simplified restriction endonuclease method for typing and

J Clin Pathol 1985;38:331-335

Simplified restriction endonuclease method for typingand subtyping adenovirusesJM DARVILLE

From the University ofBristol Medical School, Department of Virology, Bristol Royal Infirmary, Bristol

SUMMARY Restriction endonuclease digestion of viral DNA labelled in vivo with phosphorus-32has been used to type and to subtype both conventional and enteric adenoviruses. The method isa modification of that already described for typing and subtyping herpes simplex virus and, likethe latter, needs far less material than methods previously described.

The lack of serological variants of the herpes viruses(herpes simplex virus types 1 and 2, varicella-zostervirus, and cytomegalovirus) has meant that thedetailed characterisation of subtypes of these virusesand, therefore, the full study of their epidemiology,has been delayed until the development of molecu-lar techniques such as restriction endonucleaseanalysis.' -3There are 40 known serotypes of human

adenovirus forming subgroups A (types 12, 18, and31), B (3, 7, 11, 14, 16, 21, 34, and 35), C (1, 2, 5,and 6), D (8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32,33, 36, 37, and 39), E (4), F (40), and G (41)45according to their oncogenicity for newborn ham-sters and to the characterisation of their DNA.

Because of this large number of serotypesadenovirus epidemiology has not been subject to thesame degree of constraint as that of the herpesviruses. Nevertheless, the value of serological tech-niques in studying the epidemiology of these virusesis limited by the fact that serotypic intermediatesoccur,6 as do intertypic recombinants,7 at leastexperimentally. Furthermore, restriction endonu-clease analysis has shown within single adenovirusserotypes the existence of intratypic variants ofgenome types,8 which have stable and distinct elec-trophoretic profiles. This technique has been used todemonstrate subtypes of several serotypes,5 9"10 I

thus aiding the study of the epidemiology of thevirus. These studies, however, have relied on largescale virus cultures'2 and the purification of virusparticles" and, although good results have beenachieved, the methods used are too demanding inresources and too lengthy for use in a routinelaboratory.

Accepted for publication 22 October 1984

This paper reports the successful adaptation forthe subtyping of adenoviruses of a restrictionendonuclease analysis method already developed inthis laboratory for typing and subtyping herpes sim-plex virus.'3

Material and methods

VIRUSESStandard strains of adenoviruses were obtainedfrom the Virus Reference Laboratory, CentralPublic Health Laboratory, London. Otheradenoviruses were isolated in our laboratory fromclinical specimens and were typed by neutralisationor by immune electron microscopy in a methodderived from that of Svensson and von Bonsdorff.'4An enteric adenovirus was kindly provided by DrCR Madeley of the Royal Victoria Infirmary, New-castle upon Tyne.

RESTRICTION ANALYSISThe experimental procedure is essentially asreported previously,'3 although there are some vari-ations in detail which are outlined in the descriptionof the method given below.

Adenoviruses to be analysed are harvested byfreezing and thawing when their cultures show com-plete or near complete cytopathic effect on primaryisolation or on passage. Each virus isolate is inocu-lated (10 ,ul) into one well of a microculture plate(Linbro) containing 293 cells'5 (Flow) (about 0 25cm2 or 10 cells), which have been incubated inphosphate free medium. These cells have beenintroduced for the routine isolation of conventionaladenoviruses"6 and readily support the growth ofenteric adenovirus.'' Human embryo kidney (HEK)cells and HEp-2 cells may also be used. After 1 h 60,ul of phosphate free medium is added and after a

331

on June 4, 2022 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.38.3.331 on 1 M

arch 1985. Dow

nloaded from

http://jcp.bmj.com/

332

further 2 h the inoculum is removed and replacedwith 75 .ul of phosphate free medium containing 7-5gCi32P (orthophosphate in dilute HCI, Amersham).

After three or four days, when the cytopathiceffect is total or near total, the DNA is extractedwith sodium dodecyl sulphate (5% wtlvol) andphenol (chromatography grade, BDH; saturatedwith 20 mmolI Tris, 75 mmoUl NaCl, and 50 mmol/ledetic acid), precipitated with ethanol, and resus-pended in 170 ul of dilute RNA-ase (50 ug/mlRNA-ase A, 250 units/ml RNA-ase T1; Sigma). The32P content of the DNA is estimated by useof a Mini-Monitor contamination meter (Mini-Instruments Ltd).

Reaction mixtures are set up in sterile round bot-tomed microtitre plates (Flow) in a final volume of50 ul. The mixtures consist of 2 units of restriction

1 2 3 4 : 8 9 10 't 1 12 13 14

*1...... _ s:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Fig. 1 Tracks I to 14 are Pvu 1I restriction endonucleaseprofiles ofadenovirus types 1, 2, 4, 5, 6, 7, 9, 10, 11, 14, 15,31, "10/19" and an enteric adenovirus.

Darville

enzyme, 40 ,ul of DNA solution, and 8 ,ul of restric-tion buffer, the final concentrations of which are 6mmol/I of Tris HCl (pH 7.5), 6 mmoVI of MgCl2, 6mmoIIl of B-mercaptoethanol, and 800 mg/ml ofnuclease free bovine serum albumin (Sigma). Threedigests may be made from each DNA harvest, andwhile many enzymes are used for analysingadenoviruses those used in this study were Pvu IIand Bgl II (Bethesda Research Laboratories) andSst I and Pst I (Cambridge BiotechnologyLaboratories).The mixtures are incubated for 3-4 h at 37°C and

the reactions stopped with 6 ,ul of 100 mmol/I edeticacid containing 10% Ficoll 400 (Sigma) andbromophenol blue. The resultant DNA fragmentsare resolved by overnight horizontal agarose gelelectrophoresis at 2 V/cm. The gels are air dried(900) and then autoradiographed.For successful resolution of the smaller fragments

from the relatively small adenovirus genome,polyacrylamide gel electrophoresis (PAGE) mayalso been used. Good differentiation of adenovirussubtypes may usually be achieved, however, withonly the agarose gel and no PAGE results are pre-sented here.To test the stability of the adenovirus genome in

cell culture and to look for interactions betweeninfecting virus and the fragment of adenovirus type5 genome in 293 cells restriction enzyme analysiswas performed on isolates of adenovirus types 3 and4 before and after six passages in 293 and HEp-2cells. As a further test for interactions with theadenovirus type 5 fragment restriction profiles ofadenovirus types 3, 4, and 5 labelled in 293, HEK,and HEp-2 cells were compared. Furthermore,DNA from uninfected cells was analysed in the sameway as that from virus infected cells.

Results

Fig. 1 is an autoradiograph of the Pvu II profiles ofthe DNAs from 14 serotypes of adenovirus; theseare types 1, 2, 4, 5, 6, 7, 9, 10, 11, 14, 15, 31,"10/19", and an enteric adenovirus. The "10/19"isolate (track 13), which was from an ocular speci-men, had a restriction profile almost indistinguish-able from that of a standard type 37 (unpublishedobservation). In all tracks there is a relatively faintband of high molecular weight which is probably ofnon-viral origin. There are striking similarities bet-ween the profiles of types 1 and 2 (tracks 1 and 2)and to a lesser extent between these and types 5 and6 (tracks 4 and 5), all four of which are inadenovirus subgroup C. Again, similar profiles areseen with types 9, 10, and 15 (tracks 7, 8, and 11) insubgroup D. In contrast, while two of the represen-


http://jcp.bmj.com

/J C


arch 1985. Dow

nloaded from

http://jcp.bmj.com/

Simplified restriction endonuclease method for typing and subtyping adenoviruses

1 2 3 4 5 6 7 8 9 10 11 12 131l

_U

0~~_R*,!!,¶Ia do

.:.'

*1

Fig. 2 Tracks 1 to 14 are Bgl II restriction endonucleaseprofies ofadenovirus types 1, 2, 4, 5, 6, 7, 9, 10, 11, 14, 15,31, "10119" and an enteric adenovirus.

tatives of subgroup B (types 11 and 14 in tracks 9and 10) are similar, the third (type 7 in track 6) isnot.

Fig. 2 shows the profiles of the same adenovirusstrains as in Fig. 1 when digested with Bgl II. As inFig. 1 all tracks show a faint high molecular weightband, and in most of the tracks faint bands due toincomplete digestion are visible. Again, somesimilarities may be seen between the profiles ofsome of the members of the same subgroup.

In Fig. 3 the profiles are presented as tracings inorder to compensate for the running of digests ondifferent gels, for different track lengths, and for theexposure and development to different intensities.The first track shows the common profile of all type3 adenoviruses analysed with Pvu II, while the nextfour tracks show the distinct profiles of four unre-

lated isolates of this type analysed with Sst I. Thenext three tracks are Pvu II profiles of three clinicalisolates of adenovirus type 4. The next three are PvuII profiles of reference strains of types 7 and 7a andof a recent laboratory isolate of type 7, the formershowing much similarity with its fellow sub-group Bmember, type 3. Then follow two profiles of type 8which vary little. Finally, there are profiles of twoenteric adenoviruses, and while it is not clearwhether these are different serotypes or variants ofthe same serotype a comparison of their Sma Iprofiles (unpublished observations) with those pub-lished by Kidd et al'2 indicates that they are types 40and 41 respectively.The restriction of labelled but uninfected 293 cells

sometimes gives rise to a single high molecularweight band such as is seen in Figs. 1 and 2, but onother occasions no bands are seen. At no time haveany bands been seen in digests of uninfected cellswhich could be confused with bands fromadenovirus infected cells.The passage of types 3 and 4 through 293 cells or

HEp-2 cells did not result in any detectable changesin restriction profiles. The profiles of types 3, 4, and5 were identical whether virus was labelled in 293,HEp-2, or HEK cells.3 3 3 3 3 4 4 4 7 7 7 8 8 40 41

Fig. 3 Restriction endonuclease profies ofselectedadenovirus serotypes are presented as tracings. Shown hereare five profies ofadenovirus type 3 (the common Pvu IIprofie followed by four distinct Sst II profies), three Pvu IIprofies ofadenovirus type 4, three Pvu II profies ofadenovirus type 7 (7 and 7a type strains and one clinicalisolate), two Pvu II profies ofadenovirus type 8, and twoPst I profies ofenteric adenoviruses.

333

I

.:u.

I

i

40..

00 ?Oh


http://jcp.bmj.com

/J C


arch 1985. Dow

nloaded from

http://jcp.bmj.com/

334

Discussion

The value of restriction endonuclease analysis in thecharacterisation of subtypes of adenoviruses hasalready been shown.5 8-11 Published procedures forthis analysis are complex, however, and in order tomake the technique more suitable for use in routinelaboratories it is necessary to miniaturise and simp-lify the procedures making analysis cheaper andeasier to perform. Such a modification is describedin this paper.The choice of 293 cells for labelling is determined

by their requirement for the growth of the hithertouncultivable enteric adenoviruses"7 and by theirability to grow adenovirus type 8 more quickly thancells other than HEK cells (Dr APCH Roome, per-sonal communication). For other adenovirusesHEp-2 cells were satisfactory, but for conveniencethe single cell line is usually used. Despite the longand variable time taken for the adenoviralcytopathic effect to appear on primary isolation, aperiod of three to four days is nearly always ade-quate for the production of a sufficient amount oflabelled DNA, whether conventional or entericadenoviruses are being analysed.A possible objection to the use of 293 cells is their

possession of a fragment of the adenovirus type 5genome which might interact with the DNA of aninfecting virus. It was not shown, however, either byserial passage in 293 cells or by the comparison ofprofiles of DNA labelled in 293 cells with that label-led in HEp-2 or HEK cells that this type 5 fragmentaltered the restriction profile. In some circumstancesa high molecular weight band may be seen, and sincethis is also seen in profiles of uninfected cells it mustbe of cellular (or contaminant) origin. This, how-ever, is always much fainter than bands of viralorigin. Another problem, but again one which isobvious and easy to correct, is the production ofbands from incomplete digests when too much DNAor too little enzyme is added to the restriction mix-tures.The use of restriction endonuclease subtyping for

viral epidemiology relies on the profiles being stablewith passage. While the effects of long term in vivopassage are unknown, selected adenoviruses werestable during six in vitro passages (unpublishedobservations), and it has been shown that at lowmultiplicity of infection adenovirus type 16 remainsidentical with its prototype after 22 in vitro passages(Professor G Wadell, personal communication).

In this paper restriction profiles of 16 adenovirusserotypes are illustrated. In addition to these,profiles have been obtained in this laboratory fortypes 13, 16, 17, 19, 30, 31, and 37. Intratypic var-iants of types 3, 4, 7, and 8 are shown as are two

Darville

different enteric adenoviruses, probably types 40and 41 rather than variants of the same type. Itshould be noted that the type 3 in track 5 (Fig. 3)has bands common to two other subtypes as well asother exclusive bands, which suggests that this iso-late could be a mixture of subtypes. Although out-side the scope of this paper, it is interesting to specu-late on the origin of so many apparently stable sub-types of adenoviruses.The choice of enzymes to demonstrate both inter-

and intratypic differences is important. Adeno-viruses of the same subgroup tend to have similarprofiles-for example, with Pvu II type 1 resemblestype 2 and type 3 resembles type 7. Furthermore,Pvu II, which can differentiate subtypes of types 4,7, and 8, has not been shown to do so with type 3,whereas Sst I can. If the smaller fragments of DNAare resolved by PAGE, however, more intratypicdifferences may be demonstrable. Clearly,adenovirus restriction profiles must be interpretedwith caution. Indeed, with some intratypic differ-ences being so great it may be unwise to attempt toattribute type other than by serology until largenumbers of isolates are analysed with many differentenzymes and standard profiles established.

In conclusion, the method described here is acheap, efficient, and easy way of demonstrating sub-types of adenoviruses and should prove useful instudying the epidemiology of those viruses.

This work was supported by a grant from theResearch Committee of the Bristol and WestonHealth Authority.

I thank Mrs M Armstrong for technical assistanceand the staff of the Department of Virology for theirhelp.

References

Tullo AB, Shimeld C, Easty DL, Darville JM. Distribution oflatent herpes simplex virus infection in the human trigeminalganglion. Lancet 1983;i:353.

2 Pichini B, Ecker JR, Grose C, Hyman RW. DNA mappingof paired varicella-zoster virus isolates from patients withshingles. Lancet 1983;ii: 1223-5.

3Spector SA. Transmission of cytomegalovirus among infants inhospital documented by restriction-endonuclease-digestionanalyses. Lancet 1983;i:378-81.

Wadell G. Adenoviruses. In: Lycke E, Norrby E, eds. Textbookof medical virology. London: Butterworths, 1983:286-92.

Kidd AH. Genome variants of adenovirus 41 (subgroup G) fromchildren with diarrhoea in South Africa. J Med Virol1984; 14:49-59.

6 Wigand R, Fliedner D. Serological intermediate adenovirusstrains: A regular feature of group II adenoviruses. Arch GesVirusforsch 1968;24:245-56.

Sambrook J, Williams J, Sharp PA, Grodzicker T. Physical map-ping of temperatue-sensitive mutations of adenoviruses. J MolBiol 1975;97:369-90.


http://jcp.bmj.com

/J C


arch 1985. Dow

nloaded from

http://jcp.bmj.com/

Simplified restriction endonuclease method for typing and subtyping adenoviruses8Wadell G, Hammarskjold M-L, Winberg G, Varsanyi TM, Sun-

dell G. Genetic variability of adenoviruses. Ann NY Acad Sci1980; 354: 16-42.

9 Wadell G, Varsanyi TM. Demonstration of three different sub-types of adenovirus type 7 by DNA restriction site mapping.Infect Immun 1978;21:238-46.

'° Wadell G, de Jong JC, Wolontis S. Molecular epidemiology ofadenoviruses: Alternating appearance of two different genometypes of adenovirus 7 during epidemic outbreaks in Europefrom 1958-80. Infect Immun 1981;34:368-72.

"Wadell G, de Jong JC. Restriction endonucleases inidentification of a genome type of adenovirus 19 associatedwith keratoconjunctivitis. Infect Immun 1980;27:292-6.

12 Kidd AH, Banatvala JE, de Jong JC. Antibodies to fastidiousfaecal adenoviruses (species 40 and 41) in sera from children. JMed Virol 1983; 11: 333-41.

3 Darville JM. A miniaturised and simplified technique for typingand subtyping herpes simplex virus. J Clin Pathol1983;36:929-34.

Svensson L, von Bonsdorff C-H. Solid-phase immune electronmicroscopy (SPIEM) by use of protein A and its applicationfor characterisation of selected adenovirus serotypes. J MedVirol 1982; 10:243-53.

5 Graham FL, Smiley J, Russell WC, Naim R. Characteristics of a

human cell line transformed by DNA from human adenovirustype 5. J Gen Virol 1977;36:59-72.

Yirrell DL, Roome APCH, Darville JM, Ashley CR, Harbour J.

Comparison of the continuous cell line 293 human embryokidney cells and human embryo fibroblast cells for the cultiva-tion of ocular viruses. J Clin Pathol 1983;36:996-9.

Takiff HE, Straus SE, Garon CF. Propagation and in vitrostudies of previously non-cultivable enteral adenoviruses in293 cells. Lancet 1981;ii:832-4.

Requests for reprints to: Dr JM Darville, Department ofVirology, Bristol Royal Infirmary, Marlborough Street,Bristol BS2 8HW, England.

335


http://jcp.bmj.com

/J C


arch 1985. Dow

nloaded from

http://jcp.bmj.com/

simplified restriction endonuclease method for typing and

Documents