CopyrightC 1988, AmericanSociety for Microbiology
Remarkably Homogeneous
Population of Adenovirus Type 3
and
7
Genome
Types
MAX ARENSt*AND VICKIEDILWORTHt
Department of PediatricslAdolescent Medicine, St. Louis University Medical School, St. Louis, Missouri 63104
Received 29January1988/Accepted 3 May 1988
Atotal of 270 frozen adenovirus isolates, obtained from January 1981toDecember 1986,were grownin KB cells. DNAwassuccessfully prepared from 248 of theseisolates, and the prepared DNAwasdigested initially with SmaI restriction endonuclease and then analyzed by agarose gel electrophoresis. Restriction fragment patterns showed that 68 of these DNAs were either adenovirus type 3 (Ad3) or Ad7. Further analysis by digestion withBamHI showed that 24 isolateswereAd3a and 44 isolateswereAd7b. WhenAd3 isolateswere digested with SmaIorPstI, the restriction fragment patternsof the 24 isolates were identical. Ofthese 24 isolates, 20 (83%) alsogaveidentical patterns whendigested withHindIII, and the patternsof the remaining 4isolatesweredifferent from that of the 20 but identicaltoeach other.Ali Ad3 isolateswereobtained in 1982, 1983, and 1986.All the 44 Ad7b isolatesgaveidenticalfragment patterns when digested with BglIorPvuII, and 43 of 44(98%) isolatesgaveidentical patterns when digested with SstII. We obtainedatleasttwoAd7isolates during each of the 6yearsstudied. Although considerable genomeheterogeneity within adenovirus serotypes
andsubtypes has been reported,ourpopulation of isolates is remarkably homogeneous. This homogeneousset ofgenome types was obtained from diverse anatomical sites and was associated witha broad spectrumof clinicalsyndromes.
There hasbeen considerable recent worldwide interest in
the use ofrestriction endonucleases to investigate the ge-nomic variability of adenoviruses. A single base change in
the viral DNA can potentially be observed as an altered restriction enzyme fragment pattern in an agarose gel, and thus, various patterns with different enzymes have been used to define specific genomic variants orviral subtypes.
Thisabilitytodescribe subtypes is interesting and useful for epidemiological purposes in determining the presence and relationship of specific variants circulating in a community andpossibly inevaluating therelative virulence of different subtypes.
In this study, we have grown virus and prepared viral
DNAfrom 248adenovirus isolates obtainedinthe
Diagnos-tic Virology Laboratory at Cardinal Glennon Children's Hospitalin St.Louis, Mo., between 1981 and 1986. Of these isolates, 204 were typed by comparison of their restriction
enzyme fragment patterns with standard patterns. Within thisgroup,24isolatesweretypedasadenovirustype3 (Ad3)
and 44 isolates were Ad7. These isolates were further
digestedwith additional restrictionenzymesforpurposesof
subtyping.Incontrast topreviousreports,oursubtypedAd3 andAd7 populations were foundto be extremely homoge-neous.
KB cells (approximately 1 x 106) were plated in 6-cm-diameter tissueculture dishes and incubated at 37°C in 5%
C02 until confluent (24 to 48 h). The growth medium was removed,andcellswereinfected with 150to250,ulofaviral isolate and furtherincubatedat 37°C with occasional tilting
for 1 h. Cells were fed with 5 ml of medium (Dulbecco
modified Eaglemedium withnonessentialaminoacids, 10% fetal bovine serum, and 50 mg ofgentamicin per ml) and
* Correspondingauthor.
tPresentaddress: Washington UniversityMedical School,
Chil-dren'sHospital,400SouthKingshighway, St.Louis,MO 63110.
t Present address: MonsantoCorporation, St. Louis, MO63141.
incubated as described above until 50 to100% of the cells were showing cytopathic effect (48to72 h).
DNA was prepared from infected cells essentially by the method ofKemp et al. (6). Cells were harvested, washed with phosphate-buffered saline, suspended in 100 ,uI of phosphate-buffered saline, and then lysed by the addition of
0.5 ml of lysis buffer (1% sodium dodecyl sulfate, 1% 2-mercaptoethanol, 10 mM Trishydrochloride [pH 8.0], 150 mMNaCI, 5mM EDTA)andincubatedat37°C for15 min. Proteinase K (200 ,ug) was added, and incubation was continued for 60 min. This digestion mixture was then extracted with an equal volume of phenol-chloroform-isoamylalcohol (24:24:1),and the large clump ofcellDNA was removedand discardedfollowing centrifugationto sep-arate the phases. The DNA in the aqueous phase was precipitated with 2volumes of ethanolat -20°Cfor30min. The precipitate was recovered by centrifugation, washed withethanol,and then dried invacuo.DNAwasdissolvedin 0.5 ml of TES buffer (10 mMTris hydrochloride [pH 8], 1 mMEDTA, 150 mMNaCI), digestedwith RNase A(25 ,ug)
and RNase T1(25 U)at37°Cfor60min,extracted twice with
phenol-chloroform-isoamyl alcohol as describedabove, ex-tracted withether,andprecipitatedwith ethanol. The recov-ered DNAwas dissolved in 200 pI ofH20.
Purified DNA (1 to 2 ,ug) was digested with restriction enzymesaccordingtomanufacturerspecificationswith 10to 20 U ofenzyme. Digested DNA was subjected to
electro-phoresis in 1.2% agarose gels containingethidium bromide for 17 hat 30 V. Gelswere photographedunder UV light.
Some isolates were serotyped by neutralization, and re-striction patterns from these isolateswere used for
compar-ison with unknown patterns from isolates and withpublished
patterns.
A total of 270 isolates collected from January 1981 to December 1986were inoculated into KB cellsas described above. Viral DNAwas successfully purifiedfrom248(92%)
of these isolates. The yearly breakdown of isolates and the number thatgrewin KBcellsand thegenometypes ofthese 1604
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NOTES 1605 TABLE 1. Summaryof adenovirus genome types
No. ofisolates
Genome type
1981 1982 1983 1984 1985 1986
1/2 10 il 7 10 18 14
3a 13 3 8
4 1 1
4a 2
5 5 2 5 1 6
6 2 1 1
7b 4 13 2 2 8 15
il 1 1
31 4 3 3 5 2
34/35/11 2 1 1 3
37 3 5 2
41 2
Group
A 1
B 2 1
C 4 2 10 8 3 4
B/C 3
Other 2 4 1
Total 42 52 43 29 52 52
Total that grewin 40 50 37 21 49 51
KBcells
isolatesasdetermined bycomparisonwithstandardsorwith
published patterns are shown in Table 1. By far the most
common types were types 1 and 2, as would be expected
fromapediatric population (3). The genomes of thesetwo types cannot be distinguished when digested with SmaI
restriction enzyme (4). Thenext most commontypes were Ad7, which accounted for 17.7% ofthe 248 typed isolates,
and Ad3, which accounted for 9.8% of the typed isolates.
Therewere45 isolates(10%of thetotal) which couldnotbe
easily typed after digestion with SmaI, but most of these
isolatescouldbeassignedtoaserotypegrouponthebasis of theirfragmentpattern (Table 1). GroupCadenoviruses are knowntoshowconsiderablegenomevariation(1, 4, 5),and the subtypes can be identified only after the digestion of
DNA with several restriction enzymes. Seven isolates had complicatedoruncharacteristicpatternsthatdidnotseemto fitaparticulargrouppatternand werethusclassified under othergenometype in Table 1.
Figure 1 shows the yearly distribution of Ad3 and Ad7
isolatesfrom 1981 through 1986. As has been shown previ-ously (8, 9), theisolation of Ad3 and Ad7 serotypes fluctu-ates considerably from year to year. Although these two serotypesgenerally fluctuateinconcertwitheach other, Ad7
isusuallypresentevery yearwhereas Ad3 often disappears forayear or moreand thenreappears in the same popula-tion, as wasthecasewith theisolates in this study.
Table 2shows theresultsofdigestionofall24Ad3isolates from the23patients representedinFig. 1.DNAwasinitially digested with BamHI to subtype the isolate. All of our isolates were Ad3aas determined by comparison with the published fragment profilesofWadell(7). Digestion of all 24
DNAsampleswithSmaI revealed afragment pattern
iden-ticalto SmaIcleavage pattern III of Bailey and Richmond
(2).DigestionofallAd3a DNAs withPstIyieldedacleavage
pattern notpreviously described and whichwe have desig-nated pattern E. Finally, digestion of these DNAs with HindIIIgavetwodistinctpatterns,neitherof whichhasbeen previously described. DNAfrom four of the Ad3a isolates yieldedtheHindIIIpatternshownfor isolate82-1421 inFig.
2 and which is designated pattern U in Table 2. The remaining 20 HindIII DNA fragment patterns were identical (represented by the patterns for isolates 82-3053 and 86-3996 in Fig. 2), and the pattern is designated pattern V in Table 2. Thus, 4of the 24 Ad3a isolates were indistinguishable from each other bythis analysis. Theremaining 20isolates were
distinguishablefrom the first groupbytheir HindIII patterns
butwere identicalto each other.
Table 3 shows theresultsof restriction enzyme digestion of the Ad7isolates.The BamHI patternwascharacteristic of
Ad7b asdefined by Wadell (7). All 44isolates gave identical
fragment patterns when digested withBglI or PvuII restric-tion enzymes. TheBgll and PvuII patternswerepatternsB and Y,respectively, asdefinedbyBaileyandRichmond(2).
DNA digestion with SstII revealed 43 isolates with pattern
III (2) and 1 isolate with pattern IV. Figure 3 shows
representative Ad7b restriction patterns, all of whichwere
identical for each enzyme used in this analysis except for SstII.Isolate 82-1218 gaveauniquepatternwith SstII which distinguished it from the other 43 isolates. Table 4
summa-rizes the datapresentedin Tables 2and3 and illustrates the homogeneous nature ofour Ad3 and Ad7 isolates. Ofour
Ad3 isolates, 83% belonged to the newly described Ad3a7
subtype, and 98% of our Ad7 isolates belonged to the previously described Ad7b2 subtype.
Incontrast topreviousreportsofgenetic heterogeneity of
certainpopulations of adenovirus isolates,
particularly
Ad3and Ad7, our present study shows that the Ad3 and Ad7 isolates obtained at the Diagnostic Virology Laboratory at
Cardinal Glennon Children's Hospital constitute a
remark-ably homogeneous population.
Apreviousreport(2) defined nine Ad3 genomesubtypesin
a population of29 isolates from Manchester, England. By
usingthe same restriction enzymes for analysis, we found
only two genome subtypes of Ad3 for our 24 isolates
collected from 1981 to 1986 in St. Louis, Mo. Of these
isolates, 83% belonged to asingle subtype, nowdesignated
Ad3a7. Theremaining isolatesalso gaverestrictionpatterns that have not previously been described and have been
classified asbelongingto asubtype designated Ad3a6.
Forapopulation of 44 Ad7isolates, apreviousreport (2)
defined threecirculating subtypes whichwereaboutequally
represented among the isolates. In the present study, we
foundonlytwogenomevariants amongour44Ad7isolates,
and 43(98%) of thesebelonged to asingle subtype(Ad7b2).
The global distribution ofadenovirus typesand subtypes
isbecoming well characterized through the effortsof
molec-20
18- * Ad7
« 16- Ad3
1
1141
CL 12-
10-Z 4
1981 1982 1983 1984 1985 1986
FIG. 1. Numberofpatientswith Ad3 orAd7 from1981 to 1986in St. Louis, Mo.
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J.CLIN. MICROBIOL.
TABLE 2. Ad3aisolates from1981 to 1986
Isolate HindIIIrestriction
no. Specimen Diagnosis pattern
82-963 Throat Feverofunknownorigin U
82-1420 NPb U
82-1421 NP U
82-1694 Eyeswab Cellulitis V
82-1948 Conjunctiva swab Conjunctivitis U
82-2941 Throat Pharyngitis V
82-3033 NP Feverof unknownorigin V
82-3053 Throat Upperrespiratoryinfection V
82-3370 Sputum Pneumonia V
82-3412 Throat V
82-3486 Throat Feverofunknownorigin V
82-3503 Urinec Rule outcytomegalovirus V
82-3586 Throat V
83-1941 NP V
83-4029 NP Neutropenia V
83-4260 Stool Respiratorydistress V
86-3536 Lefteyeswab Rule outadenovirus V
86-3996 Conjunctiva Ruleoutadenovirus V
86-3969 NP Pharyngitis V
86-2281 NP Pneumonia V
86-3681 Throat Pancytopenia V
86-1823 Stool Feverof unknown origin V
86-1869 Throat V
86-1041 Throat V
aRestrictionpatterns aredesignatedby letters orRomannumeralsaccordingto thenomenclatureof Bailey andRichmond(2) exceptinthe casesofPstIand
HindIII digestions in whichnew patterns were observed. The restrictionpatterns forBamHI,PstI,andSmaI digestions for all isolates are3a, E, andIII,
respectively.
bNP, Nasopharyngeal.
' Catheterspecimen.
|
BamHI|
Pst|
Mmndlll
|
Sma|
I- m Ç0 Mv cc 10 - m ~D
Nt o O> N o >) N on O>a> N Mt
l cvN e a CO> 0 e
A
_*
O U6 Cc0
CSO
eNCA
|~~~~
I---
~ ~~~~~~~ ~~~~~~~~~~~~~~~~FIG. 2. Agarose gel electrophoresis of DNA fragments from restriction enzyme digestion ofrepresentative Ad3a isolates. Par-tially purified DNAs were digested with restriction enzymes, and fragments were subjected toelectrophoresis in 1.2% agarose con-taining ethidium bromide and photographed under UV light. LambdaDNA fragments are 23.1, 9.6, 6.3, 4 (notvisible), 2.3, and 2.1 kilobase pairs in size.
Sma
|BamHI
BgII
|
PvuIf SstIl
O cO 0 O 10 CO CD CO 0 °
e Nt e cmNd e N N t N N e N N
N tS N es NY N U> <D CY US
a oe X 0X 0a: 0 O O Co A
FIG. 3. Agarose gel electrophoresis of DNA fragments from restrictionenzymedigestionofrepresentativeAd7b isolates. DNA was digested, subjectedto electrophoresis in agarose, and photo-graphed under UV light. Lambda DNAfragments aredescribed in
the-egendto Fig. 2. Faintpartial digestion products arevisible in
somelanes.
1606 NOTES
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NOTES 1607 TABLE 3. Ad7b isolates from 1981 to1986
Isolate SstII restriction
no. Specimen Diagnosis pattern'
Tracheal aspirate Tracheal aspirate Throat Eye Eye drainage NPb Stool Eye Throat NP Stool Trachealaspirate NP NP NP NP Urine Eye drainage Bronchial wash Conjunctiva Conjunctivaswab Stool Throat Eye Rectal Eye swab Rectal
NP or throat or both Eyedrainage Throat NP Mucous sputum Eye Eye Eye Eye Eye Left eye Throat Lefteye Right conjunctiva Left eye Throat Eye Bilateral pneumonia Pneumonia Failure to thrive Conjunctivitis Eyedrainage Ruleout sepsis Rule outsepsis Eye infection Asthma Ruleout sepsis Gastroenteritis Gastroenteritis
Gastrointestinal bleeding Pneumonia
Apnea
Rule outconjunctivitis Seizures(mumps) Conjunctivitis Pneumonia Conjunctivitis Conjunctivitis Conjunctivitis Gastroenteritis
Diarrhea andconjunctivitis Gastroenteritis
Pneumonia Conjunctivitis
Pneumonia;rule outRSV' Pneumonia;RSV Eye infection Ruleoutadenovirus Conjunctivitis
Ruleoutadenovirus
Ruleoutadenovirus Rule out adenovirus Pneumonia
Rule outadenovirus
Rule out adenovirus Rule out adenovirus
Periorbital cellulitis
' Restrictionpatternsaredesignated by lettersorRoman numeralsaccordingtothe nomenclatureofBaileyand Richmond(2).The restrictionpatternsfor
BamHI,BgIl,andPi'ulI digestions for ail isolatesare7b, B. and Y, respectively.
NP,Nasopharyngeal.
' RSV, Respiratory syncytial virus.
ularepidemiologists. Wadell etal. (8) reportedthat between 1958 and 1969 thepredominantAd7genometypeinEurope was Ad7c. This was replaced by Ad7b as thepredominant species from 1970tothe early 1980s. Baileyand Richmond
(2) confirmed Ad7b as the major species in Manchester, England, duringa16-monthepidemic from 1983to1985 and furtherestablished thealmost equal distribution ofthe three genome subtypes. In the United States between 1962 and
1980,thepredominantstrainwasAd7b which madeupabout 65% of the isolates reported by Wadell (8). Ad7a made up about24% of the U.S. Ad7isolatesduring the sameperiod. Ad7b has recentlybecome the predominantAd7 species in Australia buthas beenreportedonly rarelyinAfricaandnot atall in China, Japan, or South America.
Theglobal,aswellaslocal,distribution of various adeno-virus subtypes presents interesting epidemiological
prob-lems which merit continuing investigation. The current
evi-dence does not support the theory that some subtypes are morevirulent thanothers,but thepossibilityexists thatnew
emergingsubtypes mightcausemore severedisease than the
ones they supplant. The genetic stability of subtypes is
TABLE 4. Summary ofAd3 and Ad7genomesubtypesisolated from1981to 1986in St. Louis, Mo.
Genomesubtype" No. of
isolates(%)
Ad3a6.. 4(16.7)
Ad3a7.. 20(83.3)
Ad7b .. 1 (2.3)
Ad7b2.. 43 (97.7)
"These designations are according to the nomenclature of Bailey and Richmond(2),who defined the restrictionenzymeprofilesforsubtypesAd3a
through Ad3a5and forsubtypesAd7bthroughAd7b3. 81-263 81-450 81-340 82-1166 82-1494 82-1495 82-1513 82-1870 82-2342 82-2925 82-3451 82-3448 82-3460 82-3462 82-3559 82-3682 83-2746 83-4190 84-4039 84-4044 85-3393 85-3266 85-3247 85-3224 85-3095 85-2905 85-2157 85-1218 85-19 86-332 86-418 86-428 86-750 86-864 86-3337 86-2485 86-3193 86-3188 86-4138 86-4091 86-4087 86-4758 86-6022 86-6797 III Ili III I'I III IlI III III Ili Ili III IlI III III III III Ili Ili Ili Ili 111 Ili IIl Ili Ili III Ili IV IIl III III III III III Ili Ili III III Ili Ili IlI IlI Ili III VOL.26, 1988
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unknown, and theease and speed withwhichnewsubtypes replace old ones are also unknown. Theworldwide hetero-geneity of adenovirus subtypes is slowly becoming under-stood, but the epidemiology in local microenvironments is apparently poorly resolved and deserves further analysis.
Wethank Ella Swierkosz for making clinical adenovirus isolates available tousandLeslie Bartonforreviewing the manuscript.
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