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Journal of Clinical Microbiology, March 1999, p. 844-847, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Use of Restriction Fragment Analysis and Sequencing
of a Serotype-Specific Region To Type Adenovirus Isolates
Quan-Gen
Li,1,2,*
Anna
Henningsson,1
Per
Juto,1
Fredrik
Elgh,1,3 and
Göran
Wadell1
Department of Virology, Umeå
University,1 and
Department of
Microbiology, Division of NBC Defence, Defence Research
Establishment,3 Umeå, Sweden, and
Institute of Infectious Diseases, Beijing,
China2
Received 8 June 1998/Returned for modification 30 September
1998/Accepted 9 December 1998
 |
ABSTRACT |
Restriction fragment analysis and sequencing of a serotype-specific
region were used to type 12 and 2 clinical isolates, respectively. Both
molecular methods produced clear-cut results that completely correlated
with that of the neutralization test.
| |
TEXT |
Some human adenovirus serotypes
(Ads), such as Ad1 to Ad8, Ad40, and Ad41, are frequently associated
with epidemic outbreaks of acute respiratory tract, gastroenteric
tract, and conjunctiva infections (10, 17, 20).
Identification of the serotype is important for the epidemiological
survey of the adenoviruses.
A serotype is defined mainly on the basis of a neutralization test
which either exhibits no cross-reaction with others or shows
a homologous/heterologous titer ratio greater than 16 in both directions. In the case of homologous/heterologous titer ratios of 8 or 16, the hemagglutination inhibition test must be applied and manifest a lack of crossreaction (19).
Repeated experiments are often required, especially for the
uncommon and unexpected serotypes. Sometimes, ambiguous results appear
because of cross-reactions between different serotypes and the
so-called intermediates. Moreover, the number of serotypes is still
increasing (11), making serotyping even more
complicated. Therefore, an alternative efficient and rapid typing
method is desired.
Restriction fragment analysis (RFA) is one of the powerful methods that
can be used to study the molecular epidemiology of adenoviruses
(1-6, 11-16, 21). A comparison of all the precise restriction patterns revealed that all the genome types of a serotype usually present similar patterns. We therefore reasoned that RFA could
be used for the identification of serotypes.
Our previous work has demonstrated that the adenovirus hexon possesses
seven hypervariable regions, of which three regions (A1, A3, and B1)
are obviously serotype specific (9, 18). Therefore, it
seemed feasible to try to identify serotypes by sequencing of a
serotype-specific region (SSR), especially the variable B1 region of
the hexon loop 2.
In Northern Sweden, 20 to 60 adenovirus isolates have been obtained
each winter since the late 1980s at the Department of Clinical
Virology, University Hospital, Umeå, Sweden. However, during winter
1994-1995, 122 adenovirus strains were isolated. The significantly
increased number of cases over the seasonal norm indicated that an
epidemic outbreak of adenovirus infections had appeared. All the
adenovirus strains were isolated in A549 cells and identified by a
direct immunofluorescence test or a latex agglutination test.
Twelve isolates were selected from the 122 strains, especially those
from patients with severe diseases (Table
1). Four standard prototypes of Ad2, Ad3,
Ad5, and Ad7 were originally obtained from the American Type Culture
Collection.
All adenovirus isolates were propagated in A549 cells. Viral DNA
preparation was performed by the method described previously (16,
22). DNA restriction enzyme cleavages were carried out according
to the manufacturer's instructions. The DNA restriction fragments were
separated and analyzed by the methods described previously
(16). The adenovirus genome types were denominated according
to the nomenclature system described previously (13, 17).
The primer pair for PCR was designed according to the alignment result
of 14 different hexons described previously (18). They were
located at the conserved region of loop 2 and were QG65 (GGAACTGCCTAATTACTGTTTTCC) and QG66
(TTGGAGTACAGAAAACTTCTCCA). The PCR fragment covered
the hypervariable and SSR B1 of the hexon. The templates were obtained
from culture medium by chloroform extraction. The PCR fragments from
strains 95355 and 95553 were ligated into the pT7-Blue plasmid and then
were transformed into NovaBlue cells. The nucleotide sequences for both
strands were determined by the dideoxynucleotide chain determination method.
The alignment of the predicted amino acid sequences of two strains,
95553 and 95355, and of the 18 serotypes was determined with the
program MegAlign (DNAStar Inc., Madison, Wis.).
Results and discussion.
According to the highest pairwise
comigrating restriction fragments (13) between a new strain
and a known serotype, the four representative strains (95553, 95355, 9544, and 9558) (Table 1) were predicted to belong to the serotypes
Ad7, Ad5, Ad3, and Ad2, respectively (Fig.
1). Strain 95553 was identical to genome type Ad7b (17). Strain 95355 shared the same patterns as Ad5 genome types with the exception of the BamHI pattern
(4, 8). It was consequently recognized as a new genome type,
which was named Ad5b. Strain 9544 shared the same patterns as Ad3a and
Ad3a1 through Ad3a8 with the exception of the BglII pattern
(14). It therefore represented a new genome type of Ad3,
which was named Ad3a9. Strain 9558 shared the same restriction patterns
as Ad2 genome types with the exception of HindIII
pattern (4, 7). It represented a new genome type of Ad2,
which was named Ad2p1.
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FIG. 1.
Diagrammatic representation of DNA restriction patterns
of four genome types digested with BamHI, BglII,
BstEII, HindIII, and SmaI. The
restriction patterns of 2p1, 3a9, 5b, and 7b were from representative
strains 9558, 9544, 95355, and 95553, respectively. Numbers on the left
indicate fragment sizes (in kilobase pairs). Double bands (indicated
with asterisks) were unable to be separated by agarose gel
electrophoresis as used in this study.
|
|
Of the 12 selected adenovirus isolates, six were determined to be
genome type Ad7b and four were Ad5b. They were the dominating
genome
types among the 12 isolates
studied.
The alignment of the amino acid sequences of the hexon B1 region from
two new isolates and 18 prototypes demonstrated serotype-specific
features. Strain 95553 and Ad7 and strain 95355 and Ad2 had distinctly
higher percentages of similarity than the mean homology between
members
of each subgenus, 72.9 and 90.4%, respectively (Table
2). Therefore, according to the highest
percentage of similarity
to a known serotype, strain 95553 was
determined to be serotype
Ad7 and strain 95355 was determined to be
Ad2.
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TABLE 2.
Amino acid sequence similarities of the B1 regions
between clinical isolates and 18 different
adenovirus serotypesa
|
|
To evaluate the two molecular methods, conventional neutralization
tests were performed. The representative strains 9558,
9544, 95355, and
95553 were identified as Ad2, Ad3, Ad5, and Ad7,
respectively. This
result completely corroborated both RFA and
SSR sequencing. The
processing of the neutralization test takes
at least 3 weeks, whereas
each of the other two methods takes
only 1
week.
The two molecular methods applied herein for typing are based on
accumulated data obtained over more than 2 decades. The two
methods, at
present, can be efficiently used only for the identification
of common
Ads, i.e., those serotypes of which sequences of the
hexon B1 region
and restriction patterns have been studied. To
type all of the known
serotypes of any clinical isolate, the restriction
patterns of as many
presently known genome types as possible are
required. Also, the
sequences of the hexon B1 region from uncommon
serotypes have to be
determined.
When the amino acid sequence of the B1 region of strain 95553 was
aligned with the sequences of two earlier characterized
Ad7b strains
(
17), an amino acid substitution at the tip of
loop 2 (
2) was discovered: 27Ser in BC30 and KCH4 was replaced
by
Tyr in 95553 (Fig.
2). This indicates
that genetic drift had
occurred within a certain genome type.
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FIG. 2.
Comparison of the hexon B1 region of prototype Ad7p and
those of three strains of genome type Ad7b (95553, BC30, and KCH4).
Unconserved amino acids are shown in boxes.
|
|
Nucleotide sequence accession numbers.
Accession numbers for
the two sequences determined in this study were obtained from GenBank,
AF51950 for strain 95553 and AF51949 for strain 95355.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Swedish Foundation for
Strategical Research and the Swedish Medical Research Council (grant
K97-06X-05688-18A).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Feng Tai Road
26, Building 5-1-9, Beijing 100039, China. Phone and fax:
86-10-63845559.
| |
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Journal of Clinical Microbiology, March 1999, p. 844-847, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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