An outbreak of epidemic keratoconjunctivitis caused by a new intermediate adenovirus 22/H8 identified by molecular typing.
ABSTRACT In a 4-week period, 12 patients contracted adenoviral keratoconjunctivitis. Eight of these patients had visited the same ophthalmologist's practice before onset of symptoms. Adenovirus was detected in swab specimens obtained from 9 patients. Sequence-based typing of 2 isolates revealed type 22/H8. This is, to our knowledge, the first report of a keratoconjunctivitis outbreak caused by an intermediate adenovirus type 22/H8.
- SourceAvailable from: Ijad Madisch[show abstract] [hide abstract]
ABSTRACT: Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization epsilon determinant (loops 1 and 2) and the hemagglutination gamma determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. epsilon and gamma determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of epsilon determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob (gamma determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 (epsilon determinant) and a speculative, not-yet-isolated HAdV prototype (gamma determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the epsilon determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, gamma determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution.Journal of Virology 12/2005; 79(24):15265-76. · 5.08 Impact Factor
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ABSTRACT: Precise typing of human adenoviruses (HAdV) is fundamental for epidemiology and the detection of infection chains. As only few of the 51 adenovirus types are associated with life- threatening disseminated diseases in immunodeficient patients, detection of one of these types may have prognostic value and lead to immediate therapeutic intervention. A recently published molecular typing scheme consisting of two steps (sequencing of a generic PCR product closely adjacent to loop 1 of the main neutralization determinant epsilon, and for species HAdV-B, -C, and -D the sequencing of loop 2 [Madisch et al., 2005]) was applied to 119 clinical samples. HAdV DNA was typed unequivocally even in cases of culture negative samples, for example in immunodeficient patients before HAdV causes high virus loads and disseminated disease. Direct typing results demonstrated the predominance of HAdV-1, -2, -5, and -31 in immunodeficient patients suggesting the significance of the persistence of these viruses for the pathogenesis of disseminated disease. In contrast, HAdV-3 predominated in immunocompetent patients and cocirculation of four subtypes was demonstrated. Typing of samples from a conjunctivitis outbreak in multiple military barracks demonstrated various HAdV types (2, 4, 8, 19) and not the suspected unique adenovirus etiology. This suggests that our molecular typing scheme will be also useful for epidemiological investigations. In conclusion, our two-step molecular typing system will permit the precise and rapid typing of clinical HAdV isolates and even of HAdV DNA in clinical samples without the need of time-consuming virus isolation prior to typing.Journal of Medical Virology 10/2006; 78(9):1210-7. · 2.37 Impact Factor
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ABSTRACT: Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was <or=15 copies/run and the linear range of quantitation 1.5 x 10(1) to 1.5 x 10(8) copies/run. TaqMan PCR gave identical results compared to an established conventional one-step PCR protocol in 218 (38 positive and 180 negative) of 234 clinical samples including blood, serum, eye swabs, and feces, and had divergent results in 16 samples (15 positive only in TaqMan PCR, all with low copy numbers, and one positive only in conventional PCR), indicating a higher sensitivity of TaqMan PCR. Adenovirus viremia was detected by TaqMan PCR in 4 of 27 (14.8%) paediatric and 8 of 93 (8.6%) adult stem cell transplant recipients but only in 5 of 306 healthy controls (blood donors, 1.6%). Virus loads of pediatric patients (median 1.7 x 10(5)) were significantly higher than in adult patients (median 2.3 x 10(3)) and than in controls (all samples <or=1.7 x 10(3) copies/ml). A few immunosuppressed children had very high virus loads (up to 1.1 x 10(10) copies/ml), which were associated with symptoms of disseminated disease. In conclusion, real-time PCR is a sensitive and quantitative procedure for the detection of adenovirus infections.Journal of Medical Virology 06/2003; 70(2):228-39. · 2.37 Impact Factor
BRIEF REPORT • CID 2006:43 (1 October) • 000
B R I E F R E P O R T
An Outbreak of Epidemic
Keratoconjunctivitis Caused by a New
Intermediate Adenovirus 22/H8
Identified by Molecular Typing
Ilka Engelmann, Ijad Madisch, Heidi Pommer, and Albert Heim
Institut fu ¨r Virologie, Medizinische Hochschule Hannover, Hannover, Germany
In a 4-week period, 12 patients contracted adenoviral kera-
toconjunctivitis. Eight of these patients had visited the same
ophthalmologist’s practice before onset of symptoms. Ade-
novirus was detected in swab specimens obtained from 9
patients. Sequence-based typing of 2 isolates revealed type
22/H8. This is, to our knowledge, the first report of a ker-
atoconjunctivitis outbreak caused by an intermediate ade-
novirus type 22/H8.
Adenoviridae are nonenveloped, double-stranded DNA viruses
with an icosahedral capsid. Human adenoviruses (HAdVs) are
classified into 6 species (HAdV-A to HAdV-F) that consist of
51 HAdV types. Ophthalmologic manifestations of adenovirus
infection include severe epidemic keratoconjunctivitis (EKC),
which is almost exclusively caused by serotypes HAdV-D8,
HAdV-D19, and HAdV-D37. Less severe pharyngoconjunctival
fever is mainly caused by serotypes HAdV-B3, HAdV-B7, and
HAdV-E4, and acute hemorrhagic conjunctivitis is mainly
caused by serotype B11 .
Classical typing of adenoviruses is performed by neutrali-
zation tests using type-specific antiserum. The ? determinant
in the hexon is responsible for the neutralization properties.
Furthermore, typing can be performed by hemagglutination
inhibition tests that reflect the properties of the hemagglutinin
(g determinant) located at the fiber knob . More-rapid and
more-precise molecular typing methods by sequencing have
recently become available by systematic analysis of molecular
phylogeny of the ? determinant .
In general, recombination events between different adeno-
Received 20 February 2006; accepted 8 June 2006; electronically published 24 August
Reprints or correspondence: Dr. Albert Heim, Institut fu ¨r Virologie, MedizinischeHochschule
Hannover, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany (Heim.Albert@mh-hannover.de).
Clinical Infectious Diseases2006;43:000–000
? 2006 by the Infectious Diseases Society of America. All rights reserved.
virus types result in intermediate types with contradictory typ-
ing results in the neutralization and hemagglutination inhibi-
tion test. Intermediate types can also be identified by extending
sequence analysis to the fiber knob (g determinant). Recom-
bination events may also result in new pathogenic properties
and contribute to the generation of new adenovirus types—
and even to the generation of new species exhibiting new pat-
terns of organo-tropism and virulence .
HAdV-D22 was first isolated from a patient with trachoma in
agent of EKC. Furthermore, we never found HAdV-D22 in a
systematic analysis of the circulation of human adenoviruses in
Germany during the past 2 years, and the analysis included 119
we cannot exclude the possibility that HAdV-D22 circulates in
Germany and causes only mild symptoms and, therefore, is not
being diagnosed. We report the first outbreak of EKC caused by
HAdV-D22. Genetic analysis demonstrated an unusual recom-
bination variant of HAdV-D22 with HAdV-D8 and HAdV-D37
that obviously resulted in high virulence.
Clinical and epidemiological information con-
cerning the patients involved in the outbreak of EKC was col-
lected retrospectively from the treating ophthalmologist and
from the local health authorities. Adenovirus from 2 different
patients was cultivated on A549 cells (isolates Hannover-2005-
IAI-1 and Hannover-2005-IAI-2).
Diagnosis of adenoviral keratoconjunctivitis was confirmed
by quantitative real-time PCR of conjunctival swab specimens
. HAdV DNA was amplified from clinical samples by a ge-
neric PCR protocol . For typing purposes, the adenovirus
hexon (? determinant) loop-2 (L2) region from 2 outbreak
patients was amplified and sequenced as described elsewhere
. After virus cultivation, the fiber knob region and thehexon
loop-1 region was amplified as described elsewhere [2, 7]. Ad-
ditionally, penton base sequencing was performed on the basis
of a newly developed PCR protocol (authors’ unpublished
data). PCR products were separated in a 2% agarose gel (1%
in the case of fiber amplicons) for 60 min at 120 V. DNA
extraction from the agarose gels was performed withtheQiagen
gel extraction kit (Qiagen), in accordance with the manufac-
Both strands of PCR amplicons were cycle sequenced with
rhodamine-labeled dideoxynucleotide chain terminators(DNA
sequencing kit; ABI) and analyzed on an ABI Prism 310 au-
tomatic sequencer (Applied Biosystems). PCR primers were
used for the sequencing reactions.
000 • CID 2006:43 (1 October) • BRIEF REPORT
loop 2 (A) and the fiber knob region (B) generated by the NeighborJoining
method, Kimura 2-parameter matrix (Molecular Evolutionary Genetics
Analysis software package, version 3.1 [Molecular Evolutionary Genetics
Phylogenetic analysis of nucleic acid sequences of thehexon
Two-step molecular typing was performed as described else-
where . A sequence diversity of !2.5% in the L2 region, com-
pared with the next homologous prototype, was used as molec-
ular typing criterion . L2 sequences were compared with
prototype sequences in GenBank by the Blast internet server.
Phylogenetic analysis was performed by using the Molecular
Evolutionary Genetics Analysis (MEGA) software package, ver-
sion 3.1 (Kumar, Tamura, Nei 2004). The phylogenetic trees
were constructed with the neighbor-joining method (Kimura-2
parameter matrix), with a transition/transversion ratio of 2.0.
The following GenBank sequences were used to generate
alignments of L2: HAdV-D8 (AB023546), HAdV-D9(AF161562),
HAdV-D10 (AB023548), HAdV-D17 (AF108105), HAdV-D19
(AF161565), HAdV-D22 (AJ745883), HAdV-D37 (AJ745892),
and fiber knob region: HAdV-D8 (AB162771), HAdV-D9
(X74659), HAdV-D10 (AJ811442), HAdV-D17 (Y14241),
HAdV-D19 (U69131), HAdV-D22 (AJ811445), and HAdV-
The index patient received a diagnosis of kerato-
conjunctivitis at an ophthalmologic practice. Subsequently,
within a period of 4 weeks, 11 other patients developed ker-
atokonjunctivitis. Nine of these patients had been to the same
ophthalmologic practice after the index patient for different
medical reasons, and 2 were household contacts. Patients were
33–73 years old. Clinically, all patients presented with lid swell-
ing, pain, photosenstitivity, and corneal subepithelialinfiltrates.
Two of the patients also presented with swelling of cervical
lymph nodes, fever, and common cold symptoms evocative of
Conjunctival swab specimens were obtained from 9 patients,
and adenovirus was identified in all of them by quantitative
real-time PCR . Of the 3 other patients, 2 lived in the same
household as the virologically diagnosed patients, and no swab
specimens were obtained, because diagnosis appeared to be
clinically and epidemiologically evident. Thesourceofinfection
for the index patient was not found. However, transmission in
the ophthalmologic practice was obvious in the 8 patients pre-
senting after the index patient, all of whom had been to the
same ophthalmologic practice before onset of their symptoms.
Cases in the other 2 patients are suspected to have been caused
by intrafamilial transmission.
Viruses recovered from 2 patients (isolates Hannover-2005-
IAI-1 and Hannover-2005-IAI-2) were analyzed by a 2-step
molecular typing system . Identification of species HAdV-D
was achieved by sequencing of the ampliconsof agenerichexon
PCR  and identification of HAdV-D22 by sequencing of the
amplicons of the highly variable L2 of the neutralization de-
terminant ?. Both L2 sequences (accession numbers DQ404182
and DQ404185) showed 100% identity to each other and to
the HAdV-D22 prototype sequences inthedatabase(figure1A).
Additional sequencing of the loop-1 loop of the neutralization
determinant ? (accession numberDQ404181)confirmedL2typ-
ing results. Results of neutralizations tests using a rabbit serum
specific for HAdV-D22 confirmed the molecular typing results.
To identify possible recombination events resulting in inter-
mediate strains, the fiber knob region (g determinant) of 2 pa-
tient isolates was also amplified and sequenced. The sequences
of the fiber knob (DQ404183 and DQ404184) region showed
100% identity to HAdV-D8 sequences in the data bank
(AB162771) (figure 1B) and displayed significant sequence di-
vergence, compared with all other human adenovirus types.
Therefore, the causativeagentoftheEKCoutbreakwasidentified
as a newly described intermediate adenovirus, type 22/H8.
Cell and organo tropism is determined by the fiber knob (g
determinant), as well as by the penton protein (RGD motif
[arginine, glycine, and aspartic acid]); therefore, we extended
sequence analysis to the penton gene. Penton base sequences
of the 2 isolates (DQ404187 and DQ404186) were identical to
penton base sequences of the EKC-associated HAdV-D37. This
result indicates a second recombination event in the phylo-
genetic origin of the strains Hannover-2005-IAI-1 and Han-
EKC is typically reported to be caused by ad-
enovirus types HAdV-D8, HAdV-D9, and HAdV-D37, which
frequently cause nosocomial outbreaks . Other adenovirus
types have rarely been associated with EKC. This is the first
description of an EKC outbreak caused by adenovirus type 22.
BRIEF REPORT • CID 2006:43 (1 October) • 000
In contrast to types HAdV-D8, HAdV-D19, and HAdV-D37,
HAdV-D22 is not clearly associated with keratoconjunctivitis,
although it has some tropism for the eye. This type was first
described in Saudi Arabia, and the first isolate came from the
eye of a patient with trachoma in 1956 . However, its as-
sociation with trachoma was probably only coincidental. Since
then, only 2 other reports mentioned HAdV-D22 with respect
to ophthalmologic manifestations [8, 9]. The first report de-
scribes a laboratory infection causedbyHAdV-D22manifesting
second report describes a few cases of keratoconjunctivitis in
Japan associated with HAdV-D22 without any evidence of an
outbreak . In the latter report, the virus had been typed by
classic methods and DNA restriction-length polymorphism as
an intermediate HAdV-D22 that had a hemagglutination de-
terminant similar to HAdV-D10, HAdV-D19, or HAdV-D37.
Because the hemagglutination determinants of HAdV-D10,
HAdV-D19, and HAdV-D37 are phylogenetically closely re-
lated , it was probably impossible to get a more precise
typing result by classical methods. However, the hemaggluti-
nation determinant of HAdV-D8 is phylogenetically distant
from that of HAdV-D19 and HAdV-D37 (figure1B).Therefore,
as the agent responsible for the EKC outbreak, is not identical
to the oculotropic HAdV-D22H10/19/37 described previously
in Japan .
Because the fiber knob mediates the attachment of the virion
to the primary receptor on the cell membrane , the ability
of the intermediate type 22/H8 virus to cause keratoconjunc-
tivitis is probably mediated by its fiber characteristics of type
8, an adenovirus type typically associated with EKC . The
hypothesis that the fiber characteristics are responsible for tro-
pism is supported by a report that an adenovirus type 5 pseu-
dotyped with the fiber of type 37 retains the ocular tropism of
type 37 .
A second receptor for virus internalization are integrins-avb3
and integrins-avb5 in the cell membrane . Their mainligand
for binding is the RGD motif in the penton base of human
adenoviruses . Because interaction with integrins may also
be important for cell tropism, we also sequencedthepentonbase
of strains Hannover-2005-IAI-1 and Hannover-2005-IAI-2. In-
terestingly, these viruses had penton base sequences identical to
that of HAdV-D37, which is also a typical causative agent of
EKC. Therefore, our results indicate the significance of both
penton base and fiber knob for cornea tropism of HAdV.
by an intermediate adenovirus type 22/H8 that may have ac-
quired its unusual organo tropism and capability of epidemic
spread by an additional recombination event with HAdV-D37.
This recombinant virus may be a future cause of keratocon-
junctivitis outbreaks. Further characterization of naturally oc-
curring multiple recombinant HAdV isolates may enlighten our
knowledge regarding organo tropism of HAdV and may also
facilitate future design of highly specific gene therapy vectors.
We thank Dr. Noack for providing us with clinical data for patients and
Dr. Schalasta for primary laboratory diagnosis of adenovirus keratocon-
junctivitis and for providing us with 3 conjunctival swab samples.
Potential conflicts of interest.
All authors: no conflicts.
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