1. Human Adenovirus (HAdV) novel recombinant analysis
BINF 704
Eric Munger & Tiange Cui
Human Adenovirus (HAdV) novel recombinant analysis

2. Project Reference

3. Outline Outline of resource paper
Introduction, analysis, and conclution
Outline of project
Materials and Methods
Results
Discussion

4. HAdV structure and characterization
① Penton Base
② Hexon Capsomeres
③ Viral Genome (ds DNA)
④ Fiber ④

5. Outline of Resource - Introduction
Human adenovirus (HAdV) genome sequences, types 1–69, are accessible as a reference data set
Through computational and comparative analyses of newly emergent HAdV pathogens
Detailed understanding of molecular mechanisms allowing these new pathogens to emerge
Examples of the genome characterization of emergent HAdV pathogens include
HAdV-D53 - highly contagious epidemic keratoconjunctivitis (EKC) pathogen
A recombinant of at least three parents;
HAdV-D56 - Emergent acute respiratory disease (ARD) and EKC pathogen
A recombinant of three parents; and
HAdV-B55 - Emergent ARD pathogen
A recombinant of two parents.
Historically curious HAdVs can be now understood at high resolution
ex: HAdV-E4 - Recombinant genome of SAdV-E26 sequences but shares a hexon gene with HAdV-B16
HAdVs are important biomedical tools
Serve as vectors for epitope and gene delivery
Simian adenoviruses (SAdVs) have been studied to try and bypass pre-existing immune responses
Has brought additional genomes into the data set

6. Outline of Resource - Introduction
Computational analysis of a pair of nearly identical SAdV genomes from a chimpanzee and a bonobo
Genomes of SAdV-B35.1 and -B35.2 are studied
It is shown that these SAdV genomes can recombine amongst each other because they,
Share large portions of thier genomes with SAdV-B21 and -B27
SAdV-B35.1 and -B35.2 also share two regions with HAdV genomes
Through recombinant events
DNA sequences can be transfered between simian and human AdV
These cross-species recombinant events include
Large sequence - Penton base and hexon genes, shared with HAdV-B21
Smaller region - Contains upstream sequences and fiber gene, shared with HAdV-B16
Both SAdV-B35 genomes contain the human host transcription factor NF-I binding site
Required for efficient HAdV replication
Since HAdV-B21 and HAdV-B16 are human respiratory pathogens and as some SAdV have been characterized as simian pathogens,
Computational results predict that SAdV-B35 may be an emergent human respiratory pathogen

7. Outline of Resource - Comparative Genomic Analysis
Nearly identical genomes of two simian adenoviruses (SAdVs) were analyzed computationally
Sample were taken from non-human primates lacated at two different primate facilities and sequenced
SAdV-B35.1 from Chimpanzee & SAdV-B35.2 from Bonobo
Nucleotide sequences were 99.7% identical
SAdV-B35.1 is 95.0% similar to SAdV-B21
Comparisons to SAdV-B35.1 with select HAdVs yield similar results, with % identities:
HAdV-B21 (89.7%);
HAdV-B50 (89.2%);
HAdV-B16 (87.8%); and
HAdV-B7 (85.5%).
The whole genome and select gene sequences were analyzed
Results reflect the high levels of similarity between SAdV-B35.1 and several SAdVs and HAdVs:
HAdV-B21, SAdV-B21 and HAdV-B16.

8. Outline of Resource - Comparative Genomic Analysis
For example, pairwise nucleotide sequence alignments of the genomes reflected the high levels of similarity and identity beyween SAdV-B35.1 and SAdVs & HAdVs
% sequence similarity showing high similarity of SAdV-B35.1 to HAdVs - Table 1
Penton base gene has a similarity of 99.3% to HAdV-B21
Hexon gene percent identity is 98.0% to HAdV-B21
An overall fiber gene similarity of 97.5% to HAdV-B16
Distal portion showing 100% identity to SAdV-B21
Table 1 - Comparison of protein % identities of SAdV-B35.1 proteins to their homologs in select adenoviruses.
Gene was divided into and reanalyzed as a proximal sequence (237 AAc) and a distal sequence (117 AAc)
Protein
Penton
Hexon
Fiber
Fiber (proximal)
Fiber (distal)
SAdV-B35.2
100
99.8
99.4
99.2
SAdV-B21
98.2
92.2
81.6
72.6
HAdV-B16
84.5
84.7
97.5
97.4
HAdV-B21
99.3 98
54.3
55.7
51.7
HAdV-B7
85.5 86
53.6
53.8
HAdV-B14
91.8
90.3
51.8
51.3
52.9

9. Outline of Resource - Sequence Recombination Analysis
A detailed examination of the SAdV-B35.1 genome sequence was completed using Simplot software
Graphical display of putative recombination events
x-axis: Genome nucleotide positions,
y-axis: % of nucleotide identities (Simplot) or permutated trees (Bootscan)
Fig.1A&B - whole genome; Fig. 1C - hexon gene; and Fig. 1D - fiber gene
The whole genome profile, Fig. 1A&B, suggested SAdV-B35.1 contains a base recombinant genome frame of simian adenoviruses, SAdV-B21 & B27 and HAdV-B16 & B21
Since SAdV-B21 appeared to contribute sequences throughout the SAdV-B35 genome, including the 5′-end region, two internal regions and the 3′-end region,
SAdV-B21 is likely the ancestral genome

10. Fig. 1 - Survey of recombination partners contributing to SAdV-B35
Fig. 1 - Survey of recombination partners contributing to SAdV-B Recombination analysis of the SAdV-B35.1 genome using Simplot software.
SimPlot analysis (A) and a Bootscan analysis (B) of the genome,
Shows lateral transfers of genome regions from SAdV-B27, HAdV-B21 and HAdV-B16 into SAdV-B21.
Closer inspection of the hexon gene (C) and the fiber gene (D)

11. Outline of Resource - Phylogenomic analysis
A larger phylogenomic analysis of all HAdV and SAdV available genomes is shown in Fig. 2.
This group of genomes was chosen to represent the relationships between
(SAdV-B35.1 and -B35.2) and (SAdVs and HAdVs)
It is noted that the simplest explanation of these sequence relationships are
The SAdV-B35 genomes share significant sequence similarities to several SAdV and HAdV genomes
The direction of lateral gene transfer could not be established, however
The thesis that there are cross-infections and co-infections of human and simian hosts by adenoviruses is supported

12. Fig. 2 - Phylogenomic analysis of SAdV-B35. 1 and - B35
Fig. 2 - Phylogenomic analysis of SAdV-B35.1 and - B35.2 genomes and select genes.
Neighbor-joining bootstrap- confirmed phylogenetic trees of the
whole-genome,
penton base,
hexon (C)
fiber,
fiber shaft, and
fiber knob
Sequences are presented, with the fiber gene (D) further dissected for the detailed analyses of two domains:
E) shaft, 716 bp, represented the proximal sequences, and
F) knob, 346 bp, representing the distal portion

13. Outline of Resource - Whole genome analysis
Fig 2D Fiber gene examination
SAdV-B35 formed a subclade with HAdV-B16
Branching from this is SAdV-B21.
These comprise a subclade containing the HAdV species B viruses.
The analyses suggested a recombination event occurred that included a junction within the fiber gene, near the putative border between the shaft and the knob sequences.
Dissecting the fiber gene into the shaft and knob regions
exact nucleotide location borders were difficult to set. Therefore,
716 bases were extracted for the analysis of the shaft (Fig. 2E) and
Abutting 346 bases extracted for the analysis of the knob (Fig. 2F).
Fig 2 E&F - Both shaft and knob regions were analyzed separately.
The shaft sequence has high similarity with HAdV-B16 and the knob has similarity with SAdV-B21.
Fig 2E - Fiber shaft sequences
SAdV-B35 forms a subclade with HAdV-B16, with a low bootstrap value of 66 indicating low confidence in the segregation. A bootstrap value of 99 provides confidence that SAdV-B35 forms a separate clade away from SAdV-B21.
Both are also contained within the HAdV species B clade.
Fig 2F - Knob sequences
In contrast, SAdV-B35 forms a subclade with SAdV-B21, but with a low bootstrap value of 52;
both are in a subclade separate from HAdV-B16, bootstrap value 99.
Again, these sequences cluster with HAdV species B.
Outline of Resource - Whole genome analysis
Phylogenetic analysis of the whole genomes showed
Fig 2A
Genomes of SAdV-B35.1 and -B35.2, noted here in this discussion collectively as “SAdV-B35”, forming a clade with SAdV-B21.
Branching away from these is the SAdV-B27 genome.
All three are contained in a larger clade with HAdV the members of species B.
All three branch's Bootstrap value was 100
Previously, when SAdV-B21 was one of only five SAdVs whose genome was sequenced, it was not included within this larger clade.
Fig 2B
To obtain higher resolution views of the gene sequences, several regions noted by the above analyses were examined in greater detail.
A phylogenetic analysis of the penton base genes showed SAdV-B35 forming a subclade with HAdV-B21, on a branch apart from SAdV-B21.
These are contained in the HAdV species B clade.
These data confirmed the global pairwise genome comparisons and the recombination analyses.
Fig 2C
An inspection of the hexon sequence phylogenetic alignment also supported the data noted above.
SAdV-B35 formed a subclade with HAdV-B21,
branched away from SAdV-B21 with the subclade contained within the HAdV species B clade.
The overall tree had a similar profile as the penton base tree (Fig. 2B),
indicating both genes were participants in one recombination event

14. Outline of Resource - Conclusion
Genomics and computational analysis provide a unique view of the possible emergence and prediction of a human viral pathogen in very high resolution
SAdV-B35 contains a recombinant genome comprising a base frame scafold with elements from SAdV & HAdV genomes, with
Sequences from both ends and internal regions highly similar to SAdV-B21, and
Penton base, hexon, fiber shaft, and a DNA replication factor binding motif sequences shared with HAdV genomes
This report of lateral genomic DNA transfers between HAdVs and SAdVs and observations of multiple recombination events is of importance
When considering SAdVs as candidate gene and epitope delivery vectors for humans.

15. JCVI HAdV Project
35 newly sequenced type B HAdVs from JCVI has already been deposited into GenBank.
But the annotation was done by automatic mass-production, so the genotypes and the serotypes are not 100% accurate.
The project is to reanalysis these HAdVs and see if there are any recombinants in this batch.

16. 2 UNVERIFIED (lack of annotation).

17. Are there any recombinant HAdVs?
The QUESTION
Are there any recombinant HAdVs?

18. Material Raw whole genome sequences downloaded directly from GenBank
Software:
Perl
GATU (Genome Annotation Transfer Utility)
MEGA (Molecular Evolutionary Genetics Analysis)
SimPlot

19. Methods Write Perl script to extract genomic region
Use GATU to annotate unverified sequences
Use NCBI BLAST search to compare a query sequence with database
Use CBRC MAFFT to align multiple sequences
Use MEGA to generate phylogenetic trees
Use SimPlot for recombinant analysis

20. Genome annotation using GATU
Ref: HAdV-B7
[UN]KF429748
Ref: HAdV-B7
[UN]KF429752
Penton, Hexon and Fiber genes

21. fasta_chopper.pl

22. BLAST search

23. Results

24. Multiple alignment by MAFFT

25. Whole genome KF268315 has 100% sequence identity as Human Adenovirus
HAdV-B68

26. Penton KF268315 has 100% sequence identity as Human Adenovirus
HAdV-B68

27. Hexon
KF has 100% sequence identity as Human Adenovirus
HAdV-B68

28. Fiber
KF has 99% sequence identity as Simian Adenovirus SAdV-B35! Not a HAdV!

29. KF268315
SAdV-B35

30. KF268315
SAdV-B35
KF – P68H68F(S35)

31. Whole genome KF633445 has 100% sequence identity as Simian Adenovirus
SAdV-B35

32. Penton KF633445 has 100% sequence identity as Simian Adenovirus
SAdV-B35

33. Hexon KF633445 has 100% sequence identity as Simian Adenovirus
SAdV-B35

34. Fiber KF633445 has 100% sequence identity as Simian Adenovirus
SAdV-B35

35. KF268315
SAdV-B35
KF – P(S35)H(S35)F(S35)

36. Whole genome KF268328 has 100% sequence identity as Human Adenovirus
HAdV-B14/55

37. Penton KF268328 has 100% sequence identity as Human Adenovirus
HAdV-B34

38. Hexon
KF has 100% sequence identity as Human Adenovirus
HAdV-B34

39. Fiber
KF has 99% sequence identity as Human Adenovirus
HAdV-B14

40. KF268328
15289
13604
18171
21026
30742
31716
Penton
Hexon
Fiber

41. KF268328 Penton - HAdV-B34 Hexon - HAdV-B34 Fiber - HAdV-B14
KF – P34H34F14

42. Discussion 2/35 recombinant HAdV are found.
“Interspecies” transmission/recombination event is found.
Since SAdV-B35 is a human respiratory pathogen, KF and KF may also cause human respiratory disease.

43. QUESTIONS?
Thank you!