1. Molecular epidemiology of HIV-1 in central region of Nepal Nirajan Bhusal 1,2, Navin Horthongkham 1, Niracha Athipanyasilp 1, Wannee Kantakamalakul 1, Ruengpung Sutthent 1 1 Department of Microbiology, Faculty of Medicine Siriraj hospital, Mahidol University, Thailand. 2 Department of Microbiology, Kathmandu University School of Medical Sciences, Kavre, Nepal

2. data sources:AIDSinfoonline.org Trend of global HIV Infection and Death 1990-2013

7. HIV Prevalence:

9. Subtypes and CRFs distribution in Asia:

10. Epidemic snapshot of HIV in Nepal

13. Subjects

14. Clinical characteristics of selected sample Sample I.DYear of Infection Location Risk GroupViral loadART status NP0032007 Nawalparasi HSR3,685NONE NP0042008 Chitwan HSR79,787AZT,3TC,NVP NP0052008 Nawalparasi HSR5,942NONE NP0072009 Chitwan IDU3,298AZT,3TC,NVP NP0082011 parsa MTCT31,940NONE NP0102001 Chitwan HSR10,413AZT,3TC,NVP NP0142011 Chitwan HSR5,942AZT,37C,NVP NP0152011 Chitwan HSR63,485NONE NP0162011 Chitwan HSR14,420AZT,3TC,NVP NP0212011 Gorkha HSR4,599AZT,3TC,EFV NP0232008 Chitwan HSR22,777AZT,3TC,NVP NP0282011 Makwanpur HSR< 40AZT,3TC,NVP NP0352011 Chitwan MTCT8,64,318d4T,3TC,EFV NP0412010 Chitwan IDU4,599AZT,3TC,NVP NP0462009 Chitwan IDU40,192NONE NP0512010 Chitwan HSR159,488AZT,3TC,NVP NP0542003 Chitwan IDU25,980AZT,3TC,NVP NP0652010 Chitwan MSM2,176NONE NP0682007 Gorkha HSR2,230NONE NP0692011 Chitwan MTCT6,067NONE NP0712009 Chitwan IDU51,218AZT,3TC,NVP NP0742011 Nawalparasi HSR86,103AZT,3TC,NVP NP0752009 Nawalparasi HSR7,314NONE NP0762009 Nawalparasi HSR9,649NONE NP0782011 Chitwan MTCT3,186NONE

15. Sample I.DYear of Infection Location Risk GroupViral loadART status NP0792011 Chitwan HSR9,649NONE NP0802005 Nawalparasi HSR21,253NONE NP0822011 Nawalparasi HSR32,235NONE NP0842008 Chitwan HSR44,285NONE NP0862011 Parsa HSR4,695 AZT,3TC,NVP NP0892001 Chitwan HSR60,480NONE NP0912007 Chitwan HSR3,535NONE NP0922006 Nawalparasi MSM21,265AZT,3TC,NVP NP0932010 Chitwan HSR5,545NONE NP0952011 Chitwan HSR<40AZT,3TC,EFV NP0972009 Nawalparasi MSM1,819NONE NP1002011 Nawalparasi MTCT9,625NONE NP1012011 Chitwan HSR5,807NONE NP1022010 Nawalparasi HSR31,794AZT,3TC,NVP NP1032010 Chitwan HSR2,612AZT,3TC,NVP NP1042010 Chitwan HSR159,488NONE NP1072003 Chitwan IDU25,980AZT,3TC,NVP NP1112010 Chitwan HSR2,176NONE NP1132003 Gorkha HSR2,230AZT,3TC,NVP NP1152011 Chitwan MSM6,067NONE NP1192011 Chitwan HSR51,218NONE NP1202008 Nawalparasi HSR86,103AZT,3TC,NVP NP1222009 Nawalparasi HSR7,314NONE NP1282009 Nawalparasi HSR9,649NONE NP1312011 Chitwan MTCT3,186NONE Clinical characteristics of selected sample

17. Siriraj hospital in bangkok

18. Ethical issues

19. Characteristics Total patientsART Naïve patientsART experienced n n n Number of patients1073 Male642 Female431 Transmission mode Heterosexual541 Homosexual110 MTCT211 IDU211 medianmin-maxmedian Age (years) 335-5730 CD4 + T cells 365.58 - 1188443 Viral load (log copies/ml) 3.31.60 – 6.003.61

23. Phylogenetic analysis of 10 HIV-1 nearly full length nucleotide sequences

24. 9 samples : 11NP004,11NP014, 11NP016, 11NP065, 11NP076, 11NP084, 11NP086, 11NP091 and 11NP115 1 sample 11NP079

25. Fragment Start Position Uncertainty Region Start - End Breakpoint Interval Start - End Fragment End PositionFragment Subtype Position in the original sequence [pred_recombinati on], [recombination_incl_UR _and_BPI], [UR_and_BPI][pred_recombinati on][recombination_incl_UR _and_BPI][UR_and_BPI] 1--789N/A 7902701 - 2716-2716C 27172717 - 3590-3590D 35913591 - 35973818 - 38653828C 3829--4902D 4903--9390C 9391--9681N/A Position based on HXB2 numbering [pred_recombinat ion] [recombination_incl_UR _and_BPI] [UR_and_BPI]HXB2 numbering[pred_recombinat ion][recombination_incl_UR _and_BPI][UR_and_BPI]

27. Sample ID HIV diagnosis year Route of infection Plasma HIV-1 RNA level (copies/ml) HIV DRM PI NRTI NNRTIDrugs 11NP0082011MTCT31,940--K103R Efavirenz 11NP0102010HSR10,413--V179D Delavirdine 11NP0682009HSR2,230--E138A Etravirine 11NP0712009IDU51,218--K103R Efavirenz 11NP822010HSR32,325A71V-- Ritonavir 11NP1032010HSR2,612A71T-- Indinavir 11NP1042010MTCT159,297--V108I Efavirenz 11NP1222011MSM58,782A71T-- Mutation pattern of amino acids and drug resistance

28. Subtype C NP015 one major mutation at position 82 one minor at 60 CRF-08BC NP008 three minor mutation at position 36,60 and 77 Position 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 B-cons T V L E E M N L P G R W K P K M I G G I G G F I K V R Q Y D C-cons..... V S... K. R............... E NP008..... D I..................... D Q NP015....... N.................... D H Position 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 B-cons A I G T V L V G P T P V N I I G R N L L T Q I G C T L N F C-cons.................. M.......... NP008...... I...................... NP015........... I................. Amino acid polymorphism in different subtypes

29. Signature sequence pattern analysis 295-----------------------------305---------------------------315 NP003- HSRT C T R P S N N T R K S I I G P G Q T F Y NP131- MSMN.... N............... ----------------------------325--------------- NP003- HSRA T G D I I G D I R Q A H C N I NP131- MSM....... A V....... 295------------------------------305-----------------------------315 NP003-HSRT C T R P S N N T R K S I I G P G Q T F Y NP128-MTCTN......... Q R...... A.. ----------------------------------325--------------- NP003- HSRA T G D I I G D I R Q A H C N I NP128-MTCT....... E.. K.....

30. 30 295-----------------------------305---------------------------315 NP003-HSRT C T R P S N N T R K S I I G P G Q T F Y NP041-IDUV. I.. N............... ---------------------------------325-------------------- NP003-HSRA T G D I I G D I R Q A H C N I NP041-IDU................ Signature sequence pattern analysis 9 variable positions amino acid sequences between different risk groups All risk group had GPGQ pattern at v3 loop.