kb pro-RT region (HXB2, nucleotides 2253 to 3392) of HIV-1 subtype B strains that were identified by our screening procedure (see Fig. 1 and Materials and Methods for details). HIV-1 subtype C sequences were used as the outgroup. Bootstrap support values that are relevant to this study are shown at the corresponding nodes. Ten out of 13 JP.MSM.B-1 variants found in the UKHIVRDB, together with one sample each from Germany and the United States, formed a distinct monophyletic subcluster [designated Global (UK)-JP.MSM.B-1] within JP.MSM.B-1. In addition, seven JP.MSM.B-1 variants were identified outside Japan in the Los Alamos HIVDB. The clusters of interest in our study are highlighted. The country of origin of each sequence is indicated by the ISO 3166 two-letter code (http://www.iso.org/iso/country_names_and_code_elements) as follows: CA, Canada; CN, China; DE, Germany; JP, Japan; TH, Thailand; TW, Taiwan; UK, United Kingdom; US, United States. Region abbreviations: E Euro, Eastern Europe; SE Asia, Southeast Asia; S Asia, South Asia. The risk factor for each United Kingdom sample (if available; see also Tables 2 and 3) (denoted in the right lower box) is indicated at the tip of each branch. Asian subtype B variants and their subclusters are indicated at the right of the tree. The countries of birth for United Kingdom samples (if available) (see also Tables 2 and 3) and the names of the strains of interest in this study are shown at the tips of the respective branches.
Our survey of three HIV sequence databases revealed a small number of hits to three minor Japanese subtype B MSM lineages (JP.MSM.B-2 through JP.MSM.B-4) that were sporadically identified outside Japan and/or among foreign MSM residing in Japan (Table 1). The JP.MSM.B-2 lineage contained one 2005 United Kingdom sample (05UK.b01) and two from Chinese MSM residing in Japan (09JP.CN.GM3061 [AB735867] and 09JP.CN.Y519 [AB735896]) (10). These two Chinese MSM sequences grouped with sequences from Japanese MSM with a bootstrap score of 99% (10). In addition, the JP.MSM.B-3 lineage contained one 2011 United Kingdom sample (11UK.c01) and one sample from a United Kingdom MSM residing in Japan (05JP.UK.GM1736). In a neighbor-joining tree, these two cases were genetically similar and grouped with a bootstrap score of 89% (Fig. 2). The JP.MSM.B-4 lineage contained one sample from the United Kingdom (06UK.d01) and one from Taiwan (09TW.36025) (Fig. 2).
In contrast, the Chinese MSM subtype B lineage (CN.MSM.B-1) was rarely detected outside China; only one CN.MSM.B-1 sequence was detected in the UKHIVRDB (2005 sample 05UK.e01) (Tables 1 and 3; Fig. 2). However, a total of 20 sequences belonging to the B′/CN.MSM.B-2 lineage were identified in the UKHIVRDB from 2004 to 2010 (Tables 1 and 3; Fig. 2). This represents 0.025% (20/∼80,000) of the HIV-1 strains in the UKHIVRDB and 0.06% (20/∼32,000) of the subtype B strains in the UKHIVRDB (Table 2). Among these 20 sequences, 5 formed a distinct phylogenetic cluster (designated B′uk) within B′/CN.MSM.B-2 with a bootstrap score of 90% (Fig. 2).
TABLE 3.
Summary of epidemiologic and demographic information about subjects identified in UKHIVRDB who harbored HIV-1 subtype B variants uniquely associated with the epidemic in Chinaa
| Chinese HIV-1 subtype B variant and strain code | Country or region of birtha | Sampling yr | Sexb | Risk factorc | Source | Accession no. | Transmission subclusterd |
|---|---|---|---|---|---|---|---|
| CN.MSM.B-1, 05UK.e01 | UK | 2005 | M | MSM/bi | UKHIVRDB | AB906921 | |
| B′/CN.MSM.B-2 | |||||||
| 05UK.f01 | UK | 2005 | F | Hetero | UKHIVRDB | AB906922 | |
| 06UK.f02 | SE Asia | 2006 | F | Hetero | UKHIVRDB | AB906923 | |
| 06UK.f03 | SE Asia | 2006 | F | Hetero | UKHIVRDB | AB906924 | |
| 06UK.f04 | UK | 2006 | F | Hetero | UKHIVRDB | AB906925 | |
| 07UK.f05 | UK | 2007 | M | MSM/bi | UKHIVRDB | AB906926 | |
| 07UK.f06 | NAe | 2007 | F | Hetero | UKHIVRDB | AB906927 | |
| 07UK.f07 | E Europe | 2007 | M | MSM/bi | UKHIVRDB | AB906928 | B′uk |
| 07UK.f08 | E Europe | 2007 | M | MSM/bi | UKHIVRDB | AB906929 | B′uk |
| 07UK.f09 | SE Asia | 2007 | F | Hetero | UKHIVRDB | AB906930 | |
| 07UK.f10 | NA | 2007 | NA | NA | UKHIVRDB | AB906931 | B′uk |
| 08UK.f11 | S Asia | 2008 | M | NA | UKHIVRDB | AB906932 | B′uk |
| 08UK.f12 | SE Asia | 2008 | M | Hetero | UKHIVRDB | AB906933 | |
| 08UK.f13 | NA | 2008 | M | Hetero | UKHIVRDB | AB906934 | |
| 08UK.f14 | NA | 2008 | NA | NA | UKHIVRDB | AB906935 | |
| 09UK.f15 | UK | 2009 | M | MSM/bi | UKHIVRDB | AB906936 | |
| 09UK.f16 | NA | 2009 | F | Hetero | UKHIVRDB | AB906937 | |
| 09UK.f17 | NA | 2009 | NA | NA | UKHIVRDB | AB906938 | |
| 09UK.f18 | NA | 2009 | NA | NA | UKHIVRDB | AB906939 | |
| 10UK.f19 | NA | 2010 | NA | NA | UKHIVRDB | AB906940 | |
| 11UK.f20 | NA | 2011 | NA | NA | UKHIVRDB | AB906941 | B′uk |
UK, United Kingdom; SE Asia, Southeast Asia; S Asia, southern Asia (India, Bangladesh, Pakistan); E Europe, Eastern Europe).
M, male; F, female.
Hetero, heterosexual; bi, bisexual.
B′uk, subtype B′ transmission subcluster identified in the United Kingdom and most likely associated with MSM/bisexual population in the United Kingdom (see the text) (Fig. 2).
NA, not available.
Time scale of the global dispersal of Asian MSM subtype B lineages.
To explore when subtype B variants associated with transmission among MSM in East Asia emerged and spread in the United Kingdom and globally, we estimated divergence times and spatial dispersal by a Bayesian relaxed molecular clock analysis (25, 31). The analyses were performed with BEAST (24) on the basis of a nucleotide sequence alignment of 1.1-kb pol (pro-RT) regions of subtype B strains; this alignment was identical to that used for the neighbor-joining phylogeny presented in Fig. 2. The estimated evolutionary rate of the pro-RT region analyzed was 2.20 (95% highest probability density [HPD], 1.88 to 2.51) ×10−3 substitutions/site/year. This rate is similar to those previously estimated for subtype B (10, 32–34).
As shown in Fig. 3, the estimated time of the most recent common ancestor (tMRCA) of lineage JP.MSM.B-1 was 1989.1 (95% HPD, 1986.5 to 1992.5) and the tMRCAs of lineages CN.MSM.B-1 and B′/CN.MSM.B-2 were 1985.6 (1981.5 to 1989.8) and 1985.3 (1981.8 to 1988.8), respectively. The estimated tMRCAs for the Global (UK)-JP.MSM.B-1 and CN-JP.MSM.B-1 clusters were 1998.9 (1995.6 to 2001.7) and 2000.0 (1997.5 to 2002.5), respectively (Table 4).
FIG 3.
Bayesian molecular clock reconstruction of HIV-1 subtype B evolutionary history in East Asia and the rest of the world. Shown is an MCC tree of the subtype B 1.1-kb pro-RT sequences estimated by a Bayesian MCMC approach (see Materials and Methods for details). The branch lengths in the MCC trees represent time, and the corresponding time scale is shown at the bottom of the tree. The 95% HPD intervals of the tMRCA estimates for phylogenetic clusters of interest are shown. Nodes marked with asterisks have strong statistical support (posterior probability, >0.95). The geographic origins of the sequences are represented by colors (see inset panel) or two-letter country codes (as described in the legend to Fig. 2).
TABLE 4.
Summary of estimated tMRCAs of HIV-1 subtype B variants associated with transmission in East Asia and the related subclustersa
| HIV-1 subtype B variant | Subcluster | tMRCA (95% HPD) | Remark(s) (reference[s]) |
|---|---|---|---|
| JP.MSM.B-1 | 1989.1 (1986.5–1992.5) | Major subtype B variant associated with transmission among MSM in Japan (10) | |
| Global (UK)-JP.MSM.B-1 | 1998.9 (1995.6–2001.7) | A subcluster within JP.MSM.B-1 that was identified in the United Kingdom and contained the strains from other regions of the world (this study) | |
| CN-JP.MSM.B-1 | 2000.0 (1997.5–2002.5) | JP.MSM.B-1 variants identified among MSM in China (10) | |
| JP.MSM.B-2 | 1988.3 (1982.7–1993.3) | One of the minor subtype B transmission clusters among MSM in Japan (10) | |
| cn-JP.MSM.B-2 | 1997.9 (1994.2–2001.6) | JP.MSM.B-2 variants found among Chinese MSM resident in Japan (10) | |
| JP.MSM.B-3 | 1987.9 (1983.7–1991.8) | One of the minor subtype B transmission clusters among MSM in Japan (10) | |
| JP.MSM.B-4 | 1989.4 (1985.1–1996.6) | One of the minor subtype B transmission clusters among MSM in Japan (10) | |
| CN.MSM.B-1 | 1985.6 (1981.5–1989.8) | Subtype B transmission cluster identified among MSM in China (10) | |
| B′/CN.MSM.B-2 | 1985.3 (1981.8–1988.8) | Subtype B′ (Thailand variant of subtype B) (35, 36); a fraction of the subtype B strains identified among MSM in China belong to the subtype B′ lineage (10) | |
| B′fpd | 1991.5 (1988.7–1994.9) | Subtype B′ subcluster responsible for the epidemic among FPDs in China (33) | |
| B′uk | 2002.6 (1999.5–2005.3) | Subtype B′ subcluster identified in the United Kingdom (mostly among MSM) (this study) |
Based on BEAST analysis under a relaxed uncorrelated lognormal molecular clock with the GTR nucleotide substitution model, a gamma-distribution model, and a nonparametric Bayesian skyride coalescent model (see Materials and Methods). Estimated substitution rate, 2.20 (95% HPD, 1.88 to 2.51) ×10−3/site/year. Effective sample size (ESS) values are >200.
In the molecular clock phylogeny, the B′uk cluster was placed among subtype B′ strains isolated from Thailand and the nearby Yunnan Province of China (Fig. 3). Other Chinese subtype B′ strains formed a separate cluster (designated B′fpd) that comprised a number of subtype B′ strains from former plasma donors (FPDs). The estimated tMRCA of the B′fpd and B′uk clusters were 1991.5 (1988.7 to 1994.9) and 2002.6 (1999.5 to 2005.3), respectively (Fig. 3; Table 4). The estimated tMRCAs of the JP.MSM.B-2, JP.MSM.B-3, and JP.MSM.B-4 lineages were 1988.3 (1982.7 to 1993.3), 1987.9 (1983.7 to 1991.8), and 1989.4 (1985.1 to 1996.6), respectively. (Fig. 3; Table 4). These estimated tMRCAs are compatible with our previous estimates (10). At the root of the molecular clock phylogeny were subtype B strains from Haiti and from Trinidad and Tobago and some U.S. strains. This is in agreement with the hypothesis that the global dispersal of pandemic subtype B took place via the United States from the Caribbean region (35, 36). All of the subtype B clusters identified in this study were statistically well supported with posterior probability support values of >0.95 (Fig. 3).
DISCUSSION
In this study, we explored the possible interplay between HIV-1 epidemics in East Asia and those in the rest of the world, with a particular focus on the United Kingdom. We carried out an extensive survey of the Los Alamos HIVDB, the UKHIVRDB, and the JP-TK HIVDB. We found that a major HIV-1 subtype B variant (JP.MSM.B-1), which is responsible for approximately one-third of the HIV infections among Japanese MSM, is also present globally. This lineage was detected in various locations in the western hemisphere and the Asia-Pacific region (n = 20): Asia (China and Taiwan) (n = 4), Europe (United Kingdom and Germany) (n = 14), and North America (the United States and Canada) (n = 2) (Table 1; Fig. 2). Intriguingly, we found that 10 of 13 JP.MSM.B-1 variants from the UKHIVRDB, together with two samples from the Los Alamos HIVDB (from Germany and the United States) formed a distinct monophyletic cluster within JP.MSM.B-1 [designated Global (UK)-JP.MSM.B-1] (Fig. 2 and 3; Table 2). Similarly, three Chinese MSM sequences were placed as a separate monophyletic cluster within JP.MSM.B-1 (designated CN-JP.MSM.B-1) (Fig. 2 and 3; Table 2). A molecular clock analysis performed with BEAST indicated that the tMRCA for the JP.MSM.B-1 lineage was ∼1989 and the tMRCAs for the Global (UK)-JP.MSM.B-1 and CN-JP.MSM.B-1 clusters within it were ∼1999 and ∼2000, respectively (Fig. 3; Table 4). All seven United Kingdom samples with known exposure category information are from MSM (Table 2). Thus, most of the JP.MSM.B-1 infections in the United Kingdom discovered in this study likely belong to the MSM risk group. Taken together, these results suggest that JP.MSM.B-1 circulated for several years in Japan before being introduced into nearby regions of Asia (China and Taiwan) and to the western hemisphere (the United States and European countries), most likely through global MSM networks (Fig. 3).
The Chinese subtype B MSM lineage (CN.MSM.B-1) was identified only once in the UKHIVRDB and never in the rest of the world, except in Japan (Table 1). In contrast, a total of 20 B′/CNMSM.B-2 variants were detected in the UKHIVRDB among samples collected in 2005 to 2010 (Table 1). HIV-1 subtype B′ (37, 38) was originally identified among injection drug users (IDUs) in Thailand and spread into neighboring Asian countries through drug use networks. This lineage also contributed the founding strain responsible for the HIV outbreaks in central China among FPDs in the early to mid-1990s (33, 39). The exposure categories of the 20 subtype B′ infections identified in the UKHIVRDB are as follows: heterosexual exposure, n = 9 (45%); MSM or bisexual exposure, n = 4 (20%); unknown exposure, n = 7 (35%). These individuals originated from the following regions: Southeast Asia, n = 4 (25%); the United Kingdom, n = 4 (25%); Eastern Europe, n = 2 (10%); Southwest Asia, n = 1 (5%); unknown, n = 9 (45%) (Table 3). We also noted that most (18 of 20) of the United Kingdom subtype B′ infections were identified before 2009 (Table 1). While the subtype B′ lineage is thought to have originated from the Bangkok IDU epidemic in the late 1980s, the B′ samples in this study do not have IDU as a risk factor (where risk information is known). The exposure categories of the samples that belonged to the B′uk subcluster (n = 5) were MSM or bisexual (n = 2) and unknown (n = 3), suggesting that the B′uk cluster is most likely related to the MSM transmission network in the United Kingdom (Table 3).
By combining the various sources of information at our disposal, we illustrate in Fig. 4 the hypothesized time scales and migration pathways of the global dissemination of the subtype B lineages associated with transmission among MSM in East Asia. Figure 4 is based on the results obtained in this and previous studies (10).
FIG 4.
Illustration of the global dispersal estimated timeline of HIV-1 subtype B variants associated with MSM transmission in East Asia. JP.MSM.B-1, a major subtype B lineage among Japanese MSM, has been disseminated globally. It formed distinct subclusters in the United Kingdom [with strains from Germany and the United States; designated Global (UK)-JP.MSM.B-1] and in northeastern China (designated CN-JP.MSM.B-1). Subtype B′ was originally identified among IDUs in Bangkok, Thailand, was disseminated widely among IDUs in neighboring countries and was responsible for the explosive HIV-1 outbreak among FPDs in central China in the early to mid-1990s. It was also identified among MSM in China (designated B′/CN.MSM.B-2) (10, 12). A relatively large number of subtype B′ infections were identified in the UKHIVRDB (see text; Table 1). Arrow thickness reflects qualitative trends in viral migration. Black arrows depict the origin of pandemic subtype B and its plausible global dispersal history (33, 36). The estimated tMRCAs (95% HPD) of each subtype B lineage and its ancestor are shown (33, 36). For country code definitions, see the legend to Fig. 2.
The strategy used here to search for virus variants was straightforward to implement and may be useful for the investigation and detection of other unknown epidemiological links among HIV epidemics in different locations and risk groups. While an initial survey with the BLAST tool is robust and convenient, its results do not always correlate with the phylogenetic relationships of any two sequences, especially when the tool is applied to rapidly evolving pathogens that are sampled sequentially over time and that exhibit evolutionary rate variation among lineages. As illustrated in Fig. 5, as intracluster genetic diversity increases over time, there is an increasing chance that genetic distance comparisons do not accurately reflect whether a given sequence belongs to a specific monophyletic cluster or not (Fig. 5). As a consequence, phylogenetic cluster definitions based on monophyly are robust when applied to temporally sampled (heterochronous) sequence alignments.
FIG 5.
Relationship between genetic distance and phylogeny. In this hypothetical tree, branch lengths are shown in arbitrary units (italicized numerals). The value at the tip of each branch is the genetic distance of each sequence from the representative reference (solid circle) strain for the hypothetical cluster. These distances are also ranked from smallest to largest, and the ranks are shown in brackets. The common ancestor of the cluster of interest is marked by the open circle, and × marks sequences that do not belong to that cluster. Crucially, sequences that do not belong to the cluster may be more genetically similar to the reference strain than sequences within the cluster.
Wertheim et al. recently proposed a technique for the rapid screening of a very large number of HIV-1 sequences to find “clusters” that are defined directly by sequence similarity, and they conclude that a threshold of 1% sequence similarity between sequences from different individuals was sensitive enough to identify many of the inferred transmission clusters reported in previous studies (40, 41). Such approaches are objective, computationally efficient, and robust to recombination, yet necessarily they do not use all of the information present in a phylogenetic representation. Nonetheless, phylogenetic and genetic distance-based cluster definitions are closely related; in our study, we found no identified BLAST hits to our target lineages among nucleotide sequences with <94 to 95% homology (Fig. 6). Further work is required to fully explore the advantages and disadvantages of the two approaches when applied to rapidly evolving heterochronous sequences.
FIG 6.
Distribution of sequence similarities (to the query sequence) of the sequences in the UKHIVRDB with the 100 highest BLAST scores. Each histogram (solid bars) shows the frequency distribution of the sequence among the 100 nucleotide sequences in the UKHIVRDB with the highest BLAST score of similarity to each query sequence. The query sequence used is shown in the upper left corner of each graph. The similarity values of sequences that clustered with each query in neighbor-joining phylogenies are shown as open bars. Open bars are superimposed on solid bars.
Our results provide new insights into the global spread of HIV-1 strains that warrant further study to elucidate the interrelationships of HIV epidemics on different continents. In the past, the HIV-1 epidemics in Japan and China have been considered as founded by “imported strains” from other locations. However, the results reported here show that virus migration is bidirectional and these countries are also exporting specific HIV lineages. This observation is particularly important for China, where the HIV epidemic continues to grow; furthermore, socioeconomic ties and travel between China and the rest of the world have increased rapidly.
Our study highlights the urgent importance of strengthening HIV monitoring efforts and the need to implement effective control measures to reduce HIV transmission on a global scale.
ACKNOWLEDGMENTS
This study was supported by a grant-in-aid for AIDS research from the Ministry of Health, Labor, and Welfare of Japan, as well as by the United Kingdom Medical Research Council (grant G0900274) and the European Community's 7th framework program (FP7/2007-2013) under the Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN; project 223131), and in part by international grants from the Daiwa Anglo-Japanese Foundation (O.G.P. and Y.T.) and from the Japan China Medical Association (Y.T.).
The collaborators contributing to the UKHIVRDB are as follows: steering committee members Celia Aitken (Gartnavel General Hospital, Glasgow), David Asboe, Anton Pozniak (Chelsea and Westminster Hospital, London), Patricia Cane (Public Health England, Porton Down), Hannah Castro, David Dunn (cochair), David Dolling, Esther Fearnhill, Kholoud Porter, Anna Tostevin (MRC Clinical Trials Unit at University College London, London), David Chadwick (South Tees Hospitals NHS Trust, Middlesbrough), Duncan Churchill (Brighton and Sussex University Hospitals NHS Trust), Duncan Clark (St. Bartholomew's and The London NHS Trust), Simon Collins (HIV i-Base, London), Valerie Delpech (Centre for Infections, Public Health England), Samuel Douthwaite (Guy's and St. Thomas' NHS Foundation Trust, London), Anna Maria Geretti (Institute of Infection and Global Health, University of Liverpool), Antony Hale (Leeds Teaching Hospitals NHS Trust), Stéphane Hué (University College London), Steve Kaye (Imperial College, London), Paul Kellam (Wellcome Trust Sanger Institute and University College London Medical School), Linda Lazarus (Expert Advisory Group on AIDS Secretariat, Public Health England), Andrew Leigh Brown (University of Edinburgh), Tamyo Mbisa (Virus Reference Department, Public Health England), Nicola Mackie (Imperial NHS Trust, London), Chloe Orkin (St. Bartholomew's Hospital, London), Deenan Pillay (cochair), Andrew Phillips, Caroline Sabin (University College London Medical School, London), Erasmus Smit (Public Health England, Birmingham Heartlands Hospital), Kate Templeton (Royal Infirmary of Edinburgh), Peter Tilston (Manchester Royal Infirmary), Daniel Webster (Royal Free NHS Trust, London), Ian Williams (Mortimer Market Centre, London), Hongyi Zhang (Addenbrooke's Hospital, Cambridge), and Mark Zuckerman (King's College Hospital, London). The centers contributing data are the Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge (Jane Greatorex); Guy's and St. Thomas' NHS Foundation Trust, London (Siobhan O'Shea, Jane Mullen); PHE-Public Health Laboratory, Birmingham Heartlands Hospital, Birmingham (Erasmus Smit); PHE-Virus Reference Department, London (Tamyo Mbisa); Imperial College Health NHS Trust, London (Alison Cox); King's College Hospital, London (Richard Tandy); Medical Microbiology Laboratory, Leeds Teaching Hospitals NHS Trust (Tracy Fawcett); Specialist Virology Centre, Liverpool (Mark Hopkins, Lynn Ashton); Department of Clinical Virology, Manchester Royal Infirmary, Manchester (Peter Tilston); Department of Virology, Royal Free Hospital, London (Claire Booth, Ana Garcia-Diaz); Edinburgh Specialist Virology Centre, Royal Infirmary of Edinburgh (Jill Shepherd); Department of Infection and Tropical Medicine, Royal Victoria Infirmary, Newcastle (Matthias L. Schmid, Brendan Payne); South Tees Hospitals NHS Trust, Middlesbrough (David Chadwick); Department of Virology, St. Bartholomew's and The London NHS Trust (Spiro Pereira, Jonathan Hubb); Molecular Diagnostic Unit, Imperial College, London (Steve Kaye); University College London Hospitals (Stuart Kirk); and the West of Scotland Specialist Virology Laboratory, Gartnavel, Glasgow (Alasdair MacLean, Celia Aitken, and Rory Gunson). The coordinating center was the Medical Research Council Clinical Trials Unit (Hannah Castro, Kate Coughlin, David Dunn, David Dolling, Esther Fearnhill, Lorraine Fradette, Kholoud Porter, Anna Tostevin, and Ellen White).
We have no conflicts of interest to report.
Footnotes
Published ahead of print 18 June 2014
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