Use of subgenomic poliovirus DNA hybridization probes to detect the major subgroups of enteroviruses

Abstract

Three nucleic acid hybridization probes were derived from DNA clones of the poliovirus type 1 genome. Used in dot hybridization experiments, the probes successfully detected members of each of the major enteroviral subgroups. The hybridization patterns obtained with the three probes suggested that a highly conserved nucleotide sequence existed among the enteroviruses tested, mapping between bases 220 and 1809 in the poliovirus genome. Two new antiviral agents capable of inhibiting enterovirus replication in tissue culture were used to demonstrate the specificity of the probes for viral RNA. * Corresponding author. saline (30 mM Tris and 150 mM NaCl [pH 7.0]), and the cells were lysed with 0.125 ml of 0.5% Nonidet P-40 (Bethesda Research Laboratories, Gaithersburg, Md.) in Tris-saline. The plates were then scraped, and the cell lysate was centrifuged at 15,000 x g at 4°C for 5 min. Samples of the supernatant fluid were immediately frozen at-70°C. For some experiments, 1 .g (final concentration) of candidate

Detection of enteroviruses using cDNA and synthetic oligonucleotide probes

“…These findings are in agreement with our recent findings on the genomic interaction between different rhinovirus serotypes using 7 rhinovirus cDNA probes (Forsyth et al, in press). They are also in general agreement with previous studies on the genomic interrelationship between enteroviruses using probes of different lengths and from different viruses (Rotbart et al, 1984;Tracy, 1985;Hyypia et al, 1988) and basically show that it would be difficult to select an optimal probe or a combination of cDNA probes that would detect all enteroviruses satisfactorily. This problem of sensitivity is further complicated by the fact that in clinical samples, these viruses are generally present in relatively low concentrations and therefore cDNA probes are unlikely to detect these viruses consistently or efficiently.…”

“…Recently the genomes of a number of picornaviruses such as the polioviruses (Kitamura et al, 1981;Racaniello and Baltimore, 1981;Nomoto et al, 1982;Stanway et al, 1983;Toyoda et al, 1984;La Monica et al, 1986), coxsackie B3, B4 and A21 (Hughes et al, 1987;Jenkins et al, 1987;Lindberg et al, 1987) and rhinovirus 14, 2, 1B and 89 (Stanway et al, 1984;Skern et al, 1985;Duechler et al, 1987;Hughes et al, 1988) have been sequenced. A number of cDNA (Rotbart et al, 1984;Tracy, 1985;Al-Nakib et al, 1986) and RNA (Rotbart et al, 1988) probes have been generated and used in hybridization assays to detect these viruses. None has been found to be universally satisfactory.…”

“…None has been found to be universally satisfactory. They show variable sensitivity for different picornaviruses, presumably due to variations in the degree of homology between the genome of the virus and that of the probe (Rotbart et al, 1984;Al-Nakib et al, 1986;Hyypia et al, 1987;Forsyth et al, in press). None has been found useful so far in detecting enteroviruses in clinical material.…”

Nucleic acid probes as potential tools in oral microbial epidemiology

“…In medicine, nucleic acid probes against a number of viruses have opened up new opportunities to study the pathogenesis of important infectious diseases. Of particular importance are probes to hepatitis B virus (39,40), and enteroviruses (41,42). Using DNA probes specific to human papilloma virus together with in situ hybridization teehnique, HENKE et al (34) managed to demonstrate the assoeiation between this virus and foeal epithelial hyperplasia of the oral tnueosa.…”

Rapid and accurate viral diagnosis

“…Different processing methods have been examined to determine the optimum for detection of enterovirus viremia (Prather et al, 1984), and the factors influencing recovery of varicella-zoster virus (VZV) have also been studied (Levin et al, 1984). A number of studies have determined the importance of centrifugation of specimens onto the monolayer.…”