Eugenia Bolanaki

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

ABSTRACT 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.

Molecular characterization of wild-type polioviruses isolated in Greece during the 1996 outbreak in Albania

ABSTRACT 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.

Partial 3D gene sequences of Coxsackie viruses reveal interspecies exchanges

The 3D region of 46 clinical Coxsackievirus strains, primarily belonging to the human enterovirus B species (HEV-B), were analyzed using nucleotide distance matrices and phylogeny software. The conclusions from previously analyzed genomic regions (VP1—2A-2B-2C) of the aforementioned strains revealed that enteroviruses’ inheritance is being guided by gene adaptation among viruses of different serotypes. In this report the comparison of partial VP1 and 3D gene phylogenies presented an obvious incongruence. Moreover, the phylogeny of 3D sequences of the strains revealed an unexpected (and for the first time reported) homology among strains of different species. The observations of our study indicate that conversion events such as multiple mutations or recombination among strains and unknown donors may occur during the evolution of circulating strains, leading, probably, to viruses with altered genome and virulence.

Nucleotide Analysis and Phylogenetic Study of the Homology Boundaries of Coxsackie A and B Viruses

Modern molecular methods use VP1 coding region as a target for RT-PCR assays followed by sequencing, in order to identify new untyped enteroviruses’ strains. In the present study, two different genomic portions of VP1 and the full length of 2A coding region of 53 clinical isolates, mostly belonging to HEV-B species, were amplified and sequenced. Nucleotide analysis of the produced sequences revealed that the values that define an unknown strains serotype vary according to the serotype and the specific part of VP1, which is investigated. The correlation, however, with the serotype was affirmed in both VP1 portions that were studied, as well as in the first 20 bases of 2A region. In the rest of 2A, no correlation with the serotype and disruption of monophyly was observed. Phylogenetic analysis of the same sequences confirmed, in most cases, the results of the nucleotide analysis.

Molecular phylogeny of VP1, 2A, and 2B genes of echovirus isolates: epidemiological linkage and observations on genetic variation

Summary.Phylogenetic relationships between 37 echovirus clinical isolates, most of them originating from an aseptic meningitis outbreak during 2001 in Greece, were investigated by RT-PCR and sequencing. The generic primers 292 and 222 were used to amplify about 300 bp of the 5′ end of VP1 while primers EUG3a, 3b, 3c, and EUC2 amplified the entire coding sequence of the 2A and 2B genes. Phylogenetic trees were constructed for each genomic region using the clinical isolates’ sequences and those of the prototype echoviruses in order to investigate the correlation of part of VP1 with the serotype as well as the genetic variation of the echovirus genome in 2A and 2B. The phylogenetic grouping pattern of the clinical isolates revealed that there is a correlation of serotype and genotype in the part of VP1 that was investigated, while this pattern is disrupted in the adjacent genomic regions that were sequenced. Sequence analysis of the adjacent 2A and 2B genes provided a different pattern of phylogenetic relationships and strong evidence of epidemiological linkage of most of the clinical isolates.

Vaccine derived bi- and multi-recombinant Sabin strains

A retrospective analysis of five Sabin intertypic recombinant strains, isolated from human feacal specimens during the time period 1978–1985 in Greece, was performed by RT-PCR, Restriction Fragment Length Polymorphism (R.F.L.P.) and sequencing. Of the studied strains, three (EPA, EPB, EPC) were found to be bi-recombinant Sabin3/Sabin2/Sabin3 (S3/S2/S3), one strain was characterized as a probable S3/S2- CAV18 or CAV21-S2/S1 multi-recombinant (EDP11) and one was identified as a tripartite one S3/S2/S1 (EDP12). Samples EPA, EPB and EPC presented a common recombination junction in the 2C genomic region. Moreover, strains EPA and EPB shared also the second recombination site in the 3D genomic region, whereas the second recombination of EPC was also determined in 3D but in a different nucleotide position. Strains EDP11 and EDP12 presented both identical recombination motifs and recombination sites. The first was detected in the 2C genomic region and the second in the 3D region. Strain EDP11 presented an interesting feature since a sequence of 120 nucleotides seems to have derived from a member of human enteroviruses species C (CAV18 or CAV21). This finding is of great importance, considering that this strain (EDP11) was isolated from an area and time period, where no Coxsackie A virus or poliovirus epidemics occurred. Our study underlines the role of specific positions and motifs of the poliovirus genomic sequences involved in recombination events and prompts that Coxsackie A viruses belonging to human enterovirus species C (genetically closely related to PV) are considered as the possible counterparts of the recombination.

Evolution of 2B and 2C Genomic Parts of Species B Coxsackie Viruses. Phylogenetic Study and Comparison with Other Regions

Modern molecular approaches on the genome of enteroviruses’ circulating strains have established new data about the mechanism and significance of its evolution. In the present study, 46 enteroviruses isolates, belonging to HEV-B species and exhibiting distinct origin in geographical or chronological terms, were investigated concerning their primary structure and phylogeny. Two regions of the aforementioned strains genome, which have not been thoroughly investigated (2B and 5′ extreme of 2C) were amplified and sequenced for the first time. Phylogenetic and nucleotide analysis of the isolates’ fragments, along with representative prototype sequences, demonstrate that the classification scheme of monophyly and accordance with the genotype, which characterizes VP1 region, is seriously disturbed. Moreover, the phylogenetic trees constructed from adjacent regions of the genome appear radically incongruent suggesting that the parameters that affect these portions are different or act in a different extent. Our study results an additional step in the study of enteroviruses evolution and inheritance, by investigating unstudied regions of newly sequenced strains and revealing that the primary structure and phylogeny of them is different not only comparably to the structural genome but also from one to another.

Cocirculation of genotypes D4 and D6 in Greece during the 2005 to 2006 measles epidemic.

One of World Health Organizations proposed methods for the establishment of measles surveillance worldwide, to achieve the elimination of measles virus by 2010, is the genetic characterization of measles wild-type virus strains. In this study, 34 measles virus strains, isolated from clinical samples during the 2005 to 2006 measles outbreak in Greece, were genotyped and studied in terms of nucleotide variation and phylogeny. Interestingly, the cocirculation of 2 different genotypes, namely, D6 and D4, was revealed. In fact, the D4 genotype has never been previously reported in Greece. Finally, although the D4 Greek strains possessed identical nucleotide sequences, the D6 isolates segregated into 3 distinct subgroups, 2 of which differed genetically and phenotypically from all GenBank deposited measles sequences. It is, thus, important to continue the epidemiologic surveillance of measles in Greece to aid future studies of measles transmission, monitor the effectiveness of measles immunization, and eventually document the elimination of the virus in our country.