Comparison of cDNA probe hybridizations and RT-PCR detection methods for the identification and differentiation of enteroviruses isolated from sea water samples

Fifteen enteroviruses (EVs) previously isolated from Tyrrhenian sea water samples were used. They were first identified by traditional dot-blot and Northern-blot hybridizations with a group of cDNA probes from cloned Poliovirus 1 and Coxsackievirus B4 and oligodeoxynucleotides complementary to echovirus 6 and 9 sequences. Using both wild viruses and known enteroviruses a reverse-PCR protocol was then set up followed by cDNA sequencing of the fragments generated. The sequences of primers were selected from a consensus of several 5′ non-coding ends of enterovirus genomes, representing highly conserved regions. The downstream (region 577–603) and the upstream (region 436–465) oligonucleotide primers carried an extra sequence in order to generate BamHI and a HindIII restriction sites at the 5′ and 3′ end respectively of the amplified cDNA fragments for directional cloning in a plasmid. The downstream 5′NC primer was 5′-biotinylated in order to allow direct sequencing of the amplicon, when possible, after strand separations on streptavidin coated magnetic beads. The PCR of reverse transcribed viral RNAs resulted in a 167–170 b.p. cDNA product on ethidium bromide-stained 2% agarose gels in all the samples and reference viruses. The test is negative on reoviruses, hepatitis A and uninfected BGM cells and detects < 1 TCID50 viral particles. Sequences of cloned fragments were compared with sequences of cloned enteroviruses stored in commercial data banks. The 5′NC region of a reference echovirus 5 was also cloned and sequenced to improve the comparison. On the basis of deduced genetic distances, three poliovirus 1, eight coxsakievirus B5, four coxsakievirus B1 were diagnosed. One poliovirus Sabin 2 was isolated together with a coxsackievirus-related strain in the same lysate sample. The reliability and sensitivity of this RT-PCR method makes it an attractive approach to virus detection in environmental samples.