Genetic Relationship between Cocirculating Human Enteroviruses Species C

Recombination events between human enteroviruses (HEV) are known to occur frequently and to participate in the evolution of these viruses. In a previous study, we reported the isolation of a panel of viruses belonging to the Human enterovirus species C (HEV-C) that had been cocirculating in a small geographic area of Madagascar in 2002. This panel included type 2 vaccine-derived polioviruses (PV) that had caused several cases of acute flaccid paralysis in humans. Previous partial sequencing of the genome of these HEV-C isolates revealed considerable genetic diversity, mostly due to recombination. In the work presented herein, we carried out a more detailed characterization of the genomes of viruses from this collection. First, we determined the full VP1 sequence of 41 of these isolates of different types. These sequences were compared with those of HEV-C isolates obtained from other countries or in other contexts. The sequences of the Madagascan isolates of a given type formed specific clusters clearly differentiated from those formed by other strains of the same type isolated elsewhere. Second, we sequenced the entire genome of 10 viruses representing most of the lineages present in this panel. All but one of the genomes appeared to be mosaic assemblies of different genomic fragments generated by intra- and intertypic recombination. The location of the breakpoints suggested potential preferred genomic regions for recombination. Our results also suggest that recombination between type HEV-99 and other HEV-C may be quite rare. This first exhaustive genomic analysis of a panel of non-PV HEV-C cocirculating in a small human population highlights the high frequency of inter and intra-typic genetic recombination, constituting a widespread mechanism of genetic plasticity and continually shifting the HEV-C biodiversity

Εμβολιακά στελέχη πολιοϊών - συσχέτιση ανασυνδυασμών με δευτεροταγείς RNA δομές

Σκοπός της παρούσας διατριβής είναι η διαλεύκανση της σχέσης που μπορεί να συνδέει την εντόπιση συγκεκριμένων τύπων ανασυνδυασμών σε συγκεκριμένες περιοχές του γενώματος (περιοχές 2C και 3D), με τις δευτεροταγείς RNA δομές που εδράζονται στις εν λόγω περιοχές των εμβολιακών στελεχών πολιοϊών. Αρχικά θα μελετηθούν κλινικά δείγματα στις γενωμικές περιοχές 2C και 3D για την ύπαρξη ανασυνδιασμών. Επίσης, θα μελετηθεί η VP1 περιοχή κλινικών δειγμάτων τόσο για την ύπαρξη ανασυνδυασμών όσο και για την ύπαρξη μεταλλάξεων. Στη συνέχεια με τη χρήση προγραμμάτων βιοπληροφορικής θα επιχειρηθεί η δημιουργία μοντέλων για τις δευτεροταγείς RNΑ δομές των 2C και 3D περιοχών. Η κατασκευή των δευτεροταγών δομών θα συνδράμει στην κατανόηση της σχέσης που μπορεί να συνδέει τη μεγάλη συχνότητα εμφάνισης συγκεκριμένων τύπων ανασυνδυασμών στις συγκεκριμένες περιοχές του ιικού γονιδιώματος, με τις δευτεροταγείς δομές που εδράζονται σε αυτές τις περιοχές. Τέλος θα επιχειρηθεί η δημιουργία ενός in vitro μοντέλου παραγωγής ανασυνδυασμένων εμβολιακών στελεχών πολιοϊών. Με αυτόν τον τρόπο θα γίνει προσπάθεια σύγκρισης των αποτελεσμάτων που αφορούν τη σχέση ανασυνδυασμών και δευτεροταγών δομών στα κλινικά δείγματα με τα αντίστοιχα αποτελέσματα που θα προκύψουν από το in vitro μοντέλο.In the first chapter, a study was conducted in order to identify recombination junctions and mutations in VP1 region of 15 poliovirus clinical isolates and assess their effects in virus fitness providing some insights into the patterns of natural evolution of polioviruses. In conclusion, some of the Sabin strains investigated in the present study displayed a different pattern of evolution in VP1 region. Non-synonymous mutations with a direct phenotypic impact to the virus fitness that will result in amino acid substitutions in structural elements of VP1 (N-Ags, canyon), and mutations associated with reversion to neurovirulence are selected, in spite of synonymous mutations with no phenotypic impact to the virus fitness. Thus, virus can replicate and evolve for a longer period of time in the host, escaping the host’s immune response. Full genomic sequencing of two isolates which present extraordinary interest, was conducted. Extensive genomic sequencing of the first strain, isolate 7/b/97, revealed 1.87% VP1 sequence divergence and a recombination event between a Sabin 1 strain and a strain belonging to the group HEV-C, in the 2A genomic region. The genomic features of isolate 7/b/97 may place this strain in the cVDPV category. The 1,87% divergence in VP1 revealed that isolate 7/b/97, was circulated in a region free from poliovirus for approximately 2 years. In the past, cVDPV strains were responsible for polio epidemics, due to their increased neurovirulence and their increased ability for transmission. Full genomic sequencing of the second strain (K/2002) revealed a Sabin 3/Sabin 2 recombination junction in VP1 genomic region. Intertypic capsid recombination is a very rare event. In our study the age of isolate K/2002, was approximately 11 months, while the age of the patient was 4 months. This means that isolate K/2002 has been circulated in the community, posing questions about the safety of OPV live vaccine

Complete nucleotide sequence analysis of the VP1 genomic region of Echoviruses 6 isolated from sewage in Greece revealed 98% similarity with Echoviruses 6 that were characterized from an aseptic meningitis outbreak 1 year later

The molecular characterization of two enterovirus strains (LR51A5 and LR61G3) isolated from the sewage treatment plant unit in Larissa, Greece, in May and June 2006 and the investigation of their relationship with enteroviruses of the same serotype isolated in Greece in 2001 and 2007 were performed by complete VP1 sequence analysis of the isolates. The close phylogenetic relationship and the high nucleotide similarity (98%) led to the conclusion that the virus isolated from sewage in 2006 was associated with that isolated from an aseptic meningitis outbreak 1 year later. Bootscan analysis of the VP1 genomic region revealed that intraserotypic multi-recombination events might have been involved in the evolutionary past history of the LR51A5 and LR61G3 isolates

Use of mutational pattern in 5 '-NCR and VP1 regions of polioviruses for molecular diagnosis

Polioviruses are members of the enterovirus genus, belonging to the Picornaviridae family. They are the causative agents of poliomyelitis, a paralytic and sometimes fatal disease in humans. The number of poliomyelitis cases caused by wild poliovirus infections has been dramatically reduced by the extensive use of two available vaccines: the inactivated poliovirus vaccine (IPV) and the oral poliovirus vaccine (OPV). Despite the importance of OPV in the reduction of poliomyelitis cases, one of the disadvantages associated with this vaccine is the rare occurrence of vaccine-associated paralytic poliomyelitis (VAPP) in vaccinees or their healthy contacts through the accumulation of mutations and/or recombination in Sabin strains genome. Thirteen clinical isolates originating from healthy vaccinees and VAPP cases were investigated in order to identify genomic modifications in 5' non-coding region (5'-NCR) and VP1 genomic regions. The analysis of samples was conducted by RT-PCR, RFLP, sequencing and bioinformatics analysis. All clinical isolates were characterized as OPV-Iike viruses. Our results showed that analysis of 5'-NCR and VP1 regions of Poliovirus Sabin strains is important in order to identify mutations that increase the neurovirulence conducting to the eventuality of emergence of VAPP cases. (C) 2007 Elsevier Ltd. All rights reserved

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 frorn 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 front enteroviral strains that circulated all over the world. (C) 2007 Elsevier Inc. All rights reserved

Molecular Characterization of Wild-Type Polioviruses Isolated in Greece during the 1996 Outbreak in Albania

During the present study three type 1 poliovirus strains isolated in Greece during the 1996 poliomyelitis outbreak in Albania were retrospectively investigated and determination of their relationship with other epidemic strains isolated in Albania or elsewhere during previous epidemics was attempted. SimPlot analysis revealed that the three Greek strains are the result of a recombination event in the VP2 coding region

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

Retrospective Characterization of a Vaccine-Derived Poliovirus Type 1 Isolate from Sewage in Greece▿

Retrospective molecular and phenotypic characterization of a vaccine-derived poliovirus (VDPV) type 1 isolate (7/b/97) isolated from sewage in Athens, Greece, in 1997 is reported. VP1 sequencing of this isolate revealed 1.87% divergence from the VP1 region of reference strain Sabin 1, while further genomic characterization of isolate 7/b/97 revealed a recombination event in the nonstructural part of the genome between a vaccine strain and a nonvaccine strain probably belonging to Enterovirus species C. Amino acid substitutions commonly found in previous studies were identified in the capsid coding region of the isolate, while most of the attenuation and temperature sensitivity determinants were reverted. The ultimate source of isolate 7/b/97 is unknown. The recovery of such a highly divergent derivative of a vaccine strain emphasizes the need for urgent implementation of environmental surveillance as a supportive procedure in the polio surveillance system even in countries with high rates of OPV coverage in order to prevent cases or even outbreaks of poliomyelitis that otherwise would be inevitable