Molecular characterization of human enteroviruses in clinical samples: Comparison between VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products

Abstract

Three nested RT-PCR assays were developed to permit sensitive typing of enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity (M-NW) indices. Comparative results obtained independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes in the VP1 region (26/26, 100% Clustal; 22/26, 85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26, 65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.

Sequencing of ‘untypable’ enteroviruses reveals two new types, EV-77 and EV-78, within human enterovirus type B and substitutions in the BC loop of the VP1 protein for known types

“…used due to the isolate containing multiple types, genetic changes of the virus or conceivably by the encounter of a 'prime' strain or a previously undescribed type. The correlation between the enterovirus VP1 sequence and serotype has been shown in several recent studies, and genetic typing of enteroviruses by complete or partial sequencing of the VP1 region may now considerably simplify the typing (Oberste et al, 1999a(Oberste et al, , b, 2000Caro et al, 2001;Casas et al, 2001;Norder et al, 2001).…”

Enteroviral Central Nervous System Infections in Children of the Region of Monastir, Tunisia: Diagnosis, Laboratory Findings of Cerebrospinal Fluid and Clinical Manifestations

“…This study was conducted with a nested RT-PCR assay described previously [10]. The major advantage of RT-PCR assay is that EV detection and characterization done directly on CSF samples overcomes some of the difficulties associated with virus cultures such as: the inoculation of specimens into non-susceptible cell lines, the inability of virus conjugated with antibodies to infect susceptible cells, and the failure of some EV to grow in cell cultures, as is the case for most of the group A Coxsackieviruses.…”

“…Generated amplicons were 609 bp of the VP1-2A enteroviral genome region [10]. Table 1 shows the sequences and the relative positions of VP1 primers in Echovirus 9 strain Barty.…”

“…Such process is, however, time consuming, and results are difficult to interpret, especially with some EV serotypes that either grow poorly or not at all in cell cultures. Furthermore, some samples, such as cerebrospinal fluid (CSF), tend to have virus titers too low to be detected by standard methods [10].…”

Papel del Laboratorio Nacional de Poliovirus en el Programa de erradicación y vigilancia de la poliomielitis

“…-Entre las responsabilidades del LNP se encuentran las de analizar todos los datos recibidos y caracterizar los aislados que, atendiendo a las recomendaciones de la OMS (apartado 5.2 del Documento EUR/ICP/CMDS 030113), utiliza tanto los métodos tradicionales como nuevos méto-dos moleculares que permiten (i) la confirmación de resultados y (ii) la detección y caracterización de PV y otros EV, incluso en muestras con cantidades que no son detectables por los tradicionales cultivos celulares, mediante las siguientes técnicas: a. Serotipado de los EV no polio, mediante técnicas de microneutralización usando las mezclas de antisueros de Melnick y/o tipificación molecular mediante la amplificación, secuenciación y estudio del extremo 3 del gen VP1 13,14 .…”