AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 8, 1992 Mary Ann Liebert, Inc., Publishers
Wide Distribution of Two
Subtypes of HIV-1
in Thailand
CHIN-YIH OU,1 YUTAKA TAKEBE,2 CHI-CHENG LUO,1 MARCIA KALISH,1 WATTANA AUWANIT,3 CLAUDIU BANDEA.' NICK DE la TORRE,1 J.L. MOORE,1 GERALD SCHOCHETMAN,1 SHUDO YAMAZAKI,2 HELENE D. GAYLE,1 NANCY L YOUNG,'4 and BRUCE G. WENIGER1'4
INTRODUCTION
and sequence determination. HIV genetic characterization was performed on specimens from HIV-1-infected persons by sequencing approximately 300 nucleotides including the C2-V3 domains of HIV-1 envelope gene and/or hybridization.
HIV-1 reported
was introduced recently into Thailand, with the first case of AIDS in 1984,1 and HIV antibody first detected among small numbers (1-3%) of male prostitutes in 1985.2"1 Seroepidemiologic surveys from 1985 through 1987 failed to detect HIV infection among intravenous drug users (IVDU).4 Widespread transmission of HIV began in 1988 with 15-20% HIV seropositivity detected in the IVDU population in Bangkok in early 1988, and reaching more than 40% by the end of 1988.5-7 The explosive nature of the HIV epidemic and its extensive geographic distribution was documented by several national sérologie surveys.5,6-8>9 Recent projections estimated that 1.6 million (or more than 1 in 40) Thais will be infected with HIV by 1995 unless immediate dramatic measures are imple-
mented.1"
Because the HI V epidemic in Thailand is so recent, one would expect to find a high degree of genetic diversity among the HIVs currently circulating in that population unless there were a large number of HIV strains that were introduced into the country and these were responsible for the epidemic. However, if only a small number of viral strains were introduced, it should be possible to determine the number of HIV strains initially responsible for the epidemic and their possible geographic origins. We describe here the identification and genetic characteristics of two predominant HIV-1 subtypes in Thailand based on the specimens collected from various high-risk groups representing all geographic regions of the country. Heparinized blood was collected from persons in various high-risk groups by staff from the seven regional laboratories of the Department of Medical Sciences of the Thailand Ministry of Public Health and was shipped, usually overnight, to the National Institutes of Health (NIH) in Bangkok for lymphocyte separation, DNA extraction, and virus culture. Extracted DNA specimens subsequently were shipped to the Centers for Disease Control (CDC), Atlanta, GA, for sérologie testing, polymerase chain reaction (PCR) using procedures described elsewhere," not
METHODS We used Kimura's method12 to analyze sequence variations between any two sequences within the same subject or between two subjects to yield intraperson and interperson variations,
respectively. Comparisons of the
amino acid sequences of the V3 loop of HIV-Is in Thailand demonstrate that there are two distinct subtypes, A and B. Subtype A appeared to be present in every risk group examined, while subtype B appeared to be present primarily in IDUs. HIV-1 strains within a subtype were closely related to each other. The average intraperson variation for subtypes A and B, was 2.0% and 2.7%, respectively. The average interperson variation within subtype A and B was 3.8% and 3.7%, respectively. This interperson variation (3.7-3.8%) was severalfold lower than those observed within Africa, Haiti, and the United States,1"1 where the HIV-1 epidemic was known to have begun at least a decade earlier than in Thailand. The average interperson variation between subtypes A and B from different persons, (i.e., the intersubtype variation), was 18.1%. Assuming that the rate of variation in the HIV V3 region is 0.5-1% per year13 and that subtypes A and B were derived from the same prototype HIV-1 which originally entered the country, it would have taken at least 18 years to have the prototype HIV-1 evolve into the two subtypes currently present in Thailand. Therefore, it is more likely that the HIV-1 subtypes A and B entered the country recently, yet independently from each other. Phylogenetic tree analysis indicated that subtype B is more closely related to North American isolates than to African isolates and that subtype A is equally distant from all the United
'Division of HIV/AIDS, Centers for Disease Control, U.S. Public Health Service. Atlanta. GA 30333. 2National Institute of Health, Tokyo, Japan. 'National Institute of Health. Bangkok, Thailand.
4HIV/AIDS Collaboration. Bangkok. Thailand.
1471
1472
OU ET AL.
States and African isolates with a range of 20-30%. Of interest is that most HIV sequences contained the GPGQ motif at the tip of the V3 loop regardless of their subtype. The GPGQ sequence is the most common motif in African HIV-1 strains11 but rarely is found in HIV-1 strains in North America. In contrast, most (>85%) of the HIV-1 strains isolated from North America, Europe, and Japan possess the GPGR motif.
analysts, to Richard George for assistance in specimen shipping and study management, Karen Potts and Gregg Orloff for their critical review of the manuscript. Y.T. was partially supported by a Japanese Foundation for AIDS Prevention. and tree
REFERENCES 1.
RESULTS Our findings suggest that two HIV-1 strains currently predominate in Thailand. It appears that although subtype A and B were responsible for the initial explosion of HIV infection in the IDU population, it is predominantly subtype A that has since spread into the heterosexual community; subtype B is still seen primarily in the IVDU population. Subtype B is more closely related to North American isolates (with —85% sequence identity) than to African isolates (—75% sequence identity), however, it possess a GPGQ motif commonly shared by most African HIV-1 isolates. The presence of a GPGQ motif at the V3 crown may present a problem in that antibodies that typically recognize HIV-1 MN strains (which contains the GPGR motif) may not efficiently react with the V3 loop from the Thailand subtype A viruses. On the other hand, the distinct V3 amino acid patterns of subtype A and B provide a basis for the differentiation of the infected population based on their antibody reactivity to the immunodominant V3 loop. Assays based on synthetic peptides derived from the V3 loop or from envelope antigen preparations should make possible the rapid identification of persons who are either infected with subtype A or B, coinfected with subtypes A and B, or infected with a different HIV subtype. It should be possible also for the first time to follow the natural history of two HIV-1 subtypes in a population to determine the relative pathogenicity and transmission efficiency of the two viruses between adults, or from mother to offspring. Also of interest will be evaluating the spread of these viruses to neighboring countries in Asia. Finally, the relative homogeneity of the HIV-1 strains in Thailand, in contrast to the vast heterogeneity of HI V-1 in the United States or Africa, offers a theoretical advantage in designing vaccines for potential large-scale clinical trials.
ACKNOWLEDGMENT
grateful to Khunying Preeya Kashemsant Na Dr. Boonluan Phanthumachinda, D. Sompop Ahandrik, and the staff of the Thailand National Institute of Health, and the Kiatisak Ragkiatsakul, Supatra Im-erb, Pathum Choorat, Napaporn Panja, Tipawan Jitaweekul, and Chalermsak Tongthanachart, and the staff of the regional laboratories of the Department of Medical Sciences for assistance in collection of specimens, to Gerald Myers for his excellent help in computer We
are
2.
3.
4.
5.
6.
7.
8.
9.
Wangroongsarb Y, and Wattanasri S: The first AIDS case in Thailand (unpublished data). Division of Epidemiology, Ministry of Public Health, Thailand. Wangroongsarb Y, Weniger BG, Wasi C. Traisupa A, Kunasol P, Rojanapithayakora W. and Fucharoen S: Prevalence of HTLV-III/ LAV antibody in selected populations in Thailand. Southeast Asian J Trop Med Public Health 1986;16:517-520. Traisupa A, Wongba C, and Taylor DN: AIDS and prevalence of antibody to human immunodeficiency virus (HIV) in high risk groups in Thailand. Genitour Med 1987:63:106-108. Suwanagool S, and Kobwanthanakun S: Prevalence of HIV infection in population at risk in Thailand (in Thai). J Infect Dis Antimicrob Agents 1988;5:47-55. Ungchusak K, Sriprapandh S, Pinichpongse S. Kunasol P, and Thanprasertsuk S: First national sentinel seroprevalence survey of HIV-1 infection in Thailand. Thai AIDS J 1989;1:57-74. Vanichseni S. Piangsringarm K, Sonchai W, Akarasewi P, and Wright NH: Prevalence rate of primary HIV infection among drug users in narcotics clinics and rehabilitation centers of Bangkok metropolitan administration in 1989. Thai AIDS J 1990;1:75-82. Weniger BG. Limpakarnjanarat K, Ungchusak K, et al: The epidemiology of HIV infection and AIDS in Thailand. AIDS '91 1991; (Suppl): (in press). Siraprapasiri T, Thanprasertsuk S. Rodkaly A. Srivanichakorn S. Sawanpanyalert P. and Temtanarak J: Risk factors for HIV among prostitutes in Chiangmai, Thailand. AIDS 1991:5:579-582. Traisupa A, Teerathaum S. Tharavanich S. and Saengam S: Seroprevalence of antibodies to human immunodeficiency virus HIV-1 in a high risk-group in four provinces with tourist attraction.
Thai AIDS J 1990:2:57-63. 10. Smith DG: Thailand: AIDS crisis looms. Lancet 1991:1:781-782. 11. Rogers M, Ou C-Y. Rayfield M, Thomas PA. Achoenbaum EE. Krainski K, Selwyn PA, Moore J, Kaul A, Grimm KT. Bamji M, and Schochetman G: New York City Collaborative Study of Maternal HIV Transmission Study Group. Polymerase chain reaction for early detection of HIV proviral sequence in infants born to seropositive mothers. N Engl J Med 1989:320-1649-1654. 12. Kimura M: A Simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980; 16:111-20. 13. Myers G, Korber B, Berzofsky JA, Smith RF, and Database and analysis staff (eds): Human Retroviruses and AIDS 1991 : A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory. Los Alamos, NY, 1991.
Ayuthaya,
Address
reprint requests
to:
Chin-Yih Ou Division of HIV/AIDS Centers for Disease Control U.S. Public Health Service Atlanta, GA 30333
Wide Distribution of Two
Subtypes of HIV-1
in Thailand
CHIN-YIH OU,1 YUTAKA TAKEBE,2 CHI-CHENG LUO,1 MARCIA KALISH,1 WATTANA AUWANIT,3 CLAUDIU BANDEA.' NICK DE la TORRE,1 J.L. MOORE,1 GERALD SCHOCHETMAN,1 SHUDO YAMAZAKI,2 HELENE D. GAYLE,1 NANCY L YOUNG,'4 and BRUCE G. WENIGER1'4
INTRODUCTION
and sequence determination. HIV genetic characterization was performed on specimens from HIV-1-infected persons by sequencing approximately 300 nucleotides including the C2-V3 domains of HIV-1 envelope gene and/or hybridization.
HIV-1 reported
was introduced recently into Thailand, with the first case of AIDS in 1984,1 and HIV antibody first detected among small numbers (1-3%) of male prostitutes in 1985.2"1 Seroepidemiologic surveys from 1985 through 1987 failed to detect HIV infection among intravenous drug users (IVDU).4 Widespread transmission of HIV began in 1988 with 15-20% HIV seropositivity detected in the IVDU population in Bangkok in early 1988, and reaching more than 40% by the end of 1988.5-7 The explosive nature of the HIV epidemic and its extensive geographic distribution was documented by several national sérologie surveys.5,6-8>9 Recent projections estimated that 1.6 million (or more than 1 in 40) Thais will be infected with HIV by 1995 unless immediate dramatic measures are imple-
mented.1"
Because the HI V epidemic in Thailand is so recent, one would expect to find a high degree of genetic diversity among the HIVs currently circulating in that population unless there were a large number of HIV strains that were introduced into the country and these were responsible for the epidemic. However, if only a small number of viral strains were introduced, it should be possible to determine the number of HIV strains initially responsible for the epidemic and their possible geographic origins. We describe here the identification and genetic characteristics of two predominant HIV-1 subtypes in Thailand based on the specimens collected from various high-risk groups representing all geographic regions of the country. Heparinized blood was collected from persons in various high-risk groups by staff from the seven regional laboratories of the Department of Medical Sciences of the Thailand Ministry of Public Health and was shipped, usually overnight, to the National Institutes of Health (NIH) in Bangkok for lymphocyte separation, DNA extraction, and virus culture. Extracted DNA specimens subsequently were shipped to the Centers for Disease Control (CDC), Atlanta, GA, for sérologie testing, polymerase chain reaction (PCR) using procedures described elsewhere," not
METHODS We used Kimura's method12 to analyze sequence variations between any two sequences within the same subject or between two subjects to yield intraperson and interperson variations,
respectively. Comparisons of the
amino acid sequences of the V3 loop of HIV-Is in Thailand demonstrate that there are two distinct subtypes, A and B. Subtype A appeared to be present in every risk group examined, while subtype B appeared to be present primarily in IDUs. HIV-1 strains within a subtype were closely related to each other. The average intraperson variation for subtypes A and B, was 2.0% and 2.7%, respectively. The average interperson variation within subtype A and B was 3.8% and 3.7%, respectively. This interperson variation (3.7-3.8%) was severalfold lower than those observed within Africa, Haiti, and the United States,1"1 where the HIV-1 epidemic was known to have begun at least a decade earlier than in Thailand. The average interperson variation between subtypes A and B from different persons, (i.e., the intersubtype variation), was 18.1%. Assuming that the rate of variation in the HIV V3 region is 0.5-1% per year13 and that subtypes A and B were derived from the same prototype HIV-1 which originally entered the country, it would have taken at least 18 years to have the prototype HIV-1 evolve into the two subtypes currently present in Thailand. Therefore, it is more likely that the HIV-1 subtypes A and B entered the country recently, yet independently from each other. Phylogenetic tree analysis indicated that subtype B is more closely related to North American isolates than to African isolates and that subtype A is equally distant from all the United
'Division of HIV/AIDS, Centers for Disease Control, U.S. Public Health Service. Atlanta. GA 30333. 2National Institute of Health, Tokyo, Japan. 'National Institute of Health. Bangkok, Thailand.
4HIV/AIDS Collaboration. Bangkok. Thailand.
1471
1472
OU ET AL.
States and African isolates with a range of 20-30%. Of interest is that most HIV sequences contained the GPGQ motif at the tip of the V3 loop regardless of their subtype. The GPGQ sequence is the most common motif in African HIV-1 strains11 but rarely is found in HIV-1 strains in North America. In contrast, most (>85%) of the HIV-1 strains isolated from North America, Europe, and Japan possess the GPGR motif.
analysts, to Richard George for assistance in specimen shipping and study management, Karen Potts and Gregg Orloff for their critical review of the manuscript. Y.T. was partially supported by a Japanese Foundation for AIDS Prevention. and tree
REFERENCES 1.
RESULTS Our findings suggest that two HIV-1 strains currently predominate in Thailand. It appears that although subtype A and B were responsible for the initial explosion of HIV infection in the IDU population, it is predominantly subtype A that has since spread into the heterosexual community; subtype B is still seen primarily in the IVDU population. Subtype B is more closely related to North American isolates (with —85% sequence identity) than to African isolates (—75% sequence identity), however, it possess a GPGQ motif commonly shared by most African HIV-1 isolates. The presence of a GPGQ motif at the V3 crown may present a problem in that antibodies that typically recognize HIV-1 MN strains (which contains the GPGR motif) may not efficiently react with the V3 loop from the Thailand subtype A viruses. On the other hand, the distinct V3 amino acid patterns of subtype A and B provide a basis for the differentiation of the infected population based on their antibody reactivity to the immunodominant V3 loop. Assays based on synthetic peptides derived from the V3 loop or from envelope antigen preparations should make possible the rapid identification of persons who are either infected with subtype A or B, coinfected with subtypes A and B, or infected with a different HIV subtype. It should be possible also for the first time to follow the natural history of two HIV-1 subtypes in a population to determine the relative pathogenicity and transmission efficiency of the two viruses between adults, or from mother to offspring. Also of interest will be evaluating the spread of these viruses to neighboring countries in Asia. Finally, the relative homogeneity of the HIV-1 strains in Thailand, in contrast to the vast heterogeneity of HI V-1 in the United States or Africa, offers a theoretical advantage in designing vaccines for potential large-scale clinical trials.
ACKNOWLEDGMENT
grateful to Khunying Preeya Kashemsant Na Dr. Boonluan Phanthumachinda, D. Sompop Ahandrik, and the staff of the Thailand National Institute of Health, and the Kiatisak Ragkiatsakul, Supatra Im-erb, Pathum Choorat, Napaporn Panja, Tipawan Jitaweekul, and Chalermsak Tongthanachart, and the staff of the regional laboratories of the Department of Medical Sciences for assistance in collection of specimens, to Gerald Myers for his excellent help in computer We
are
2.
3.
4.
5.
6.
7.
8.
9.
Wangroongsarb Y, and Wattanasri S: The first AIDS case in Thailand (unpublished data). Division of Epidemiology, Ministry of Public Health, Thailand. Wangroongsarb Y, Weniger BG, Wasi C. Traisupa A, Kunasol P, Rojanapithayakora W. and Fucharoen S: Prevalence of HTLV-III/ LAV antibody in selected populations in Thailand. Southeast Asian J Trop Med Public Health 1986;16:517-520. Traisupa A, Wongba C, and Taylor DN: AIDS and prevalence of antibody to human immunodeficiency virus (HIV) in high risk groups in Thailand. Genitour Med 1987:63:106-108. Suwanagool S, and Kobwanthanakun S: Prevalence of HIV infection in population at risk in Thailand (in Thai). J Infect Dis Antimicrob Agents 1988;5:47-55. Ungchusak K, Sriprapandh S, Pinichpongse S. Kunasol P, and Thanprasertsuk S: First national sentinel seroprevalence survey of HIV-1 infection in Thailand. Thai AIDS J 1989;1:57-74. Vanichseni S. Piangsringarm K, Sonchai W, Akarasewi P, and Wright NH: Prevalence rate of primary HIV infection among drug users in narcotics clinics and rehabilitation centers of Bangkok metropolitan administration in 1989. Thai AIDS J 1990;1:75-82. Weniger BG. Limpakarnjanarat K, Ungchusak K, et al: The epidemiology of HIV infection and AIDS in Thailand. AIDS '91 1991; (Suppl): (in press). Siraprapasiri T, Thanprasertsuk S. Rodkaly A. Srivanichakorn S. Sawanpanyalert P. and Temtanarak J: Risk factors for HIV among prostitutes in Chiangmai, Thailand. AIDS 1991:5:579-582. Traisupa A, Teerathaum S. Tharavanich S. and Saengam S: Seroprevalence of antibodies to human immunodeficiency virus HIV-1 in a high risk-group in four provinces with tourist attraction.
Thai AIDS J 1990:2:57-63. 10. Smith DG: Thailand: AIDS crisis looms. Lancet 1991:1:781-782. 11. Rogers M, Ou C-Y. Rayfield M, Thomas PA. Achoenbaum EE. Krainski K, Selwyn PA, Moore J, Kaul A, Grimm KT. Bamji M, and Schochetman G: New York City Collaborative Study of Maternal HIV Transmission Study Group. Polymerase chain reaction for early detection of HIV proviral sequence in infants born to seropositive mothers. N Engl J Med 1989:320-1649-1654. 12. Kimura M: A Simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980; 16:111-20. 13. Myers G, Korber B, Berzofsky JA, Smith RF, and Database and analysis staff (eds): Human Retroviruses and AIDS 1991 : A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory. Los Alamos, NY, 1991.
Ayuthaya,
Address
reprint requests
to:
Chin-Yih Ou Division of HIV/AIDS Centers for Disease Control U.S. Public Health Service Atlanta, GA 30333
