Absence of HIV-1 Evolution in the Gut-Associated Lymphoid Tissue from Patients on Combination Antiviral Therapy Initiated during Primary Infection

English version České info

Mucosal mononuclear (MMC) CCR5+CD4+ T cells of the gastrointestinal (GI) tract are selectively infected and depleted during acute HIV-1 infection. Despite early initiation of combination antiretroviral therapy (cART), gut-associated lymphoid tissue (GALT) CD4+ T cell depletion and activation persist in the majority of HIV-1 positive individuals studied. This may result from ongoing HIV-1 replication and T-cell activation despite effective cART. We hypothesized that ongoing viral replication in the GI tract during cART would result in measurable viral evolution, with divergent populations emerging over time. Subjects treated during early HIV-1 infection underwent phlebotomy and flexible sigmoidoscopy with biopsies prior to and 15–24 months post initiation of cART. At the 2^nd biopsy, three GALT phenotypes were noted, characterized by high, intermediate and low levels of immune activation. A representative case from each phenotype was analyzed. Each subject had plasma HIV-1 RNA levels <50 copies/ml at 2^nd GI biopsy and CD4+ T cell reconstitution in the peripheral blood. Single genome amplification of full-length HIV-1 envelope was performed for each subject pre- and post-initiation of cART in GALT and PBMC. A total of 280 confirmed single genome sequences (SGS) were analyzed for experimental cases. For each subject, maximum likelihood phylogenetic trees derived from molecular sequence data showed no evidence of evolved forms in the GALT over the study period. During treatment, HIV-1 envelope diversity in GALT-derived SGS did not increase and post-treatment GALT-derived SGS showed no substantial genetic divergence from pre-treatment sequences within transmitted groups. Similar results were obtained from PBMC-derived SGS. Our results reveal that initiation of cART during acute/early HIV-1 infection can result in the interruption of measurable viral evolution in the GALT, suggesting the absence of de-novo rounds of HIV-1 replication in this compartment during suppressive cART.

Vyšlo v časopise: Absence of HIV-1 Evolution in the Gut-Associated Lymphoid Tissue from Patients on Combination Antiviral Therapy Initiated during Primary Infection. PLoS Pathog 8(2): e32767. doi:10.1371/journal.ppat.1002506
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002506

Souhrn

Zdroje

1. MellorsJWRinaldoCRJrGuptaPWhiteRMToddJA 1996 Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 272 1167 1170

2. DaarES 1998 Virology and immunology of acute HIV type 1 infection. AIDS Res Hum Retroviruses 14 Suppl 3 S229 234

3. RosenbergESAltfeldMPoonSHPhillipsMNWilkesBM 2000 Immune control of HIV-1 after early treatment of acute infection. Nature 407 523 526

4. MohriHPerelsonASTungKRibeiroRMRamratnamB 2001 Increased turnover of T lymphocytes in HIV-1 infection and its reduction by antiretroviral therapy. J Exp Med 194 1277 1287

5. ChunTWCarruthLFinziDShenXDiGiuseppeJA 1997 Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387 183 188

6. CDC 2006 Epidemiology of HIV/AIDS–United States, 1981–2005. MMWR Morb Mortal Wkly Rep 55 589 592

7. PalellaFJJrDelaneyKMMoormanACLovelessMOFuhrerJ 1998 Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 338 853 860

8. ZhangZQNotermansDWSedgewickGCavertWWietgrefeS 1998 Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection. Proc Natl Acad Sci U S A 95 1154 1159

9. AutranBCarcelainGLiTSBlancCMathezD 1997 Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science 277 112 116

10. PakkerNGNotermansDWde BoerRJRoosMTde WolfF 1998 Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: a composite of redistribution and proliferation. Nat Med 4 208 214

11. WongJKGunthardHFHavlirDVZhangZQHaaseAT 1997 Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure. Proc Natl Acad Sci U S A 94 12574 12579

12. FinziDHermankovaMPiersonTCarruthLMBuckC 1997 Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278 1295 1300

13. WongJKHezarehMGunthardHFHavlirDVIgnacioCC 1997 Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278 1291 1295

14. VolberdingPADeeksSG 1998 Antiretroviral therapy for HIV infection: promises and problems. JAMA 279 1343 1344

15. MarkowitzMJinXHurleyASimonVRamratnamB 2002 Discontinuation of antiretroviral therapy commenced early during the course of human immunodeficiency virus type 1 infection, with or without adjunctive vaccination. J Infect Dis 186 634 643

16. FinziDBlanksonJSilicianoJDMargolickJBChadwickK 1999 Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 5 512 517

17. ChunTWEngelDMizellSBEhlerLAFauciAS 1998 Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J Exp Med 188 83 91

18. MowatAM 2003 Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol 3 331 341

19. KimSKReedDSHeathWRCarboneFLefrancoisL 1997 Activation and migration of CD8 T cells in the intestinal mucosa. J Immunol 159 4295 4306

20. SchieferdeckerHLUllrichRHirselandHZeitzM 1992 T cell differentiation antigens on lymphocytes in the human intestinal lamina propria. J Immunol 149 2816 2822

21. VeazeyRSDeMariaMChalifouxLVShvetzDEPauleyDR 1998 Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 280 427 431

22. Smit-McBrideZMattapallilJJMcChesneyMFerrickDDandekarS 1998 Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes. J Virol 72 6646 6656

23. HarouseJMGettieATanRCBlanchardJCheng-MayerC 1999 Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs. Science 284 816 819

24. MattapallilJJDouekDCHillBNishimuraYMartinM 2005 Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 434 1093 1097

25. VeazeyRSMansfieldKGThamICCarvilleACShvetzDE 2000 Dynamics of CCR5 expression by CD4(+) T cells in lymphoid tissues during simian immunodeficiency virus infection. J Virol 74 11001 11007

26. NabelGBaltimoreD 1987 An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 326 711 713

27. PolesMAElliottJTaingPAntonPAChenIS 2001 A preponderance of CCR5(+) CXCR4(+) mononuclear cells enhances gastrointestinal mucosal susceptibility to human immunodeficiency virus type 1 infection. J Virol 75 8390 8399

28. AntonPAElliottJPolesMAMcGowanIMMatudJ 2000 Enhanced levels of functional HIV-1 co-receptors on human mucosal T cells demonstrated using intestinal biopsy tissue. AIDS 14 1761 1765

29. BrenchleyJMSchackerTWRuffLEPriceDATaylorJH 2004 CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 200 749 759

30. MehandruSPolesMATenner-RaczKHorowitzAHurleyA 2004 Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 200 761 770

31. GuadalupeMReayESankaranSPrindivilleTFlammJ 2003 Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol 77 11708 11717

32. MehandruSPolesMATenner-RaczKJean-PierrePManuelliV 2006 Lack of mucosal immune reconstitution during prolonged treatment of acute and early HIV-1 infection. PLoS Med 3 e484

33. MehandruSPolesMATenner-RaczKManuelliVJean-PierreP 2007 Mechanisms of gastrointestinal CD4+ T-cell depletion during acute and early human immunodeficiency virus type 1 infection. J Virol 81 599 612

34. ChunTWEngelDBerreyMMSheaTCoreyL 1998 Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection. Proc Natl Acad Sci U S A 95 8869 8873

35. PalmerSWiegandAPMaldarelliFBazmiHMicanJM 2003 New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 41 4531 4536

36. RamratnamBRibeiroRHeTChungCSimonV 2004 Intensification of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication. J Acquir Immune Defic Syndr 35 33 37

37. LewinSRVesanenMKostrikisLHurleyADuranM 1999 Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J Virol 73 6099 6103

38. ChunTWNickleDCJustementJSLargeDSemerjianA 2005 HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir. J Clin Invest 115 3250 3255

39. SharkeyMETeoIGreenoughTSharovaNLuzuriagaK 2000 Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy. Nat Med 6 76 81

40. ZhangLRamratnamBTenner-RaczKHeYVesanenM 1999 Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med 340 1605 1613

41. BuzonMJMassanellaMLlibreJMEsteveADahlV 2010 HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med 16 460 465

42. BenitoJMLopezMLozanoSMartinezPGonzalez-LahozJ 2004 CD38 expression on CD8 T lymphocytes as a marker of residual virus replication in chronically HIV-infected patients receiving antiretroviral therapy. AIDS Res Hum Retroviruses 20 227 233

43. GunthardHFWongJKIgnacioCCGuatelliJCRiggsNL 1998 Human immunodeficiency virus replication and genotypic resistance in blood and lymph nodes after a year of potent antiretroviral therapy. J Virol 72 2422 2428

44. MartinezMACabanaMIbanezAClotetBArnoA 1999 Human immunodeficiency virus type 1 genetic evolution in patients with prolonged suppression of plasma viremia. Virology 256 180 187

45. ShiuCCunninghamCKGreenoughTMuresanPSanchez-MerinoV 2009 Identification of ongoing human immunodeficiency virus type 1 (HIV-1) replication in residual viremia during recombinant HIV-1 poxvirus immunizations in patients with clinically undetectable viral loads on durable suppressive highly active antiretroviral therapy. J Virol 83 9731 9742

46. GunthardHFFrostSDLeigh-BrownAJIgnacioCCKeeK 1999 Evolution of envelope sequences of human immunodeficiency virus type 1 in cellular reservoirs in the setting of potent antiviral therapy. J Virol 73 9404 9412

47. BaileyJRSedaghatARKiefferTBrennanTLeePK 2006 Residual human immunodeficiency virus type 1 viremia in some patients on antiretroviral therapy is dominated by a small number of invariant clones rarely found in circulating CD4+ T cells. J Virol 80 6441 6457

48. DinosoJBKimSYWiegandAMPalmerSEGangeSJ 2009 Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy. Proc Natl Acad Sci U S A 106 9403 9408

49. KiefferTLFinucaneMMNettlesREQuinnTCBromanKW 2004 Genotypic analysis of HIV-1 drug resistance at the limit of detection: virus production without evolution in treated adults with undetectable HIV loads. J Infect Dis 189 1452 1465

50. ShenLSilicianoRF 2008 Viral reservoirs, residual viremia, and the potential of highly active antiretroviral therapy to eradicate HIV infection. J Allergy Clin Immunol 122 22 28

51. SedaghatARSilicianoJDBrennanTPWilkeCOSilicianoRF 2007 Limits on replenishment of the resting CD4+ T cell reservoir for HIV in patients on HAART. PLoS Pathog 3 e122

52. SedaghatARSilicianoRFWilkeCO 2008 Low-level HIV-1 replication and the dynamics of the resting CD4+ T cell reservoir for HIV-1 in the setting of HAART. BMC Infect Dis 8 2

53. JoosBFischerMKusterHPillaiSKWongJK 2008 HIV rebounds from latently infected cells, rather than from continuing low-level replication. Proc Natl Acad Sci U S A 105 16725 16730

54. SigalAKimJTBalazsABDekelEMayoA 2011 Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy. Nature 477 95 98

55. PalmerSKearneyMMaldarelliFHalvasEKBixbyCJ 2005 Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis. J Clin Microbiol 43 406 413

56. ShrinerDRodrigoAGNickleDCMullinsJI 2004 Pervasive genomic recombination of HIV-1 in vivo. Genetics 167 1573 1583

57. SimmondsPBalfePLudlamCABishopJOBrownAJ 1990 Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1. J Virol 64 5840 5850

58. SimmondsPBalfePPeuthererJFLudlamCABishopJO 1990 Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol 64 864 872

59. ButlerDMPacoldMEJordanPSRichmanDDSmithDM 2009 The efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool. J Virol Methods 162 280 283

60. FangGZhuGBurgerHKeithlyJSWeiserB 1998 Minimizing DNA recombination during long RT-PCR. J Virol Methods 76 139 148

61. MeyerhansAVartanianJPWain-HobsonS 1990 DNA recombination during PCR. Nucleic Acids Res 18 1687 1691

62. YangYLWangGDormanKKaplanAH 1996 Long polymerase chain reaction amplification of heterogeneous HIV type 1 templates produces recombination at a relatively high frequency. AIDS Res Hum Retroviruses 12 303 306

63. Salazar-GonzalezJFBailesEPhamKTSalazarMGGuffeyMB 2008 Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing. J Virol 82 3952 3970

64. HazenbergMDOttoSAvan BenthemBHRoosMTCoutinhoRA 2003 Persistent immune activation in HIV-1 infection is associated with progression to AIDS. AIDS 17 1881 1888

65. Salazar-GonzalezJPhamKKeeleBMcPhersonD 2007 Standard Operating Procedure for: Single Genome Amplification of HIV-1 Envelope [SOP#: CHAVI-MBSC-1]. University of Alabama at Birmingham

66. LearnGHJrKorberBTFoleyBHahnBHWolinskySM 1996 Maintaining the integrity of human immunodeficiency virus sequence databases. J Virol 70 5720 5730

67. AlcantaraLCCassolSLibinPDeforcheKPybusOG 2009 A standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences. Nucleic Acids Res 37 W634 642

68. de OliveiraTDeforcheKCassolSSalminenMParaskevisD 2005 An automated genotyping system for analysis of HIV-1 and other microbial sequences. Bioinformatics 21 3797 3800

69. ChoeHFarzanMSunYSullivanNRollinsB 1996 The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85 1135 1148

70. ConnorRISheridanKECeradiniDChoeSLandauNR 1997 Change in coreceptor use correlates with disease progression in HIV-1–infected individuals. J Exp Med 185 621 628

71. BrummeZLDongWWYipBWynhovenBHoffmanNG 2004 Clinical and immunological impact of HIV envelope V3 sequence variation after starting initial triple antiretroviral therapy. AIDS 18 F1 9

72. JensenMALiFSvan't WoutABNickleDCShrinerD 2003 Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences. J Virol 77 13376 13388

73. RichmanDDBozzetteSA 1994 The impact of the syncytium-inducing phenotype of human immunodeficiency virus on disease progression. J Infect Dis 169 968 974

74. MooreJPKitchenSGPugachPZackJA 2004 The CCR5 and CXCR4 coreceptors–central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection. AIDS Res Hum Retroviruses 20 111 126

75. MargolisLShattockR 2006 Selective transmission of CCR5-utilizing HIV-1: the ‘gatekeeper’ problem resolved? Nat Rev Microbiol 4 312 317

76. WhitcombJMHuangWFransenSLimoliKTomaJ 2007 Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor tropism. Antimicrob Agents Chemother 51 566 575

77. KeeleBFGiorgiEESalazar-GonzalezJFDeckerJMPhamKT 2008 Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A 105 7552 7557

78. TamuraKDudleyJNeiMKumarS 2007 MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24 1596 1599

79. RosePPKorberBT 2000 Detecting hypermutations in viral sequences with an emphasis on G→A hypermutation. Bioinformatics 16 400 401

80. VartanianJPMeyerhansASalaMWain-HobsonS 1994 G→A hypermutation of the human immunodeficiency virus type 1 genome: evidence for dCTP pool imbalance during reverse transcription. Proc Natl Acad Sci U S A 91 3092 3096

81. MartinezMAVartanianJPWain-HobsonS 1994 Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations. Proc Natl Acad Sci U S A 91 11787 11791

82. LiHBarKJWangSDeckerJMChenY 2010 High Multiplicity Infection by HIV-1 in Men Who Have Sex with Men. PLoS Pathog 6 e1000890

83. ShankarappaRMargolickJBGangeSJRodrigoAGUpchurchD 1999 Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection. J Virol 73 10489 10502

84. SaitouNNeiM 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4 406 425

85. RonquistFHuelsenbeckJP 2003 MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19 1572 1574

86. HudsonRRKaplanNL 1985 Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111 147 164

87. RozasJSanchez-DelBarrioJCMesseguerXRozasR 2003 DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19 2496 2497

88. MaydtJLengauerT 2006 Recco: recombination analysis using cost optimization. Bioinformatics 22 1064 1071

89. MensHKearneyMWiegandAShaoWSchonningK 2010 HIV-1 continues to replicate and evolve in patients with natural control of HIV infection. J Virol 84 12971 12981

90. AchazGPalmerSKearneyMMaldarelliFMellorsJW 2004 A robust measure of HIV-1 population turnover within chronically infected individuals. Mol Biol Evol 21 1902 1912

91. HudsonRRBoosDDKaplanNL 1992 A statistical test for detecting geographic subdivision. Mol Biol Evol 9 138 151

92. KearneyMSpindlerJShaoWMaldarelliFPalmerS 2011 Genetic diversity of simian immunodeficiency virus encoding HIV-1 reverse transcriptase persists in macaques despite antiretroviral therapy. J Virol 85 1067 1076

93. SlatkinMMaddisonWP 1989 A cladistic measure of gene flow inferred from the phylogenies of alleles. Genetics 123 603 613

94. PondSLFrostSDMuseSV 2005 HyPhy: hypothesis testing using phylogenies. Bioinformatics 21 676 679

95. GanttSCarlssonJHeathLBullMEShettyAK 2010 Genetic analyses of HIV-1 env sequences demonstrate limited compartmentalization in breast milk and suggest viral replication within the breast that increases with mastitis. J Virol 84 10812 10819

96. HeathLFoxAMcClureJDiemKvan't WoutAB 2009 Evidence for limited genetic compartmentalization of HIV-1 between lung and blood. PLoS One 4 e6949

97. BullMLearnGGenowatiIMcKernanJHittiJ 2009 Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations. PLoS One 4 e7122

98. BullMELearnGHMcElhoneSHittiJLockhartD 2009 Monotypic human immunodeficiency virus type 1 genotypes across the uterine cervix and in blood suggest proliferation of cells with provirus. J Virol 83 6020 6028

99. HatanoHHayesTLDahlVSinclairELeeTH 2011 A randomized, controlled trial of raltegravir intensification in antiretroviral-treated, HIV-infected patients with a suboptimal CD4+ T cell response. J Infect Dis 203 960 968

100. GandhiRTZhengLBoschRJChanESMargolisDM 2010 The effect of raltegravir intensification on low-level residual viremia in HIV-infected patients on antiretroviral therapy: a randomized controlled trial. PLoS Med 7 pii: e1000321

101. YuklSAShergillAKMcQuaidKGianellaSLampirisH 2010 Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS 24 2451 2460

102. ImamichiHDegrayGDewarRLMannonPYaoM 2011 Lack of compartmentalization of HIV-1 quasispecies between the gut and peripheral blood compartments. J Infect Dis 204 309 314

103. LernerPGuadalupeMDonovanRHungJFlammJ 2011 Gut mucosal viral reservoir in HIV infected patients is not the major source of rebound plasma viremia following HAART interruption. J Virol 85 4772 4782

104. AndersonJAArchinNMInceWParkerDWiegandA 2011 Clonal Sequences Recovered From Residual HIV-1 Viremia In Patients On Intensified Antiretroviral Therapy Are Identical To Replicating Viral RNAs Recovered From Circulating Resting CD4+ T Cells. J Virol 85 5220 5223

105. McMahonDJonesJWiegandAGangeSJKearneyM 2010 Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy. Clin Infect Dis 50 912 919

106. SilicianoRF 2005 Scientific rationale for antiretroviral therapy in 2005: viral reservoirs and resistance evolution. Top HIV Med 13 96 100

107. FiebigEWWrightDJRawalBDGarrettPESchumacherRT 2003 Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS 17 1871 1879

108. Salazar-GonzalezJFPhamKTKeeleBFMcPhersonD 2007 Standard Operating Procedure for: Single Genome Amplification of HIV-1 Envelope [SOP#: CHAVI-MBSC-1]. University of Alabama at Birmingham

109. HuangXMadanA 1999 CAP3: A DNA sequence assembly program. Genome Res 9 868 877

110. LarkinMABlackshieldsGBrownNPChennaRMcGettiganPA 2007 Clustal W and Clustal X version 2.0. Bioinformatics 23 2947 2948

111. TamuraKNeiM 1993 Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10 512 526

112. GuindonSGascuelO 2003 A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52 696 704