Human Adenovirus (HAdV) novel recombinant analysis.

Human Adenovirus (HAdV) novel recombinant analysis

BINF 704

Eric Munger & Tiange Cui

Project Reference

Outline

• Outline of resource paper• Introduction, analysis, and conclution

• Outline of project

• Materials and Methods

• Results

• Discussion

HAdV structure and characterization

• ① Penton Base

• ② Hexon Capsomeres

• ③ Viral Genome (ds

DNA)

• ④ Fiber

④

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

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

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.

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

Protein Penton Hexon Fiber Fiber (proximal)

Fiber (distal)

SAdV-B35.2

100 99.8 99.4 99.2 100

SAdV-B21 98.2 92.2 81.6 72.6 100

HAdV-B16 84.5 84.7 97.5 97.5 97.4

HAdV-B21 99.3 98 54.3 55.7 51.7

HAdV-B7 85.5 86 53.6 53.6 53.8

HAdV-B14 91.8 90.3 51.8 51.3 52.9

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)

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

o x-axis: Genome nucleotide positions, o 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, o SAdV-B21 is likely the ancestral genome

Fig. 1 - Survey of recombination partners contributing to SAdV-B35. Recombination analysis of the SAdV-B35.1 genome using Simplot software.

o 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.

o Closer inspection of the hexon gene (C) and the fiber gene (D)

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

Neighbor-joining bootstrap-confirmed phylogenetic trees of the

A.whole-genome,

B.penton base,

C.hexon (C)

D.fiber,

E.fiber shaft, and

F. 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

Fig. 2 - Phylogenomic analysis of SAdV-B35.1 and -B35.2 genomes and select genes.

Outline of Resource - Whole genome analysisPhylogenetic 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

● 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 - 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.

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.

2 UNVERIFIED (lack of

annotation).

The QUESTION

Are there any recombinant HAdVs?

Material

• Raw whole genome sequences downloaded directly from

GenBank

• Software:

Perl

GATU (Genome Annotation Transfer Utility)

MEGA (Molecular Evolutionary Genetics Analysis)

SimPlot

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

Genome annotation using GATU

Ref: HAdV-B7

[UN]KF429748

Ref: HAdV-B7

[UN]KF429752

Penton, Hexon and Fiber genes

fasta_chopper.pl

BLAST search

Results

Multiple alignment by MAFFT

Whole genome

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

Penton


Hexon


Fiber

KF268315 has 99% sequence identity as Simian Adenovirus SAdV-B35! Not a HAdV!

KF268315

SAdV-B35

KF268315

SAdV-B35

KF268315 – P68H68F(S35)

Whole genome

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

Penton


Hexon


Fiber


KF268315SAdV-B35

KF633445 – P(S35)H(S35)F(S35)

Whole genome

KF268328 has 100% sequence identity as Human Adenovirus HAdV-B14/55

Penton


Hexon


Fiber


1360415289

Penton

18171 21026

Hexon

30742 31716

Fiber

KF268328

KF268328

Penton - HAdV-B34

Hexon - HAdV-B34

Fiber - HAdV-B14

KF268328 – P34H34F14

Discussion

• 2/35 recombinant HAdV are found.

• “Interspecies” transmission/recombination event is

found.

• Since SAdV-B35 is a human respiratory pathogen,

KF268315 and KF633445 may also cause human

respiratory disease.

QUESTIONS?

Thank you!

Human Adenovirus (HAdV) novel recombinant analysis.

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