Molecular epidemiology of adenovirus strains isolated from patients with ocular disease in the area of Thessaloniki, Greece (1998-2002).

Abstract

Thirty strains of adenovirus (Ads) associated with ocular disease have been isolated over a period of 4 years in Thessaloniki, Northern Greece. Eleven strains were isolated from sporadic patients with conjunctivitis or keratoconjunctivitis in Thessaloniki city between 1998 and 2000. Nineteen strains were isolated from patients with keratoconjunctivitis during an outbreak of Ads in the area of Thessaloniki (Thessaloniki and Serres cities) in 2002. PCR-sequence method using primers targeted against the hypervariable regions (HVRs) of hexon gene, as well as the neutralization test were used for typing the Ad isolates and assessing a possible relation among these strains, and their genetic variability. Ad4 with very close homology to variant Z-G 95-873 was the most frequent genotype causing sporadic conjunctivitis over a period of 4 years. Two other strains, one Ad2, and one Ad3 were similar to the prototype ones, and a third one shows close homology to the variant of prototype Ad15, the Morrison strain. The genome typing of twenty two Ad8 isolates showed very close homology in their amino acid and nucleotide sequences to the variant of Ad8, strain 1127 (accession no. X74663). Four were isolated from patients with keratoconjunctivitis in 1998, 1999, 2000 and 18 during the outbreak in 2002. As far as strain 1127 is concerned, all the Ad8 isolates showed the same changes in the HVR 1 and HVR 2 except one isolate in 1998, which showed some changes outside the HVRs. During the outbreak of Ad8 keratoconjunctivitis, it was not possible to identify the exact source of infection (nosocomial or/and outpatients). Finally, Ad4 variant Z-G 95-873 and Ad8 which is closely related to the strain 1127, were found to be the predominant adenoviruses circulating in Northern Greece during 1998-2002.

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Journal of Medical Virology 75:440–446 (2005)
Molecular Epidemiology of Adenovirus Strains
Isolated From Patients With Ocular Disease in
the Area of Thessaloniki, Greece (1998–2002)
Filanthi Frantzidou,1* Aikaterini Pavlitou,1Asimina Mataftsi,2Kamal Dumaidi,1
and Nikolaos Georgiadis2
1A’ Department of Microbiology, School of Medicine, Aristotle University of Thessaloniki, Greece
2Eye Clinic, AHEPA Hospital, School of Medicine, Aristotle University of Thessaloniki, Greece
Thirtystrainsofadenovirus(Ads)associatedwith
ocular disease have been isolated over a period
of 4 years in Thessaloniki, Northern Greece.
Eleven strains were isolated from sporadic
patients with conjunctivitis or keratoconjunctivi-
tis in Thessaloniki city between 1998 and 2000.
Nineteen strains were isolated from patients
with keratoconjunctivitis during an outbreak of
Ads in the area of Thessaloniki (Thessaloniki and
Serres cities) in 2002. PCR-sequence method
using primers targeted against the hypervariable
regions (HVRs) of hexon gene, as well as the
neutralization test were used for typing the Ad
isolates and assessing a possible relation among
these strains, and their genetic variability. Ad4
with very close homology to variant Z-G 95-873
was the most frequent genotype causing spora-
dic conjunctivitis over a period of 4 years. Two
other strains, one Ad2, and one Ad3 were similar
to the prototype ones, and a third one shows
close homology to the variant of prototype Ad15,
theMorrisonstrain.Thegenometypingoftwenty
two Ad8 isolates showed very close homology
in their amino acid and nucleotide sequences to
the variant of Ad8, strain 1127 (accession no.
X74663). Four were isolated from patients with
keratoconjunctivitis in 1998, 1999, 2000 and 18
during the outbreak in 2002. As far as strain 1127
is concerned, all the Ad8 isolates showed the
samechangesinthe HVR1and HVR2exceptone
isolate in 1998, which showed some changes
outside the HVRs. During the outbreak of Ad8
keratoconjunctivitis, it was not possible to iden-
tify the exact source of infection (nosocomial or/
and outpatients). Finally, Ad4 variant Z-G 95-873
and Ad8 which is closely related to the strain
1127, were found to be the predominant adeno-
viruses circulating in Northern Greece during
1998–2002. J. Med. Virol. 75:440–446, 2005.
? 2005 Wiley-Liss, Inc.
KEY WORDS:
Greece; adenovirus; (kerato)-
conjunctivitis; outbreak; poly-
merase chain reaction with
sequencing analysis; genome
typing
INTRODUCTION
To date, 51 different serotypes of adenoviruses (Ads)
have been identified [de Jong et al., 1999]. They are
classifiedintosixsubgenera,A–F,onthebasisofnucleic
acid differences, fiber protein characteristics, and bio-
logical properties [Wadell et al., 1986; Murphy et al.,
1995]. The principal adenovirus types 2, 3, 4, 6, 7, 8, 19,
and37havebeenfoundtoberesponsibleforthemajority
of ocular infections. Acute conjunctivitis may occur as
part of a pharyngo-conjunctivitis syndrome or as sepa-
rate entity. The most frequently encountered types are
adenovirus 3 (subgenus B), and adenovirus 4 (subgenus
E).Othertypessuchasadenovirus1,2,and6(subgenus
C) and 9, 10, 15, 17, 20, 22, 29 (subgroup D) and ad-
enovirus7,16ofsubgroupBhavebeenreportedtocause
conjunctivitis.Adenovirustype4isasignificantcauseof
outbreakofrespiratorydiseaseamongmilitaryrecruits,
but it has also been associated with follicular conjuncti-
vitis [Aoki et al., 1982; Cooper et al., 1993; Schepetiuk
et al., 1993]. In Western countries adenoviral conjuncti-
vitis is frequently reported as swimming pool conjuncti-
vitis or nosocomial conjunctivitis. Certain members of
subgenus D, namely the Ad8, Ad19a, and Ad37, cause
outbreaks of keratoconjunctivitis and rapid identifi-
cation of these serotypes can help in prevention and
*Correspondence to: Filanthi Frantzidou, A’ Department of
Microbiology, School of Medicine, Aristotle University of Thessa-
loniki, Thessaloniki 54124. E-mail: filanthi@med.auth.gr
Accepted 25 November 2004
DOI 10.1002/jmv.20286
Published online in Wiley InterScience
(www.interscience.wiley.com)
? 2005 WILEY-LISS, INC.

Page 2

control[Jawetzetal.,1955;Darougaretal.,1977;Kemp
et al., 1983].
Diagnosis of adenoviral infections is based on virus
isolation in tissue culture, antibodies studies or antigen
detection by immunofluoerescence [Wadell et al., 1999].
However, virus isolation in tissue culture is laborious,
time consuming, especially as Ad 8 [Hanna and Jawetz,
1962;Wigandetal.,1983],Ad40andAd41[deJongetal.,
1992] are fastidious with slow and inefficient growth in
cell culture. The need for rapid and sensitive detection
has led to polymerase chain reaction (PCR) being the
best established among all other methods. Adenovirus
serotyping can be performed by neutralization or
hemagglutination inhibition test or by sequencing and/
or restriction fragment length polymorphism analysis
(RFLP). The neutralization test is a type specific
method, since it based on the use of antibodies directed
against epitope(s) on the hexon [Norby, 1969]. RFLP is
also a powerful and practical tool to differentiate the
adenoviral serotypes. Recently, PCR assay and sequen-
cing analysis have been used for the diagnosis and
serotypingofAds,especiallythefastidiousserotypes,for
which DNA restriction analysis is not possible [Morris
et al., 1995; Cooper et al., 1999; Takeuchi et al., 1999].
The combination of PCR and sequencing analysis is
more sensitive and rapid than virus isolation in tissue
culture [Pring-A˚kerblom and Adrian, 1994]. RFLP may
fail in serotyping a virus in the presence of a point
mutation on the restriction site, whereas sequencing
analysis overcomes this disadvantage. Studies on sequ-
encescodingforthehexonproteinsinseveraladenoviral
strains showed that while most of the pedestal regions
are conserved, variable regions exist in L1 and L2
[Kinlochetal.,1984;Pring-A˚kerblomandAdrian,1994].
Seven discrete hypervariable regions (HVRs) on the
hexon that make up serotype-specific loops on the
surface of the protein have been identified [Adrian
et al., 1989; Toogood et al., 1989; Crawford-Miksza and
Schnurr,1996a].SixvariableregionsexistinL1andone
in L2. Mutations in the HVRs can be extensive and may
lead to antigenic shift, drift and the evolution of a new
serotype [Wadell et al., 1980; Crawford-Miksza and
Schnurr, 1996b].
The numbers of studies that have looked into the
epidemiology of adenoviral eye infections in Greece
are scarce. In an outbreak of keratoconjunctivititis
in Northern Greece (1992–1993), Ad8 was the only
serotype (typed by neutralization test) to be isolated
[Frantzidou et al., 1994]. During the last few years, an
increase in the isolation of Ads at the Thessaloniki
University Virus Laboratory was observed, and most
isolates originated from local eye hospitals. This study
reports the detection and genetic characterization of
adenovirus strains isolated from patients with ocular
infectioninThessalonikicityinNorthernGreece,during
the period 1998–2000, as well as findings from an out-
break of Ad8 that occurred in the Thessaloniki area
(ThessalonikiandSerres)fromMarchtoDecember2002.
The neutralization test and PCR-gene sequence
method using primers targeting HVRs of hexon were
carried out in order to type the adenovirus isolates,
assess their genetic variability and possible relation
among the strains.
MATERIALS AND METHODS
Patients and Virus Strains
During the years 1998–2002, 70 clinical ocular
samples were studied for Ads. The samples originated
from patients with acute conjunctivitis or keratoconjuc-
tivitis and sent to the laboratory from AHEPA Hospital,
Saint Dimitrios Public Hospital in Thessaloniki, and a
private ophthalmology clinic in Serres, a city 80 km
away from Thesssaloniki. Twenty-five samples came
from sporadic cases in the city of Thessaloniki during
the period from March 1998 to December 2000, and
45samplesfromanoutbreakthatoccurredintheareaof
Thessaloniki from March to December 2002.
The outbreak lasted 10 months, but was recognized
at the end of April. During the outbreak 91 affected
subjects were identified, of which: (1) 57 were out-
patients seen at the AHEPA hospital, 3 being members
of the same family. One patient was subjected to eye
examination in the AHEPA hospital 10 days before his
infection. (2) Six were doctors working at the eye clinic,
(3) three were inpatients in the neurosurgical clinic of
the AHEPA hospital, probably representing nosocomial
infections, and (4) 25 were outpatients seen in a private
eyeclinicinSerres.Clinicalandepidemiologicaldatafor
patients were derived from the hospital files and from
theprivateophthalmologist.Outof91reportedpatients
presenting with keratoconjunctivitis during the out-
break samples were obtained and investigated in only
45 cases.
Thessaloniki and Serres are two cities located in
northern part of Greece. Thessaloniki is a large city and
considered as the capital of North with a population of
1.5 million, and Serres is a city located to the north of
Thessaloniki with 50,000 inhabitants. The people in the
two cities are in a regular and continuous movement.
Thirty strains of adenovirus (11 from sporadic cases
and 19 from the outbreak) were isolated and investi-
gated further. Each isolate was obtained from different
patient. Patients who proved negative for an adenoviral
infection were investigated for other pathogens (Herpes
simplex, Chlamydia trachomatis, and other common
bacteria).
Virus Culture—Neutralization Test
Cotton-tipped conjunctival swabs from patients sus-
pected to have an adenoviral infection were collected in
virus transport medium and inoculated onto Hep-2 and
A-549 cells. Infected cells were identified by immuno-
fluorescent-antibody technique with murine monoclo-
nal antibody, specific to a common epitope on the
adenovirus hexon protein, conjugated to FITC (Daco
Ltd.,Ely,Cambridge,UK).OnehundredTCID50ofeach
isolate was used for virus neutralization tests, which
were performed on A549 cells. Anti-rabbit sera used in
Molecular Epidemiology of Adenovirus Strains 441

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this study were obtained from Denka Seiken Co. Ltd.
(Tokyo, Japan).
PCR- Sequence Method
DNA Extraction
Samples preparation and DNA extraction from
infected cells were done using the QIAamp blood Kit
protocol (QIAGEN Cmbh, Hilden, Germany) according
to the instructions of the manufacturer.
Primers and PCR Procedure
Adenovirus is a double-stranded DNA virus, with
the hexon accounting for about 2.8 kbp [Kinloch et al.,
1984]. The set of primers included HX5-1 (forward
primer), 50-AAGATGGCCACCCCCTCGATGATGCCG-
CAGT-30, and HX3-1 (reverse primer), 50-CACTTATG-
TGGTGGCGTTGCCGGCCGAGAACGG-30(Invitrogen
Ltd., Paisley, Scotland). These primers amplify the
region that corresponds to 1–2,829 bp in the hexon base
sequence of adenovirus type 3 [Takeuchi et al., 1999].
The PCR protocol of Takeuchi was modified to account
for the Herculase enzyme used. Briefly, 2 ml DNA
extractedfrom infectedcellswasusedin atotalreaction
volume of 30 ml of PCR. The reaction solution contained
1xHerculasebuffer(Stratagene,Jolla,CA),400mMcon-
centrationsofeach deoxynucleoside
(dNTPs), (Gibco BRL, Inchiman,UK), 50 pmolesof each
primer, and 2.5 U of Herculase DNA polymerase.
Thermal cycling was performed in Peltier Thermal
Cycler PTC-200 (MJ Research, Watertown, MA) for a
total of 40 cycles of denaturing at 948C for 30 sec,
annealing at 688C for 5 sec, and extension at 728C for
10 min. Five to 10 ml of PCR product was analyzed in a
1.5% TBE agarose gel, stained with ethidium bromide.
triphosphate
Sequence Analysis
PCR products of samples were purified by using
standard protocol (Concert Rapid Purification System,
GibcoBRL)and analyzedby meansofanauto sequencer
(MWG Biotech, Ebersberg, Germany) and the follow-
ing primers were used for sequencing analysis: sense
primersS-29:50-GCCAGCACRTWCTTTGACAT-30,and
S-51: 50CCCAACAGACCCAAYTACA T-30, and anti-
sense primer S-54: 50-CCAGCATTGCGGTGGTGRTT-
30[Takeuchi et al., 1999]. These primers were success-
fully used tosequence a nucleotide portionof 1,630 bpof
the hexon gene that contains all seven HVRs. The
nucleotide sequence of amplified hexon gene for each
isolate of Ads was analyzed using BLAST program of
NBCI (National center for Biotechnology Information).
The genotype determined by the PCR-sequence
method was based on homology of our sequence data
with published sequences in GenBank.
RESULTS
Overall, 30 strains of Ads were isolated. All isolates
were obtained from distinct patients. Patients from
whom adenovirus was not isolated were also found
negative for infection by Herpes simplex virus, Chlamy-
dia trachomatis, and other common bacteria. From
1998 to 2000, eleven strains of adenovirus were isolated
from 25 sporadic cases of conjunctivitis or keratocon-
junctivitis in Thessaloniki city. The year of isolation,
hospital, symptoms, results of typing by neutralization
test and sequencing analysis are given in Table I.
The portion of the hexon gene used for nucleotide
analysis was 1,630 bp and the estimation of genetic
variation was restricted to the hexon gene region
sequenced and analyzed. The eleven strains from 1998
to2000showthe followinghomology in theiraminoacid
and nucleotide sequences with adenovirus isolates
published in GenBank: adenovirus type 2 (Ad2) shows
94% and 99% homology with Ad2 possessing the ac-
cession no. JO1917 in GenBank, respectively. Adeno-
virus type 3 (Ad3) shows 91.8% and 98.6% homology
with Ad3 possessing accession no. X76549,respectively.
The isolate that was not typed by neutralization test,
duetounavailabilityofantiserumshows84%and95.5%
homology with adenovirus type 15H9 (Morrison) pos-
sessing accession no. X76707, respectively. The four
strains of adenovirus type 4 (Ad4) that were isolated
from patients with conjunctivitis or follicular conjuncti-
vitis show 99.6% and 99.7 % homology respectively with
the isolate Z-G95-873 (accession no. F065064). In these
strains of Ad4 in relation to isolate Z-G95-873, a
TABLE I. Sporadic Cases of Ocular Disease Investigated for Adenoviruses in Thessaloniki City in 1998–2000
Patient
number
Year of
isolationHospital Symptoms
Neutralization
test (NT)
Nucleotide homology with
strains published in GenBank
1
2
3
4
5
6
7
8
9
1998
1998
1999
1999
1999
1999
1999
2000
2000
2000
2000
AHEPA
AHEPA
S. Dimitrios
S. Dimitrios
S. Dimitrios
S. Dimitrios
S. Dimitrios
S. Dimitrios
S. Dimitrios
AHEPA
AHEPA
Follicular conjunctivitis
Keratoconjunctivitis
Conjunctivitis
Follicular conjunctivitis
Conjunctivitis
Conjunctivitis
Keratoconjunctivitis
Conjunctivitis
Conjunctivitis
Keratoconjunctivitis
Keratoconjunctivitis
Ad4
Ad8
Ad4
Ad4
Ad4
NDa
Ad8
Ad3
Ad2
Ad8
Ad8
99.7% with AF065064
98.7% with X74663
99.7% with AF065064
99.7% with AF065064
99.7% with AF065064
95.5 % with X76707
98.7% with X74663
98.6% with X76549
99% with J01917
98.8% with X74663
98.8% with X74663
10
11
aAntiserum not available.
442Frantzidou et al.

Page 4

replacement of 428 amino acid in HVR1 (P 428 T) and
another replacement of 457 amino acid outside the
HVRs were observed. Among the four strains of ad-
enovirustype8thathadbeenisolatedduringtheabove-
mentioned period, one isolate in 1998 shows 89.7% and
98.7% homology in its amino acid and nucleotide
sequence respectively with the isolate 1127 (accession
no. X74663), while the isolates of Ad8 in 1999 and 2000
show 91% and 98.8% homology with the isolate 1127,
respectively.
During the outbreak of keratoconjunctivitis in 2002
in the area of Thessaloniki, 19 strains of Ads were
isolatedfrom45ocularsamples.Exceptfromoneisolate
of adenovirus type 4, which is identical to Ad4 strains
isolated in the previous years, the remaining 18 isolates
were typed by neutralization test as adenovirus type 8.
The month of isolation, distribution, patient’s class, and
homology in nucleotide sequence with the isolate 1127
(accession no. X74633) are given in Table II. Among 18
isolates 13 came from the AHEPA hospital in Thessa-
loniki and 5 from a private clinic in Serres. The isolates
from Thessaloniki and Serres cities share 91% and
98.7%–98.8% homology in their amino acid and nucleo-
tide sequences respectively with the isolate 1127, and
were identical to Ad8 isolates in 1999 and 2000.
Figure 1 shows the comparison of the deduced amino
acid sequences of seven HVRs among four representa-
tivestrainsofadenovirustype8isolatedinThessaloniki
(1998, 2000, and 2002) and Serres (2002).
The nucleotide sequences of the four isolates of
adenovirus type 8 showed 98.7%–98.8% homology with
the isolate 1127 (X74663). Furthermore, the deduced
amino acid sequences starting from position 22 of the
isolate 1127 (X74663) showed 89.7%–91% homology.
All four analyzed strains showed the same amino acid
changes in the HVR1 and HVR2. In addition to these
changes, the Ad8 (1998) showed some changes outside
the HVRs in hexon gene.
DISCUSSION
In this study, 30 isolates of adenovirus from different
patients with ocular disease over a period of 4 years
in the Thessaloniki area were typed by the neutraliza-
tion test and PCR-sequence method using primers
targeted to the HVRs of hexon gene. PCR is more
sensitiveandrapidthanvirusisolationintissueculture
for diagnosis [Pring-A˚kerblom and Adrian, 1994].
Although, RFLP analysis could better reflect genomic
variations among isolates belonging to a same serotype,
thesequencemethodwasappliedinthisstudybecausea
variety of adenovirus isolates belonging to different
serotypes had to be analyzed. Sequencing analysis has
an advantage on RFLP since a point mutation in the
restriction site may lead to failure of serotyping the
respective virus by RFLP. From 1998 to 2000, eleven
strainsofadenovirusbelongingtotypes2,3,4,8,and15
were isolated from sporadic cases of conjunctivitis.
During the epidemic of keratoconjunctivitis in 2002, 18
strains Ad8 and 1 strain Ad4 were isolated.
Adenovirus type 4 (Ad4) is a known cause of con-
junctivitis.OutbreaksofAd4havebeenreportedinAsia,
Europe, and Australia [Aoki et al., 1982; Cooper et al.,
1993; Schepetiuketal.,1993]. Twostrikinglydissimilar
genome types of Ad4 prototype and Ad4a have been
demonstrated by DNA restriction enzyme analysis
(RFLP) to be implicated [Li and Wadell, 1988a; Itakura
etal.,1991].TheAd4agenometypeisisolatedcommonly
in Japan as cause of conjunctivitis [Aoki and Tagara,
2002].Inthisstudy,Ad4wasisolatedfromonepatientin
1998, three patients in 1999, and one patient in 2002.
TABLE II. Cases of Ocular Disease Investigated for Adenoviruses During an Outbreak of Keratoconjunctivitis in Thessaloniki
Area From March to December 2002
Patient
number
Month of
isolationLocation Patient’s class
Neutralization
test
Nucleotide homology with
strains published in GenBank
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
May
April
April
April
April
April
April
April
May
May
June
September
September
September
September
September
October
November
December
Serresa
Thessalonikib
Thessalonikib
Thessalonikib
Thessalonikib
Thessalonikib
Serresa
Serresa
Serresa
Serresa
Thessalonikib
Thessalonikib
Thessalonikib
Thessalonikib
Thessalonikib
Thessalonikib
Thessalonikib
Thessalonikib
Thessalonikib
Outpatient
Outpatient
Doctor in eye clinic
Doctor in eye clinic
Inpatient
Inpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Outpatient
Ad4
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
Ad8
99.7% with AF065064
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.7% with X74663
98.7% with X74663
98.7% with X74663
98.7% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
98.8% with X74663
aPrivate clinic.
bAHEPA hospital.
Molecular Epidemiology of Adenovirus Strains 443

Page 5

Ocular symptoms due to Ad4 showed a moderate
severity and ranged from mild to severe. All isolates of
Ad4 were identical to each other and showed 99.6% and
99.7% homology in their amino acid and nucleotide
sequences with the variant Z-G 95-873 (accession no.
AF065064),respectively.Inastudyofstrainvariationof
Ad4 serotype it was found that the evolution of Ad4 was
complex, with continuous genetic drift punctuated by
replacement with a new strain [Crawford-Miksza et al.,
1999]. The strain Z-G 95-873 appeared in the United
Statesin1995andallisolatesofAd4recoveredsincethat
time were this new strain. The variant Ad4 AF065064
wasfoundtoresideinnorthernGreeceduringtheperiod
1998–2002. However, because of absence of data con-
cerning adenovirus type 4 before 1998, it is not possible
to know when this variant first appeared in the area.
Adenovirus type 3 (Ad3) is a common cause of con-
junctivitis worldwide, and an alternation of different
genotypes of Ad3 has occurred in different countries
[Guo et al., 1988; NIH, 1995; Itoh et al., 1999; Saitoh-
Inagava et al., 1999]. More than twenty genotypes of
Ad3 have been identified by RFLP analysis [Li and
Wadell, 1988b; Itoh et al., 1999]. The Ad3 genotype
isolated in this study was related closely to Ad3 isolate
with accession no. Ad3X76549. O’ Donnell et al. [1993]
reported that Ad3 prototype was isolated more fre-
quently from patients with conjunctivitis than other
Ad3 genotypes during the period from 1981 to 1988 in
Glasgow, except in 1986 when Ad3a was the most fre-
quent Ad3 genotype that had been isolated. In addition,
the Ad3 prototype has been reported to be predominant
in Europe, Brazil, Africa, and Australia [Wadell, 1990].
The adenovirus type 2 (Ad2) isolate in our series was
closely related to Ad2 with accession no. JO1917. This
strain of Ad2 was isolated from a child with pharyngo-
conjunctivitis. The association of Ads with this clinical
Fig. 1. Comparison of deduced amino acid sequences of seven hypervariable regions (HVRs) among the
genometypesofadenovirustype8isolatedinThessaloniki(The1998,The2000,The2002)andSerres(Ser
2002). The sequences of loop 1 and loop 2 have been aligned to obtain maximal homology. Deduced amino
acidsofAd8isolate1127accessionnumberX74663wasobtainedfromGenBank.Theshadedregionsshow
the changes of amino acid.
444Frantzidou et al.

Page 6

presentation has been reported in the 50s [Bell et al.,
1955].Finally,theAd15isolatewasavariantoftheAd15
prototype and showed 84% and 95% homology with
Ad15H9withaccessionno.X76707(Morrison).ThisAd15
was isolated from a sporadic case of mild conjunctivitis.
Ad8 is a classic and very contagious agent causing
epidemic keratoconjunctivitis, which may be trans-
mitted by ocular examination instruments [Richmond
etal.,1984].Ad8hasamuchhighertropismforconjunc-
tival cells than Ad19 and Ad37 and produces severe
clinical manifestations and pathological alterations.
This type persists in the population and causes sporadic
epidemics in many countries [Kemp and Heirholtzer,
1986; Chastel et al., 1988; de Jong et al., 1992; Corsaro
et al., 1999; Tanaka et al., 2000; Adhikary et al., 2003].
Manyinvestigatorshaveusedgenomiccharacterization
by different restriction endonucleases to establish
genomic variants of Ad8, and around 20 genotypes have
been identified [Fujii et al., 1983, 1984; Kemp et al.,
1983; Wadell, 1984]. Genome typing can be also done
with subgenus- or type-specific PCR primers [Adrian
et al., 1994; Tanaka et al., 2000; Adhikary et al., 2003].
Recently, some methods have suggested that the com-
bination of PCR and RFLP analysis facilitate the typing
procedure [Kidd et al., 1996; Saitoh-Inagava et al.,
1996].However,thesemethodsareincompleteorlimited,
with results difficult to interpret. Although the RFLP
analysiscouldbetterreflectthevariationamongisolates
belonging to the same serotype, in this study, the PCR-
sequence method was used for various reasons; not all
neutralizing antisera for Ads were available; no refer-
ence strain for all the Ads was available; a variety
of adenovirus isolates belonging to different serotypes
hadtobeanalyzed.Finally,dataconcerningtheAdsthat
circulate in this geographical region was unavailable.
The DNA sequence of hexon HVRs of all our Ad8
isolates (18 strains in the 2002 outbreak and 4 strains
in 1999 and 2000) were identical and showed 98.7%–
98.8% homology to Ad8 strain 1127 (accession no.
X74633). The isolate 1127, which is closely related to
Ad8A, Ad8B, and Ad8E [Adhikary et al., 2003], was
first isolated from patients with keratoconjunctivitis
in Germany by Wigand et al. [1983] and was later
deposited in GenBank under the accession no. X74663
[Pring-A˚kerblom and Adrian, 1994].
Duringtheoutbreak,itwasnotpossibletoidentifythe
exact source of infection (nosocomial or outpatients),
because Ad8 was isolated from doctors working in the
eye clinic, inpatients in another clinic at the AHEPA
hospital, as well as from outpatients with keratocon-
junctivitis presented at the AHEPA hospital and at a
privateclinicinSerres,allatthesametime.However,it
is worthwhile mentioning that not all patients who
presented with keratoconjunctivitis to the eye clinics
were investigated virologically. The transmission within
the eye clinic in AHEPA hospital was monitored once
controlmeasureswereapplied.ThessalonikiandSerres
cities are located in the northern part of Greece. The
people in the two cities are in a regular and continuous
movement.Thus,itispossiblethattheoutbreakstarted
in one city and transferred to another by a patient or
health workers, e.g. doctors, nurses.
AnadenovirusstrainidenticaltoprototypeAd8(Trim
strain) was isolated from three patients with kerato-
conjunctivitisresidinginAthens,Greece,in1980–1982,
by using a neutralization test and restriction enzyme
length polymorphism analysis [Kemp and Heirholtzer,
1986]. In 1992 to 1993 an outbreak of keratoconjuncti-
vitis due to Ad8 occurred in the area of Thessaloniki
[Frantzidou et al., 1994]. In this outbreak, Ad8 was iso-
latedintissueculturefromeightpatientsandidentified
only by serum neutralization test, therefore, we cannot
know if this strain was the prototype of Ad8. The isolate
Ad8 in this study, which is very closely related to strain
1127 of Ad8, was firstly identified to be endemic in the
area of Thessaloniki, in 1998 by our group. The isolates
ofAd8in1999,2000,and2002duringtheoutbreakwere
identical. The isolate in 1998 showed some changes
outside the HVRs. As it is well known that the genomic
variants of Ad8 may allow a virus to persist in the
population over a long period [Kemp and Heirholtzer,
1986; Ishiik et al., 1987; Adrian et al., 1994], there is
a possibility that the strains of the outbreak of Ad8
keratoconjunctivitis in 1992–1993 were identical or
similar to strain 1127 (accession no. X74633).
In conclusion, this study showed that the predomi-
nanttypesofAdsinpatientswithocularinfectionsovera
4-year period were: (1) a variant of Ad4, strain Z-G 95-
853 (accession no. AF064065) in sporadic cases of
conjunctivitis, and (2) a variant of Ad8, which showed
very close homology to the strain 1127 (accession no.
X74663) was the causative agent in sporadic cases of
keratoconjunctivitis in 1998, 1999, 2000 and in an
outbreak of keratoconjunctivitis in 2002. The PCR-
sequence method is an efficient, accurate, and rapid
meansofdiagnosisandtypingoftheadenovirusandhas
significant clinical and epidemiological implications.
Finally, this study provides important information on
the relationships between the existing adenoviral
serotypes, their HVRs sequences, and presence of
sporadic cases and of a keratoconjunctivitis outbreak
that occurred in this region of the world.
REFERENCES
AdhikaryAK,NumagaJ,KaburakiT,KawashimaH,AraieM,IkedaY,
Ogino T, Suzuki E, Ushijima H, Mukoyama A, Matsuno S, Inada T,
Okabe N. 2003. Genetic characterization of adenovirus type 8
isolated in Hiroshima city over a 15 years period. J Clin Pathol
56:120–125.
Adrian T, Best B, Hierholzer JC, Wigand R. 1989. Molecular epidemio-
logy and restriction site mapping of adenovirus type 3 genome
types. J Clin Microbiol 27:1329–1334.
AdrianT,WolfU,LauerHG,WigandR.1994.Restrictionsitemapping
of adenovirus type 8 genome types. Res Virol 141:611–624.
Aoki K, Tagara T. 2002. A twenty-one years surveillance of adenoviral
conjunctivitis Sapporo, Japan. Int Ophthalmol Clin 42:49–54.
Aoki K, Kato M, Ohtsuka H, Ishii K, Nakazono N, Sawada H. 1982.
Clinical and etiological study of adenoviral conjunctivitis, with
special reference to adenovirus types 4 and 19 infections. Br J
Ophthalmol 66:776–780.
Bell JA, Rowe WB, Engler JI, Parrott RH, Huebner RJ. 1955.
Pharyngoconjunctivitis fever: Epidemiological studies of a recently
recognized disease entity. JAMA 175:1083–1092.
Molecular Epidemiology of Adenovirus Strains445

Page 7

Chastel C, Adrian T, Demazure M, Legrand-Quillien MC, Colim J,
Wigand R. 1988. Molecular epidemiology of two consecutive
outbreak of adenovirus 8 keratoconjunctivitis. J Med Virol 24:
199–204.
Cooper RJ, Bailey AS, Kikkough R, Richmond SJ. 1993. Genome
analysisof adenovirus 4isolated over asix-year period. JMed Virol
39:62–66.
Cooper RJ, Yeo CY, Bailey AS, Tullo AB. 1999. Adenovirus polymerase
chain reaction assay for rapid diagnosis of conjunctivitis. Invest
Ophathalmol Vis Sci 40:90–95.
Corsaro D, Gut JP, Le Fau a. 1999. Molecular epidemiology of ocular
isolates of adenovirus 8 obtained over nine years. J Clin Pathol
52:860–861.
Crawford-Miksza LK, Schnurr DP. 1996a. Analysis of 15 adenovirus
hexon proteins reveals the location and structure of seven
hypervariable regions containing serotype specific residues. J Clin
Microbiol 70:1836–1844.
Crawford-Miksza LK, Schnurr DP. 1996b. Adenovirus serotype evolu-
tion is driven by illegitimate recombination in the hypervariable
regions of the hexon protein. Virology 224:357–367.
Crawford-Miksza LK,NangRN,Schnurr DP.1999.Strainvariationin
adenovirus serotypes 4 and 7a causing acute respiratory disease.
J Clin Microbiol 37:1107–1112.
Darougar S, Quinlan MP, Gibson JA, Jones BR, McSwiggan DA. 1977.
Epidemic keratoconjunctivitis and chronic papillary conjunctivitis
in London due to adenovirus type 19. Br J Opthalmoll 61:76–85.
de Jong JC, Demazure M, Legrant-Quillien MC, Le Lay G, Golin J,
Wermenbol AG, Verweij-Uy ¨terwaal MW, van der Avoort HG,
Chastel C. 1992. New developments in molecular epidemiology of
adenovirus 8 keratoconjunctivitis. J Med Virol 38:102–107.
deJongJC,WermenbolAG,Verweij-UyterwaalMW,KeesW,Slaterus
KW, Van Doornum GJJ, Wertheim-van Dillen P, Van Doornum
GJJ, Khoo SH, Hierholzer JC. 1999. Adenoviruses from human
immunodeficiencyvirus-infectedindividuals,includingtwostrains
that represent new candidate serotypes Ad50 and Ad51 of species
B1 and D, respectively. J Clin Microbiol 37:3940–3945.
Frantzidou F, Kyriazopoulou B, Souliou E, Diza E. 1994. An outbreak
of Adenovirus type 8 keratoconjunctivitis in the region of Thessa-
loniki. Acta Microbiol Hellenica 39:644–650.
Fujii S, Nakazono N, Sawada H, Ishii K, Kato M, Aoki K, Ohtsuka H,
Fujinapa K. 1983. Restriction endonuclease cleavage analysis of
adenovirus 8: Two new subtypes from patients with epidemic
keratoconjunctivitis inSapporo,Japan. JpnJMedSciBiol36:307–
313.
FujiiS,NakazonoN,IshiiK,LinC,SheuM,ChenC,FujinagaK.1984.
Molecular epidemiology of adenovirus 8 (Ad8) in Taiwan: Four
subtypes recovered during the period 1980–1981 from patients
withepidemickeratoconjunctivitis.JapJMedSciBiol37:161–169.
Guo DF, Shibata R, Shinagawa M, Sato G, Aoki K, Sawada H. 1988.
Genomic comparison of adenovirus type 3 isolates fro patients with
acute conjunctivitis in Japan, Australia, and the Philippines.
Microbiol Immunol 32:833–842.
HannaL,JawetzE.1962.Attempttoenchanceinfectivityofadenovirus
type 8 for cell culture. Proc Soc Exp Biol 110:707–709.
IshiikK,NakazonoN,FujinagaK,FugiiS,KatoM,OhtsukaH,AokiK,
ChenCW,LinKH,OumBS,LeeSH,ChunCH,YoshiiT,Yamazaki
S.1987.Comparativestudiesonaetiologyofviralofthreecountries
of East Asia-Japan, Taiwan, South Korea. Int J Epidemiol 16:98–
103.
ItakuraS,AokiK,SawadaH,IshiguroN,ShinagawaM.1991.Changes
in subgenome types of adenovirus type 4 isolated from patients
with ocular disease between 1985 and 1989 in Sapporo, Japan.
J Clin Microbiol 29:1740–1743.
ItohN,KeicoT,AokiK,HinokumaR,NakagawaH,TackeuchiS,Uchio
E, Shiao S, Ohno S. 1999. Four new genotypes of adenovirus type 3
isolated from patients with conjunctivitis in Japan. J Med Virol
59:73–77.
Jawetz E, Kimura SJ, Hanna L, Coleman VR, Thygeson P, Nicholas A.
1955. Studies in the etiology of epidemic keratoconjunctivitis.Am J
Ophtalmol 40:200–211.
KempMC,HeirholtzerJC.1986.Threeadenovirustype8genometypes
defined by restriction enzyme analysis: Prototype stability in
geographically separated populations. J Clin Microbiol 23:469–
474.
Kemp MC, Heirholtzer JC, Cabradilla CP, Obijeski JF. 1983. The
changing etiology of epidemic keratoconjunctivitis: Antigenic and
restriction enzyme analysis of adenovirus types19 and 37 isolated
over a ten-year period. J Infect Dis 148:24–33.
Kidd AH, Jo ¨nsson M, Garwicz D, Kajon A, Wermenbol AG, Verweij
MW, de Jong JC. 1996. Rapid subgenus identification of human
adenovirus isolates by a general PCR. J Clin Microbiol 34:622–
627.
Kinloch R, Mackay N, Mautner V. 1984. Adenovirus hexon sequence
comparisonofsubgroupCserotypes2and5.JBiolChem259:6431–
6436.
Li Q, Wadell G. 1988a. The degree of genetic variability among
adenovirus type 4 strains isolated from man and chimpanzee. Arch
Virol 101:65–77.
Li QG, Wadell G. 1988b. Comparison of 17 genome types of adenovirus
type3identifiedamongstrainsrecoveredfromsixcontinents.JClin
Microbiol 26:1009–1015.
Morris DJ, Bailey AS, Cooper RJ, Turner PC, Jackson R, Corbitt G,
Tullo AB. 1995. Polymerase chain reaction for rapid detection of
ocular adenovirus infection. J Med Virol 46:126–132.
Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW,
Martelli GP, Mayo MA, Summers MD, editors. 1995. Virus
taxonomy:Classificationandnomencultureofviruses.Sixthreport.
Vienna, Austria: Springer-Verlag. pp 128–133.
NIH. 1995. Viruses isolated from the eye, Japan 1990–1994. Infect
agents surveillance Rep 16:97–98.
Norby E. 1969. The structural and functional diversity of adenovirus
capsid components. J Gen Virol 5:221–236.
O’ Donnell B, McCruden EA, Desselberger U. 1993. Molecular
epidemiology of adenovirus conjunctivitis in Glasgow 1881–1991.
Eye 7:8–14.
Pring-A˚kerblomP,AdrianT,1994.Typeandgroupspecificpolymerase
chain reaction for adenovirus detection. Res Virol 145:25–35.
Richmond S, Burman R,Crosdale E,Cropper L, Longson D, Enoch BE,
Dodd CL. 1984. A large outbreak of keratoconjunctivitis due to
adenovirus type 8. J Hyg 93:285–291.
Saitoh-InagavaW,OshimaA,AokiK,ItohN,IsobeK,UchioE,OhnoS,
Nakajima H, Hata K, Ishiko H. 1996. Rapid diagnosis of adenoviral
conjunctivitis by PCR and restriction fragment length polymor-
phism analysis. J Clin Microbiol 34:2113–2116.
Saitoh-Inagava W, Aoki K, Uchio E, Itoh N, Ohno S. 1999. Ten years’
surveillance of viral conjunctivitis in Sapporo, Japan. GraefesArch
Clin Exp Ophthalmol 237:35–38.
Schepetiuk SK, Norton R, Kok T, Irving LG. 1993. Outbreak of
adenovirus type 4 conjunctivitis in South Australia. J Med Virol
41:316–318.
Takeuchi S, Itoh N, Uchio E, Aoki K, Ohno S, 1999. Serotyping of
Adenoviruses on conjunctival scrapings by PCR. J Clin Microbiol
3:1839–1845.
Tanaka K, Itoh N, Saitoh-Inagawa W, Uchio E, Takeuchi S, Aoki K,
SorianoE,NishiM,DurigonEL,StewienKE,OhnoS.2000.Genetic
characterization of adenovirus strains from patients with acute
conjunctivitis in the city of Sa ˘o Paulo, Brazil. J Med Virol 61:143–
149.
Toogood CIA. Murali R, Burentt RM, Hay Rt. 1989. The adenovirus
type 40 hexon: Sequence, predicted structure and relationship to
other adenovirus hexon. J Gen Virol 70:3203–3214.
Wadell G. 1984. Molecular epidemiology of human adenoviruses.
Curr Top Microbiol Immunol 110:191–220.
Wadell G, 1990. Adenoviruses. In: Zuckerman AJ, Banatvala JE,
Pattison JR, editors. Principles and practice of clinical virology.
Chichester: John Wiley and Sons Ltd. pp 286–287.
Wadell G, Hammarsjold ML, Winberg G, Varsanyi T, Sundell G. 1980.
Genetic variability of adenoviruses. Ann NY Acad Sci 354:16–42.
Wadell G, Allard A, Evander M, Li Q. 1986. Genetic variability and
evolution of adenoviruses. Chem Scripta 26B:325–335.
Wadell G, Allard A, Hierholzer JC. 1999. Adenvoviruses, p970-982. In:
Murray PR, Baron EJ, Pflaller MA, Tenover F, Yolken N, editors.
Manual of clinical microbiology, 17th edn. Washinton, DC: Amer-
ican Society for Microbiology.
WigandR,GeiderblomH,OzelM,DistlerH,AdrianT.1983.Character
stics of Mastadenovirus h8, the causative agent of epidemic
keratoconjunctivitis. Arch Virol 76:307–319.
446Frantzidou et al.