Previous Article | Next Article 
Journal of Clinical Microbiology, June 1999, p. 1839-1845, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Serotyping of Adenoviruses on Conjunctival
Scrapings by PCR and Sequence Analysis
Satoshi
Takeuchi,1,*
Norihiko
Itoh,1
Eiichi
Uchio,1
Koki
Aoki,2 and
Shigeaki
Ohno1
Department of Ophthalmology, Yokohama City
University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama
236-0004,1 and Aoki Eye Clinic, 2-1 Kita, 6 Hondori, Shiroishi-ku, Sapporo
003-0027,2 Japan
Received 2 November 1998/Returned for modification 14 January
1999/Accepted 10 February 1999
 |
ABSTRACT |
To detect and identify adenovirus (Ad), we investigated
hypervariable regions (HVRs) of Ad by using a combination of PCR and direct sequencing (PCR-sequence) method. Primers for nested PCR to
amplify the conserved region in the hexon protein containing HVRs were
designed based on hexon gene sequences derived from GenBank. These two
primer sets amplified a DNA fragment of 7 HVRs from 16 prototypes of
Ad, which were divided into five subgenera, including seven serotypes
that are the predominant causative agents of acute conjunctivitis in
Japan, and from 31 recent conjunctival scraping specimens from patients
with adenoviral conjunctivitis. HVR DNA sequences were determined by
means of universal sequence primers. Analysis of the predicted amino
acid homology of HVRs among Ad prototypes suggested three regions,
HVR4, -5, and -7, to be candidates for the neutralization epitopes. The
clinical serotype of specimens was determined by the PCR-sequence
method with reference to these three HVRs. The serotype determined
according to this method was identical to that obtained by culture
isolation and the neutralization test (NT) in all scraping samples,
whereas the results of this method did not match PCR and restriction
fragment length polymorphism (PCR-RFLP) analysis in five samples. It
took only three days to detect Ad and to identify the serotype, in contrast to culture isolation-NT, which took at least 2 weeks. These
findings indicate that our newly developed PCR-sequence method is
applicable for the detection and serotyping of human Ads.
| |
INTRODUCTION |
Adenoviruses (Ads) are characterized
by reproducing well in specific cell lines (32), and Ad
serotypes have been determined based on the fact that immune sera
specifically inhibit their reproduction (9, 24, 38). At
present there are 49 serotypes of Ad (15, 31, 38), and they
have been classified into six subgenera, A to F, based on DNA homology
and the physical, biochemical, and biological properties of the viruses
(36). However, differences in virus reproduction rates in
cultured cells (14, 28) and ease of adsorption and
penetration of host cells according to Ad serotype (11, 37)
create problems in attempting to identify virus serotypes. In addition,
determination of neutralization and determination of virus titer
endpoints depend on the evaluation of cytopathic effects (CPE) and are
subjective and time-consuming (9). For these reasons,
standard sera cannot always be expected to perform neutralization under
stable reproducible conditions and so details concerning the epitopes
that govern Ad neutralization still remain unknown. Furthermore, many
of the serotypes produce proteins that inhibit host cell resistance
(10, 39, 40), and this modifies the pathological picture,
which is not only related to neutralization but to both tissue affinity
for the host and host resistance as well.
We have previously developed a PCR and restriction fragment length
polymorphism (PCR-RFLP) analysis based on PCR of the hexon region
combined with restriction fragment patterns (30). However, this method has limitations, since it targets the downstream conserved region of the hexon structural gene which has not been specified as a
region determining the serotype.
Recently, it has been reported that by hexon X-ray crystallography
analysis (3, 29) and sequencing (19, 26, 27, 34,
35), hypervariable regions (HVRs) that participate in type-specific neutralization have been found to exist on the surface of
Ads (6, 7). We therefore focused our attention on these HVRs
and, by combining nested PCR, which amplifies these regions, with the
direct sequencing method (PCR-sequence method), we have developed a new
method that detects Ads in conjunctival scrapings and identifies their
serotype. We compared the results of this method with those obtained by
conventional serotype identification methods (9,
30).
| |
MATERIALS AND METHODS |
Study group and specimens.
The Ad prototypes used
were obtained from the American Type Culture Collection (ATCC),
Rockville, Md. A total of 16 serotypes were studied. They consisted of
the seven strains that cause Ad conjunctivitis in Japan: Ad types 3, 4, 7, 8, 11, 19, and 37 (1, 2, 12, 16) and, in addition, Ad
types 11, 14, 34, and 35, which belong to subgenus B2; Ad types 1, 2, 5, and 6 in subgenus C; and Ad types 40 and 41 in subgenus F. The study
materials consisted of 31 conjunctival scraping specimens collected
from patients with Ad conjunctivitis in Japan from 1994 through 1998. The lower palpebral conjunctiva was scraped with two cottonwool swabs,
and the virus was extracted by immediately placing each sample in 1.5 ml of Eagle minimal essential medium (MEM) solution (Nissui, Tokyo,
Japan). One swab was used for culture isolation, and the other was used
for PCR.
Culture isolation-neutralization test (NT).
Swabs from the
conjunctiva were collected and inoculated onto HEp-2 cells and HEL
cells. The viruses were passaged in cell cultures, maintained under
Eagle MEM with 2% fetal calf serum, and subpassaged as described
previously (13). Infected cells were identified by the
immunofluorescent-antibody technique with the mouse monoclonal antibody
of Ad (Chemicon International, Inc., Temecula, Calif.). All virus
preparations were tested in a preliminary logarithmic serial dilution
to determine an optimum working dilution of 100 50% tissue culture
infective doses by using four replicate wells per dilution
(5). Neutralization tests were performed on HEp-2 cells and
HEL cells in 96-well microtiter plates to provide an index of CPE.
Antiserum used in this study was obtained from the ATCC.
PCR-RFLP analysis.
Serotype identification was performed by
PCR-RFLP analysis of all of the specimens prior to carrying out the
PCR-sequence method. We described the details of PCR-RFLP analysis
previously (30). Briefly, the 1,004-bp conserved region for
the hexon was amplified in the first-step PCR and, in the second step,
nested PCR was performed to amplify the 956-bp DNA fragment.
Differences in the restriction patterns obtained with three restriction
enzymes, EcoT14I, HaeIII, and HinfI,
were then evaluated, and the serotypes of the clinical specimens were
identified by comparisons with the Ad prototypes.
PCR-sequence method. (i) DNA extraction from prototypes and
clinical specimens.
A 500-µl sample of each of the
prototype-infected cell suspensions and conjunctival scrapings
extracted with MEM solution was collected in a 1.5-ml microtube and
centrifuged at 12,000 × g (High-Speed Microcentrifuge
MRX-15; Tomy Seiko Co., Ltd., Tokyo, Japan) at 4°C for 15 min. The
pellets were used as samples, and DNA was extracted with SepaGene
(Sanko Junyaku Co., Ltd., Tokyo, Japan). After the addition of 50 µl
of Tris buffer, 50 µl of guanidine thiocyanate, 350 µl of
chloroform containing a protein absorbant, and 200 µl of sodium
acetate to the pellet with stirring, the mixture was centrifuged at
12,000 × g at 15°C for 10 min. The supernatant was
recovered, 24 µl of acetate buffer and 600 µl of 100% ethanol were
added, and the solution was centrifuged at 12,000 × g
at 4°C for 10 min. The supernatant was discarded, and 380 µl of
70% ethanol was added to the pellet and, after centrifugation at
12,000 × g at 4°C for 1 min, the supernatant was
discarded again. When the DNA pellet had dried, it was dissolved in 25 µl of sterile water.
(ii) PCR primers.
Ad is a double-stranded DNA virus, and its
full length is approximately 36 kbp (33), with the hexon
accounting for about 2.8 kbp (19). The primers to amplify
the hexon HVRs were designed for the sites with the highest homology.
The first set of primers included HX5-1 (forward primer),
5'-AAGATGGCCACCCCCTCGATGATGCCGCAGT-3', and HX3-1 (reverse
primer), 5'-CACTTATGTGGTGGCGTTGCCGGCCGAGAACGG-3', which were
designed to amplify the region that corresponds to 1 to 2,829 bp in the
hexon base sequence of Ad type 3. The second PCR primer set included
HX5-3 (forward primer), 5'-CACATCGCCGGACAGGATGCTTCGGAGTA-3', and HX3-4 (reverse primer),
5'-GTGTTGTGAGCCATGGGGAAGAAGGTGGC-3'. In the second-step PCR,
the primers were designed to amplify the region that contains all seven
HVRs, which corresponds to bp 40 to 1847 in the Ad type 3 hexon base
sequence (Fig. 1).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 1.
Ad genome. Gene map of the regions surrounding the Ad
hexon protein showing the seven HVRs, the primers, and the regions
sequenced.
|
|
(iii) Nested PCR.
In the first-step PCR, 1 µl of a
100-fold dilution was used when the sample was an Ad prototype whose
template DNA was extracted from infected cells, and 2 µl was used
when the sample was a clinical specimen extracted from conjunctival
scrapings. A 20-µl volume of PCR reaction solution contained Long and
Accurate (LA) PCR Buffer II (LA PCR Kit, Ver. 2; Takara Shuzo
Biomedicals Co., Ltd., Shiga, Japan), deoxynucleoside triphosphate
(dNTP; i.e., dATP, dGTP, dCTP, and dTTP) mixture containing 400 µM
concentrations of each, 0.2 µM concentrations of each primer, and 1 U
of LA Taq DNA polymerase (Takara Shuzo Biomedicals). Shuttle
PCR consisting of a total of 40 cycles of denaturing at 98°C for
10 s, with annealing and extension at 65°C for 6 min, was
carried out (Gene Amp PCR System 9600; Perkin-Elmer, Norwalk, Conn.).
In the second-step PCR, the DNA amplified in the first-step PCR (1 µl
for the prototypes and 2 µl for the clinical specimens) was placed in
20 µl of PCR solution. The reaction solution contained buffer (10 mM
Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2), dNTP mixture
containing 200 µM concentrations of each, 0.2 µM concentrations of
each primer, and 1 U of Taq DNA polymerase (TaKaRa
Taq; Takara Shuzo Biomedicals). The reaction conditions consisted
of conventional PCR in a thermal sequencer (Thermal Sequencer TSR-300;
Iwaki Glass Co., Ltd., Chiba, Japan) with 40 cycles of denaturing at
94°C for 1 min, annealing at 40°C for 1 min, and extension at
72°C for 2 min.
Each PCR test included a sample of double-distilled water that was
treated identically to the virus samples throughout as
a negative
control. As positive controls, 10 pg of Ad2 or Ad41
DNA was used.
Furthermore, to avoid the potential for contamination
for DNA
amplification, DNA-free handling for reaction mixture
preparation and
specimen addition for first- and second-step PCRs
were performed in
separate rooms. Plugged pipette tips, tubes,
and other materials used
for PCR were all
disposable.
(iv) Purification of PCR products.
A 1-µl sample of the
nested-PCR amplification product was subjected to 1.5% agarose gel
electrophoresis and, after being stained with 0.5 mg of ethidium
bromide per ml, an approximately 1.8-kbp band of Ad DNA fragment was
confirmed under UV light. The confirmed amplified DNA in the PCR
products of the specimens was concentrated with Microcon-100 (Amicon,
Inc.), and the unreacted primers were removed. After the buffer was
completely replaced with deionized water, the purified DNA was
electrophoresed, and the DNA concentration was adjusted to
approximately 100 ng/ml based on the density of the band.
(v) Sequence primers.
The sequences of the 12 serotypes
whose base sequences upstream of the hexon had already been elucidated
among the 16 standard serotype strains that were the subject of the
study were compared, and universal primers were designed in the
conserved region present between the individual HVRs (Fig. 1). A total
of six primers was used: for sense, S-28 (5'-ACCCACGATGTGACCAC-3';
bp 152 to 172 in the base sequence of Ad type 3), S-29
(5'-GCCAGCACRTWCTTTGACAT-3'; bp 289 to 308), and S-51
(5'-CCCAACAGACCCAAYTACAT-3'; bp 937 to 956); and for antisense, S-52
(5'-CCCATGTTGCCAGTGCTGTTGTARTACA-3'; bp 986 to 1013), S-53
(5'-AAGGGGTTGACGTTGTCCAT-3'; bp 1555 to 1574), and S-54
(5'-CCAGCATTGCGGTGGTGRTT-3'; bp 1576 to 1595). The melting points
(Tms) of all of the primers were calculated from
their percent GC content and primer length, and they were designed to
have a Tm of ~60°C.
(vi) Cycle sequence.
A total volume of 20 µl, consisting
of 8.0 µl of Terminator Ready Reaction Mix (DNA Sequencing Kit;
Perkin-Elmer Applied Biosystems, Warrington, Great Britain), 1.0 µl
(3.2 µM) of primers for each sequence, 1.0 µl of purified DNA, and
10 µl of sterile water, was placed in a 0.2-ml Gene Amp microtube,
and a cycle sequence with a total of 25 cycles at 96°C for 10 s,
50°C for 5 s, and 60°C for 4 min was carried out (Gene Amp PCR
System 9600). The samples were analyzed by means of an autosequencer
(ABI Prism 310 Genetic Analyzer; Perkin-Elmer).
Analysis of PCR-sequence method. (i) Homology between
prototypes.
The sequences of a total of 16 standard serotype
strains were compared. The sequences of prototypes previously reported
were derived from GenBank. DNASIS (Hitachi Software Engineering Co., Ltd., Tokyo, Japan) was used for sequence alignment and analysis. After
referring to the HVR sites described by Crawford-Miksza et al.
(7), we compared the HVRs of the 16 serotypes, and the range
of each of the new HVRs was reestablished. The rate of homology of each
HVR was evaluated.
(ii) Serotype identification of clinical specimens.
The
predicted amino acid sequences of the clinical specimens were aligned
according to the sequences of the 16 serotype prototypes. The homology
rate between clinical specimens and Ad prototypes was determined. The
serotypes of the clinical specimens were determined by comparing the
homology rate. The serotypes identified by the PCR-sequence method were
compared with the results of serotype identification by culture
isolation-NT and PCR-RFLP analysis.
(iii) Nucleotide sequence accession numbers.
Sequence data
from this article have been deposited with the GenBank/EMBL/DDBJ data
libraries under the following accession numbers: hexon genes Ad11
(AB018424), Ad14 (AB018425), Ad34 (AB018426), and Ad35 (AB018427). The
amino acid sequences of these residues were deduced.
| |
RESULTS |
PCR of Ad hexons and determination of HVR sequences.
The
entire approximately 2.8-kbp hexon region of all 16 prototypes could be
amplified by LA PCR with the first-step PCR primer set. In addition,
the approximately 1.8-kbp region that includes the hexon HVR1 to HVR7
regions could be amplified by conventional PCR with the second-step PCR
primer set (Fig. 2). The size of the
amplified product of the second-step PCR matched the size predicted
from the known sequence (corresponding to 1,808 bp in the base sequence
of Ad type 3).
View larger version (67K):
[in this window]
[in a new window]
|
FIG. 2.
Electrophoresis of PCR-amplified products. Agarose gel
electrophoresis shows the specific band of ca. 2.8 kbp obtained in the
first PCR (a) and of ca. 1.8 kbp obtained in the second PCR (b) from
DNA samples of all 16 prototypes. Numbers above the lanes correspond to
the Ad serotypes, and lane M contains molecular weight standards
(pHY).
|
|
In addition, it was also possible to determine the HVR sequences of all
of the newly sequenced Ad prototypes (Ad types 11,
14, 34, and 35) and
the clinical specimens by using the six sequence
primers described
above. The region containing seven HVRs was
amplified and sequenced
directly from as few as 10
3 virus particles by two-step PCR
(data not shown). On the other
hand, by using single amplification
products, it was not possible
to obtain complete or clear sequences of
these regions, particularly
in clinical specimens (data not shown). The
minimum limit of detection
of 10
3 copies is sufficient
because the nested PCR assay is highly
sensitive.
Comparison of sequences between Ad prototypes.
The amino acid
sequences derived from the nucleotide sequences of the 16 serotype
prototypes are shown in Fig. 3, divided into two loops: loop 1 and loop 2. The results of aligning the 16 different serotypes of the prototype Ad strains revealed that six of
the seven HVRs, HVR6 being the only exception, had changed. As an
example of the reason for this, in HVR1 the sequence was expanded by
four amino acids upstream, but the homology of the prototypes of the 16 serotypes at these four amino acids was at least 25%. In contrast, the
homology of the four amino acids immediately preceding them was at
least 50%. In HVR2, the sequence was expanded by six amino acids
downstream. The homology of this region in the prototypes of the 16 serotypes was low (16.7%). In contrast, at least 83.3% homology was
conserved in the next six amino acids. The newly designed HVRs were as
follows (amino acids): HVR1 (132 to 189), HVR2 (195 to 211), HVR3 (219 to 230), HVR4 (254 to 272), HVR5 (279 to 300), HVR6 (317 to 328), and
HVR7 (430 to 474) (amino acid numbers according to Crawford-Miksza et
al. [7].
View larger version (116K):
[in this window]
[in a new window]
|
FIG. 3.
Comparison of predicted amino acid sequences of seven
hexon HVRs among 16 prototypes. The sequences of the region of loop 1 and loop 2 have been aligned to obtain maximal homology. Deduced amino
acid sequences taken from previous reports were as follows (accession
number): Ad1 (X67709) (25), Ad2 (J 01917) (17),
Ad5 (X76550) (19), Ad6 (X67710) (25), Ad3
(X76549) (27), Ad7 (X76551) (26), Ad8 (X74663),
Ad19 (X98359), Ad37 (X98360), Ad4 (X84646) (27), Ad40
(X51782) (35), and Ad41 (X51783) (34).
|
|
The amino acid homology rate in all seven HVRs among the prototypes are
shown in Table
1. The six pairs of
serotypes that
showed highest amino acid homology in whole HVRs were:
Ad3 and
Ad7, 77.5%; Ad11 and Ad35, 74.0%; Ad14 and Ad34, 69.0%, Ad11
and
Ad34, 58.0%; Ad11 and Ad14, 57.5%; and Ad1 and Ad2, 56.3%. Each
of these prototypes belongs in the same subgenus. The homology
rate of
each HVRs, that is, HVR1 to HVR7, among the prototypes
is shown
in Tables
1 to
4.
Serotype identification of clinical specimens.
The results of
serotyping by the culture isolation-NT, PCR-RFLP, and PCR-sequence
methods for 31 clinical specimens are shown in Table
5. The serotype determined by the
PCR-sequence method was based on homology with the prototypes in HVR
regions 4, 5, and 7. With the exception of the three specimens in
subgenus D, in which the results of the culture isolation-NT and
PCR-RFLP analysis conflicted, the homology of the clinical specimens in this study was 83.1% for Ad7 and 90% or more for the other serotypes. In the 26 specimens, the results of serotyping by culture isolation-NT and PCR-RFLP analysis and those obtained by PCR-sequence method were
identical. In the five specimens in which there was a discrepancy between the results of the culture isolation-NT and PCR-RFLP analysis, the serotype determined by the PCR-sequence method was identical to
that determined by culture isolation-NT.
View this table:
[in this window]
[in a new window]
|
TABLE 5.
Percent homology between clinical isolates and prototypes
in HVR4, -5, and -7 by the PCR-sequence methoda
|
|
| |
DISCUSSION |
In this study we developed a method of rapidly and accurately
identifying the serotype of Ad by using a new technique, the PCR-sequence method. The serotypes identified by comparing the hexon
HVR sequences were identical to the results obtained by the
neutralization assay, which is the conventional serotyping method. This
new method saves time and labor compared to culture isolation-NT, and
it is a more objective and accurate method than PCR-RFLP analysis.
The results obtained by recent molecular biological approaches have now
demonstrated the structure of the Ad hexon (3, 29) and the
existence of HVRs (6, 7), and serotype-specific HVRs are
thought to be important regions for serotype identification. The fact
that serotype-specific sequences are restricted to seven independent
HVRs (6, 7) means that the neutralization epitopes are
located in one to several sites in these regions. The comparison of
hexon HVRs in this study revealed homology within the same subgenus to
be high. These results mean that the HVRs reflect the subgenus, which
is a group that has similar DNA properties (36). In
particular, in subgenus B, whose sequence we elucidated in this study,
B1 and B2 were both restricted to within their respective individual
subgenus. B1 is known to cause acute respiratory tract infections, and
B2 is said to be common in urinary tract infection (20). Our
results suggested that the hexon that contains the HVRs may also be
associated with disease specificity.
Neutralization epitopes are the optimal regions for serotype
identification by the PCR-sequence method and therefore it was necessary to elucidate the regions where neutralization is governed in
the seven HVRs. Neutralization epitopes may be regions where there is
low homology. The mean maximum homology rate between any two serotypes
was 74.0% for HVR3 and 66.9% for HVR6. This indicates that HVR3 and
HVR6 may not be involved in the neutralization regions which should
reflect the serotype specificity, and this finding is consistent with
the report by Crawford-Miksza et al. (6), who compared HVRs
in subgenus D. HVR3 had the highest mean maximum homology rate with
other serotypes, 74.0%, but it was demonstrated that high rates are
restricted to within the same subgenus. Actually, both types Ad3 and
Ad7 and types Ad34 and Ad35 had 100% homology in HVR3. Since subgenera
are groups that have similar genomic properties (36),
members of the same subgenus tend to show high homology. In HVR2, high
homology was observed outside the subgenus, as shown by the 66.7%
homology between Ad7 (subgenus B) and Ad37 (subgenus D), the 62.5%
homology between Ad7 (subgenus B) and Ad41 (subgenus F), and the 52.6% homology between Ad19 (subgenus D) and Ad6 (subgenus C). HVR6 had high
homology outside the subgenus, in particular with subgenera B, D, and
E, where it was greater than 50%. Collectively, HVR2 and HVR6 are not
candidates for neutralization epitopes. In contrast, HVR4, -5, and -7 are the HVRs with the mean maximum homologies with other
serotypes
54.3, 55.9, and 58.0%, respectivelyand there were few
serotypes with a maximum homology outside the subgenus. These findings
suggest that these three HVRs are serotype specific.
Homology can be compared by substituting amino acids for nucleic acids,
but since neutralization is based on the recognition of proteins, it
seems valid to make comparisons based on amino acids. Moreover, at
present, since it is not clear whether neutralization epitopes are
involved within HVRs alone, further assessment is needed to determine
whether the PCR-sequence method, which only compares hexon HVRs, can be
used to determine serotype. However, Ad serotypes are not only
determined by NT of the antigenic determinant 
, which is present on
the external, exposed portion of the hexon, but by hemagglutination
inhibition tests for the antigenic determinant 
, which is present at
the outer end of the fibers (22, 24). Recently, molecular
biological approaches examining the fiber regions were reported
(8), and we are currently analyzing the serotype-specific
regions within fibers (23).
While 49 human Ad serotypes are currently known (15, 31,
38), the clinical specimens in this study were compared with 16 different prototypes. Our objective was to detect Ad in the conjunctival scrapings of conjunctivitis patients and to develop a new
method of identifying the associated serotypes. There have been seven
principal etiologic serotypes of conjunctivitis in Japan recently: Ad
types 3, 4, 7, 8, 11, 19, and 37 (1, 2, 12, 16). In the
western hemisphere, subgenus C Ad serotypes 1, 2, 5, and 6 are also
known to cause acute conjunctivitis (4, 18, 21). Application
of the PCR-sequence method to specimens collected from Ad
conjunctivitis patients in Asia and western countries is currently
being attempted, but the Ad prototypes prepared in this study are
almost enough for the identification of ordinary serotypes that cause
adenoviral conjunctivitis. However, an extremely rare virus to cause
conjunctivitis, Ad34, was discovered in a conjunctivitis patient in
Japan in this study. In the future, the HVR sequences of all 49 serotypes currently known should be determined for accurate
identification of serotypes that have not been known as causes of
conjunctivitis in the past. Ad is also a cause of acute pharyngitis,
acute hemorrhagic cystitis, and infantile intussusception
(20). The PCR-sequence method is not restricted to the field
of ophthalmology but should also prove useful as a method of Ad
detection and serotyping in specimens related to a variety of diseases.
LA PCR and conventional PCR were compared in the first-step PCR, but
there was more nonspecific amplification in the low-molecular-weight region with conventional PCR than with LA PCR (data not shown). Furthermore, since the reaction conditions of LA PCR included a higher
Tm value when the primers were long and the
difference of temperatures between annealing and DNA extension was
smaller, there was less nonspecific reaction with shuttle PCR, which
uses two different temperatures, than with conventional PCR, which uses
three (data not shown).
These findings show that the results obtained by the PCR-sequence
method coincided with the results of culture isolation-NT, and it was
demonstrated that the PCR-sequence method is useful as a method of
serotype identification. The series of processes, i.e., sequence
alignment, comparison of HVR homology, and serotype identification,
were all performed manually in this study, but it took only 3 days for
the determination of the serotype. If advances could be made to
simplify the process in order to reduce the time required for
sequencing and if new analysis software that enables calculation of the
homology based on amino acid alignment is developed, serotype
identification will require even less time. The PCR-sequence method
targeting HVR4, -5, and -7 could become a standard method of detecting
and identifying Ads in clinical specimens.
| |
ACKNOWLEDGMENTS |
The skillful technical assistance by Akira Oshima, from
Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., is gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Ophthalmology, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan. Phone: 81-45-787-2683. Fax: 81-45-781-9755. E-mail:
takeuchi{at}med.yokohama-cu.ac.jp.
| |
REFERENCES |
| 1.
|
Aoki, K.,
M. Kato,
H. Ohtsuka,
K. Ishii,
N. Nakazono, and H. Sawada.
1982.
Clinical and aetiological study of adenoviral conjunctivitis, with special reference to adenovirus type 4 and 19 infections.
Br. J. Ophthalmol.
66:776-780[Abstract/Free Full Text].
|
| 2.
|
Aoki, K.,
R. Kawana,
I. Matsumoto,
G. Wadell, and J. C. de Jong.
1986.
Viral conjunctivitis with special reference to adenovirus type 37 and enterovirus 70 infection.
Jpn. J. Ophthalmol.
30:158-164[Medline].
|
| 3.
|
Athappilly, F. K.,
R. Murali,
J. J. Rux,
Z. Cai, and R. M. Burnett.
1994.
The refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 Å resolution.
J. Mol. Biol.
242:430-455[Medline].
|
| 4.
|
British Journal of Ophthalmology.
1977.
Adenovirus keratoconjunctivitis.
Br. J. Ophthalmol.
61:73-75[Free Full Text]. (Editorial.)
|
| 5.
|
Crawford-Miksza, L., and D. P. Schnurr.
1994.
Quantitative colorimetric microneutralization assay for characterization of adenoviruses.
J. Clin. Microbiol.
32:2331-2334[Abstract/Free Full Text].
|
| 6.
|
Crawford-Miksza, L. K., and D. Schnurr.
1996.
Adenovirus serotype evolution is driven by illegitimate recombination in the hypervariable regions of the hexon protein.
Virology
224:357-367[Medline].
|
| 7.
|
Crawford-Miksza, L., and D. P. Schnurr.
1996.
Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues.
J. Virol.
70:1836-1844[Abstract].
|
| 8.
|
Eiz, B.,
T. Adrian, and P. Pring-Åkerblom.
1995.
Immunological adenovirus variant strains of subgenus D: comparison of the hexon and fiber sequences.
Virology
213:313-320[Medline].
|
| 9.
|
Fife, K. H.,
R. Ashley,
A. F. Shields,
D. Salter,
J. D. Meyers, and L. Corey.
1985.
Comparison of neutralization and DNA restriction enzyme methods for typing clinical isolates of human adenovirus.
J. Clin. Microbiol.
22:95-100[Abstract/Free Full Text].
|
| 10.
|
Gooding, L. R.
1992.
Virus proteins that counteract host immune defenses.
Cell
71:5-7[Medline].
|
| 11.
|
Greber, U. F.,
M. Willetts,
P. Webster, and A. Helenius.
1993.
Stepwise dismantling of adenovirus 2 during entry into cells.
Cell
75:477-486[Medline].
|
| 12.
|
Guo, D. F.,
M. Shinagawa,
K. Aoki,
H. Sawada,
S. Itakura, and G. Sato.
1988.
Genome typing of adenovirus strains isolated from conjunctivitis in Japan, Australia, and the Philippines.
Microbiol. Immunol.
32:1107-1118[Medline].
|
| 13.
|
Hierholzer, J. C.,
N. O. Atuk, and J. M. Gwaltney.
1975.
New human adenovirus isolated from a renal transplant recipient: description and characterization of candidate adenovirus type 34.
J. Clin. Microbiol.
1:366-376[Abstract/Free Full Text].
|
| 14.
|
Hierholzer, J. C.,
Y. O. Stone, and J. R. Broderson.
1991.
Antigenic relationships among the 47 human adenoviruses determined in reference horse antisera.
Arch. Virol.
121:179-197[Medline].
|
| 15.
|
Hierholzer, J. C.,
R. Wigand,
L. J. Anderson,
T. Adrian, and J. W. M. Gold.
1988.
Adenoviruses from patients with AIDS: a plethora of serotypes and a description of five new serotypes of subgenus D (type 43-47).
J. Infect. Dis.
158:804-813[Medline].
|
| 16.
|
Ishii, K.,
N. Nakazono,
K. Fujinaga,
S. Fujii,
M. Kato,
H. Ohtsuka,
K. Aoki,
C. W. Chen,
C. C. Lin,
M. M. Sheu,
K. H. Lin,
B. S. Oum,
S. H. Lee,
C. H. Chun,
T. Yoshii, and S. Yamazaki.
1987.
Comparative studies on aetiology and epidemiology of viral conjunctivitis in three countries of east Asia-Japan, Taiwan and South Korea.
Int. J. Epidemiol.
16:98-103[Abstract/Free Full Text].
|
| 17.
|
Jörnvall, H.,
G. Akusjärvi,
P. Aleström,
H. von Bahr-Lindström,
U. Pettersson,
E. Appella,
A. V. Fowler, and L. Philipson.
1981.
The adenovirus hexon protein. The primary structure of the polypeptide and its correlation with the hexon gene.
J. Biol. Chem.
256:6181-6186[Abstract/Free Full Text].
|
| 18.
|
Kinchington, P. R.,
S. E. Turse,
R. P. Kowalski, and Y. J. Gordon.
1994.
Use of polymerase chain amplification reaction for the detection of adenoviruses in ocular swab specimens.
Invest. Ophthalmol. Vis. Sci.
35:4126-4134[Abstract/Free Full Text].
|
| 19.
|
Kinloch, R.,
N. Mackay, and V. Mautner.
1984.
Adenovirus hexon: sequence comparison of subgroup C serotypes 2 and 5.
J. Biol. Chem.
259:6431-6436[Abstract/Free Full Text].
|
| 20.
|
Mei, Y.-F., and G. Wadell.
1995.
Molecular determinants of adenovirus tropism.
Curr. Top. Microbiol. Immunol.
199(Pt. 3):213-228.
|
| 21.
|
Morris, D. J.,
A. S. Bailey,
R. J. Cooper,
P. C. Turner,
R. Jackson,
G. Corbitt, and A. B. Tullo.
1995.
Polymerase chain reaction for rapid detection of ocular adenovirus infection.
J. Med. Virol.
46:126-132[Medline].
|
| 22.
|
Norby, E.
1969.
The structural and functional diversity of adenovirus capsid components.
J. Gen. Virol.
5:221-236[Abstract/Free Full Text].
|
| 23.
|
Oshima, A.,
N. Itoh,
K. Tanaka,
H. Ishiko,
S. Ohno, and K. Aoki.
1996.
The sequence analysis of fiber knob genomic region on adenovirus types 19 and 37.
Invest. Ophthalmol. Vis. Sci.
37:S1030.
|
| 24.
|
Philipson, L.
1984.
Structure and assembly of adenoviruses.
Curr. Top. Microbiol. Immunol.
109:1-52[Medline].
|
| 25.
|
Pring-Åkerblom, P., and T. Adrian.
1993.
The hexon genes of adenoviruses of subgenus C: comparison of the variable regions.
Res. Virol.
144:117-127[Medline].
|
| 26.
|
Pring-Åkerblom, P.,
F. E. J. Trijssenaar, and T. Adrian.
1995.
Hexon sequence of adenovirus type 7 and comparison with other serotypes of subgenus B.
Res. Virol.
146:383-388[Medline].
|
| 27.
|
Pring-Åkerblom, P.,
F. E. J. Trijssenaar, and T. Adrian.
1995.
Sequence characterization and comparison of human adenovirus subgenus B and E hexons.
Virology
212:232-236[Medline].
|
| 28.
|
Roba, L. A.,
R. P. Kowalski,
A. T. Gordon,
E. G. Romanowski, and Y. J. Gordon.
1995.
Adenoviral ocular isolates demonstrate serotype-dependent differences in in vitro infectivity titers and clinical course.
Cornea
14:388-393[Medline].
|
| 29.
|
Roberts, M. M.,
J. L. White,
M. G. Grüter, and R. M. Burnett.
1986.
Three-dimensional structure of the adenovirus major coat protein hexon.
Science
232:1148-1151[Abstract/Free Full Text].
|
| 30.
|
Saitoh-Inagawa, W.,
A. Oshima,
K. Aoki,
N. Itoh,
K. Isobe,
E. Uchio,
S. Ohno,
H. Nakajima,
K. Hata, and H. Ishiko.
1996.
Rapid diagnosis of adenoviral conjunctivitis by PCR and restriction fragment length polymorphism analysis.
J. Clin. Microbiol.
34:2113-2116[Abstract].
|
| 31.
|
Schnurr, D. P., and M. E. Dondero.
1993.
Two new candidate adenovirus serotypes.
Intervirology
36:79-83[Medline].
|
| 32.
|
Takiff, H. E.,
S. E. Straus, and C. F. Gordon.
1981.
Propagation and in vitro studies of previously non-cultivable enteral adenoviruses in 293 cells.
Lancet
ii:832-834.
|
| 33.
|
Toogood, C. I. A.,
J. Crompton, and R. T. Hay.
1992.
Antipeptide antisera define neutralizing epitopes on the adenovirus hexon.
J. Gen. Virol.
73:1429-1435[Abstract/Free Full Text].
|
| 34.
|
Toogood, C. I. A., and R. T. Hay.
1988.
DNA sequence of the adenovirus type 41 hexon gene and predicted structure of the protein.
J. Gen. Virol.
69:2291-2301[Abstract/Free Full Text].
|
| 35.
|
Toogood, C. I. A.,
R. Murali,
R. M. Burnett, and R. T. Hay.
1989.
The adenovirus type 40 hexon: sequence, predicted structure and relationship to other adenovirus hexons.
J. Gen. Virol.
70:3203-3214[Abstract/Free Full Text].
|
| 36.
|
Wadell, G.
1984.
Molecular epidemiology of human adenoviruses.
Curr. Top. Microbiol. Immunol.
110:191-220[Medline].
|
| 37.
|
Wickham, T. J.,
P. Mathias,
D. A. Cheresh, and G. R. Nemerow.
1993.
Integrins  v 3 and v5 promote adenovirus internalization but not virus attachment.
Cell
73:309-319[Medline].
|
| 38.
|
Wigand, R.,
T. Adrian, and F. Bricout.
1987.
A new human adenovirus of subgenus D: candidate adenovirus type 42.
Arch. Virol.
94:283-286[Medline].
|
| 39.
|
Wold, W. S. M., and L. R. Gooding.
1991.
Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions.
Virology
184:1-8[Medline].
|
| 40.
|
Wold, W. S. M.,
A. E. Tollefson, and T. W. Hermiston.
1995.
E3 transcription unit of adenovirus.
Curr. Top. Microbiol. Immunol.
199(Pt. 1):237-274.
|
Journal of Clinical Microbiology, June 1999, p. 1839-1845, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Zhu, Z., Zhang, Y., Xu, S., Yu, P., Tian, X., Wang, L., Liu, Z., Tang, L., Mao, N., Ji, Y., Li, C., Yang, Z., Wang, S., Wang, J., Li, D., Xu, W.
(2009). Outbreak of Acute Respiratory Disease in China Caused by B2 Species of Adenovirus Type 11. J. Clin. Microbiol.
47: 697-703
[Abstract]
[Full Text]
-
Russell, W. C.
(2009). Adenoviruses: update on structure and function. J. Gen. Virol.
90: 1-20
[Abstract]
[Full Text]
-
Miura-Ochiai, R., Shimada, Y., Konno, T., Yamazaki, S., Aoki, K., Ohno, S., Suzuki, E., Ishiko, H.
(2007). Quantitative Detection and Rapid Identification of Human Adenoviruses. J. Clin. Microbiol.
45: 958-967
[Abstract]
[Full Text]
-
Selvaraj, G., Kirkwood, C., Bines, J., Buttery, J.
(2006). Molecular epidemiology of adenovirus isolates from patients diagnosed with intussusception in melbourne, australia.. J. Clin. Microbiol.
44: 3371-3373
[Abstract]
[Full Text]
-
Ebner, K., Rauch, M., Preuner, S., Lion, T.
(2006). Typing of Human Adenoviruses in Specimens from Immunosuppressed Patients by PCR-Fragment Length Analysis and Real-Time Quantitative PCR.. J. Clin. Microbiol.
44: 2808-2815
[Abstract]
[Full Text]
-
Lee, J. A, Kim, N. H., Kim, S. J., Choi, E. H., Lee, H. J.
(2005). Rapid Identification of Human Adenovirus Types 3 and 7 from Respiratory Specimens via Multiplex Type-Specific PCR. J. Clin. Microbiol.
43: 5509-5514
[Abstract]
[Full Text]
-
Moya-Suri, V, Schlosser, M, Zimmermann, K, Rjasanowski, I, Gurtler, L, Mentel, R
(2005). Enterovirus RNA sequences in sera of schoolchildren in the general population and their association with type 1-diabetes-associated autoantibodies. J Med Microbiol
54: 879-883
[Abstract]
[Full Text]
-
Chmielewicz, B., Nitsche, A., Schweiger, B., Ellerbrok, H.
(2005). Development of a PCR-Based Assay for Detection, Quantification, and Genotyping of Human Adenoviruses. Clin. Chem.
51: 1365-1373
[Abstract]
[Full Text]
-
Fujimoto, T., Okafuji, T., Okafuji, T., Ito, M., Nukuzuma, S., Chikahira, M., Nishio, O.
(2004). Evaluation of a Bedside Immunochromatographic Test for Detection of Adenovirus in Respiratory Samples, by Comparison to Virus Isolation, PCR, and Real-Time PCR. J. Clin. Microbiol.
42: 5489-5492
[Abstract]
[Full Text]
-
Sarantis, H., Johnson, G., Brown, M., Petric, M., Tellier, R.
(2004). Comprehensive Detection and Serotyping of Human Adenoviruses by PCR and Sequencing. J. Clin. Microbiol.
42: 3963-3969
[Abstract]
[Full Text]
-
Li, L., Shimizu, H., Doan, L. T. P., Tung, P. G., Okitsu, S., Nishio, O., Suzuki, E., Seo, J. K., Kim, K. S., Muller, W. E. G., Ushijima, H.
(2004). Characterizations of Adenovirus Type 41 Isolates from Children with Acute Gastroenteritis in Japan, Vietnam, and Korea. J. Clin. Microbiol.
42: 4032-4039
[Abstract]
[Full Text]
-
Lin, B., Vora, G. J., Thach, D., Walter, E., Metzgar, D., Tibbetts, C., Stenger, D. A.
(2004). Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses. J. Clin. Microbiol.
42: 3232-3239
[Abstract]
[Full Text]
-
Adhikary, A. K., Inada, T., Banik, U., Numaga, J., Okabe, N.
(2004). Identification of Subgenus C Adenoviruses by Fiber-Based Multiplex PCR. J. Clin. Microbiol.
42: 670-673
[Abstract]
[Full Text]
-
Wang, D., Coscoy, L., Zylberberg, M., Avila, P. C., Boushey, H. A., Ganem, D., DeRisi, J. L.
(2002). Microarray-based detection and genotyping of viral pathogens. Proc. Natl. Acad. Sci. USA
99: 15687-15692
[Abstract]
[Full Text]
-
Saitoh-Inagawa, W., Tanaka, K., Uchio, E., Itoh, N., Ohno, S., Aoki, K.
(2001). Genome Typing of Adenovirus Type 34 Isolated in Two Cases of Conjunctivitis in Sapporo, Japan. J. Clin. Microbiol.
39: 4187-4189
[Abstract]
[Full Text]
-
Imai, Y., Kameya, S., Ohkoshi, M., Yamaki, K., Sakuragi, S.
(2001). Identification of the Hexon Region of an Adenovirus Involved in a New Outbreak of Keratoconjunctivitis. J. Clin. Microbiol.
39: 2975-2977
[Abstract]
[Full Text]
-
Adhikary, A. K., Numaga, J., Kaburaki, T., Kawashima, H., Kato, S., Araie, M., Miyata, K., Shimizu, H., Yagyu, F., Suzuki, E., Ushijima, H.
(2001). Rapid Detection and Typing of Oculopathogenic Strain of Subgenus D Adenoviruses by Fiber-Based PCR and Restriction Enzyme Analysis. IOVS
42: 2010-2015
[Abstract]
[Full Text]
-
Shen, J., Taylor, N., Duncan, L., Kovesdi, I., Bruder, J. T, Forrester, J. V, Dick, A. D
(2001). Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium. Br J Ophthalmol
85: 861-867
[Abstract]
[Full Text]
-
Allard, A., Albinsson, B., Wadell, G.
(2001). Rapid Typing of Human Adenoviruses by a General PCR Combined with Restriction Endonuclease Analysis. J. Clin. Microbiol.
39: 498-505
[Abstract]
[Full Text]
-
Xu, W., McDonough, M. C., Erdman, D. D.
(2000). Species-Specific Identification of Human Adenoviruses by a Multiplex PCR Assay. J. Clin. Microbiol.
38: 4114-4120
[Abstract]
[Full Text]
-
Elnifro, E. M., Cooper, R. J., Klapper, P. E., Bailey, A. S.
(2000). PCR and Restriction Endonuclease Analysis for Rapid Identification of Human Adenovirus Subgenera. J. Clin. Microbiol.
38: 2055-2061
[Abstract]
[Full Text]
-
Takeuchi, S., Itoh, N., Uchio, E., Tanaka, K., Kitamura, N., Kanai, H., Isobe, K., Aoki, K., Ohno, S.
(1999). Adenovirus Strains of Subgenus D Associated with Nosocomial Infection as New Etiological Agents of Epidemic Keratoconjunctivitis in Japan. J. Clin. Microbiol.
37: 3392-3394
[Abstract]
[Full Text]
Top
Abstract
Introduction
