computational analysis of four human adenovirus type 4 genomes reveals molecular evolution through...
TRANSCRIPT
Computational analysis of four human adenovirus type 4 genomes reveals molecular evolution
through two interspecies recombination events
Amy SmithJudy Chu
Kemi Abolude
September 17th, 2013
Shoaleh Dehghan, Jason Seto, Elizabeth B.Liu , Michael P.Walsh, David W.Dyer, James Chodosh, Donald Seto
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Outline
• Introduction• Materials & Methods• Results• Discussion• Conclusion
IntroductionMaterials & Methods
ResultsDiscussionConclusion
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• 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
TerminologiesIntroduction
Materials & MethodsResults
DiscussionConclusion
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• Computational analysis of human adenovirus type 4 (HAdV-E4)
• Provides insight into it’s 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
OverviewIntroduction
Materials & MethodsResults
DiscussionConclusion
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• 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
AdenovirusesIntroduction
Materials & MethodsResults
DiscussionConclusion
1. penton capsomeres2. hexon capsomeres3. viral genome (linear dsDNA)Image from: Pico en el Ojo, 2006
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• Medium-sized (90-100nm)
• Contains linear, non-segmented double-stranded DNA
• Size: 26-46Kbp (22-46 coding genes)
AdenovirusesIntroduction
Materials & MethodsResults
DiscussionConclusion
Source: http://www.difossombrone.it
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Function ProteinStructural proteins capsid 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 proteins E1A, E1B 19K, E1B 55K, …
Adenovirus coding genesIntroduction
Materials & MethodsResults
DiscussionConclusion
Some examples of AdV proteins:
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• 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 - ITRsIntroduction
Materials & MethodsResults
DiscussionConclusion
*Image in public domain
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• 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 AdenovirusesIntroduction
Materials & MethodsResults
DiscussionConclusion
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• Total of 57 serotypes classified into seven:
Human AdenovirusesIntroduction
Materials & MethodsResults
DiscussionConclusion
A 12, 18, 31B 3, 7, 11, 14, 16, 21, 34, 35, 50, 55C 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 4F 40, 41G 52
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• 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-E4Introduction
Materials & MethodsResults
DiscussionConclusion
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MATERIALS & METHODS
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• 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)
StockIntroduction
Materials & MethodsResults
DiscussionConclusion
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• 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
StockIntroduction
Materials & MethodsResults
DiscussionConclusion
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• The viruses were grown in A-549 cells (ATCC CCL-185), with the virus growth and DNA purification by Virapur, LLC
DNA PrepIntroduction
Materials & MethodsResults
DiscussionConclusion
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• 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 & AnnotationIntroduction
Materials & MethodsResults
DiscussionConclusion
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• 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 MIT’s GenScan webserver, and confirmed by comparisons to previously annotated type 4 genomes
Sequencing & Annotation Introduction
Materials & MethodsResults
DiscussionConclusion
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• 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 AnalysisIntroduction
Materials & MethodsResults
DiscussionConclusion
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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 AlignmentIntroduction
Materials & MethodsResults
DiscussionConclusion
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• 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 AnalysisIntroduction
Materials & MethodsResults
DiscussionConclusion
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Obesity Virus?
Image Courtesy of Rob Rogers, quickweightloss123.com
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Infectious Humor
Image Courtesy of Jeremy Schneider, Student enrolled in Humans and Viruses course taught by Prof. Robert Seigel, Stanford University
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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 Institute
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Comparative Genomics● HAdV-E4 Strain Information● Sequence ComparisonsProtein HomologyPhylogeny● Whole Genome● Hexon● Hexon: Variable Region● Hexon: Constant Region
Sequence Recombination● Bootscan● SimplotInverted 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
ResultsIntroduction
Materials & MethodsResults
DiscussionConclusion
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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
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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
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Comparative Genomics● HAdV-E4 Strain Information● Sequence ComparisonsProtein HomologyPhylogeny● Whole Genome● Hexon● Hexon: Variable Region● Hexon: Constant Region
Sequence Recombination● Bootscan● SimplotInverted Terminal Repeats● Entire Sequence● First 8 nucleotides● Core Origin● Intervening sequence between
Core Origin & Nuclear Factor I● Nuclear Factors I and III
Protein Homology
ResultsIntroduction
Materials & MethodsResults
DiscussionConclusion
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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
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Comparative Genomics● HAdV-E4 Strain Information● Sequence ComparisonsProtein HomologyPhylogeny● Whole Genome● Hexon● Hexon: Variable Region● Hexon: Constant Region
Sequence Recombination● Bootscan● SimplotInverted 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
ResultsIntroduction
Materials & MethodsResults
DiscussionConclusion
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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
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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
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Results: Phylogeny
Hexon: Variable RegionNucleotides 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
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Results: Phylogeny
Hexon: Constant RegionNucleotides 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
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Comparative Genomics● HAdV-E4 Strain Information● Sequence ComparisonsProtein HomologyPhylogeny● Whole Genome● Hexon● Hexon: Variable Region● Hexon: Constant Region
Sequence Recombination● Bootscan● SimplotInverted 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
ResultsIntroduction
Materials & MethodsResults
DiscussionConclusion
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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
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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
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Sequence Recombination● Bootscan● SimplotInverted 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 ComparisonsProtein HomologyPhylogeny● Whole Genome● Hexon● Hexon: Variable Region● Hexon: Constant Region
ResultsIntroduction
Materials & MethodsResults
DiscussionConclusion
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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
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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
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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
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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
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Comparative Genomics● HAdV-E4 Strain Information● Sequence ComparisonsProtein HomologyPhylogeny● Whole Genome● Hexon● Hexon: Variable Region● Hexon: Constant Region
Sequence Recombination● Bootscan● SimplotInverted Terminal Repeats● Entire Sequence● First 8 nucleotides● Core Origin● Intervening sequence between
Core Origin & Nuclear Factor I● Nuclear Factors I and III
ResultsIntroduction
Materials & MethodsResults
DiscussionConclusion
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DISCUSSION
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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 dataIntroduction
Materials & MethodsResults
DiscussionConclusion
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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 lightIntroduction
Materials & MethodsResults
DiscussionConclusion
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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
IntroductionMaterials & Methods
ResultsDiscussionConclusion
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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
IntroductionMaterials & Methods
ResultsDiscussionConclusion
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Recombination between HAdV-B and E, a novel observation
2nd 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 RecombinationIntroduction
Materials & MethodsResults
DiscussionConclusion
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HAdV-E4 A clearer pictureIntroduction
Materials & MethodsResults
DiscussionConclusion
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Pathoepidemiology of HAdV-E4Introduction
Materials & MethodsResults
DiscussionConclusion
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Gene Therapy?Introduction
Materials & MethodsResults
DiscussionConclusion
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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
ConclusionsIntroduction
Materials & MethodsResults
DiscussionConclusion
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References
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THANK YOU!
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QUESTIONS
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Why has E4 been a problem in the military population and not in the civilian population?
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Using simian non-human adenoviruses as alternatives to HAdVs…
…what concerns should we have (if any)?
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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?