Evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis.

Evidence of Molecular Evolution Driven by
Recombination Events Influencing Tropism in a Novel
Human Adenovirus that Causes Epidemic
Keratoconjunctivitis
Michael P. Walsh1, Ashish Chintakuntlawar2, Christopher M. Robinson2, Ijad Madisch3, Bala´zs Harrach4,
Nolan R. Hudson5, David Schnurr6, Albert Heim3, James Chodosh2, Donald Seto1, Morris S. Jones5*
1Department of Bioinformatics and Computational Biology, George Mason University, Manassas, Virginia, United States of America, 2Howe Laboratory, Massachusetts
Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States of America, 3 Insitut fu¨r Virologie, Medizinische Hochschule, Hannover, Germany,
4 Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary, 5Clinical Investigation Facility, David Grant USAF Medical Center, Travis AFB,
California, United States of America, 6 Viral and Rickettsial Disease Laboratory, California Department of Public Health, Richmond, California, United States of America
Abstract
In 2005, a human adenovirus strain (formerly known as HAdV-D22/H8 but renamed here HAdV-D53) was isolated from an
outbreak of epidemic keratoconjunctititis (EKC), a disease that is usually caused by HAdV-D8, -D19, or -D37, not HAdV-D22.
To date, a complete change of tropism compared to the prototype has never been observed, although apparent
recombinant strains of other viruses from species Human adenovirus D (HAdV-D) have been described. The complete
genome of HAdV-D53 was sequenced to elucidate recombination events that lead to the emergence of a viable and highly
virulent virus with a modified tropism. Bioinformatic and phylogenetic analyses of this genome demonstrate that this
adenovirus is a recombinant of HAdV-D8 (including the fiber gene encoding the primary cellular receptor binding site),
HAdV-D22, (the e determinant of the hexon gene), HAdV-D37 (including the penton base gene encoding the secondary
cellular receptor binding site), and at least one unknown or unsequenced HAdV-D strain. Bootscanning analysis of the
complete genomic sequence of this novel adenovirus, which we have re-named HAdV-D53, indicated at least five
recombination events between the aforementioned adenoviruses. Intrahexon recombination sites perfectly framed the e
neutralization determinant that was almost identical to the HAdV-D22 prototype. Additional bootscan analysis of all HAdV-
D hexon genes revealed recombinations in identical locations in several other adenoviruses. In addition, HAdV-D53 but not
HAdV-D22 induced corneal inflammation in a mouse model. Serological analysis confirmed previous results and
demonstrated that HAdV-D53 has a neutralization profile representative of the e determinant of its hexon (HAdV-D22) and
the fiber (HAdV-D8) proteins. Our recombinant hexon sequence is almost identical to the hexon sequences of the HAdV-D
strain causing EKC outbreaks in Japan, suggesting that HAdV-D53 is pandemic as an emerging EKC agent. This documents
the first genomic, bioinformatic, and biological descriptions of the molecular evolution events engendering an emerging
pathogenic adenovirus.
Citation: Walsh MP, Chintakuntlawar A, Robinson CM, Madisch I, Harrach B, et al. (2009) Evidence of Molecular Evolution Driven by Recombination Events
Influencing Tropism in a Novel Human Adenovirus that Causes Epidemic Keratoconjunctivitis. PLoS ONE 4(6): e5635. doi:10.1371/journal.pone.0005635
Editor: Wanda Markotter, University of Pretoria, South Africa
Received January 7, 2009; Accepted April 8, 2009; Published June 3, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: The work reported herein was performed under United States Air Force Surgeon General-approved Clinical Investigation No. FDG20040024E, and
partially supported by Hungarian Research Fund grant K72484, U.S. Public Health Service NIH grants EY013124 and T32 A1007633, and a Research to Prevent
Blindness Physician-Scientist Merit Award (to JC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: drmorrisj@yahoo.com
Introduction
Epidemic keratoconjunctivitis (EKC), characterized by inflam-
mation of the conjunctiva and cornea, produces a sudden onset of
acute follicular conjunctivitis and stromal keratitis and is a
worldwide problem causing significant and sometime lasting
morbidity [1]. Human adenoviruses (HAdVs) HAdV-D8, -D19,
and -D37 are the most common pathogens causing EKC [1].
Adenoviruses were first isolated from civilians and military
trainees who had respiratory disease in the early 1950s [2,3]. They
were the first respiratory viruses to be isolated and characterized.
Epidemiological studies confirmed that adenoviruses are the cause
of acute febrile respiratory disease among military recruits [4,5]
and have been persistent in the global population. Since then, 52
human adenovirus (HAdV) genotypes have been characterized
and classified according to their immunochemical properties,
nucleic acid similarities, hexon and fiber protein characteristics,
biological properties, and phylogenetic analysis, and placed in the
genus Mastadenovirus [6,7]. These 52 adenovirus genotypes that
infect humans are classified into seven species (Human adenovirus A
to G) [6,8] and are known to cause a range of diseases specific to
the tropisms of the viruses: keratoconjunctivitis (HAdV-D8,
HAdV-D19, and HAdV-D37) [9,10], gastroenteritis (HAdV-
A31, HAdV-F40, HAdV-F41, and HAdV-G52) [6], acute
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respiratory disease (HAdV-B3, HAdV-E4, HAdV-B7, HAdV-
B14, and HAdV-B21) [5], and perhaps obesity (HAdV-D36) [11].
Adenoviruses have linear double-stranded DNA genomes that
generally range from 26 to 45 kb and are encapsidated in an
icosahedral protein shell that ranges from 70 to 100 nm [8]. The
primary components of the protein shell are the hexon, penton
base, and fiber proteins. Through genome sequence analysis, it has
been demonstrated that the genomes of all human adenoviruses
have similar genetic organization [12,13,14].
In the past, human adenovirus serotype and species classifica-
tion were defined by reactivity of outer coat proteins to
discriminating antibodies (e.g., immunochemistry/virus neutrali-
zation) as well as by other biological properties (e.g. oncogenic
potential, hemagglutination properties). Today, given the avail-
ability of DNA sequencing and analysis technology, phylogenetics
(based on comparative nucleic acid and amino acid sequence
analysis of informative viral proteins or/and their genes, as well as
analysis of genomic organization) is a highly quantitative, cost-
effective, expedient method and the preferred and reliable method
for classifying adenoviruses. It is a preferred and reliable method
for demonstrating how viruses are related through molecular
evolution as it provides and relies on the primary sequence data
[15,16,17,18].
In this study we sought to characterize a unique intermediate
recombinant HAdV isolate, at the molecular level. This novel
strain was isolated from a patient who, along with eleven other
patients, presented with highly contagious EKC outbreak in
Germany was described [19]. Since HAdV-D22 was never
associated with EKC, we performed whole genome sequencing,
complemented with bioinformatics, including phylogenetic and in
silico proteome analysis, as well as in vivo studies in a mouse model
to characterize this unique recombinant virus. To reflect this novel
and different genome and because of the multiple recombination
events and several unique sequence segments in the genome of this
virus, we renamed this virus HAdV-D53.
Results
Amplification and sequencing of the new adenovirus
Initial and partial sequencing of HAdV-D53 (previously HAdV-
D22/H8) demonstrated that portions of the penton and fiber
genes were similar to HAdV-D37 and HAdV-D8, respectively
[19]; thus suggesting that this disease causing virus was the result
of recombination. To understand clearly the genetic characteristics
and the nature of HAdV-D53, the entire genome has been
sequenced and analyzed.
Physical features of new adenovirus genome
The genome length of HAdV-D53 is 34,909 base pairs, with a
base composition of 23% A, 20.8% T, 28.2% G, 28% C and the
GC content was 56.2%. The GC content is consistent with
members of species Human adenovirus D (HAdV-D) (57.0% mean).
The organization of the 36 open reading frames (ORF’s) that were
found had a genome organization similar to other mastadeno-
viruses (Fig. 1). The inverted terminal repeat (ITR) sequences for
HAdV-D53 were determined to be 212 bp in length.
The nucleotide and amino acid identities for selected genes in
the genome of HAdV-D53 to its nearest relatives are shown in
Tables 1 and 2, respectively. Interestingly, the pVII and protein V
genes were dissimilar to homologous genes in any adenovirus
species with 83 and 87% nucleotide identity, respectively to
HAdV-D37 (Table 1), the nearest relative in that region of the
HAdV-D53 genome. For pVII, the low nucleotide identity is
partially due to a 99 bp deletion, resulting in a 33 amino acid
deletion of the predicted pVII protein. When compared to HAdV-
D37, the protein V gene contains 2 deletions. The first deletion is
18 bp and the second is 93 bp.
Genomic recombination analysis
To determine if recombination occurred within the HAdV-D53
genome, several software tools were applied. A bootscanning
Figure 1. Genome organization of HAdV-D53. Genome is represented by a central black horizontal line marked at 5-kbp intervals. Protein-
encoding regions are shown as boxes. Boxes above the black line represent open reading frames (ORFs) that are encoded on the forward (or upper)
strand. Boxes underneath the black line represent ORFs that are encoded on the reverse (or lower) strand. The colors of the boxes correspond to
which adenovirus the protein is most likely descended from: red – HAdV-D8, aqua – HAdV-22, orange – HAdV-D37, white – dissimilar to all known
adenoviruses.
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program [20] was used to determine the relationship of HAdV-D53
to all of the fully sequenced HAdV-D genotypes. According to the
alignments, several regions indicated recombination events; nucle-
otides (as genome coordinates) 1–1000, 1500–3250 (E1A, E1B,
55K), 8500–15,750 (52K, pIIIa, penton base), 17,000–17,750, (pX,
pVI) and 19,500–25,000 (second half of hexon, protease, DBP,
100K, 22K) showed a strong relationship to HAdV-D37; nucleotides
17,750–19,500 (first half of hexon) showed a strong relationship to
HAdV-D22; nucleotides 27,375–29,750 (CR1-b) and 30,500–
34,909 (14.7K, fiber, E4 ORFs) showed a strong relationship to
HAdV-D8 (Fig. 2). Although the bootscan analysis showed that
nucleotides 29750–30,500 have a strong relationship to HAdV-D49,
we believe that this region comes from an unsequenced HAdV-D8
strain, because that region (RIDb) in a partially sequenced HAdV-
D8 strain has 100% amino acid identity to the HiroshimaHAdV-D8
isolate (Table 2). These relationships were confirmed by comparison
with nucleotide identity in Table 1, as well as BLASTP similarity
analysis of the proteins (Table 2). In contrast, nucleotides 1000–
1500, 3250–8500, 15,750–17,000, and 25,000–27,375 showed
slightly lower similarity to several known adenoviruses, suggesting
that this region of species HAdV-D adenoviruses are both well
conserved and so far unique for the studied strain. Thus, bootscan
analysis of the HAdV-D53 genome shows evidence of multiple
recombination events.
Hexon recombination analysis
The results of our whole genome bootscan indicated that a
recombination event occurred inside the hexon gene. The hexon
contains loops 1 (L1) and 2 (L2), which are the most important
determinants of neutralization via antibodies as well as immune
escape. Since L1 and L2 are the most relevant for serotyping, we
performed bootscan analysis to pinpoint where the recombination
events occurred in the hexon gene of HAdV-D53. The results of
the bootscan analysis shown in Figure 3A and 3B reveal that a
recombination event occurred between nucleotide 380 and 1400–
1620 which are the amino terminus of L1 and the conserved C
terminus of the highly variable L2, respectively (Table 3). Thus,
the complete neutralization epitope e, which is nearly identical to
the sequenced HAdV-D22, is framed by non HAdV-D22
sequences in the recombinant strain HAdV-D53.
Based on the nucleotide identity of HAdV-D53 to other
adenoviruses, we believe that the previous name HAdV-D22/H8
is not appropriate due to the fact that the fully sequenced genome
and the bioinformatic analyses demonstrate that HAdV-D53 is the
Table 1. Percent identities of the nucleotide coding sequences of selected HAdV-D53 proteins and their homologsa.
HAdV-D8pb HAdV-D19 HAdV-D22 HAdV-D37 HAdV-D48 HAdV-D49
E1B 19K 94.9 99.3 99 99.5 97.8 97.8
E1B 55K 96 99.2 98.3 99.3 97.4 96.9
IX 95.1 96.1 98.5 96.1 97.1 97.1
IVa2 96.7 98 97.4 98 98.3 91.3
DNA polymerase 95.2 98.1 97.6 98 98 98
pTP 93.3 97.6 96.8 97.9 96.4 96.8
52K 95.7 98 98.1 100 97.9 98.3
penton base 89.2 91.2 92.4 100 90.5 90.1
pVII 79.2 80.4 80.4 83.2 80 80.1
V 85.2 87 87.5 87.2 87.7 87.3
pX 95.1 100 100 100 98.7 99.6
hexon 89.4 90.2 98.4 90.5 90.6 90.4
protease 95.4 96 95.4 99.8 96.5 96.5
22K 89.9 100 98.8 100 99.3 99
pVIII 95.6 98.4 98.5 98.4 98.3 97.7
12.2K 93.2 96 97.5 96 96.9 96.9
CR1-a 52.5 80.5 97 80.5 75.4 77.6
18.4K 95.2 91.1 98.1 91.1 96.2 94.1
CR1-b 100 74.5 85.1 74.5 64.5 58
CR1-c 86.4 75.3 Ndc 75.3 75.1 80.5
RID-a 93.5 94.2 94.2 94.2 98.2 98.2
RID-b 90.6 87.4 93.2 87.4 94.2 99.2
14.7K 96.7 95.2 97.2 95.2 96.7 97.7
fiber 100 75.1 67.6 75 69 67.6
dTPase 100 48.4 88.9 48.4 85.1 85
Standard nomenclature has been applied so that orthologs have the same name (Davison et al., 2003). Numbers in bold reflect the proposed origin. Italics note the
gene with supposed double origin.
aPercent identities and similarities were determined by global alignment using the EMBOSS needle program with a gap penalty of 10.0 and a gap extension penalty of
5.0.
bNot present in the genome.
cPrototype HAdV-D8 strain is Trim isolate – ATCC VR-1604.
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product of multiple recombinations of known and perhaps
undiscovered and/or yet unsequenced adenoviruses. Taken togeth-
er, we propose the name ‘‘HAdV-D53’’ for this novel recombinant
adenovirus, reflecting its genome divergence from other human
adenoviruses. We also believe this ‘‘genome type’’ designation is
more appropriate in light of the current and future DNA sequencing
Figure 2. Whole genome (A) bootscan and (B) simplot of HAdV-D53 compared to fully sequenced HAdV-D genomes.
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and analysis technology, superseding the importance of the previous
classifications based on serology (e.g., serotypes).
Hexon recombination is common in species Human
adenovirus D
To determine whether or not this phenomenon was common in
other adenoviruses, the available hexon genes of all HAdV-D
genotypes were cross-examined. Recombination events at similar
nucleotide locations of the hexon gene in HAdV-D13, HAdV-
D32, and HAdV-D39 (Fig. 3C–E) were found. The HAdV-D13
recombination is especially interesting regarding the present study,
as HAdV-D13 acquired L1 and L2 from HAdV-D37 and the
same region in HAdV-D53 was presumably exchanged for L1 and
L2 of HAdV-D22. To demonstrate the validity of our recombi-
nation predictions, we included the bootscan analysis of the
HAdV-D49 hexon gene, which does not show evidence of any
recombination events (Fig. 3F). Taken together, these data suggest
that adenoviruses in HAdV-D species are susceptible to recom-
bination events at the amino terminus of L1 and the carboxy
terminus of L2 of the hexon gene; implicating a mechanism which
allows adenoviruses to switch neutralization epitopes.
In vivo HAdV-D53 induced keratitis
Since HAdV-D53 was isolated from a patient with EKC and
appeared to be corneotropic [19], we tested its ability to induce
corneal innate immune responses in a previously described mouse
model of adenovirus keratitis, in which EKC viruses induce a
keratitis similar to human EKC, but without viral replication [21].
HAdV-D53 infection induced a clinically evident keratitis (corneal
opacity) as early as 1 day post-infection (dpi) that peaked by 3–4
dpi (Fig. 4A). In contrast, mock and HAdV-D22 injection did not
induce corneal opacity at any time post-infection. Neither virus
replicated in the mouse cornea (data not shown). Hematoxylin and
eosin staining of corneal cross sections at 4 dpi with HAdV-D53
showed thinning of the epithelial cell layer, stromal edema, and
infiltration by leukocytes (Fig. 4B). In contrast, HAdV-D22
infection induced only modest cellular infiltration. We next
assessed corneal myeloperoxidase (MPO) levels after infection as
a measure of the presence of infiltrating neutrophils and
monocytes [22,23]. HAdV-D53 infection induced significantly
higher levels of MPO when compared to HAdV-D22 and mock
infected corneas (Fig. 4C). By flow cytometry, corneal infection
with HAdV-D53 caused a significantly greater number of
infiltrating neutrophils (Gr1+F4/802) [24,25], similar to previous
studies with HAdV-D37 [21], than with HAdV-D22 infection.
Inflammatory monocytes (Gr1+F4/80+) [26,27] and resident
macrophages (Gr1-F4/80+) [28] did not increase significantly
after infection with either virus (Figs. 4F and G). Because
neutrophils appeared by histology and flow cytometry to be the
predominant infiltrating cell in HAdV-D53 keratitis, we also tested
Table 2. Percent identities of selected amino acid sequences of HAdV-D53 proteins and their homologs.
HAdV-D8 HAdV-D8p HAdV-D19 HAdV-D22 HAdV-D37 HAdV-D48 HAdV-D49
E1B19K 92 99 99 100 97 97
E1B55K 96 99 98 99 97 96
IX 95 97 99 97 97 98
IVa2 97 99 99 99 99 98
DNA polymerase 96 98 98 98 99 98
pTP 94 97 97 98 97 97
52K 96 99 99 100 98 98
penton base 89 91 92 100 89 89
pVII 79 80 80 81 79 80
V 84 87 87 87 87 87
pX 100 100 100 100 100 100
hexon 92 90 99 90 92 90
protease 97 100 99 100 100 100
DBP 96 99 98 100 97 97
22K 81 100 99 100 99 98
pVIII 100 97 98 99 98 98 98
12.5K 100 95 95 99 95 98 98
CR1-a 99 54 73 94 73 67 71
18.4K 97 95 91 97 91 94 92
CR1-b 97 100 74 81 74 48 44
CR1-c 100 81 62 65 62 65 73
RID-a 100 94 29 96 96 100 100
RID-b 100 89 92 93 92 89 97
14.7K 100 96 94 97 94 97 98
fiber 100 100 74 62 74 63 59
dUTPase 100 92 87 92 84 82
Numbers in bold reflect the proposed origin. Italics note the gene with supposed double origin.
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the expression of neutrophil chemokines CXCL1 and CXCL2
[29,30]. Both CXCL1 (Fig. 4D) and CXCL2 (Fig. 4E) were
expressed at significantly higher levels after infection with HAdV-
D53 than with HAdV-D22.
Phylogenetic analysis
Detailed phylogenetic analysis of selected proteins, performed
with nucleotide data and deduced amino acid sequences confirmed
that HAdV-D53 was an unusual recombinant adenovirus. The tree
topology of HAdV-D53 was different depending on which protein
was tested. The penton base had the closest relationship to HAdV-
D37, whereas the fiber gene was closest to HAdV-D8 (Fig. 5).
Interestingly, DNA polymerase, protein V, and pVII genes did not
cluster tightly with any other virus, which reflects their unique
sequences. As expected the L1 and L2, which are responsible for the
neutralization e determinant, clustered with HAdV-D22. In L1,
HAdV-D22 was 1.8% distant to HAdV-D53 and L2 was identical
to HAdV-D53. In the b-determinant, HAdV-D53, supported by a
strong bootstrap value (83%), clustered to HAdV-D37. Using
sequence data that was available in GenBank, the hexon sequences
of HAdV-D53 clustered tightly with the Japanese isolates 1/
Yamaguchi/2004, C075/Matsuyama/2003, and FS161/Fukui/
2004 suggesting that HAdV-D53 and the Japanese isolates
represent different isolates of the same HAdV genotype.
Viral neutralization
Since our sequence analysis shows that HAdV-D53 is
genetically similar to HAdV-D8, -D22, and -D37, we wanted to
determine its serum neutralization profile. Antisera to HAdV-D8
and HAdV-D22 neutralized HAdV-D53 at dilutions of 1:128 and
1:256, respectively. In contrast, antisera to HAdV-D37 was unable
to neutralize HAdV-D53 at a dilution of equal to or less than 1:8.
These results confirm previous results and demonstrated that
HAdV-D53 has a neutralization profile representative of its hexon
and fiber proteins whereas the penton base did not contribute to
neutralization.
Figure 3. Bootscan of HAdV-D species hexon genes demonstrating recombination events. Comparison of HAdV-D53 by (A) bootscan and
(B) simplot with the HAdV-D types which have a fully sequenced genome. (C) HAdV-D13, (D) HAdV-D32, (E) HAdV-D39, and (F) HAdV-D49 were
compared to all hexon genes in species HAdV-D by bootscan analysis.
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Discussion
The initial report of HAdV-D53 described that this novel,
possibly emergent disease-causing, strain comprised a HAdV-D22
strain that had recombined with HAdV-D8 and HAdV-D37 [19].
Full genome sequencing of this isolate, HAdV-D53, and bioinfor-
matic analysis have demonstrated that this genome is so different
both from HAdV-D22 and all the other officially accepted
serotypes that it must be seen as a novel human adenovirus which
we have re-named HAdV-D53 based on its primary sequence data
and analyses.
This genome is based on a highly probable homologous
recombination between HAdV-D37 and HAdV-D8; however
(probably after the initial recombination event), other parts of the
genome have been replaced by genome parts from several known or
unknown HAdVs. Altogether, we assume the occurrence of at least
five major recombination events: (1) recombination of HAdV-D37
and HAdV-D8 (occurring between the end of the HAdV-D37 22K
gene and the beginning of the HAdV-8 pVIII gene); (2) exchange
with an unknown or unsequenced adenovirus from species HAdV-
D from the beginning of the protein IX gene to the end of the pTP
gene; (3) replacement of the pVII and protein V genes with the same
genome fragment from an unknown or unsequenced adenovirus
from species HAdV-D; (4) exchange of L1 and L2 between HAdV-
D22 and the recombinant virus; (5) and replacement of the 18.4K
gene from an unknown source.
Here we presented evidence that the neutralization epitope e of
HAdV-D53, highly homologous to HAdV-D22, was generated by
two recombination events which brought about the complete
exchange of L1 and L2. This phenomenon is apparent in three
other adenoviruses from HAdV-D (HAdV-D13, -D32, and -D39),
as well as HAdV-B16, which is a member of species HAdV-B [31].
This detailed analysis at the complete genome level demonstrates
that recombination may be a common event within adenoviruses,
especially in species HAdV-D, as a general mechanism driving
molecular evolution and immunogenicity. The neutralization
epitope is framed by highly conserved sequences, which are also
used for generic detection of most HAdVs by PCR [32,33,34].
These conserved sequences allow homologous recombination
when a cell is infected with two different adenovirus types. Our
results demonstrate that within HAdV-D, the neutralization
epitopes e are exchangeable in nature leading to immune escape
of a highly virulent and prevalent HAdV type. This resembles the
antigenic shift mechanism of influenza A viruses which is caused
by reassortment, a more efficient way of gene transfer.
To date, this is the first fully sequenced recombinant adenovirus
to be associated with EKC. Bootscan analysis showed that several
regions of HAdV-D53 (IVa2, DNA polymerase, pTP, pVII, V,
and 18.4K) were dissimilar to any known adenovirus. These
sequences are either from an undiscovered adenovirus or a known
yet unsequenced HAdV-D isolate. Additional whole genome
sequencing studies of adenoviruses will shed light on this important
question.
In light of its association with EKC, it seems significant that
experimental corneal infection with HAdV-D53 induced inflam-
mation, while infection with HAdV-D22, a virus not associated
with EKC but highly related to HAdV-D53, did not. Those areas
of the genome unique to EKC-causing viruses represent likely
sources of corneal tropism. Full genome sequencing, bioinfor-
matics analysis, and genome wide comparisons between EKC and
non-EKC inducing HAdV-D strains are beginning to yield clues
to corneal tropism and pathogenesis [8,9]. Further experiments
recombining different adenovirus genes will determine which
genes are crucial for EKC.
Early genotyping of HAdV-D53 by sequencing of the hexon (the
major neutralization determinant) and other determinants (fiber and
penton) gave results of a recombinant strain HAdV-D22/H8 [19].
Thus, HAdV-D53 fulfilled the hexon L1 and L2 criteria for typing as
HAdV-D22 [18], with a fiber knob (hemagglutination determinant)
sequence identical to HAdV-D8. In contrast to the classical concept
of a recombinant strain, HAdV-D53 was cross reactive with a
HAdV-D8 specific antiserum (Table 4). This confirms that some of
the neutralization antibodies in the HAdV-D8 antiserum bind to the
HAdV-D8-like fiber of HAdV-D53 and block infectivity by
interfering with virus/primary cellular receptor interaction.
Phylogenetic analysis of the complete genomic sequence of
HAdV-D53 showed similar genetic distances to the other available
HAdV-D types (6.1% to 9.3% nucleic acid sequence divergence) as
observed between other prototypes of species HAdV-D (6.0% to
9.5%) (Fig. 5). This supports the idea that HAdV-D53 is the
prototype of a new genotype. Therefore, phylogeny deduced from
complete genomic sequence data supports that HAdV-D53 is a new
prototype. However, HAdV-D53 is a recombinant virus and its
genome is not of monophyletic origin. For most parts of its genome
the ancestors of its sequence (HAdV-D8, -D22, -D37) could be
identified by bootscan analysis and confirmed by building
phylogenetic trees of the corresponding sequence stretches. For
example, L1 and L2 of the neutralization determinant e are highly
variable and evolved rapidly by immune escape mechanisms. L1
and L2 of HAdV-D53 were (except for a single point mutation)
identical to HAdV-D22 suggesting a recent recombination event in
the phylogeny of HAdV-D53. However, bootscan analysis
suggested that several regions of HAdV-D53 (IX, IVa2, DNA
polymerase, pTP, pVII, protein V, and 18.4K) were dissimilar to all
Table 3. An excerpt from the plot values of a Simplot
Bootscan of the HAdV-22D and HAdV-D37 hexons.
Center Pos HAdV-D22 hexon HAdV-D37 hexon
1320 100 0
1340 100 0
1360 100 0
1380 100 0
1400 100 0
1420 100 0
1440 100 0
1460 80 0
1480 18 1
1500 1 12
1520 0 7
1540 0 7
1560 0 3
1580 0 2
1600 0 46
1620 0 51
1640 0 45
1660 0 51
1680 0 60
1700 0 57
1720 0 50
1740 0 80
doi:10.1371/journal.pone.0005635.t003
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known adenoviruses. Construction of phylogenetic trees supported
that these parts of the genome are either from an undiscovered
adenovirus or a known yet unsequenced HAdV-D isolate. However,
these genome regions are well conserved in HAdV-D and thus led
to low, non significant bootstrap values (see Fig. 5 polymerase,
protein V and pVII). Additional whole genome sequencing studies
of adenovirus prototypes may elucidate whether some of these parts
of the HAdV-D53 genome are also derived from recombination
Figure 4. HAdV-D53 induces keratitis. (A) Clinical appearance of HAdV-D53 keratitis. Virus-free buffer (mock), 104 TCID of HAdV-D22, or HAdV-
D53 was injected in the corneal stroma of C57BL/6 mice (n = 8 corneas/group). Corneas were examined under a surgical microscope up to 4 days
post-infection. One representative picture from each group is shown at the indicated time points. (B) Histopathology of HAdV-D53 keratitis.
Representative histopathological sections at 4 days post-infection of mouse corneas injected with buffer, HAdV-D22, or HAdV-D53 are shown
(hematoxylin and eosin stain; scale bar = 50 m). (C) Myeloperoxidase (MPO) expression in HAdV-D53 keratitis. Mock, HAdV-D22, and HAdV-D53
infected corneas were analyzed by ELISA at 24 hours post-infection for the expression of myeloperoxidase enzyme. (D, E) Chemokine expression in
HAdV-D53 keratitis. Expression of CXCL1 (D) and CXCL2 (E) in mock, HAdV-D22, and HAdV-D53 infected corneas were analyzed by ELISA at 16 hpi.
Data is mean6SEM from three individual experiments (n = 9 corneas/group). (F, G) Phenotypic analysis of inflammatory cells in HAdV-D53 keratitis.
Mock, HAdV-D22, and HAdV-D53 infected corneas at 24 hours post-infection were homogenized and single cell preparations were stained with anti-
CD45, anti-Gr1, and anti-F4/80 antibodies. Cells were gated on CD45-positive staining. (F) Representative dot plots or (G) quantification of three
separate experiments is shown for each group (mean cells/cornea6SEM, n= 9 corneas/group). In all experiments statistical significance is denoted by
*, P,.05 as determined by ANOVA with Scheffe’s multiple comparison test.
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Figure 5. Phylogenetic analysis of HAdV-D53. Analysis of HAdV-D53 is based on the nucleic acid sequence of (A) complete genomes, as well as
the predicted amino acid sequences of (B) polymerase, (C) L1 and (D) L2 of the hexon protein penton, (E) b-determinant, (F) c-determinant, (G) pV
and (H) pVII. Numbers denote human adenovirus serotypes. HAdV-D53 (in bold) shows the new isolate. The numbers close to the nodes represents
bootstrap pseudoreplicates.
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events. Interestingly, protein V, a minor capsid protein, was
significantly smaller than the homologous proteins of all other
members of HAdV-D (e.g. 297 aa vs. 334 aa in HAdV-D46).
Moreover, pVII also contained several deletions, nevertheless
phylogenetic trees clearly supported clustering of HAdV-D53
protein V and pVII with species HAdV-D in spite of these deletions.
The 59-ITR sequence contains highly conserved critical motifs
that are required for adenovirus replication [37]. These motifs
include the canonical ‘core origin,’ defined as the minimal DNA
requirement for the initiation of replication, binding the terminal
protein-DNA polymerase complex [38], and several host transcrip-
tion factor binding sequences which are required for efficient
adenovirus replication [39,40]. For example, it has been shown that
Oct-1 binds to the NF III motif to stimulate transcription by 6–8
fold [41]. Within most HAdV species, both NF I and NF III binding
sites are conserved except for species HAdV-E as seen in HAdV-E4
and HAdV-E4 vaccine strain [42,43] and simian AdV-21 (SAdV-
B21), SAdV-E22 through E25 (unpublished observations) which
lack the NF I binding site. Significantly, HAdV-D53 is also missing
the NF I motif, like the other members of the sequenced HAdV-D
types (Fig. 6). Previous annotations of the sequenced HAdV-D
members do not remark upon this absence of the NF I. Perhaps this
absence of an NF I site is an indication of a different evolutionary
line of origin for species HAdV-D, as opposed to the other HAdVs
with both NF I and NF III motifs. The latter half of the ITR
contains motifs for binding Sp1 (GGG GGT GG) and ATF (TGA
CGT). These motifs are also reported to contribute to the efficiency
of viral DNA replication [44]. While the Sp1 motif seems to be less
conserved (GGG CGg/t gg), they are similar for HAdV-D types.
The ATF motif is conserved and present in HAdV-D (Fig. 6).
As new strains of adenoviruses appear and are isolated, usually
with an accompanying pathology, initial attempts at understanding
the clinical relevance involves characterizing the isolate with respect
to structural features. These include the traditional serological
methods and reagents. However, in some cases the isolates are
difficult to culture and/or the reagents are not readily available. In
the past, the isolate is either characterized as much as possible or
archived in a laboratory as an unculturable, yet interesting isolate.
Today, when an interesting adenovirus isolate arises, full-genome
sequencing, phylogenetic analysis, and other state-of-the-art
methodology and technology provide alternatives to these limita-
tions. As a recent example, when HAdV-G52 was discovered, it was
found that the virus grew too slowly in tissue culture to be ‘properly’
serotyped. This and the lack of readily available serotyping reagents
limited a ‘traditional’ characterization. However, phylogenetic
analysis, only made possible through whole genome sequencing,
demonstrated that it was a novel adenovirus isolate that was quite
divergent from all other species of human adenoviruses [6].
Similarly, if serology and limited sequence analysis, e.g., limited
hexon, penton and fiber data, were the only tools that we had
available for the original characterization of this proposed HAdV-
D53, the reported original conclusion in regards to HAdV-D53,
that it is a variant of HAdV-D22 albeit with minor genetic
modifications in the penton and fiber genes [19], would have been
and remained incorrect. In order to conclusively characterize a
suspected novel adenovirus, whole genome sequencing and
bioinformatics analysis of the resultant and complete reference
primary nucleotide sequence should be performed.
The fact that the genes associated with serum neutralization are
from known viruses raises a central question, ‘‘What are the
criteria for defining and naming a new ‘‘type’’ of adenovirus?’’
Although serology has been crucial in the pre-genomic era, it can
not be used as the gold-standard for the typing of novel
adenoviruses that will be sequenced and characterized in the
future. If serology was the only tool that we had in our typing
toolbox, we would not have determined that HAdV-D53 was due
to several recombinations of known and perhaps unknown
adenoviruses. In the past, the ‘‘serotype’’ designation was used
to distinguish different and separate adenoviruses. However, due
to the fact that there are about 200 known adenovirus types, this
approach is impractical. Moreover, the neutralization of recom-
binants such as HAdV-B16, and -D53 would yield inconclusive
data. Full-genome sequencing and bioinformatic analyses should
be the primary methods used when proclaiming novel adenovirus
genotypes as it is quicker and a less cumbersome alternative for
adenovirus typing, especially given the cost-effective technology to
obtain genome sequences rapidly and the growing array of
bioinformatics tools, along with the growing adenovirus database.
We propose using ‘‘genotype’’ rather than ‘‘serotype’’ as a
means for identifying, characterizing and differentiating adenovi-
ruses, based on genome sequence analyses. This fits into the
currently accepted classification of adenovirus ‘‘genome types,’’ in
which substrains of adenoviruses are designated by lower case
alphabetic designations in addition to their primary designation,
e.g., HAdV-7a, b, c…, if their restriction enzyme digestion
patterns differ from the reference prototype genome, ‘‘HAdV-7p.’’
Recently, partial genome sequences from HAdV-D strains
causing EKC outbreaks in Japan were published [35,36]. These
were almost identical to HAdV-D53 (including the intrahexon
recombination sites) suggesting that HAdV-D53 has already spread
around the globe as an emerging EKC agent, reflecting the
epidemiology of a globally connected population and a newly
emergent pathogen.
Materials and Methods
Ethics Statement
The animals involved in this study were procured, maintained,
and used in accordance with the Laboratory Animal Welfare Act
of 1966, as amended, and NIH 80-23, Guide for the Care and Use
of Laboratory Animals, National Research Council.
Nucleotide sequence accession numbers
The HAdV-D53 genome and annotation have been deposited
in GenBank prior to manuscript submission; accession number
FJ169625. The following HAdV genomes (GenBank accession
numbers) were used: HAdV-A12 (AC_000005), HAdV-B7
(AY594255), HAdV-D8 (AB110079), HAdV-B11 (AY163756),
HAdV-C5 (AC_000008), HAdV-E4 (AY599837), HAdV-D49
(DQ393829), HAdV-D53 (FJ169625), HAdV-D9 (AJ854486),
HAdV-B16 (AY601636), HAdV-D17 (AC_000006), HAdV-D19
(ER121005), HAdV-D22 (FJ404771) HAdV-D26 (EF153474),
HAdV-D37 (DQ900900), HAdV-D46 (AY875648), HAdV-D48
(EF153473), HAdV-D22 (unpublished genome sequence), HAdV-
D8 (published partial sequences (AB110079) and unpublished
whole genome sequence).
Table 4. Neutralization of HAdV-D53 with hyper immune
serum.
Antiserum HAdV-D53 HAdV-D8 HAdV-D22 HAdV-D37
aHAdV-D8 1/128 1/1024 ,1/8 ,1/8
aHAdV-D22 1/256 ,1/8 1/128 ,1/8
aHAdV-D37 ,1/8 ,1/8 ,1/8 1/4096
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Figure 6. Analysis of the HAdV-D53 inverted terminal repeat (ITR). NF I, NF III, SpI, and pTP binding motifs are marked. The ATF binding site
is TGACGT.
doi:10.1371/journal.pone.0005635.g006
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Amplification of the HAdV-D53 genome
To amplify regions of HAdV-D53 flanking the sequences
described by Engelmann et al. [19], we designed primers based on
conserved adenovirus sequences of types in HAdV-D. All
amplicons were then sequenced using primer walking.
Viruses, cells and neutralization test
Viral neutralization assays were run as previously described
[45]. Rabbit antisera to prototype strains were standardized in
cross-neutralization tests against adenovirus prototype viruses 1–
49. Prototype viruses were from archives maintained at the State
of California, Department of Public Health, Viral and Rickettsial
Disease Laboratory.
Nucleic Acid Isolation
Viral DNA was extracted from tissue culture and processed
stool samples using the MagNA Pure LC DNA Isolation Kit I
(Roche, Indianapolis, IN) according to the manufacturers’
recommendations for the MagNA Pure LC automated nucleic
acid extraction system.
Bioinformatics
Percent idenitities for HAdV-53 genes/proteins. The global
alignment were performed using the EMBOSS [46] needle
program. The proteins and genes of HAdV-53 were compared
to homologs in other HAdV-D genomes. In cases were a genome
lacked sufficient annotation, genes and proteins were found
manually using the Artemis [47] annotation program. The percent
identities for the proteins (Table 2) of the HAdV-D sequences were
obtained via BLASTP [48]. The percent identities for the
nucleotide sequences (Table 2) that code for these proteins were
determined using a BioJava [49] implementation of a Needleman-
Wunsch algorithm.
Recombination analysis of hexon genes (Figure 3). Hexons
genes from the HAdV-D genomes were aligned using ClustalW
[50] alignment option available in the MEGA 4 program [51].
The default gap opening and gap extension penalties were used
(15.0 and 6.66). SimPlot [20] software was used to complete a
bootscan analysis of the aligned hexon genes of the available
HAdV-D genomes. The default settings for window size, a step
size, replicates used, gap stripping, distance model, and tree model
were, respectively, 200, 20, 100, ‘‘on’’, ‘‘Kimura’’, and ‘‘Neighbor
Joining’’. The HAdV-53 hexon was chosen as the reference
sequence for the analysis.
Recombination analysis of HAdV-D whole genomes (Figure 2).
The available HAdV-D genomes were aligned using the MAFFT
[52] alignment method which is available through a web interface
at http://www.ebi.ac.uk/Tools/mafft/. The default parameters
for gap open penalty, gap extension penalty, and perform fft were
used (1.53, 0.12, ‘‘localpair’’).
Simplot [20] software was used to complete a bootscan analysis
of the aligned HAdV-D genomes. The default parameters for
window size and step size were altered (1000, 200). All other
default parameters were left unchanged.
Recombination Analysis
Two groups of hexon coding nucleotide sequences were analyzed
for recombination events. The first group consisted of the hexon
genes of the human adenovirus D species (HAdV-D8, -D9, -D17, -
D22, -D26, -D37, -D46, -D48, -D49, -D53). This group is referred
to as the HAdV-D53 hexon group. The second group consisted of
hexon genes from HAdV-B16, -C5, -E4, -B7, -B11, and -C2. The
following accession numbers were used for the hexon recombina-
tion analyses. HAdV-A12 (AC_000005), HAdV-B7 (AY594255),
HAdV-B11 (AY163756), HAdV-C5 (AC_000008), HAdV-E4
(AY599837), HAdV-D49 (DQ393829), HAdV-D22, (AB330103),
HAdV-D53 (FJ169625), HAdV-D9 (AJ854486), HAdV-16/B1
(AY601636), HAdV-D17 (AC_000006), HAdV-D26 (EF153474),
HAdV-D37 (DQ900900), HAdV-D46 (AY875648), HAdV-D48
(EF153473). The two groups of sequences were aligned using the
ClustalW [50] alignment option available in the MEGA 4 program
[51]. The default gap opening and gap extension penalties were
used. Those penalties were 15.0 and 6.66 respectively.
Two different programs were used to analyze the two
alignments for recombination events. The first program is SimPlot
[20]. The bootscan option of SimPlot was used to analyze the
alignments. The default settings were used. These included a
window size = 200, a step size = 20, replicates used= 100, gap
stripping = ‘‘on’’, distance model = ‘‘Kimura’’, tree model = ‘‘-
Neighbor Joining’’. The HAdV-D53 hexon was chosen as the
reference sequence HAdV-D53 hexon group. HAdV-D16’s hexon
was chosen as the reference in the HAdV-16 hexon group.
The second program is the Recombination Detection Program
(RDP) [53]. This program uses several different algorithms
(including bootscanning) to determine the presence of recombi-
nation events. 1 of the ‘‘general recombination detection options’’
was changed so that the program would recognize that the
sequences in the alignment were linear and not circular. No other
default options were changed.
Phylogenetic analysis of HAdV-D53
DNA polymerase, penton base (b-determinant), pVII, protein V,
L1 and L2 of the hexon, and fiber knob (c-determinant) nucleotide
sequences were compared by sequential pairwise alignment with the
Clustal Algorithm implemented in the BioEdit software package
(version 6.0.5) and adjusted manually to conform to the optimized
alignment of deduced amino acid sequences. Phylogenetic relation-
ships were inferred from the aligned nucleic acid as well as from the
amino acid sequences by the neighbour-joining method imple-
mented in the programs DNAdist and Neighbor integrated in the
MEGA software package (version 3.1) using the Kimura two-
parameter substitution model and a transition/transversion ratio of
10. Support for specific tree topologies was estimated by bootstrap
analysis with 1000 pseudoreplicate data sets.
In vivo model of adenovirus keratitis
Eight to 12 week old C57BL/6J mice (stock # 000664) were
purchased from Jackson Laboratory (Bar Harbor, ME). Animal
housing and care were in accordance with Animal Care and Use
Committee guidelines. Mice were anesthetized for virus infection by
intramuscular injection of ketamine (85 mg/kg) and xylazine
(14 mg/kg) and later euthanized by CO2 inhalation. For infection,
1 microliter of virus-free dialysis buffer, cesium chloride gradient
purified HAdV-D22, or purified HAdV-D53 (104 tissue culture
infectious dose) was injected in the central corneal stroma as
previously described [21]. After euthanasia, corneas were removed
and fixed in 10% neutral buffered formalin, embedded in paraffin,
and sections cut at 5 m thick prior to staining. For ELISA, corneas
were harvested at indicated time points and homogenized using
phosphate buffered saline (PBS) with protease inhibitors, and the
reactions performed as per manufacturer’s instructions (R&D
Systems, Minneapolis, MN). ELISA plates were analyzed on a
microplate reader (Molecular Devices, Sunnyvale, CA) with limits
of detection of ,2 pg/mL for CXCL1 and ,1.5 pg/mL for
CXCL2. Flow cytometry was performed as described by Carr and
coworkers [54]. Corneas were dissected at indicated time points,
and digested with 1 mg/ml collagenase type I (Sigma, St. Louis,
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MO). Non-specific binding was blocked by anti-mouse Fc (BD
Pharmingen, San Diego, CA) and 5% normal rat serum (Jackson
Immuno Research, West Grove, PA). Cells were labeled with
FITC-conjugated anti-mouse F4/80 (clone CI:A3-1), phycoery-
thrin-Cy5-conjugated anti-CD45 (clone 30-F11), and PE-conjugat-
ed anti-mouse Gr-1 (clone RB6-8C5) (all from BD Biosciences, San
Jose, CA). and incubated in the dark on ice for 30 min, washed 36
with PBS/1% BSA, resuspended in PBS containing 1% parafor-
maldehyde, and incubated overnight. CountBright absolute
counting beads (Invitrogen, Eugene, OR) were added (21,600
beads/sample), cell suspensions gated on CD45high labeled cells,
and the numbers of each cell type determined at this gate setting. A
second gate was established to count the number of beads that
passed through during each run (300 sec). The absolute number of
cells per cornea was determined by calculating the number of input
beads/21,6006number of cells in the CD45high-gated sample.
Acknowledgments
We thank Sarah Torres and Lisa Thrasher for help with sequencing
HAdV-D8 and HAdV-D22.
The animals involved in this study were procured, maintained, and used
in accordance with the Laboratory Animal Welfare Act of 1966, as
amended, and NIH 80-23, Guide for the Care and Use of Laboratory
Animals, National Research Council.
The views expressed in this material are those of the authors, and do not
reflect the official policy or position of the U.S. Government, the
Department of Defense, or the Department of the Air Force.
Author Contributions
Conceived and designed the experiments: IM DS AH JC DS MSJ.
Performed the experiments: MPW AVC CMR IM NRH. Analyzed the
data: MPW AVC CMR IM BH DS AH JC DS MSJ. Contributed
reagents/materials/analysis tools: DS AH JC DS MSJ. Wrote the paper:
MPW AVC CMR IM BH DS AH JC DS MSJ.
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