JOURNAL OF CLINICAL MICROBIOLOGY, JUIY 1992, p. 1703-1710 0095-1137/92/071703-08$02.00/0
Vol. 30, No. 7
Copyright X3 1992, American Society for Microbiology
Conserved Sequences of the Adenovirus Genome for Detection of All Human Adenovirus Types by Hybridization TIM H. SCOTT-TAYLOR AND GREGORY W. HAMMOND* Cadham Provincial Laboratory and Department of Medical Microbiology, Canada University of Manitoba, Winnipeg, Manitoba R3C
3Y1,
Received 5 November 1991/Accepted 15 April 1992
The application of DNA hybridization directly to clinical specimens has the potential of improving the diagnosis of fastidious types of adenovirus. In this study, the genome of one adenovirus type from each human subgenus (A to F) was systematically evaluated by hybridization for homologous sequences to find the optimal common probe for detection of all human adenovirus types. The area of cross-hybridization, most closely defined with adenovirus type 2 (Ad2), mapped from map units 11.4 to 16.1 and 26.9 to 29.7 and, principally, to a central area of the genome between map units 47.5 and 65.2. The last area, enclosing the hexon gene, was highly conserved. Cloned probes generated from this area demonstrated the greatest homology to heterologous types by hybridization analysis. A HindIII-BgIII clone containing the hexon gene of Ad2 within narrow confines reacted most evenly with all adenoviral types and detected the DNA of all other subgenera with a sensitivity 2 logs greater than that of a complete genomic Ad2 probe. The most homologous adenoviral gene sequences were observed in genes involved with DNA replication or intimately connected to the hexon in the early capsid formation. These results show that the hexon gene constitutes the best single region of the adenovirus genome for use as a genus-specific probe for the diagnosis of all human adenoviral subgenera by DNA hybridization. have relied on the reactivity of randomly cloned fragments of the adenovirus genome as reagents. The following work systematically evaluated the cross-reactivity of all genomic sequences and tested a battery of cloned fragments to find the optimal common probe for detection of all human adenovirus types. Studies of the melting temperature of interspecies genomic DNA hybrids have indicated a high degree of genomic sequence homology between adenovirus types within one subgenus (16). One adenovirus type from each subgenus, therefore, was chosen for analysis to represent all the human species.
Adenoviruses are common and ubiquitous pathogens. Extensive epidemiological surveys have placed an adenoviral agent as the cause of 5 to 10% of the total juvenile pneumonias and respiratory infections of North American children (5, 12, 13). Respiratory infection with adenoviruses is regularly responsible for a number of infant deaths (30) and considerable morbidity in certain adult populations (21). The improved health of military recruits administered a vaccine of live adenovirus type 4 (Ad4) and Ad7 implemented to counter acute respiratory disease reduced health costs by nearly twice the $4.83 million cost of vaccine development within 2 years (9). Adenovirus particles are seen in some 5 to 17% of cases of gastroenteritis in children up to 5 years of age, a rate second only to that of rotavirus as a cause of pediatric diarrhea (38). Allowing for the fact that only half of the adenoviral infections contacted develop into illness (5), Chanock (8) has estimated from serological prevalence studies that the average individual undergoes a minimum of two or three clinical episodes of adenoviral infection during childhood. Enteric adenovirus types are commonly at a high concentration, up to 1011 particles per g of stool (15). The majority of isolates observed by the electron microscope in cases of adenoviral gastroenteritis are the fastidious enteric types Ad4O and Ad41, which cannot be reliably cultured for subsequent identification. The electron microscope is estimated to detect adenoviruses only when they are present at a concentration of >106 particles per g and possibly fails to detect many adenovirus isolates for most of the period of illness (11). A variety of more sensitive tests have therefore been devised to improve adenovirus diagnosis in fecal specimens. Monoclonal antibody immunoassays developed to date (17, 31) have targeted epitopes prone to variation because of immunological selection (29). DNA hybridization studies *
MATERIALS AND METHODS Cell culture and virus strains. Cell lines HEp-2, A549, and 293 and the prototype strains of adenovirus species from each subgenus A to F were obtained from the American Type Culture Collection (Rockville, Md.). A single type representative of each subgenus (Ad3l 1315, Ad7 Gomen, Ad2 Adenoid 6, Ad8 Trim, Ad4 RI-67, and Ad4l Tak of serogroups A, B, C, D, E, and F, respectively) was chosen for the availability of restriction endonuclease maps (6, 19, 33-35). Ad2 maps were generated by Pustell Sequence Analysis software (IBI, New Haven, Conn.) on a genomic sequence recorded by GenBank (National Institutes of Health). Ad4l was grown in 293 cells, and other types were propagated in HEp-2 or A549 cells. Batches of 10 150-cm2 flasks with semiconfluent monolayers were infected with the virus suspended in Eagle's minimum essential medium or, later, L15 medium (no. 10-511; Flow Laboratories, Inc., Mississauga, Ontario, Canada) at a multiplicity of 1 to approximately 10 50% tissue culture infective doses per cell. The viral overlay was replaced after 1 to 2 h with medium containing 2% fetal bovine serum and changed every third day. Viral titer was assessed as described by Reed and Muench (26) from application of log dilutions of stock culture supernatants to cells in 4 duplicate wells of 24-well plates.
Corresponding author. 1703
1704
SCOTT-TAYLOR AND HAMMOND
Preparation and treatment of viral DNA. Infected cells were monitored daily with an inverted microscope for the appearance of cytopathic effect. Cells showing the typical swelling and grapelike clustering of adenovirus infection were harvested and subjected to several cycles of freezethawing. Cell lysate was clarified by centrifugation, and virus was concentrated at 30,000 rpm for 90 min in a type 35 (no. 330926; Beckman, Burnaby, British Columbia, Canada) rotor over a cushion of 1.8 g of CsCl per cm3. The interphase was homogenized with an equal volume of Freon (trichlorotrifluoroethane; Fisher Scientific) in an omnimixer (Sorvall Ltd., Norwalk, Conn.) to remove lipid. Virions were then purified by density gradient centrifugation, first in a preformed 1.2- to 1.5-g/cm3 continuous CsCl gradient and last by isopycnic centrifugation in 1.344 g of CsCl per cm3. Virus bands were collected by a fractionator (Beckman no. 343890), dialyzed against 100 mM Tris-100 mM MgCl, and treated with 50 ,ug of DNase and RNase per ml to purify the intact virions of cellular components. Treated cells were then digested with 1% sodium dodecyl sulfate (SDS) and 50 ,ug of proteinase K per ml for 1 h at 37°C. Viral protein was removed by several extractions with equal volumes of phenol and chloroform, and the DNA was precipitated with 0.1 volume of 5 M NaCl and 2 volumes of ethanol. DNA concentration and purity were assessed spectrophotometrically and visually by electrophoretic comparison with lambda phage DNA standards. These DNAs gave no reaction when hybridized with HEp-2 cell DNA, and the restriction profiles accorded with the patterns of prototype strains (1). Cloning and plasmid extraction. Hindlll fragments of Ad2 DNA were ligated to plasmid pGem 3Z (Promega Biotec, Mississauga, Ontario, Canada) and added to competent Escherichia coli JM109. Transformants were distinguished as white colonies on Luria-Bertani plates supplemented with ampicillin, isopropyl thio-D-galactoside, and 5-bromo-4chloro-3-indolyl-I3-D-galactopyranoside (X-Gal; BRL, Gaithersburg, Md.), indicating that the P-galactosidase gene in the plasmid was disrupted by insertion of viral DNA. Transformants were stored as broth cultures supplemented with 15% glycerol at -70°C and were plated out every 6 months. Subcultures were chipped from stored aliquots without defrosting. Plasmids containing viral DNA inserts (Ad2 HindIll fragments A, B, C, D, E, G, H, I, and J) were differentiated by ampicillin (50 ,ug/ml; Sigma Chemical Co.) susceptibility after replica plating. The identities of inserted fragments were found by matching quick plasmid preparation (3) bands with Ad2 genomic DNA digested with the same restriction enzymes. Large plasmid DNA preparations were purified by isopycnic centrifugation in 1 g of CsCl per ml at 45,000 rpm for 36 h in an SW50.1 rotor (Beckman no. 340079). BglII subclones of HindlIl plasmids were ligated to the compatible BamHI site in plasmid pBR322. Electrophoresis and blotting. Purified adenoviral DNA (1 to 2 ,ug) was digested with at least 10 U of restriction enzyme in buffers specified by the manufacturer (Boehringer Mannheim). The 20-pl reaction volumes were mixed with 10 ,u of tracking dye, consisting of 0.07% bromophenol blue, 0.7% SDS, and 33% glycerol, and pipetted into wells in 150-ml slabs of 0.8% agarose submerged in 89 mM Tris-borate-10 mM EDTA (pH 8) buffer containing 0.5 jig of ethidium bromide per ml. Lambda phage DNA (Boehringer Mannheim no. 208396) digested with EcoRI and/or HindIll and a small quantity of Ad4l DNA digested with SmaI were included in each agarose gel as molecular weight markers and control. Electrophoresis was carried out at 1.7 V/3.5
J. CLIN. MICROBIOL.
mA/cm of gel overnight in a Sub Cell (Bio-Rad, Mississauga, Ontario, Canada) apparatus. As the dye front approached the end of the agarose, the gel was transferred to a UV transilluminator (Ultraviolet Products Inc., San Gabriel, Calif.) and the fluorescent DNA bands were photographed through a red filter with a Polaroid camera and type 57 film. An alkaline adaptation (25) of the Southern blot was used to transfer the DNA to nylon membranes (0.45-,um mesh; Micron Separations Inc.), which were stored at 4°C after being baked at 68°C for 6 to 12 h. Slot blot analysis of Ad2 probes. Adenoviral, lambda, and cellular DNAs, diluted from 10 ,ug to 10 pg/ml, were denatured with the addition of 0.1 volume of 3 M NaOH, neutralized with 0.1 volume of 3 M ammonium acetate after 30 min of incubation, and equilibrated to physiological salt conditions with the addition of 0.3 volumes of 20x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M Na citrate). A 150-,u volume of each dilution was applied by slot blot apparatus (no. 03431; Schleicher & Schuell, Keene, N.H.) under very low vacuum to nylon membrane prewetted in 6x SSC. Membranes were washed twice in 6x SSC, air dried, and baked at 65°C for 8 h prior to hybridization. Hybridization. Membranes were sealed in a plastic envelope (Sears Seal-a-Meal apparatus) and prehybridized at 68°C for 3 to 6 h in a rocking water bath in a buffer of 0.5% SDS, 6x SSC, and 5x Denhardt's solution (lx Denhardt's solution is 0.3% Ficoll 400, 0.3% bovine serum albumin, and 0.3% polyvinylpyrrolidone) containing 100 mg of sheared, denatured herring sperm DNA per ml. The prehybridization solution was discarded through a clipped corner of the envelope and replaced with 1 ml of the same buffer per cm2 of membrane; the buffer also contained 100 mg of freshly denatured herring sperm DNA and at least 107 cpm of nick-translated probe, prepared with commercial reagents (kit no. N500; Amersham, Oakville, Ontario, Canada), per ml. Hybridization under the same stringent temperature conditions was performed overnight. Membranes were subsequently given two pairs of 30-min washes at 68°C, first in 2x SSC-0.1% SDS and then in 0.2x SSC-0.1% SDS. After excess moisture was blotted, membranes were resealed in plastic envelopes and enclosed with Kodak XAR-5 film between intensifying screens (Dupont Quanta III, no. 224032; Picker International, Winnipeg, Manitoba, Canada) at -70°C until the homologous Ad4l DNA control lane was showing strongly. The comparative intensity of hybridization was assessed by densitometer (model GS-300; Hoefer Scientific Instruments; directed by an IBM GS360 Data System program) set at 100% gain on the homologous control and expressed as a proportion of the total absorption per lane. Probes were removed from membranes after autoradiography by two 15-min washes, first in 250 ml of 0.4 M NaOH and then in 250 ml of 0.1 x SSC-0.1% SDS-0.2 M Tris (pH 7.5) in a rocking water bath at 45°C. The membranes were checked for residual radioactivity before being resealed in envelopes and stored. Nylon membranes could be hybridized two or three times with little loss in intensity of the DNA bands. RESULTS Hybridization of adenovirus subgenera. Areas of sequence conservation in the adenovirus genome were examined by hybridization of the restriction endonuclease fragments of one type of each subgenus with a genomic Ad4l probe. Ad2 DNA fragments cleaved with a variety of restriction enzymes were separated by gel electrophoresis (Fig. 1A). Clal,
VOL. 30, 1992
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ADENOVIRUS CROSS-HYBRIDIZATION X
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FIG. 1. Hybridization of Ad2 DNA with a genomic Ad4l probe. (A) Electrophoresis of 2-,ug aliquots of Ad2 DNA digested with a variety of restriction enzymes. Ad41 DNA (0.5 pug) was included in the outside lane as a control. (B) Appearance of cross-reactive Ad2 fragments on film after blotting and hybridization of gel in panel A with genomic Ad4l DNA.
used in conjunction with a number of other enzymes in the genomic digests of Ad2 DNA, liberated fragments of 3,000 to 4,000 bp closely enclosing the hexon gene. The gel was hybridized under stringent conditions with an Ad4l genomic DNA probe. A 48-h autoradiograph registering those Ad2 DNA fragments containing sequences with a high degree of homology to Ad4l is shown in Fig. 1B. Despite application of only a quarter of the quantity of DNA used for each Ad2 digest, the Ad4l control lane reacted strongly with the homologous probe. Transfer of sufficient DNA in small fragments was indicated by the strong signal from the 670-bp fragment in the XhoI and ClaI dual-digest lane. Each of the liberated hexon gene bands was evident. Fragments that did not hybridize can be discounted as not containing any sequence of identity with Ad4l under the experimental conditions of stringency. Fragments observed in Fig. 1B were denoted by underscoring their positions on the Ad2 genome in the restriction maps in Fig. 2. A repetitive pattern was discerned, three sequences being principally represented in the cross-reaction of every lane. When all those fragments that did not appear in the film were deducted, sequences of homology between Ad4l and Ad2 were most closely defined by the overlap of the positive fragments and were enclosed in two anterior areas between map units 11.4 to 16.1 and 27.4 to 29.7 and in a central area between map units 47.5 and 65.2. A value for each discernible fragment, figured as a proportion of the total absorbance of the lane, was assigned by densitometric analysis of autoradiographs and attributed to the position of the fragment on the genomic maps in Fig. 2. The individual values of comigrating fragments could not be distinguished. The three homologous areas reacted disproportionately; the anterior areas consistently registered less strongly than the central area. This was most clearly illustrated with the PvuI digest of Ad2, in which a small fragment between map units 52 and 60 was responsible for more than half of the total absorbance of that lane. Hybridization of subgenera A, B, D, and E. The process of
blotting, hybridization, and autoradiography was repeated with Ad4, Ad7, Ad8, and Ad3l electrophoresis gels, representing subgenera E, B, D, and A, respectively, and can be followed in Fig. 3. The areas of positive fragment overlap, denoting areas of consistent hybridization, of all five types tested are compared in Fig. 4, and it is evident that the hybridizing fragments of subgenera A, B, D, and E combined to describe a wide area of each genome that includes each of the sequences of Ad2 homology with Ad41. A fourth area of marginal hybridization was inconsistently seen in some lanes of Ad8 and Ad4 and repetitively with Ad7 but was not observed when hybridized as a separate fragment of the Ad31 genome. As with Ad2, the fragments reacted with different levels of intensity, and three separate regions, where the enzymes separated areas appropriately, could be distinguished in at least one lane of each type. The relative absorbance values for the positive fragments of one digest of each type are plotted in Fig. 5, and it can be seen that the cross-reactive sequences of different subgenera react with Ad4l probes in a disproportionate fashion similar to the three-tier pattern given by areas of the Ad2 genome. Two anterior areas of sequence identity reacted with 5 to 20% of the total absorbance per lane in each case. The greatest intensity of reaction was displayed by a central fragment of each genome between 50 and 60 map units; this region accounted for over half of the total absorbance of each
digest. Evaluation of various Ad2 fragments as probes. The abilities of various areas of the Ad2 genome, isolated as electroeluted fragments or cloned as HindlIl plasmids, to perform as probes in the detection of adenoviral DNAs of different subgenera were tested on slot blots. The reaction of the whole genome as a probe is visualized in the first panel of Fig. 6, where the homologous reaction was detected up to five dilutions at 100 pg of Ad2 DNA. Reactions with other subgenera were variable; 10 or 100 ng of Ad4, Ad7, and Ad8 DNA were detected, concentrations 100- to 1,000-fold higher
1706
J. CLIN. MICROBIOL.
SCOTT-TAYLOR AND HAMMOND B 22-6%
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A 113% 713 762 836 898 594 A B iFi D IE 1C 71.7% 18 3% 26 519 594 713 762 836 898 A B ,F ID'E IG 1D/E Hi C 282% 381 527% 191% 799 78 173 32.3 379 418 509 734 797 895 972 B F aC i A H II E IG IK inJa D I 77%c'D 15 6% M 7 7%C/D 768% L 428 57 398 475 619 720 237 935 II H I B D E I C G1 A IF I 89% 186% 661% 64% 42.8 26 57 237 398 475 519 619 720 935 C F B 1J IG IH I D/EI D/E i A K11I1 I 8 2% 177% 59-3/E 148% 17.3 22-0 51.4 59.2 5.2 34.4 40-2 C pF D A B HI 1E I aI 69% 167% 137% 531% 96% 26.3 27-4 46 6
D 116% 149% 73.5% 2.8 1d 9 18 3 36 3 40 2 52 6 56-8 76 1 76 7 u J1 E 11 F , I I B C D I HI A Sma MV 8-3% 53-9%A/s 14,7% 23-0% 53.9%A/8 --L 16 1 22 9 26-9 53-8 66.6 82-9 D C l F GII A E I I Xho I 6-8% 57.4% 35-8% 16-1 22-9 26-9 2.6 51.9 53-8 66-6 82.9 D A C Hi I F Gi III E I I Xho I tCIa 50 7%D/E 29 0% 20 3% 507ND/E Map units 1,0 50 70 60 80 210 310 410
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~~~ II KB 10 20 5 15 30 25 35-9 FIG. 2. Restriction fragments of Ad2 DNA with homology to the Ad4l genome. The intensities of bands in the autoradiograph in Fig. 1B were assessed by densitometer. The proportions of the total optical density, expressed as percentages of the absorbance of each lane, are presented below the cross-reactive (underlined) fragments on the restriction map. II
than in the homologous reaction, while not even 1 p.g of Ad31 DNA was seen with the genomic probe in this time frame. HindlIl fragments from either genomic terminus gave relatively weak reactions with the other subgenera DNAs, as exemplified by the electroeluted HindIII K fragment probe in the second panel. This fragment from the right terminus reacted highly specifically, detecting Ad2 DNA 3 to 4 log dilutions less concentrated than the DNA of other types. Probes containing either of the anterior regions of the Ad2 genome found homologous in the hybridization with Ad4l, such as the HindlIl B and C plasmids or electroeluted Sacl G and SmaI F fragments, gave reactions intermediate between those of the whole genome and the specific termini. The SacI-G probe in the third panel reacted weakly with Ad31 and Ad4 DNA. The most even reaction of any Ad2 probe was given by the HindIlI A-fragment plasmid in the fourth panel, which showed consistent detection of heterologous DNA 2 to 3 log concentrations higher than the detection level of homologous DNA. This plasmid, from map units 42 to 70 of the Ad2 genome, was subcloned, and a single BglII plasmid retained the equilateral cross-reaction of the parent plasmid. A small fragment called HP II, closely enclosing the Ad2 hexon gene, was excised with enzymes
I
HindIll and PvuII. This electroeluted fragment of 3,453 bp, starting 532 bp upstream of the hexon gene and cut by PvuII 25 bp after the hexon terminus, hybridized with Ad3l DNA with a sensitivity comparable to that of other subgenera. The relative difference between homologous and heterologous reactions with an HP II probe in the last panel of Fig. 6 was within approximately 2 log dilutions with all species of DNAs, including Ad31.
DISCUSSION Genomic adenovirus DNAs were hybridized to locate genetic sequences common to all the human types of the Mastadenovirms genus. The accuracy of definition of areas of nucleotide identity was determined by the availability of known restriction sites; consequently, the cross-reactive areas of species other than type Ad2 are broad and show some variation. The left termini of the Ad3l and perhaps the Ad8 genomes, unlike that of Ad2, hybridize to Ad4l when isolated as fragments. A fourth area of hybridization was inconsistently observed near map unit 72 in some types, i.e., beyond the area of Ad2 DNA hybridization with Ad4l. However, these differences from the Ad2 pattern of frag-
VOL. 30, 1992
ADENOVIRUS CROSS-HYBRIDIZATION
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Vol. 30, No. 7
Copyright X3 1992, American Society for Microbiology
Conserved Sequences of the Adenovirus Genome for Detection of All Human Adenovirus Types by Hybridization TIM H. SCOTT-TAYLOR AND GREGORY W. HAMMOND* Cadham Provincial Laboratory and Department of Medical Microbiology, Canada University of Manitoba, Winnipeg, Manitoba R3C
3Y1,
Received 5 November 1991/Accepted 15 April 1992
The application of DNA hybridization directly to clinical specimens has the potential of improving the diagnosis of fastidious types of adenovirus. In this study, the genome of one adenovirus type from each human subgenus (A to F) was systematically evaluated by hybridization for homologous sequences to find the optimal common probe for detection of all human adenovirus types. The area of cross-hybridization, most closely defined with adenovirus type 2 (Ad2), mapped from map units 11.4 to 16.1 and 26.9 to 29.7 and, principally, to a central area of the genome between map units 47.5 and 65.2. The last area, enclosing the hexon gene, was highly conserved. Cloned probes generated from this area demonstrated the greatest homology to heterologous types by hybridization analysis. A HindIII-BgIII clone containing the hexon gene of Ad2 within narrow confines reacted most evenly with all adenoviral types and detected the DNA of all other subgenera with a sensitivity 2 logs greater than that of a complete genomic Ad2 probe. The most homologous adenoviral gene sequences were observed in genes involved with DNA replication or intimately connected to the hexon in the early capsid formation. These results show that the hexon gene constitutes the best single region of the adenovirus genome for use as a genus-specific probe for the diagnosis of all human adenoviral subgenera by DNA hybridization. have relied on the reactivity of randomly cloned fragments of the adenovirus genome as reagents. The following work systematically evaluated the cross-reactivity of all genomic sequences and tested a battery of cloned fragments to find the optimal common probe for detection of all human adenovirus types. Studies of the melting temperature of interspecies genomic DNA hybrids have indicated a high degree of genomic sequence homology between adenovirus types within one subgenus (16). One adenovirus type from each subgenus, therefore, was chosen for analysis to represent all the human species.
Adenoviruses are common and ubiquitous pathogens. Extensive epidemiological surveys have placed an adenoviral agent as the cause of 5 to 10% of the total juvenile pneumonias and respiratory infections of North American children (5, 12, 13). Respiratory infection with adenoviruses is regularly responsible for a number of infant deaths (30) and considerable morbidity in certain adult populations (21). The improved health of military recruits administered a vaccine of live adenovirus type 4 (Ad4) and Ad7 implemented to counter acute respiratory disease reduced health costs by nearly twice the $4.83 million cost of vaccine development within 2 years (9). Adenovirus particles are seen in some 5 to 17% of cases of gastroenteritis in children up to 5 years of age, a rate second only to that of rotavirus as a cause of pediatric diarrhea (38). Allowing for the fact that only half of the adenoviral infections contacted develop into illness (5), Chanock (8) has estimated from serological prevalence studies that the average individual undergoes a minimum of two or three clinical episodes of adenoviral infection during childhood. Enteric adenovirus types are commonly at a high concentration, up to 1011 particles per g of stool (15). The majority of isolates observed by the electron microscope in cases of adenoviral gastroenteritis are the fastidious enteric types Ad4O and Ad41, which cannot be reliably cultured for subsequent identification. The electron microscope is estimated to detect adenoviruses only when they are present at a concentration of >106 particles per g and possibly fails to detect many adenovirus isolates for most of the period of illness (11). A variety of more sensitive tests have therefore been devised to improve adenovirus diagnosis in fecal specimens. Monoclonal antibody immunoassays developed to date (17, 31) have targeted epitopes prone to variation because of immunological selection (29). DNA hybridization studies *
MATERIALS AND METHODS Cell culture and virus strains. Cell lines HEp-2, A549, and 293 and the prototype strains of adenovirus species from each subgenus A to F were obtained from the American Type Culture Collection (Rockville, Md.). A single type representative of each subgenus (Ad3l 1315, Ad7 Gomen, Ad2 Adenoid 6, Ad8 Trim, Ad4 RI-67, and Ad4l Tak of serogroups A, B, C, D, E, and F, respectively) was chosen for the availability of restriction endonuclease maps (6, 19, 33-35). Ad2 maps were generated by Pustell Sequence Analysis software (IBI, New Haven, Conn.) on a genomic sequence recorded by GenBank (National Institutes of Health). Ad4l was grown in 293 cells, and other types were propagated in HEp-2 or A549 cells. Batches of 10 150-cm2 flasks with semiconfluent monolayers were infected with the virus suspended in Eagle's minimum essential medium or, later, L15 medium (no. 10-511; Flow Laboratories, Inc., Mississauga, Ontario, Canada) at a multiplicity of 1 to approximately 10 50% tissue culture infective doses per cell. The viral overlay was replaced after 1 to 2 h with medium containing 2% fetal bovine serum and changed every third day. Viral titer was assessed as described by Reed and Muench (26) from application of log dilutions of stock culture supernatants to cells in 4 duplicate wells of 24-well plates.
Corresponding author. 1703
1704
SCOTT-TAYLOR AND HAMMOND
Preparation and treatment of viral DNA. Infected cells were monitored daily with an inverted microscope for the appearance of cytopathic effect. Cells showing the typical swelling and grapelike clustering of adenovirus infection were harvested and subjected to several cycles of freezethawing. Cell lysate was clarified by centrifugation, and virus was concentrated at 30,000 rpm for 90 min in a type 35 (no. 330926; Beckman, Burnaby, British Columbia, Canada) rotor over a cushion of 1.8 g of CsCl per cm3. The interphase was homogenized with an equal volume of Freon (trichlorotrifluoroethane; Fisher Scientific) in an omnimixer (Sorvall Ltd., Norwalk, Conn.) to remove lipid. Virions were then purified by density gradient centrifugation, first in a preformed 1.2- to 1.5-g/cm3 continuous CsCl gradient and last by isopycnic centrifugation in 1.344 g of CsCl per cm3. Virus bands were collected by a fractionator (Beckman no. 343890), dialyzed against 100 mM Tris-100 mM MgCl, and treated with 50 ,ug of DNase and RNase per ml to purify the intact virions of cellular components. Treated cells were then digested with 1% sodium dodecyl sulfate (SDS) and 50 ,ug of proteinase K per ml for 1 h at 37°C. Viral protein was removed by several extractions with equal volumes of phenol and chloroform, and the DNA was precipitated with 0.1 volume of 5 M NaCl and 2 volumes of ethanol. DNA concentration and purity were assessed spectrophotometrically and visually by electrophoretic comparison with lambda phage DNA standards. These DNAs gave no reaction when hybridized with HEp-2 cell DNA, and the restriction profiles accorded with the patterns of prototype strains (1). Cloning and plasmid extraction. Hindlll fragments of Ad2 DNA were ligated to plasmid pGem 3Z (Promega Biotec, Mississauga, Ontario, Canada) and added to competent Escherichia coli JM109. Transformants were distinguished as white colonies on Luria-Bertani plates supplemented with ampicillin, isopropyl thio-D-galactoside, and 5-bromo-4chloro-3-indolyl-I3-D-galactopyranoside (X-Gal; BRL, Gaithersburg, Md.), indicating that the P-galactosidase gene in the plasmid was disrupted by insertion of viral DNA. Transformants were stored as broth cultures supplemented with 15% glycerol at -70°C and were plated out every 6 months. Subcultures were chipped from stored aliquots without defrosting. Plasmids containing viral DNA inserts (Ad2 HindIll fragments A, B, C, D, E, G, H, I, and J) were differentiated by ampicillin (50 ,ug/ml; Sigma Chemical Co.) susceptibility after replica plating. The identities of inserted fragments were found by matching quick plasmid preparation (3) bands with Ad2 genomic DNA digested with the same restriction enzymes. Large plasmid DNA preparations were purified by isopycnic centrifugation in 1 g of CsCl per ml at 45,000 rpm for 36 h in an SW50.1 rotor (Beckman no. 340079). BglII subclones of HindlIl plasmids were ligated to the compatible BamHI site in plasmid pBR322. Electrophoresis and blotting. Purified adenoviral DNA (1 to 2 ,ug) was digested with at least 10 U of restriction enzyme in buffers specified by the manufacturer (Boehringer Mannheim). The 20-pl reaction volumes were mixed with 10 ,u of tracking dye, consisting of 0.07% bromophenol blue, 0.7% SDS, and 33% glycerol, and pipetted into wells in 150-ml slabs of 0.8% agarose submerged in 89 mM Tris-borate-10 mM EDTA (pH 8) buffer containing 0.5 jig of ethidium bromide per ml. Lambda phage DNA (Boehringer Mannheim no. 208396) digested with EcoRI and/or HindIll and a small quantity of Ad4l DNA digested with SmaI were included in each agarose gel as molecular weight markers and control. Electrophoresis was carried out at 1.7 V/3.5
J. CLIN. MICROBIOL.
mA/cm of gel overnight in a Sub Cell (Bio-Rad, Mississauga, Ontario, Canada) apparatus. As the dye front approached the end of the agarose, the gel was transferred to a UV transilluminator (Ultraviolet Products Inc., San Gabriel, Calif.) and the fluorescent DNA bands were photographed through a red filter with a Polaroid camera and type 57 film. An alkaline adaptation (25) of the Southern blot was used to transfer the DNA to nylon membranes (0.45-,um mesh; Micron Separations Inc.), which were stored at 4°C after being baked at 68°C for 6 to 12 h. Slot blot analysis of Ad2 probes. Adenoviral, lambda, and cellular DNAs, diluted from 10 ,ug to 10 pg/ml, were denatured with the addition of 0.1 volume of 3 M NaOH, neutralized with 0.1 volume of 3 M ammonium acetate after 30 min of incubation, and equilibrated to physiological salt conditions with the addition of 0.3 volumes of 20x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M Na citrate). A 150-,u volume of each dilution was applied by slot blot apparatus (no. 03431; Schleicher & Schuell, Keene, N.H.) under very low vacuum to nylon membrane prewetted in 6x SSC. Membranes were washed twice in 6x SSC, air dried, and baked at 65°C for 8 h prior to hybridization. Hybridization. Membranes were sealed in a plastic envelope (Sears Seal-a-Meal apparatus) and prehybridized at 68°C for 3 to 6 h in a rocking water bath in a buffer of 0.5% SDS, 6x SSC, and 5x Denhardt's solution (lx Denhardt's solution is 0.3% Ficoll 400, 0.3% bovine serum albumin, and 0.3% polyvinylpyrrolidone) containing 100 mg of sheared, denatured herring sperm DNA per ml. The prehybridization solution was discarded through a clipped corner of the envelope and replaced with 1 ml of the same buffer per cm2 of membrane; the buffer also contained 100 mg of freshly denatured herring sperm DNA and at least 107 cpm of nick-translated probe, prepared with commercial reagents (kit no. N500; Amersham, Oakville, Ontario, Canada), per ml. Hybridization under the same stringent temperature conditions was performed overnight. Membranes were subsequently given two pairs of 30-min washes at 68°C, first in 2x SSC-0.1% SDS and then in 0.2x SSC-0.1% SDS. After excess moisture was blotted, membranes were resealed in plastic envelopes and enclosed with Kodak XAR-5 film between intensifying screens (Dupont Quanta III, no. 224032; Picker International, Winnipeg, Manitoba, Canada) at -70°C until the homologous Ad4l DNA control lane was showing strongly. The comparative intensity of hybridization was assessed by densitometer (model GS-300; Hoefer Scientific Instruments; directed by an IBM GS360 Data System program) set at 100% gain on the homologous control and expressed as a proportion of the total absorption per lane. Probes were removed from membranes after autoradiography by two 15-min washes, first in 250 ml of 0.4 M NaOH and then in 250 ml of 0.1 x SSC-0.1% SDS-0.2 M Tris (pH 7.5) in a rocking water bath at 45°C. The membranes were checked for residual radioactivity before being resealed in envelopes and stored. Nylon membranes could be hybridized two or three times with little loss in intensity of the DNA bands. RESULTS Hybridization of adenovirus subgenera. Areas of sequence conservation in the adenovirus genome were examined by hybridization of the restriction endonuclease fragments of one type of each subgenus with a genomic Ad4l probe. Ad2 DNA fragments cleaved with a variety of restriction enzymes were separated by gel electrophoresis (Fig. 1A). Clal,
VOL. 30, 1992
A
ADENOVIRUS CROSS-HYBRIDIZATION X
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FIG. 1. Hybridization of Ad2 DNA with a genomic Ad4l probe. (A) Electrophoresis of 2-,ug aliquots of Ad2 DNA digested with a variety of restriction enzymes. Ad41 DNA (0.5 pug) was included in the outside lane as a control. (B) Appearance of cross-reactive Ad2 fragments on film after blotting and hybridization of gel in panel A with genomic Ad4l DNA.
used in conjunction with a number of other enzymes in the genomic digests of Ad2 DNA, liberated fragments of 3,000 to 4,000 bp closely enclosing the hexon gene. The gel was hybridized under stringent conditions with an Ad4l genomic DNA probe. A 48-h autoradiograph registering those Ad2 DNA fragments containing sequences with a high degree of homology to Ad4l is shown in Fig. 1B. Despite application of only a quarter of the quantity of DNA used for each Ad2 digest, the Ad4l control lane reacted strongly with the homologous probe. Transfer of sufficient DNA in small fragments was indicated by the strong signal from the 670-bp fragment in the XhoI and ClaI dual-digest lane. Each of the liberated hexon gene bands was evident. Fragments that did not hybridize can be discounted as not containing any sequence of identity with Ad4l under the experimental conditions of stringency. Fragments observed in Fig. 1B were denoted by underscoring their positions on the Ad2 genome in the restriction maps in Fig. 2. A repetitive pattern was discerned, three sequences being principally represented in the cross-reaction of every lane. When all those fragments that did not appear in the film were deducted, sequences of homology between Ad4l and Ad2 were most closely defined by the overlap of the positive fragments and were enclosed in two anterior areas between map units 11.4 to 16.1 and 27.4 to 29.7 and in a central area between map units 47.5 and 65.2. A value for each discernible fragment, figured as a proportion of the total absorbance of the lane, was assigned by densitometric analysis of autoradiographs and attributed to the position of the fragment on the genomic maps in Fig. 2. The individual values of comigrating fragments could not be distinguished. The three homologous areas reacted disproportionately; the anterior areas consistently registered less strongly than the central area. This was most clearly illustrated with the PvuI digest of Ad2, in which a small fragment between map units 52 and 60 was responsible for more than half of the total absorbance of that lane. Hybridization of subgenera A, B, D, and E. The process of
blotting, hybridization, and autoradiography was repeated with Ad4, Ad7, Ad8, and Ad3l electrophoresis gels, representing subgenera E, B, D, and A, respectively, and can be followed in Fig. 3. The areas of positive fragment overlap, denoting areas of consistent hybridization, of all five types tested are compared in Fig. 4, and it is evident that the hybridizing fragments of subgenera A, B, D, and E combined to describe a wide area of each genome that includes each of the sequences of Ad2 homology with Ad41. A fourth area of marginal hybridization was inconsistently seen in some lanes of Ad8 and Ad4 and repetitively with Ad7 but was not observed when hybridized as a separate fragment of the Ad31 genome. As with Ad2, the fragments reacted with different levels of intensity, and three separate regions, where the enzymes separated areas appropriately, could be distinguished in at least one lane of each type. The relative absorbance values for the positive fragments of one digest of each type are plotted in Fig. 5, and it can be seen that the cross-reactive sequences of different subgenera react with Ad4l probes in a disproportionate fashion similar to the three-tier pattern given by areas of the Ad2 genome. Two anterior areas of sequence identity reacted with 5 to 20% of the total absorbance per lane in each case. The greatest intensity of reaction was displayed by a central fragment of each genome between 50 and 60 map units; this region accounted for over half of the total absorbance of each
digest. Evaluation of various Ad2 fragments as probes. The abilities of various areas of the Ad2 genome, isolated as electroeluted fragments or cloned as HindlIl plasmids, to perform as probes in the detection of adenoviral DNAs of different subgenera were tested on slot blots. The reaction of the whole genome as a probe is visualized in the first panel of Fig. 6, where the homologous reaction was detected up to five dilutions at 100 pg of Ad2 DNA. Reactions with other subgenera were variable; 10 or 100 ng of Ad4, Ad7, and Ad8 DNA were detected, concentrations 100- to 1,000-fold higher
1706
J. CLIN. MICROBIOL.
SCOTT-TAYLOR AND HAMMOND B 22-6%
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A 113% 713 762 836 898 594 A B iFi D IE 1C 71.7% 18 3% 26 519 594 713 762 836 898 A B ,F ID'E IG 1D/E Hi C 282% 381 527% 191% 799 78 173 32.3 379 418 509 734 797 895 972 B F aC i A H II E IG IK inJa D I 77%c'D 15 6% M 7 7%C/D 768% L 428 57 398 475 619 720 237 935 II H I B D E I C G1 A IF I 89% 186% 661% 64% 42.8 26 57 237 398 475 519 619 720 935 C F B 1J IG IH I D/EI D/E i A K11I1 I 8 2% 177% 59-3/E 148% 17.3 22-0 51.4 59.2 5.2 34.4 40-2 C pF D A B HI 1E I aI 69% 167% 137% 531% 96% 26.3 27-4 46 6
D 116% 149% 73.5% 2.8 1d 9 18 3 36 3 40 2 52 6 56-8 76 1 76 7 u J1 E 11 F , I I B C D I HI A Sma MV 8-3% 53-9%A/s 14,7% 23-0% 53.9%A/8 --L 16 1 22 9 26-9 53-8 66.6 82-9 D C l F GII A E I I Xho I 6-8% 57.4% 35-8% 16-1 22-9 26-9 2.6 51.9 53-8 66-6 82.9 D A C Hi I F Gi III E I I Xho I tCIa 50 7%D/E 29 0% 20 3% 507ND/E Map units 1,0 50 70 60 80 210 310 410
92-1 98 4 I G IK
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~~~ II KB 10 20 5 15 30 25 35-9 FIG. 2. Restriction fragments of Ad2 DNA with homology to the Ad4l genome. The intensities of bands in the autoradiograph in Fig. 1B were assessed by densitometer. The proportions of the total optical density, expressed as percentages of the absorbance of each lane, are presented below the cross-reactive (underlined) fragments on the restriction map. II
than in the homologous reaction, while not even 1 p.g of Ad31 DNA was seen with the genomic probe in this time frame. HindlIl fragments from either genomic terminus gave relatively weak reactions with the other subgenera DNAs, as exemplified by the electroeluted HindIII K fragment probe in the second panel. This fragment from the right terminus reacted highly specifically, detecting Ad2 DNA 3 to 4 log dilutions less concentrated than the DNA of other types. Probes containing either of the anterior regions of the Ad2 genome found homologous in the hybridization with Ad4l, such as the HindlIl B and C plasmids or electroeluted Sacl G and SmaI F fragments, gave reactions intermediate between those of the whole genome and the specific termini. The SacI-G probe in the third panel reacted weakly with Ad31 and Ad4 DNA. The most even reaction of any Ad2 probe was given by the HindIlI A-fragment plasmid in the fourth panel, which showed consistent detection of heterologous DNA 2 to 3 log concentrations higher than the detection level of homologous DNA. This plasmid, from map units 42 to 70 of the Ad2 genome, was subcloned, and a single BglII plasmid retained the equilateral cross-reaction of the parent plasmid. A small fragment called HP II, closely enclosing the Ad2 hexon gene, was excised with enzymes
I
HindIll and PvuII. This electroeluted fragment of 3,453 bp, starting 532 bp upstream of the hexon gene and cut by PvuII 25 bp after the hexon terminus, hybridized with Ad3l DNA with a sensitivity comparable to that of other subgenera. The relative difference between homologous and heterologous reactions with an HP II probe in the last panel of Fig. 6 was within approximately 2 log dilutions with all species of DNAs, including Ad31.
DISCUSSION Genomic adenovirus DNAs were hybridized to locate genetic sequences common to all the human types of the Mastadenovirms genus. The accuracy of definition of areas of nucleotide identity was determined by the availability of known restriction sites; consequently, the cross-reactive areas of species other than type Ad2 are broad and show some variation. The left termini of the Ad3l and perhaps the Ad8 genomes, unlike that of Ad2, hybridize to Ad4l when isolated as fragments. A fourth area of hybridization was inconsistently observed near map unit 72 in some types, i.e., beyond the area of Ad2 DNA hybridization with Ad4l. However, these differences from the Ad2 pattern of frag-
VOL. 30, 1992
ADENOVIRUS CROSS-HYBRIDIZATION
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