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Journal of Virology, February 2005, p. 2559-2572, Vol. 79, No. 4
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.4.2559-2572.2005

Genomic and Bioinformatics Analysis of HAdV-4, a Human Adenovirus Causing Acute Respiratory Disease: Implications for Gene Therapy and Vaccine Vector Development

Anjan Purkayastha,1,2,3 Susan E. Ditty,2,4,{dagger} Jing Su,1,2,3 John McGraw,2,4 Ted L. Hadfield,2,4,{dagger} Clark Tibbetts,2,3 and Donald Seto1,2,3*

Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, Manassas,1 HQ USAF Surgeon General Office, Directorate of Modernization,3 Epidemic Outbreak Surveillance Consortium, Falls Church, Virginia,2 Division of Microbiology, Department of Infectious and Parasitic Diseases Pathology, Armed Forces Institute of Pathology, Washington, D.C.4

Received 16 June 2004/ Accepted 13 October 2004


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ABSTRACT
 
Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD.


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AdVs.
 
Adenoviruses (AdVs) are members of the Adenoviridae family, which comprises five genera and infects hosts across the broad spectrum of vertebrates (4, 14). There are 51 serotypes of human AdVs (HAdVs) making up the genus Mastadenovirus. These are subdivided into six species, A to F (formerly subgroups or subgenera), based on differentiating properties including antigenic as well as genomic differences (14). The first HAdV to be isolated was a cytopathic agent from adenoid tissue, described in 1953 (60). Coincidentally, HAdV-4 was isolated and identified within the same time period as cytopathogenic respiratory illness agent RI-67 (34). Together with HAdV-1, these were the basis for the group name of "adenoviruses" (19).


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AdV-associated diseases.
 
HAdVs are recognized as the etiologic agents in a number of diseases, ranging from acute respiratory disease (ARD) to gastroenteritis. For example, HAdV-C species (serotypes HAdV-1, -2, -5, and -6) cause typically benign respiratory and gastrointestinal infections in an endemic pattern among human hosts in early childhood (8). In contrast, a different pattern of epidemiology and pathogenesis is presented by HAdV-E species (HAdV-4). HAdV-4 and HAdV-B1 species (HAdV-3, -7, and -21) are causative agents in epidemic outbreaks of ARD. In addition to ARD, HAdV-4 causes acute viral conjunctivitis (66, 70, 76). Although affecting the civilian population (10, 70, 71), these viruses are of more importance to the U.S. Department of Defense (11, 27, 33, 63, 64). HAdV-4 was originally isolated from a U.S. Army trainee diagnosed with acute respiratory illness from an epidemic in the winter of 1952 to 1953 at Ft. Leonard Wood, Mo. (34). The morbidity has been and is still a major concern. In addition to high rates of morbidity, mortality due to both HAdV-4 and HAdV-7 has been noted (18, 47, 65).


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Military HAdVs.
 
HAdVs are responsible for 60 to 80% of the ARD cases in military training camps, leading to hospitalization in approximately 20% of diagnosed cases (17, 26). HAdV-4 and HAdV-7 were the predominant serotypes identified as causative agents of ARD (26). Successful control of AdV-caused ARD was achieved with live enteric-coated oral vaccines against HAdV-4 and HAdV-7a. These lowered HAdV morbidity by 95 to 99% and total ARD morbidity by 50 to 60% (9, 28, 83). However, despite the success of this vaccination program, the production of the vaccines was discontinued in 1996; supplies were exhausted in 1999, leading to an increase of HAdV-associated ARD cases to pre-vaccine-era levels (28). Surveillance of HAdV infections in training bases by the U.S. Naval Health Research Center (NHRC; San Diego, Calif.) shows the continued prevalence of HAdV-4 and HAdV-7 among military recruits (2, 28). Recent isolates collected in the past few years documented the rise and predominance of the HAdV-4 serotype as the major determinant in ARDs (11, 28, 42, 47). The reemergence of HAdV-related ARD has spurred HAdV vaccine development initiatives. This gap in vaccine coverage is an opportunity to develop and validate new technologies and methods for monitoring and diagnosing viral agents causing ARD.


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Advanced molecular diagnostics.
 
With the advent of molecular and DNA analysis technologies, protocols and instrumentation developed for the Human Genome Project, and the convergence of the need to monitor for infectious disease and biothreat agents, an opportunity to develop rapid, high-throughput, and nonbiased molecular technology-based diagnostics is available. These options include microarray-based assays, as well as multiplex PCR and real-time PCR assays (21, 38, 84, 90). The USAF Surgeon General's Office, Directorate of Modernization, is sponsoring a consortium (Epidemic Outbreak Surveillance [EOS]) to develop and validate these advanced molecular diagnostic platforms for monitoring viral and bacterial agents causing ARD in a real-world test bed. The determination of these genomes is one step in identifying unique pathogen DNA signatures for use as diagnostic probes. As a lagniappe, this applied biology interest allows further insight into the basic biology and natural history of AdVs.


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Evolutionary considerations and origins of HAdV-4.
 
HAdV-4 is the sole member of the HAdV-E species. Its evolutionary origins have been a subject of speculation. This singularity and the lack of genomic diversity of HAdV-4 strains (10, 20, 48, 70) imply this HAdV species may be the product of a relatively recent evolutionary event and is an archetype HAdV (29, 48). Early restriction enzyme digestion analyses of HAdV-4 and two chimpanzee AdVs (Pan 7 and 9) indicate the HAdV-4 may be distantly related to these simian adenoviruses (SAdVs) (48, 86). These studies suggest tentatively that the HAdV-E species may have originated from a chimpanzee-to-human zoonotic event. There is precedence for this, given that human viral lineages originating from interspecies transmission are well-documented in other viruses, such as human immunodeficiency virus (HIV) and human T-cell leukemia/lymphotropic virus (HTLV) (25, 43). However, this hypothesis was difficult to prove due to the previous scarcity of whole-genome sequence data on both HAdV-4 and SAdVs and the lack of molecular techniques at the time. An equally plausible hypothesis suggests HAdV-4 originated from a recombination event between two HAdV species, B and C (29). Viral recombination has been demonstrated in the AdVs (12, 52, 77-79), as well as other viruses, particularly the segmented viruses (49, 57, 58). These questions are now resolved with the whole-genome analysis and data of the HAdV-4 reported here. Evidence is presented for a recombination event between B-like and C-like AdVs, followed by a zoonotic event.


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Vectors for gene therapy and vaccine delivery.
 
There is a growing interest in using chimpanzee AdV-based vectors for human gene therapy and perhaps also for vaccine delivery. To that end, genome sequencing of several chimpanzee AdV isolates, also known as SAdV, was recently completed (22, 61). Supporting data for this application included sero-neutralization studies which suggest there is no cross-reaction between SAdV proteins and the human immune response (61). This implies SAdV-based vectors are safe and could be used multiple times; however, conflicting neutralization data have been reported (48). The recent genome reannotation of SAdV-21 (BK000412) (14) and the recent sequencing and annotation of Pan 9 (AF394196) (22) and BK000413 (14)—as well as Pan 5, 6, and 7 (AY530876, AY530877, and AY530878, respectively) (61)—have yielded additional data to complement and enhance the genome and bioinformatics analysis of HAdV-4. The data presented here have relevance in vector development.

In this report, the complete and annotated genome sequence of HAdV-4 (GenBank accession no. AY594253) is presented. This genome of HAdV-4 is 35,990 bp in length, and a comprehensive annotation identifies 49 coding sequences, along with numerous other biological features. Organization of the HAdV-4 genome is similar to that of other members of Mastadenoviruses. The bioinformatics and phylogenetic studies on this and other AdV genomes provide insight into the biology of HAdV-4, as well as the evolution of the HAdV-E species, and raise interesting questions about the use of putatively related SAdV genomes as vectors in human gene therapy and vaccine delivery.


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MATERIALS AND METHODS
 
Cells, virus stocks, and DNA preparation. HAdV-4 (ATCC VR-4; strain RI-67) was obtained from the American Type Culture Collection (ATCC). Virus stocks were expanded in A-549 cells (ATCC CCL-185), a human lung tumor cell line used earlier for HAdV-1 virion production (44). Viral DNA was prepared essentially as described by Le et al. (45).

PCR methodology. Standard PCR methodologies were used to amplify regions to be sequenced. Pfu Turbo DNA polymerase (Stratagene, Inc.) was optimal for PCR amplifications.

Leveraged primer-walking DNA-sequencing strategy. Genes and DNA sequences from HAdV-4 are archived in GenBank. These were used as scaffolds for developing minimally tiled and overlapping primers for PCR amplification and DNA sequencing. Additionally, SAdV-25 (NC_003266) sequences were used to design primers for tiling this minimally overlapping three-fold coverage. This was listed as the member of HAdV species E in GenBank. Gaps in sequence coverage were closed by PCR amplifying across the gap and sequencing the amplicon. Amplicons were purified with the Montage DNA gel extraction kit (Millipore Corp., Billerica, Mass.) to purify target amplicons.

PCR fragments were sequenced with either the PCR or sequencing primers, using the ABI Prism BigDye Terminator v3.1 Cycle Sequencing Ready Reaction kit on an ABI 3100 DNA sequencer (Applied Biosystems, Inc., Foster City, Calif.). Postreaction products were purified with the Montage SEQ96 sequencing reaction cleanup kit and a Millipore Multiscreen384 vacuum manifold (Millipore Corp.).

Direct sequencing of ITR ends. For sequencing of the inverted terminal repeat (ITR) ends, the ends of this double-stranded DNA linear genome were determined by direct sequencing off the purified DNA. Primers were designed from newly determined internal sequences. Template DNA (0.2 to 1.0 µg per reaction) was purified further by passing through a MicroSpin G-50 column (Amersham Biosciences, Piscataway, N.J.) and sequenced.

Genome assembly, annotation, and sequence analysis. DNA sequences were assembled with Sequencher 4.1.1 (Gene Codes Corporation, Inc., Ann Arbor, Mich.). Features of the DNA sequence were revealed by the Wisconsin GCG package (SeqWeb v.2).

The genome sequence was annotated by parsing into 1-kb nonoverlapping segments and querying each segment. This was identical to the annotation algorithm optimized for HAdV-1 analysis (44). These were queried systematically against the nonredundant National Center for Biotechnology Information database, using the BLASTX program of the BLAST suite sequence-alignment software (1). The searches used the default parameters of a word size of 3 and expectation of 10, with the BLOSUM62 substitution matrix and with gap penalties of 11 (existence) and 1 (extension). Low-complexity sequences were filtered out of the queries.

GenomeScan was used for theoretical gene predictions. This was useful for identifying exons from the coding sequences where exon-intron borders were difficult to determine. To enable this, the algorithm uses exon-intron identification combined with similarity searches to a sequence database in order to predict coding sequences in a given DNA fragment (91). Novel sequences or "hypothetical proteins" were also identified by using another gene prediction software, GeneMark (6). During the course of this annotation, while GeneMark had a slightly higher accuracy than GenomeScan, neither was completely accurate nor comprehensive in generating a list of putative genes. To visualize the progress, the web-accessible annotation tool Artemis was used to expedite genome annotation (5).

Multiple sequence alignment was performed with CLUSTALX software (81). All sequence alignments were performed with default parameters (for pairwise alignment, gap-opening and extension penalties of 10 and 0.1, respectively, and the Gonnet 250 protein weight matrix; for multiple alignment, gap-opening and extension penalties of 10 and 0.2, respectively, and the Gonnet series of protein weight matrices). Phylogenetic trees were constructed by the neighbor-joining method (67). Bootstrapping was performed with 1,000 resampling iterations to assess the robustness of the trees.


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RESULTS
 
Genome sequencing with a sparsely tiled primer array. Given the partial or complete genome sequences of related HAdVs either in GenBank (including archived partial genomic and gene sequences) or the database of HAdV genomes newly sequenced by the USAF EOS consortium, PCR and DNA sequencing primers were identified and optimized for efficient and low-cost genome sequencing of the HAdV-4 genome based on a sparsely tiled overlap of PCR and sequencing primers. Either bracketing PCR primers or internal primers were used as sequencing primers to obtain overlapping and complementing sequences for a redundancy of threefold coverage (minimum) with the "2 + 1" rule, which calls for complementary reads. This method of rapid genome determinations of the HAdV genomes validates the notion of a rapid sequencing method for examining and understanding related and similar genomes of immediate and urgent interest.

It should be noted that initial genomic assembly and analysis of the ATCC-archived HAdV-4 showed the presence of HAdV-3. The reported sequence is derived from a plaque-purified sample. Recent work suggests that the original isolate could have contained a coinfection of at least two HAdV serotypes (EOS; unpublished data).

Genome annotation. HAdV-4 genome sequence is 35,990 bp in length and has an overall base composition of 21.95% A, 28.96% C, 28.7% G, and 20.36% T. The GC content of 57.67% is within the 57-to-59% range noted in the literature for HAdV-E (73). Like the other Mastadenoviruses, the HAdV-4 genome is organized into early, intermediate, and late transcription regions. Forty-nine coding sequences were identified in the genome sequence, including those of six hypothetical proteins.

Noncoding features. (i) Sequence motifs. Noncoding DNA sequence motifs on the HAdV-4 genome are listed in Table 1. Genome location, putative function, and functional orientation are indicated.


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  		TABLE 1. HAdV-4 genome noncoding motif annotationa

Canonical ITRs are located at either end of the linear sequence (13); they are 116 bp in length. The extreme termini are missing the canonical CAT box that is present in all other HAdVs. This HAdV-4 sequence CTATCTATAT differs significantly from the HAdV consensus motif of CATCATCAAT (77). This observation reconfirms earlier reports on the unique HAdV-4 ITRs (82).

Contained within the ITR are DNA sequence motifs that are required for viral replication as well as gene activation and transcription. The core origin of DNA replication, ATAATATACC, that binds the preterminal protein-DNA polymerase complex, was present at bp 9 to 18 (80). In addition to the DNA polymerase and pTP, HAdVs also require a set of host cellular factors for efficient replication. These are reflected in the cellular transcription factor DNA-binding motifs. The ITR region of HAdV-4 has an NFIII/Oct-1 recognition site (TATGCAAATAA) at (bp 41 to 51) and an Sp1 binding site (GGGGATGGGGC) at (bp 65 to 75). The NFI/CTFI recognition site was not present. This is all consistent with earlier work reporting the DNA sequences required for HAdV-4 replication (30). In vivo and in vitro studies showed that while HAdV-2 requires both NFI and NFIII for efficient DNA replication, HAdV-4 apparently does not need these two host cellular factors. The HAdV-4 ITR contains a NFIII recognition site but lacks an NFI recognition site.

(ii) VA RNA. Non-protein-coding RNA sequences, known as the virus-associated (VA) RNA species, repress the antiviral activity of host interferons and thus play a role in host response to infection (51). HAdV VA RNA genes have been studied and compared with one another in the literature (41). As reported, HAdV-A, -B2, and -F species members contain one such gene; HAdV-B1, -C, -D, and -E species members contain two such genes. It was also reported that the VA RNA I gene of HAdV-16 is much more closely related (98.7% identity) to its HAdV-4 counterpart than to the counterparts from other members of the HAdV-B1 subspecies (HAdV-3 and -7). Additionally, the VA RNA genes of prototype HAdV-4 differ in length and composition from a "wild" variant, HAdV-4a, and from several other "wild" variants, which may or may not be HAdV-4a. A 65-base deletion in HAdV-4a VA RNA II eliminates part of one promoter element (element A) and all of another (element B); its significance is unknown (85).

These earlier literature observations are borne out in the current in-depth genome analyses. The HAdV-4 VA RNA I and II coding sequences are located at bp 10356 to 10514 and 10575 to 10743, respectively. BLAST analysis shows that VA RNA I is greater than 95% identical to its counterparts in HAdV-7, -16, and -21, as well as approximately 95% identical to SAdV-21, -23, -24, and -25. VA RNA II returned scores of 94, 82, and 90% against HAdV-7 (three regions of identity), 83% each against HAdV-16 and -21, as well as greater than 90% identity against SAdV-22 through -25. This closeness to the chimpanzee AdVs has also been reported in the literature, as both VA RNA I and II genes from SAdV-21 through -25 are all highly related to their HAdV-4 counterparts.

Gene coding features. Table 2 displays the annotation of coding genes found in HAdV-4, along with their locations along the genome. The coding orientation is also detailed.


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  		TABLE 2. HAdV-4 genome gene coding annotationa

(i) Early genes. (a) E1A. The E1A gene is the first transcription unit to be expressed after infection. The alternative splicing of a common RNA precursor transcribed from a constitutively active promoter generates multiple proteins derived from the E1A transcript. These E1A proteins function as transcriptional regulators within the host cell, modulating both viral and cellular gene expression (23). The proteins lack sequence-specific DNA-binding activity (96); they control gene expression by interacting with cellular elements of the transcription machinery. Three putative E1A proteins with sizes of 6.8, 24.6, and 28.1 kDa were identified in the HAdV-4 genome.

(b) E1B. Five coding sequences were identified in the E1B region. The early 20-kDa protein has high identity to the small T antigen that is conserved in other HAdVs. The 54.2-kDa protein has identity to the large T antigen protein, which inhibits the cellular p53-mediated host defense mechanisms (92). The large T antigen protein also plays a role in regulating viral late gene expression. The 8.1- and the 16.7-kDa proteins show significant identity to their counterparts in the SAdV genome sequences. These have identities to the 1.26- and 1.31-kb mRNA products identified in the HAdV-C species.

(c) E2. The E2 transcriptional unit encodes proteins required for viral DNA replication. HAdV-4 replication requires three virus-encoded factors—terminal protein precursor, DNA polymerase, and DNA binding protein—as well as additional human cellular proteins (15). The E2 transcription unit is divided into two regions, E2A and E2B. A 57.4-kDa DNA binding protein was identified within E2A. In the E2B region, a 135.2-kDa DNA polymerase and a 73.7-kDa terminal protein precursor were located.

(d) E3. The E3 region of HAdVs encodes proteins antagonistic to the host immune mechanism (89). These proteins are not required for efficient viral growth in vitro. The HAdV-4 E3 gene region encodes nine proteins of the following sizes: 12.0, 23.3, 19.3, 24.7, 6.31, 29.7, 10.4, 16.7, and 14.9 kDa. The 12.0-kDa protein has significant homology to an immunomodulating E3 protein in HAdV-2. BLAST alignments suggest that HAdV-4 23.3-kDa protein is homologous to the SAdV genome-encoded CR1-alpha 1 protein; its homologs are found in other HAdVs. The 19.3-kDa protein appears to be a homolog of the E3-gp 19-kDa major histocompatibility class I antigen-binding glycoprotein found in HAdV-7. The 24.7-kDa protein has identity to the CR1 (conserved region 1)-containing proteins in the E3 region of other HAdVs (16). A function for this 80-amino-acid (aa) domain has not been identified. Interestingly, the 29.7-kDa protein also contains a CR1 domain and has a high identity to its counterpart in the SAdV-25 genome (CR1-delta 1 protein). This is not found in other HAdVs. The 10.9-kDa protein has significant identity to an E3 protein that plays a role in down-regulating the epidermal growth factor receptor. The 16.7-kDa protein has identity to an HAdV E3 protein known to protect virus-infected cells against tumor necrosis factor-induced cytolysis (37).

(e) E4. Members of the E4 transcription unit perform a range of functions (46). For example, the E4 proteins are involved in viral RNA export and stabilization. The E4 Orf6 protein combines with the E2 55-kDa protein to inhibit cellular p53. E4 Orf6/7 regulates the cellular transcription factor E2F, while E4 Orf4 controls protein phosphorylation in infected cells. Seven putative coding sequences were identified in the HAdV-4 E4 region. These include a 13.5-kDa protein with identity to the E4 protein Orf1, a 14.6-kDa Orf2 protein, a 13.6-kDa nuclear binding Orf3 protein, a 14-kDa Orf4 protein, a 15.8-kDa Orf6/7 protein, a 34.6-kDa Orf6 protein, and a 7.3-kDa Orf7-like protein. In HAdV-9 (species D), the E4 Orf1 coding sequence has a dUTPase domain and is reported to be an oncogenic determinant (87). The pathway of oncogenic transformation was partly elucidated when it was shown that Orf1 activates phosphatidylinositol 3-kinase, at the host cell membrane, thus initiating a cascade of downstream events that eventually lead to cell transformation (24).

(ii) Intermediate genes. (a) IX. The intermediate transcript-derived protein IX (pIX) is a minor component of the AdV capsid. In addition to functioning as a transcriptional regulator, it is partially responsible for virion stability; virions lacking pIX are heat labile and lose their infectivity if the packaged DNA exceeds 35 kb in size (69). In HAdV-5, IX also acts as a transcriptional activator for the major late promoter (MLP) as well as other viral promoters, including those enhancing expression from the E1A, E4, and major late promoters, as well as cellular promoters. The physiological role of IX as a transcriptional regulator is not clearly understood (68). Recent literature indicates that, although pIX can affect transcription from a variety of viral promoters, it does not appear to play a significant role in the activation of AdV promoters during normal AdV replication (69). An open reading frame (ORF) encoding a 14.4-kDa pIX was identified at bp 3441 to 3869.

(b) IVa2. The second intermediate transcript-derived protein, IVa2, plays a serotype-specific role in packaging viral DNA during AdV assembly (95). The IVa2 protein binds the "A repeats" sequence, at the left end of the genome during the packaging process (55, 94). It is speculated that the virions are assembled around the DNA rather than the DNA being packaged into a preassembled viral capsid (94). The IVa2 protein also functions as a transcription factor for the major late genes (7). An HAdV-4 IVa2 protein coding sequence was identified at bp (3930 to 5554)c, where the attached letter "c" represents coding sequence transcribed from the complementary strand.

(iii) Late genes. The HAdV late genes are transcribed from a single promoter, the MLP. Multiple poly(A) signals are utilized to produce the various distinct mRNA species (72). The core elements of MLP were identified from extensive studies of the HAdV-2 genome sequence (93). Based on sequence comparison, all of the regulatory elements in the HAdV-4 MLP were identified: inverted CAAT box (bp 5803 to 5812), upstream element (bp 5823 to 5832), TATA box (bp 5854 to 5860); and MAZ/Sp1 binding sites flanking the TATA box at bp 5844 to 5853 and 5861 to 5871. The initiator element, which includes the transcription start site for the late transcription unit, is located at bp 5883 to 5889. Two downstream elements recognizing the IVa2 protein were identified at bp 5970 to 5980 (DE1) and 5985 to 6000 (DE2a and DE2b). The late transcription unit encodes the major AdV structural proteins and is subdivided further into regions L1 to L5, each region being expressed as a distinct mRNA species.

(a) L1. In the L1 region, the 52-kDa protein (bp 10765 to 11937) and protein IIIa (bp 11961 to 13736) were identified. The 52-kDa protein serves as a scaffold for capsid assembly during virus assembly (31). The IIIa protein is found on the outer surface of the virus and reportedly has a function in holding the virus facets together (68).

(b) L2. Four coding sequences were catalogued in the L2 region of the HAdV-4 genome. The penton base protein III, which is found at the 12 virion vertices, is located at bp 13815 to 15422. The penton protein binds to the host integrins via a conserved Arg-Gly-Asp (RGD) motif to trigger virus internalization (88). The RGD motif in the HAdV-4 penton is located at bp 14772 to 14780. Coding sequences for proteins VII and V, found at the viral core, are located at bp 15426 to 16007 and 16055 to 17080, respectively. A coding sequence for an 8.4-kDa pX protein was identified at bp 17103 to 17336. This pX protein, also known as the mu protein, has no known function.

(c) L3. Three coding sequences were identified in the L3 region of HAdV-4: the minor capsid protein precursor, pVI; hexon; and 23-kDa protease. The minor capsid protein is probably found on the inner capsid surface and may play a role as a structural intermediate between the capsid and the viral core. In HAdV-4, the coding sequence for the pVI precursor is located at bp 17368 to 18141. The coding sequence for the 105.2-kDa HAdV-4 hexon is located at bp 18248 to 21058. The hexon protein is the major structural component of the AdV capsid, constituting nearly 63% of the virion mass. Its length is 936 aa. In comparison with the other HAdV hexons, the HAdV-4 hexon is 75% identical to the HAdV-2 hexon, 92% identical to the HAdV-16 hexon, and between 82 and 83% identical to the hexons of HAdV-3, -7, and 21. The hexon monomer is made of two eight-stranded ß-barrels and three extended loops. A CLUSTAL-based multiple sequence alignment revealed four major regions of variation (variable regions [VRs] A to D) between the hexons of HAdV-2, -4, -5, and -7 (Fig. 1). When mapped onto the three-dimensional structure of the HAdV-2 hexon, all four regions mapped onto a series of outer loops. These four variable loops probably represent the serotype-specific epitopes. The hexon amino acid sequence is highly conserved outside the VRs. The last coding sequence in the HAdV-4 L3 region encodes a 23-kDa protease and was located at bp 21082 to 21702. This protease is required for the cleavage of viral proteins during virus maturation and assembly.



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  		FIG. 1. Multiple sequence alignment of the hexon proteins of HAdV serotypes 2, 4, 5, and 7. CLUSTALX alignment of the amino acid sequences of the hexons of HAdV-2, -4, -5, and -7 reveals four major regions of variation (noted as VR A through D). All VRs map onto a series of loops in the three-dimensional structure of the HAdV-2 hexon. CLUSTAL notes amino acid alignments as follows: an asterisk indicates a con-served amino acid, a single dot indicates either size or hydropathy is conserved, and stacked dots (:) indicate both size and hydropathy are conserved.

(d) L4. The L4 region of HAdV-4 contains four coding sequences. These correspond to the 100-kDa protein (bp 23341 to 25716), 22-kDa protein (bp 25439 to 25978), 33-kDa protein (bp 25439 to 25756 and 25926 to 26249), and pVIII protein (bp 26318 to 27001). The splice sites of the 33-kDa protein at bp 25756 (donor) and 25926 (acceptor) were identical to the GT-AG consensus. Mutational studies suggest the 100-kDa nonstructural protein has a role in hexon assembly and is required for the efficient translation of the late viral mRNAs (32, 54). The functions of the 22- and 33-kDa proteins have yet to be identified. The pVIII protein is found on the interior of the capsid and forms a bridge between the capsid and the viral core elements (68). Like other AdV homologs, the HAdV-4 pVIII is rich in proline, arginine, and lysine (~11.4%) and probably has a highly disordered structure.

(e) L5. The L5 region of HAdV-4 encodes the 45.1-kDa fiber protein at bp 31645 to 32922. A trimeric fiber assembly protrudes from the vertices of the icosahedral AdV capsid. The N-terminal domain attaches noncovalently to the penton base protein, while the globular C-terminal "knob" domain binds host cells. A study of the crystal structure of the HAdV-12 knob domain bound to the coxsackievirus and AdV receptor (CAR) revealed the key fiber amino acid residues required for CAR binding (39). These residues include Asp415, Pro417, and Pro418. Another important residue, Lys429, is conserved throughout all HAdV species, except HAdV-F. Multiple sequence alignment of the fiber sequences of the HAdV-B and -C species, as well as HAdV-12 and HAdV-4, shows the aspartate residue is present in HAdV-2, -4, and -12 but is replaced with an alanine in HAdV-5 and either asparagine or lysine in HAdV-B, as shown in Fig. 2. Pro417 is substituted for by serine in HAdV-4 and all of the HAdV-C species members; it is replaced with either glutamic acid or threonine in HAdV-B. Lys429 is conserved across all the fiber sequences.



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  		FIG. 2. Multiple sequence alignment of fiber proteins of HAdV species A, B, C, and E. Amino acid sequences of the fiber protein from species B (HAdV-3 and -7), C (HAdV-2 and -5), and E (HAdV-4) are aligned with the HAdV-12 (species D) homologous sequence. The three- dimensional structure of the HAdV-12 fiber was solved, and the key residues involved in CAR binding were mapped. Some of these key residues are marked by numbers at the top of the alignment showing the conservation among the CAR binding species A, C, and E AdVs relative to HAdV-12: D415 (no. 1), P417 (no. 2), P418 (no. 3), and K429 (no. 4). CLUSTAL notes amino acid alignments as follows: an asterisk indicates a conserved amino acid, a single dot indicates either size or hydropathy is conserved, and stacked dots (:) indicate both size and hydropathy are conserved.

(iv) Miscellaneous proteins. Seven hypothetical proteins were identified in the HAdV-4 genome. One of these putative proteins was identified by the GeneMark gene identification software. The rest were identified as homologs of hypothetical AdV proteins archived in GenBank. Four of these proteins lie in the ~5-kb stretch between the MLP initiator element and the L1 52-kDa protein coding sequence. E1B encodes two additional hypothetical proteins, which are presumably expressed from 1.26- and 1.31-kb mRNAs. Both putative proteins have partial identity to the E1B 55-kDa protein. The hypothetical protein predicted by GeneMark is encoded at bp 35331 to 35426 and has no identity to any protein in the GenBank database. The presence of these putative coding sequences strongly suggests the complete set of proteins encoded by the AdV genome sequence has not been identified.

The evolutionary origins of HAdV4. HAdV4 and HAdV7 are the etiological agents of ARD. However, unlike HAdV-7, which is a member of a species with several distinct serotypes, HAdV4 is the sole member of species E. This and the lack of a large number of HAdV4 genome types imply that this AdV species is the product of a relatively recent evolutionary event. The nature of this event, however, has been a matter of debate. One hypothesis suggests that HAdV-4 originated from a recombination event between two HAdV species (29). A second hypothesis indicates that HAdV-4 originated from a chimpanzee-human interspecies transmission event (48).

Serotype and species origins through genome recombination. Genome recombination occurs in AdVs. It was observed within a single serotype: HAdV-12 (79). Interserotypic recombinants, both laboratory-generated and naturally occurring strains, have also been documented (40, 52, 78). Illegitimate recombination was shown to be a factor in serotype evolution (12). Improved technology, such as rapid high-throughput DNA sequencing and analysis, permits the exact identification of recombination sites and clarification of such molecular events (52). The hypothesis of evolution of new serotypes through recombination is further supported by the recent determination of the complete genome of HAdV-11. Multiple sequence alignment and analyses imply that HAdV-11 is possibly the product of a recombination event between HAdV-7-like and HAdV-35-like genomes (77).

In a study of the sequences from HAdV4 fiber gene and its immediate neighbors, it was concluded that HAdV-E arose as a recombination event between a species B genome and a species C genome (29). The authors left open the possibility of finding the sites of recombination to prove this hypothesis with more extensive DNA sequencing, especially of the intergenic flanking sequences. To investigate this, the immediate 5'-upstream fiber-flanking 100-bp sequence of HAdV-4 was aligned with sequences from both the HAdV-B and HAdV-C species. A similar alignment was performed with the 100-bp sequence immediately downstream of the fiber stop codon (data not shown). There were conserved segments in the upstream region, but there were no regions of identity downstream of the fibers.

The full-length genome data of HAdV-4 along with several other HAdV genomes allow a comprehensive evaluation of all genes across these species along the entire genome. As shown in Table 3, the percent identities do not reflect a recombination event between species B and species C. In fact, a higher percent identity is seen with genes corresponding to an SAdV, SAdV-25.


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  		TABLE 3. Percent identities of select HAdV-4 proteins to their homologs in other HAdV speciesa

Serotype and species origin through an interspecies jump (zoonosis). Li and Wadell, using restriction analyses data, indicated that HAdV-4 may be closer to the SAdVs than to the HAdVs (48). This suggests a simian origin for HAdV-4. The "zoonotic event" hypothesis was tested by a performing a detailed analysis of the genes spanning the entire AdV genome (Table 3). The genes coding for the following proteins were selected: E1A 32-kDa protein, E1B 55-kDa protein, L1 55-kDa protein, L2 penton, L3 hexon, L5 fiber, and E4 34-kDa protein. These genes span the Mastadenovirus genome sequentially; therefore, phylogenetic analyses allow insights into the evolutionary associations among these AdVs across the entire length of the genome sequences. HAdV serotypes 3, 7 and 11 (representing species B); serotypes 1, 2, and 5 (representing species C); serotype 4 (representing species E); and serotype 40 (representing species F, an "outgroup" for reference) were included in the study. The sequences of the recently characterized SAdVs SAdV-22, -23, -24, and -25 were included in the analyses (14, 25, 61).

The amino acid sequences of each of the proteins from all the aforementioned HAdVs and SAdVs were aligned by using CLUSTALX (81). Figure 3 displays the phylogenetic relationships among these viruses. The gross topologies of the trees constructed with the genes coding for E1A 32-kDa protein, E1B 55-kDa protein, L1 55-kDa protein, L2 penton, L3 hexon, and E4 34-kDa protein were similar. There were three main groupings among the HAdVs and SAdVs. The first cluster comprises HAdV-4 plus SAdV-22, -23, -24, and -25. This group is closest to the second cluster, which comprised species B members (HAdV-3, -7, and -11). The third cluster comprised species C (HAdV-1, -2 and -5). In contrast, the phylogenetic tree constructed from the L5 fiber sequences had a distinctly different topology. Based on the fiber amino acid sequence, the HAdV-4, SAdV-22, SAdV-23, SAdV-24, and SAdV-25 cluster is closer to species C than to species B. This may be explained by an earlier recombination event prior to a split between the SAdVs and HAdV-4. HAdV-4, however, still maintains its close similarity to the SAdVs. In four of the six trees (genes coding for E1A 32-kDa protein, E1B 55-kDa protein, L1 52-kDa protein, and L5 fiber), SAdV-25 is most closely related to HAdV-4; this was the SAdV used for the earlier restriction enzyme digestion analysis suggesting these two AdVs were "distantly related" (48). Additional phylogenetic analyses (data not shown and unpublished data) show a close relationship between SAdV-21 and the HAdV-B. This agrees with the recent assignment of SAdV-21 to the Mastadenovirus HAdV-B species by Harrach (www.vmri.hu/~harrach/ADENOSEQ.HTM).



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  		FIG. 3. Phylogenetic analyses of selected HAdV-4 proteins. The amino acid sequences of six AdV proteins from eight HAdVs and four SAdVs were aligned by CLUSTALX using default parameters. The sequences of HAdV serotypes 3, 7, and 11 were used to represent the B subgroup, while the sequences of serotypes 1, 2, and 5 were chosen to represent the C subgroup. The sequences of HAdV-40 were used as outgroups in each of the trees. The unrooted trees were constructed by the neighbor-joining method (67). The robustness of the trees was measured by bootstrapping (1,000 replications). Numbers indicate bootstrap values in support of the adjacent node.

A comparison of the coding and noncoding features of HAdVs and SAdVs. Detailed pairwise sequence analyses of various coding sequences and noncoding biological features were performed to gather additional evidence for this "zoonotic event" hypothesis. Further proof of the close evolutionary relationship between HAdV4 and the SAdVs come from a BLAST analysis of the individual genes across the Mastadenovirus genome. For example, Table 4 presents a summary of selected genes, encoding the capsid proteins, across the genomes demonstrating the closeness of the HAdV-4 genes to the SAdV genes. Of importance for vector development and applications in humans, the outer viral proteins of HAdV-4, which are the putative determinants for immunogenicity, have high percent identities to their simian counterparts: for example (HAdV-4 reference and SAdV-25 query), penton, 90%; hexon, 88%; and fiber, 86%. In contrast, HAdV-C has lower percent identities to HAdV-4: penton, 68%; hexon, 75%; and fiber, 53% (Tables 3 and 4).


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  		TABLE 4. Percent identities of select HAdV-4 structural proteins to their homologs in SAdV speciesa

Fiber gene analysis. A detailed comparison of the fiber genes, shown in Table 5, reinforces this view as the HAdV-4 fiber is 425 aa in length and is nearly identical or very similar to the fibers from SAdV-25 (425 aa), SAdV-24 (443 aa), SAdV-23 (443 aa), and SAdV-22 (443 aa). In contrast, HAdV-C (HAdV-1, -2, and -5) fibers are approximately 582 aa in length. BLAST scores and percent identities obtained with HAdV-4 as the reference and the other AdVs as queries support this argument as well: SAdV-25 (BLAST score, 739; identity, 86%), SAdV-24 (BLAST score, 586; identity, 67%), SAdV-23 (BLAST score, 474; identity, 57%), and SAdV-22 (BLAST score, 474; identity, 57%). For comparison, using HAdV-4 as the reference, the HAdV-C data are as follows: BLAST score of 303 and identities of 43, 32, and 34% for HAdV-1 (there were three regions that gave BLAST scores); BLAST score of 304 and identities of 41, 30, and 30% for HAdV-2; and BLAST score of 310 and identities of 41, 32, and 31% for HAdV-5. HAdV-4 fiber has a BLAST score of 804 and a 94% identity when compared against itself. This is due to the low-complexity filtering algorithm of BLAST that removes stretches of contiguous but noninformative amino acids from the analysis (e.g., MQQAAAAAMQ in the penton protein).


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  		TABLE 5. Comparison of the fiber lengths and amino acid sequence identities between HAdV-4 and selected HAdVs and SAdVsa

E3 region. Presented in Fig. 4 is a comparison of gene order between HAdV-4 and SAdV-25 along with representative human species, including HAdV-C (serotype 5) and HAdV-B (serotype 7) species, showing the E3/L5 gene region. Upstream of the fiber CDS, HAdV-4 shares a homolog with SAdV-25 that is not found in any other human serotype. The HAdV-4 29.7-kDa protein is 62% identical to the SAdV-25 delta-1 protein. This gene is also present in SAdV-22, -23, and -25 but not in SAdV-21 (data not shown). HAdV-4, HAdV-7, and SAdV-25 also share two E3 coding sequences, the 19- and 24.8-kDa protein coding sequences. Distinguishing HAdV-4 from SAdV-25 is the absence in HAdV-4 of the CR1-gamma1 protein coding sequence. This sequence is also absent from the other HAdVs.



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  		FIG. 4. Arrangement of coding sequences in the E3 and L5 regions. Genome maps of HAdV serotypes 4, 7, and 5 and SAdV-25, showing the arrangement of coding sequences in the E3 and L5 regions, are presented. Displayed are schematics of each double-stranded linear genome sequence in the E3/L5 region, along with the three forward frames of translation. The color scheme for the coding sequences is as follows: brown = E3 19-kDa protein; dark green = E3 24.8-kDa protein; purple = E3 6.3-kDa protein; red (hatched) = E3 29.7-kDa protein (also called the CR1-delta-1 in SAdVs); blue = E3 10.4-kDa protein; light green = E3 14.5-kDa protein; red (solid) = E3 14.7-kDa protein; light blue = fiber; blue (hatched) = E3 CR1-gamma1 protein (in SAdV-25); and green (hatched) = 7.7-kDa protein (in HAdV-7). A comparison of gene order and synteny in the E3 region, among HAdVs and chimpanzee AdVs, highlights unique conserved features across species.

Comparison of the hypothetical genes in HAdV-4 and SAdV-25. A second key difference between HAdV-4 and SAdV-25 is the unique presence of the hypothetical L1 14.1-kDa protein coding sequence in the HAdV-4 genome sequence. However, there are three hypothetical coding units common between HAdV-4 and SAdV-25: those for the 29.7-, 19-, and 24.8-kDa proteins.

Noncoding genome landmarks. The noncoding genome landmark identities are complemented by identities from comparisons of noncoding motifs and regions. For example, the critically important ITR of HAdV-4 has the highest identity (BLAST score of 50) to the ITR of SAdV-22 rather than to other HAdV ITRs (highest BLAST score among the HAdVs = 42). Among the HAdV species, the HAdV-4 ITR is most related to those of the B species (HAdV-3, -7, and -21; BLAST score for each = 42). But, as noted in the annotation section, the HAdV-4 ITR diverges considerably from the HAdV "canonical" sequence. For reference, the SAdV-25 ITR has a lower match with HAdV-4 but also contains regions of identity to HAdV-4 (BLAST score of 36).

Human and simian VA RNA genes have been studied in detail (41). One observation is that HAdV-E, -B1, -C, and -D species contain two VA RNA genes, whereas the HAdV-B2, -A, and -F species contain only one. In addition, it was noted that the chimpanzee AdVs (SAdV-21 through -25) have two VA RNA genes, whereas AdVs isolated from monkeys apparently have either one or zero VA RNA genes. As noted, both VA RNA genes of SAdV-22 to -25 are all highly related to their HAdV-4 counterparts (41).

The current study supports and extends this earlier observation. VA RNA I coding sequences of HAdV-4 and SAdV-25 have two mismatches between them (BLAST score of 147), while the VA RNA II coding sequences have five mismatches and 10 gaps (BLAST score of 114). Among the HAdVs, the only VA RNA coding sequence in HAdV-11 had some identity to the HAdV-4 VA RNA I (BLAST score of 44). The VA RNA genes of HAdV-7 had equally low BLAST scores against HAdV-4, scoring 33 (VA RNA I) and 33 (VA RNA II) against their homologs.

Taken together, these data support a model in which HAdV-4 (species E) arose from a zoonotic event involving a SAdV-25-like virion. They also complement earlier observations with restriction enzyme digestion analyses (48, 86).


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DISCUSSION
 
Advanced molecular diagnostics: applications of rapid genome determinations. There is considerable interest in developing rapid, accurate, and high-throughput assays for monitoring infectious diseases, in particular ARD (44), as well as biothreat agents. As one of several identified pathogens causing ARD, HAdVs are important as a model organism in the development and validation of molecular diagnostic techniques such as multiplex PCR, real-time PCR, and microarray-based assays. Present methods for clinical diagnoses include serum neutralization, microneutralization, and hemagglutination. These clinical assays have acknowledged limitations such as throughput or reliance on antibodies, which may be variable in response and are in short supply. These tests are often tedious and not readily applicable as a general screen for more than one serotype routinely.

To this end, the complete genome of HAdV-4 has been sequenced, annotated, and analyzed for the first time. Due to the modest-sized genome of the HAdVs (ca. 36 kb), scaffolds of primers derived from existing genome data and coupled with the rapid methods for DNA sequencing can produce complete genome sequences. Up-to-date field strains can be sequenced rapidly, and unique DNA sequence signature-based arrays may be generated to survey unambiguously and simultaneously all of the important serotypes and strains of HAdV (and other causative agents). This algorithm of genome-based diagnostics is a basis for the detection of other pathogens: e.g., severe acute respiratory syndrome-causing coronavirus (50, 62).

This underscores the importance and effectiveness of the "leveraged primer-walking" genome-sequencing strategy using archived genomes and partial genomes to generate pathogen DNA sequence data for rapid turnaround in microarray assay design and deployment. The unique ARD-related pathogen signatures have been incorporated into a respiratory pathogen microarray (RPM) chip developed by EOS. These RPM chips (versions 1 and 2) are undergoing validation in a real-world test bed.

AdV evolution and phylogeny. The study of viral origins and evolution is in its infancy (53). Continued improvements in genome-sequencing and analysis methodology and technology are resulting in more and complete viral genomes being deposited into databases, especially in the context of emerging human pathogens. With the availability of genome sequence data, viral and host evolution can be viewed through horizontal transfer of genes among hosts and through genome recombination of related viruses within a host. These events are likely to play major roles in the evolution of viruses as human pathogens.

Based on the presented genome data and phylogenetic analyses, HAdV-4 is evolutionarily closest to the SAdVs. It is plausible that the HAdV-E species resulted from an interspecies transmission event. The notion that new viral species in humans may arise from zoonotic infections has precedence. There is evidence in the literature suggesting that the two strains of HIV, HIV-1 and HIV-2, represent cross-species infections from the chimpanzee (HIV-1) and the sooty mangabey monkey (HIV-2) (25, 36). Similarly, phylogenetic data on HTLV and simian T-cell leukemia/lymphotropic virus (STLV) indicate that HTLV I and II originated from separate interspecies transfers between simian species and humans (74).

The phylogenetic data presented, however, also suggest a recombination at the fiber region between B- and C-like AdVs. This recombination event may have preceded the zoonotic transmission of HAdV-4 from chimpanzees to humans. It is borne out by the fact that HAdV-4 and SAdV-23 through -25 show a closer similarity to the HAdV-Cs at the fiber but are closer to the HAdV-Bs at other regions of the genome, both upstream and downstream of the fiber coding sequence.

Origins of the chimpanzee AdVs. Due to the intriguing bioinformatics analysis of HAdV-4 in the context of the chimpanzee AdVs and the apparent phylogenetic closeness, it may be questionable whether the chimpanzee AdVs are originally of chimpanzee origin or represent a cross-species jump from humans (animal handlers) to chimpanzees. The literature reports at least three independent isolations of chimpanzee AdVs, with the latest being the source of the four sequenced genomes (59).

First reports described isolation of AdVs from chimpanzee throat washings and fecal specimens (35, 75). This was followed by studies in the late 1960s and early 1970s of experimental kuru syndrome and Creutzfeldt-Jakob (CJ) disease in a laboratory colony of chimpanzees. This work resulted in the isolation of more than 100 strains of latent viruses (3, 59). Tissues were aseptically removed from nine sacrificed chimpanzees (Pan satyrus) experimentally inoculated with kuru prions 1 to 3 years previously. Each isolate was given a chimpanzee virus number in the order of its appearance (59). Distinct virus types defined from these strains were tentatively called "Pan" viruses (in laboratory nomenclature) to distinguish the latent viruses they found in chimpanzees (3). Pan 5, 6, 7, and 9 were proven to be new AdVs (3): Pan 5 was isolated from mesenteric lymph nodes of a chimpanzee experimentally infected with kuru; Pan 6 was from the mesenteric nodes of another chimpanzee; and Pan 7 was isolated from the inguinal nodes of yet another similarly infected chimpanzee. Pan 9 was isolated from the mesenteric nodes of a chimpanzee infected with CJ disease. All four isolates had the biochemical and physical characteristics of AdVs. Complement fixation tests, hemagglutination, and neutralization tests were performed. These four viruses were defined as new AdV types, as they were not neutralized by antisera to known AdVs of human or simian origin (3). Antisera included "human [sero]types 1-33." The sera of nine normal chimpanzees that were bled when they entered the colony were also tested against the Pan AdVs. Three of the nine had antibodies to one or more of them. Sera taken from animal handlers when they came on duty at the chimpanzee colony and again after 1 to 3 years did not have antibodies for these Pan viruses (3). It is likely these cataloged and archived AdVs are indeed of chimpanzee origin.

Chimpanzee AdV-derived vectors as vaccine and gene therapy vectors in the context of HAdV-4 bioinformatics. The importance of whole-genome determinations and bioinformatics analyses is far-reaching. There is interest in understanding the genomics and biology of SAdVs, especially in the context of the chimpanzee AdVs being considered as alternative vectors for gene therapy and for vaccine delivery development. Currently, vectors used for gene therapy are based on HAdV-5. However, HAdV-5-derived vectors cause problems in human gene therapy protocols, including fatality (56). In general, host preexisting immunity to HAdV is enough of a concern to stimulate the development of alternative AdV vectors to which neutralizing antibodies would be rare in the human population. The lack of neutralizing antibodies against chimpanzee AdV in human serum samples suggests vectors derived from them will be useful as vaccine and gene therapy vectors (61).

However, contrasting neutralization data have also been presented in the literature. According to Li and Wadell (48), SAdVs are "distantly related" to HAdV-4 based on restriction enzyme digestion profiles. The same report noted SAdVs were neutralized by rabbit antisera against HAdV-4 prototype virion (48). The bioinformatics analyses presented here of the HAdV-4 genome along with the SAdV genomes suggest a stronger link may exist, along with a more worrisome potential and theoretical host immune response, as in cross-reaction of the SAdV-derived vectors with the human host.

These observations may impact the development of vectors for applications in gene therapy and vaccine delivery as these vectors may be administered to the patient multiple times.


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ACKNOWLEDGMENTS
 
We thank Mary Lou Gibson (Virapur, Inc., San Diego, Calif.) for virus growth and DNA preparation and Jason Seto and Brenda Desjardins for critically reading the manuscript. D.S. thanks Menas Kafatos, SCS/George Mason University, for support and postdoctoral funding (A.P.).

Research support was provided through a grant (DAMD17-03-2-0089) from the U.S. Army Medical Research and Material Command (USAMRMC). Partial support was also provided through the Epidemic Outbreak Surveillance Project (EOS), funded through HQ USAF Surgeon General Office, Directorate of Modernization (SGR), and the Defense Threat Reduction Agency.

The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense.

During the course of this work, the EOS Consortium included the following members: Peter F. Demitry and Theresa Lynn Difato, Department of USAF/SGR; Jerry Diao, Kenya Grant, Rosana R. Holliday, Cheryl J. James, Chris Olsen, and Kathy Ward, USAF/SGR (Ctr); John Gomez, Margaret Jesse, Kindra Nix, Jose J. Santiago, Curtis White, and Sue A. Worthy, Lackland AFB, Tex.; Eric H. Hanson and Robb K. Rowley, The George Washington University (IPA); Elizabeth A. Walter, Texas A&M University—San Antonio (IPA); Russell P. Kruzelock, Virginia Tech (IPA); Jennifer Weller, George Mason University (IPA); Robert Crawford, Armed Forces Institute of Pathology; Baochuan Lin, David A. Stenger, Dzung Thach, Gary J. Vora, and Zheng Wang, Naval Research Laboratory; Brian K. Agan and Michael Jenkins, Wilford Hall Medical Center; Linda Canas, Air Force Institute for Operational Health; and David Metzgar, Kevin Russell, and Jianguo Wu, Navy Health Research Center.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Computational Sciences, George Mason University, 10900 University Blvd., MSN 5B3, Manassas, VA 20110. Phone: (703) 993-8403. Fax: (703) 993-8401. E-mail: dseto{at}gmu.edu. Back

{dagger} Present address: Midwest Research Institute, Palm Bay, FL 32909. Back


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Journal of Virology, February 2005, p. 2559-2572, Vol. 79, No. 4
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.4.2559-2572.2005




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