Original Paper
Intervirology 1992;33:86-90
Department of Microbiology, School of Medicine, University of Buenos Aires, Argentina Department of Virology, University of Umea, Sweden
Key Words Acute respiratory infection Adenovirus 7 Pairwise comigrating restriction fragments
Characterization of Adenovirus Genome Type 7h: Analysis of Its Relationship to Other Members of Serotype 7
Summary Ad7h is a newly identified genome type associated with severe lower acute respiratory infections and has so far been isolated only in South America. To obtain a clue to its possible origin, the degree of restriction enzyme site homology between adenovirus genome type 7h and those representative of the three described genomic clusters (GC) for serotype 7 was studied by analysis of pairwise comigrating DNA restriction fragments (PCRF) after digestion with Bam HI, BgR, Bglll, BstEU, EcoRl, ///«dill, Hpal, Sail, Smal, Xbal, and Xhol. The most closely related genome types, Ad7b, Ad7d, and Ad7g, displayed 85.7,83.8, and 82.8% comigrating fragments, respectively. Ad7p (GC1) was clearly distant with only 68.6% PCRF. No remarkable close genetic relationships (%PCRF>90) were observed with any of the genome types grouped within GC3 or with Ad7g (the only member of GC2), so since Ad7h seems to be related to both members of GC2 and GC3, it could equally be considered to represent a new cluster or to end up grouped in either GC2 or GC3, depending on the results of further analysis.
Introduction Adenovirus type 7 (Ad7) is the serotype which is most frequently associated with severe and even fatal respiratory disease [1, 2], Since
Received: March II, 1991 Accepted : June 24, 1991
the isolation of the prototype strain Gomen of Ad7 (Ad7p) by Berge et al. [3], a total of 19 genome types designated Ad7p, pi, a, al— 5, b, bl, c, cl, d, dl, e, el, f, and g have been described and their worldwide distribution reported
Prof. Göran Wadell Department of Virology University of Umeâ S-901 85 Umeâ (Sweden)
©1992 S. Karger AG, Basel 0300-5526/92/ 0332 0086S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 1:40:24 PM
Adriana E. Kajona-b Goran Wadellb
Material and Methods Virus Strains Adenovirus strains representative of genome types belonging to the three different genomic clusters de scribed by Li and Wadell [5] were selected for the analysis. The origin of all virus strains is shown in table I. Adenovirus strain 87/922 was chosen as representa tive of genome type 7h. This strain and several others showing identical restriction profiles were previously identified as corresponding to genome type 3f [8]. They were reanalyzed in neutralization assays using reference horse antisera (Center for Disease Control, Atlanta, Ga., USA) and confirmed to belong to serotype 7.
DNA Restriction Analysis Viral DNA was extracted from infected A-549 cells by the method of Shinagawa et al. [9]. Aliquots of DNA were then cleaved with endonucleases Asnl, Bam HI.
Bell, Bfrl. Bgll, BgH\. BstEU. Dial, EcoRl, Hindlll,
Table 1. Origin of Ad7 strains
Genomic Genome Representative Place and year of cluster type strain isolation 1 2 3
7
P g a b c d e f h
Gomen BC25 SI058 KCH4 37300 4492 B762 AI8787 87/922
USA (1954) China (1958) USA (1958) England (1973) Sweden (1964) Chinaf 1981-1984) Brazil (1981) Australia (1980) Argentina (1987)
Hpal, Nrul. Pstl, Sail, Smal, SspI, Xbal,and AVtol under conditions specified by the manufacturers (Boehringer Manheim, FRG). The DNA fragments were separated by electrophoresis in 0.8-1.2% agarose horizontal gels. The bands were stained with 25 ¡lg/ml of ethidium bromide and photographed on Polaroid 4 x 5 Land Film type 55 or 57. Determination o f PCRF The approximate size of restriction fragments was determined, and the degree of relationship expressed as percentage of PCRF was calculated. Sizes of fragments obtained with BamHl, Bell. Bgll, BgM, B.s/EII, EcoRl, ///«dill. Hpal. Sail, Smal, Xbal, and Xhol for Ad7p. a, b, c, d, e, f, and g were taken from the literature [5,6]. All data were stored and compared using the COMIG program for comigration analysis developed by Hambraeus and described elsewhere [10].
Terminology Employed Genoiny type: A distinct viral entity within a serotype identified by DNA restriction patterns ]2. II]. Genomic cluster: A group of genome types that are closely related. A statistically significant difference should be demonstrated in comparison between the mean PCRF within a group and the mean PCRF be tween the groups [5,12].
Results To estimate the relationship of genome type 7h with those representative of the three de-
87
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 1:40:24 PM
[4-6]. Recently, the isolation and characteriza tion of a new genome type of Ad7 designated Ad7h has been reported by Niel et al. [7], In addition, the study in depth of some strains of adenovirus isolated in Buenos Aires from chil dren with severe acute lower respiratory in fections revealed that genome type 7h has been circulating in Argentina at least since 1984 (Kajon and Wadell, in preparation]. Some of these strains were originally wrongly serotyped as Ad3 and their restriction profiles with 12 en donucleases published [8]. The analysis of the degree of concurrence of restriction enzyme sites between genome types of Ad7 by Li and Wadell [5] allowed their division into three gen omic clusters: G O , GC2, and GC3. The con cept of genomic clusters was established based on the analysis of many restriction sites in the entire adenovirus DNA. Genomic cluster I contains Ad7p and Ad7pl; Ad7g is the only member of GC2, and all the other genome types are grouped into GC3. This report describes the analysis of pair wise comigrating (DNA) restriction fragments (PCRF) of the newly identified genome type 7h and other genomic variants of serotype 7.
Asn I
Bfrl
Oral
M b h g b h g b h g
Ssp I
b h g M
Hindlll N r ul
Pst I
Sma I X h o l
b h g b h g b h g b h g b h g
Fig. 1. Restriction profiles of adenovirus genome types 7b, 7g, and 7h after digestion with Asti I, Bfrl, Dial. and Sspl. * = kHindlll fragments; ~ =
Intervirology 1992;33:86-90
Department of Microbiology, School of Medicine, University of Buenos Aires, Argentina Department of Virology, University of Umea, Sweden
Key Words Acute respiratory infection Adenovirus 7 Pairwise comigrating restriction fragments
Characterization of Adenovirus Genome Type 7h: Analysis of Its Relationship to Other Members of Serotype 7
Summary Ad7h is a newly identified genome type associated with severe lower acute respiratory infections and has so far been isolated only in South America. To obtain a clue to its possible origin, the degree of restriction enzyme site homology between adenovirus genome type 7h and those representative of the three described genomic clusters (GC) for serotype 7 was studied by analysis of pairwise comigrating DNA restriction fragments (PCRF) after digestion with Bam HI, BgR, Bglll, BstEU, EcoRl, ///«dill, Hpal, Sail, Smal, Xbal, and Xhol. The most closely related genome types, Ad7b, Ad7d, and Ad7g, displayed 85.7,83.8, and 82.8% comigrating fragments, respectively. Ad7p (GC1) was clearly distant with only 68.6% PCRF. No remarkable close genetic relationships (%PCRF>90) were observed with any of the genome types grouped within GC3 or with Ad7g (the only member of GC2), so since Ad7h seems to be related to both members of GC2 and GC3, it could equally be considered to represent a new cluster or to end up grouped in either GC2 or GC3, depending on the results of further analysis.
Introduction Adenovirus type 7 (Ad7) is the serotype which is most frequently associated with severe and even fatal respiratory disease [1, 2], Since
Received: March II, 1991 Accepted : June 24, 1991
the isolation of the prototype strain Gomen of Ad7 (Ad7p) by Berge et al. [3], a total of 19 genome types designated Ad7p, pi, a, al— 5, b, bl, c, cl, d, dl, e, el, f, and g have been described and their worldwide distribution reported
Prof. Göran Wadell Department of Virology University of Umeâ S-901 85 Umeâ (Sweden)
©1992 S. Karger AG, Basel 0300-5526/92/ 0332 0086S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 1:40:24 PM
Adriana E. Kajona-b Goran Wadellb
Material and Methods Virus Strains Adenovirus strains representative of genome types belonging to the three different genomic clusters de scribed by Li and Wadell [5] were selected for the analysis. The origin of all virus strains is shown in table I. Adenovirus strain 87/922 was chosen as representa tive of genome type 7h. This strain and several others showing identical restriction profiles were previously identified as corresponding to genome type 3f [8]. They were reanalyzed in neutralization assays using reference horse antisera (Center for Disease Control, Atlanta, Ga., USA) and confirmed to belong to serotype 7.
DNA Restriction Analysis Viral DNA was extracted from infected A-549 cells by the method of Shinagawa et al. [9]. Aliquots of DNA were then cleaved with endonucleases Asnl, Bam HI.
Bell, Bfrl. Bgll, BgH\. BstEU. Dial, EcoRl, Hindlll,
Table 1. Origin of Ad7 strains
Genomic Genome Representative Place and year of cluster type strain isolation 1 2 3
7
P g a b c d e f h
Gomen BC25 SI058 KCH4 37300 4492 B762 AI8787 87/922
USA (1954) China (1958) USA (1958) England (1973) Sweden (1964) Chinaf 1981-1984) Brazil (1981) Australia (1980) Argentina (1987)
Hpal, Nrul. Pstl, Sail, Smal, SspI, Xbal,and AVtol under conditions specified by the manufacturers (Boehringer Manheim, FRG). The DNA fragments were separated by electrophoresis in 0.8-1.2% agarose horizontal gels. The bands were stained with 25 ¡lg/ml of ethidium bromide and photographed on Polaroid 4 x 5 Land Film type 55 or 57. Determination o f PCRF The approximate size of restriction fragments was determined, and the degree of relationship expressed as percentage of PCRF was calculated. Sizes of fragments obtained with BamHl, Bell. Bgll, BgM, B.s/EII, EcoRl, ///«dill. Hpal. Sail, Smal, Xbal, and Xhol for Ad7p. a, b, c, d, e, f, and g were taken from the literature [5,6]. All data were stored and compared using the COMIG program for comigration analysis developed by Hambraeus and described elsewhere [10].
Terminology Employed Genoiny type: A distinct viral entity within a serotype identified by DNA restriction patterns ]2. II]. Genomic cluster: A group of genome types that are closely related. A statistically significant difference should be demonstrated in comparison between the mean PCRF within a group and the mean PCRF be tween the groups [5,12].
Results To estimate the relationship of genome type 7h with those representative of the three de-
87
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 1:40:24 PM
[4-6]. Recently, the isolation and characteriza tion of a new genome type of Ad7 designated Ad7h has been reported by Niel et al. [7], In addition, the study in depth of some strains of adenovirus isolated in Buenos Aires from chil dren with severe acute lower respiratory in fections revealed that genome type 7h has been circulating in Argentina at least since 1984 (Kajon and Wadell, in preparation]. Some of these strains were originally wrongly serotyped as Ad3 and their restriction profiles with 12 en donucleases published [8]. The analysis of the degree of concurrence of restriction enzyme sites between genome types of Ad7 by Li and Wadell [5] allowed their division into three gen omic clusters: G O , GC2, and GC3. The con cept of genomic clusters was established based on the analysis of many restriction sites in the entire adenovirus DNA. Genomic cluster I contains Ad7p and Ad7pl; Ad7g is the only member of GC2, and all the other genome types are grouped into GC3. This report describes the analysis of pair wise comigrating (DNA) restriction fragments (PCRF) of the newly identified genome type 7h and other genomic variants of serotype 7.
Asn I
Bfrl
Oral
M b h g b h g b h g
Ssp I
b h g M
Hindlll N r ul
Pst I
Sma I X h o l
b h g b h g b h g b h g b h g
Fig. 1. Restriction profiles of adenovirus genome types 7b, 7g, and 7h after digestion with Asti I, Bfrl, Dial. and Sspl. * = kHindlll fragments; ~ =
