High genetic diversity of HIV-1 viruses in Macao, China

Journal of Infection (2010) 61, 164e172

www.elsevierhealth.com/journals/jinf

High genetic diversity of HIV-1 viruses in Macao, China Yi Tan a, Desmond Chan a, Denise Chan a, Peng Kei Ip d, Chong Lam e, Nga Yin Fong c, Stephen Kwok-Wing Tsui c, Shui Shan Lee a,b,* a

Stanley Ho Centre for Emerging Infectious Diseases, School of Public Health, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong, China b Department of Microbiology, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong, China c School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong, China d Public Health Laboratory, Macao, China e Macao CDC, Macao, China Accepted 30 April 2010 Available online 20 May 2010

KEYWORDS BF genotype; Molecular epidemiology; Injection drug users; IDU; Macao; China

Summary Objective: To investigate the molecular epidemiology of recently diagnosed HIV-1 infection in Macao for better understanding the epidemiology in this Chinese city, in context of its relationship with other countries in Asia and the rest of the world. Methods: Serum samples of HIV positive cases reported between 2005 and 2007 were collected from the Macao Public Health Laboratory. HIV genotype was determined by phylogenetic analysis of sequences from gag, RT, and env regions. Results: A total of 30 HIV positive samples were genotyped. The HIV-1 viruses circulating in Macao were characterized by their relatively high genetic diversity. CRF01_AE was predominant (56%), followed by subtype B (13%), CRF12_BF (10%), G/CRF12_BF, A1/CRF10_AD and CRF07_BC, of which CRF12_BF and G/CRF12_BF were first reported in Southeast Asia. Phylogenetic analysis showed that there was no clear clustering of CRF01_AE strains but a distinct CRF12_BF cluster associated with injection drug use could be delineated. Conclusion: The results suggested that there were multiple introductions of HIV strains in Macao that have been circulating for an extended period of time, superimposed by an outbreak in injection drug users. ª 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. 2/F Postgraduate Education Centre, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China. Tel.: þ852 2252 8812; fax: þ852 2635 4977. E-mail address: [email protected] (S.S. Lee). 0163-4453/$36 ª 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2010.04.012

HIV in Macao

165

Introduction Globally, there were an estimated 33 million people living with human immunodeficiency virus (HIV) as of 2007. The HIV epidemic has stabilized, albeit with unacceptably high levels of new HIV infections and AIDS deaths.1 The pandemic currently is contributed largely by HIV-1, while HIV2 is restricted to Western and Central Africa.2 The diversity of HIV-1 with its division into subtypes (AeD, FeH, J and K of Group M) and 43 circulating recombinant forms (CRFs) offers a unique opportunity for mapping the spread of the infection geographically.3 In Asia, HIV transmission is gaining momentum, the molecular epidemiology of which would be useful for tracking its spread in the continent. CRF01_AE circulates in major parts of Asia, particularly Southeast Asia,4e6 a pattern unlike that in Western Europe and Americas where Subtype B has always been the main epidemic component.7,8 Globally, Subtype C is at the heart of the pandemic, accounting for 60% of HIV infections worldwide.9,10 There are disparate epidemic trends in Asia, home to 5 million people with HIV. Although the prevalences in Cambodia, Myanmar and Thailand have shown declines,1 new infections are increasing steadily in China, especially in the southern provinces.11,12 Macao, a small Chinese city of 28.6 km2 in the Pearl River Delta (PRD), is one of the linkages between China and countries in and outside Asia. Macao has been a territory of Portugal and was both the first and last European colony in China. However, little information is available about the epidemiology of HIV-1 in this small city. According to the World Health Organization (WHO) and Joint United Nations Programme on HIV/AIDS (UNAIDS), the epidemic state of HIV in Macao is described as low, as HIV prevalence is generally less than 0.1%.1 Macao is however facing the challenges of maintaining the low HIV infection level. Injection drug use and sexual activities in the region, for example, can lead to an increase of HIV transmission and the introduction of new genetic forms. There are no studies addressing the emergence of HIV genetic variants in Macao. The objective

Table 1

of this study is, therefore, to investigate the molecular epidemiology of HIV-1 infection in Macao, so as to contribute to effective surveillance as well as to improve prevention and treatment.

Materials and methods Study participants This is a cross-sectional study on the molecular epidemiology of HIV-1 infections in Macao, which forms an extended part of HIV surveillance under the disease notification mechanism. Left-over sera from reported HIV cases were collected from Macao Public Health Laboratory from June 2006 to November 2007. Serum samples with viral load at least 50 copies per ml and volume not less than 500 ml were archived. All laboratory tests were performed at Stanley Ho Centre for Emerging Infectious Diseases, the Chinese University of Hong Kong, as part of a collaborative arrangement.

HIV RNA extraction, reverse transcription, nestedPCR amplification, and sequencing HIV RNA was extracted from 200 mL of serum by using QIAamp Viral RNA Mini Kit (Qiagen Inc., Hiden, Germany), followed by standard protocols. Complementary DNA (cDNA) was synthesized from 5 mL extracted RNA with First Strand Synthesis System for RT-PCR (Invitrogen Corporation, San Diego, CA). Primers for gag and env genes were obtained from the Centers for Disease Control and Prevention, United States (US CDC). The primers for the RT gene were designed in-house (Table 1). First round PCR using primer F1 and R1 was conducted with the following conditions: 95  C for 5 min, then 39 cycles of 95  C for 1 min, 45  C for 1 min and 72  C for 1 min 45 s. The PCR ended with a final extension of 72  C for 10 min. Second round PCR using primer F2 and R2 was conducted with the same

List of the PCR primers used in the study.

Region

Name

Sequences (50 e30 )

Positionb

Gene

gag

Rv-typing-gag-F1 Rv-typing-gag-F2 Rv-typing-gag-R1 Rv-typing-gag-R2

GCG AGA GCG TCA RTA TTA AGI GG GGG AAA AAA TTC GGT TAA GGC C TCT GAT AAT GCT GWR AAC ATG GG CTT CTA YTA CTT TYA CCC ATG C

796e818 836e857 1319e1297R 1271e1249R

P17 and P24

env

Rv-typing-env-F1 Rv-typing-env-F2 Rv-typing-env-R1 Rv-typing-env-R2

ACA GTR CAR TGY ACA CAT GG CTG TTI AAT GGC AGI CTA GC CAC TTC TCC AAT TGT CCI TCA RAT GGG AGG RGY ATA CAT

6954e6973 7002e7021 7668e7648R 7541e7524R

gp120

RT

BF_1740F1 BF_1745F2 BF_2718R1 BF_2619R2

GGA AAA TGG AAA CCA AAA ATG A ATG GAA ACC AAA AAT GAT AGG G TTT CCC ACT AAC TTC TGT ATG TC GTT CAT AAC CCA TCC AAA GGA A

2370e2391 2375e2396 3337e3315R 3249e3228R

Protease and P51RT

a

a b

Genomic regions amplified. Nucleotide positions with reference to the numbering of HIV HXB2 strain (Genbank accession number K03455).

166

Y. Tan et al.

condition except that the annealing temperature was 55  C. The purified second round PCR products were used as templates for the sequencing reaction. Automatic sequencing was performed by the ABI Prism 3100/3100-Avant Genetic Analyzer with Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems Ltd., Foster City, CA).

HIV genotyping and phylogenetic analyses Using the NCBI viral genotyping tool (http://www.ncbi.nlm. nih.gov/projects/genotyping/formpage.cgi), the sequences of gag, RT, and env regions were submitted for assessment. HIV genotype was determined by combining the results of three regions. Genotyping reference sequences were downloaded from Los Alamos HIV Sequence Database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage. html) and genotyping was confirmed by phylogenetic analysis. Sequences were aligned by CLUSTAL_X and edited by BioEdit

Table 2 Sample

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M22 M23 M24 M25 M27 M28 M29 M30 M31 M32 No. typed a b c

program. Phylogenetic analysis was performed using MEGA 4. 0.2 (http://www.megasoftware.net/). The neighbor-joining method was used with 1000 bootstrap replications (Kimura 2parameter Substitution Model). To determine their genetic relatedness, phylogenetic tree was also constructed using the Maximum Likelihood approach implemented in PhyML 3.412 with 1000 bootstrap replications.13 A general-time-reversal (GTR) substitution model with gamma distributed rate ˜4) and a proportion of heterogeneity of 4 rate categories (A invariable sites suggested by Modeltest was used.

Results Demography of study participants and HIV genotype distribution A total of 31 HIV positive samples were collected, a majority of which (n Z 26, 83.9%) were younger than 40 years old. A

Genotyping results of Macao HIV-1 isolates. Genotyping resultsa gag

RT

env

CRF01_AE B CRF01_AE CRF01_AE CRF01_AE F G G G B CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE C F CRF01_AE B C A1 CRF01_AE F CRF01_AE 27

CRF01_AE B CRF01_AE CRF01_AE CRF01_AE B CRF12_BF G G CRF12_BF B CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF07_BC CRF12_BF CRF01_AE B C A1 CRF01_AE CRF12_BF 28

B CRF01_AE CRF01_AE CRF01_AE B F G G F B CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE B C CRF10_CD CRF01_AE F CRF01_AE 25

Genotype/recombinant

Transmission routeb

Country of residence

CRF01_AE B CRF01_AE CRF01_AE CRF01_AE B CRF12_BF G G G/CRF12_BFc B CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF07_BC CRF12_BF CRF01_AE B C A1/CRF10_CDc CRF01_AE CRF12_BF CRF01_AE 30

Heterosexual MSM – – IDU MSM IDU IDU IDU IDU – IDU Heterosexual Heterosexual MSM Heterosexual Heterosexual – Heterosexual Heterosexuall – IDU – MSM MSM Heterosexual – IDU IDU IDU

Vietnam Macao/China Macao/China Macao/China Macao/China Macao/China China Macao/Portugal Macao/Portugal Macao/Portugal Sierra Leone China China Thailand Macao Macao/China Macao/China Macao/China Macao/China Burma Macao/Portugal Macao/China Peru Macao/China Macao/China South Africa Tanzania China China China

“-” Representing failure of amplification. “–” Transmission route not known from the surveillance records. G/CRF12_BF recombinant and A1/CRF10_CD recombinant need to be confirmed by full genome sequence.

HIV in Macao

167

total of 20 (64.4%) were Chinese and 4 (12.9%) were Portuguese. About half (n Z 18, 58%) were Macao residents, while others had originated from other countries in Southeast Asia and Africa. About one-third (n Z 11, 35%) were infected through injection drug use, 29% by heterosexual activity, and 16% through sex between men. None of gag, RT, or env region, was amplified for sample M26 due to the very low viral load (249 copies/mL). The genotyping results of 30 isolates for genes gag, RT and env are listed in Table 2. Fig. 1 shows the phylogenetic tree of gag, RT, and env genes. The genotype of individual isolate was assigned based on the agreement between results of two or three genes, in accordance with NCBI viral genotyping tool and CRF. Some samples gave different genotyping results in different regions. The RT and gag of sample M29 showed subtype A1, whereas env region showed CRF10_CD. On the other hand, RT and env of M10 suggested CRF12_BF, while gag region suggested subtype G. For M20, only one region could be amplified. The overall genotype distribution of the Macao HIV-1 isolates is shown in Table 3. In summary, CRF01_AE, a recombinant strain in Southeast Asia, was predominant, accounting for 17 out of the 30 typed isolates (56%), followed by genotype B (13%) and CRF12_BF (10%). Genotype G, G/CRF12_BF, A1/CRF10_AD, C, and CRF07_BC were also isolated in this Macao cohort.

Phylogenetic analysis CRF01_AE was the commonest subtype in this Macao cohort. The virus was transmitted mainly by heterosexual activities (41%), followed by injection drug use (IDU, 24%) and men who have sex with men (MSM, 12%) (Table 2). There was however no statistically significant correlation between specific genotype and transmission category. All sequences of CRF01_AE available in the database were then aligned with CRF01_AE from Macao and phylogenetic analysis was performed (Fig. 2). From the phylogenetic tree, M22, M12, M18, M13, M5, and one sequence from China, were relatively close, but there was no welldefined cluster, and the bootstrap value was below 70%. Patient M14 came from Thailand and the sequence was also closer to those isolated from Thailand. Again the bootstrap value was very low and no clustering was observed. Overall, there was no other obvious clustering of CRF01_AE between Macao samples and the sequences from other countries. HIV subtype B, G, CRF12_BF, and the suspected G/CRF12_BF recombinant together accounted for 10 out of the 30 isolates (33%). All subtype B cases were MSM, while G, CRF12_BF, and G/CRF12_BF were identified as injection drug users (Table 2). Sequences of genotype B, G, CRF12_BF, G/CRF12_BF isolated from Asia, Europe, Africa, and South America available in the database were aligned with the Macao sequences and phylogenetic analysis was performed (Fig. 3). There was a clear cluster of CRF12_BF (M10, M7, and M31) in the phylogenetic tree with high bootstrap values of about 70%. M2, M8, and M9 were closer to those sequences from Spain. The sequences from M24 (from Peru), M7, M31, and M10 were close to those from South America, such as Argentina, Uruguay, and Brazil.

Figure 1 Neighbor-joining phylogenetic trees of (a) HIV-1 gag, (b) RT, and (c) env genes. Sequences from Macao are marked as black dots.

168

Y. Tan et al.

Figure 1

(Continued)

HIV in Macao

169 Table 3 Distribution of different genotypes among Macao HIV-1 isolates.

Figure 1

(Continued)

Discussion This is the first study that documents the molecular epidemiology of HIV-1 in recently diagnosed patients in Macao. The phylogenetic analyses showed high genetic diversity with CRF01_AE being the most prevalent (57%), followed by subtype B (13%) and CRF12_BF (10%). Subtype

Genotype/recombinant

No.

Percent (%)

CRF01_AE B CRF12_BF G G/CRF12_BF recombinant A1/CRF10_CD recombinant C CRF07_BC

17 4 3 2 1 1 1 1

57% 13% 10% 6% 3% 3% 3% 3%

Total

30

100%

G, C, and CRF07_BC were present in small proportions. We also identified two potential new recombinants which need to be further confirmed, G/CRF12_BF and A1/CRF10_CD. In the phylogenetic analysis using RT region, there was no clear CRF01_AE cluster, nor was there clustering between Macao sequences and reference sequences from other countries in Southeast Asia. CRF12_BF, an uncommon strain in Southeast Asia was identified. Phylogenetic analysis suggested the presence of a CRF12_BF cluster in Macao, and CRF12_BF strains were closely related to the reference strains from South America. The HIV-1 viruses circulating in Macao were characterized by the relatively high genetic diversity, which is different from other countries or regions in Southeast Asia, e.g. Thailand, Myanmar, and other provinces in Southern China.4,14,15 CRF01_AE, subtype B, and subtype C are dominant in Asia.16 Subtype C is the most prevalent globally, which is found predominantly in India.17 Subtype B circulates mainly in Japan, Taiwan, and Northern China. CRF01_AE, a strain that originated from Central Africa, was first identified in Thailand and has since dominated Southeast Asian countries such as Cambodia, Myanmar, and Vietnam.18 In Southern China, CRF07_BC and CRF08_BC have been spreading through injection drug use.19,20 CRF01_AE and subtype B are also the main HIV-1 strains in Macao. Other recombinant forms such as CRF12_BF, G/ CRF10_BF A1/CRF10_CD, and subtype G were seen, which have otherwise not been reported in Asian countries.21e23 The cultural and historic background of Macao, currently an important casino city, could be behind the complex diversity of the HIV epidemic in this city. In our study, CRF12_BF is specifically associated with injection drug use. The diverse HIV-1 genotype distribution could be a result of population exchanges between Macao and other parts of the world, through the flourishing gambling and tourist industry. Furthermore, Macao and South America have both been ruled by Portugal and Spain. There has clearly been high level of human traffic among Macao, Europe, and South America. HIV-1 genetic diversity reflects the paths of virus transmission and the infection time. Except for outbreaks, older epidemics are usually associated with multiple introductions of viruses and characterized by higher genetically diverse strains. Recent infections from a single introduction

170

Y. Tan et al. of the virus can produce strains with shorter genetic distance, i.e. lower genetic diversity.24 In our study, the high genetic diversity of HIV-1 strains suggested that there were multiple introductions of HIV strains in Macao that have been circulating in the city for an extended period of time. The circulating CRF01_AE strains did not show any clustering, but were instead characterized by their scattered distribution in the phylogenetic tree. Among the other strains, CRF10_CD was first reported in Tanzania.25, 26 Our patient with A1/CRF10_CD did come from Tanzania, who might have brought the virus directly to Macao. Likewise, BF recombinant strains have emerged and become dominant in South America. In the study, one BF strain from a patient had come from Peru in South America. The strain showed the highest similarity with other sequences from Argentina.21 It is therefore speculated that CRF12_BF was introduced to Macao from South America. Separately, subtype B might have come from Europe, while subtype C and A1 were introduced to Macao from Africa, whereas CRF07_BC have originated from mainland China. Like other countries in Asia, HIV epidemic in Macao was driven by sexual transmission and injection drug use. In Macao, heterosexual HIV spread began in the 1980s. Since the efficiency of sexual transmission of HIV is relatively low, the viruses could have been circulating in Macao at a low prevalence without causing any outbreak so far. Injection drug use is an important transmission route by which HIV spreads much faster. The discovery of a cluster of CRF12_BF strains in IDUs is a cause for concern. Its presence, as supported by high bootstrap value, might suggest an outbreak of CRF12_BF in IDUs in the territory. The circulation of CRF12_BF and other subtypes may be due to intersubtype recombination in Macao. One patient was tentatively classified as a BF/G recombinant, the identity of which would need to be confirmed by full-length sequencing. Macao is a unique city and a special administrative region of China. From 1986 to March 2008, a total of 391 HIV infections were reported to the government. Most of them were female, adults, non-Chinese, and non-local residents. After excluding non-local residents, the local cases were predominantly male. One potential pitfall of the current study is that the samples might not be representative of the overall HIV situation in Macao; nevertheless, they did account for a majority of all recently diagnosed HIV infections in the past years. Molecular epidemiological study of HIV-1 infection in Macao has allowed us to keep track of the spread of the different subtypes in the community, detect clustering and the emergence of unusual or novel clades. These are the keys to early investigation and the introduction of appropriate control measures. A comprehensive surveillance system supplemented by molecular characterization is useful in describing the HIV epidemiology in any locality irrespective of its size, as illustrated in our study in Macao.

Figure 2 Evolutionary relationships of Macao HIV-1 CRF01_AE with other CRF01_AE reference sequences from GenBank depicted on a maximum likelihood phylogenetic tree of RT gene. Sequences from Macao are marked as black dots.

Names of some sequences are not shown (detailed information available upon request). Codes for sources of samples: CN Z China, JP Z Japan, HK Z Hong Kong, TH Z Thailand.

HIV in Macao

171

Acknowledgements The authors thank staff of Macao CDC and Public Health Laboratory for assistance in the conduction of studies and staff of Stanley Ho Centre for Emerging Infectious Diseases for administrative support. This research project was funded by Dr. Stanley Ho Medical Development Foundation, which was not involved in the study design, data collection, interpretation and the writeup of the manuscript.

Conflicts of interest The authors declare that there is no conflict of interest.

References

Figure 3 Evolutionary relationships of Macao HIV-1 subtype B, G/CRF14_BG, and CRF12_BF with reference sequences from Asia, Europe, Africa, and South America depicted on a maximum likelihood phylogenetic tree of RT gene. Sequences from Macao are marked as black dots. Names of some sequences are not shown (detailed information available upon request). Codes for sources of samples: CN Z China, JP Z Japan, HK Z Hong Kong, TH Z Thailand, FR Z France, KR Z Korea, BR Z Brazil, ES Z Spain.

1. UNAIDS. Report on the global AIDS epidemic. Geneva: UNAIDS. Available from: http://www.unaids.org/en/ KnowledgeCentre/HIVData/globalReport/2008/2008_global_ report.asp; 2008. 2. Requejo HI. Worldwide molecular epidemiology of HIV. Rev Saude Publica 2006;40:331e45. 3. Robertson DL, Anderson JP, Bradac JA, Carr JK, Foley B, Funkhouser RK, et al. HIV-1 nomenclature proposal. Science 2000;288:55e6. 4. Ananworanich J, Phanuphak N, de Souza M, Paris R, Arroyo M, Trichavaroj R, et al. Incidence and characterization of acute HIV-1 infection in a high-risk Thai population. J Acquir Immune Defic Syndr 2008;49:151e5. 5. Yam WC, Chen JH, Wong KH, Chan K, Cheng VC, Lam HY, et al. Clinical utility of genotyping resistance test on determining the mutation patterns in HIV-1 CRF01_AE and subtype B patients receiving antiretroviral therapy in Hong Kong. J Clin Virol 2006;35:454e7. 6. Perrin L, Kaiser L, Yerly S. Travel and the spread of HIV-1 genetic variants. Lancet Infect Dis 2003;3:22e7. 7. Wainberg MA. HIV-1 subtype distribution and the problem of drug resistance. AIDS 2004;18(Suppl. 3):S63eS68. 8. Tatt ID, Barlow KL, Clewley JP, Gill ON, Parry JV. Surveillance of HIV-1 subtypes among heterosexuals in England and Wales, 1997e2000. J Acquir Immune Defic Syndr 2004;36:1092e9. 9. Takebe Y, Kusagawa S, Motomura K. Molecular epidemiology of HIV: tracking AIDS pandemic. Pediatr Int 2004;46:236e44. 10. Taylor BS, Hammer SM. The challenge of HIV-1 subtype diversity. N Engl J Med 2008;359:1965e6. 11. Piyasirisilp S, McCutchan FE, Carr JK, Sanders-Buell E, Liu W, Chen J, et al. A recent outbreak of human immunodeficiency virus type 1 infection in southern China was initiated by two highly homogeneous, geographically separated strains, circulating recombinant form AE and a novel BC recombinant. J Virol 2000;74:11286e95. 12. Saksena NK, Wang B, Steain M, Yang RG, Zhang LQ. Snapshot of HIV pathogenesis in China. Cell Res 2005;15:953e61. 13. Guindon S, Gascuel O. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003;52:696e704. 14. Kalish ML, Baldwin A, Raktham S, Wasi C, Luo CC, Schochetman G, et al. The evolving molecular epidemiology of HIV-1 envelope subtypes in injecting drug users in Bangkok, Thailand: implications for HIV vaccine trials. AIDS 1995;9: 851e7. 15. Subbarao S, Vanichseni S, Hu DJ, Kitayaporn D, Choopanya K, Raktham S, et al. Genetic characterization of incident HIV type 1 subtype E and B strains from a prospective cohort of

172

16.

17.

18. 19.

20.

Y. Tan et al. injecting drug users in Bangkok, Thailand. AIDS Res Hum Retroviruses 2000;16:699e707. Osmanov S, Pattou C, Walker N, Schwardla ¨nder B, Esparza J. WHOeUNAIDS Network for HIV isolation and characterization. Estimated global distribution and regional spread of HIV-1 genetic subtypes in the year 2000. J Acquir Immune Defic Syndr 2002;29:184e90. Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, et al. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 1999;73:152e60. Ruxrungtham K, Brown T, Phanuphak P. HIV/AIDS in Asia. Lancet 2004;364:69e82. Tee KK, Pybus OG, Li XJ, Han X, Shang H, Kamarulzaman A, et al. Temporal and spatial dynamics of human immunodeficiency virus type 1 circulating recombinant forms 08_BC and 07_BC in Asia. J Virol 2008;82:9206e15. McClutchan FE, Carr JK, Murphy D, Piyasirisilp S, Gao F, Hahn B, et al. Precise mapping of recombination breakpoints suggests a common parent of two BC recombinant HIV type 1 strains circulating in China. AIDS Res Hum Retroviruses 2002;18: 1135e40.

21. Pando MA, Eyzaguirre LM, Carrion G, Montano SM, Sanchez JL, Carr JK, et al. High genetic variability of HIV-1 in female sex workers from Argentina. Retrovirology 2007;4:58. 22. Aulicino PC, Holmes EC, Rocco C, Mangano A, Sen L. Extremely rapid spread of human immunodeficiency virus type 1 BF recombinants in Argentina. J Virol 2007;81:427e9. 23. Monteiro JP, Alcantara LC, de Oliveira T, Oliveira AM, Melo MA, Brites C, et al. Genetic variability of human immunodeficiency virus-1 in Bahia state, Northeast, Brazil: high diversity of HIV genotypes. J Med Virol 2009;81:391e9. 24. Ba ´rtolo I, Rocha C, Bartolomeu J, Gama A, Marcelino R, Fonseca M, et al. Highly divergent subtypes and new recombinant forms prevail in the HIV/AIDS epidemic in Angola: new insights into the origins of the AIDS pandemic. Infect Genet Evol 2009;9:672e82. 25. Koulinska IN, Ndung’u T, Mwakagile D, Msamanga G, Kagoma C, Fawzi W, et al. A new human immunodeficiency virus type 1 circulating recombinant form from Tanzania. AIDS Res Hum Retroviruses 2001 Mar 20;17(5):423e31. 26. Kiwelu IE, Koulinska IN, Nkya WM, Shao J, Kapiga S, Essex M. Identification of CRF10_CD viruses among bar and hotel workers in Moshi, Northern Tanzania. AIDS Res Hum Retroviruses 2005 Oct;21(10):897e900.