HAdV-4: Adaptive evolution in

1
HAdV-4 Adaptive evolution in a re-emerging
pathogen
2
Adenovirus
3
GEIS studyAcute Respiratory Disease Outbreaks
among Basic Military Trainees
4
Then and Now
Germs do not respect boundaries
5
Epidemic Outbreak SurveillanceMaking the
Difference
Delay until Clinical Diagnosis
Detection via Medical Surveillance
100
80
60
40
Casualties
20
0
D0
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D1
DAYS TO RECOGNITION
6
Advanced Molecular Diagnostics Operations and
Deliverables
7
Zebra ChipAd4 versus Ad7 tiles

• Single test distinguishes biowarfare agents of
  flu-like syndrome
• Total DNA amplification (no specific primers!)
• Assay on EOS Respiratory Pathogen Microarray
  (RPM) v. 1, v.2, v.3
• Approaching objective Decision-quality
  information in 2 hours

Ad4 Tiles
Ad7 Tiles
Chip Navigation
8
Genome data mining Pathogen sequence signatures
From genome data
To signatures data
TAG TAA TGA
ATG
PROMOTER
Applied versus Basic
9
Genome data miningRegulatory and other
non-coding features
Basic versus Applied
10
(No Transcript)
11
Genome data mining Genes and other coding units
in sequence
From sequencing data
To biological data
ATG
TAG TAA TGA
POLY A SIGNAL
PROMOTER
INTRON
GT
AG
EXON
EXON
12
Annotation of coding regions
13
Genome Sequence Annotation Adenovirus Finished
Genes, ORFs and Features Map
From Stone et al, 2003.

• GENSCAN and GeneMark analysis to identify
• hypothetical genes
• Identify non-coding features- promoters, polyA
• signals, regulatory sites, with reference to
• archived adenoviral annotations in GenBank,
• including third-party annotations (TPAs)

Transcription units
Early Intermediate Late
14
Adenovirus Genome Sequences Summary of Finished
Genomes
15
Analysis of hexon proteins
16
Analysis of fiber proteins
17
Comparative gene/protein analysis and adenovirus
evolution HAdV-E from B and/or C
Questions..
Gruber et al., 93
18
HAdV-4 zoonosis versus recombination

• old data are pre-Simian
• E closer to B1s than C
• except for fiber

19
Phylogenetic relationships
20
Comparison with simian adenoviral proteins
21
Comparative genomics of loci
22
Evaluation of fiber genes
23
In progress (Nov. 04)..

• HAdV-1 JGenVirol
• HAdV-4 JVirol
• HAdV-7 Virol
• Vaccines JCM (in press)
• Field strains ?
• B1/B2
• HAdV-6
• Phylogeny
• ----gtHAdV-40 ?

24
New questionsBreakThrough strains or
co-infections
ATCC repository

• Contamination or co-infection?
• Genome sequence
• Limited gene sequence
• Microarray hyb data
• immunochemistry data

• NHRC GEIS collection
• BT4/5 5, 21, 4 Earlier vaccinated indiv.
• Originally co-infected
• Implications for vaccine development
• Implications for pathogen agent mutations

25
Distribution of HAdV serotypes causing ARD
26
Genomics and bioinformatic analyses of current
field strains
evolving gold standards
27
Comparative genomics
28
HAdV-4 prototype compared with current field
strains
29
HAdV-7 differences in genomes
30
Comparative genomics
31
Mutation rates in genomes
32
Multiple sequence alignments of ITRs
by eye
33
Another MSA of ITRs
34
ITR phylogenetic analysis
35
Finer phylogenetic analysis of ITRs
36
Sequence details of ITRs
37
Plus downstream
38
cont..
39
cont..
40
Evaluation of txn factor binding motifs
not Transfac
41
Reemerging pathogen