Amplification of Echoviruses genomic regions by different RT-PCR protocols - a comparative study

In the present report, the results of a comparative study in the detection of all Echoviruses reference strains as well as of 38 clinical isolates are presented. Using RT-PCR with already published primer pairs (UG(52)-UC53, 292-222, 012-011 and EUG2a, 2b, 2c-EUC2) from the 5’UTR, the VP1 region as well as a long genomic fragment including the VP1 3’ end, the entire coding sequence of 2A, 2B, and the 5’ moiety of the 2C-coding region amplification was effective with all reference and clinical Echovirus isolates with primer pair UG52-UC53 while with 292-222 and 012-011 were amplified 27/28 reference Echovirus strains and all clinical isolates. As far as EUG2a,2b,2c-EUC2 is concerned, the RT-PCR gave a positive result for 26/28 reference Echovirus strains and 34/38 clinical isolates. The sequence analysis of a large part of the 5’UTR has revealed that there is no correlation between 5’UTR identity and the currently recognized human enterovirus species. It has been suggested that part of VP1 coding sequence would correlate well with serotype since a number of important neutralization epitopes, as well as receptor recognition sequences, lie within the VP1 coding sequence. Therefore, UG52-UC53 and 292-222 primer pairs seem to be the most appropriate for Echovirus detection and, moreover, UG52-UC53 is useful for the classification of enteroviruses into genetic clusters (sub-groups) while 292-222 for the identification of enteroviruses by amplicon sequencing. (C) 2004 Elsevier Ltd. All rights reserved

Amplification of Echoviruses genomic regions by different RT-PCR protocols - a comparative study

In the present report, the results of a comparative study in the detection of all Echoviruses reference strains as well as of 38 clinical isolates are presented. Using RT-PCR with already published primer pairs (UG(52)-UC53, 292-222, 012-011 and EUG2a, 2b, 2c-EUC2) from the 5'UTR, the VP1 region as well as a long genomic fragment including the VP1 3' end, the entire coding sequence of 2A, 2B, and the 5' moiety of the 2C-coding region amplification was effective with all reference and clinical Echovirus isolates with primer pair UG52-UC53 while with 292-222 and 012-011 were amplified 27/28 reference Echovirus strains and all clinical isolates. As far as EUG2a,2b,2c-EUC2 is concerned, the RT-PCR gave a positive result for 26/28 reference Echovirus strains and 34/38 clinical isolates. The sequence analysis of a large part of the 5'UTR has revealed that there is no correlation between 5'UTR identity and the currently recognized human enterovirus species. It has been suggested that part of VP1 coding sequence would correlate well with serotype since a number of important neutralization epitopes, as well as receptor recognition sequences, lie within the VP1 coding sequence. Therefore, UG52-UC53 and 292-222 primer pairs seem to be the most appropriate for Echovirus detection and, moreover, UG52-UC53 is useful for the classification of enteroviruses into genetic clusters (sub-groups) while 292-222 for the identification of enteroviruses by amplicon sequencing. (C) 2004 Elsevier Ltd. All rights reserved

Evaluation of seroneutralization and molecular diagnostic methods for echovirus identification

In this study we compared the identification results of 41 echovirus clinical isolates using RIVM pools (National Institute for Public Health and the Environment RIVM, Bilthoven, The Netherlands) and reverse transcription-polymerase chain reaction (PCR) assays. Primer pair UG(52)-UC53 amplified a 433-bp segment in the 5' untranslated region. Restriction enzyme HpaII was used for subgrouping of our isolates into 2 different genetic clusters. Amplification of 315 bp that is located in 5' end of VP1 gene as well as of a long genomic fragment (1452 bp) including the VP1 3' end, the entire coding sequence of 2A, 2B, and the 5' moiety of the 2C-coding region was achieved by the application of PCR protocols with primers 292-222 and EUG2a, 2b, 2c-EUC2, respectively. Phylogenetic trees were constructed for the 5' end as well as for the 3' end of VP1 gene using nucleotide sequences derived from sequencing of clinical isolates and homologous sequences of all echovirus serotypes. The phylogenetic grouping pattern of the clinical isolates revealed a correlation of serotype and genotype either in the 5' or in the 3' end of the VP1 gene that was investigated in the present study claiming that they can be either used for molecular typing of echoviruses. (c) 2005 Elsevier Inc. All rights reserved

Evaluation of seroneutralization and molecular diagnostic methods for echovirus identification

In this study we compared the identification results of 41 echovirus clinical isolates using RIVM pools (National Institute for Public Health and the Environment RIVM, Bilthoven, The Netherlands) and reverse transcription-polymerase chain reaction (PCR) assays. Primer pair UG(52)-UC53 amplified a 433-bp segment in the 5’ untranslated region. Restriction enzyme HpaII was used for subgrouping of our isolates into 2 different genetic clusters. Amplification of 315 bp that is located in 5’ end of VP1 gene as well as of a long genomic fragment (1452 bp) including the VP1 3’ end, the entire coding sequence of 2A, 2B, and the 5’ moiety of the 2C-coding region was achieved by the application of PCR protocols with primers 292-222 and EUG2a, 2b, 2c-EUC2, respectively. Phylogenetic trees were constructed for the 5’ end as well as for the 3’ end of VP1 gene using nucleotide sequences derived from sequencing of clinical isolates and homologous sequences of all echovirus serotypes. The phylogenetic grouping pattern of the clinical isolates revealed a correlation of serotype and genotype either in the 5’ or in the 3’ end of the VP1 gene that was investigated in the present study claiming that they can be either used for molecular typing of echoviruses. (c) 2005 Elsevier Inc. All rights reserved

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. © Springer Science+Buisness Media, Inc. 2006

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

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. © Springer-Verlag 2006

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. © 2005 Springer Science+Business Media, Inc

A comparative amplification of five different genomic regions on Coxsackie A and B viruses. Implications in clinical diagnostics

Modern molecular approaches in Human Enterovirus detection rely on the designing of generic and often degenerate primers in order to amplify specific sequences within the enterovirus genome. In the present study a comparative application of primer sets targeting 5’UTR, the VP1 region, the 3D region as well as a long genomic fragment including the 3’end of VP1, the full length of 2A and 2B, and the 5’ moiety of the 2C-coding region was attempted, in order to evaluate their specificity and suitability. The best amplification results from the investigation of 21 CAV reference strains, all six CBV reference strains and 44 clinical strains varying in origin and time of isolation, arose using primer sets 292-222 and UC53-UG52. Based on the above results we conclude that some of the published protocols need to be improved so as to fulfill the demands of an accurate detection and typing of Coxsackie A and B viruses. Contrarily, two of the protocols applied were proved to be more accurate in terms of specificity and general applicability, suggesting that RT-PCR followed by a simple RFLP assay in the case of primer pair UC53-UG52 or by sequencing and sequence analysis in the case of primer set 292-222 should constitute alternative means of modern typing and diagnostics against conventional immunological classification methods. (C) 2004 Elsevier Ltd. All rights reserved