Restriction site mapping of adenovirus type 8 genome types

(~) |NSTITUT PASTEUR/ELSEVIER Paris 1990

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v• r ;I !, . .L.l ln,

1990, 141, 611-624

RESTRICTION SITE MAPPING OF ADENOVIRUS TYPE 8 GENOME TYPES T. Adrian (1)(.), U. Wolf (e), H.J. Lauer (2) and R. Wigand (2) (1) Nationales Referenzzentrum fiir Adenoviren, lnstitut fiir Virologie und

Seuchenhygiene, Medizinische Hochschule, 3000 Hannover (Germany)

(2) Abteilung fiir Virologie der Universitiitskliniken, 6650 Homburg/Saar (Germany)

SUMMARY

The DNA of 60 adenovirus type 8 (AV8) isolates (collected during the period 1961 to 1982, mostly in Western Germany) was analysed by 6 endonucleases and revealed 6 different genome types, thus implyfi:g that the variability of AV8 is relatively low. It was found that 45 isolates belonged to the genome type D1. Restriction site maps of a prototype D1 and of all deviating restriction variants were elaborated for enzymes BamHI, BglII, Hind-III and SalI. KEY-WORDS :

Adenovirus, DNA: Genome type 8, Restriction site mapping~

variability.

INTRODUCTION

Adenovirus type 8 (AV8) is the main causative agent of epidemic keratoconjunctivitis in man. The virus has biological and epidemiological characteristics which are different from all the other 22 prototypes of subgenus D (Wigand et al., 1983). Likewise, the virion polypeptides (Wadell, Hammarskj61d et aL, 1980) and DNA restriction patterns (Adrian et al., 1986) are also relatively different. A further characteristic feature is a slow and inefficient growth in cell culture, most striking with the prototype strain Trim (Wigand et al.,

Submitted March 30, 1990, accepted October 23, 1990. (*) Corresponding author. Address: Nationales Referenzzentrum fiir Adenoviren, Institut fiir Virologie und Seuchenhygiene, Medizinische Hochschule, Postfach 610180, 3000 Hannover (Germany).

T. A D R I A N E T A L .

612

1983) but also observed in many field strains (FuGuo et al., 1988; own observations). Restriction site mapping of the genome of AV8 is the first step in studying the pathogenicity on a molecular basis and for developing diagnostic D N A probes of defined specificity. Even though several studies of D N A restriction analysis of AV8 (Wigand et al., 1983; Takacs et al., 1983; Kemp and Hierholzer, 1986; Adrian et al., 1986) and of various AV8 genome types (Takacs et al., 1983; Fujii et al., 1983, 1984; Kemp and Hierholzer, 1986; Ishii et al., 1987; FuGuo et al., 1988) have been reported, physical mapping has only been attempted for an AV8 genome type by Takacs et al. (1983). However, we came to the conclusion that the results of these authors were 0nly partially correct. Hence, after analysis of 60 AV8 isolates with 6 endonucleases, we were able to construct physical maps of the AV8 genome by biochemical methods. We also localized altered restriction sites on the genomes of the 6 genome types.

MATERIALS AND METHODS Virus strains.

All strains were isolated from conjunctival material of patients with conjunctivitis or keratoconjunctivitis. As AV8 strains can be difficult to propagate, some strains were multiplied in human embryonic kidney cells and the rest in HeLa cell cultures. A number of strains had to be eliminated as no satisfactory DNA could be prepared. From a total of 60 AV8 strains, isolated between 1961 and 1982, the DNA was analysed by 6 restriction endonucleases (see below). Of the isolates studied 54, came from Germany and 6 from other countries (see below). From the prototype strain Trim (Jawetz et al., 1955), only low quality DNA could be obtained, despite a considerable effort to extract it (see below). All strains were identified by conventional means, both by neutralization and haemagglutination-inhibition. DNA restriction analysis.

DNA was extracted from infected HeLa cells (Wadell, Varsanyi et ai., 1980) and analysed using 6 endonucleases (BamHI, BglII, HindIII, KpnI, Sail, and Smal) according to the protocols of Boehringer Mannheim, Germany. The DNA fragments were separated on 0.8 to 1.5 °70 agarose gels. The BamHI and SmaI fragments of AV2 served as molecular standards. From the gel photographs, schematic drawings were prepared as previously described (Adrian et aL, 1986). When purified DNA fragments were needed, the fragments were separated in lowmelting agarose gels (Seaplaque agarose), then cut out of the gel and the DNA purified by phenol extraction plus an additional phenol/chloroform/isoamylalcohol (25/24/1) extraction.

A V 8 = a d e n o v i r u s t y p e 8.

I

m.u.

=

map unit.

M A P P I N G OF A D E N O V I R U S TYPE 8 GENOAI'ES

6i3

Restriction site mapping. Terminal fragments were determined by exonuclease digestion with the enzyme Bal31 (Legerski et al., 1978). Further investigations included double digestion with two distinct restriction endonucleases and hybridization techniques. Appropriate D N A fragments were labelled in vitro with 32p (Rigby et al., 1977) and hybridized with restriction fragments that were transferred onto nitrocellulose sheets (Southern, 1975). Hybridizations were carried out for 20h at 42°C, and the labelled D N A fragments were made visible by autoradiography (Kodak X-Omat film).

RESULTS

DNA restriction patterns. The various restriction patterns are shown as schematic drawings in figure l. HindIII showed the greatest diversity, whereas KpnI patterns were identical for all strains. From the obvious difference between the AV8 pattern in HindIII and the typical distribution of restriction fragments, it is clear that AV8 is very different from all the other prototypes of subgenus D (Adrian et al., 1986).

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F]o. 1. - - DNA restriction patterns of A V8 with indicated enzymes. Schematic drawings are presented in a logarithmic scale. The numbers of restriction variants correspond to the enzyme code in table I. Ordinate = molecular weight in kilobase pairs; x = unresolved double bands; * = triple band.

614

T. A D R I A N E T AL.

TABLE I. - - Genome types, representative strains and enzyme codes of AV8. Genome type

Strain code

Place

Origin Year

Enzyme code(*)

DI D2 D3 D4 D5 D6

Koja Ijima Budapest 91 124 440

Japan Japan Hungary Vienna Freiburg Homburg

'61 '61 '61 '73 '77 '77

1 1 2 3 2 1

1 1 1 1 2 1

1 2 3 4 4 5

1 1 1 1 1 1

1 1 2 1 3 1

1 1 1 2 1 1

Number of isolates 45 1 3 2 8 1

(*) Enzyme order: BamHI, Bg/II, Hindlll, Kpld, Sail, SmaI. DNA restriction patterns correspond to those shown in figure 1.

Genome types.

Six different genome types were identified and denominated, as described previously (Adrian et al., 1985; van der Avoort et al., 1986) (table I). The majority (45 strains) belonged to genome type D 1, which prevailed in Western Europe from 1970 to 1982, apart from the early 1961 isolate (Koja) from Japan. The D3 strain was isolated in Budapest in 1961 (Takacs et al., 1983); two further strains of this genome type were isolated in G6ttingen, Germany, in 1966. The two D4 isolates were collected in 1973 from two patients with conjunctivitis in Vienna. Six of the 8 D5 isolates were collected in Homburg in 1980 and 1981, 4 of these from nosocomial infections, and another i was isolated in Australia in 1981. Comigration analysis.

The pairwise analysis of comigrating fragments for the 6 genome types showed that there was a close relationship between D1, D2 and D6 (94 to 97 °70 comigrating fragments), whereas D4 and D5 were further apart (72 to 79 070 compared to all the others), and D3 was intermediate (84 to 89 °70, in relation with the D1 group). The mean number of restriction fragments for all genome types was 53.7 _+ 1.2. Prototype strain Trim.

All attempts to obtain satisfactory DNA from this virus strain (Wigand et al., 1983), including passages from fresh material obtained from the American Type Culture CcAiec:~:3~_~, failed. The restriction patterns of Trim were almost certainly identical with those of the D 1 genome type for BglII, KpnI, and SalI, whereas minor differences could not be ruled out for BamHI, Hind-

?dAPPING OF A D E N O V i R U S T Y P E 8 GENOh4ES

615

III, and SmaI. Results of other authors who claimed to have analysed Trim (Fujii et al., 1983, 1984; Kemp and Hierholzer, 1986) did not show convincing gel photographs of the Trim strain. No difference in the restriction patterns between Trim and the D1 we used have ever been reported (see below). Even if Trim may be "trimmed" or "forced" to yield sufficient DNA, the necessity of passing the virus repeatedly with a high multiplicity or with a high proportion of incomplete particles may lead to mutation. Hence, as all laboratories have the same difficulty, we strongly suggest that the Trim strain as prototype be substituted for an easily-grown strain of the predominating genome type DI. This genome type, and not Trim, hasbeen well characterized (Wigand et al., 1983; Adrian et aL, 1986; this study).

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FIG. 2. - - Bal31 digestion o f strain Budapest (genome type 11)3). Mapping of BamHI fragments. C • = BamHI fragment pattern (control); 1 to 5 = 5-, 10-, 15-, 20- and 30-min Bai31 digestions. The C and D fragments vanished first, as marked by arrows.

616

T. A D R I A N E T AL.

Restriction site mapping (see figure 6).

The published physical maps of the Budapest strain D3 (Takacs et al., 1983) for the enzymes BamHI, HindIII, and SalI proved to be only partially correct. The fragment patterns were similar in both studies; however, the Hungarian authors did not find the smallest fragment in either HindIII or SalI. Hence mapping of D 1 and other restriction variants was started de novo for 4 of the 6 endonucleases we used. Exonuclease Bal31 was best suited to determine terminal and adjacent fragments of the genome. Figure 2 shows that the BamHI fragments C and D vanished first, then subsequently fragments B and A. Therefore, fragment D and not B (as claimed by Takacs et al., 1983) is the right terminal fi-agment.

C® 1 2

C® 1 2

FIG. 3. - - Hybridization of Bgl/l fragments of variants 1 and 2 (VI, V2), with labelled

Hindlll-G* fragment of strain Budapest (V3).

C • = HindIII control; 1 and 2, see figure 1. Labelled fragments are marked by arrows.

M A P P I N G OF A D E N O VIR US T Y P E 8 G E N O M E S

617

Great differences were evident with regard to the HindlII map published by these authors. The fragments J and E are terminal fragments as was shown by exonuclease digestion. These correspond to the left part of A and to J + F of the AV8 prototype (fig. 6). We could not map HindlII fragments B, C and D of strain Budapest separately, since these fragments could not be

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FIG. 4. - - Hybridization of Hindlll fragments of variants VI-V3 with labelled Bglll-E

fragment of strain Budapest.

1, 2, and 3, see figure 1 ; • - C / D fragments of VI correspondin~ to the B / C / D fragments of V3; ~ • = fragment K of V2 which has the same map pomion as fragment L of Vl and V3 (see fig. 6).

618

T. A D R I A N E T A L .

resolved in our gel system and appeared as a triple fragment (fig. 1). The linear arrangement o f internal HindIII fragments was based either on hybridization experiments or was deduced from double-digest patterns, using a second restriction enzyme with known cleavage sites. T w o examples are shown in figures 3 and 4. Fragment G must map between 0.19 and 0.25 map u ~ t s (m.u.) as it hybridized with the BgIII-D and BglII-A fragments (fig. 3), and the Hind-

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MAPPING OF ADEN T v P= E •8 c:r;'~roA~rr'e . O . V . !. R. I.I q . U L . , z w~..11vI.L:,J

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. . . . . . -,T T, T ,l:',,,t~,,~,,t~ L and H must map between 0.6 and 0.7 m.u. as they hybridized with the BglII-E fragment ( 0 . 6 - 0 . 6 6 m.u. ; fig. 4).

The physical map of SalI fragments from Takacs et ai., (1983) corresponded with our results with regard to the map positions of fragments A to D, but not with the linear arrangement of fragments G to F. Fragments G and F (Budapest strain) are located between 0.42 and 0.52 m.u., as shown by hybridization with ~its HindIII-F fragment (fig. 5). The resulting restriction site maps for AV8 prototype D 1 and the restriction site alterations for all other variant patterns are shown in figure 6 for the 4 enzymes B a m H I , BgllI, HindlII and SalI. Five altered restriction sites were found! in HindlII and 7 sites altogether in the other 3 enzymes. These were randomly distributed over the genome. Figure 7 shows the restriction site alterations for the 5 genome types in comparison with D 1. Genome type D5 with its 8 different sites showed the greatest genetic distance from D1.

620

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FIG. 7. - - Restriction site alterations o f A V8 g e n o m e types. T o p line = all restriction sites f o u n d in D I . Lower lines = c o m p a r i s o n o f all other g e n o m e types with DI. ! = new a n d ! = lost restriction sites; C) = B a m H I , • = BglII, A = H i n d I I I , A = SalI ; x = site o f 2 u n r e s o l v a b l e e n z y m e s .

DISCUSSION

The genetic variability of AVS, as revealed in this study and in previous investigations (see table II), is relatively low. The same was found for the two other conjunctivitis-causing virus types of subgenus D, namely AV19a (WadeU and de Jong, 1980; Adrian and Wigand, 1989) and AV37 (Adrian et al., 1988). The genetic relatedness among the AV8 genome types, as determined by analysis of comigrating fragments, showed that some were closely related (D 1, D2, D6) and others more distantly related (D4, D5). From other studies, "AV8D" (Kemp and Hierholzer, !986) was found to be fairly different from all others (see table II). More comprehensive restriction analysis with more enzymes and more strains of AV8 might enable the grouping of AV8 genome types into genomic clusters, as was done for AV3 and 7 (Li and Wadell, 1988). Genome type D 1, which we consider to be the prototype, prevailed in our study. We identified a further 11 isolates from two consecutive outbreaks of conjunctivitis in Brest, France, in 1983 and 1984 (Chastel et al, 1988)o Strains identical to D1 in all comparable enzymes were reported by Fujii et al. (1983; 1984), Kemp and Hierholzer (1986) (see table II), and by FuGuo et al. (1988). For epidemiological studies it might also be advantageous to apply restriction enzymes cutting tetranucleotides to further subdivide isolates of this common genome type. Strains like our D2 and D4 genome types were also found by Fujii et al. (1983; "SA") and Kemp and Hierholzer (1986; "8C"), respectively.

27

70

Fujii et al. (1984) Taiwan '80-'81

Kemp and Hierholzer (1986)

5

5

4

4 6(*)

C D

1 1 1 1

1 2 2

Hind III

1

4(*) 4(*) 1 4(*)

1 1 1

Bam HI

P

C D E F

P A B

Genome types

1 2(*)

1

1 4(*)

1

1 1 1 1

1 1 2

1 3(*)

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Restriction enzymes Kpn Sal Sma I I I

2 3

1

Sac I

2 1 1 3

Sst I

D4

DI

DI

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DI D2

Comparison

Genome types are as denominated by the authors. Restriction enzymes omitted: BglII (not used by the authors), PstI (used by Fujii et al., t983, 1984; identical patterns found for all strains). Comparison made with the genome types of this study (see table I). Restriction patterns were either identical or probably identical with 1Lhisstudy (see fig. 1) or, for Sacl and SstI, not studied here; patterns marked by (*) were not found by us.

USA '66-'85, Taiwan, Greece '81-'83

25

No. o f : strains enzymes

Japan '75-'81

Fujii et al. (1983)

Ref.

Location, time interval

TABLE II. - - G e n o m e types of AV8 reported by other workers compared with our results.

622

T. A D R I A N E T A L .

The genetic relationship was clearly higher among the AV8 genome types (72 °70 comigrating fragments or more) than Letween AV8 and the other prototypes of subgenus D (Adrian et al., 1986" 38 to 52 070). However, a serological atypical " A V 8 prime" strain had only 31 °70 comigrating fragments with the AV8 prototype (Adrian et al., 1987), whereas all strains in this study were serologically typical. This observation stresses the need to apply D N A restriction analysis and serology for full strain characterization. The physical mapping of AV8 is the fiist step in studying the unusual pathogenicity of this serotype on a molecular level. Its physical map was similar to those found for AV9 and 15 in BglII (Adrian et al., 1989), less so far BarnHI and not at all for HindIII.

RI~SUMI~ CARTOGRAPHIE DES SITES DE RESTRICTION DES TYPES GI~NOMIQUES D'ADI~NOVIRUS 8

Des 6tudes ont 6t6 r6alis6es afin de d6terminer les profils de restriction du g6nome de 60 souches d'ad6novirus type 8 isol6es de 1961/l 1982, en majorit6 en Allemagne de l'Ouest. Les g6notypes ont 6t6 d6termin6s a l'aide de 6 enzymes de restriction. Six types g6nomiques diff6rents ont 6t6 trouv6s. Les r6sultats indiquent que la variation entre les souchcs d'ad6novirus type 8 est relativement faible. Sur les souches isol6es, 45 ont 6t6 class6es dans le g6notype DI. Les cartes g6nomiques du prototype D 1 et des variants ont 6t6 r6alis6es/L l'aide des enzymes BamHI, BgllI, HindlII et Sail. MOTS-CLi~S:Ad6novirus, ADN; G6notype 8, Cartographic de restriction, Variabilit6.

ACKNOWLEDGEMENTS

The technical a,nistance of B. Best, D. Keller, I. Maurer, and G. Sch/ifer is gratefully acknowledged. We thank Mrs. I. Meusel for expert photographic work. The work was aided by grams from the following German institutions: Deutsche Forschungsgemeinschaft (Wi 3-21), Bundesministerium fiir Jugend, Familie, Frauen und Gesundheit, and F6rdererverein der Deutschen Vereinigung zur Bek~impfung der Viruskrai~kheiten, Miinc~en.

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M A P P I N G OF A D E N O VIR US T Y P E 8 GENO_MES

623

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