我国部分地区HIV-1毒株的分离培养与基因序列特征研究
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摘要
分离我国流行的HIV毒株并进行相关基因序列特征的分析是建立我国HIV毒种库,认识我国HIV的生物学特征、分析其流行和变异重组规律的重要基础;全基因组的序列测定有助于阐明毒株的进化特征,可以为有关病毒流行特征等方面的研究提供一个完整、清楚的遗传学和分子生物学背景。本研究首先对我国部分地区流行的HIV-1毒株进行分离培养,对其中的部分毒株进行了gag, pol, env基因特征进行分析,对2株病毒进行了全基因组序列测定和研究。
目的分离我国部分地区流行的HIV-1毒株,了解分离毒株的生物学及基因序列特征。
方法用外周血单核细胞(PBMCs)体外共培养的方法从我国HIV-1感染者中分离原代HIV-1毒株,分析毒株的体外生长动力学特征和融合诱导性。提取病毒基因组RNA,反转录PCR扩增病毒的gag,pol,env区基因并进行序列测定,使用软件Clustal,BioEdit version,MEGA3.1对基因序列特征进行分析。提取NX2、SH52两个HIV-1毒株的前病毒基因组DNA,套式PCR方法扩增全长基因组并分段测序,使用SimPlot软件进行基因序列重组分析。
结果将本室冻存的67株初步分离呈阳性反应的毒株复苏传代,得到37株稳定传代的病毒;从5个省市的270名HIV-1感染者外周血中分离出58株HIV-1原代毒株。分离株的体外生长动力学呈现快/高、慢/高、慢/低三种类型。病毒载量越高分离的阳性率(HC=78.959,χ~2_((1)0.01)=6.63.P<0.01)越高、同时病毒分离株呈快高型生长的比例增大。
52株分离株的序列呈现B(欧美B、泰国B’)、CRF01_AE重组、CRF_BC重组和G 4种亚型;以B亚型分布最广,CRF01_AE重组株主要分布在广西,上海发现G亚型病毒。V3环顶端四肽GPGR广泛的分布于B亚型和CRF_AE亚型中。CRF01_AE亚型毒株的V3环平均净电荷数略高于B亚型;基因离散率分析显示,env基因离散率远高于gag和pol基因;CRF01_AE亚型病毒pol和env区基因离散率均在三种亚型中最高。表型预测显示多数为利用CCR5辅助受体、M嗜性的毒株。
扩增得到NX2 08BC重组亚型和SH52 G亚型全长基因组序列。NX2 CRF08_BC重组亚型在3个位置发生重组,分别位于gag,pol和nef区,重组位点位于1000nt,2600nt和9200nt。
结论传代分离出我国部分地区的HIV-1流行毒株95株;毒株呈现多种体外复制动力学特征,并与血浆HIV-1病毒载量水平相关;毒株呈现多种亚型(B、CRF01_AE重组、CRF_BC和G),在不同传播途径和地区具有不同的分布;env基因离散率远高于gag和pol基因, HIV-1毒株多数是不致细胞病变的M嗜性、NSI表型;V3环序列变化较大。得到2株HIV-1毒株的全长基因组序列,亚型分别为CRF08_BC和G亚型。
Isolations and sequences analyses of HIV-1 strains circulating in China afford a solid basis to build the HIV strains stocks, acquire their biological features and analyze the rules of pandemic, diversities and recombinations.Full-length genomes sequencing play a important role in clarifying evolving characteristics of different clades, and supply a complete, clear genetics and molecular background to the virus epidemiology characters.In the research descried this article, we isoalted and cultured several HIV-1 strains from partional regions in China and analyze their important structural genes sequences such as gag, pol, env, in particular, full-length genome sequences analyses were also performed on the rare circulating isolates .
Objective To isolate HIV-1 strains circulating in some regions of China, then learn their biological and sequence feature.
Methods Primary HIV-1 strains were isolated from HIV-1 infected persons using PBMCs coculture method, then their growth dynamics feature and capacity of inducing syncytium were identified. Meanwhile, RNA was prepared and gag、pol、env gene was amplified by RT-PCR. Then the sequence was determined and sequence was analyzed by Clustal,BioEdit version,MEGA3.1 .The DNA of NX2 and SH52 were prepared and the full-length sequence was amplified by nested-PCR, sequence was analyzed by SimPlot.
Results In 67 HIV-1 strains, 37 virus were subculturied steadily. 58 primary strains were isolated from 270 HIV-1 infected persons. It showed us three types growth dynamics that were rapid/high、slow/high、slow/low. It suggested that strains in samples of higher viral loads get a higher isolating rat(eH_C=78.959,χ~2_((1)0.01)=6.63.P<0.01),and the proportion of rapid/high strains in samples of higher viral loads were bigger than that in samples of lower viral loads. The sequence of 52 strains turned out to be 4 subtypes of B、CRF01_AE、CRF_BC and G. The strains of subtype B existed in the widest area. The subtype CRF01_AE strains mostly existed in Guangxi.Subtype G was found in shanghai.V3 loop central motif-GPGR mostly existed in the subtype B and subtype CRF01_AE The number of net charge in V3 loop in the subtype CRF01_AE is the higher in the subtype B.Genic diversity in env is bigger than gag and pol. Genic diversity in pol and env in subtype CRF01_AE is the biggest in three subtypes.Majority of the isolated strains were predicted to be M-tropism and CCR5 using strains.The full-length sequence of NX2 (subtype CRF_BC) and SH52(subtype G)was amplified by PCR.NX2 CRF08_BC showed three breakpoints located at positions at 1000 in gag、2600 in pol and 9200 in nef.
Conclusion 95 HIV-1 strains circulating in some regions of China were subculturied and isolated; The replication dynamics of the isolated strains varied and related to HIV-1 viral loads in plasma; There existed many kinds of subtype as B、CRF01_AE、CRF07_BC and G, and the distribution of the subtype varied according to different regions and different transmission path; Genic diversity in env is bigger than gag and pol. Majority of the isolated strains were predicted to be M-tropism and NSI strains; The sequence variation of V3 regions were apparent.Two full-length sequence of subtype 08BC and G were amplified.
目的分离我国部分地区流行的HIV-1毒株,了解分离毒株的生物学及基因序列特征。
方法用外周血单核细胞(PBMCs)体外共培养的方法从我国HIV-1感染者中分离原代HIV-1毒株,分析毒株的体外生长动力学特征和融合诱导性。提取病毒基因组RNA,反转录PCR扩增病毒的gag,pol,env区基因并进行序列测定,使用软件Clustal,BioEdit version,MEGA3.1对基因序列特征进行分析。提取NX2、SH52两个HIV-1毒株的前病毒基因组DNA,套式PCR方法扩增全长基因组并分段测序,使用SimPlot软件进行基因序列重组分析。
结果将本室冻存的67株初步分离呈阳性反应的毒株复苏传代,得到37株稳定传代的病毒;从5个省市的270名HIV-1感染者外周血中分离出58株HIV-1原代毒株。分离株的体外生长动力学呈现快/高、慢/高、慢/低三种类型。病毒载量越高分离的阳性率(HC=78.959,χ~2_((1)0.01)=6.63.P<0.01)越高、同时病毒分离株呈快高型生长的比例增大。
52株分离株的序列呈现B(欧美B、泰国B’)、CRF01_AE重组、CRF_BC重组和G 4种亚型;以B亚型分布最广,CRF01_AE重组株主要分布在广西,上海发现G亚型病毒。V3环顶端四肽GPGR广泛的分布于B亚型和CRF_AE亚型中。CRF01_AE亚型毒株的V3环平均净电荷数略高于B亚型;基因离散率分析显示,env基因离散率远高于gag和pol基因;CRF01_AE亚型病毒pol和env区基因离散率均在三种亚型中最高。表型预测显示多数为利用CCR5辅助受体、M嗜性的毒株。
扩增得到NX2 08BC重组亚型和SH52 G亚型全长基因组序列。NX2 CRF08_BC重组亚型在3个位置发生重组,分别位于gag,pol和nef区,重组位点位于1000nt,2600nt和9200nt。
结论传代分离出我国部分地区的HIV-1流行毒株95株;毒株呈现多种体外复制动力学特征,并与血浆HIV-1病毒载量水平相关;毒株呈现多种亚型(B、CRF01_AE重组、CRF_BC和G),在不同传播途径和地区具有不同的分布;env基因离散率远高于gag和pol基因, HIV-1毒株多数是不致细胞病变的M嗜性、NSI表型;V3环序列变化较大。得到2株HIV-1毒株的全长基因组序列,亚型分别为CRF08_BC和G亚型。
Isolations and sequences analyses of HIV-1 strains circulating in China afford a solid basis to build the HIV strains stocks, acquire their biological features and analyze the rules of pandemic, diversities and recombinations.Full-length genomes sequencing play a important role in clarifying evolving characteristics of different clades, and supply a complete, clear genetics and molecular background to the virus epidemiology characters.In the research descried this article, we isoalted and cultured several HIV-1 strains from partional regions in China and analyze their important structural genes sequences such as gag, pol, env, in particular, full-length genome sequences analyses were also performed on the rare circulating isolates .
Objective To isolate HIV-1 strains circulating in some regions of China, then learn their biological and sequence feature.
Methods Primary HIV-1 strains were isolated from HIV-1 infected persons using PBMCs coculture method, then their growth dynamics feature and capacity of inducing syncytium were identified. Meanwhile, RNA was prepared and gag、pol、env gene was amplified by RT-PCR. Then the sequence was determined and sequence was analyzed by Clustal,BioEdit version,MEGA3.1 .The DNA of NX2 and SH52 were prepared and the full-length sequence was amplified by nested-PCR, sequence was analyzed by SimPlot.
Results In 67 HIV-1 strains, 37 virus were subculturied steadily. 58 primary strains were isolated from 270 HIV-1 infected persons. It showed us three types growth dynamics that were rapid/high、slow/high、slow/low. It suggested that strains in samples of higher viral loads get a higher isolating rat(eH_C=78.959,χ~2_((1)0.01)=6.63.P<0.01),and the proportion of rapid/high strains in samples of higher viral loads were bigger than that in samples of lower viral loads. The sequence of 52 strains turned out to be 4 subtypes of B、CRF01_AE、CRF_BC and G. The strains of subtype B existed in the widest area. The subtype CRF01_AE strains mostly existed in Guangxi.Subtype G was found in shanghai.V3 loop central motif-GPGR mostly existed in the subtype B and subtype CRF01_AE The number of net charge in V3 loop in the subtype CRF01_AE is the higher in the subtype B.Genic diversity in env is bigger than gag and pol. Genic diversity in pol and env in subtype CRF01_AE is the biggest in three subtypes.Majority of the isolated strains were predicted to be M-tropism and CCR5 using strains.The full-length sequence of NX2 (subtype CRF_BC) and SH52(subtype G)was amplified by PCR.NX2 CRF08_BC showed three breakpoints located at positions at 1000 in gag、2600 in pol and 9200 in nef.
Conclusion 95 HIV-1 strains circulating in some regions of China were subculturied and isolated; The replication dynamics of the isolated strains varied and related to HIV-1 viral loads in plasma; There existed many kinds of subtype as B、CRF01_AE、CRF07_BC and G, and the distribution of the subtype varied according to different regions and different transmission path; Genic diversity in env is bigger than gag and pol. Majority of the isolated strains were predicted to be M-tropism and NSI strains; The sequence variation of V3 regions were apparent.Two full-length sequence of subtype 08BC and G were amplified.
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70. Cornelissen M, Kampinga G, Zorgedrager F, er al. Human immunodeficiency virus type 1 subtypes defines by env show high frequency of recombinant gag genes. THE UNAIDS Network for HIV Isolation and Characterization. J Virol. 1996, 70(11):8209-12
71. ZHU GW, Liu ZQ, Joag SV, et al. Pathogenesis of lymphocyte-tropic andmacrophage-tropic SIV mac infection in the brain. J Neurovirol. 1995 Mar; 1(1):78-91
72. Graham NM. Metabolic disorders among HIV-infected patients treated with protease inhibitors: a review. J Acquir Immune Defic Syndr ,2000 :25 (Suppl1) :S4-11
73. Kijak GH and McCutchan FE: HIV diversity, molecular epidemiology, and the role of recombination. Curr Infect Dis Rep 2005; 7:480-488.
74. Dario A, Alehandro M, Leonardo L, et al. HIV type 1 genetic diversity surveillance among newly diagnosed individuals from 2003 to 2005 in Buenos Aires, Argenitna. AIDS Res Hum Retroviruses 2007; 23:1201-1207
75. Qiu Z, Xing H, Wei M, et al. Characterization of five nearly full-length genomes of early HIV type 1 strains in Ruili city: Implications of the genesis of CRF07_BC and CRF08_BC circulating in China. AIDS Res Hum Retroviruses 2005; 21:1051-1056.
76. Parren P, Gauduin MC, Koup RA, et al. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol. Lett. 57:105-112.
77. Flynn NM, Forthal DN, Harro CD, et al. Placebocontrolled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection.2005; J. Infect. Dis. 191:654-665.
78. Dacheux L, Moreau A, Ataman-Onal Y, et al. Evolutionary dynamics of glycan shield of the human immuodeficiency viruns envelope during natural infecgtion and implications for exposure of the 2G12 epitope. J. Virol.78:12625-37.
1. Ronberson DL, Anderson JP, Bradac JA, et al. HIV-1 nomenclature proposal. Science 2000;288:55-56.
2. Fabio T, Filippa B, Anna MP, et al.Genetic diversity of HIV-1 non-B strains in Sicily: evidence of intersubtype recombinants by sequence analysis of gag, pol, and env genes. AIDS Res Hum Retroviruses.2007;23:1131-1138.
3. AJ Kandathil, S Ramalingam, R Kannangai , et al. Molecular epidemiology of HIV. Indian Journal of Medical Research , 2005 ,121 ,1664, Pg333 ,12pgs.
4. Kanki PJ, Harnel DJ, Sankale JL, et al. Human immunodeficiency virus type 1 subtypes differ in disease progression. J Infect Dis ,1999 , 179 :68-73.
5. Cccilia D, Kulkarni SS, Tripathy SP, et al. Absence of coreceptor switch with disease progression in human immunodeficiency virus infections in India.Virology, 2000, 271: 253-258.
6. Mehendale SM, Bollinger RC, Kulkarni SS, et al. Rapid disease progression in human immunodeficiency virus type-1 infected seroconverters in India. AIDS Res Hum Retroviruses, 2002, 18 : 1175 -1179.
7. Vasan A, Renjifo B, Hertzmark E, et al. Different rates of disease progression of HIV type 1 infection in Tanzania based on infection subtype. Clin Infect Dis 2006;42:843-852
8. Baeten JM, Chohan B, Lavreys L, et al. HIV-1 Subtype D infection is associated with faster disease progression than A in spite of similar plasema HIV-1 load. J Infect Dis 2007; 195:1177-1180.
9. Donald JH, Jean-louis S, Geoffrey E, et al. Twenty years of prospective molecular epidemiology in Senegal: change in HIV diversity. AIDS Res Hum Retroviruses 2007;23:1189-1196.
10. Kaul R, T Dong, Plummer FA, et al. CD8+ lymphocytes respond to different HIV epitopes in seronegative and infected subjects. J. Clin. Investig. 2001;107:1303-1310.
11. Skurnick JH, Palumbo P, et al. Correlates of nontransmission in US women at high risk of human immunodeficiency virus type 1 infection through sexual exposure. J Infect Dis. 2002;185:428–438.
12. Takaichi H, Kazuhiro M, Yurina H, et al. A single-nucleotide synonymous mutation in the gag gene controlling human immunodeficiency virus type 1 virion production. J Virol. 2007;81:1528-1533
13. June KM, Melissa B, Keri LS, et al. Degeneracy and repertoire of human HIV-1 gag p1777-85 CTL response. J Immunol. 2006.6690-6701.
14. Fatkenheuer G, Theisen A, Rockstroh J, et al. Virological treatment failure of protease inhibitor therapy in an unselected cohort of HIV-infected patients. AIDS.1997 Nov 15;11(14):F113-6.
15. Boden D, Hurley A, Zhang L, et al. HIV-1 drug resistance in newly infected individuals. JAMA. 1999 Sep 22-29;282(12):1135-1141.
16. Ibá?ez A, Clotet B, Martínez MA. Human immunodeficiency virus type 1 population bottleneck during indinavir therapy causes a genetic drift in the env quasispecies. J Gen Virol. 2000 Jan;81(Pt 1):85-95.
17. Misse D, Gajardo J, Oblet C, et al. Soluble HIV-1 gp120 enhances HIV-1 replication in non-dividing CD4+ T cells, mediated via cell singaling and Tatcofactor over expression. AIDS. 2005; 19(9):897-905.
18. Shankarappa R, Margolick JB, Gange SJ, et al. Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection. J Virol. 1999;73:10489-10502.
19. Cole KS, Steckbeck JD, Rowles JL, et al. Removal of N-linked glycosylation sites in the V1 region of simian imunodeficiency virus gp120 results in redirection of B-cell responses to V3. J Virol .78:1525-1539.
20. Draenert R, Allen TM, Liu Y, et al. Constrains on HIV-1evolution and immunodominance revealed in monozygotic adult twins infected with the same virus. J Exp Mcd. 2006 203:529-539.
21. Forst SD, Dumaurier MJ, Hobson S, et al. Neutralizing antibody responses drive the evolution of human immunodeliciency virus type 1 envelope during recent HIV infection. Proc Natl Acad Sci. USA 98:6975-6980.
22. Nijhuis M, Boucher CA, Schipper P, et al. Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy. Proc Natl Acad Sci USA. 1998 Nov 24;95(24):14441-14446.
23. Rouzine IM, Coffin JM. Linkage disequilibrium test implies a large effective population number for HIV in vivo. Proc Natl Acad Sci USA. 1999 Sep 14;96(19):10559-10561.
2. 国家卫生部, 《二零零七年中国艾滋病防治联合评估报告》
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69. urke DS. Recombination in HIV: an important viral evolutionary strategy. Emerg Infect Dis.1997 Jul-Sep; 3(3):253-9
70. Cornelissen M, Kampinga G, Zorgedrager F, er al. Human immunodeficiency virus type 1 subtypes defines by env show high frequency of recombinant gag genes. THE UNAIDS Network for HIV Isolation and Characterization. J Virol. 1996, 70(11):8209-12
71. ZHU GW, Liu ZQ, Joag SV, et al. Pathogenesis of lymphocyte-tropic andmacrophage-tropic SIV mac infection in the brain. J Neurovirol. 1995 Mar; 1(1):78-91
72. Graham NM. Metabolic disorders among HIV-infected patients treated with protease inhibitors: a review. J Acquir Immune Defic Syndr ,2000 :25 (Suppl1) :S4-11
73. Kijak GH and McCutchan FE: HIV diversity, molecular epidemiology, and the role of recombination. Curr Infect Dis Rep 2005; 7:480-488.
74. Dario A, Alehandro M, Leonardo L, et al. HIV type 1 genetic diversity surveillance among newly diagnosed individuals from 2003 to 2005 in Buenos Aires, Argenitna. AIDS Res Hum Retroviruses 2007; 23:1201-1207
75. Qiu Z, Xing H, Wei M, et al. Characterization of five nearly full-length genomes of early HIV type 1 strains in Ruili city: Implications of the genesis of CRF07_BC and CRF08_BC circulating in China. AIDS Res Hum Retroviruses 2005; 21:1051-1056.
76. Parren P, Gauduin MC, Koup RA, et al. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol. Lett. 57:105-112.
77. Flynn NM, Forthal DN, Harro CD, et al. Placebocontrolled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection.2005; J. Infect. Dis. 191:654-665.
78. Dacheux L, Moreau A, Ataman-Onal Y, et al. Evolutionary dynamics of glycan shield of the human immuodeficiency viruns envelope during natural infecgtion and implications for exposure of the 2G12 epitope. J. Virol.78:12625-37.
1. Ronberson DL, Anderson JP, Bradac JA, et al. HIV-1 nomenclature proposal. Science 2000;288:55-56.
2. Fabio T, Filippa B, Anna MP, et al.Genetic diversity of HIV-1 non-B strains in Sicily: evidence of intersubtype recombinants by sequence analysis of gag, pol, and env genes. AIDS Res Hum Retroviruses.2007;23:1131-1138.
3. AJ Kandathil, S Ramalingam, R Kannangai , et al. Molecular epidemiology of HIV. Indian Journal of Medical Research , 2005 ,121 ,1664, Pg333 ,12pgs.
4. Kanki PJ, Harnel DJ, Sankale JL, et al. Human immunodeficiency virus type 1 subtypes differ in disease progression. J Infect Dis ,1999 , 179 :68-73.
5. Cccilia D, Kulkarni SS, Tripathy SP, et al. Absence of coreceptor switch with disease progression in human immunodeficiency virus infections in India.Virology, 2000, 271: 253-258.
6. Mehendale SM, Bollinger RC, Kulkarni SS, et al. Rapid disease progression in human immunodeficiency virus type-1 infected seroconverters in India. AIDS Res Hum Retroviruses, 2002, 18 : 1175 -1179.
7. Vasan A, Renjifo B, Hertzmark E, et al. Different rates of disease progression of HIV type 1 infection in Tanzania based on infection subtype. Clin Infect Dis 2006;42:843-852
8. Baeten JM, Chohan B, Lavreys L, et al. HIV-1 Subtype D infection is associated with faster disease progression than A in spite of similar plasema HIV-1 load. J Infect Dis 2007; 195:1177-1180.
9. Donald JH, Jean-louis S, Geoffrey E, et al. Twenty years of prospective molecular epidemiology in Senegal: change in HIV diversity. AIDS Res Hum Retroviruses 2007;23:1189-1196.
10. Kaul R, T Dong, Plummer FA, et al. CD8+ lymphocytes respond to different HIV epitopes in seronegative and infected subjects. J. Clin. Investig. 2001;107:1303-1310.
11. Skurnick JH, Palumbo P, et al. Correlates of nontransmission in US women at high risk of human immunodeficiency virus type 1 infection through sexual exposure. J Infect Dis. 2002;185:428–438.
12. Takaichi H, Kazuhiro M, Yurina H, et al. A single-nucleotide synonymous mutation in the gag gene controlling human immunodeficiency virus type 1 virion production. J Virol. 2007;81:1528-1533
13. June KM, Melissa B, Keri LS, et al. Degeneracy and repertoire of human HIV-1 gag p1777-85 CTL response. J Immunol. 2006.6690-6701.
14. Fatkenheuer G, Theisen A, Rockstroh J, et al. Virological treatment failure of protease inhibitor therapy in an unselected cohort of HIV-infected patients. AIDS.1997 Nov 15;11(14):F113-6.
15. Boden D, Hurley A, Zhang L, et al. HIV-1 drug resistance in newly infected individuals. JAMA. 1999 Sep 22-29;282(12):1135-1141.
16. Ibá?ez A, Clotet B, Martínez MA. Human immunodeficiency virus type 1 population bottleneck during indinavir therapy causes a genetic drift in the env quasispecies. J Gen Virol. 2000 Jan;81(Pt 1):85-95.
17. Misse D, Gajardo J, Oblet C, et al. Soluble HIV-1 gp120 enhances HIV-1 replication in non-dividing CD4+ T cells, mediated via cell singaling and Tatcofactor over expression. AIDS. 2005; 19(9):897-905.
18. Shankarappa R, Margolick JB, Gange SJ, et al. Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection. J Virol. 1999;73:10489-10502.
19. Cole KS, Steckbeck JD, Rowles JL, et al. Removal of N-linked glycosylation sites in the V1 region of simian imunodeficiency virus gp120 results in redirection of B-cell responses to V3. J Virol .78:1525-1539.
20. Draenert R, Allen TM, Liu Y, et al. Constrains on HIV-1evolution and immunodominance revealed in monozygotic adult twins infected with the same virus. J Exp Mcd. 2006 203:529-539.
21. Forst SD, Dumaurier MJ, Hobson S, et al. Neutralizing antibody responses drive the evolution of human immunodeliciency virus type 1 envelope during recent HIV infection. Proc Natl Acad Sci. USA 98:6975-6980.
22. Nijhuis M, Boucher CA, Schipper P, et al. Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy. Proc Natl Acad Sci USA. 1998 Nov 24;95(24):14441-14446.
23. Rouzine IM, Coffin JM. Linkage disequilibrium test implies a large effective population number for HIV in vivo. Proc Natl Acad Sci USA. 1999 Sep 14;96(19):10559-10561.