1. HIV-1 Diversity and Related Retroviruses Dr. Matthew D. Marsden, Ph.D. UCLA, Department of Medicine mmarsden@ucla.edu

2. Types of Viruses (Baltimore Classification) I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc) Herpesviridae (Herpes, CMV, EBV), Poxviridae (Smallpox, Chickenpox, Vaccinia), Papilloma virus, Adenovirus II: Single-stranded (+) sense DNA (Parvoviruses) Erythema infectiosum, Phages III: Double-stranded RNA (Reoviruses; Birnaviruses) Rotavirus, Reovirus IV: Single-stranded (+) sense RNA (Picornaviruses; Togaviruses, etc) Polio, SARS, Hep A, Hep C, Rubella, Yellow fever V: Single-stranded (-) sense RNA (Orthomyxoviruses, Rhabdoviruses, etc) Rubella, Influenza, Rabies, Measles, Mumps, Ebola VI: Single-stranded (+) sense RNA with DNA intermediate (Retroviruses) HTLV, HIV VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses) Hep B

4. Classification of Retroviruses: Divided into 3 subfamilies based primarily on pathogenicity rather than genome relationships. Subfamilies Oncovirinae Lentivirinae Spumavirinae

5. Classification of Retroviruses: Subfamilies are further divided based on: 1.Virion structure (types A-D) 2.Utilization of particular cell receptors 3.Lifestyle: whether endogenous or exogenous 4.Presence or absence of an oncogene 5.Other pathogenic properties When nucleotide sequences and genome structure are considered, 7 groups (genus) emerge.

6. Classification of Retroviruses: SubfamilyGenusIsolates LentivirinaeLentivirusHIV-1 HIV-2 SIV FIV Visna/maedi EIAV CAEV

7. Lentivirus All Retroviruses contain gag, pol, pro* and env Lentiviruses are more complex: Two regulatory genes: tat and rev Accessory genes: HIV-1 nef, vif, vpr, vpu HIV-2 and SIV lack vpu and have vpx *pro is sometimes contained within pol (HIV)

9. Diversity resulting from base mis- incorporation (clades: resemble each other across the genome)

10. Lacks proofreading function 1000-fold higher rate of nucleotide substitutions than seen with replication of viral DNA genomes 1 nucleotide change is introduced each time provirus is synthesized This results in quasispecies formation Reverse transcriptase

11. HIV Life cycle http://preprod.www.tibotec.com/content/backgrounders/www.tibotec.com/hiv_lifecycle.html

12. HIV Life cycle http://preprod.www.tibotec.com/content/backgrounders/www.tibotec.com/hiv_lifecycle.html HIV mutates every time it replicates HIV replicates in billions of cells simultaneously every day HIV therefore evolves around 1 MILLION TIMES faster than mammalian genes

13. http://www.nature.com/embor/journal/v4/n6s/fig_tab/embor857_F1.html Comparison of HIV genetic variation with flu virus (longer lines mean more mutations)

14. http://www.nature.com/embor/journal/v4/n6s/fig_tab/embor857_F1.html Comparison of HIV genetic variation with flu virus (longer lines mean more mutations)

15. http://www.nature.com/embor/journal/v4/n6s/fig_tab/embor857_F1.html Comparison of HIV genetic variation with flu virus (longer lines mean more mutations)

16. http://www.nature.com/embor/journal/v4/n6s/fig_tab/embor857_F1.html Comparison of HIV genetic variation with flu virus (longer lines mean more mutations)

17. http://www.avert.org/hiv-types.htm HIV “family tree”

18. Genetic subtypes of HIV-1 MON A-H, J (9) F1 &F2 Groups Clades or Subtypes Subclades Strains or isolates JR-CSF 8 full, and 5 partial, Genome sequences P 1 infected person Reported ‘09 Limited Spread 2nd infected person Reported ‘10

19. Groups (env sequences): M, O, N, P M: Main, O: Outlier, N: New or Non-M-Non-O, P follows O Subtypes or clades (env sequences): A-H, J Equidistant env (25-30% a.a. differences) Up to 20% a.a. sequence differences within a clade Full length gene sequences required with no evidence for recombination Strains or isolates: e.g., HIV IIIb, HIV RF Genetic subtypes of HIV

20. Reasons For Genetic Diversity Of HIV: 1.Zoonotic transmission from at least 3 sources: Chimpanzees and Sooty Mangabees and Gorillas on at least ten different occasions. 2. Rapid rate of mutation due to: Reverse transcriptase (~1 error per genome) and fast turnover in humans. 3. Recombination events

21. http://what-when-how.com/medical-microbiology-and-infection/zoonoses-systemic-infection/ Zoonosis = Cross-species transmission event

22. Phylogeny of lentiviruses. Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 ©2011 by Cold Spring Harbor Laboratory Press

23. Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841

24. Where did HIV come from? HIV entered the human population from primates, which harbor a related virus known as SIV (simian immunodeficiency virus). This probably occurred during the butchering and consumption of monkey meat in Africa. http://www.avert.org/hiv-types.htm HIV “family tree”

25. Where did HIV come from? HIV entered the human population from primates, which harbor a related virus known as SIV (simian immunodeficiency virus). This probably occurred during the butchering and consumption of monkey meat in Africa. http://www.avert.org/hiv-types.htm HIV “family tree” http://www.awf.org/content/wildlife/detail/chimpanzee HIV-1 group M, which is the most prevalent HIV strain, jumped from chimpanzees into humans.

26. Where did HIV come from? HIV entered the human population from primates, which harbor a related virus known as SIV (simian immunodeficiency virus). This probably occurred during the butchering and consumption of monkey meat in Africa. http://www.avert.org/hiv-types.htm HIV “family tree” HIV-1 group M, which is the most prevalent HIV strain, jumped from chimpanzees into humans. http://www.awf.org/content/wildlife/detail/chimpanzee HIV-2 originated in sooty mangabeys and is responsible for fewer infections than HIV-1 New viruses are still being transferred from primates to humans, and have the potential to cause new diseases and epidemics. http://pin.primate.wisc.edu/factsh eets/entry/sooty_mangabey

29. Origins of human AIDS viruses. Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 ©2011 by Cold Spring Harbor Laboratory Press

30. Source: Adapted from Atlas of Infectious Diseases, Mandell & Mildvan (ed.), pp. 2.3 HIV and SIV Hosts VirusHostNaturalPathogenicOrigin HIV-1HumansNoYesAfrica SIVcpzChimpanzeesYes ?No ?Africa HIV-2HumansNoYesAfrica SIVmacMacaquesNoYesAsia SIVsmSooty MangabeesYesNoAfrica SIVagmAfrican Green MonkeysYesNoAfrica SIVmndMandrillsYesNoAfrica SIVsykSykes MonkeysYesNoAfrica GorillasSIVgorYesAfrica ?

31. Cross Species Transmission of SIV cpz SIVcpz and HIV-1 are identical in genomic organi- zation. Only two lentiviruses to contain vpu. Sequence of SIVcpz suggests HIV-1 transmission from P. t. troglodytes. Observed natural infection of P. t. troglodytes with isolates more closely related to SIVcpzUS and YBF30 (group N) than any other HIV-1/SIV.

32. Origins of HIV-1 West Equatorial Africa is the only location where M, N, and O co-circulate, and where P. t. troglodytes is infected with closely related viruses. Group M appears to originate from SIVcpzPtt in South Central Cameroon. Group N most likely originated in southeastern Cameroon as N sequences are highly related to SIVcpzPtt in that region.

33. Infection of Gorillas and Chimpanzees in Cameroon with viruses closely related to Group O HIV. Although sequences suggest chimpanzee transmission to Gorillas, it’s unclear which animal is responsible for transmission to humans. Gorilla transmission to one woman in Cameroon resulted in group P Greatest diversity of group M is observed in Cameroon. Origins of HIV-1

35. Geographic distribution of SIVcpz and SIVgor infections in sub-Saharan Africa. Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 ©2011 by Cold Spring Harbor Laboratory Press

36. HIV-1 origins. Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 ©2011 by Cold Spring Harbor Laboratory Press

37. HIV-1 origins. Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 ©2011 by Cold Spring Harbor Laboratory Press Likely independent transmission events into humans

38. HIV-2 origins. Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 ©2011 by Cold Spring Harbor Laboratory Press

39. HIV-2 origins. Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 ©2011 by Cold Spring Harbor Laboratory Press Likely independent transmission events into humans

40. When did HIV first jump into humans?

41. M Worobey et al. Nature 455, 661-664 (2008) doi:10.1038/nature07390 Maximum clade credibility topology inferred using BEAST v1.4.7 under a Bayesian skyline plot tree prior.

42. The strain of HIV responsible for the majority of global infections (HIV-1 Group M) probably jumped into humans in western Africa sometime between 1884 and 1924. From there it then spread throughout the world.

43. Origin and Spread of HIV

44. Groups (env sequences): M, O, N, P M: Main, O: Outlier, N: New or Non-M-Non-O Subtypes or clades (env sequences): A-H, J Equidistant env (20-30% a.a. differences) Up to 10-15% a.a. sequence differences within a clade Full length gene sequences required with no evidence for recombination Strains or isolates: e.g., HIV IIIb, HIV RF Genetic subtypes of HIV

45. Group M (1959 [1910 – 1950]) Arose from one cross-species transmission event. Most prominent group worldwide with 11 subtypes or clades: A-K Of these only 9 are true clades, E and I appear to be recombinants. G clade has accessory genes that resemble clade A but it is otherwise distinct. Are some unclassified clades.

46. Group M cont. Has four subclades: F1, F2, B and D F diverged into F1 and F2 and divergence between these two subclades is not much greater than between other clades. B and D show the same extent of divergence but for historical reasons continue to be classified as clades, not subclades. Clade B is the most common clade in North America and Europe.

47. Evolutionary relationships among non-recombinant HIV-1 group M clades based on near-full length genome sequences. Peeters and Sharp AIDS 2000, 14 (Suppl 3): S129-S140.

48. Spira et al. J. of Antimicrobial Chemotherapy 2003, 51: 229-240

49. Group O (1963) First described in ~1990 in Camaroon, France and Gabon Highly divergent from group M with only ~50% homology to M in env. Represents a minority of infections in those regions, ~2-5%. Has no subtypes which may be the result of slow and limited spread, although with more sequencing of genomes, subtypes may be found.

50. Group N More recently identified (1995) ~12 confirmed infections all in Camaroon Sequences suggest this group is mosaic 5’ half distantly related to M, 3’ half more closely resembles SIVcpzus

51. 1st known infection (seropositive 2004, reported 2009) Woman in Paris, France recently emigrated from Cameroon Sequences suggest SIVgor was responsible Cannot detect by PCR using M or O primers Group P

52. 2 nd individual identified in Camerron HIV-seropositive male hospital patient Screening of 1,736 HIV-seropositive suggest group is rare ~ only 0.06% of HIV- infections Reported 2010 Vallari et al. J.V. Nov. 17 th

53. Diversity due to recombination (mosaics: genes derived from more than one parental strain)

54. Nature Reviews Immunology 6, 930-939 (December 2006)

55. Mechanism(s) For Recombination ABAB ABAB

56. http://www.hiv.lanl.gov/content/se quence/HIV/REVIEWS/hahn.html

57. Recombinants or Mosaics Eg. MAL: one of the first African HIV-1 isolates characterized. 1994: first multiply infected individual identified. ~10-20% of newly characterized strains. Identified by discrete breakpoints in genomic regions. Isolated in regions where both parental strains are found.

58. Recombinants or Mosaics (cont.) Similar breakpoints reflect common ancestry. Designated ‘circulating recombinant forms: (CRF) CRFs are given identifying numbers followed by letters reflecting the parental clades: CRF01-AE If more than three clades are parental, the CRF is followed by cpx for ‘complex’. CRF01-CPX

59. Evolutionary relationships of CRFs, CRF01-AE and CRF02-AG. Based on full length gag, 3’ end of pol, and gp120 sequences. Peeters and Sharp AIDS 2000, 14 (Suppl 3): S129-S140.

60. Peeters and Sharp AIDS 2000, 14 (Suppl 3): S129-S140. Evolutionary relationships of CRFs, CRF01-AE and CRF02-AG. Based on full length gag, 3’ end of pol, and gp120 sequences.

61. Peeters and Sharp AIDS 2000, 14 (Suppl 3): S129-S140. Geographical distribution of predominant groups, clades and CRF’s.

62. Importance of CRFS Successful transmission of SIV to Chimpanzees and humans, and subsequent human to human transmission, required viral adaptation. Recombination increases the odds of “successful diversity”. Indeed, transmission of SIV to Chimpanzees is thought to have included a recombinant event. CRF01_AE is Southeast Asia and CRF02_AG in West Africa are the fastest-spreading epidemic strains. Subtypes A, C and CRF02_AG now account for 75% of the 14,000 estimated new daily infections worldwide. Heeney et al. Science 2006, 313:462

63. Why is understanding diversity important? Key to understanding the path of the epidemic. Diagnostic tests: mainly available for clade B, and diversity can affect sensitivity. Antiretroviral drugs: mainly available for clade B only. Group O and HIV-2 are naturally resistant to NNRTs.Group M shows variation in drug susceptibilities. Transmission and rates of disease progression differ between HIV-1 and HIV-2.

64. Why is understanding diversity important? Biological differences among groups and clades: Virus or host factors? Vaccines: nAb and CTL may be clade specific – or not. Implications are that conserved regions must be included. Vaccines and gene therapy: Recombination events are a concern. Endogenous retro- viruses and infectious viruses must be considered.

65. So, How Did Chimpanzees Get Infected? Chimpanzees are predatory animals. Prey of chimpanzees are also infected with SIV Infection was with a recombinant virus, an ancestor of viral strains currently infecting red-capped Mangabeys and the greater spot-nosed monkeys. Both of these monkeys have overlapping ranges with P.t. troglodytes in west and central Africa. Origin of infection of P. t. schweinfurthii is not currently clear.

66. So, How Did gorillas Get Infected? Gorillas are not predatory animals. Share home range with chimpanzees who are also infected with SIV Route of infection is currently unclear

67. Origins of HIV: Some major take home points Different strains of HIV have been independently transmitted into humans from animals (Chimpanzee, Sooty mangabey, or Gorilla) on over 10 separate occasions. The most common strain of HIV (HIV-1, group M), which is responsible for over 90% of global infections, originated in chimpanzees (Pan troglodytes). This likely occurred in Cameroon (West Africa) approximately 100 years ago. Bushmeat (hunting monkeys for food) is the most likely source of these transmissions of AIDS viruses into humans. Mutation and recombination have shaped the HIV epidemic, and are important for immune evasion, drug resistance, vaccine development etc.

68. General References: Sharp P M, and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841 Publication Date: August 1, 2013 | ISBN-10: 0199641145 | ISBN-13: 978-0199641147 | Edition: 1

69. More References: 1.Hybrid origin of SIV in Chimpanzees Bailes et al. Science 2003, 300:1713 1.Origins of HIV and the Evolution of Resistance to AIDS. Heeney et al. Science 2006, 313:462 3.The Black Death and AIDS: CCR5-D32 in genetics and history Q.J.Med 2006, 99:497 4.Chimpanzee Reservoirs of Pandemic and Nonpandemic HIV-1 Keele et al. Science 2006, 313:523

70. More References Molecular Epidemiology of Simian Immunodeficiency Virus Infection in Wild- living Gorillas Neel C, Etienne L, Li Y, Takehisa J, Rudicell RS, et. Al. J Virol. 2009 Nov 11. [Epub ahead of print]PMID: 19906908 [PubMed - as supplied by publisher] Origin and Biology of Simian Immunodefiency Virus in Wild-Living Western Gorillas Takehisa J, Kraus MH, Ayouba A, Bailes E, Van Heuverswyn F, Decker JM, Li Y, Rudicell RS, Learn GH, Neel C, Ngole EM, Shaw GM, Peeters M, Sharp PM, Hahn BH. J Virol. 2009 Feb;83(4):1635-48. Epub 2008 Dec 10.PMID: 19073717