Unique sequence features of the Adenovirus 31 complete genomic sequence are conserved in clinical isolates.

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ssBioMed CentBMC Genomics
Open AcceResearch article
Unique sequence features of the Human Adenovirus 31 complete
genomic sequence are conserved in clinical isolates
Soeren Hofmayer1, Ijad Madisch1,2, Sebastian Darr1, Fabienne Rehren1 and
Albert Heim*1
Address: 1Institut für Virologie, Medizinische Hochschule Hannover, Hannover, Germany and 2Current address: Department of Radiology,
Massachusetts General Hospital Harvard Medical School 100 Charles River Plaza, Suite 400 Boston, Massachusetts 02114, USA
Email: Soeren Hofmayer - mail@s-hofmayer.de; Ijad Madisch - imadisch@partners.org; Sebastian Darr - SebastianDarr@gmx.de;
Fabienne Rehren - rehren.fabienne@mh-hannover.de; Albert Heim* - heim.albert@mh-hannover.de
* Corresponding author
Abstract
Background: Human adenoviruses (HAdV) are causing a broad spectrum of diseases. One of the
most severe forms of adenovirus infection is a disseminated disease resulting in significant
morbidity and mortality. Several reports in recent years have identified HAdV-31 from species A
(HAdV-A31) as a cause of disseminated disease in children following haematopoetic stem cell
transplantation (hSCT) and liver transplantation. We sequenced and analyzed the complete
genome of the HAdV-A31 prototype strain to uncover unique sequence motifs associated with its
high virulence. Moreover, we sequenced coding regions known to be essential for tropism and
virulence (early transcription units E1A, E3, E4, the fiber knob and the penton base) of HAdV-A31
clinical isolates from patients with disseminated disease.
Results: The genome size of HAdV-A31 is 33763 base pairs (bp) in length with a GC content of
46.36%. Nucleotide alignment to the closely related HAdV-A12 revealed an overall homology of
84.2%. The genome organization into early, intermediate and late regions is similar to HAdV-A12.
Sequence analysis of the prototype strain showed unique sequence features such as an
immunoglobulin-like domain in the species A specific gene product E3 CR1 beta and a potentially
integrin binding RGD motif in the C-terminal region of the protein IX. These features were
conserved in all analyzed clinical isolates. Overall, amino acid sequences of clinical isolates were
highly conserved compared to the prototype (99.2 to 100%), but a synonymous/non synonymous
ratio (S/N) of 2.36 in E3 CR1 beta suggested positive selection.
Conclusion: Unique sequence features of HAdV-A31 may enhance its ability to escape the host's
immune surveillance and may facilitate a promiscuous tropism for various tissues. Moderate
evolution of clinical isolates did not indicate the emergence of new HAdV-A31 subtypes in the
recent years.
Published: 25 November 2009
BMC Genomics 2009, 10:557 doi:10.1186/1471-2164-10-557
Received: 26 June 2009
Accepted: 25 November 2009
This article is available from: http://www.biomedcentral.com/1471-2164/10/557
© 2009 Hofmayer et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 14
(page number not for citation purposes)
Background
Adenoviridae are non-enveloped, double-stranded DNA
viruses with an icosahedral capsid [1]. Human Adenovi-
ruses (HAdV) belong to the genus Mastadenovirus and are
classified into six species (HAdV-A to HAdV-F) that were
defined historically as subgenera on the basis of hemag-

BMC Genomics 2009, 10:557 http://www.biomedcentral.com/1471-2164/10/557
glutination properties [2,3]. Subsequently, oncogenic
properties in rodents and DNA homology were also used
to define the subgenus (species) [1]. Recently a new strain
of HAdV was discovered and has been classified as HAdV-
52, representing a putative new species G [4].
Human adenoviruses have long been recognized as path-
ogens causing a broad spectrum of different diseases
depending on the type-related organotropism and viru-
lence. For example, infections of the upper respiratory
tract are caused by HAdV-C1, -C2, -C5, -B3, and -B7 [5,6],
the more dangerous infections of the lower respiratory
tract mainly by HAdV-B3, -B7, -B21, and -E4 [7-9]. The
types HAdV-D8, -D19, and -D37 are closely associated
with severe epidemic keratoconjunctivitis. Gastroenteritis
and diarrhoea are caused by the enteric adenoviruses
HAdV-F40,-F41 and HAdV-A31, which are frequently
found in infants and children.
Moreover, immunocompromised patients can develop a
sepsis-like, disseminated adenovirus syndrome that is
associated with high levels of immunosuppression (for
example, lymphocyte counts <300/μl) as a crucial risk fac-
tor [10]. A wide range of organs can be affected and an
effective antiviral therapy is not yet available. Conse-
quently, mortality rates of up to 60% were reported [10-
12]. Disseminated disease is mainly caused by species C
adenoviruses. However, in recent decades, HAdV-A31 has
been increasingly reported as a etiologic agent for dissem-
ination in immunosuppressed children following allo-
genic haematopoietic stem cell transplantation [10,13-
15].
One of the essential sequence features for HAdV types
causing dissemination may be the viral RGD (integrin
binding) motif of the penton base protein, because
HAdV-F types lacking the RGD motif have never caused a
disseminated infection (with exception of a single case
report) in spite of their high prevalence [16-18]. In addi-
tion, recent studies have shown that the binding of blood
coagulation factor (F) X to the HAdV-C5 hexon protein
facilitates infection of the liver and could also foster virus
dissemination [19]. Similar to HAdV-C5, F IX binding
may promote HAdV-A31 infection of epithelial cells [20].
The outstanding clinical relevance of HAdV-A31 is also
documented by its significant association with immuno-
suppressed patients in comparison to immunocompetent
patients [16]. This high incidence may be explained by
reactivations of latent (or persisting) HAdV infections,
which have been recently described for species C HAdV
[21]. A similar mechanism may be suspected for HAdV-
A31 although any conclusive data on its persistence is still
even in the same clinical centre, suggesting reactivations
rather than infection chains of de novo HAdV-A31 infec-
tions [14,22]. However, HAdV-A31 may also be transmit-
ted easily in a nosocomial setting between
immunosuppressed patients, as high amounts of HAdV-
A31 are spread with faeces [23].
In spite of this increasing clinical relevance of HAdV-A31,
the virus had not yet been completely sequenced. There-
fore, we determined the complete nucleotide sequence of
the HAdV-A31 prototype strain in order to search for
unique sequence motifs which may be associated with its
high virulence. In addition, we compared several viru-
lence associated gene regions (E1A, E3, E4, fiber knob,
penton base, protein IX, and pX) of seven clinical isolates
and the HAdV-A31 prototype.
Results
General properties
The complete genomic sequence of HAdV-A31 is 33,763
base pairs in length and was submitted to GenBank as
AM749299. The plus strand has a base composition of
22.89% G, 23.48% C, 27.49% A and 26.14% T. The GC
content is 46.36%. As other Mastadenoviruses, HAdV-A31
is organized into four early, one intermediate and five late
transcription units. We identified 34 coding regions that
are homologue to previously described gene products of
other human adenoviruses (Figure 1). The annotation of
the predicted coding gene regions is listed in Table 1.
Phylogeny
Phylogenetic analysis of the whole genomic sequence of
HAdV-A31 was performed by using the neighbor-joining
method. Representative members of all HAdV species
were included in the analysis. HAdV-A31 clustered as
expected to the species HAdV-A, close to HAdV-A12 (Fig-
ure 2).
A global pairwise alignment was constructed using the
mVISTA Limited Area Global Alignment of Nucleotides
(LAGAN) [24] in order to compare the predicted whole
genomic sequence of HAdV-A31 to the representative
types of each species. The graphical alignments showed
the close relationship between HAdV-A31 and -A12 with
the exception of the coding regions for immunogenic
determinants (hexon, fiber). Interestingly, the early tran-
scription unit E3, which is not under selective pressure by
the immune system, also showed a significant divergence
between HAdV-A31 and HAdV-A12 (Figure 3).
ITR
HAdV's inverted terminal repeats (ITR) and its flanking
DNA regions exhibit several binding sites for viral pro-Page 2 of 14
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lacking. So far, genetic analysis of HAdV-A31 strains iso-
lated from hSCT patients showed significant differences
teins and a set of cellular factors for efficient adenoviral
DNA replication [25]. HAdV-A31's ITRs are 148 bp in

BMC Genomics 2009, 10:557 http://www.biomedcentral.com/1471-2164/10/557
length. A nuclear factor III (NF-III) binding site described
as conserved among most human adenoviruses as a
5'ATGNNAATGA 3' sequence motif (ATGCAAATAA in
HAdV-E4) [26] was not identified in the HAdV-A31 ITR,
but a 5'ATGAAGTGGG 3' sequence at position bp 46-55
may function as a binding site for NF-III. HAdV-A12 also
lacks the classical NF-III binding motif but has the same
5'ATGAAGTGGG 3' motif as HAdV-A31 at bp 46-55. The
conserved NF-I binding site (5'TGGACTTGAGCCAA 3')
was predicted for HAdV-A31 at position 25-38.
HAdV-A31 ITRs also revealed binding sites for the tran-
scription factors SP1 and ATFs. The binding of ATFs to a
5'TGACGT 3' motif has been shown to be important for
efficient viral growth of HAdV-C5 [27]. Whereas all other
human adenoviruses (including HAdV-A12) reveal
exactly this ATF binding motif, HAdV-31's putative single
ATF binding site between bp 113 - 117 showed a C instead
the ITRs had the common 5'CTATCTATAT 3' motif that is
required for viral replication and also protects the viral
genome by ORP-A binding from DNAse-I digestion. The
core origin of DNA replication, which binds the complex
of preterminal protein (pTP) and DNA polymerase, was
present in the highly conserved motif 5' ATAATATACC 3'
between bp 9 and 18 in HAdV-A31.
Human elongation factor 1-alpha (EF-1A) is known to be
an efficient factor for enhancement of E1A gene region
transcription. HAdV-A31 revealed only two putative EF-
1A binding motifs (5'GCCGGATGT 3'), which are located
within the ITR region at bp 105-113 and 136-144,
whereas the five EF-1A binding sites are located upstream
from the E1A promoter region of HAdV-C5 [28].
E1A coding region
E1A is the first gene expressed after adenoviral infection
Table 1: HadV-A31 annotation of coding regions
Region Common name Product Location Length in aa
E1A n.n. 29.4K 479-1045 & 1124-1348 266
E1B 19 K small t-antigen 18.3 K 1488-1955 156
55 K large t-antigen 53.1 K 1793-3208 472
Intermediate IX 14.9 K 3290-3721 144
IVa2 50.9 K c3757-5093 & c5372-5384 449
E2B DNA Polymerase 134.6 K c4866-8042 & c13193-13201 1184
pTP 73.1 K c8222-9982 & c13193-13201 633
L1 52/55 K 41.8 K 10307-11413 368
pIIIa 64.4 K 11435-13171 578
L2 III (Penton Protein) 57.1 K 13246-14763 505
V 39.8 K 15373-16416 347
pVII 20.4 K 14777-15340 187
pX 7.9 K 16440-16658 72
L3 pVI 28.5 K 16734-17516 260
Hexon 103.7 17575-20343 922
Protease 23.3 K 20370-20981 203
E2A DNA binding Protein 55.3 K c21065-22522 485
L4 100 K 86.8 K 22551-24878 775
22 K 20.2 K 24616-25146 176
33 K 23 K 24616-24913 & 25092-25393 199
pVIII 25.2 K 25452-26153 233
E3 12.5 K 12.1 K 26153-26470 105
CR 1 alpha 28.6 K 26424-27212 250
CR 1 beta 29.4 K 27215-27967 262
RID alpha 10.5 K 27999-28274 91
RID beta 12.5 K 28271-28600 109
14.7 K 14.6 K 28593-28979 128
L5 Fiber 58.9 K 29157-30827 556
E4 ORF 1 13.9 K c33046-33429 127
ORF 2 14.9 K c32619-33014 131
ORF 3 13.4 K c32272-32622 116
ORF 4 13.7 K c31904-32266 120
ORF 5 33.9 K c31102-31971 289
ORF 6/7 13.9 K c30861-31082 & c31834-31971 119Page 3 of 14
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of the commonly observed T at the end of the motif. An
SP1 binding site was present as a GC-rich region between
bp 52 - 61 (5' TGGGCGGAGT 3'). The extreme termini of
[3]. The predicted gene product of the HAdV-A31 E1A
ORF was 266 amino acids in length with a molecular
weight of 29.4 K. Most of the functional sites of E1A pro-

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teins are located in conserved regions CR1 - CR4 [29];
homologue amino acid sequences at corresponding posi-
tions were found in HAdV-A31. The predicted E1A protein
of HAdV-A31 revealed a sequence (EQDENGMAH-
VSAAAAAAAANRER) at position 122 - 144 that is homo-
logue to the E1A protein of HAdV-A12. The latter was
previously identified as a repressor for the presentation of
MHC class I molecules on the surface of infected cells.
Essential for this function of HAdV-A12 is a stretch of 23
amino acids, which could not be found in the E1A pro-
teins of other HAdV species [30].
HAdV-A31 revealed the essential Rb-protein binding
motif as a LLCYE sequence at residues 107-111. A C-ter-
minal binding protein (CtBP) interacting motif (PVDLS),
which is probably equivalent to PLDLS in other HAdV
types, was found near the C-terminus of the E1A protein.
Two zinc finger motifs CSLC and CKSC, both essential for
transactivation of transcription [31] are located in the CR3
region.
dicted 18.3 K protein was 156 amino acids in length and
homologue to the small t-antigen, which is known to
have anti-apoptotic features [32]. While the stretch of the
first 145 amino acids of the HAdV-A31 E1B 18.3 K protein
shared a homology of 93.1% to the corresponding gene
product of HAdV-A12, the C-terminus is highly divergent
(only 27.7% identity).
The second E1B gene product was a predicted 53.1 K pro-
tein, 472 amino acids in length and homologue to the
large t-antigen. It defends the virus against the p53 medi-
ated antiviral host cell response by binding to the DNA
linked p53 protein directly and repressing its function as
a transcriptional activator. In addition to this direct inter-
action, a complex comprising the large t-antigen, the E4
ORF 6 gene product and a set of cellular cofactors build an
E3-ligase-complex that also degrades the p53 protein
[33,34]. Essential for the stability of this complex is a BC-
Box binding motif ((A,P,S,T)LxxxCxxx(A,I,L,V)), present
in the predicted E1B 53 K gene product of HAdV-A31 as
an ALRPDCTYKI motif at amino acid position 156-164.
Map of the genome organization and transcription units of HAdV-A31Figure 1
Map of the genome organization and transcription units of HAdV-A31. Early and late transcription units are repre-
sented in different colors, intermediate gene products in white. The block arrows represent the predicted protein, titled either
by protein name or predicted molecular size. Orientation of the arrows indicates the direction of transcription.Page 4 of 14
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E1B coding region
Two ORF in the E1B gene region coded for predicted pro-
teins of 18.3 K and 53.1 K molecular weight. The pre-
Amino acid comparison with the predicted large t-antigen
of HAdV-A12 revealed a high sequence divergence (only

BMC Genomics 2009, 10:557 http://www.biomedcentral.com/1471-2164/10/557
55.5% identity) between residues 38 - 100. However, the
C-terminus of large t-antigen, which was described as
essential for repressing the function of p53 in HAdV-A12
infected cells, showed a high sequence homology to
HAdV-A12.
E2 coding region
E2 is divided into two transcriptional units, E2A and E2B.
Transcription of the E2 region is controlled by a well char-
acterized RNA polymerase II promoter (major promoter)
on the complementary strand, which is transactivated by
E1A and E4 ORF6/7 gene products. The E2 promoter
region of HAdV-A31 was located between bp c25299-
c25431. It lacked at least one of the typical E2F (bp
c25422-25429, TTTCCCGC) binding motifs as described
for HAdV-C2 and -C5. However, a putative binding motif
for E4F1 (base pair c25430-c25436, ACGTCAC) was pre-
gene products [35]. Moreover, the E2 promoter of HAdV-
A31 had a binding motif for ATF (bp c25431 - c25439,
TGACGTCAC) and a TATA-like sequence for binding TBP
(bp c25381-c25386, TTAAGC). Within the E2A region an
ORF for a predicted protein of 55.3 K was identified. The
product was homologue to the DNA binding protein
(DBP) and revealed a zinc-binding domain at amino acid
position 229-242 (HxC8CxH).
The E2B region of HAdV-A31 encoded a predicted 134.6
K protein homologue to the HAdV DNA polymerase pro-
tein, and a 73.1 K gene product homologue to the precur-
sor of the terminal protein (pTP), both on the
complementary strand. The common nuclear localization
signal of pTP was present as RLPVRRRRRRLP motif at
amino acid position 351-362.
E3 coding region
The E3 region is known to code for a set of proteins that
are not essential for virus replication in vitro but are
important factors for interfering with the host immune
response [36,37]. Six ORFs were identified in the E3 tran-
scription unit of HAdV-A31, encoding putative gene prod-
ucts of the following sizes: 12.1 K, 28.6 K, 29.4 K, 10.5 K,
12.5 K, and 14.6 K. The organization is similar to the E3
region of the closely related HAdV-A12 [38].
The 12.1 K predicted protein was homologue to a 12.5 K
protein which is present in all HAdV types beside the
enteric HAdV-F40 and -F41. The sequence identity
between HAdV-A31 and -A12 was comparatively high
(96.1%).
The second and third reading frames, coding for a 28.6 K
(CR1 alpha) and a 29.4 K (CR1 beta) protein, revealed
comparatively low identities of 76.1% and 72.1% to
homologue E3 gene products of HAdV-A12. CR1 alpha
and beta were described as species HAdV-A specific gene
products [39]. Prediction of transmembrane domains
suggested that both gene products were type Ia transmem-
brane proteins. Protein Blast search of CR1 beta showed
homologies between the putative gene product of HAdV-
A31 and proteins of the SLAM (signalling lymphocyte
activation molecule) family. Patterns of predicted func-
tional domains of HAdV-A31 in comparison to HAdV-
A12 and -F40 are shown in Figure 4A.
The 10.5 K and 12.5 K ORF of HAdV-A31 were identified
to be homologue to the known RID (receptor internaliza-
tion and degradation) alpha and beta proteins, which are
present in the E3 transcription units of all HAdV. Both
proteins are non-covalently associated integral membrane
proteins [40]. The N-terminal phosphorylation sites pat-
Phylogenetic analysis of all available complete genomic HAdV sequences representing a l human adenovirus species (A to G), including th newly generated HAdV-A31 equ nceFig re 2
Phylogenetic analysis of all available complete
genomic HAdV sequences representing all human
adenovirus species (A to G), including the newly gen-
erated HAdV-A31 sequence. The tree was generated
with MEGA 3.1 using neighbor-joining method, bootstrap val-
ues (%) were generated with 1,000 pseudoreplicates. For
nucleotide accession numbers see Methods section.Page 5 of 14
(page number not for citation purposes)
dicted. E4F1 is another cellular transcription factor that
stimulates the transcription of E4 genes mediated by E1A
tern of HAdV-A31 RID-beta was more similar to HAdV-
C5, whereas HAdV-A12 lacked these phosphorylation

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sites completely (Figure 4B). YxxO motifs and proline rich
sequence stretches near the C-terminus, which are con-
served among all HAdV and may be part of a protein inter-
acting domain [39], were identified in the predicted RID
beta protein of HAdV-A31 (Figure 4B). YxxO motifs func-
tion as signals for transport and internalization into lyso-
RID alpha is a hydrophobic protein and appears in two
isoforms [39]. Depending on cleavage of the signal pep-
tide, it either functions as a type I or a type II transmem-
brane protein. An analogue cleavage pattern was predicted
for the RID alpha protein of HAdV-A31 by using the web
based TMHMM v. 2.0 software. As described for other
Global pairwise sequence alignment of the HAdV-A31 genome with representative types of each HAdV speciesFigure 3
Global pairwise sequence alignment of the HAdV-A31 genome with representative types of each HAdV spe-
cies. The x axis shows the genome position, the y axis shows the sequence conservation in percent. Arrows on top display the
transcription units and the direction of their transcription.Page 6 of 14
(page number not for citation purposes)
somes/endosomes. human adenoviruses dileucine, dileucine-like and YxxO
motifs are also present in the cytoplasmatic portion of the
HAdV-A31 RID alpha gene product.

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The last ORF of the E3 region encodes a 14.6 K protein
that is homologue with a sequence identity of 88.2% to
the 14.7 K protein of HAdV-A12. A corresponding protein
is present in all species of HAdV species [41]. It has been
shown to be located in the cytosol and nucleolus, func-
tioning as an inhibitor of TNF mediated cell lysis. Struc-
ture and function analysis of the 14.7 K of HAdV-C5
indicate that its biological function does not depend on
single conserved subdomains but that critical amino acids
are distributed throughout the entire protein. It has been
shown that a set of three cysteine residues between amino
acid 40 and amino acid 120 are essential for the function
of the protein [36,37]. Corresponding cysteine residues
were identified in the predicted 14.6 K protein of HAdV-
A31.
E4 coding region
Transcription of the E4 region on the complementary
strand is stimulated by the E1A gene product and the cel-
lular transcription factor E4F1. An E4F1 binding motif
Corresponding to the E4 transcription units of other
human adenoviruses (including the closely related HAdV-
A12), six ORFs were predicted within the genome of
HAdV-A31. These ORFs encoded for respective proteins of
13.9 K, 14.9 K, 13.4 K, 13.7 K, 33.9 K and 13.9 K (spliced
gene product ORF6/7). The predicted gene product of the
HAdV-A31 ORF6/7 exhibited only a single BC-Box like
motif (SAWPECNSLT) slightly different to the consensus
motif sequence (A,P,S,T)LxxxCxxx(A,I,L,V). This is in con-
trast to HAdV-C5, which shows two BC-Box motifs that
are both required for degradation of p53 [34].
Virus associated RNA
Sequence coding for the VA RNA was predicted by com-
parison with HAdV-A12 and is located at bp10146 -
10286 in the genome of HAdV-A31.
Intermediate genes
Two proteins are encoded in the adenoviral intermediate
gene region, IX and IVa2. The protein IX of HAdV-A31 was
predicted as a 14.9 K protein of 144 amino acids in length.
It is a structural component of the virus and influences
hexon-hexon interaction. A stretch of 32 amino acids is
conserved among all HAdVs and is supposed to be crucial
for the incorporation of the protein IX into the capsid.
Corresponding amino acids were identified in the protein
IX of HAdV-A31 at position 14-45. Surprisingly, we iden-
tified an RGD motif in the amino acid sequence of HAdV-
A31 protein IX at amino acid position 102-104 (Figure 5).
This RGD motif is only 40 amino acids distant from the
C-terminus that is assumed to be exposed on the surface
of the virus [42]. Moreover, the RGD motif is located at
the N- terminal end of a coiled coil region predicted by the
COILS web based software [43]. This prediction suggested
that the RGD motif is possibly solvent exposed and may
be functional in binding to alpha integrins.
The IVa2 ORF is transcribed from the complementary
strand and codes for a 50.9 K protein of 449 amino acids
in length. The protein IVa2 of human adenoviruses binds
the A-repeat sequences at the left end of the genome and
is involved in the process of viral DNA packaging and
virus assembly. Furthermore, it is assumed to have a func-
tion as a transcriptional activator of the late adenoviral
genes [44].
Late genes
The major late transcription unit (MLTU) encodes the
majority of the virus structural proteins and is organized
into five subregions L1-L5. The initiation of late gene tran-
scription is controlled by a promoter region that is present
in all human adenoviruses and termed as major late pro-
moter (MLP) [3]. Based on sequence comparison, the
Schematic view of the predicted E3 CR1 beta (A) and the E3 RID beta (B) pr teins of HAdV-A 1, -A12 and -F40 or -C5, respectivelyFigur 4
Schematic view of the predicted E3 CR1 beta (A) and
the E3 RID beta (B) proteins of HAdV-A31, -A12 and
-F40 or -C5, respectively. (A): a V-Set domain (red box)
was only predicted within the N-terminal region of the E3
CR1 beta protein of HAdV-A31. (B): N-terminal phosphor-
ylation sites (small red boxes) were predicted both for the
HAdV-A31 and -C5 RID beta proteins, but not for HAdV-
A12. Domain predictions were carried out using web based
Pfam, ProSite and BLASTp.Page 7 of 14
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ACGTCAC (of the consensus sequence ACGTMAC) was
identified in HAdV-A31 at bp c33513-c33519 located
upstream of the putative TATA box at bp c33479-c33502.
putative inverted CAAT box (TGATTGGTT) was identified
at bp 5633-5641 and the TATA box at position 5684-5690
in the genome of HAdV-A31. The L1 transcription unit

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encodes the 52/55 K and the pIIIa protein. An ORF coding
for a 41.8 K protein as a homologue of 52/55 K that is
known to be functionally relevant in the process of virion
assembly was predicted. A second ORF encodes the puta-
tive pIIIa protein of 64.4 K that is associated with the
hexon protein and present on the outer surface of the vir-
ion.
Four ORFs within the L2 region were identified: coding
for the penton base protein (III), proteins V, pVII and the
pX. The predicted penton base protein of HAdV-A31 is
505 amino acids in length and exhibits an integrin (αvβ3
and αvβ5) binding RGD motif at amino acid position 301-
303. In addition to the RGD sequence, an LDV motif was
present at amino acid position 285-287 of HadV-A31.
LDV motifs were described as interacting with another
group of integrins (α4β1 and α4β7) [45-47].
The predicted homologue of protein V has a molecular
weight of 39.8 K and is 347 amino acids in length. A 20.4
K gene product is homologue to the pVII, and a predicted
product of 7.9 K represents the pX protein of HAdV-A31.
All three proteins are described as being core proteins and
are associated with the virus DNA [48]. The sequence gen-
erated for pX protein of HAdV-A31 was highly divergent
from the HAdV-A31 pX sequence previously available in
GenBank [GenBank: U14653] (nucleic acid sequence
homology 70.4%, amino acid 53.4%). Therefore, this
region was re-sequenced a second time and additionally
result was confirmed. Most likely, the databank sequence
relates to a subtype of HAdV-A31 or is mislabelled.
The L3 transcription unit encodes three proteins; we
could identify ORF for pVI, the hexon protein (II) and the
virus encoded protease. The predicted pVI protein of
HAdV-A31 is 260 amino acids in length and has a molec-
ular weight of 28.5 K. It revealed two nuclear localization
signals (KRPRP at amino acid 136-140 and KRRR at
amino acid 255-258) close to its C-terminus. Correspond-
ing motifs of HAdV-C2 play an important role in directing
cytoplasmatic proteins to the nucleolus and thus might be
functionally active as nuclear localization signals [49].
The mature pVI protein is a minor capsid component.
The predicted hexon protein of HAdV-A31 has a molecu-
lar weight of 103.7 K and a length of 922 amino acids.
Due to its serotype defining main neutralization determi-
nant ε, it was highly divergent from the closely related
HAdV-A12.
The predicted ORFs of the L4 region of HAdV-A31 encode
putative proteins of 86.8 K (as equivalent to100 K), 20.2
K (as equivalent to 22 K), 23 K (as equivalent to 33 K),
25.2 K (as equivalent to pVIII), respectively.
Only one ORF is present in the L5 region of HAdV-A31,
encoding a 58.9 K protein of 556 amino acids in length,
representing the fiber protein (IV), a major structural pro-
Multiple alignment of the protein IX amino acid sequences of HAdV-A31, -A12, -C5, -F40 and -G52Figure 5
Multiple alignment of the protein IX amino acid sequences of HAdV-A31, -A12, -C5, -F40 and -G52. The RGD
motif found in the protein IX of HAdV-A31 is highlighted.Page 8 of 14
(page number not for citation purposes)
sequenced for all seven clinical isolates; our previous tein with the highly variable hemagglutination determi-
nant in its terminal knob structure. The fiber shaft was

BMC Genomics 2009, 10:557 http://www.biomedcentral.com/1471-2164/10/557
found to be 31 amino acids (two pseudorepeats) shorter
than the fiber shaft of HAdV-A12. We detected a deletion
in the 3rd non-consensus β-repeat of HAdV-A31 fiber shaft
and a KLGXGHXFS motif in the penultimate repeat
instead of the classical KLGXGLXFD/N flexibility consen-
sus motif of other adenovirus serotypes. This could affect
the function of the flexibility regions and influence the
cell attachment.
Comparison of clinical isolates to the prototype sequence
The coding regions of the E1A, E3, E4, penton base, fiber
knob, protein IX and pX genes of seven clinical isolates
were sequenced and compared. All strains have been iso-
lated from paediatric patients with disseminated infec-
tions following hSCT. Overall, the pX, IX, E3 RID alpha,
E4 ORF 3 and ORF 4 nucleic acid sequences of all isolates
were 100% identical to the prototype. The lowest amino
acid identity was observed in the E3 CR1 beta gene with
99.2%. In Table 2, gene product and amino acid substitu-
tions of clinical strains are listed. Lowest S/N ratios were
calculated for the E3 CR1 beta (2.36), whereas only syn-
onymous mutations were observed in the penton base
protein of several clinical isolates. Two isolates revealed
an amino acid substitution in the penton protein at posi-
tion 305 (tyrosine residue instead of phenylalanine),
which is close to the functional RGD motif and may influ-
ence integrin binding. Interestingly, all wild type strains
revealed the additional RGD motif in protein IX of the
HAdV-A31 prototype strain. All motifs described for the
HAdV-A31 prototype strain were conserved in the clinical
isolates.
Discussion
We determined the complete 33,763 base pair genome of
the HAdV-A31 prototype strain and identified 34 putative
genes. HAdV-A31 is a highly significant pathogen, which
has been frequently isolated from severely affected hSCT
recipients, and frequently presents itself as a disseminated
disease, which is usually caused by species HAdV-C [10].
Experimental data strongly suggested that species HAdV-C
types have the ability to establish latent infections in
mucosal lymphocytes and that stimulation of those cells
can cause viral reactivation in cases of immunosuppres-
sion [21]. Especially the early gene products (e.g. E3),
which counteract host anti-viral defence mechanisms,
might play a key role in the process of persistence and
reactivation [39,50,51]. A similar mechanism of persist-
ence and reactivation can be suspected in case of HAdV-
A31, which would explain its high incidence in immuno-
suppressed patients. Therefore, the early coding regions
E1A, E1B and E3 of the newly generated HAdV-A31 proto-
type sequence were analyzed in detail for functional
motifs. Moreover, these genome regions of seven HAdV-
A31 wild type strains isolated from immunosuppressed
patients were also sequenced in order to clarify whether a
highly pathogenic subtype of HAdV-A31 was circulating
in recent years. However, analysis of nucleic acid and pre-
dicted amino acid sequences of seven HAdV-A31 clinical
Table 2: Sequence comparison of clinical isolates to the HAdV-A31 prototype sequence
Clinical
isolate
V04-03789 0105019310 2006001610 95/8866 95/6956 96/783 95/6315
Origin Regensburg,
Germany
Hannover,
Germany
Hannover,
Germany
Nancy, France Nancy, France Nancy, France Nancy, France
Year 2004 2001 2006 1995 1995 1996 1995
E1A S56C, V222N
(5)
E3 12.1 E34Q (1) A5T (3)
E3 CR1 α Q71E, N159S
(9)
E3 CR1 β Q164E, T242I
(2)
T242I (7) Q164E, T242I
(3)
D130N, Q164E
(2)
Q164E (3) Q164E, T242I
(4)
Q164E, T242I
(2)
E3 RID β M13V, E107G
(2)
E107G (1) E107G (1) E107G (1) E107G (1) E107G (1)
E3 14.7 T106N (3) T74A, T106N
(4)
T106N (4) T106N (5) T106N (5) T106N (3)
PB F259Y (7) F259Y(8)
FK V40L, R62Q (4) R62Q (4) R62Q (3) R62Q (3) R62Q (3) V40L, T53A,
R62Q (5)
E4 ORF1 P119T (1) V59L (2)
E4 ORF2 L84P (1)
E4 ORF5 R51K (6)
E4 ORF6/7 K105N, I115S
(3)Page 9 of 14
(page number not for citation purposes)
The coding regions which showed amino acid substitutions in at least one isolate are listed. For each isolate, the sequence positions of amino acid
substitution are noted. Additionally, the number of mutations in the nucleotide sequence is indicated in brackets.

BMC Genomics 2009, 10:557 http://www.biomedcentral.com/1471-2164/10/557
isolates revealed high identity to the prototype strain
(Table 2). Non-synonymous mutations in clinical isolates
clustered in the E3 region, but did not affect previously
described and predicted functional sites and motifs (Fig-
ure 4) [37,39]. An S/N ratio of 2.36 within the species
HAdV-A specific CR1 beta protein suggested selection of a
potentially highly functional E3 protein, which is
assumed to interact with the immunosurveillance of ade-
novirus infected cells [39]. Unfortunately, experimental
data about the functions of the E3 gene products CR1
alpha and beta were not available. Therefore, in silico pro-
tein analysis of the predicted E3 CR1 alpha and beta pro-
teins of HAdV-A31 were performed. Interestingly, the E3
CR1 beta protein of the HAdV-A31 prototype and all ana-
lyzed clinical isolates were predicted to exhibit an immu-
noglobulin-like (V-set) domain, which was predicted
neither for the closely related HAdV-A12 nor for the cor-
responding E3 gene products of the related enteric species
F adenoviruses. Immunoglobulin-like domains are
described to be involved in cell-cell interaction of the
immune system [52]. A similar domain was previously
described as involved in a novel feature of the soluble 49
K E3 gene product of HAdV-D19a adenoviruses [37].
Functional studies of the 49 K protein of HAdV-D19a
demonstrated proteolytic processing and secretion of the
type Ia transmembrane protein [37]. Furthermore, a NK
cell binding activity was detected and the immunoglobu-
lin-like domain of the HAdV-D19a 49 K protein was
assumed to interact directly with NK cells, protecting
infected cells against lyses [37]. For comparison, in silico
protein analysis of HAdV-A31 CR1 beta also predicted a
type Ia transmembrane domain, a (signalpeptide-)cleav-
age probability of ~83%, a C-terminal sorting motif and
various glycosylation sites, all of which are analogous to
the confirmed predictions for HAdV-D19a E3 49 K pro-
tein.
In contrast to these predictions for CR1 beta, potential
functionality of the predicted CR1 alpha protein of HAdV-
A31 have remained obscure, since all performed analyses
and predictions did not reveal similarities with functional
sites or motifs of any E3 counterparts of other HAdV spe-
cies. Overall, CR1 alpha and beta amino acid sequence
comparison between HAdV-A31 and -A12 showed a par-
ticularly low identity of only 76.1% and 72.1%, respec-
tively. This is in considerable contrast to corresponding E3
gene products of HAdV-F40 and -41, which had a high
intraspecies homology of 98.8% and 99.2%, respectively.
Significant differences between both species A adenovi-
ruses were also identified in the theoretical molecular
weight and isoelectric point (pI) for CR1 alpha and beta
gene products computed and visualized in virtual 2D gel
analysis (data not shown). These considerable differences
quently indicated differences in protein function and
might be an important feature in explaining the described
higher virulence of HAdV-A31.
Comparison of the other E3 gene products, 12.5 K, RID
alpha, RID beta and 14.7 K, revealed sequence identities
between 80 and 96% with HAdV-A12. With the exception
of predicted phosphorylation sites of RID beta (Figure
4B), previously described functional sites and motifs are
conserved in HAdV-A31 and HAdV-A12, suggesting a
comparable functionality. As determined for HAdV-A12
and species F adenoviruses, the E3 transcription unit of
HAdV-A31 also lacked the extensively studied GP 19 K
protein, which down regulates the expression of MHC I
molecules and NK activation receptors [53-55]. As a sub-
stitute for this important immune escape mechanism, the
E1A gene product of HAdV-A12 was identified to down
regulate the expression of MHC class I molecules by inter-
fering with the transcription of MHC I gene products [56].
This unique feature can be assumed for the E1A protein of
HAdV-A31 as well, because amino acid stretches of HAdV-
A12, which have been identified as essential for this
mechanism, were identified in the predicted E1A gene
product of HAdV-A31 at corresponding positions. While
the E1A protein is known to have pro-apoptotic features,
the E1B 19 K gene product of HAdV-C5 shares homology
with the cellular anti-apoptotic Bcl-2 protein and inter-
feres with a set of different cellular pro-apoptotic proteins
(Bak, Bax and Nbk/Bik), thus protecting infected cells
against apoptosis [32]. Comparison of the E1B 19 K small
t- antigen homologue of HAdV-A31 with HAdV-A12
revealed high divergences of the C-terminus. Since the
E1B 19 K C-terminus of HAdV-C5 has been identified as
exhibiting a functional domain that influences the lateral
viral spread of HAdV-C5 by interfering with cellular apop-
totic pathways [57], the observed divergence between
HAdV-A31 and -A12 might have functional relevance.
In addition to the capability of persistence and of reactiva-
tion by interfering with the host immune response, the
capacity for dissemination seems to be essential for a
highly pathogenic HAdV subtype. For example, an out-
break of the strictly enterotropic HAdV-F41 did not cause
any fatalities in paediatric hSCT recipients [17], which
might be due to the missing integrin binding RGD motif
within the penton protein of species F adenoviruses. Since
fatal dissemination of adenoviruses affects various
organic systems, a more promiscuous behaviour in cell
attachment and entry can be assumed to be favourable
[58]. Interestingly, the protein IX of HAdV-A31 revealed
an RGD motif at amino acid position 102 - 106, which is
conserved within all seven analyzed clinical isolates. This
RGD motif is only 40 residues distant from the C - termi-Page 10 of 14
(page number not for citation purposes)
between HAdV-A31 and -A12 in the primary structure,
protein size, pI and predicted functional domains conse-
nus, and it is unique among all sequenced human adeno-
viruses. As described previously, the C-terminus of HAdV

BMC Genomics 2009, 10:557 http://www.biomedcentral.com/1471-2164/10/557
protein IX is exposed on the outer surface of the virion
[42,59]. It was shown for protein IX of BAdV-3, which is
125 amino acids in length that the N-terminus (13 - 32)
and the central region (61 - 80) have immunogenic sites
but are not exposed on the outer surface of the virion [60].
So far, no structural data about the exposed region of the
C-terminus of protein IX of HAdV-A31 is available, but
the observations for BAdV-3 indicate that the amino acid
stretch of the protein IX of HAdV-A31 that contains the
RGD motif might be present on the outer surface of the
virion. As the HAdV-A31 fiber shaft is shorter and proba-
bly less flexible than the fiber shaft of HAdV-A12, a func-
tional binding of the secondary cellular receptor to the
RGD motif of protein IX instead of the RGD motif in the
penton base may be possible or even preferred. Moreover,
the construction of adenoviral vectors with an incorpo-
rated RGD motif within the C - terminus of protein IX has
demonstrated that the additional RGD motif resulted in a
significant augmentation of fiber independent infection
of CAR-deficient cell types [60]. Therefore, the additional
RGD motif within the protein IX HAdV-A31 might lead to
a more effective targeting and internalization, and could
be a factor in increased transmission and infectivity of the
virus.
In addition to protein IX, sequence comparison of the cel-
lular receptor binding sites of the penton protein of the
clinical isolates with the HAdV-A31 prototype revealed an
amino acid substitution (F305Y) in two clinical isolates.
This substitution is close to the functional RGD motif and
may influence integrin binding. Moreover, a low S/N ratio
of 2.78 (with exception of clinical isolate number: 95/
8866) for the fiber knob indicated selective pressure on a
major structural protein of the clinical isolates.
Overall, the sequence divergence of the isolated clinical
strains in comparison to the prototype sequence was
determined to range between 99.2 and 100%. These
results suggested that all isolated strains were closely
related to the prototype; a single outbreak subtype associ-
ated with severe disease in stem cell transplant recipients
was not identified (Table 2). This is in congruence with
previous results of RFLP analysis of 79 HAdV-A31 wild
type isolates from immunocompromised and immuno-
competent hosts, where a wide variety of slightly geneti-
cally different subtypes of HAdV-A31 was described [22].
Conclusion
Overall, studying the HAdV-A31 prototype seemed to be
sufficient to elucidate the high incidence and disease bur-
den in immunosuppressed patients because HAdV-A31
strains recently circulating in immunocompromised
patients were closely related to the prototype. Unique
additional RGD motif of protein IX may promote promis-
cuous tropism for various tissues and enhance dissemina-
tion. Further studies of its biological relevance in vivo/in
vitro are necessary to clarify its potentially unique charac-
teristics.
Methods
Sequencing strategy
Previously published partial nucleotide sequences of
HAdV-A31 and the genomic HAdV-A12 sequence were
used to design the PCR primer for the production of
genome fragments of the HAdV-A31 up to 5000 base pairs
in length. Depending on the size of the amplicon, these
fragments were either cloned and sequenced subsequently
or sequenced directly from the amplicon. Both strands
were sequenced by primer walking with overlapping
sequencing reactions. Furthermore, we resequenced the
HAdV-A31 sequences already available in GenBank (with
exception of the hexon gene [61]). On the basis of the
newly generated complete nucleotide sequence of HAdV-
A31, we designed the PCR and sequencing primers for
sequencing the E1A, E3, E4, fiber knob and penton base
coding regions of seven clinical HAdV-A31 isolates.
HAdV prototype strain, wild type strains and cells
The HAdV-A31 (ATTC) prototype strain was obtained
from the American Type Culture Collection (ATCC). Wild
type strains of HAdV-A31 had been isolated between 1995
- 2004 from cases of severe HAdV disease in France and
Germany (Table 2) [14].
All viruses were propagated on the human lung cancer cell
line A549 (ATCC, CCL-185) on 75-cm2 or 25 cm2 culture
flasks using DMEM medium with 5% of FBS (Biowest,
Nuaillé, France) and 1% Penicillin/Streptomycin
(Cytogen, Sinn, Germany) added. When the cytopatho-
genic effect was above 50%, cells were washed with PBS-
(Cytogen) and lysed using Trypsin/EDTA (Cytogen). The
DNA was extracted with the Qiagen blood kit (Qiagen,
Hilden, Germany).
PCR amplification and cloning of DNA fragments
For the amplification of fragments up to 500 bp we used
the HotStar Mix (Qiagen, Hilden, Germany) in a total vol-
ume of 50 μl with 1 μM of each primer and 5 μl of the
purified genomic HAdV-A31 DNA. The PCR program
began with the activation of the "hot start" DNA polymer-
ase for 15 min at 95°C, followed by 40 cycles consisting
of denaturation at 94°C for 20 s, a primer annealing tem-
perature between 52 - 56°C depending on the composi-
tion of the used pair of primers for 20 s and elongation at
72°C for 40 s, followed by a final extension step of 72°C
for 5 min. All fragments of larger size were amplified usingPage 11 of 14
(page number not for citation purposes)
motifs of the HAdV-A31 E1 and E3 regions may provide
immune modulation and perhaps virus persistence. The
the Expand High Fidelity PCR System from Roche Applied
Science (Mannheim, Germany) which achieves a higher

BMC Genomics 2009, 10:557 http://www.biomedcentral.com/1471-2164/10/557
transcriptional fidelity by a proofreading activity. PCR
reactions were performed in a total volume of 50 μl with
1 μM of each primer and 5 μl of the purified genomic
DNA. The program starts with an initial denaturation step
at a temperature of 94°C for 2 min, followed by 10 cycles
consisting of denaturation at 94°C for 15 s, annealing,
depending on the used primer pair, at temperatures
between 52 - 56°C for 30 s and an elongation at 68°C for
1 - 4 min depending on the fragment length. These cycles
were followed by 20 cycles with the same conditions but
with 5 s extension of the elongation phase for each succes-
sive cycle. All PCR reactions were performed in a T-per-
sonal 48 thermocycler (Biometra, Goettingen, Germany).
Gel electrophoresis
PCR products were separated in a 1% agarose gel for 60
min at 120 V. DNA extraction from the agarose gels was
performed with the Qiagen gel extraction kit according to
the manufacturer's recommendations.
Cloning of PCR products
Depending on their length, extracted DNA fragments were
either cloned before sequencing or they were sequenced
directly. We performed AT-cloning reactions using the
pGEM - T Easy Vector System I (Promega Corporation,
Madison, WI, USA) and transfected replication competent
E.coli bacteria. Plasmid DNA was extracted using the Qia-
gen mini prep kit according to the manufacturer's recom-
mendations.
Sequencing
Cycle sequencing of both DNA strands was performed
with rhodamine-labeled dideoxynucleotide chain termi-
nator (DNA sequencing kit; ABI, Warrington, England)
and analyzed on an ABI Prism 310 automatic sequencer
(Applied Biosystems, Foster City, CA, USA). PCR primers
were used for the sequencing reactions.
DNA and protein analysis
Sequence assembly was carried out with the program Seq-
Man 5.00 from the DNASTAR software package. DNA and
protein homology searches were performed using the
NCBI BLAST program. DNA and protein sequence align-
ment were carried out using the ClustalW algorithm
implemented in the BioEdit package (version 7.0.4.1).
Genome annotation, analysis of non coding DNA motifs
and functional protein motifs were performed by using
the web based gene prediction software GENEMARK
http://exon.biology.gatech.edu, the DNASIS MAX
2.00.002.002 software and by sequence comparison.
Phylogenetic analysis was performed with the MEGA soft-
ware package (version 3.1). The phylogenetic trees were
graphical alignments were performed with LAGAN 2.0
software http://lagan.stanford.edu/lagan_web/
index.shtml[24].
In silico protein analysis and motif prediction were per-
formed with the web based Pfam http://
pfam.sanger.ac.uk[62] and proSITE http://
www.expasy.ch/prosite software [63]. Transmembrane
domains were predicted with the TMHMM software http:/
/www.cbs.dtu.dk/services/TMHMM[64]. Virtual 2D Gel
analysis was carried out using the JVirGel Version 2.0 soft-
ware http://www.jvirgel.de[65,66]. Prediction of coiled
coil regions was performed using the COILS software
http://www.ch.embnet.org/software/
COILS_form.html[43].
Nucleotide sequence accession numbers
HAdV-1 [GenBank: AF534906], HAdV-2 [GenBank:
NC_001405], HAdV-3 [GenBank: DQ086466], HAdV-4
[GenBank: AY599837], HAdV-5 [GenBank: AC_000008],
HAdV-7 [GenBank: AC_000018], HAdV-8 [GenBank:
AB448768], HAdV-9 [GenBank: AJ854486], HAdV-11
[GenBank: AC_000015], HAdV-12 [GenBank:
AC_000005], HAdV-16 [GenBank: AY601636], HAdV-17
[GenBank: AC_000006], HAdV-19 [GenBank:
AB448771], HAdV-21 [GenBank: AY601633], HAdV-22
[GenBank: FJ404771], HAdV-26 [GenBank: EF153474],
HAdV-34 [GenBank: AY737797], HAdV-35 [GenBank:
AY128640], HAdV-37 [GenBank: DQ900900], HAdV-40
[GenBank: L19443], HAdV-41 [GenBank: DQ315364],
HAdV-46 [GenBank: AY875648], HAdV-48 [GenBank:
EF153473], HAdV-49 [GenBank: DQ393829], HAdV-50
[GenBank: AY737798], HAdV-52 [GenBank: DQ923122].
The previously sequenced HAdV-31 hexon protein's
accession number is GenBank: DQ149611, pX is Gen-
Bank: U14653.
Authors' contributions
SH carried out the laboratory work, molecular genetic
studies, genome annotation, bioinformatic analysis and
drafted the manuscript. IM participated in the design of
the study and performed the phylogenetic analysis. SD
participated in sequencing and analysis of the fiber gene
regions. FR participated in sequencing and bioinformatic
analysis. AH conceived the study, and participated in its
design and coordination and helped to draft the manu-
script. All authors read and approved the final manu-
script.
Acknowledgements
We would like to thank Anneliese Plentz (Institut für Medizinische Mikro-
biologie, Universität Regensburg) and Veronique Venard (Laboratoire de
Bactériologie-Virologie, Faculté de Médecine) for providing us with clinical Page 12 of 14
(page number not for citation purposes)
constructed with the neighbor-joining method. Bootstrap
analysis was performed with 1,000 pseudoreplicates. The isolates of HAdV-A31, Heidi Deppe and Gabi Harste for excellent technical
assistance, and Hannah Elmer for critical reading of the manuscript.

BMC Genomics 2009, 10:557 http://www.biomedcentral.com/1471-2164/10/557
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