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We aimed to investigate whether the sequence length of HIV-1 increases over time. We performed a longitudinal analysis of full-length coding region sequences (FLs) during an HIV-1 outbreak among patients with hemophilia and local controls infected with the Korean subclade B of HIV-1 (KSB). Genes were amplified by overlapping RT-PCR or nested PCR and subjected to direct sequencing. Overall, 141 FLs were sequentially determined over 30 years in 62 KSB-infected patients. Phylogenetic analysis indicated that within KSB, two FLs from plasma donors O and P comprised two clusters, together with 8 and 12 patients with hemophilia, respectively. Signature pattern analysis of the KSB of HIV-1 revealed 91 signature nucleotide residues (1.1%). In total, 48 and 43 signature nucleotides originated from clusters O and P, respectively. Six positions contained 100% specific nucleotide(s) in clusters O and P . In-depth FL analysis for over 30 years indicated that the KSB FL significantly increased over time before combination antiretroviral therapy (cART) and decreased with cART. This increase occurred due to the significant increase in env and nef genes, originating in the variable regions of both genes. The increase in sequence length of HIV-1 over time suggests an evolutionary direction.

We determined the earliest full-length HIV-1 gag gene sequences in 110 patients with HIV-1, including 20 hemophiliacs (HPs) and 90 local controls (LCs). The gag gene from stored sera was amplified using RT-PCR, and was subjected to direct sequencing. Phylogenetic analysis indicated that 94 and 16 sequences belonged to the Korean subclade of HIV-1 subtype B (KSB) and subtype B, respectively. A total of 12 signature pattern amino acids were found within the KSB, distinct from the worldwide consensus of subtype B. Within the KSB, the gag gene sequences from donors O and P and those from the 20 HPs comprised two subclusters. In particular, sequences from donor O strongly clustered with those of eight HPs. Moreover, signature pattern analysis indicated that 14 signature nucleotides were shared between the HPs and LCs within KSB ( p < 0.01). Among the 14 nucleotides, positions 9 and 5 belonged to clusters O and P, respectively. In conclusion, signature pattern analysis for the gag gene revealed 12 signature pattern residues within the KSB and also confirmed the previous conclusion that the 20 HPs were infected with viruses due to incompletely inactivated clotting factor IX. This study is the first genetic analysis of the HIV-1 gag gene in Korea.

There have been no studies related to groups at the highest risk for HIV-1 infection in Korea before 1993. In this study, for the first time, we report the distribution of HIV subtypes in overseas sailors (OSs) and prostitutes who worked in brothels near U.S. military bases in Korea. We retrospectively determined the sequences of nef in 131 patients using reverse transcription polymerase chain reaction (RT-PCR). These patients composed of 102 OSs, 14 OS spouses, and 15 prostitutes. Phylogenetic analysis was performed using 128 Korean OSs, OS spouses, and prostitutes. The distribution of non-B subtypes (n = 105) was as follows: 39, CRF02&#95;AG; 15, CRF01&#95;AE; 7, A1; 7, A2; 6, D; 2, CRF06&#95;cpx; 3, C; 6, G; 11, untypable; and 1 each for CRF09&#95;cpx, CRF12&#95;BF, CRF50&#95;A1D, A3, AFG, H, F1, F2, and A. Of the 116 OSs and OS spouses, 101 (87%), 11 (9%), and 4 (3%) subjects had non-B, Western B, and Korean subclade B (KSB) HIV-1s, respectively. Among the 15 prostitutes, 10 had Western B (67%), 4 non-B (27%), and 1 KSB (7%) HIV-1s. All 14 couples, each comprising of an OS and his spouse, had the same subtype. KSB (5%) was detected in OSs and prostitutes in 1990 and 1994, respectively. Of the 131 patients analyzed in this study, 105 (80%), 21 (16%), and 5 (4%) were infected with the non-B, Western B, and KSB subtypes of HIV, respectively. In future, these data may provide an important foundation for analysis of HIV-1 subtypes in Korea.

The epidemiological link in the hypervariable env gene between viruses infecting HIV-positive hemophiliacs (HPs) and plasma donors was not studied. We determined full-length env gene sequences in 20 HPs, 3 plasma donors whose plasma was used for domestic clotting factor (DCF) production, and 54 local controls (LCs). Env genes from viruses in frozen stored sera obtained 1-3 years after diagnosis and from samples collected several years after infection were amplified via RT-PCR and subjected to direct sequencing. Phylogenetic analysis indicated that all sequences were subtype B, including 133 sequences from 77 cases (20 HPs, 3 plasma donors, and 54 LCs) belonging to the Korean subclade B (KSB) and 6 sequences from 5 cases that did not belong to the KSB. Env gene sequences from donors O and P and those of the 20 HPs comprised 2 subclusters within the KSB, although phylogenetic analysis did not support significant bootstrap values. In contrast, signature pattern analysis indicated signature nucleotides at 43 positions between the HPs and LCs (P < 0.05). In particular, specific signature nucleotides at 4 positions were fully conserved in the HPs, but not in the LCs (P < 0.0001). Furthermore, there were 26 signature residues within the KSB and were distinct from the worldwide consensus for subtype B. In conclusion, signature pattern analysis for the hypervariable env gene revealed an epidemiological link that the 20 HPs in this study had been infected with viruses from the DCF used for treatment, consistent with our previous finding.

Insertion mutations at codon 69 (T69-ins insertion) of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase confer full resistance to all approved nucleoside reverse transcriptase inhibitors. To date, nearly all reports on T69-ins insertions have described subtypes B and rarely subtypes A, C, and F of HIV-1. Here, we provide the first report of a T69-ins insertion in circulating recombinant form (CRF) 06&#95;cpx in a patient who had been treated with a zidovudine/didanosine combination for 18 months and then shifted to lamivudine, stavudine, and nelfinavir for 76 months. Thereafter, the patient was additively administered Korean red ginseng. This is the first report on the appearance of the T69-ins insertion mutation in CRF HIV-1.

The Korean subclade of subtype B (KSB) is the most prevalent HIV-1 strain found in Korea. To date, only two near full-length HIV-1 sequences from Korean patients have been reported. Here, we analyzed a total of 24 near full-length genomes of HIV-1 strains that were isolated from 17 antiretroviral therapy (ART)-naive patients and four ART-exposed patients. Proviral DNA from peripheral blood mononuclear cells was PCR amplified and directly sequenced. Phylogenetic analyses were used to classify viruses from 19 patients as KSB, from one patient as subtype B, from one patient as subtype D, and three viruses from one patient as CRF02&#95;AG. All KSB viruses demonstrated TAAAA instead of TATAA at the TATA box in the LTR. Of the 19 KSB patients, their sequence identities at the nucleotide level ranged from 89.8% to 97.1% from the lowest env gene to the highest pol gene. Other than the CRF02&#95;AG viruses, no recombination events were noted in any of the 19 KSB patients, which is consistent with our previous studies on the pol, vif, and nef genes. Except for one strain, all of the strains were classified as non-syncytium-inducing strains. This is the first report to describe near full-length KSB.

There was an outbreak of HIV-1 transmission among 20 out of 122 Korean hemophiliacs from 1990. We assessed the genetic relationships among HIV-1 viruses found in three cash-paid plasma donors whose preseroconversion plasma was used to produce Korean-made clotting factor, 20 hemophiliacs infected with HIV-1 in Korea, three hemophiliacs infected with HIV-1 from clotting factor manufactured outside Korea, and 71 local control patients infected with the Korean subclade of HIV-1 subtype B (KSB). Full-length pol gene sequences (2841 bp) of viruses from frozen stored serum, samples obtained 1-3 years after diagnosis, were amplified by RT-PCR and sequenced by direct DNA sequencing. Phylogenetic and signature pattern analyses were used to investigate the relationships among the sequences. Donors O and P were associated with two clusters, of 8 and 12 hemophiliacs, respectively, which were demarcated from the 71 KSB-infected local control patients and donor R. These data confirm that HIV-1 transmission to 20 hemophiliacs occurred through infusion of Korean-made clotting factor.

To determine whether Korean red ginseng (KRG) has beneficial effects on human immunodeficiency virus type 1 (HIV-1)-infected patients administered highly active antiretroviral therapy (HAART), we analyzed the CD4 T-cell count, viral load, and resistance mutations to HAART in 46 individuals. Thirteen patients harbored resistance mutations at baseline. The study population was divided into two groups: specifically, a group treated with a combination of HAART plus KRG (23 patients) and a group treated with HAART alone (23 patients). The annual increase in CD4 T-cell count in the combination group was significantly higher than that in the group treated with HAART alone (P < 0.05). Overall, 21 patients harbored resistance mutations after 3 years of therapy. Following exclusion of 13 patients displaying baseline resistance mutations, 7.1% of patients (1/14) in the combination group and 42.1% (8/19) in the HAART group were identified with resistance mutations. One patient with baseline resistance mutations in the combination group did not display resistance mutations 3 years after HAART therapy. High-level resistance mutations were significantly lower in the combination group than in the group treated with HAART alone. Five patients showed no improvement in viral copy number (26.3% [5/19]) in the combination group and 9 (45.0% [9/20]) showed no improvement in the HAART-only group. Our data support the clinical utility of KRG intake during HAART therapy.

Abstract We have shown that Korean red ginseng (KRG) intake is associated with gross deletions in the 5' LTR/gag (gDeltaLTR/gag) and nef genes (gDeltanef) of patients infected with subtype B of HIV-1. Here, we investigated these effects in three long-term survivors (LTSs) of subtype CRF02&#95;AG of HIV-1. The three LTSs were diagnosed with HIV in 1987, 1988, and 1989, and have been treated with KRG for 7-15 years. Thirty-two samples of peripheral blood mononuclear cells were obtained from the subjects and used to amplify the 5' LTR/gag and nef genes via nested PCR. We obtained 88 amplicons in 5' LTR/gag and 128 amplicons in nef. The frequency of gDeltaLTR/gag was significantly higher (37.5%) in three LTSs than in control patients (8.6%, p < 0.01). Eight amplicons (9.5%) contained premature stop codon(s) in the gDeltaLTR/gag in three LTSs. Fourteen of the 128 nef amplicons (10.9%) contained the gDeltanef, which was present in only two (7.7%) of the 26 amplicons from control subjects. Interestingly, gDeltanef was detected 7 years after the reinitiation of KRG intake in an LTS and, coincidently, CD4 T cell counts and CD4/CD8 ratios rapidly increased. These data indicate that long-term intake of KRG has the therapeutic potential to induce gross deletions in HIV-1.

Twenty hemophiliacs were infected with Korean subclade B (KSB) of HIV-1 from two cash-paid plasma donors in Korea in 1990. Our previous studies revealed that Korean red ginseng (KRG) intake increases the frequency of gross deletion in the nef gene (gDeltanef). We investigated whether KRG and highly active antiretroviral therapy (HAART) affected the frequency of gDeltanef in the 20 hemophiliacs who share common characteristics of the HIV-1 source, mode of transmission, and infection time. Over a 10-year period, we obtained 522 nef amplicons by nested PCR using 172 samples of peripheral blood mononuclear cells. Of the 522 nef amplicons, 69 (13.2%) were gDeltanef. Despite a 2-fold higher monthly dose of KRG, the frequency of gDeltanef detection (3.2%) was significantly reduced during HAART compared with that prior to HAART (20.6%) (p < 0.001). gDeltanef was detected significantly more in patients treated with a monthly KRG intake of more than 60 g (26.8%) than in patients treated with a monthly KRG intake of less than 60 g (10.5%) (p < 0.05). These finding suggest that the frequency of gDeltanef is dependent on the amount of KRG intake, although further study is needed. These data might provide a new perspective on the pathogenesis of HIV-1.

Our previous studies have shown that gross deletions in the nef gene as well as slow decreases in CD4 T cell numbers are associated with Korean red ginseng (KRG) intake in HIV-1-infected patients. To determine whether there might be an association between KRG intake and occurrence of gross deletions (gDelta) in the 5' LTR and gag regions, we examined the 1125 base pair (bp) sequences encompassing these regions in 10 long-term survivors (LTSs) treated with KRG (total of 13,364 +/- 5364 g) for > 12 years, and in 8 LTS control patients with no or minimal (total of 1436 +/- 1027 g) KRG intake (LTS controls). In the 10 LTSs, 189 PCR products were obtained from 80 peripheral blood mononuclear cell (PBMC) samples. In total, 44 of the 80 PBMC samples (55%) and 71 of the 189 PCR products (37.6%) displayed gDelta. While 55% of PBMC samples and 37.6% of PCR products showed gDelta in the 10 LTSs, the corresponding figures for the eight LTS controls were 30.3% and 14.8%. These differences were significant (p < 0.05 and p = 0, respectively). In addition, the proportions of 28 patients in the general population (without KRG intake) displaying PBMC and PCR gDelta were 13.3% and 8.3%, respectively. Our data strongly suggest that gDelta occurrence in the HIV-1 5' LTR and gag regions is associated with KRG intake.

A nested PCR and direct sequencing methods were used to define human immunodeficiency virus type 1(HIV-1) reverse transcriptase codons 41 to 219 in DNA from 127 peripheral blood mononuclear cell samples obtained from 35 patients treated with nucleoside reverse transcriptase inhibitors (NRTI). The follow-up period after the initiation of NRTI therapy was 61.8 +/- 31 months (mean and standard deviation). In addition to NRTI therapy, 32 of 35 patients were simultaneously treated with Korean red ginseng. The annual decrease in the CD4(+) T-cell count over 5 years was 13.2/microl. Twenty-eight (80%) of the 35 patients had mutations conferring resistance to NRTI. The frequencies of K70R, T215S/Y/F (i.e., mutation of T at codon 215 to S, Y, or F), D67N/E, K219Q, T69N/S/A, M41L, and L210W mutations conferring resistance to zidovudine were 57.6, 36.4, 36.4, 27.2, 24.2, 21.2, and 12.1%, respectively. Mutations conferring resistance to didanosine and lamivudine were detected in 2 (L74V and M184I; 14.2%) of 11 patients tested and in 4 (M184V; 57%) of 7 patients tested, respectively. In particular, the frequency of T69N/S/A increased sharply after more than 48 months of zidovudine monotherapy. However, Q151M was not detected. As the first report on the frequency of NRTI resistance mutations in Korea, our data suggest that genotypic antiretroviral drug testing should be considered for the design of better drug regimens to improve the management of HIV-1-infected patients.