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1.  Dominance of HIV-1 Subtype CRF01_AE in Sexually Acquired Cases Leads to a New Epidemic in Yunnan Province of China 
PLoS Medicine  2006;3(11):e443.
Background
Dating back to the first epidemic among injection drug users in 1989, the Yunnan province has had the highest number of human immunodeficiency virus type 1 (HIV-1) infections in China. However, the molecular epidemiology of HIV-1 in Yunnan has not been fully characterized.
Methods and Findings
Using immunoassays, we identified 103,015 accumulated cases of HIV-1 infections in Yunnan between 1989 and 2004. We studied 321 patients representing Yunnan's 16 prefectures from four risk groups, 11 ethnic populations, and ten occupations. We identified three major circulating subtypes: C/CRF07_BC/CRF08_BC (53%), CRF01_AE (40.5%), and B (6.5%) by analyzing the sequence of p17, which is part of the gag gene. For patients with known risk factors, 90.9% of injection drug users had C/CRF07_BC/CRF08_BC viruses, whereas 85.4% of CRF01_AE infections were acquired through sexual transmission. No distinct segregation of CRF01_AE viruses was found among the Dai ethnic group. Geographically, C/CRF07_BC/CRF08_BC was found throughout the province, while CRF01_AE was largely confined to the prefectures bordering Myanmar. Furthermore, C/CRF07_BC/CRF08_BC viruses were found to consist of a group of viruses, including C, CRF08_BC, CRF07_BC, and new BC recombinants, based on the characterization of their reverse transcriptase genes.
Conclusions
This is the first report of a province-wide HIV-1 molecular epidemiological study in Yunnan. While C/CRF07_BC/CRF08_BC and CRF01_AE are codominant, the discovery of many sexually transmitted CRF01_AE cases is new and suggests that this subtype may lead to a new epidemic in the general Chinese population. We discuss implications of our results for understanding the evolution of the HIV-1 pandemic and for vaccine development.
This is a molecular epidemiology study of circulating HIV strains and subtypes in Yunnan province, which has China's largest number of HIV-infected individuals.
Editors' Summary
Background.
The first human immunodeficiency virus (HIV) cases in China were seen in 1989 in Yunnan, a region of south-western China. This area borders Myanmar, Laos, and Vietnam, and is a major entry point for illegal drugs into China. The initial HIV outbreak in this area was in injecting drug users, but HIV is beginning to affect other groups of people in the Yunnan province and is becoming more common across China. There is still not much known about the different types of HIV virus in China and which parts of the population are most likely to be infected. This knowledge is important because it can help people to understand how the epidemic started and how it is likely to spread in the future, and because it helps direct efforts for HIV education and prevention. It is also necessary for the future design of appropriate HIV vaccines.
Why Was This Study Done?
The Yunnan province has the highest rate of HIV-infected individuals in China. It is an important entry point of new HIV virus types into China, and some of the HIV types found in patients in other parts of China appear to have spread from Yunnan. A group of researchers from the United States and China wanted to look at the different types of HIV virus that were infecting people in the Yunnan province and to work out how these types had evolved over the course of the HIV epidemic in China. They focused on human immunodeficiency virus type 1 (HIV-1), the most common form of HIV virus worldwide and also the most infectious. There are at least nine distinct subtypes of HIV-1, and the virus continues to evolve and to form new subtypes.
What Did the Researchers Do and Find?
They collected blood samples from 321 HIV-infected individuals who represented a broad cross-section of the population of Yunnan (people from all geographic parts of the province, 11 ethnic populations, different occupations, etc.) and analyzed the genetic information of the viruses found in these blood samples. Because HIV evolves very rapidly, the genetic information differs between different virus subtypes, and the researchers could therefore tell which subtypes were infecting which subsets of the population. The researchers identified three distinct subtypes of HIV-1: “B” (in about 6.5% of the samples), a group of “C” variants (C/CRF07_BC/CRF08_BC in 53% of the samples), and CRF01_AE (in 40.5%). The CRF01_AE subtype had not previously been reported at such high levels in the Chinese population, and people who were thought to have been infected with HIV through sexual contact (as opposed to contaminated needles) were more likely to be infected with that particular subtype.
What Do These Findings Mean?
The results show a dynamic and evolving pattern of HIV types in the Yunnan province, segregating among different parts of the population. Sexual transmission appears to be on the rise, suggesting that the epidemic could spread rapidly from high-risk groups such as drug users to the general population. HIV/acquired immunodeficiency syndrome (AIDS) education and prevention efforts in the general population are therefore urgently needed. It is also likely that some of the developments of the HIV epidemic in the Yunnan province will be similar in other parts of China as the various subtypes spread. The results of the study also have implications for future HIV vaccine development. Given the range of subtypes, it will be necessary either to develop vaccines that can protect against all the circulating subtypes, or to have a cocktail of several vaccines that each protects against some of them.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030443.
Information from AVERT, an international AIDS charity on HIV subtypes and HIV in China
The UNAIDS on AIDS in Asia
The China AIDS Network—a charity devoted to AIDS research in China
doi:10.1371/journal.pmed.0030443
PMCID: PMC1635743  PMID: 17105339
2.  Phylodynamics of the HIV-1 Epidemic in Cuba 
PLoS ONE  2013;8(9):e72448.
Previous studies have shown that the HIV-1 epidemic in Cuba displayed a complex molecular epidemiologic profile with circulation of several subtypes and circulating recombinant forms (CRF); but the evolutionary and population history of those viral variants remains unknown. HIV-1 pol sequences of the most prevalent Cuban lineages (subtypes B, C and G, CRF18_cpx, CRF19_cpx, and CRFs20/23/24_BG) isolated between 1999 and 2011 were analyzed. Maximum-likelihood analyses revealed multiple introductions of subtype B (n≥66), subtype C (n≥10), subtype G (n≥8) and CRF18_cpx (n≥2) viruses in Cuba. The bulk of HIV-1 infections in this country, however, was caused by dissemination of a few founder strains probably introduced from North America/Europe (clades BCU-I and BCU-II), east Africa (clade CCU-I) and central Africa (clades GCU, CRF18CU and CRF19CU), or locally generated (clades CRFs20/23/24_BG). Bayesian-coalescent analyses show that the major HIV-1 founder strains were introduced into Cuba during 1985–1995; whereas the CRFs_BG strains emerged in the second half of the 1990s. Most HIV-1 Cuban clades appear to have experienced an initial period of fast exponential spread during the 1990s and early 2000s, followed by a more recent decline in growth rate. The median initial growth rate of HIV-1 Cuban clades ranged from 0.4 year−1 to 1.6 year−1. Thus, the HIV-1 epidemic in Cuba has been a result of the successful introduction of a few viral strains that began to circulate at a rather late time of the AIDS pandemic, but then were rapidly disseminated through local transmission networks.
doi:10.1371/journal.pone.0072448
PMCID: PMC3767668  PMID: 24039765
3.  Characterization of HIV-1 gag and nef in Cameroon: further evidence of extreme diversity at the origin of the HIV-1 group M epidemic 
Virology Journal  2013;10:29.
Background
Cameroon, in west central Africa, has an extraordinary degree of HIV diversity, presenting a major challenge for the development of an effective HIV vaccine. Given the continuing need to closely monitor the emergence of new HIV variants in the country, we analyzed HIV-1 genetic diversity in 59 plasma samples from HIV-infected Cameroonian blood donors. Full length HIV gag and nef sequences were generated and phylogenetic analyses were performed.
Findings
All gag and nef sequences clustered within HIV-1M. Circulating recombinant form CRF02_AG predominated, accounting for 50% of the studied infections, followed by clade G (11%), clade D and CRF37_cpx (4% each), and clades A, F, CRF01_AE and CRF36_cpx (2% each). In addition, 22% of the studied viruses apparently had nef and gag genes from viruses belonging to different clades, with the majority (8/10) having either a nef or gag gene derived from CRF02_AG. Interestingly, five gag sequences (10%) and three (5%) nef sequences were neither obviously recombinant nor easily classifiable into any of the known HIV-1M clades.
Conclusion
This suggests the widespread existence of highly divergent HIV lineages in Cameroon. While the genetic complexity of the Cameroonian HIV-1 epidemic has potentially serious implications for the design of biomedical interventions, detailed analyses of divergent Cameroonian HIV-1M lineages could be crucial for dissecting the earliest evolutionary steps in the emergence of HIV-1M.
doi:10.1186/1743-422X-10-29
PMCID: PMC3560183  PMID: 23339631
HIV-1 diversity; West central Africa; RDP3; Maximum likelihood; PHYML
4.  CRF22_01A1 is involved in the emergence of new HIV-1 recombinants in Cameroon 
Cameroon is a West African country where high genetic diversity of HIV-1 has been reported. The predominant CRF02_AG is involved in the emergence of more complex intersubtype recombinants. In this study, we sequenced the full-length genome of a novel unique recombinant form (URF) of HIV-1, 02CAMLT04 isolated in blood donors in urban Cameroon. Phylogenetic tree and bootscan analysis showed that 02CAMLT04 was complex and appeared to be a secondary recombinant derived from CRF02_AG and CRF22_01A1. The genomic composition of 02CAMLT04 strain showed that it is composed of three segments; twenty four percent of the genome is classified as CRF02_AG, spanning most of the envelope gene. The remaining seventy six percent of the genome is classified as CRF22_01A1. In addition, the sequence analysis of 13 full-length sequences from HIV-1 positive specimens received from Cameroon between 2002 and 2010 indicated that five specimens are pure CRF22_01A1 viruses, and six others have homology with CRF22_01A1 sequences in either gag, pol or env region where as 6% of strains contain portions of CRF22_01A1. Further study demonstrated that CRF22_01A1 is a primary prevalence strain co-circulating in Cameroon and is involved in complex intersubtype recombination events with subtypes (D or F), subsubtypes (A1 or F2) and CRFs (CRF01_AE or CRF02_AG). Our studies show that novel recombinants between CRF22_01A1 and other clades and recombinant forms may be emerging in Cameroon that could contribute to the future global diversity of HIV-1 in this region and world wide.
doi:10.1097/QAI.0b013e318258c7e3
PMCID: PMC3392544  PMID: 22549382
HIV-1; recombinant; genetic diversity; phylogenetic analysis; CRF22_01A1; CRF02_AG; Cameroon
5.  Identification of new, emerging HIV-1 unique recombinant forms and drug resistant viruses circulating in Cameroon 
Virology Journal  2011;8:185.
Background
The HIV epidemic in Cameroon is characterized by a high degree of viral genetic diversity with circulating recombinant forms (CRFs) being predominant. The goal of our study was to determine recent trends in virus evolution and emergence of drug resistance in blood donors and HIV positive patients.
Methodology
Blood specimens of 73 individuals were collected from three cities and a few villages in Cameroon and viruses were isolated by co-cultivation with PBMCs. Nested PCR was performed for gag p17 (670 bp) pol (840 bp) and Env gp41 (461 bp) genes. Sequences were phylogenetically analyzed using a reference set of sequences from the Los Alamos database.
Results
Phylogenetic analysis based on partial sequences revealed that 65% (n = 48) of strains were CRF02_AG, 4% (n = 3) subtype F2, 1% each belonged to CRF06 (n = 1), CRF11 (n = 1), subtype G (n = 1), subtype D (n = 1), CRF22_01A1 (n = 1), and 26% (n = 18) were Unique Recombinant Forms (URFs). Most URFs contained CRF02_AG in one or two HIV gene fragments analyzed. Furthermore, pol sequences of 61 viruses revealed drug resistance in 55.5% of patients on therapy and 44% of drug naïve individuals in the RT and protease regions. Overall URFs that had a primary HIV subtype designation in the pol region showed higher HIV-1 p24 levels than other recombinant forms in cell culture based replication kinetics studies.
Conclusions
Our results indicate that although CRF02_AG continues to be the predominant strain in Cameroon, phylogenetically the HIV epidemic is continuing to evolve as multiple recombinants of CRF02_AG and URFs were identified in the individuals studied. CRF02_AG recombinants that contained the pol region of a primary subtype showed higher replicative advantage than other variants. Identification of drug resistant strains in drug-naïve patients suggests that these viruses are being transmitted in the population studied. Our findings support the need for continued molecular surveillance in this region of West Central Africa and investigating impact of variants on diagnostics, viral load and drug resistance assays on an ongoing basis.
doi:10.1186/1743-422X-8-185
PMCID: PMC3118203  PMID: 21513545
6.  A Newly Emerging HIV-1 Recombinant Lineage (CRF58_01B) Disseminating among People Who Inject Drugs in Malaysia 
PLoS ONE  2014;9(1):e85250.
The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main) comprising of 11 subtypes and sub-subtypes (A1, A2, B–D, F1, F2, G, H, J, and K) and to date 55 circulating recombinant forms (CRFs). In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes—subtype B (including subtype B′, the Thai variant of subtype B), CRF01_AE, and CRF33_01B—have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs) in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B′ and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%), CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258). Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand) in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention.
doi:10.1371/journal.pone.0085250
PMCID: PMC3898983  PMID: 24465513
7.  HIV-1 recombinants with multiple parental strains in low-prevalence, remote regions of Cameroon: Evolutionary relics? 
Retrovirology  2010;7:39.
Background
The HIV pandemic disseminated globally from Central West Africa, beginning in the second half of the twentieth century. To elucidate the virologic origins of the pandemic, a cross-sectional study was conducted of the genetic diversity of HIV-1 strains in villagers in 14 remote locations in Cameroon and in hospitalized and STI patients. DNA extracted from PBMC was PCR amplified from HIV(+) subjects. Partial pol amplicons (N = 164) and nearly full virus genomes (N = 78) were sequenced. Among the 3956 rural villagers studied, the prevalence of HIV infection was 4.9%; among the hospitalized and clinic patients, it was 8.6%.
Results
Virus genotypes fell into two distinctive groups. A majority of the genotyped strains (109/164) were the circulating recombinant form (CRF) known to be endemic in West Africa and Central West Africa, CRF02_AG. The second most common genetic form (9/164) was the recently described CRF22_01A1, and the rest were a collection of 4 different subtypes (A2, D, F2, G) and 6 different CRFs (-01, -11, -13, -18, -25, -37). Remarkably, 10.4% of HIV-1 genomes detected (17/164) were heretofore undescribed unique recombinant forms (URF) present in only a single person. Nearly full genome sequencing was completed for 78 of the viruses of interest. HIV genetic diversity was commonplace in rural villages: 12 villages each had at least one newly detected URF, and 9 villages had two or more.
Conclusions
These results show that while CRF02_AG dominated the HIV strains in the rural villages, the remainder of the viruses had tremendous genetic diversity. Between the trans-species transmission of SIVcpz and the dispersal of pandemic HIV-1, there was a time when we hypothesize that nascent HIV-1 was spreading, but only to a limited extent, recombining with other local HIV-1, creating a large variety of recombinants. When one of those recombinants began to spread widely (i.e. became epidemic), it was recognized as a subtype. We hypothesize that the viruses in these remote Cameroon villages may represent that pre-epidemic stage of viral evolution.
doi:10.1186/1742-4690-7-39
PMCID: PMC2879232  PMID: 20426823
8.  Spatiotemporal Dynamics of the HIV-1 Subtype G Epidemic in West and Central Africa 
PLoS ONE  2014;9(6):e98908.
The human immunodeficiency virus type 1 (HIV-1) subtype G is the second most prevalent HIV-1 clade in West Africa, accounting for nearly 30% of infections in the region. There is no information about the spatiotemporal dynamics of dissemination of this HIV-1 clade in Africa. To this end, we analyzed a total of 305 HIV-1 subtype G pol sequences isolated from 11 different countries from West and Central Africa over a period of 20 years (1992 to 2011). Evolutionary, phylogeographic and demographic parameters were jointly estimated from sequence data using a Bayesian coalescent-based method. Our analyses indicate that subtype G most probably emerged in Central Africa in 1968 (1956–1976). From Central Africa, the virus was disseminated to West and West Central Africa at multiple times from the middle 1970s onwards. Two subtype G strains probably introduced into Nigeria and Togo between the middle and the late 1970s were disseminated locally and to neighboring countries, leading to the origin of two major western African clades (GWA-I and GWA-II). Subtype G clades circulating in western and central African regions displayed an initial phase of exponential growth followed by a decline in growth rate since the early/middle 1990s; but the mean epidemic growth rate of GWA-I (0.75 year−1) and GWA-II (0.95 year−1) clades was about two times higher than that estimated for central African lineages (0.47 year−1). Notably, the overall evolutionary and demographic history of GWA-I and GWA-II clades was very similar to that estimated for the CRF06_cpx clade circulating in the same region. These results support the notion that the spatiotemporal dissemination dynamics of major HIV-1 clades circulating in western Africa have probably been shaped by the same ecological factors.
doi:10.1371/journal.pone.0098908
PMCID: PMC4053352  PMID: 24918930
9.  Identification and Genetic Characterization of a Novel CRF22_01A1 Recombinant Form of HIV Type 1 in Cameroon 
AIDS Research and Human Retroviruses  2010;26(9):1033-1045.
Abstract
Cameroon is a country in West Central Africa in which all four groups of HIV-1 (M, N, O, and P), some circulating recombinant forms (CRFs) and unique recombinant forms (URFs) are prevalent. The CRF22 was initially identified through a novel URF strain, 01CM53122, and later defined from two additional sequences; however, the genomic properties of CRF22 have never been demonstrated in detail. In this study, we describe the characterization of five CRF22_01A1 strains, 02CMLT72, 01CM1867LE, 01CM001BBY, 02CM3097MN, and 02CM1917LE, identified in Cameroon without apparent epidemiological links. A typical CRF22_01A1 strain contains five fragments that can be assigned to the CRF01_AE and subsubtype A1 radiations. Forty-eight percent of the genome is classified as CRF01_AE, spanning the entire region of the gag gene, part of the pol gene, and accessory genes as well as the beginning and the end of the env gene and nef gene. Fifty-two percent of the genome is subsubtype A1 including regions mostly in the pol, vif, and env genes. The five CRF22_01A1 viruses formed a deep branch outside the groups of CRF01_AE and displayed similar mosaic structure but were moderately different from the original strain of CRF22_01A1, 01CM53122. Further analysis of the 01CM53122 genome showed that this virus represents a diverse set of mosaic genomes from CRF22_01A1, including a 446-nt segment of 01CM53122 in the env region, but unlike other CRF22 strains, clustered with CRF01_AE rather than the A1 sequence, suggesting that the 01CM53122 strain is a recombinant of CRF22_01A1 and CRF01_AE.
doi:10.1089/aid.2009.0197
PMCID: PMC2931544  PMID: 20812894
10.  Molecular Diversity of HIV-1 among People Who Inject Drugs in Kuala Lumpur, Malaysia: Massive Expansion of Circulating Recombinant Form (CRF) 33_01B and Emergence of Multiple Unique Recombinant Clusters 
PLoS ONE  2013;8(5):e62560.
Since the discovery of HIV-1 circulating recombinant form (CRF) 33_01B in Malaysia in the early 2000 s, continuous genetic diversification and active recombination involving CRF33_01B and other circulating genotypes in the region including CRF01_AE and subtype B′ of Thai origin, have led to the emergence of novel CRFs and unique recombinant forms. The history and magnitude of CRF33_01B transmission among various risk groups including people who inject drugs (PWID) however have not been investigated despite the high epidemiological impact of CRF33_01B in the region. We update the most recent molecular epidemiology of HIV-1 among PWIDs recruited in Malaysia between 2010 and 2011 by population sequencing and phylogenetic analysis of 128 gag-pol sequences. HIV-1 CRF33_01B was circulating among 71% of PWIDs whilst a lower prevalence of other previously dominant HIV-1 genotypes [subtype B′ (11%) and CRF01_AE (5%)] and CRF01_AE/B′ unique recombinants (13%) were detected, indicating a significant shift in genotype replacement in this population. Three clusters of CRF01_AE/B′ recombinants displaying divergent yet phylogenetically-related mosaic genomes to CRF33_01B were identified and characterized, suggestive of an abrupt emergence of multiple novel CRF clades. Using rigorous maximum likelihood approach and the Bayesian Markov chain Monte Carlo (MCMC) sampling of CRF33_01Bpol sequences to elucidate the past population dynamics, we found that the founder lineages of CRF33_01B were likely to have first emerged among PWIDs in the early 1990 s before spreading exponentially to various high and low-risk populations (including children who acquired infections from their mothers) and later on became endemic around the early 2000 s. Taken together, our findings provide notable genetic evidence indicating the widespread expansion of CRF33_01B among PWIDs and into the general population. The emergence of numerous previously unknown recombinant clades highlights the escalating genetic complexity of HIV-1 in the Southeast Asian region.
doi:10.1371/journal.pone.0062560
PMCID: PMC3646884  PMID: 23667490
11.  Origin and Epidemiological History of HIV-1 CRF14_BG 
PLoS ONE  2011;6(9):e24130.
Background
CRF14_BG isolates, originally found in Spain, are characterized by CXCR4 tropism and rapid disease progression. This study aimed to identify the origin of CRF14_BG and reconstruct its epidemiological history based on new isolates from Portugal.
Methodology/Principal Findings
C2V3C3 env gene sequences were obtained from 62 samples collected in 1993–1998 from Portuguese HIV-1 patients. Full-length genomic sequences were obtained from three patients. Viral subtypes, diversity, divergence rate and positive selection were investigated by phylogenetic analysis. The molecular structure of the genomes was determined by bootscanning. A relaxed molecular clock model was used to date the origin of CRF14_BG. Geno2pheno was used to predict viral tropism. Subtype B was the most prevalent subtype (45 sequences; 73%) followed by CRF14_BG (8; 13%), G (4; 6%), F1 (2; 3%), C (2; 3%) and CRF02_AG (1; 2%). Three CRF14_BG sequences were derived from 1993 samples. Near full-length genomic sequences were strongly related to the CRF14_BG isolates from Spain. Genetic diversity of the Portuguese isolates was significantly higher than the Spanish isolates (0.044 vs 0.014, P<0.0001). The mean date of origin of the CRF14_BG cluster was estimated to be 1992 (range, 1989 and 1996) based on the subtype G genomic region and 1989 (range, 1984–1993) based on the subtype B genomic region. Most CRF14_BG strains (78.9%) were predicted to be CXCR4. Finally, up to five amino acids were under selective pressure in subtype B V3 loop whereas only one was found in the CRF14_BG cluster.
Conclusions
CRF14_BG emerged in Portugal in the early 1990 s soon after the beginning of the HIV-1 epidemics, spread to Spain in late 1990 s as a consequence of IVDUs migration and then to the rest of Europe. CXCR4 tropism is a general characteristic of this CRF that may have been selected for by escape from neutralizing antibody response.
doi:10.1371/journal.pone.0024130
PMCID: PMC3182163  PMID: 21969855
12.  Circulation of HIV-1 CRF02_AG among MSM Population in Central Italy: A Molecular Epidemiology-Based Study 
BioMed Research International  2013;2013:810617.
Introduction. The evolutionary and demographic history of the circular recombinant form CRF02_AG in a selected retrospective group of HIV-1 infected men who have sex with men (MSM) resident in Central Italy was investigated. Methods. A total of 55 HIV-1 subtype CRF02_AG pol sequences were analyzed using Bayesian methods and a relaxed molecular clock to reconstruct their dated phylogeny and estimate population dynamics. Results. Dated phylogeny indicated that the HIV-1 CRF02_AG strains currently circulating in Central Italy originated in the early 90's. Bayesian phylogenetic analysis revealed the existence of a main HIV-1 CRF02_AG clade, introduced in the area of Rome before 2000 and subsequently differentiated in two different subclades with a different date of introduction (2000 versus 2005). All the sequences within clusters were interspersed, indicating that the MSM analyzed form a close and restricted network where the individuals, also moving within different clinical centers, attend the same places to meet and exchange sex. Conclusions. It was suggested that the HIV-1 CRF02_AG epidemic entered central Italy in the early 1990s, with a similar trend observed in western Europe.
doi:10.1155/2013/810617
PMCID: PMC3863479  PMID: 24369538
13.  Subtype Classification of Iranian HIV-1 Sequences Registered in the HIV Databases, 2006-2013 
PLoS ONE  2014;9(9):e105098.
Background
The rate of human immunodeficiency virus type 1 (HIV-1) infection in Iran has increased dramatically in the past few years. While the earliest cases were among hemophiliacs, injection drug users (IDUs) fuel the current epidemic. Previous molecular epidemiological analysis found that subtype A was most common among IDUs but more recent studies suggest CRF_35AD may be more prevalent now. To gain a better understanding of the molecular epidemiology of HIV-1 infection in Iran, we analyzed all Iranian HIV sequence data from the Los Alamos National Laboratory.
Methods
All Iranian HIV sequences from subtyping studies with pol, gag, env and full-length HIV-1 genome sequences registered in the HIV databases (www.hiv.lanl.gov) between 2006 and 2013 were downloaded. Phylogenetic trees of each region were constructed using Neighbor-Joining (NJ) and Maximum Parsimony methods.
Results
A total of 475 HIV sequences were analyzed. Overall, 78% of sequences were CRF_35AD. By gene region, CRF_35AD comprised 83% of HIV-1 pol, 62% of env, 78% of gag, and 90% of full-length genome sequences analyzed. There were 240 sequences re-categorized as CRF_AD. The proportion of CRF_35AD sequences categorized by the present study is nearly double the proportion of what had been reported.
Conclusions
Phylogenetic analysis indicates HIV-1 subtype CRF_35AD is the predominant circulating strain in Iran. This result differed from previous studies that reported subtype A as most prevalent in HIV- infected patients but confirmed other studies which reported CRF_35AD as predominant among IDUs. The observed epidemiological connection between HIV strains circulating in Iran and Afghanistan may be due to drug trafficking and/or immigration between the two countries. This finding suggests the possible origins and transmission dynamics of HIV/AIDS within Iran and provides useful information for designing control and intervention strategies.
doi:10.1371/journal.pone.0105098
PMCID: PMC4154867  PMID: 25188443
14.  Phylogeographic Analyses Reveal a Crucial Role of Xinjiang in HIV-1 CRF07_BC and HCV 3a Transmissions in Asia 
PLoS ONE  2011;6(8):e23347.
Background
China faces an increasing prevalence of two HIV-1 circulating recombinant forms (CRFs) 07_BC and 08_BC. Both CRFs_BC were previously demonstrated to originate in Yunnan and spread to Liaoning from Yunnan via injection drug use (IDU) in China. Supposing it is true, we are unable to answer why only CRF07_BC, rather than both CRFs_BC together, was transmitted to Xinjiang.
Methodology/Principal Findings
We investigated the phylogeography of CRF07_BC and CRF08_BC using multiple HIV-1 genomic regions with Bayesian phylogeography method. Phylogenetic reconstructions showed that all CRF07_BC sequences were divided into two clades, Yunnan and Xinjiang, and all strains from other regions of mainland China clustered within the Xinjiang clade. Significant geographic diffusion links of Xinjiang with other regions (including Liaoning, Beijing, Jiangsu and Guangdong) were supported by Bayes factor tests. The temporal dynamics analyses showed that CRF07_BC spread from Xinjiang to Liaoning in 1996.10, and to Jiangsu in 2000.9. The analyses of CRF08_BC not only confirmed the previous conclusion on temporal and spatial dynamics of CRF08_BC, but also indicated that the CRF08_BC strains from Guangdong and Shanghai originated from Yunnan. The analyses of HCV 3a showed that it was introduced into Xinjiang in the early 1980s, and spread from Xinjiang to Yunnan in 1990.10 and to Jiangsu in 1999.2, and further from Yunnan to Guangxi in 1995.3. The temporal and spatial dynamics of HCV 3a were similar to some extent to that of HIV-1 CRF07_BC and/or CRF08_BC, suggesting a possible association in migration patterns between HCV and HIV-1 through IDU. In addition, HCV 3a spread from Xinjiang to Pakistan, implying a drug trafficking route linking them.
Conclusions/Significance
Xinjiang, as the most important transfer station for drug trafficking from Golden Crescent to other regions of China, plays a very crucial role in the transmission of viruses (e.g., HIV-1 and HCV) through IDU in Asia.
doi:10.1371/journal.pone.0023347
PMCID: PMC3155551  PMID: 21858079
15.  Phylodynamic Analysis Reveals CRF01_AE Dissemination between Japan and Neighboring Asian Countries and the Role of Intravenous Drug Use in Transmission 
PLoS ONE  2014;9(7):e102633.
Background
One major circulating HIV-1 subtype in Southeast Asian countries is CRF01_AE, but little is known about its epidemiology in Japan. We conducted a molecular phylodynamic study of patients newly diagnosed with CRF01_AE from 2003 to 2010.
Methods
Plasma samples from patients registered in Japanese Drug Resistance HIV-1 Surveillance Network were analyzed for protease-reverse transcriptase sequences; all sequences undergo subtyping and phylogenetic analysis using distance-matrix-based, maximum likelihood and Bayesian coalescent Markov Chain Monte Carlo (MCMC) phylogenetic inferences. Transmission clusters were identified using interior branch test and depth-first searches for sub-tree partitions. Times of most recent common ancestor (tMRCAs) of significant clusters were estimated using Bayesian MCMC analysis.
Results
Among 3618 patient registered in our network, 243 were infected with CRF01_AE. The majority of individuals with CRF01_AE were Japanese, predominantly male, and reported heterosexual contact as their risk factor. We found 5 large clusters with ≥5 members and 25 small clusters consisting of pairs of individuals with highly related CRF01_AE strains. The earliest cluster showed a tMRCA of 1996, and consisted of individuals with their known risk as heterosexual contacts. The other four large clusters showed later tMRCAs between 2000 and 2002 with members including intravenous drug users (IVDU) and non-Japanese, but not men who have sex with men (MSM). In contrast, small clusters included a high frequency of individuals reporting MSM risk factors. Phylogenetic analysis also showed that some individuals infected with HIV strains spread in East and South-eastern Asian countries.
Conclusions
Introduction of CRF01_AE viruses into Japan is estimated to have occurred in the 1990s. CFR01_AE spread via heterosexual behavior, then among persons connected with non-Japanese, IVDU, and MSM. Phylogenetic analysis demonstrated that some viral variants are largely restricted to Japan, while others have a broad geographic distribution.
doi:10.1371/journal.pone.0102633
PMCID: PMC4099140  PMID: 25025900
16.  Development, Evaluation, and Validation of an Oligonucleotide Probe Hybridization Assay To Subtype Human Immunodeficiency Virus Type 1 Circulating Recombinant Form CRF02_AG 
Journal of Clinical Microbiology  2004;42(4):1428-1433.
We have developed and validated an oligonucleotide probe hybridization assay for human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) CRF02_AG. In the p17 coding region of the gag gene, a CRF02_AG-specific signature pattern was observed. Five working probes were designed to discriminate CRF02_AG infections from infections by all other documented subtypes and CRFs in an enzyme-linked immunosorbent assay-based oligonucleotide probe hybridization assay. Nucleic acids were extracted from a panel of HIV-1-positive plasma samples from Cameroon, Bénin, Côte d'Ivoire, Kenya, Zambia, and Belgium and from blood spots from The Gambia. CRF02_AG (n = 147) and non-CRF02 (n = 100) samples were analyzed to evaluate and validate the oligonucleotide probe hybridization assay. The CRF02_AG-specific oligonucleotide probe hybridization assay has a high sensitivity and specificity, with good positive and negative predictive values in regions of high and low prevalence. A validation of the assay with West and West Central African samples indicated a sensitivity of 98.4% and a specificity of 96.7%. The oligonucleotide probe hybridization assay as a diagnostic tool will allow for rapid screening for CRF02_AG. This could be used to track the HIV epidemic in terms of documenting the real prevalence of CRF02_AG strains and will complement efforts in vaccine development. Moreover, this technology can easily be applied in laboratories in developing countries.
doi:10.1128/JCM.42.4.1428-1433.2004
PMCID: PMC387545  PMID: 15070984
17.  The rapidly expanding CRF01_AE epidemic in China is driven by multiple lineages of HIV-1 viruses introduced in the 1990s 
AIDS (London, England)  2013;27(11):1793-1802.
Objectives:
We sought to comprehensively analyze the origin, transmission patterns and sub-epidemic clusters of the HIV-1 CRF01_AE strains in China.
Methods:
Available HIV-1 CRF01_AE samples indentified in national molecular epidemiologic surveys were used to generate near full-length genome (NFLG) sequences. The new and globally available CRF01_AE NFLG sequences were subjected to phylogenetic and Bayesian molecular clock analyses, and combined with epidemiologic data to elucidate the history of CRF01_AE transmission in China.
Results:
We generated 75 new CRF01_AE NFLG sequences from various risk populations covering all major CRF01_AE epidemic regions in China. Seven distinct phylogenetic clusters of CRF01_AE were identified. Clusters 1, 2 and 3 were prevalent among heterosexuals and IDUs in southern and southwestern provinces. Clusters 4 and 5 were found primarily among MSM in major northern cities. Clusters 6 and 7 were only detected among heterosexuals in two southeast and southwest provinces. Molecular clock analysis indicated that all CRF01_AE clusters were introduced from Southeast Asia in the 1990s, coinciding with the peak of Thailand's HIV epidemic and the initiation of China's free overseas travel policy for their citizens, which started with Thailand as the first destination country.
Conclusion:
China's HIV-1 epidemic of sexual transmissions, was initiated by multilineages of CRF01_AE strains, in contrast to the mono-lineage epidemic of B′ strain in former plasma donors and IDUs. Our study underscores the difficulty in controlling HIV-1 sexual transmission compared with parenteral transmission.
doi:10.1097/QAD.0b013e328360db2d
PMCID: PMC3819312  PMID: 23807275
China; CRF01_AE; HIV-1; near full-length genome; phylogenetic cluster; risk population
18.  Emerging Variability in HIV-1 Genetics among Recently Infected Individuals in Yunnan, China 
PLoS ONE  2013;8(3):e60101.
Background
Yunnan has the longest endured Human Immunodeficiency Virus-1 (HIV-1) epidemic in China, and the genetic diversity of HIV-1 constitutes an essential characteristic of molecular epidemiology in this region. To obtain a more comprehensive picture of the dynamic changes in Yunnan’s HIV-1 epidemic, a cross-sectional molecular epidemiological investigation was carried out among recently infected individuals.
Methodology/Principal Findings
We sequenced partial gag (HXB2∶781–1861) and env (HXB2∶7002–7541) genes from 308 plasma samples of recently infected patients. With phylogenetic analysis, 130 specimens generated interpretable genotyping data. We found that the circulating genotypes included: CRF08_BC (40.8%), unique recombinant forms (URFs, 27.7%), CRF01_AE (18.5%), CRF07_BC (9.2%), subtype B (2.3%) and C (1.5%). CRF08_BC was the most common genotype, and was predominant in both intravenous drug users (IDUs) and heterosexually transmitted populations. CRF08_BC and CRF07_BC still predominated in eastern Yunnan, but CRF08_BC showed increasing prevalence in western Yunnan. Strikingly, the URFs raised dramatically in most regions of Yunnan. Seven different types of URFs were detected from 12 prefectures, suggesting that complicated and frequent recombination is a salient feature of Yunnan’s HIV-1 epidemic. Among URFs, two BC clusters with distinctive recombination patterns might be potential new CRF_BCs. CRF01_AE was no longer confined to the prefectures bordering Myanmar, and had spread to the eastern part of Yunnan, especially the capital city of Kunming, with a large number of infections in the transient population. The ratios of the main genotypes showed no statistical differences between infected IDUs and heterosexually transmitted infections.
Conclusions/Significance
The changing patterns of the dominant HIV-1 genotypes in Yunnan indicate the complex evolving dynamic nature of the epidemic. Understanding new trends in molecular epidemiology of HIV-1 infection is critical for adjusting current prevention strategies and vaccine development in Yunnan.
doi:10.1371/journal.pone.0060101
PMCID: PMC3608604  PMID: 23555898
19.  Identification and Characterization of CRF02_AG, CRF06_cpx, and CRF09_cpx Recombinant Subtypes in Mali, West Africa 
Abstract
Multiple HIV-1 subtypes and circulating recombinant forms (CRFs) are known to cocirculate in Africa. In West Africa, the high prevalence of CRF02_AG, and cocirculation of subtype A, CRF01_AE, CRF06_cpx, and other complex intersubtype recombinants has been well documented. Mali, situated in the heart of West Africa, is likely to be affected by the spread of recombinant subtypes. However, the dynamics of the spread of HIV-1 recombinant subtypes as well as nonrecombinant HIV-1 group M subtypes in this area have not been systematically assessed. Herein, we undertook genetic analyses on full-length env sequences derived from HIV-1-infected individuals living in the capital city of Mali, Bamako. Of 23 samples we examined, 16 were classified as CRF02_AG and three had a subsubtype A3. Among the remaining HIV-1 strains, CRF06_cpx and CRF09_cpx were each found in two patients. Comparison of phylogenies for six matched pol and full-length env sequences revealed that two strains had discordant subtype/CRF designations between the pol and env regions: one had A3polCRF02_AGenv and the other had CRF02_AGpolA3env. Taken together, our study demonstrated the high prevalence of CRF02_AG and complexity of circulating HIV-1 strains in Mali. It also provided evidence of ongoing virus evolution of CRF02_AG, as illustrated by the emergence of more complex CRF02_AG/A3 intersubtype recombinants in this area.
doi:10.1089/aid.2008.0111
PMCID: PMC2981380  PMID: 19182920
20.  HIV-1 Molecular Epidemiology in Guinea-Bissau, West Africa: Origin, Demography and Migrations 
PLoS ONE  2011;6(2):e17025.
The HIV-1 epidemic in West Africa has been dominated by subtype A and the recombinant form CRF02_AG. Little is known about the origins and the evolutionary history of HIV-1 in this region. We employed Maximum likelihood and Bayesian methods in combination with temporal and spatial information to reconstruct the HIV-1 subtype distribution, demographic history and migration patterns over time in Guinea-Bissau, West Africa. We found that CRF02_AG and subsubtype A3 were the dominant forms of HIV-1 in Guinea-Bissau and that they were introduced into the country on at least six different occasions between 1976 and 1981. These estimates also corresponded well with the first reported HIV-1 cases in Guinea-Bissau. Migration analyses suggested that (1) the HIV-1 epidemic started in the capital Bissau and then dispersed into more rural areas, and (2) the epidemic in Guinea-Bissau was connected to both Cameroon and Mali. This is the first study that describes the HIV-1 molecular epidemiology in a West African country by combining the results of subtype distribution with analyses of epidemic origin and epidemiological linkage between locations. The multiple introductions of HIV-1 into Guinea-Bissau, during a short time-period of five years, coincided with and were likely influenced by the major immigration wave into the country that followed the end of the independence war (1963–1974).
doi:10.1371/journal.pone.0017025
PMCID: PMC3041826  PMID: 21365013
21.  Evolutionary History of HIV-1 Subtype B and CRF01_AE Transmission Clusters among Men Who Have Sex with Men (MSM) in Kuala Lumpur, Malaysia 
PLoS ONE  2013;8(6):e67286.
HIV-1 epidemics among men who have sex with men (MSM) continue to expand in developed and developing countries. Although HIV infection in MSM is amongst the highest of the key affected populations in many countries in Southeast Asia, comprehensive molecular epidemiological study of HIV-1 among MSM remains inadequate in the region including in Malaysia. Here, we reported the phylodynamic profiles of HIV-1 genotypes circulating among MSM population in Kuala Lumpur, Malaysia. A total of n = 459 newly-diagnosed treatment-naïve consenting subjects were recruited between March 2006 and August 2012, of whom 87 (18.9%) were self-reported MSM. Transmitted drug resistance mutations were absent in these isolates. Cumulatively, phylogenetic reconstructions of the pro-rt gene (HXB2∶2253–3275) showed that HIV-1 subtype B and CRF01_AE were predominant and contributed to approximately 80% of the total HIV-1 infection among MSM. In addition to numerous unique transmission lineages within these genotypes, twelve monophyletic transmission clusters of different sizes (2–7 MSM sequences, supported by posterior probability value of 1) were identified in Malaysia. Bayesian coalescent analysis estimated that the divergence times for these clusters were mainly dated between 1995 and 2005 with four major transmission clusters radiating at least 12 years ago suggesting that active spread of multiple sub-epidemic clusters occurred during this period. The changes in effective population size of subtype B showed an exponential growth within 5 years between 1988 and 1993, while CRF01_AE lineage exhibited similar expansion between 1993 and 2003. Our study provides the first insight of the phylodynamic profile of HIV-1 subtype B and CRF01_AE circulating among MSM population in Kuala Lumpur, Malaysia, unravelling the importance of understanding transmission behaviours as well as evolutionary history of HIV-1 in assessing the risk of outbreak or epidemic expansion.
doi:10.1371/journal.pone.0067286
PMCID: PMC3688664  PMID: 23840653
22.  Analysis of the Origin and Evolutionary History of HIV-1 CRF28_BF and CRF29_BF Reveals a Decreasing Prevalence in the AIDS Epidemic of Brazil 
PLoS ONE  2011;6(3):e17485.
Background
HIV-1 subtype B and subtype F are prevalent in the AIDS epidemic of Brazil. Recombinations between these subtypes have generated at least four BF circulating recombinant forms (CRFs). CRF28_BF and CRF29_BF are among the first two BF recombinants being identified in Brazil and they contributed significantly to the epidemic. However, the evolution and demographic histories of the CRFs are unclear.
Methodology/Principal Findings
A collection of gag and pol sequences sampled within Brazil was screened for CRF28_BF-like and CRF29_BF-like recombination patterns. A Bayesian coalescent framework was employed to delineate the phylogenetic, divergence time and population dynamics of the virus having CRF28_BF-like and CRF29_BF-like genotype. These recombinants were phylogenetically related to each other and formed a well-supported monophyletic clade dated to 1988–1989. The effective number of infections by these recombinants grew exponentially over a five-year period after their emergence, but then decreased toward the present following a logistic model of population growth. The demographic pattern of both recombinants closely resembles those previously reported for CRF31_BC.
Conclusions
We revealed that HIV-1 recombinants of the CRF28_BF/CRF29_BF clade are still circulating in the Brazilian population. These recombinants did not exhibit a strong founder effect and showed a decreasing prevalence in the AIDS epidemic of Brazil. Our data suggested that multiple URFs may also play a role in shaping the epidemic of recombinant BF HIV-1 in the region.
doi:10.1371/journal.pone.0017485
PMCID: PMC3046974  PMID: 21390250
23.  Human Immunodeficiency Virus Type 1 Group M Protease in Cameroon: Genetic Diversity and Protease Inhibitor Mutational Features 
Journal of Clinical Microbiology  2002;40(3):837-845.
To establish a baseline for monitoring resistance to protease inhibitors (PIs) and examining the efficacy of their use among persons in Cameroon infected with human immunodeficiency virus type 1 (HIV-1), we analyzed genetic variability and PI resistance-associated substitutions in PCR-amplified protease (PR) sequences in strains isolated from 110 HIV-1-infected, drug-naïve Cameroonians. Of the 110 strains, 85 were classified into six HIV-1 PR subtypes, A (n = 1), B (n = 1), F (n = 4), G (n = 7), H (n = 1), and J (n = 7), and a circulating recombinant form, CRF02-AG (n = 64). PR genes from the remaining 25 (23%) specimens were unclassifiable, whereas 2% (7 of 301) unclassifiable PR sequences were reported for a global collection. Two major PI resistance-associated mutations, 20M and 24I, were detected in strains from only two specimens, whereas secondary mutations were found in strains from all samples except one strain of subtype B and two strains of CRF02-AG. The secondary mutations showed the typical PI resistance-associated pattern for non-subtype B viruses in both classifiable and unclassifiable PR genes, with 36I being the predominant (99%) mutation, followed by 63P (18%), 20R (15%), 77I (13%), and 10I or 10V (11%). Of these mutations, dual and triple PI resistance-associated substitutions were found in 38% of all the Cameroonian strains. Compared with classifiable PR sequences, unclassifiable sequences had significantly more dual and triple substitutions (64% versus 30%; P = 0.004). Phenotypic and clinical evaluations are needed to estimate whether PI resistance during antiretroviral drug treatment occurs more rapidly in individuals infected with HIV-1 strains harboring multiple PI resistance-associated substitutions. This information may be important for determination of appropriate drug therapies for HIV-1-infected persons in Cameroon, where more than one-third of HIV-1 strains were found to carry dual and triple minor PI resistance-associated mutations.
doi:10.1128/JCM.40.3.837-845.2002
PMCID: PMC120267  PMID: 11880402
24.  Near Full-Length Sequence Analysis of HIV Type 1 BF Recombinants from Italy 
Abstract
Recombination between HIV-1 subtypes B and F has generated several circulating and unique recombinant forms, particularly in Latin American areas. In Italy, subtype B is highly prevalent while subtype F is the most common pure non-B subtype. To investigate the recombination pattern in Italian BF recombinant viruses, we characterized full-length sequences derived from 15 adult patients, mostly Italian and infected by the heterosexual route. One of the BF mosaics was a CRF29, three sequences clustered with low bootstrap values with CRF39, CRF40, and CRF42. With the exception of the CRF29-like sequence, the other recombination patterns were unique, but two possible clusters were identified. Analysis of the gp120 V3 domain suggested a possible link with subtype F from Eastern Europe rather than from Latin America, favoring the hypothesis of local recombination between clade B and F viruses over that of import of BF recombinants from Latin America. HIV-1 subtypes B and F appear prone to generation of unique recombinants in Italy, warranting epidemiological surveillance and investigation of a possible clinical significance.
doi:10.1089/aid.2011.0002
PMCID: PMC3292755  PMID: 21740272
25.  HIV-1 Subtype F1 Epidemiological Networks among Italian Heterosexual Males Are Associated with Introduction Events from South America 
PLoS ONE  2012;7(8):e42223.
About 40% of the Italian HIV-1 epidemic due to non-B variants is sustained by F1 clade, which circulates at high prevalence in South America and Eastern Europe. Aim of this study was to define clade F1 origin, population dynamics and epidemiological networks through phylogenetic approaches. We analyzed pol sequences of 343 patients carrying F1 subtype stored in the ARCA database from 1998 to 2009. Citizenship of patients was as follows: 72.6% Italians, 9.3% South Americans and 7.3% Rumanians. Heterosexuals, Homo-bisexuals, Intravenous Drug Users accounted for 58.1%, 24.0% and 8.8% of patients, respectively. Phylogenetic analysis indicated that 70% of sequences clustered in 27 transmission networks. Two distinct groups were identified; the first clade, encompassing 56 sequences, included all Rumanian patients. The second group involved the remaining clusters and included 10 South American Homo-bisexuals in 9 distinct clusters. Heterosexual modality of infection was significantly associated with the probability to be detected in transmission networks. Heterosexuals were prevalent either among Italians (67.2%) or Rumanians (50%); by contrast, Homo-bisexuals accounted for 71.4% of South Americans. Among patients with resistant strains the proportion of clustering sequences was 57.1%, involving 14 clusters (51.8%). Resistance in clusters tended to be higher in South Americans (28.6%) compared to Italian (17.7%) and Rumanian patients (14.3%). A striking proportion of epidemiological networks could be identified in heterosexuals carrying F1 subtype residing in Italy. Italian Heterosexual males predominated within epidemiological clusters while foreign patients were mainly Heterosexual Rumanians, both males and females, and South American Homo-bisexuals. Tree topology suggested that F1 variant from South America gave rise to the Italian F1 epidemic through multiple introduction events. The contact tracing also revealed an unexpected burden of resistance in epidemiological clusters underlying the need of public interventions to limit the spread of non-B subtypes and transmitted drug resistance.
doi:10.1371/journal.pone.0042223
PMCID: PMC3410915  PMID: 22876310

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