1. Computational analysis of four human adenovirus type 4 genomes reveals molecular evolution through two interspecies recombination events Amy Smith Judy Chu Kemi Abolude September 17th, 2013 Shoaleh Dehghan, Jason Seto, Elizabeth B.Liu, Michael P.Walsh, David W.Dyer, James Chodosh, Donald Seto

2. Outline Introduction Materials & Methods Results Discussion Conclusion Introduction Materials & Methods Results Discussion Conclusion 1

3. AdV: Adenovirus, HAdV: Human Adenovirus Gene therapy: using DNA as a pharmaceutical agent to treat disease Lateral gene transfer: the transfer of genetic material between species Zoonosis: an infectious disease that is transmitted between species, from other animals to humans or vice versa ITR: Inverted Terminal Repeats Terminologies Introduction Materials & Methods Results Discussion Conclusion 2

4. Computational analysis of human adenovirus type 4 (HAdV-E4) Provides insight into its zoonotic origin and molecular adaptation to a new host First report of interspecies recombination event for HAdVs, and first documentation of a lateral partial gene transfer from chimpanzee AdV Important because chimpanzee AdVs could be used as candidate gene delivery vectors for human patients instead of HAdVs due to concerns of seroprevalence Overview Introduction Materials & Methods Results Discussion Conclusion 3

5. First isolated in 1953 from adenoid Found in many vertebrate populations ranging from fish to human Non-enveloped with an icosahedral (20-sided) nucleocapsid structure Heavily reliant on host cell for survival and replication Adenoviruses Introduction Materials & Methods Results Discussion Conclusion 1. penton capsomeres 2. hexon capsomeres 3. viral genome (linear dsDNA) Image from: Pico en el Ojo, 2006 4

6. Medium-sized (90-100nm) Contains linear, non- segmented double- stranded DNA Size: 26-46Kbp (22-46 coding genes) Adenoviruses Introduction Materials & Methods Results Discussion Conclusion Source: http://www.difossombrone.it 5

7. FunctionProtein Structural proteinscapsid proteins II (hexon), III (penton base), IIIa, IV (fiber), VI, VIII, and IX; and core proteins V, VII, X, and terminal protein TP Encapsidation proteins (involved in assembly of capsids) IVa2, 52K, and L1, and hexon assembly protein 100K Control proteinsE1A, E1B 19K, E1B 55K, … Adenovirus coding genes Introduction Materials & Methods Results Discussion Conclusion Some examples of AdV proteins: 6

8. Inverted Terminal Repeats (ITRs) occur at both ends of the DNA strand Comprises 145 bases each Important for recombination and circularization of AdV genomes, and integration with the host cell genome Adenovirus Genome - ITRs Introduction Materials & Methods Results Discussion Conclusion 7 *Image in public domain

9. Causes diseases involving respiratory, GI, occular, genitourinary systems as well as metabolic disorder (obesity) Most infections are asymptomatic and persistent Found to co-infect with other HAdVs, with up to four viruses characterized in some patients Important biomedical tools as vectors in vaccination and gene therapy Human Adenoviruses Introduction Materials & Methods Results Discussion Conclusion 8

10. Total of 57 serotypes classified into seven: Human Adenoviruses Introduction Materials & Methods Results Discussion Conclusion A 12, 18, 31 B 3, 7, 11, 14, 16, 21, 34, 35, 50, 55 C 1, 2, 5, 6, 57 D 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, 51, 53, 54, 56 E 4 F 40, 41 G 52 9

11. Relatively high similarity to several chimpanzee adenoviruses Origins thoroughly probed using several protocols and instruments Recombinant genome; contains the hexon loops of HAdV-B16 in genome chasis of SAdV- E26 (simian adenovirus) HAdV-E4 Introduction Materials & Methods Results Discussion Conclusion 10

12. MATERIALS & METHODS 11

13. 2 Field strains: – HAdV-E4FS1 obtained from Naval Health Research Center, CA – HAdV-E4FS2 obtained from Brooks AFB, TX 2 Strains from > 50 years ago: HAdV-E4p & HAdV-E4vac Strains confirmed as type 4 by molecular typing (PCR and microarray analysis) Stock Introduction Materials & Methods Results Discussion Conclusion 12

14. Strains were further distinguished as HAdV- E4a2 and HAdV-E4a1 using restriction enzyme analysis Kajon A E et al. J Infect Dis. 2007;196:67-75 Stock Introduction Materials & Methods Results Discussion Conclusion 13

15. The viruses were grown in A-549 cells (ATCC CCL-185), with the virus growth and DNA purification by Virapur, LLC DNA Prep Introduction Materials & Methods Results Discussion Conclusion 14

16. Sanger sequencing method was used Sequences assembled with DNA Sequencher (coverage: average 5-fold, minimum 3-fold) Supplemented with PCR amplification and re- sequencing of questionable areas Annotation was performed using the Genome Annotation Transfer Utility (GATU), and recorded and visualized using Artemis (genome viewer) Sequencing & Annotation Introduction Materials & Methods Results Discussion Conclusion 15

17. For quality control, annotation was compared to other type 4 genome data Open reading frames were compared against GenBank entries for confirmation and protein similarity Splice sites were predicted using MITs GenScan webserver, and confirmed by comparisons to previously annotated type 4 genomes Sequencing & Annotation Introduction Materials & Methods Results Discussion Conclusion 16

18. Multiple whole-genome alignments using MAFFT (Multiple Alignment using Fast Fourier Transform) Pairwise alignments, comparisons, genome visualization using zPicture ITR alignment using Clustal Omega, then other gaps/mismatches edited manually (more) Computational Analysis Introduction Materials & Methods Results Discussion Conclusion 17

19. Results: Inverted Terminal Repeats Method Clustal Omega-generated alignment of nucleotide sequences of ITRs (58 bases) from 5 termini of five HAdV-E4 genomes with HAdVs and SAdVs. Three sequence motifs identified: 1) Core Origin and two host transcription factors in HAdVs 2) nuclear factors I and 3 (NF-I, NF-III). ITR Alignment Introduction Materials & Methods Results Discussion Conclusion 18

20. Sequence percent identity analysis of regions L1 and L2 Genome and gene recombination analysis using Simplot and Bootscan Phylogenetic analysis of whole genome and hexon regions. Phylogenetic trees were constructed from aligned sequences using Molecular Genetic Analysis Software MEGA4.0.2 Computational Analysis Introduction Materials & Methods Results Discussion Conclusion 19

21. Obesity Virus? Image Courtesy of Rob Rogers, quickweightloss123.com 20

22. Infectious Humor Image Courtesy of Jeremy Schneider, Student enrolled in Humans and Viruses course taught by Prof. Robert Seigel, Stanford University 21

23. Results Vijay S. Reddy, S. Kundhavai Natchiar, Phoebe L. Stewart, and Glen R. Nemerow. Crystal Structure of Human Adenovirus at 3.5 Å Resolution. Science, 27 August 2010: 1071-1075 DOI: 10.1126/science.1187292. Scripps Research Institute10.1126/science.1187292 22

24. Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons Protein Homology Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region Sequence Recombination Bootscan Simplot Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons Results Introduction Materials & Methods Results Discussion Conclusion 23

25. Results: Comparative Genomics HAdV-E4 Strain Information Genomes of two HAdV-E4 field strains (FS) came from two U.S. military basic trainees presenting with Acute Respiratory Disease at separate geographic locations (Table 1). Sequence alignments of HAdV-E4 FS1 and FS2 with Sequencher and MEGA show 46 base substitutions and seven insertion deletions (indels): five 1- base indels and two 3-base indels. HAdV-E4 FS1 and FS2 are similar, but also show divergence, to HADV-E4p and –E4vac genomes (prototype and vaccine) isolated ~50 years earlier. Table 1. Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons 24

26. Results: Comparative Genomics Sequence Comparisons Global Pairwise nucleotide sequence alignments of genomes with zPicture (Fig. 1). HAdV-E4 FS1 and FS2 are nearly identical (top), and both are highly similar to HAdV-E4p (middle) and HAdV-E4vac (bottom). Differences appear in inverted terminal repeat (ITR), E1B, and E3 regions. Genome Percent Identities: FS1 vs. FS2 (99.9%), FSs vs. –E4p (95.1%), FSs vs. –E4vac (95.1%) Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons 25

27. Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons Protein Homology Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region Sequence Recombination Bootscan Simplot Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III Protein Homology Results Introduction Materials & Methods Results Discussion Conclusion 26

28. Results: Protein Homology Protein Homology Percent identities of selected HADV-E4FS1 proteins compared to SAdVs and HAdV-B16 with EMBOSS (Table 2). For hexon, L1 is one of the two variable loop regions and C is the complementary constant region. Hexon: Highest identity with HAdV-B16 (93.5%), constant region is nearly identical in all AdVs, and L1 has higher similarity to HAdV-B16 (94.8%) than SAdVs (ca. 64%). Fiber: SAdV-E26 has the highest similarity (93.2%), not HAdV-B16 (29.5%). Table 2. Protein Homology 27

29. Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons Protein Homology Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region Sequence Recombination Bootscan Simplot Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region Results Introduction Materials & Methods Results Discussion Conclusion 28

30. Results: Phylogeny Whole Genome Species E Clade: High levels of similarity between HAdV-E4 and five of six SAdVs. HAdV-E4 genomes subclade: (bootstrap = 100). Two subclades: 1) field strains and 2) prototype and vaccine. Suggests common lineage given different isolation times, not separate lineages caused by independent zootonic events. Species B Clade: HAdV-B16 in clade with subspecies B1; Clade for B2 subspecies; includes SAdV-B21. Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region B1 B2 E 29

31. Results: Phylogeny Hexon HAdV-E4 strains form a clade distinct from SAdVs (bootstrap = 100), but HAdV- B16 is excluded despite sequence similarity. Hexon Examined in greater detail as previous reports state variable region as candidate for recombination generating new genome types and novel pathogens. Divided hexon into two halves, variable region and constant region. Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region HAdV-E4 SAdVs 30

32. Results: Phylogeny Hexon: Variable Region Nucleotides 1 – 1557 Proximal variable region includes L1 and L2 – epitopes for serum neutralization. HAdV-B16 form a subclade with HAdV-E4 strains. SAdV-E23 is the SAdV closest related to HAdV-E4, but bootstrap of 72 is below the threshold of 80. Species E SAdVs subclade. Species B HAdVs clade includes SAdV-B21. Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region SAdVs B 31

33. Results: Phylogeny Hexon: Constant Region Nucleotides 1558 - 3072 HAdV-B16, which is part of the subspecies B1, forms its own clade off subspecies B2 (Bootstrap = 100). Unique bipartite observation may account for the 93.5% identity of HAdV-B16 to HAdV-E4FS1 in the hexon. Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region B2 32

34. Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons Protein Homology Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region Sequence Recombination Bootscan Simplot Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III Sequence Recombination Bootscan Simplot Results Introduction Materials & Methods Results Discussion Conclusion 33

35. Results: Sequence Recombination Bootscan Analysis of whole genome (Fig. A) and hexon (Fig. B) of HAdV- E4FS1. Done individually for each SAdV as similar genomes compete out signal. Only SAdV- E26 shown. HAdV-E4FS1 is nearly identical to HAdV-B16 in first third of hexon, revealing lateral transfer from HAdV-B16. Hexon recombination region is less similar to parental sequences (Fig. B), suggesting recombination is not recent and mutations led to divergence. Sequence Recombination Bootscan Simplot 34

36. Results: Sequence Recombination Simplot Analysis of whole genome (Fig. C) and hexon (Fig. D) of HAdV-E4FS1. Uses entire set of SAdV-E22 through –E26 sequences. Hexon recombination region located from nucleotides 306 to 1257, representing 897 bases or 31.9% of the hexon (2,811 bases) and includes the epitopes for serum neutralization, L1 and L2. Recombinant comprises of 2.5% of the HAdV-E4FS1 genome (35,956 bases). Sequence Recombination Bootscan Simplot 35

37. Sequence Recombination Bootscan Simplot Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons Protein Homology Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region Results Introduction Materials & Methods Results Discussion Conclusion 36

38. Results: Inverted Terminal Repeat Entire Sequence ITRs from HAdV-E4 Field Strains 1 & 2 and a contemporary field strain (Jax78) (Houng et al., 2006) are identical Divergent from HAdV-E4p and HAdV-E4vac genomes HAdV-E4p and HAdV-E4vac also diverge from each other at first eight nucleotides Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III 37

39. Results: Inverted Terminal Repeats First Eight Nucleotides HAdV-E4vac (CATCATCA) identical to sequences in SAdV-B21, -E22, -E23, - E24, -E26. SAdV-E25 has the same extra C at 5 terminus as SAdV-B21 and a single base difference at nucleotide 6. HAdV-E4p sequence (CTATCTAT) identical in counterparts HAdV-B3, - B7, -B21, and -D9. Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III 38

40. Results: Inverted Terminal Repeats Core Origin & Intervening Core Origin was identical for all HAdVs and SAdVs with the exception of the first base in some HAdVs (T instead of A). Intervening Sequence between Core Origin and NF-I motif – sequence in HAdV-E4 field strains (TTATAGA) was unlike the one found in HAdV-E4p and HAdV-E4vac (TTATTTTT) but identical to counterparts in species B. Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III 39

41. Results: Inverted Terminal Repeats Nuclear Factors I and III HAdV-E4p and -E4vac identical at NF-I and NF-III motifs; slightly deviated from SAdVs at NF-1 (TTTGTGTGAGTTAA) and nearly identical to SAdVs at NF-III. The three HAdV-E4 field strains are identical to HAdV species B at NF-1 (TGGAATGGTGCCAA). At NF-III, the three HAdV-E4 field strains are identical to subspecies B2, HAdV-B14; slightly different from subspecies B1 viruses (CATGTAAATGA). Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III 40

42. Comparative Genomics HAdV-E4 Strain Information Sequence Comparisons Protein Homology Phylogeny Whole Genome Hexon Hexon: Variable Region Hexon: Constant Region Sequence Recombination Bootscan Simplot Inverted Terminal Repeats Entire Sequence First 8 nucleotides Core Origin Intervening sequence between Core Origin & Nuclear Factor I Nuclear Factors I and III Results Introduction Materials & Methods Results Discussion Conclusion 41

43. DISCUSSION 42

44. Intellectual challenge Limited sequencing: Not recognized as separate species Novel virus resulting from recombination of species B and C Low-resolution genomic data: Suggests relationship with chimpanzee adenoviruses %GC content supports E as separate from B and C HAdV-E4 before high resolution data Introduction Materials & Methods Results Discussion Conclusion 43

45. Data: complete genome sequences, including additional simian adenovirus genomes HAdV-E4 resembles SAdV-E26 with partial hexon gene from HAdV-B16 High-resolution data shed light Introduction Materials & Methods Results Discussion Conclusion 44

46. Traditional species differentiation: serum neutralization Theory: centered on hexon L1 and L2 E4FS1 and B16 hexon L1 are 94.8% identical, so they should have a close antigenic relationship. Do they? Not necessarily. Cross-reaction mostly one-sided in multiple studies Two-sided neutralization with SAdV-E25, sometimes Reconciling serology and DNA sequencing Introduction Materials & Methods Results Discussion Conclusion 45

47. Whole genome tree is not the same as the L1 and L2 trees SN epitope alone is not enough to understand the relationships between HAdV-E4 and -B16 CCTAGCTTCAAACC... Vellinga, et al. Reconciling serology and DNA sequencing Introduction Materials & Methods Results Discussion Conclusion 46

48. Recombination between HAdV-B and E, a novel observation 2 nd independent recombination event between species E and a different B Revisit belief that species serve as recombination barriers Recombination between a human adenovirus and chimpanzee adenovirus, a novel observation among adenoviruses *author unknown; published in The Hornet in 1871 Interspecies Recombination Introduction Materials & Methods Results Discussion Conclusion 47

49. HAdV-E4 A clearer picture Introduction Materials & Methods Results Discussion Conclusion 48

50. Pathoepidemiology of HAdV-E4 Introduction Materials & Methods Results Discussion Conclusion 49

51. Gene Therapy? Introduction Materials & Methods Results Discussion Conclusion 50

52. Two independent, time-separated recombination events found in current HAdV-E4 strains This view of the optimization of a virus to a new host illustrates the value of genomic analysis in the study of infectious disease agents This report of lateral transfer between HAdVs and SAdVs serves as a caveat when considering chimpanzee adenoviruses as gene delivery vectors for human patients Conclusions Introduction Materials & Methods Results Discussion Conclusion 51

53. Dehghan, S., et al., Computational analysis of four human adenovirus type 4 genomes reveals molecular evolution through two interspecies recombination events. Virology (2013), http://dx.doi.org/10.1016/j.virol.2013.05.014i. Harrach B, Benkö M, Both GW, Brown M, Davison AJ, Echavarria M, Hess M, Jones MS, Kajon A, Lehmkuhl HD, Mautner V, Mittal SK, Wadell G (2011) Family Adenoviridae. King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, San Diego pp 125-141 Hatfield, L., Hearing, P., 1991. Redundant elements in the adenovirus type 5 inverted terminal repeat promote bidirectional transcription in vitro and are important for virus growth in vivo. Virology 184, 265–276. Hatfield, L., Hearing, P., 1993.The NFIII/OCT-1 binding site stimulates adenovirus DNA replication in vivo and is functionally redundant with adjacent sequences. J. Virol. 67, 3931–3939. Houng, H.S., Clavio,S., Graham,K., Kuschner,R., Sun,W., Russell,K.L., Binn,L.N., 2006. Emergence of a new human adenovirus type 4 (Ad4) genotype: identification of a novel inverted terminal repeated (ITR) sequence from majority of Ad4 isolates from US military recruits. J. Clin. Virol. 35, 381–387. Kajon,A.E., Moseley, J.M.,Metzgar, D.,Huong,H.S., Wadleigh, A.,Ryan, M.A., Russell, K.L.2007. Molecular epidemiology of adenovirus type 4 infections in US military recruits in the postvaccination era (1997–2003). J. Infect. Dis. 196, 67–75 Liu, E.B., Wadford, D.A., Seto, J., Vu, M., Hudson, N.R., Thrasher, L., Torres, S., Dyer, D. W., Chodosh, J., Seto, D., Jones, M.S., 2012. Computational and serologic analysis of novel and known viruses in species human adenovirus D in which serology and genomics do not correlate. PLoS One 7, e33212. References 52

54. Madisch, I., Harste,G.,Pommer,H.,Heim,A.,2005.Phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adeno- virus prototypes as a basis for molecular classification and taxonomy. J. Virol. 79, 15265–15276. Matsushima, Y., Shimizu, H., Phan,T.G., Ushijima,H., 2011. Genomic characterization of a novel human adenovirus type 31 recombinant in the hexon gene. J. Gen. Virol. 92, 2770–2775. Mul, Y.M., Verrijzer, C.P., vanderVliet, P.C., 1990. Transcription factors NFI and NFIII/ oct-1 function independently, employing different mechanisms to enhance adenovirus DNAreplication.J.Virol.64, 5510–5518. Pruijn, G.J.,van Miltenburg, R.T., Claessens, J.A., vander Vliet,P.C., 1988. Interaction between the octamer-binding protein nuclear factor III and the adenovirus origin of DNA replication. J. Virol. 62, 3092–3102. Purkayastha, A., Ditty, S.E., Su,J., McGraw,J., Hadfield, T.L., Tibbetts,C., Seto,D., 2005a. Genomic and bioinformatics analysis of HAdV-4, a human adenovirus causing acute respiratory disease: implications for gene therapy and vaccine vector development. J. Virol. 79, 2559–2572. Vellinga, J., Van der Heijdt, S., & Hoeben, R. C. (2005). The adenovirus capsid: major progress in minor proteins. The Journal of general virology, 86(Pt 6), 1581–8. Walsh, M.P., Chintakuntlawar, A.,Robinson, C.M.,Madisch, I., Harrach,B.,Hudson, N.R., Schnurr,D.,Heim,A.,Chodosh,J.,Seto,D.,Jones,M.S.,2009.Evidenceof molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis. PLoS One 4, e5635. Walsh,M.P.,Seto,J.,Jones,M.S.,Chodosh,J.,Xu,W.,Seto,D.,2010.Computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen. J. Clin. Microbiol. 48, 991–993. References 53

55. THANK YOU! 54

56. QUESTIONS 55

57. Why has E4 been a problem in the military population and not in the civilian population? 56

58. Using simian non-human adenoviruses as alternatives to HAdVs… …what concerns should we have (if any)? 57

59. Which AdV is the current vector for gene therapy and vaccines derived from and what benefits does using SAdVs as vectors have over the current vector? 58