エイズ/HIV 感染症
Contents
1. 6 2 HIV 12. 4 1 9 BLAST Basic Local Alignment Search Tool 2 HIV http www hiv lanl gov content sequence HIV mainpage html HIV BLAST Nested DNA
3. HIV 1 2 2 HIV 1 HIV 2 HIV 1 HIV PCR HIV 1RNA KK TaqMan 2 4 HIV 2 HIV 2 HIV 2 10 HIV 1 HIV2
4. yy hK DNA 41 0 2nd PCR dNTP QIAamp PCR Purification Kit QIAGEN 1 PCR 5 PB 2 SLIDE CNN SN e 3 13 000 rpm 1 J7 5 13 000rpm 1 6 HC 13 000 rpm uc 8 13 000rpm 1 9 ii
5. 1 QIAamp Viral Mini Kit QIAGEN 52904 High Pure Viral RNA Kit Roche 1858882 QIAamp Viral RNA Mini Kit 1 140 uL 560 uL buffer AVL 2 15 25 C 10 3 4 560 pL PUCAS M VES 5 QIAamp 630 uL 8 000rpm 6 000 xg 1 09 2 QIAamp 2mL m
6. 13 4 1 1 HIV 1 1 gzy TO C2 VS 1 RT PCR 2nd PCR 2 RT PCR 1 R 1 HITV 1 pol PR RT env C2 ID IXB2 ID IAB2 RT PCR 509A 5 AGA CAG GYT AAT TTT TTA GGG A 3 2074 2095 573A 5 ATA TGT ACC CCR GG 3 6867 6887 512B 5 TAC TTC TGT TA
7. 2 2 HIV 0 3 1 HIV 0 3 2010 EIA PA 2 2 1 1 PA EIA IC PA EIA or IC
8. 2 HIV HIV 1 RNA KK TaqMan 5 2 KK TaqMan PCR HITV 1RNA 50 mL 50 500 000 mL 500 000 mL PCR 7900HT StepOnePlus ABI 7500 O Light Cycler 480
9. 17 CRE 4 2 HIV 1 HIV 1 Neighbor joining NJ 1E
10. Premix Taq Ex Taq version 1 D W RNase free 18 0u L 20uM sense primer 10 1 20uM anti sense primer 1 0 1 Premix Taq 25 0uL 3 2 RTPCR SgL 3a Bochum 3 s 94C 1 min l 94C 30sec 55C 30 n 30 cycles 126 min 15 J 7 min l 4C soak 4 1 5 2nd PCR HIV 1
11. Bd 4 97 B 2 B JRFL pol 2168 2591 HXB2 99 B CN RLA42 99 WEAUTB D 94061141 77 D ELI D NDK F1 vl850 100 F1 93BR020 1 70 1 5E7253 98 1 92115037 023 17 99 01 90CF 402 70 01 90CF 407 1 70 01 93TH253 gz r 01 CM240 D1 7CNGX11F C 92BR025 C ETH2220 99 C 98 012 C 951321068 100 C 9313905 G DRCBL 30 G 5E6165 G HH8793 H 991 90 997 73 90 056 J SE7887 100 J 5E7022 CPZ CPZGAB 19 9 4 2 3 4 1 3
12. BigDye terminator v3 1 Applied Biosystems ce Em ed Nuclease free water 8 0 uL BigDye terminator v3 1 3 0 uL 5x sequencing buffer 3 0 uL Primer 1g M 3 0 uL 3 pmol PCR product 3 0 uL 30 90 ng Total volume 20 0 uL 1 96 C 15 sec 96 10 60 50 sec 25 cycles b dNTPs 1abeled ddNTPs AutoSeq G 50 GE Healthcare 27 5340 BigDye X Terminator 31 Applied Biosystems 4376486 AutoSeq 6G 50 1 100mM EDTA pH8 0 10 vortex 2 2 1 4 3 1 5 4 600rpm 1 4 1 5 mL
13. HIV 1 M 1 Ed 1 RT nested PCR nested PCR DNA 4 1 RNase DW
14. 4 MEGA ver 5 http www megasoftware net 4 2 1 http www hiv lanl gov content sequence HIV mainpage html EASTA 2 fas fasa MEGA FASTA abl MEGA 2 FASTA gt CPZ CPZGAB CCTCAAATCACTCTTTGGCAGCGACCCCTCATCCCAGTAAAAGTAGAAGGGCAACTATGTGAA
15. HIV Y y Away Molecular BioProducts 7010 SE C3EB d EHI zs R EOD ZU 5 4 1 Kondo M Sudo Tanaka Sano T Sagara Iwamuro S Takebe Y Imai M Kato S Quantitation of HIV 1 group M proviral DNA using TaqMan MGB real time PCR J Virol Methods 157 2 141 146 2009 2 PCR HIV 1 HIV 18 7 20 p302 312 2009 26 6 HIV 1 6 1 1997 HIV 3 HIV 1 combination Antiretroviral Therapy cART HIV
16. MAGIC 5A HIV Long PCR JUS MAGIC 5A eb Qn e HIV MAGIC 5A KK TaqMan RT PCR MAGIC 5A
17. 6 D W PON equ mee 7 3 000 rpm 2 8 9 3 000 rpm 2 204 L 10 Vacuum concentrator 11 20uL Hi Diformamide 95 2 Genetic analyzer 4 1 8 Genetic analyzer
18. gag p24 Los Alamos HIV HTV 1 M CRE degenerate ME B A C D F G CREFO1_AE TagMan 5 3 KK TaqMan 1 EDTA CPD PCR 5001 2 HIV 1RNA HIV 1 cell line SES KK TaqMan SES HIV 1 Biosafety level 2 http www atcc org SC1318 EXA Liquid 3 Normal 8BE
19. 20 C 2400rpm 540 15211 7 2 3 PBMC PBMC 30mL PBS 4 20C 1300rpm 240 g 3 PBMC 2 5 5 FCS RPMI 1640 2 PBMC 6 0 1 PHA Difco 50mL 20 FCS RPMI 1640 PBMC 3 4 7 JUJI FIELD 10 10 8 C 13 PBMC 8 EDTA 10 20mL 9 PBMC PBMC 10 5 FCS RPMI 1640 2
20. 30 50 Recent seroconversion Aware BED EIA HIV 1 Incidence Test Calypte Co LTD http wywwr calypte com 20 L 2 3 Recent infection CD4 B gp41 A C F 2 5 2 BED Y I BED 4 BED HIV 1 Peptide SA HRP 20 8
21. washbuffer F 2 0 421 212 10 000rpm 10 20 wash buffer 0 4 mL 10 000rpm 1 wash buffer elution buffer cap 100 uL 10 000rpm 1 2 GTC 15 TRIZOL LS Invitrogen 10296 010 RNA 0 25mL 0 75mL 3 TRIZOL LS 5 0 2mL 2 15 15 000rpm 10 4 CK 1 0 1mg tRNA tRNA yeast Invitrogen 16051 039 0 5 mL
22. 2 IAS USA HP http www iasusa org Stanford HP http hivdb stanford edu 1 ide analogue RT inhibitor NRTI tenofovir TDF 1 K65R 2 T69ins T215Y M41L A62V K7OR L210W 2 1 T69S lamivudine 916 Bmtricitabine 1 M184V I 2 T69ins T215Y MAIL A62V K7OR L210W 2 1 T69ins T215Y 1 E44D A V1181 2 Q151M complex 3 4 T69ins abacabir ABC 1 M41L D67N L74V M184V I L210W T215Y F 2 Q151M A62V V75I F116Y 1 3 T69ins T215Y MAIL A62V K7OR L210W 2 1 M184V K65R L74V Y115F 2 2 T69ins T215Y 32 3 Q151M F77L 1 M41L E
23. informative site SeqPage For informative analysis Query Refl 22 Other Finesites HXB2 HXB2 HXB2 IBI Gnformative site HXB2 xxxx yyyy nt xxxx zz nt Subregion tree analysis 1
24. MMLV PrimeScript One Step RT PCR Kit 1 D W RNase free 30 5uL 10X one step RNA PCR buffer 5 0 1 One Step Enhancer Solution l 0uL 10mM dNTP 20uL 20uM Primer 509A l 0u L 20uM Primer 512B l 0u L 20uM Primer 573A l 0u L 20uM Primer 004B l 0u L RNase inhibitor l OuL Primescript RTase 0 5uL TaKaRa EX Taq HS l 0L u L 0 9011 7 SuL 3 50C 94C 2 min l 94C 30sec 55C 30sec 30 72 3 l 722 7 l 4C soak 4 1 4 2nd PCR 1 30 35 PCR RTPCR 2nd PCR
25. antisense DNA DNA PCR DNA HXB2 6 3 1 EDTA PCR 200 500 uL 2 HIV 1 RNA RNA
26. 20 mL 20 mL Ficoll Paque Plus 7 BE Dynabeads M 450 CD8 pi CD8 CD8 CD8 PBMC RYK CD8 ES 5x10 PBMC 5 mL 5x10 5 mL I CD3 ie 3 EEPO P24 2 2 3 3 MAGIC 5A HIV HIV 1 2 PHA Mitogen IL 2 ERERZ 8 HIV 1 24 gag i
27. HIV NAT WB NAT 24735 HIV 1 8 HITV 1 HIV 1 2 HIV 2 WX p24 gag pol env 3
28. 400 600
29. RT PCR UC mque 55 95 PCR 95 C 52 72 20 10 45 min min 1 TaKaLa LA 1 10x LA PCR buffer II 25 mM MgCl 2 0 uL 2 5 uL p 40 cycles nested PCR TaKaLa LA TaKaLa RRO002A KOD DNA polymerase TOYOBO KOD 101 Hot Start Ex Taq TAKARA RRO06A 29 dNTP mixture 2 5 mM 4 0 uL for primer 10 uM 1 25uL Final conc 0 54 M rev primer 10 M 1 25uL Final conc 0 54 M Nuclease free water 12 75uL TaKaRa LA Taq 5 U y 1 0 25uL RT PCR product 0 5 uL Total volume 20 0 uL PCR 247 5 94 3 min 94 C 15 sec 50 C 30 sec x 40 cycles 10 C 2 min 2 KOD DNA polymerase 1 Nuclease free water 33 0 uL 10x KOD buffer 1 5 0uL 25mM MgCl 2 0uL 1 mM 2mM dNTPs 5 0 uL 0 2 mM for Primer 100 u M 0 4 uL 40 pmol rev primer 100 uM 0 4 uL 40 pmol KOD 2 5 U u 1 0 4 uL 1 0 U RT PCR product 3 8 uL Total volume 50 O uL 94 C 15 sec 94 C 5 sec 60 C J se
30. 4 600rpm 1 5 speed vac loading buffer loading buffer formamide 25 mM EDTA pH8 0 blue dextran 50 mg mL 5 1 ABI 377 ABI 310 BigDye X Terminator 1 20 uL SAM 90 uL BigDye X Terminator 20 uL 2 30 SHAFT A 3 1000 g 2 4 gem CK s 6
31. 10 20 1 Spli Down 10 2 CPE SHAT D E TCHR MAGIC 5A MAGIC SA CPE 107 copies mL 10 copies lmL 3
32. HITV 1 HIV 2 4 3 HIV 1 2 PA 3 4 4 p24 gag 10 pg ml COI 1 HIV 1 2
33. env V1 V2 artifact Edit Delete Data Save Session MEGA 18 Data Export Alignment MEGA Format 3 aminoacid HXB2 R R I R I 0 RG P 0 R A F I 6 HXB2 AGA AAA AGA ATC CGT ATC CAG AGA GGA CCA GGG AGA GCA TTT GTT ACA ATA GGA B JRFL sno ool A A xd waa aai wd au vro 95IN21068 seansie ue Res rot Bus sE 93 905 6 ios oss 6 01 240 coa vs uu 46 M M en 01 93TH253 C G CA A idco ee e
34. 45 C 5 Similarity plot Bootscanning plot B C Simplot http sray med som jhmi edu SCRoftware HIV 1 FASTA xxx Simplot SeqPage File Open FASTA Similarity plot
35. PBMC 75mL 37C CO gt 20mL 20 FCS 20 50U mL 2 RPMI 1640 11 5 PBMC 12 3 4 1 1 80C 13 MAGIC A HIV 1 6 AO 40 70 p24 3 2 HIV
36. RIE HIV 20 mL 1 25 PBMC CD8 1 x 108 mL 1 3 00 f S amp X iL dier 13 HIVDNA 2 1 5mL PBMC 1 x 10 mL 5mL PBMC 1 10 mL 2 40gL CD3 50 EER 3 37 4 H H 15 mL 2 1 000 20 10 3 80C 4 PBMC RPMI 1640 30 FCS 0
37. 1 6 1x10 mL 5 100 u L 96 2 25L A 25xL B 11 UL 48 72 3 100zL Flip Out 4 100zL 1 Flip Out 200uL 5 100 HIV 1 Real time RT PCR 5 1 0
38. IC dde dori add dat dd Su Nee TEES MSN MD IAS HIV Ag Ab HIV 1 3 4 3 5 7 8 HIV 1 p24 gag 4 2000 4 4 EIA HIV Ag
39. Streptavidin HRP Conjugate SA HRP 1 1001 FART 5 1 X Wash Buffer 300L well 4 SA HRP 2 100 1 37C 1 30 1XWash Buffer 300 L well 4 10 R 100uL Eis CORC 19 12 100zL 13 EER 450nm 630 650nm OD I 24 cir ED D NC 2 CAL LPC HPC OD CAL LPC HPC ODn ue D CAL OpDn OD
40. HIV 1 M A B C D F G H J 9 A F A2 F1 F2 Circulating Recombinant Form CREF Unique Recombinant Form URP 2011 12 51 EO CRF http www hiv lanl gov content sequence HIV mainpage html HIV2 HIV 2 A G HITV 1 7 A B HIV 2 B CRFO1_AB 1 3
41. HIV 2 HIV 2 3 HIV 1 2 HIV 2 HIV 2 2 5 BED BED EIA HIV 1 Incidence Test 2 5 1 BBED HIV Recent seroconversion Long term seroconversion CDC ELISA BLISA WB NAT BED IgG HIV gp41 ODn 0 8 Recent seroconversion L Panel 155 OD n gt 0 8 Long term seroconversion
42. EIA X1 HIV 2011 12 HIV 1 2 HIV 1 2 PA PA HIV 1 2 CLIA HIV 1 2 CLIA Centaur HIV 1 2 CLIA HIV 1 2 CLIA HIV 1 2 HIV 1 2 HISCL HIV Ab CLIA HIV 1 2 HIV 1 2 PA aA 1 2 1 WB T 2 WB HIV Ag Ab FLIA HIV
43. 4 17 PCR HIV 1 quasispecies mis incorporation 2nd PCR Genetic analyzer Centrisep 5 LA FIZ BigDye
44. BED Y 1 HIV HIV 1 2 PA EIA 1C EIA ICF C gt HIV 1 2 2 HIV B 21 amp nE 2 3 4 WB 2 HIV 2 1 HIV HIV 2 1
45. 0200uL 37 96well 450nm 630 650Onm CA RR RA 1 5mL 15mL 500mL 1 1 101 BED 10u L 2 BED 376 1 3 HITV 1 BED Peptide 1 1001 4 XWash Buffer 300 L well 4 5 HIV 1 BED Peptide 100zL 37 C 1 EN A us
46. 6 10x10 mLMAGIC 5A 0 5 mL 24 RIEF 2 Viro Adembeads 1 10 25uL 1 5 ml Eppendorf Tube Adem Mag SV 1 3 Binding Buffer Adem Mag SV 1mL 20 5 Adem Mag SV Tube 6 1 mL Adem Mag SV 1 7 0 5 mL MAGIC 5A 1 8 9 PBS
47. MAGIC 5A LTR HIV HeLa4 5 LTR nEGFP HIV SIV MAGIC SA MAGIC 5A HIV 1 subtype Imdie C1 X Gal MAGIC 5A 2 3E tatsu nih so D 03 5285 1111 2320 12 4 HIV HIV HIV 1 HIV 2 HIV 1 M N O P 4
48. PA EIAJ IC HIV 1 2 EIA HIV 1 2 HIV 1 RNN 1 yielded E ibn quii 1 2 HITV 1 2 amp dv Cv SEE HIV 1 gp41 2 gp36 HIV 1 E HIV 2 3 HIV 1 2 HIV 1 2 PA
49. Chemicell com 9009 e 2 0 2 formaldehyde 0 04 glutaraldehyde in PBS e XX Gal SuL 0 5M potassium ferrocyanide SuL 0 5M potassium ferricyanide 8u L 0 25 M MgCl SuL 40mg mL X gal mL PBS 5 4 0 2 ug mL G418 Sigma 0 1 ug mL hygromycin Sigma 1 0 pL mL blasticidin S hydrochloride Funakoshi 10 1 0 mL puromycin Sigma Cell Banker 80C BSL3 1
50. 15 13 2 x 10 mL PBSC 2 FBS 16 1 7 Dynabeads M 450 CD8 Dynabeads 2 FBS 17 4 C 30 3 18 PBS 2 5 5 mL 19 MPC 2 Dynabeads 20 15 mL ELECT 21 1000rpm 20 10 22 PBMC 5 mL RPMI 1640 3096 THS A 23 24 1 x mL RPMI 1640 3096 FBS 25 37 C
51. 2 HIV 1 HIV 2 WB RNN HIV 2 2 HIV 1 HIV 2 WB HIV 1 HIV 2WB 2 HIV 1 2 HTV 1 2 2 HIV 2 HIV 1 2 gt HIV 1 2 HIV k HIV 2 HIV 1 HERE 2 2 F HIV 2 2 2 HIV 1
52. 13 000rpm 1 min 3 DI 1 5 mL eppendorf Buffer EB H O TE 50uL 1min 13 000rpm C 1 min DNA QIAquick PCR purification kit PCR 5 buffer PB 2 6 2 tuber qct 99 14 000rpm 30 buffer PE 0 75 14 000rpm 30 14 000rpm 30 1 5 mL 50 400uL O buffer HO TE 14 000rpm 30 DNA 5 PCR a ddNTP dye terminator ddNTP
53. 2 3 80 HIV AIDS highly active antiretroviral therapy HAART AIDS HIV 1987 HIV 1993 HIV HIV
54. 20 CTH 1 4 15 000rpm 10 SHE OT A 80 50 uL KNase free water 55 60C T 10 3 gag ezy RT PCR nested PCR 1 99 1 240 1 2 1 1 RT PCR fidelity SuperScript One Step RT PCR for Long Templates Invitrogen 11922 028 Takara One Step RNA PCR Kit Takara RRO24A
55. 2012 3 1 HIV 1 No i HIV 2 HIV 2WB HIV 1 HIV 2 HIV 12 1 2 HIV 2 HIV 2 HIV 2 HIV 2 HIV 2 HIV 2 HIV 2 HIV 2 2 HIV 2 2
56. Simplot http sray med som jhmi edu SCRoftware 1 3 DSimilarity plot Bootscanning plot S Informative site analysis Subregion tree analysis CRF branch 5
57. 28 1 SuperScript One Step RT PCR for 2x Reaction Mix for primer 10 M rev primer 10 M RNase free water RT Platinum Taq HiFi Mix Template RNA Total volume Negative control template Long Templates 1 I2 20 5 uL 7oOLL TuL 5 uL 5 uL 0 uL 0 uL Final conc 0 34 M Final conc 0 34 M RNA RNase free water RT PCR 50 94 PCR 94 50 68 30 15 30 min min min x 40 cycles 2 Takara One Step RNA PCR Kit 1 RNase free water 10x AMV RT PCR buffer 25mM MaCl 10mM dNTPs for primer 100 M rev primer 100 u M RNase Inhibitor 40 U 1 AMV RTase XL 5 U wu 1 AMV Optimized 5 U u 1 Template RNA Total volume 17 D 10 D 0 0 0 0 0 10 50 5 uL 0 uL 0 uL 0 uL 5 uL oO uL 5 uL OLL OLL 0 0 uL b mM 1 mM 50 pmol 50 pmol 20 0 2 5 U 2 5 U Negative control template RNA RNase free water
58. HIV CRFO1_AE CRFO1_AE 4 3 HIV 2 14 18 HIV 2 1 2 21 5 HIV2 3 4 3 2 3 4 HIV 2
59. HIV 1 2 23 HIV a po 2 3 24 HIV 2 2 5 BED 3 1 PBMC 32 PBMC HIV 33 MAGIC SA HIV HIV 41 42 4 3 HIV 2 HIV 1 KK TaqMan HIV 1 1 AIDS Human Immunodeficiency Virus HIV 1981 AIDS 6 000 HIV 1
60. HIV SIV MAGIC 5A HIV MAGIC 5A HIV SIV Coreceptors 1996 HeLa CD4 HIV 1 LTR SV40 Large T Antigen B Galactosidase HeLa CD4 LTR eLa Kimpton J Emerman M J Virol 66 2232 1992 pEFBOSbsrHuCCR5 Coreceptor CCRS CD4 pEFBOSpacCD4 HIV 1 Michael Emerman HeLa CD4 LTR B gal X gal Multinuclear
61. Viral Kit RNA 1 400uL Working Solution 200u L AM 2 10 000rpm 8 000 g 15 3 450zrL WashBuffe due 4 10 000rpm 8 000 g 15 5 450pL WashBuffer m 6 10 000rpm 8 000 g C 15 12 000rpm 10 PH F Wash Buffer T S30L Elution Buffer Eau US 8 10 000rpm 8 000 lt 15 RNA 14 4 1 3 1 PCR RNase
62. Ev Fy 20 uL i Buffer AR 300 uL x Xx1 1 60C Protease K 20 nuLX X1 1 Zmz kl APTA Buffer AR Protease K 320 uL 251 carrier 5 6 uL Adv 2 3 vortex l 5 vortex 3 5 A ETE RED l l 40C 10 5 5 vortex 2 10 5 vortex 3 l RE VAU l Buffer AB 300 uL 10 vortex l 6 000 rpm 1 l AW1 500 uL 8 000 rpm 1 2100 1 4 5 AWO 500 uL 14 000 rpm 3 SEHRE bT 5 l 0 7 mLa 7 14 000 rpm 1 l 1 5m AVE 30 uL 8 000 rpm 1
63. gt 1 Q23 17 CCTCAAATCACTCTTTGGCAACGACCTCTTGTCACAGTAAGAATAGGGGGACAGCTAAAAGAA Al SE7253 CCTCAAATCACTCTTTGGCAACGACCTCTTGTCACAGTAAAAATAGGGGGTCAGCTAAGAGAA FASTA MEGA File 2 6 Open A File Session Analyze or Align File Align Alignment Explorer Alignment Explorer Data Open Retrieve Sequences From File Z IRL FASTA ClustalW Alignment Align By ClustalW 3 Move selected block left right 2c fi 5 C
64. 1 CD8 PBMC ROURE PBMC CDS CD3 THIV 1 EE i Cd o TRES HIVRNA 1000 copies ml 80 2 2 3 4 Ova CPD NP SC0507 PBS Invitrogen GIBCO PBS 2 FBS PBS 296 FBS Ficoll Paque PLUS GE Healthcare Dynabeads M 450 CD8 Dynal rIL 2 329 RPMI 1640 Medium Invitrogen GIBCO RPMI 1640 3096 FBS RPMI 1640 Medium 0 1 ug mL 100 mL rIL 2 70 mL FBS 30 Pi CD3 CLB CD3 PeliCluster PBS 50 500 uL DET EG CO MPC Magnetic Particle Concentrator Dynal HIV 1 p24 mini VIDAS SysmexBiomerieux 1 HE PBMC 1 2 3 4 5 6 7 8 9 10 11 12
65. 2 60 uL b 60 uL l 3M 6uL 200 nuL X1 1 200 pL 3 vortex l 4C 15 000 rpm 10 24 l l 7096254 7 7v 100 uL 3 vortex i 4C 15 000 rpm 2 2119 5 fF x 7OBUGBE AGX AFANC ARRE Eum 5 10 iJ 70 YvA 24 RT PCR Master 50 nuL 20 nL 10 vortex 1 4 A RT PCR PCR SuperScript III Platinum One step Quantitative RT PCR System with ROX Invitrogen No 11745 100 EXPRESS One Step
66. 5 6 7 HIV 2 HIV 2 HIV 2 HIV 1 2 HIV2 21 R 2HIV 2 gag env ID SIVMM ID SIVMM Ibe 4 RT PCR gagA 5 AGG CGG AAA A 3 1110 1134 PFDI 5 GGT CTA TTW CAG AGA AGG CAG 3 5200 5220 gagB 5 CCT TGA GCC GTC TC 3 2116 2085 LTR9574 5 TGG TGA G 3 10074 10056 2nd PCR gagC 5 AGT ACA TGT TAA AAC ATG TAG TAT GGG C 3 1135 1162 EB2 5 TCA TGT GAY AAR CAY TAT TGG G 3 1258 1219 gagF 5 CCT TAA 1970 1941 5 5 CTC TGC TAG AC 3 7795 7776 Kusagawa Takebe RT PCR
67. 6 Step 5 24 10 QIAamp A 500 uL Buffer 8 000rpm 6 000 g T1 23 fist 3 4 QIAamp 2 mL 2mL QIAamp 5004L Buffer AW2 14 000rpm 20 000 1 pom 2mL QIAamp 14 000rpm ca 20 000 g 1 SHR T A QIAamp 1 5 mL 60 uL O Buffer AVE 1 8 000rpm ca 6 000 g G1 fig A Buffer AVE QIAamp High Pure Viral RNA Kit E 7760 9 mL 0 4 mL 2 O binding buffer cap EA OCTET EVRLEEAE IIVA edge Lb Ww 0000rpm ca 8 000 10 20 RNA inhibitor removal buffer cap 0 4 mL 10 000rpm 107 20
68. MAGIC A HIV 3 amp HIV HIV 1 RNA KK TaqMan HIV 1 kondo dvqy pref kanagawa jp 3 amp tatsu nih so jp Kenji_Sadamasu member metro tokyo jp kawahata iph pref osaka jp kusagawa nih go jp m takebe nih so jp wsugiura nnh hosp go jp 37 Veni Veni
69. 2 005A 5 TGG GGC GTC 3 1047 1073 2 051A 5 CCC GGA GGA TAG TGC 3 8264 8290 2 055BB 5 TGC AGC CTT CTG AGA GTG CCT G 3 1596 1575 2 052B 5 GAG AAT GGC TAG TT 3 8780 8758 2nd PCR 2 00 5 AGG TGA A 3 1079 1103 2 053A 5 CCG TCT GGG GAA CGA AAA ACC TCC AG 3 8348 8373 2 056BB 5 AAT GGC ACT ACT GCC CC 3 1573 1551 2 054B 5 CTT TGG GC 3 8832 8854 1 Kusagawa S Imamura Y Yasuoka A Oka S Takebe Y Identification of HIV type 2 subtype B transmission in East Asia AIDS Res Hum Retroviruses 19 1045 1049 2003 2 Utsumi T Nagakawa H Uenishi R Kusagawa S Takebe Y An HIV 2 infected Japanese man who was a long term nonprogressor for 36 years AIDS 21 1834 1835 2007 3 HIV 2 G3 IASR 27 343 2006 4 HIV 2 CRFOI AB IASR 31 232 233 2010 5 Ibe S Yokomaku Y Shiino T Tanaka R Hattori J Fujisaki S Iwatani Y Mamiya N Utsumi M Kato S Hamaguchi M and Sugiura W HIV 2 CREO1_AB first circulating recombinant form of HIV
70. 7 fi darunavir DRV 1 150V V32I 147V 2 I50V I54L L76V 3 L33F I54M 147V I84V 4 V11I V321 L33F I47V I50V IBAL M L76V I84V L89V 4 1 V11I V321 L33F I47V I50V I54L M L76V I84V L89V 3 Integrase Strand Transfer Inhibitor INSTI xaltes tavit 1 Y143C H R 2 G140S Q148R H K 3 N155H 7 33 1 HIV 2 3 4 5 6 7 HIV Name DRPRO5 DRPRO2L DRPROIM DRPROG DRRTIL DRRTAL DRRT7L DRRT6L 172A 73b 174A 175 106 17 14A 10B DRINO1 DRINO2 DRINOS DRIN04 Sequence 5 3 AGACAGGYTAATTTTTTAGGGA TATGGATTTTCAGGCCCAATTTTTGA AGAGCCAACAGCCCCACCAG ACTTTTGGGCCA
71. Ab ULT ELISA 4 WB Qs HIV pu pu 0296 0 5
72. SimploC Commands Query Start Scan Bootscanningplot BootScan Query Start Bootscan File Save as Metafile Copy Metafile 20 Bd 5 98 C 9511421068 C 9313905 C 9813012 C 92BRD25 C ETH2220 Sample pem 90CF 056 991 1997 B SimPlot Query Sample BootScan Query Sample 10 B GN RL42 100 951421068 01 97CNGXTTF 0 98 90 0 96 an 0 94 8 70 gt 0 60 B t 0 9 E 50 088 a 40 086 s 30 0 84 20 082 10 0 0 100 201 300 4050 500 600 0 800 0 100 200 300 400 500 600 700 800 Position Position Window 200 bp Step 20 bp GapStrip On Kimura parameter T t 20 Window 200 Step 20 GapStrip Reps 100 Kimura Z parameter TE 20 Neghbor Joimn
73. SuperScript qRT PCR Kits Universal Invitrogen No11791 200 HPLC deSK145 S AGTRGGGGGACAYCARGCAGCHATGCARAT 3 deSKCCIB 5 TACTAGTAGTTCCTGCTATRTCACTTCC 3 e deKK MGB probe 5 FAM 3 MGB 5 ATCAATGARGARGCTGCAGAATGGGA 3 D ABI 7900HT SuperScript III Platinum One step Quantitative RT PCR System with 1 Master mixture 2 X Reaction Mix with ROX 25 0 uL 20 uM deSK145 1 25 uL 20 uM deSKCC1B 1 25 uL 5 uM deKK MGB 2 0 uL Water 19 5 uL SuperScript III RT Platinum Mix 10 uL Total 500 uL D ABI Step One plus EXPRESS One Step SuperScript qRT PCR Kits Universal 1 Master mixture Express qPCR SuperMix with Premixed ROX 10 0 uL 10 uM deSK145 1 0 uL 10 uMdeSKCC1B 1 0 uL 4 uM deKK MGB 1 0 uL Water 5 0uL Express SuperScript Mix for One Step qPCR 2 0 uL Total 20 0uL RT PCR 1 2 23 50 15 95 2 95 C 15 f 60C 1 40 cycles 5 slope 3 80 91 5 3 32 100 TaqMan pr
74. Terminater Cycle Sequencing Kit ver3 1 Genetic analyzer 1 D W 2 9 4T Purified PCR product 2 9 1 6 uM Primer l 0u L Sequencing buffer 2 0 1 16 Sequencing reagent premix 20uL 2 or or ecc T dpa bus os 96 C 10sec 50 5 cycles 60C 4min 3 800u L DW 30 4 5
75. 000 3000 4000 5000 6000 7000 8000 9000 9719 172A 173B DRIN01 lt DRIN02 174A 175B DRIN05 amp 4 DRIN04 DRPRO5 amp 4 DRPRO2L 106A gt 4 17B DRPRO1M 4 DRPRO6 14A 9 4 10B 2002 HIV 1 602 703 710 Hirsch M S Brun Vezinet T D Aquila S M Hammer V Johnson D Kuritzkes C Loveday J W Mellors B Clotet B Conway L M Demeter S Vella D M Jacobsen and D D Richman 2000 Antiretroviral drug resistance testing in adult HIV 1 infection recommendations of an International AIDS Society USA Panel 283 2417 26 Sugiura W 2001 Effect of introduction of highly active antiretroviral treatment and the changes in patterns of drug resistant HIV 1 Japan J Infect Chemother 7 127 32 36 Ta L 25 Zl Zu 234 2 9 3 2 3 3 4 5 6 HIV CBE ERR CBE ER HIV 2 BED PBMC PBMC HIV
76. 2 J Acquir Immune Defic Syndr 54 241 247 2010 1024001 o HIV 2 7 0811001 HIV 2 8 0203001 HIV 2 22 5 HIV 1 KK TaqMan 1X 5 1 HIV 1 HIV 1 HIV 1 HIV 1 HIV 1 2008 4 PCR TaqMan HIV 1 TaqMan PCR m HIV 1
77. 20 mL 8i 2 1 E LJ 5 mL 2 000 rpm 960 g 10 PBS 2 3mL tube Ficoll Paque Plus ger 5mL 3 724 15 mL X 2A E C ZO EZ PBS Ficoll 1 500 rpm 540 g 20 30 PBMC 4 15 mL REI 5 mL PBS Ficoll Paque Plus 1 5 mL 10 mL PBS 2 KORE DDOD PBMC 1 200 rpm 350 g 20 15 5 mL PBS 1 000 rpm 240 g 20 10 2 AA OR 1 S3 mL RATA 13 LOO ISI ID uxo CJ BE 2 3 2 12 1000 rpm 20 10
78. 44D D67N L210W T215Y F 2 M41L D67N V118I L210W T215Y F 3 M41L D67N L210W T215Y F 4 Q151M complex b T69ins non nucleoside RT inhibitor NNRTI Efavirenz EFV 1 L100I K103N V106M Y188L G190C Q T S 1 1 G190E M230L etravirine 1 90 A986 1 1001 KIOIE H I P R V106I V179D F 1 L M T 4 2 Y181C I G190A S M230L 3 L1001 K103N Y181C 4 V179F Y181C 5 Y181C H221Y 6 Y181V 1 L1001 V179F I Y181C 190 M230L Y318F 1 protease inhibitor PI atazanavir ATV gt 1 L10I V F K20R M T L241 L33I F V M36I L V MA6I L 648 154V L L63P ATIV T I G73C S T A V82A F S T I84V L90M 5 lopinavir LPV 1 I50V 154A M S T V V82A F S 1 LIOF I GI6E K20I M V321 L33F E34Q K43T MA6I L I47V G48M V I50V 154A M S T V Q58E L63T G73T 745 V82A1F S L89I M 5 2 LIOF I 616 K20I M V321 L33F E34Q K43T M46I L 147 G48M V 150V 154A M S T V Q58E L63T G73T T74S V82A F S L89I M
79. Activation of a Galactosidase Indicator Assay MAGI 1996 Edward A Berger CXCR4 CCRS Coreceptor Science 272 872 1996 202 HeLa CXCR4 MAQI CCRS MAGIC 5A MAGI CCRS A e e RT30mini 5 Dulbecco s MEM 5 FCS SP Dulbecco s MEM Dulbecco s PBS Trypsin EDTA MAGIC SA Viro Adembeads Ademtech com 07010 http www ademtech com products aspx7id 104 id_p S8 Protocol e Adem Mag SV 1 5 mL 20101 e MagsnetoFACTOR 24 24
80. Ag Ab CLIA HIV Ag Ab ULT ELISA HIV ELISA HIV DUOI ELFA HIV 1 2 HIV1 HIV combi ECLIA HISCL HIV Ag Ab CLIA HIV Ag Ab CLIA HIV Ag Ab CLIA HIV Ag Ab 1C Y N AE Chia 1 HIV 1 P24 CLIA TaqMan HIV 1 v 2 0 RT PCR HIV 1 RNA ME 1 RT PCR 2 3 WB
81. G TGC TTT GGT TCC 3 3425 3402 004B 5 GTC TGG GTA CCG TCA GCG 3 7851 7831 Zn 1 PCR 510A 5 AGA GCC ACC AG 3 2148 2167 007A 5 ACA C 3 6945 6969 504B 5 ACT TTT GGG CCA TCC ATT CC 3 2611 2592 575B 5 ATG GGA GGG GC 3 7540 7521 513A 5 GAC CTA CAC CTG TCA ACA TAA TTG G 3 2485 2509 574A 5 TTA ATT GTA GAG GGG AAT TTT TCT AYT G 3 7351 7378 530B 5 TGG AAT GGT CC 3 3031 3012 521 B 5 GCC CAT TGC TGC TCC 3 1816 1193 532A 5 AAC TCA AGA CTT CTG GGA AGT 3 2198 2818 514B 5 TAA CAT AAA TTG 3 3372 3348 4 1 2 EL HAART PBMC DNA DNA Nested DNA High
82. M 4 1 4 2 2 44 X4 MEGA OR Phylogeny Construc Test Neighbor Joining Tree MEGA Analysis Preference Test Phylogeny Bootstrap Method No ofrep 100 ModelMethod Tajiima Nei Compute File P Save Current Session Image Copy to Clipboard
83. S 523 OO 271 500 000 50 000 5 000 500 150 50 3 mL 1 5mL 500 L 80C ERR 500 000 5 000 15015 mL 3 50000 500 50 aE mL 3 RNA RNA QIAamp UltraSens Virus Kit QIAGEN 53704 1 5m 1 5mL A 1000P 200P 20P a 23 500 pL 1 5mL l 4C 15 000rpm 1 ERE EGT 5 1 5mL l 20 uL 15 000 rpm 5
84. S x 10 mL 5 10 mL 6 37 C CO 7 507 0528 p24 HIV p24 II SysmexBiomerieux fH cm n uil 8 PBMC 80 C 2 ERU 5 p24 1 2 5 1 HIV 13 165 174 2002 2 Marijke Th L Roos Maria Prins Maarten Koot Frank de Wolf Margreet Bakker Roel A Coutinho Frank Miedema Peter Th A Schellekens Low T cell responses to CD3 plus CD28 monoclonal antibodies are predictive of development of AIDS AIDS 12 1745 1751 1998 3 Dynabeads Dyanbeads Letter http www invitrogen jp tech DynabeadsLetter DL 19 pdf HIV
85. TCCATTCC ATGATAGGGGGAATTGGAGGTTT TACTICTGTTAGTGCTTTGGTTCC GACCTACACCTGTCAACATAATTGG TAATCCCTGCATAAATCTGACTTGC ATCTCTAGCAGTGGCGCCCGAACAG CTGATAATGCTGAAAACATGGGTAT CTCTCGACGCAGGACTCGGCTTGCT CCCATGCATTCAAAGTTCTAGGTGA CATACATTATTGTGCCCCGGCTGG AGAAAAATTCCCCTCTACAATTAA AATGTCAGCTCAGTACAATGCACAC ATTTCTGGGTCCCCTCCTGAGG CAGACTCACAATATGCATTAGG CCTGTATGCAGACCCCAATATG CTGGCATGGGTACCAGCACACAA TAGTGGGATGTGTACTTCTGAAC HXB2 Accession No K0345 zx 2074 gt 2116 gt 2148 gt 9 7 29990 2 7 3425 gt 2485 gt pof 625 gt 1319 083 gt T2597 6866 1374 gt 6945 gt 336 gt 4039 gt 0264 gt 4146 gt 34 Location in HXB2 2095 2091 2167 2592 2410 3402 2509 3348 649 1294 07 1251 6889 1351 6969 1315 4060 0243 4168 5195 Orientation and usage PR outer PR outer PR inner PR inner RT outer RT outer RT inner RT inner Gag outer Gag outer Gag inner Gag inner Env outer Env outer Env inner Env inner Integrase outer Integrase outer Integrase inner Integrase inner forward reverse forward reverse forward reverse forward reverse forward reverse forward reverse forward reverse forward reverse forward reverse forward reverse SE 1 PCR pat f rev RNase H pol vpr env 5 LTR PR ME gp120 gp41 3LTR EE 0 1000 2
86. c x 30 cycles 74 5 sec 2 G 7 min 3 Hot Start Ex Taq 1 Nuclease free water 36 0 uL 10x Ex Taq buffer 5 0 uL dNTP mixture 2 5 mM 4 0 uL for primer 100 M 0 5 uL rev primer 100 M 0 5 uL TaKaRa Ex TaqHS 5 U u 1 0 5 uL RT PCR product 3 5 uL Total 50 0 uL 98 15 sec 98 C 10 sec 60 C 30 sec x 30 cycles 72 60 sec CZ 4 PCR dNTPs QIAquick Gel Extraction kit QIAGEN 28704 30 QIAquick PCR purification kit QIAGEN 28104 1 QIAquick Gel Extraction kit 1 2 3 4 elc 2 920215 PCR 3 buffer 06 1 C PCR 1 isopropanol 13 000rpm 1 min buffer PE 650uL 13 000rpm 1 5
87. n OD ODcAr 2 ODn gt 1 2 Long term seroconversion ODn 1 2 3 ODn i 20 8 Long term seroconversion ODn 0 8 Recent seroconversion 3 3 1 PBMC PHA PBMC HIV PBMC EDTA 10 20mL 1 PBMC 50mL 1 3 PBS 10mL 30mL 20mL 2 10mL PBS 2
88. obe EB 1 HIV 1 HIV 1 50 mL 1 50 mL WB 2 HIV 1 50 mL lt 50 1t mL 50 mL 6 PCR
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