One million people worldwide are infected with HIV-2. The distribution of HIV-2, unlike the global epidemic of HIV-1, is still mainly restricted to West Africa and several European and Asian countries.1-4 HIV-2 has been characterized as less pathogenic than HIV-1,5-11 with more than 75% of HIV-2-infected cases remaining asymptomatic throughout their clinical course.4 HIV-2 can be genetically classified into 8 groups, A to H, which have equivalent genetic distances to those of HIV-1 groups but not subtypes, with groups A and B circulating in the human population.12-16 In addition, 2 different AB recombinants (7312A and 510-03) have been identified in West Africa,12,13,17-19 but their circulation has not been identified to date.
In Japan, only 2 HIV-2-infected cases have been reported, but both were infected abroad.20,21 Inside the country, there has been no evidence of HIV-2 transmission and circulation. Here we report 5 HIV-2-infected cases recently identified in Japan. Of these 5 cases, 3 were shown by full-length genomic analysis to be infected with the same type of recombinant virus determined to be the first circulating recombinant form (CRF) of HIV-2.
HIV-2 Samples and Quantification of HIV Plasma Viral Loads
Among 843 HIV/AIDS cases registered at the Nagoya Medical Center (NMC), Japan from 1994 to 2008 (for demographic characteristics, see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A49), 5 cases (3 males and 2 females) were diagnosed serologically as HIV-2 infected. To better understand the molecular epidemiology of HIV-2 infection in Japan, we analyzed the HIV-2 genetic groups of the 5 cases.
Plasma HIV-1 viral loads were measured by the Cobas Amplicor HIV-1 monitor test v1.5 (Roche Diagnostics, Tokyo, Japan) or the Cobas TaqMan HIV-1 test (Roche Diagnostics), whereas plasma HIV-2 viral loads were measured by an in-house quantification assay, the Poisson quantification method described elsewhere.22,23 In brief, total RNA was extracted from 500 μL of plasma sample using the QIAamp UltraSens Virus Kit (QIAGEN, Tokyo, Japan). Reverse transcription (RT) and nested polymerase chain reaction (PCR) (RT-nested PCR) were performed using serially diluted RNA samples, and HIV-2 viral loads were statistically calculated using results from samples diluted to near the endpoint. (For details of RT-nested PCR reaction mixtures and thermal programs, see Table, Supplemental Digital Content 2, http://links.lww.com/QAI/A50).
Genomic DNA Sequencing
HIV-2 proviral DNAs were purified from peripheral blood mononuclear cells using the DNA blood mini kit (QIAGEN). To determine HIV-2 genetic groups, gag (777 bps: 1163 to 1939 according to SIVmac239) and env (454 bps: 7300 to 7753) gene fragments were amplified by nested PCR using LA Taq polymerase (Takara Bio, Shiga, Japan) and previously reported13,24 primers: gagA, gagB, gagC, and gagF for gag, and PFD1, LTR9574, EB2, and EB5 for env. To determine full-length genomic sequences, 4 DNA fragments containing (1) 5′ long terminal repeat (LTR) (915 bps: 31 to 945), (2) gag to nef genes (9122 bps: 899 to 10020), (3) 3′ LTR (791 bps: 9463 to 10252), and (4) the joining point of the circular 2 LTR form (597 bps: 10085 to 10279 and 1 to 402) were amplified by nested PCR using 8 primer pairs (see Table, Supplemental Digital Content 3, http://links.lww.com/QAI/A51). The following PCR program was used: denaturation (2 minutes at 94°C) followed by 40 cycles of PCR (94°C: 15 seconds, 60°C: 30 seconds, and 70°C: 1 minute/1000 bps). Sequencing was performed using a 3730 DNA Analyzer (Applied Biosystems, Tokyo, Japan).
Phylogenetic Tree Analysis and Determination of Recombinant Genome Structures
Multiple sequence alignment was performed using CLUSTAL W, and genetic distances were calculated based on the maximum composite likelihood model using MEGA software v4.25 Phylogenetic trees were constructed using the neighbor-joining method.
Complete full-length genomic sequences of 4 HIV-2 group A strains (ALI, BEN, CAM2CG, and UC2), 3 HIV-2 group B strains (D205, EHO, and UC1), and SIVmac239, (a rhesus macaque-adapted simian immunodeficiency viral isolate) were used as reference sequences. After realigning the sequence set, recombinant breakpoints were determined by similarity plotting, bootscanning, and informative site analysis using SimPlot software, v22.214.171.124
Estimated Times of the Most Recent Common Ancestors
Evolutionary rates, chronological phylogenies, and other evolutionary parameters were estimated from 17 full-length or near full-length HIV-2/SIV genomic sequences (see Table, Supplemental Digital Content 4, http://links.lww.com/QAI/A52) using the Bayesian Markov chain Monte Carlo (MCMC) method implemented in BEAST v126.96.36.199 The alignment data for the full-genome sequences were processed into 2 subsets consisting of sequences corresponding to the group A or B region of HIV-2 AB-recombinant virus. Bayesian MCMC analyses were performed using a relaxed molecular clock model.28 The nucleotide substitution model was evaluated by the hierarchical likelihood ratio test using PAUP v4.0 beta29 with MrModeltest (Nylander JAA. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University), and the general time-reversible model30 was adopted with both invariant sites and gamma-distributed site heterogeneity for 4 rate categories. The coalescent model used in the analyses was a logistically growing population because the population size of HIV-2 seemed constant in the early phase followed by exponential growth in the recent period.31 Each Bayesian MCMC analysis was run for 40 million states and sampled every 10,000 states. Posterior probabilities were calculated with a burn-in of 4 million states and checked for convergence using Tracer v1.4. The posterior distribution of the substitution rate obtained from the heterochronous sequences was subsequently incorporated as a prior distribution for the evolutionary rate of HIV-2 genome regions A and B, thereby adding a timescale to the phylogenetic histories of the HIV-2 strains and enabling the times of most recent common ancestor (tMRCAs) to be estimated.32
Nucleotide sequences have been registered as #AB499685 to AB499695 in the DNA databank of Japan.
HIV-2 Infection Confirmed by Nucleotide Amplification in Four AIDS Cases
Profiles of 5 HIV-2-seropositive cases are summarized in Table 1. The 3 males were from West African countries, a major endemic area for HIV-2, and suspected as seropositive before arriving in Japan. However, 2 females, both Japanese, were suspected to be recently infected within Japan based on their interviews. All their risk factors were heterosexual contacts, and no personal connection was confirmed among any of these cases. Thus, these 5 cases were independently infected with HIV-2 on different occasions. Notably, 4 cases (NMC307, NMC716, NMC786, and NMC842) were found at advanced stage AIDS with low CD4+ cell counts and high HIV-2 viral loads, accompanied by opportunistic infections (Table 1). One case (NMC678) was found at an asymptomatic stage with high CD4+ cell count and undetectable viremia. HIV-1 RNAs were undetectable in all 5 cases, indicating that they were infected by HIV-2 alone.
The First Circulating Recombinant Form Discovered in HIV-2: HIV-2 CRF01_AB
HIV-2 genetic groups were determined by both gag and env sequences. We were successful in analyzing 4 AIDS cases, however, we failed to amplify these 2 genes and analyze in asymptomatic case NMC678. One isolate (NMC786) was clearly classified into group A in phylogenetic tree analysis (Fig. 1A, B). On the other hand, isolates NMC307, NMC716, and NMC842 formed an independent cluster with a reference AB recombinant isolate 7312A (Fig. 1A, B). To better understand the detailed genomic structures of the 3 suspected AB recombinants, full-length genomic sequences of the 3 cases were analyzed. In the phylogenetic tree with full-length or near full-length reference sequences (Fig. 1C), NMC307, NMC716, NMC842, and 7312A formed an independent cluster with a high bootstrap value of 100%, suggesting these 4 isolates are the same type of AB-recombinant virus.
We next compared their genomic structures. As shown in Fig. 2A, similarity plotting and bootscanning analyses revealed that the recombinant breakpoints of our 3 isolates perfectly matched those of 7312A. This finding was supported by subregion phylogenetic analyses (Fig. 2B). In conclusion, NMC307, NMC716, and NMC842 are AB-recombinant forms with a mosaic genome structure identical to that of 7312A, demonstrating that they are the same type of HIV-2 AB-recombinant form.
The minimum requirement for declaring a new CRF, as proposed by the Los Alamos HIV sequence database in 1999, is at least 3 cases with no direct linkage, accompanied with near full-length sequences.33,34 These CRF nomenclature requirements are perfectly fulfilled with full-length genomic sequence information for 4 cases independently infected on different occasions with the AB recombinant identified by us and others.12,13,19 Our data were carefully reviewed by editors of the Los Alamos HIV sequence database and confirmed as the first CRF discovered in HIV-2. They decided that the least confusing and most consistent way to name this new strain was to call it HIV-2 CRF01_AB.
The genomic structure of CRF01_AB is shown in Fig. 3. Interestingly, all 4 recombinant breakpoints of the CRF were located near or within the env gene (Fig. 3A). Further detailed analysis revealed that CRF01_AB possessed a chimeric gp120 containing a backbone of group A and a partial C2V3 fragment of group B and a chimeric gp41 containing extracellular and transmembrane domains of group A and a cytoplasmic domain of group B (Fig. 3B).
CRF01_AB Emerged Approximately in the Mid 20th Century
To estimate the time of CRF01_AB emergence, the time of the most recent common ancestor (tMRCA) of the recombinant was calculated by the Bayesian MCMC method. The mean substitution rates per year for the group A and B regions were estimated as 2.22 × 10−3 and 1.64 × 10−3, respectively (Table 2), and the mean tMRCAs for groups A and B were estimated from 1921 to 1929, and from 1909 to 1948, respectively (Table 3). Similar results31,37 validate our estimations. Finally, the mean tMRCA of CRF01_AB was estimated from 1964 to 1973. As the emergent times for groups A and B were estimated in the early 20th century, several decades seem to have been required for CRF01_AB to emerge. Concerning the geographical origin of the recombinant form, 3 of 4 isolates (7312A, NMC307, and NMC716) were identified in West Africans from Côte d'Ivoire and Nigeria. As these 2 countries were reported as sites of an epidemic in HIV-2 group A and B strains,38,39 the most likely geographical origin of CRF01_AB is the south coastal area of West Africa.
In this study, we identified 3 HIV-2 AB recombinants with the same recombination pattern as 7312A, an isolate reported in Côte d'Ivoire in 1990.12,13,19 These 4 isolates are determined as the first CRF of HIV-2, named CRF01_AB. It is noteworthy that all 3 of our cases infected with CRF01_AB were found at the AIDS stage. Considering that more than 75% of HIV-2-infected cases have a prognosis of remaining asymptomatic throughout their lifetimes4 and that few HIV-2-seropositive cases were reported in Japan in the last 2 decades, 3 HIV-2 cases in the AIDS stage infected with the same CRF and identified in the past 5 years is highly unusual. Regarding the incubation periods for AIDS development in the 3 cases, not much information was available except for NMC842. This case was found to be seronegative for HIV-1/2 when tested in 2000. Thus, this case seems to have developed AIDS at most within 8 years, same as the median incubation period for AIDS development in HIV-1 infections (7.7-12.3 years).40-45 As for the other 2 cases (NMC307 and NMC716), they developed AIDS at 28 and 36 years old (Table 1), which is significantly younger than age 65, reported as the peak of death by HIV-2 infections.46,47 Though the number of cases identified is still small, we are concerned that the CRF01_AB might have acquired higher pathogenicity through recombination and adaptation to humans. As shown in Figure 3B, CRF01_AB has a recombination in the C2V3 region, the site of the major determinant for anti-envelope host immune responses and a functional domain for the chemokine receptor-binding site. The chimeric structure in the C2V3 region may confer advantages in host immune escape and viral replication capacity.
According to tMRCA analysis of the 4 isolates, CRF01_AB is estimated to have emerged sometime between 1964 and 1973. Interestingly, the mean tMRCA of the 3 isolates collected at NMC was estimated from 1982 to 1995 (Table 3), a later estimate than that of the 4 isolates, suggesting ongoing selection and evolution of CRF01_AB through transmission which has been taking place from the era of the 7312A isolate to the NMC isolates.
In conclusion, we report here the first CRF of HIV-2, CRF01_AB. Although national borders worldwide have become more porous than ever, it is still surprising that the same recombinant strain was harvested in Japan, an island nation remote from the original endemic area, West Africa. This ectopic observation of the virus outside its endemic area suggests an ongoing global spread of HIV-2 CRF01_AB.
We thank Dr. Thomas Leitner and editors of the Los Alamos HIV sequence database for discussing our data and naming the new HIV-2 circulating recombinant form. We thank Dr. Koya Ariyoshi for critical reading of our article and Claire Baldwin for her help in preparing the article.
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circulating recombinant form; CRF01_AB; HIV-2; molecular epidemiology
Supplemental Digital Content
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