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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.34 n.2 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000200010 

Identification of Adenovirus 7h Heterogeneity in the E3 Region

MARISOL BRUZZONE, MARIO BARRO, EUGENIO SPENCER

Laboratorio de Virología, Departamento de Ciencias Biológicas, Facultad de Química y Biología, Universidad de Santiago de Chile. Alameda 3363 casilla 40 Correo33, Santiago, Chile

Corresponding author: Eugenio Spencer. FAX: 562-6812108. email: espencer@lauca. usach .cl

Received: February 28, 2001. In revised form: April 12, 2001. Accepted: April 18, 2001

ABSTRACT

Adenovirus genotype 7h was previously reported to be originated from a recombination event between adenovirus genotypes 7p and 3p. Based on those findings, further characterization of other adenovirus 7h strains become important to determine whether all adenovirus 7h strains arose from a single recombinational event. To explore such a possibility, 160 clinical isolates were studied after developing a PCR assay using a primer set designed to amplify the region corresponding to E3-7,7 Kd of adenovirus ADV 7p and E3-9 Kd of adenovirus 3p. The assay was able to differentiate most of the subgenus B strains from adeno 7h with the genotype 3d. The study of several adenovirus 7h clinical isolates revealed the existence of three variants of adeno7h. One of the variants, 7h3, shows a high degree of similarity with gene E3-9 Kd of ADV 3p, but lacks the corresponding AUG codon. Our results suggest that more than one recombination event may explain the detection of three different types of adenovirus 7h. The genotype variants of adeno 7h were detected in different years, indicating that the recombination events took place independently from each other. The study of the recombination region may allow further understanding of the function of several viral polypeptides in the immune response, and understanding the mechanism involved in virulence associated to adenovirus 7h.

Key terms: adenovirus virus recombination

INTRODUCTION

Acute respiratory diseases are associated with infection by several pathogens, including viruses (Mallet et al. 1966). Adenovirus is one of the agents that causes the highest morbidity and mortality among children from 0 to five years old (Hierholzer et al. 1980; Matumoto et al.1958 ; Steen-Johnsen et al.1969; Wadell et al. 1980b). The adenoviruses frequently associated with acute respiratory syndrome are those belonging to subgenera B and C (Steen-Johnsen et al 1969., Wadell et al. 1980). In several countries, including those in the Southern Cone of America, a number of reports indicate that the serotypes 1-3; 5-7; 14 and 21 are more frequently associated with those cases (Kajon & Suarez 1990; Kajon & Wadell 1994). Diagnosis of the adenovirus genotypes is based on the study of viral DNA by restriction enzyme analysis using a well-established panel of enzymes (Kajon & Suarez 1990). The procedure makes it possible to identify several genomic variants among adenovirus genotypes 7, such as 7b through 7 g, indicating that those may correspond to unique genotypes that determine the clonal origin of the isolate (Li & Wadell 1986). Epidemiological surveys indicate that the adenovirus serotypes in most of the more severe cases belong to genotype 7, and of those, to genotype 7h (Kajon & Suarez 1990). PCR using specific primers has become an important tool for epidemiological survey, since it allows the determination of several differences among the clinical isolates (Allard et al. 1990).

Based on the available data of sequence analysis, it was suggested that the adenovirus genotype 7h may be a recombinant between adenovirus prototypes of serotypes 7 and 3. The recombination event involves the E3 region, where a 7,7 Kd-ORF is encoded in the adenovirus 3p and a 9Kd-ORF in the adenovirus7p (Kajon & Wadell 1996). As a result of the recombination event, genotype 7h shares the fiber gene sequence with genotype 3p and not with 7p. Furthermore, adenovirus 7h contains a deletion that involves an important portion of the fiber gene and a point mutation that eliminates an initiation codon of a potential ORF (Kajon & Wadell 1996). The recombinational region among different clinical isolates was studied to determine whether all the 7h isolates came from one original genotype.

The recombination region was studied using different clinical isolates to determine whether all the isolates could result from more than one recombinational event yielding viruses that exhibit a genotype identical to the 7h. To characterize the adenovirus strains of genotype 7h, isolates obtained from patients with acute respiratory syndrome were examined using PCR. A set of primers representing sequences located in the recombinational region were used for this purpose, and those isolates that showed differences in the migrations of the PCR product were sequenced.

MATERIAL AND METHODS

Cell and virus strains

All the adenovirus strains were grown in Hep cells (ATCC CCL 23) as described elsewhere (Kajon et al. 1994)]. The virus strains were ADV B 1 7h (87-922, Argentina, 1987), 7p (prototype, Gomen, USA, 1954), 7b (KCH4, England, 17973), 7c (37300, Sweden, 1964), 7e (B-762, Brazil, 1981), 7g (BC-25, China, 1958), 3p (prototype GB, USA, 1953), 3c (TC3449, Japan, 1983), 3d (8543, Australia, 1982), ADV B-2:14p (prototype, DeWitt). ADV subgenus C: 1p (prototype, Ad 71), 2p (prototype, Ad 6), 5p (prototype, Ad 75), ADV subgenus E: 4p (prototype, RI-67).

Clinical specimens

One hundred sixty nasopharyngeal aspirates were collected from 1988 to 1995 at the

Hospital Roberto del Río, Santiago, Chile. All the patients were children under two years of age and clinically diagnosed with acute respiratory syndrome. The samples were taken 48 hrs after admission and kept in 2 ml of PBS buffer containing 100 U/ml de penicillin and 0.1 mg/ml de streptomycin sulfate. The samples were centrifuged 1000x g for 15 min. and maintained at -20C until the PCR assay was performed. A portion of the sample was also used for indirect inmuno fluoresce (IIF) as described elsewhere (Kajon & Suarez 1990).

Primer design for PCR

One set of primers (REC1 and REC2) was designed based on a previously-published sequence of the recombination region of 7 [Kajon & Wadell 1996]. The primer REC 1, 5'-TGTACTACGCCTGCTGCT-3', hybridizes to the upstream sequence the coding region of the ADV TP E3-7,7 kd gene. The primer REC 2, 5'-GTGGTATGA GTAGAC AGG-3', hybridizes to the upstream sequence of the coding region of the ADV 3p E3-9 kd gene. The predicted size of the PCR products is 337 bp based on the published sequence [Kajon & Wadell 1996].

Clinical samples obtained as aspirates and infected culture supernatants were diluted in 10 mM Tris-HCl (pH 8.0) buffer containing 55 mM KCl, 1.65 mM Mg2Cl 1 mM EDTA and 0.5% Tween 20. The diluted samples were treated with 0.1 mg/ml of proteinase K at 50°C for 1 h. After inactivation of protease K at 101°C, the samples were subjected to PCR amplification using a Gibco BRL taq polymerase kit. As a molecular weight marker, a 1KB ladder was used.

Sequencing of amplified PCR products

The PCR products were sequenced as previously described (Innis et al.1988) using the Gibco-BRL "dsDNA cycle sequencing system" kit. The sequences were analyzed using FASTA and BLAST programs, using the previously-published sequence (Kajon & Wadell 1996).

RESULTS

A silver nitrate stained polyacrylamide gel showing the electrophoretic mobility of the amplified fragments for each of the adenovirus prototype strains is shown in Figure 1. The amplified products of the adenovirus genomic variants examined in the study (7h, 7p, 7b, 7c, 7e, 7g, 3p, 3c, 3d, 14p, 1p, 2p, 5p, 4c) vary in size. The migration pattern allowed the identification of all the strains of subgenus B except those of genotype 3d because its amplified product has a migration identical to adenovirus 7h. The strains of subgenus C and E were not amplified under these conditions. The results indicate that there are differences in the size of the amplified product of adenovirus 7h as compared to 7p and 3p strains. The size of their respective fragments was 337, 394, and 394, respectively. This result agrees with the expected size based on the reported sequences and allows the differentiation of all the isolates of the genotype 7 from adenovirus 7h and from the isolates of other adenovirus subgenus. The electrophoretic migrations of the amplified products were compared by coelectrophoresing the PCR products of the different 7 and 3 genotypes mixed with molecular size markers as shown in Figure 2.

 

Figure 1: PCR assay of the recombinational region in the E3 gene. The amplified products obtained using primers of REC-1 and REC-2 are shown using purified DNA from the indicated adenovirus serotypes and genomic variants. The marker of molecular size (MTM) is the1Kb DNA ladder. As controls, samples were amplified using uninfected cell extracts (C)

 

Figure 2: Coelectrophoresis of PCR products obtained from different adenovirus serotypes with molecular weight markers. The PCR products shown in Figure 1 were subjected to electrophoresis with and without the ladder markers to define the size differences among the viral strains. The size of the markers is indicated

 

TABLE I

Isolation of ADV 7 h variants in clinical samples obtained from 1988 to 1995

Year
ADV 7h-1
ADV 7h-2
ADV 7h-3
1988
3
2
0
1989
5
1
0
1990
7
1
0
1991
9
0
1
1992
12
0
0
1993
4
0
1
1994
2
1
0
1995
4
1
0
Total
46
6
2


To determine the characteristics of the adenovirus 7h obtained in recent years, 54 isolates of adenovirus 7 h were selected and amplified using primers REC 1 and REC-2 as shown in Figure 3. The amplified products were heterogeneous in size, and three migration patterns were observed corresponding to sizes of 337bp to 315bp; they were named 7h1, 7h2 and 7h3. The PCR amplification was done with five isolates of each variant to minimized the possibility of error. In order to show that they corresponded to genotype 7h, they were analyzed for their restriction pattern. As shown in Figure 4, all the isolates exhibit an identical pattern of genomic DNA corresponding to that described for the 7h genotype. These isolates were obtained in different years and seasons, suggesting that they resulted from more than one single event of recombination. Of all the isolates examined, 46 corresponded to adenovirus genotype 7h1, 6 to adenovirus 7h2, and 2 to adenovirus 7h3 (Table I). The sequence of the amplified products from different types of adenovirus 7h was determined after purification of the viral DNA. To lower the degree of sequence variation, 5 isolates of each of the variants were independently sequenced, and the results show a complete sequence similarity among the isolates of the three variants. The sequences of the adenovirus 7h1, 7h2, and 7h3 are shown in Figure 5. Those sequences were compared with those previously reported by Kajon and Wadell in 1996. As shown in Figure 5, the sequence of adenovirus7h1 is identical to that of the adenovirus 7h prototype. The sequence of the adenovirus 7h 2 seems to be completely different from that reported for both adenovirus 7h and adenovirus 3p. The region does not contain the initiation codon that could account for the E3-9Kd gene located in this section of the viral genome. On the other hand, the sequence of the adenovirus 7h3 is very similar to that of adenovirus 3p, which lacks the initiation codon for the E3-9Kd gene.

 

Figure 3: Identification of variants of adenovirus 7h. PCR products using primers REC-1 and REC-2 in clinical isolates of adenovirus 7h. The different PCR products obtained are indicated as variants 7h1, 7h2, and 7h3.

 

Figure 4: Restriction analysis of genomic DNA obtained from the different variants of genotype 7. DNA was subjected to digestion with BamH 1, Sma I, XhoI, and Hind III. The products were phenol extracted, ethanol precipitated, subjected to electrophoresis in 10% polyacrylamide gel and silver stained.

DISCUSSION

The characterization of adenovirus 7h from clinical isolates showed the presence of three genotype variants. The most abundant isolates corresponded to the one previously defined as 7h (Kajon & Wadell 1992; 1996). Surprisingly, we found isolates with a characteristic 7h restriction pattern exhibited PCR products different in size from those of 7h (Fig. 4). This difference was further confirmed by direct sequencing of the PCR products of these isolates (Fig. 5). The sequence analysis of the adenovirus 7h2 indicated that it differs from the rest of the adenovirus strains (Fig. 5). In the case of the adenovirus 7h3, it shares a high degree of similarity with the gene E3-9 Kd of ADV 3p, but lacks the corresponding AUG codon. These results indicate that more than one recombination event may explain the detection of three different types of adenovirus 7h. Recombination among adenoviruses has been previously reported not to be an unusual event (Sambrook et al. 1980, Boursnell & Mautner 1981). It is interesting to point out that those variants of adenovirus 7h were isolated in different years, indicating that the recombination events took place more than once. Adenovirus 7h has a deletion in the E3 region of a gene present in the prototype strains of adenovirus 7 and 3. Other adenovirus serotypes, such as adenovirus 35, also lack the same gene (Scaria et al. 1992; Kajon & Wadell 1996). The genes of the E3 region, coding for the proteins 15.2Kd and 15.3 Kd, in adenovirus 2, seem to be associated with protection against the cytolytic action of TNF (Chroboczek et al. 1992; Chroboczek et al. 1992, Basler & Horwitz 1996). It has been suggested that this region codes for a gene product that may regulate in cis the overall expression of the E3 region (Kvist et al. 1978 Scaria & Wold 1994). Therefore, changes in this region may have an extensive effect on the expression of the entire region, affecting products such as gp19KD, altering the anti-TNF function, since it has been also associated with the sequestering of the MHC class I antigen (Flomenberg et al. 1988, Kvist et al. 1978; Wold et al. 1995). This effect on the synthesis of the gp 19 KD could explain the virus evasion from the host immune response. The study of this recombinational region may allow further understanding of the function of several viral polypeptides on the immune response and the mechanisms involved in virulence associated with adenovirus 7h.

There is high degree of sequence similarity in the nucleotide sequence between the genes E3-7,7 Kd of ADV 7p and E3-9 Kd of ADV 3p, while the polypeptides have major differences due to the small deletion in both genes (Bhat et al. 1986, Defer et al. 1990, Kajon & Wadell 1996). The sequencing results indicate the existence of some deletions as well as point mutations that eliminate the initiation codon for those genes in adenovirus 7h (Figure 5). The PCR assay was developed using primers that amplified region E3-7,7 Kd of adenovirus ADV 7p and E3-9 Kd of adenovirus 3p where the recombination event took place. Those primers were able to amplify only adenovirus strains of the subgenus B. The size of the fragment varies among the different viral strains. Adenovirus 7h could be differentiated from the subgenus B strains except in the case of adenovirus virus 3d. These results suggest that this region may be important and requires definition of the role of the hypothetical polypeptide coded in the missing gene. This gene is also absent in other adenovirus virus strains such adenovirus 11,14, 34, and 35 of the subgenus B2 (Mei & Wadell 1992, Flomenberg et al. 1988). In other adenoviruses, such as adenovirus of subgenous C like 2 and 5, this region codes for a membrane glycoprotein of 11.6 kd, which is expressed in different concentrations during the infectious cycle and seems to be involved in virus release from infected cells (Scaria et al. 1992, Wold et al. 1995). This glycoprotein does not exhibit any substantial degree of similarity with E3-9 Kd of adenovirus 3p ([Signäs et al. 1985).

The primers used in this study might provide a fast and reliable method for epidemiological studies involving the adenovirus 7h and the associated acute respiratory syndrome.

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