JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1988, p. 2652-26560095-1137/88/122652-05$02.00/0Copyright C 1988, American Society for Microbiology

Vol. 26, No. 12

Nucleic Acid Hybridization for Detection ofCell Culture-Amplified Adenovirus

CINNIA HUANG* AND RUDOLF DEIBEL

Wadsworth Center for Laboratories and Research, New York State Department of Health, Box 509,Albany, New York 12201-0509

Received 28 April 1988/Accepted 18 August 1988

A number of recombinant plasmids containing genomic segments of adenovirus were constructed. Sevencloned probes, as well as total adenovirus type 2 (Ad2) and Adl6 genomic DNA, were tested by a nucleic acidhybridization technique for sensitivity and specificity in detecting adenoviruses in infected cells. AdenovirusDNA was spotted onto a nitrocellulose filter and hybridized with 32P-labeled DNA probes. The probes, totalAd2 genomic DNA, and plasmid pAd2-H (containing the hexon gene from Ad2 DNA) all detected 10 referenceserotypes of five genomic subgroups (A through E) with similar sensitivities. However, plasmid pAd2-Hrequired less preparation time than did total Ad2 DNA. Probes pAd2-F (containing the fiber gene from Ad2)and pAdl6-BD (containing the BamHI D fragment from Adl6) hybridized only with reference serotypes fromthe homologous subgroups (C and B, respectively). Of 101 patient isolates amplified in cells, pAd2-H detected100% of ali isolates from both the homologous and the heterologous subgroups. The detection rates for pAd2-Fwere 100% (subgroup C) and 3.6% (subgroups A, B, and D), and those for pAdl6-BD were 100% (subgroupB) and 9.4% (subgroups A, C, and D). A commercial biotinylated product (Pathogene II) was also included inthis study for comparison.

Nucleic acid hybridization techniques have been appliedto detect DNA viruses (3, 9, 12, 17, 18, 23, 26, 28, 30-32, 34)and RNA viruses (6, 19). In this study, viral nucleic acidsequences were detected by using radioactively labeledprobes. The method was found to be specific, sensitive, andrelatively rapid.Human adenovirus isolates are classified in genome sub-

groups A through F on the basis ofDNA sequence homologyand have been distinguished by antigenic determinants onthe hexon, fiber, and penton base. The hexon and fiber genesof adenovirus 2 (Ad2) from subgroup C have been mapped inthe viral genome (1, 4). Recombinant DNA technology hasyielded cloned probes for rapid detection of adenovirus ininfected cells. We describe here some clones containing thehexon and fiber genes and demonstrate their sensitivity andspecificity.Specimen preparation and DNA hybridization. Ten refer-

ence serotypes (serotype 12 from subgroup A; serotypes 3,7, and 11 from subgroup B; serotypes 1, 2, 5, and 6 fromsubgroup C; serotype 29 from subgroup D; and serotype 4from subgroup E) were propagated in KB cells. When acytopathic effect was observed in .50% of cells, the super-natant fluids were collected and stored at -20°C untilassayed. A total of 101 patient isolates (31 nasopharyngeal,27 stool, 23 eye, 12 urine, 3 lung, 2 sputum, 1 cerebrospinalfluid, 1 brain, and 1 not specified) amplified in humanembryonic lung cells were prepared in the same way.Sample fluids (25 ,u) were treated with 25 pi of proteinase

K (400 p.g/ml) for 30 min at 37°C followed by nucleic aciddenaturation with a hot, saturated solution of sodium iodide(50 pi at 2.5 mg/ml) (8). The strong binding of nucleic acidsto the nitrocellulose filter in sodium iodide permits omissionof the baking step. The treated samples (100 vtl) wereincubated at 95°C for 20 min and spotted onto a nitrocellu-lose filter in a 96-well manifold apparatus (Schleicher &Schuell, Inc., Keene, N.H.). The filter was air dried and

* Corresponding author.

prehybridized for 4 h at 65°C in 6x SSC (1x SSC is 0.15 MNaCI plus 0.015 M sodium citrate)-1 x Denhardt solution (5)supplemented with 100 ptg of denatured sonicated salmonsperm DNA per i. i. One microgram of the DNA probe waslabeled with 32P by nick translation (27) to a specific activityof 108 cpm/jxg and added to the hybridization buffer in 2xSSC-1x Denhardt solution-O.1% sodium dodecyl sulfate-10% dextran sulfate. The filter was hybridized overnight at65°C and washed three times in 2x SSC-0.1% sodiumdodecyl sulfate for 15 min at room temperature and threetimes in 0.2x SSC-0.1% sodium dodecyl sulfate for 30 minat 65°C. The results were recorded by autoradiography(XAR-2; Eastman Kodak Co., Rochester, N.Y.) at -70°Cfor various times in the presence of an enhancing screen.DNA preparation and plasmid construction. Recombinant

plasmids were prepared from pBR322 and pUC8 (22, 33).Ad2 and Adl6 DNAs were kindly provided by NandoChatterjee. Both viruses had been purified by CsCI gradientcentrifugation (20) before DNA extraction (2). DNA frag-ments were isolated from agarose gels by electrophoresisonto a DEAE membrane (35). The cloning procedures wereas described elsewhere (21).A BamHI C fragment (map position, 42 to 59.5) of Ad2

DNA was inserted into the BamHI site of pUC8 to createplasmid pAd2-2 (Fig. 1A). A 3.3-kilobase HindIII fragmentencoding the hexon gene (1) (map position, 50.1 to 59.5) wasexcised from pAd2-2 and inserted into the HindIII site ofpUC8 to generate pAd2-H (Fig. 1A). A 2.5-kilobase HpaI-SmaI fragment (map position, 85 to 91.9) containing theentire fiber gene (4) of Ad2 was cloned into pBR322, and theresulting recombinant plasmid, pAd2-F, was propagated andpurified (Fig. 1A). With the assumption that the fiber genesof Ad2 (subgroup C) and Adl6 (subgroup B) are similarlyarranged in the right-hand portion of the genomes, we clonedthe BamHI D fragment (map position, 82 to 92.6) of Adl6DNA into pUC8 (Fig. 1B).

Selection of probe. To study whether the cloned recombi-nant plasmid could detect adenovirus, we analyzed repre-

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DNA 0 20 40 60

--- -

lb ~̀~ ~ ~~~'l_- ,

,- ,21 %

-_ - BamHI C , -/ lIEXNE1- E// z////GENE//

(42.0) (50.1) (59.5) (50.1) (59.5)

I I

80 1001

iFIBER1 91

11GENE,1(8...........(9.9).....:::.:::::

A Ad-12 3 7 Il 1 2 5 6 29 4zoI * ***!1L-

TOTALAd2 -DNA

PROBE: TOTAL Ad2 DNA

(42.0) (50.)

0 20 40.. . . .~~~~~~~~~~~~~~~~~~~~~~~

(85.0)

60 80 109

BAd-12 3 7 1I 2 5 6 29 4

z

* * ~I

`* r

oB * *..0*,:.`-YW . .....w`*,

:*TOTAL i.`Ad2 --`? ;*ONA

PROBE: pAd 2-H

FIG. 2. Detection of adenovirus DNA with 32P-labeled probes.Total Ad2 DNA was 2.5, 1.25, 0.625, or 0.25 ng. Lane numbersindicate adenovirus types.

(92 6)

FIG. 1. Construction of recombinant plasmids containing Ad2(A) and Adl6 (B) DNA fragments. Top lines represent the total Ad2and Adl6 DNA genomes. Kb, Kilobase pairs.

A 1O-FOLD DILUTION

sentative viruses of subgroups A through E, using purifiedtotal Ad2 DNA or cloned recombinant plasmids as probes todetect DNA sequences. Among the cloned probes, plasmidpAd2-H contained the entire hexon gene. The hexons havebeen reported to carry the antigen responsible for adeno-virus neutralization (11, 25). The amino acid compositionsfor hexon proteins from human adenoviruses differ but little,suggesting that the hexon is a well-conserved entity. Thesensitivity of the dot blot hybridization for adenovirus de-tection with total Ad2 DNA as probe resembled that ob-served when pAd2-H was used as probe (Fig. 2). In contrastto Kidd et al. (17), we observed nonspecific hybridization ofpBR322 DNA in fecal specimens examined for rotavirus(data not shown). Therefore, the insert BamHI C fragment,which is virus specific, and recombinant plasmid pAd2-2were used as comparison probes. No significant difference ofsensitivity was found between recombinant plasmid pAd2-2and the cloned insert, the BamHI C fragment (Fig. 3). Thesamples examined were amplified in tissue culture cells, notclinical specimens.

Identification of group- and subgroup-specific probes.Probe pAd2-H could detect the serotypes from five genomicsubgroups (Fig. 2). The lengths of the fibers in the subgroupsdiffer (A, 28 to 31 nm; B, 9 to 11 nm; C, 23 to 31 nm; D, 12to 13 nm; and E, 17 to 18 nm) (24). Therefore, fiber geneswere thought suitable for subgroup-specific probes. TheDNA sequence of human Ad2 (map position, 70.7 to 100) hasbeen determined (7, 13-15, 29), and the sequence encodingthe fiber gene has been mapped in the genome (map position,

Ad-2

zo

g-w-J

ta-N

TOTALAd 2:-*DNA

Ad-29

a

PROBE: pAd 2-2

B

z

g--

:D a

ta-a

TOTALAd2:PJDNA

10-FOLD DILUTION

Ad- 2 Ad-29~~~lA~~~~~

PROBE: BamHI C FRAGMENT

FIG. 3. Detection of adenovirus DNA with 32P-labeled probes.Total Ad2 DNA was 25, 2.5, or 0.25 ng.

AAdeno-2

(36 kt

BADENO-16 DNA

(36 kb)

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2654 NOTES

ASUBGROUP: A B

Ad- 2 3 7 Ilzo-J

-J

ILi

(\J1

C D E-A*2 5 6 10 19 294

*m«`«:e:~~~~~~~~~~~~~~~1.: ..::..

.b~ ^ 6

. S

TOTALo.Ad2~

DNAPROBE: pAd2-H

BSUBGROUP: A B c D F

Ad-12 3 7 Il 1 2 5 6 O 19 29 4

TABLE 1. Serotypes of 101 patient isolates

Genome No. of isolates St No. of isolatessubgroup in subgroup eroype with serotype

A 4 31 4

B 37 3 147 10il 621 334 135 3

C 45 1 132 255 7

D) 15 8 99 119 229 230 1

z

-J

Ô)-J

(4i

TOTALAd2ONA

PRc

SUBCROUP: A

Ad-12zo

t--

:Z)-J

o-J

N1

TOTALAd2DNA

wl-.1

e*1"

tOBE:pAd2-F

B

3 7 r

*e.le le.

PROBE: pAd 16- B

FIG. 4. Detection of representativewith 32P-labeled probes. All adenovirstrains amplified in KB cells. Total Ad20.05 ng. Lane numbers indicate adenov

85.9 to 90.8). Plasmid pAd2-F was cgentire fiber gene of Ad2. The fiber îbeen determined, but restrictionreported (35). The BamHI D fragm92.6) of Adl6 was assumed to contacloned. The sequence homology osubgroup is reported (10) to be 81between different subgroups is onl

whether the probes prepared from Ad2 DNA (subgroup C)and from Adl6 (subgroup B) showed different levels ofcross-reactivity. Probes pAd2-H and pAd2-F proved to

i* * detect different members within subgroup C with similar* *# sensitivity (Fig. 4A and B). Probes pAd2-F and pAdl6-BD

detected the serotypes from subgroups C and B, respectivelyW* (Fig. 4B and C). These results suggest that pAd2-H is a

group-specific probe and that pAd2-F and pAdl6-BD aresubgroup-specific probes.

Patient isolates amplified in human embryonic lung cellsand serotyped by neutralization (Table 1) provided materialsfor dot blot hybridization with three probes, pAd2-H, pAd2-F, and pAdl6-BD. Some hybridization results with 32p-labeled probes are shown in Fig. 5. Three samples (b4 fromsubgroup D and d7 and b8 from subgroup B [Fig. SA]) were

C D E scored positive in the original X-ray film. A fecal specimen5 6 10 19 29 4 (arrows in Fig. 5) positive by electron microscopy for

adenovirus was deposited on the filter by using the sameprocedure without DNA extraction and assayed with threeprobes. The results revealed adenovirus DNA, but not fromsubgroups B and C. A commercial Pathogene II kit (EnzoBiochem, Inc.) containing biotinylated total Ad2 DNA wasincluded to compare sensitivity and specificity. pAd2-Hhybridized with all samples, whereas Pathogene II detectedadenoviruses with less sensitivity (82.8%) (Table 2). ProbespAd2-F and pAdl6-BD, as well as Pathogene Il, hybridizedwith the samples belonging to the homologous subgroups.Low levels of cross-reactivity were observed in two samplestested with probe pAd2-F (specificity, 96.4%) and in six

D samples tested with pAdl6-BD (specificity, 90.6%). Fordetecting members of the homologous subgroup, the positive

viruses of five subgroups predictive values were 95.7 and 86.1%, respectively; theuses tested were reference negative predictive values and sensitivity were 100% forDNA was 2.5, 1.25, 0.5, or both probes.irus types. Conclusion. Application of DNA hybridization to detect

adenoviruses is based on determinations (10) that viruses ofonstructed to contain the different subgroups share limited DNA sequence homology.gene of Adl6 has not yet Several laboratories have successfully used cloned recombi-site mapping has been nant plasmids for diagnosis of respiratory (9, 16, 18, 26, 34)ent (map position, 82 to and enteric (12, 17, 23, 32) adenovirus infections.in the fiber gene and was In this study, we constructed probes to detect adenovirusf adenoviruses within a DNA in infected cells. We aim to construct both a singleO to 90%, whereas that probe to detect viruses from different subgroups (A throughIy 10 to 15%. We tested F) and subgroup-specific probes. For this purpose, several

J. CLIN. MICROBIOL.

t* 1

2

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NOTES 2655

TOTAL Ad2 DNA

2 3 4 5 6 7 8 9 10 11 12

b 0 *

de*e C)-a- *0f r

* a * *h * IDC

PROBE: pAd2-H

B J 2 3 4

a le Obac .

d "

e

f O9h *

PROBE: pAd2-F

5 6 7 8 9 10

e

% !I

TOTALAdE DNA

Il 12

eee

TOTAL Ad2 ONA

7 8 9 10 Il 12

f

C.

a

PROBE: Ad 16 BamHI D FRAGMENTFIG. 5. Detection of viral nucleic acid after short-term amplifi-

cation of isolates in human embryonic lung cells. Total Ad2 DNAwas 2.5, 1.25, 0.625, or 0.25 ng. Arrows indicate fecal specimen.Lane numbers indicate adenovirus types.

TABLE 2. Comparison of Pathogene Iia and 32P-labeled probesNo. positive/no. studied with probe:

Genomicsubgroup Pathogene pAd2-H pAd2-F Total Adl6 BamHI-D

pAiH pA2F Tta d6 of Adl6

A 1/4 4/4 1/4 1/4 1/4B 26/37 37/37 0/37 37/37 37/37C 45/45 45/45 45/45 10/45 1/45D 10/13 15/15 1/15 9/15 4/15

a Contains Ad2 Bio-Probe; product of Enzo Biochem, Inc.

(map position, 50.1 to 72.8), respectively, was demonstratedby Hyypia (16). The advantage of the cloned probe is that alarge quantity can be easily prepared. Because of the com-plex composition of many clinical materials and our obser-vation of nonspecific hybridization between clinical speci-mens and pBR322, the use of the insert as probe is highlyrecommended.Gomes et al. (9) reported that the cloned plasmid carrying

the BamHI C and D fragments (map position, 29 to 59.5) ofAd2 detected adenoviruses of subgroups B, C, and E.Similarly, a HindIII D fragment (map position, 41 to 50.1) ofAd2 detected viruses of subgroups A, B, and C (28). Ourprobe pAd2-H (map position, 50.1 to 59.5) detected se-rotypes from five subgroups (A through E). Use of a smallerfragment (map position, 42 to 45.3) as probe (34) yieldedfalse-negatives. Therefore, the genomic locations and sizesof the probes are critical for the sensitivity of the hybridiza-tion assay. Evidently the probe containing a well-conservedhexon gene (pAd2-H) recognizes a subset of adenoviruses.Whether this probe can detect Ad40 and Ad4l remains to bedemonstrated.For subgroup specificity, two probes, pAd2-F (map posi-

tion, 85 to 91.9) and pAdl6-BD (map position, 82 to 92.6),were used and shown to hybridize with the reference se-rotypes from the homologous subgroups (C and B, respec-tively). Our results agree with those of Lehtomaki et al. (18)and Virtanen et al. (34). Their cloned probe of Ad3 (mapposition, 83.1 to 89.7) detected Ad3 and Ad7 of subgroup Bbut not Ad2 of subgroup C. All of these data suggest that theright-hand part of the genome shows diversity among virusesof different subgroups. The specificities were calculated as96.4% for pAd2-F and 90.6% for pAdl6-BD when patientisolates amplified in cells were examined. The size of theprobe is deemed responsible for the cross-reactivity. Thefiber lengths for Ad2 and Adl6 are 23 to 31 and 9 to 11 nm,respectively. The fiber gene of Ad2 contains 4.9% of thegenome (map position, 85.9 to 90.8). In our constructs, 6.9%of the genomic DNA is present in pAd2-F and 10.6% ispresent in pAdl6-BD. The presence of an additional DNAsequence other than the fiber gene of Ad2 may causecross-hybridization with the heterologous subgroups. Weare trying to reduce the viral DNA sequence of Adl6 in therecombinant plasmid to increase the specificity.

We thank Beena Ehu for her technical assistance.

probes were 32p labeled and used to detect viral nucleic acidin the sample. Total Ad2 DNA, cloned probes, and arestriction fragment from Ad2 gave positive signals withsimilar sensitivity (Fig. 2 and 3). Under the conditions used,10 pg of homologous DNA could be detected by overnightincubation (data not shown). Detection of 10 and 100 pg ofadenovirus DNA with total Ad2 DNA and the cloned probe

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c

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a

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