Full-genome sequence analysis of a multirecombinant echovirus 3 strain isolated from sewage in Greece.

An echovirus 3 (Echo3) strain (strain LR31G7) was isolated from a sewage treatment plant in Greece in 2005. Full-genome molecular, phylogenetic, and SimPlot analyses were conducted in order to reveal the evolutionary pathways of the isolate. Nucleotide and phylogenetic analyses of part of the VP1 genomic region revealed that the isolated strain correlates with Echo3 strains isolated during the same year in France and Japan, implying that the same virus circulated in Europe and Asia. LR31G7 was found to be a recombinant that shares the 3' part of its genome with an Echo25 strain isolated from asymptomatic infants in Norway in 2003. Nucleotide and SimPlot analyses of the VP1-2A junction, where the recombination was located, revealed the exact recombination breakpoint (nucleotides 3357 to 3364). Moreover, there is evidence that recombination events had occurred in 3B-3D region in the evolutionary history of the isolate. Our study indicates that recombination events play major roles in enterovirus evolution and that the circulation of multirecombinant strains with unknown properties could be potentially dangerous for public health.

Molecular identification and full genome analysis of an echovirus 7 strain isolated from the environment in Greece.

Two enteroviruses from river water and four from sewage treatment plant were isolated in Larissa, Greece, that all shared the same sequence. A full genome analysis was conducted in an attempt to reveal the evolutionary pathways of one of the isolated strains (LR11F7). VP1 nucleotide and phylogenetic analysis revealed that the isolated strain had 78% homology with the echovirus 7 prototype strain Wallace. Full genome analysis revealed that LR11F7 P1 region is related to echoviruses 7 and that P2 and P3 regions are originating from contemporary enteroviruses isolated in South Asia. Two recombination events were shown to be involved into the evolutionary history of LR11F7, the one event concerning 3A, 3B, and 2C, and the other concerning 3D genomic region, both with new types of HEV-B. The contribution of recombination to enterovirus evolution is substantial, giving rise to new genetic lineages with unknown properties.

Growth kinetic analysis of bi-recombinant poliovirus vaccine strains.

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and in rare cases may cause vaccine-associated paralytic poliomyelitis (VAPP). Mutations at specific sites of the genome and recombination between Sabin strains may result in the loss of the attenuated phenotype of OPV (Oral Poliovirus Vaccine) strains and the acquisition of traits characteristic of wild polioviruses, such as increased neurovirulence and loss of temperature sensitivity. In this study, we determined the phenotypic traits such as temperature sensitivity and growth kinetics of eight OPV isolates (six bi-recombinant and two non-recombinant). The growth phenotype of each isolate as well as of Sabin vaccine strains in Hep2 cell line at two different temperatures (37 and 40 degrees C) was evaluated using two different assays, RCT test (Reproductive Capacity at different Temperatures) and one-step growth curve analysis. Moreover, the nucleotide and amino acid positions in the genomes of the isolates that have been identified as being involved in the attenuated and thermo sensitive phenotype of Sabin vaccine strains were investigated. Mutations that result in loss of the attenuated and thermo sensitive phenotype of Sabin vaccine strains were identified in the genomes of all isolates. Both mutations and recombination events correlated well with the reverted phenotypic traits of OPV-derivatives. In the post-eradication era of wild polioviruses, the identification and the characterization (genomic and phenotypic) of vaccine-derived polioviruses become increasingly important in order to prevent cases or even outbreaks of paralytic poliomyelitis caused by neurovirulent strains.

Direct extraction and molecular characterization of enteroviruses genomes from human faecal samples.

Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several enteroviruses serotypes are not easily isolated in cell cultures system used and routinely more than one passage in cell culture is performed. A total of 54 archived faecal samples were examined. The heterogeneous nature of faecal samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. PCR inhibitors are frequently encountered in stool specimens. From the three methods initially compared for extraction of viral RNA, QIAamp Viral RNA Mini Kit was retained as it yielded the highest amount of viral RNA without the interference of RT-PCR inhibitors. Evaluation of 54 archived stool specimens by RT-PCR and cell culture resulted in a higher frequency of detection by RT-PCR. With the use of RT-PCR we were able to detect two additional samples otherwise considered negative for enterovirus isolation if only the cell culture standard methodology was employed. RNA extraction with QIAamp Viral RNA Mini Kit coupled with RT-PCR in the 5'NCR (subgrouping into distinct genetic clusters of all enteroviruses) and VP1 (reliable serotyping by sequencing) is a rapid and sensitive technique of direct poliovirus/non-polio enteroviruses recovery and molecular characterization from human faecal specimens without further passage in cell culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Complete genomic characterization of an intertypic Sabin 3/Sabin 2 capsid recombinant.

The genetic properties of strain K/2002, isolated from fecal samples of a 7-month-old child who had received his first oral poliovirus vaccine (OPV) dose at the age of 3 months, are described. Preliminary sequencing characterization of isolate K/2002 revealed an S3/S2 recombination event at the 3' end of the VP1 coding region. A recombination event resulted in the introduction of six Sabin 2 amino acid residues in a Sabin 3 genomic background. Furthermore, mutations associated with loss of the attenuated phenotype of Sabin 3 strains have been identified in the genome of isolate K/2002. The data presented here emphasize the need for careful planning of vaccination strategies, which involve stopping OPV administration in regions that are certified to be polio-free.

Possible recombination and gene adaptation exchanges among clinical echovirus strains: crossing the temporal and topological barriers.

Six echovirus strains belonging to serotypes echovirus 6, 13, and 30 were investigated in the present work by sequencing of the whole 2C gene and about 560 nt of the 5' part of 3-dimensional genomic region. Four of the 6 echovirus strains were epidemics, whereas 2 were from sporadic cases. The whole procedure was carried out by using nucleotide distance matrices and phylogeny software. The sequences obtained strengthen the observation that recent echovirus isolates differ significantly from prototype strains in the downstream regions of the genome and provides further evidence that nonstructural enterovirus genes are ubiquitous and may combine freely adapting genomic sequences that are not restricted from the place of isolates' origin. For diagnostic purposes, particular emphasis is given on the utility of sequencing downstream genes and comparison of them with corresponding genomic regions from enteroviral strains that circulated all over the world.