Molecular Investigation of Human Immunodeficiency Virus Type 2 Subtype A Cases in South Korea

Abstract

We investigated the molecular characteristics of human immunodeficiency virus type 2 (HIV-2) subtype A isolates to clarify the transmission mode of HIV-2 within Korea. These findings indicated that the viruses from the six patients infected within Korea formed a distinct subcluster in the phylogenetic tree and might have been transmitted from one source.

Human immunodeficiency virus type 2 (HIV-2) infection is restricted primarily to West Africa, Brazil, and Portugal (14). In Asia, 95% of the reported HIV-2 cases came from India (7, 13). Molecular epidemiology studies have led to classification of HIV-2 into eight specific subtypes, subtypes A to H (5). Molecular epidemiological analysis of nucleotide sequence data has been used in several investigations of HIV-1 transmission (1, 2, 10, 11, 12). Although the rate of sexual transmission of HIV-2 is very low relative to that of HIV-1, HIV-2 appears to be transmitted by the same routes as HIV-1 (6). Molecular evidence of homosexual transmission of HIV-2 in Spain has been reported (4).

In addition to the five HIV-2-infected individuals reported in our previous paper (8), five new HIV-2-infected people were identified from 2000 to 2002. Four of these additional five HIV-2-infected individuals were women between 19 and 21 years of age and were infected within Korea (Table 1). Of interest was that 9 of the 10 HIV-2-infected individuals resided in the southern part of Korea (the “SP” region), and domestic transmission was accounting for a large portion of the cases of HIV-2 transmission in Korea. Therefore, we conducted molecular epidemiological research on the HIV-2 isolates to find why HIV-2 infection was located in a specific region, to analyze the scale of HIV-2 infection, and to determine the future transmission trend for Korea.

TABLE 1.

Epidemiological histories of individuals infected with HIV-2

Subject	Yr of HIV detection	Age (yr)/sexa	Most likely place of infection	Residenceb (city)	Probable mode of transmission	Disease status (reference)
KRC2235	2002	19/F	Korea	D	Heterosexual contact	Asymptomatic
KRC2080	2002	46/M	Congo	S	Heterosexual contact with foreigner	Tuberculosis (9)
KRC2044	2002	19/F	Korea	B	Heterosexual contact	Asymptomatic
KRC0115	2000	21/F	Korea	B	Heterosexual contact	Asymptomatic
KRC0047	2000	21/F	Korea	B	Heterosexual contact	Asymptomatic
KRB8091	1998	18/M	Korea	B	Homosexual contact	Asymptomatic
KRB7051d	1997	55/M	Las Palmas de Gran Canaria	B	Heterosexual contact with foreigner	Oral candidaisis, herpes zoster rash
KRB4075	1994	40/F	Korea	B	Heterosexual contact	Asymptomatic
KRB4063	1994	47/M	Senegal	B	Heterosexual contact with foreigner	Asymptomatic
KRB1010c	1991	29/M	Las Palmas de Gran Canaria	B	Heterosexual contact with foreigner	Asymptomatic

^aF, female; M, male.

^bS, B, and D, initials of city names in Korea. The geographical distances between S and B, between S and D, and between B and D are 395 km, 280 km, and 115 km, respectively.

^cNot detected by PCR.

^dSubject died of AIDS.

This retrospective study was conducted with 10 HIV-2-infected individuals who had shown specific HIV-2 antibody reactivity by Western blot and particle agglutination assays from 1991 to 2004. According to Korea's monitoring policy for HIV-infected and AIDS patients, we measured CD4 and CD8 lymphocyte numbers every 6 months to monitor disease progression. We collected data on the epidemiological and clinical aspects of HIV-infected and AIDS patients, such as demographic characteristics and transmission routes (Table 1).

Of the 10 HIV-2-infected Koreans, the C2 to C5 region of HIV-2 env gene was analyzed for 9 of them. We conducted sequence analysis of two or three clones from them, as in the previous study (8). As shown in Fig. 1, based on the env gene alignment, isolates from eight patients clustered within the known subtype A. However, isolate KRC2080 belonged to subtype B. The patient with subtype B had engaged in unprotected heterosexual contact with prostitutes in Congo, which led to the introduction of the HIV-2 subtype B isolates into Korea. Interestingly, the earliest identified HIV-2-infected individual (subject KRB7051), a coresident of subject KRB7051 (subject KRB8091), and four female cases (subjects KRC0047, KRC0115, KRC2044, and KRC2235) formed a cluster whose sequences were distinguishable from the foreign reference sequences (bootstrap value = 100) (Fig. 1).

FIG. 1.

Phylogenetic analysis of the C2 to C5 region of the HIV-2 env gene obtained from nine Korean isolates and 35 reference sequences. Multiple alignments of the sequences were constructed by the ClustalW method in MegAlign by using Lagergene software (DNASTAR Inc.). A phylogenetic tree was constructed by the neighbor-joining method using the PAUP* program (version 4.0b10; Florida State University). Bootstrap resampling (1,000 data sets) of the multiple alignments was done to test the statistical robustness of the tree. We used 35 reference sequences sourced from GenBank for comparison with the Korean sequences: 25 subtype A isolates, 19 of which belonged to Africans and 6 to Europeans; 8 subtype B isolates; 1 subtype AB isolate; and 1 subtype G isolate. The genetic diversities of the subject clones were 0.6% (subject KRB7051), 1.6% (subject KRB8091), 0.1% (subject KRC0047), 2.0% (subject KRC0115), 1.7% (subject KRC2044), and 0.2% (subject KRC2235). The scale bar indicates 10% nucleotide distance. The suffix “1” in “KRB7051-1” denotes the clone number.

We randomly selected one clone sequence from each patient and analyzed the genetic diversity, divergence, and similarity. The interpatient sequence variability of the “SP” subcluster was 3.8 ± 1.0, an extremely low variability between sequences compared with the variability within the eight domestic Korean isolates (8.4 ± 5.4), African isolates (14.1 ± 2.8), and European isolates (12.8 ± 2.4) (Table 2). Furthermore, the divergence of the C2 to C5 region sequences between Korean and African isolates was lower than that between Korean and European isolates. This indicates that the nucleotide sequences of HIV-2 subtype A circulating in Korea are close to those of the African strain used in the reference sequences. These results are consistent with the epidemiological histories of the HIV-2-infected patients.

TABLE 2.

Diversity and divergence of nucleotide sequences of HIV-2 env gene obtained from Korean isolates and references

Parameter and subject	C2 to C5		V3
	Mean ± SD (%)	Range (%)	Mean ± SD (%)	Range (%)
Diversity within HIV-2 subtype A isolates
Korean (n = 8)	8.4 ± 5.4	2.3-15.5	6.6 ± 3.3	2.0-11.9
SPa subcluster (n = 6)	3.8 ± 1.0	2.3-5.4	4.1 ± 1.4	2.0-6.3
African (n = 19)	14.1 ± 2.8	0.2-19.8	14.9 ± 6.0	0.0-31.6
European (n = 6)	12.8 ± 2.4	10.5-16.7	13.6 ± 5.2	6.2-21.8
Divergence between HIV-2 subtype A isolates
Korean/SP subcluster	14.5 ± 0.7	13.3-15.5	9.9 ± 1.6	7.3-11.9
Korean/African	14.1 ± 2.0	10.1-19.0	13.2 ± 5.3	6.2-25.1
Korean/European	15.9 ± 1.5	12.5-18.5	13.1 ± 3.8	5.1-20.5
African/European	15.3 ± 2.3	7.8-20.5	15.9 ± 5.1	5.1-29.9

^aSP, southern part of Korea. City B and city D in Table 1 belong to SP.

In addition, we investigated each patient's similarity with subject KRB7051, the earliest diagnosed patient in the “SP” subcluster (Table 3). The similarity of the sequence of the isolate from subject KRB7051 to that of the isolate from subject KRB8091 was 97.7%, followed by that to the isolate from subject KRC0047 with 97.0%, that to the isolate from subject KRC2044 with 96.8%, that to the isolate from subject KRC0115 with 96.3%, and that to the isolate from subject KRC2235 with 95.3%. The similarities of the sequence of the isolate from subject KRB7051 to those of the isolates from subjects KRB4063 and KRB4075, which were not in the “SP” subcluster, were 86.4% and 86.9%, respectively. Among the four female cases in the “SP” subcluster, two had had multiple sexual partners, and the other two recorded that they had had multiple contacts with an unidentified male.

TABLE 3.

Similarity of nucleotide sequences of HIV-2 env gene obtained from Korean isolates

Subtype and subject	Similarity (%) to KRB7051 sequence
	C2 to C5	V3
Subtype A
SPa cluster
KRB8091	97.7	97.1
KRC0047	97.0	96.1
KRC0115	96.3	94.1
KRC2044	96.8	96.1
KRC2235	95.3	97.1
Non-SP cluster
KRB4063	86.4	90.2
KRB4075	86.9	90.2
Subtype B, KRC2080	75.3	76.5

^aSP, southern part of Korea. City B and city D in Table 1 belong to SP.

HIV-2 infection is mostly confined to West Africa and is rarely discovered in Asia, except in India (7). In Korea, HIV-2 infection was first detected in the early 1990s in a seafarer infected overseas and his wife (8); however, cases of domestic infection have been identified since 2002. In particular, two infected females were in a potentially high-risk group for heterosexual transmission, which indicated a need for research on HIV-2 transmission routes in Korea.

Subject KRB7051 was the earliest patient in the “SP” subcluster detected and was followed up five times since being identified in 1997. His CD4 count stayed at less than 200 cells/μl, and he died of AIDS in 1999. We could not find any information on subject KRB8091. Four infected women were followed up for about 24 to 48 months and kept relatively high CD4 counts of more than 500 cells/μl, with no reports of specific opportunistic infections. Subject KRC2080, who was infected with subtype B, was detected in 2002 and was reported by a Japanese team (9). Extensive investigation of his history showed that he had experienced sexual contacts with prostitutes in Africa (1980 to 1984), Korea, and Japan (1989 to 2002). He also had a history of tuberculosis, and despite treatment with antiretroviral drugs, his CD4 counts gradually decreased to less than 250 cells/μl.

The genetic diversity of the HIV-2 isolates in the eight patients infected with subtype A was 8.4%, which is consistent with the values of 11.0 to 29.0% reported in other countries (3) and 12.8 to 14.1% in the reference sequences. The genetic diversity of the isolates from the six patients belonging to the “SP” subcluster was 3.8%, which is extremely low compared with the genetic diversity of 5.6% in a homosexual group in Spain (4). Furthermore, the V3 region genetic diversity in our Korean “SP” subcluster was 4.1%, which is lower than the interpatient variability range of 8.2 to 14.0% for unrelated viruses (15).

These results indicate that the viruses from the six patients infected inside Korea were closely related and might have been transmitted from one source. Although it was not revealed in the epidemiological investigation, there was suspicion of homosexual activity between two male patients (subjects KRB7051 and KRB8091) who once shared the same residence. HIV-2 infection and transmission inside Korea are rare and are restricted to specific areas.