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 us… Show more
“…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).…”
The N-terminal part of VP1 was sequenced for 43 enterovirus isolates that could not initially be neutralized with LBM pools or in-house antisera. Most isolates were found to belong to human enterovirus type A (HEV-A) and HEV-B (18 isolates of each). All HEV-A isolates could be typed by sequencing, with CV (coxsackievirus)-A16 and EV (enterovirus)-71 being dominant (nine and seven isolates, respectively). These types thus seem to have diverged more from their prototypes than the other types. Among the HEV-B isolates, E-18 dominated with five isolates that became typable after filtration. The virus type obtained by molecular typing was verified for 28 of the other patient isolates by neutralization using high-titre monovalent antisera or LBM pools. Twenty-two of the other 30 'untypable' isolates had substitutions in the VP1 protein within or close to the BC loop. Two closely related HEV-B isolates diverged by 19?4 % from E-15, the most similar prototype. Two non-neutralizable HEV-C isolates split off from the CV-A13/CV-A18 branch, from which they diverged by 15?7-18?2 %. Three of the six non-neutralizable isolates, W553-130/99, W543-122/99 and W137-126/99, diverged by >24?2 % from the most similar prototype in the compared region. The complete VP1 was therefore sequenced and found to diverge by >29 % from all prototypes and by >28 % from each other. Strains similar to W553-130/99 that have been identified in the USA are tentatively designated EV-74. The two other isolates fulfil the molecular criterion for being new types. Since strains designated EV-75 and EV-76 have been identified in the USA, we have proposed the tentative designations EV-77 and EV-78 for these two new members of HEV-B.
“…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).…”
The N-terminal part of VP1 was sequenced for 43 enterovirus isolates that could not initially be neutralized with LBM pools or in-house antisera. Most isolates were found to belong to human enterovirus type A (HEV-A) and HEV-B (18 isolates of each). All HEV-A isolates could be typed by sequencing, with CV (coxsackievirus)-A16 and EV (enterovirus)-71 being dominant (nine and seven isolates, respectively). These types thus seem to have diverged more from their prototypes than the other types. Among the HEV-B isolates, E-18 dominated with five isolates that became typable after filtration. The virus type obtained by molecular typing was verified for 28 of the other patient isolates by neutralization using high-titre monovalent antisera or LBM pools. Twenty-two of the other 30 'untypable' isolates had substitutions in the VP1 protein within or close to the BC loop. Two closely related HEV-B isolates diverged by 19?4 % from E-15, the most similar prototype. Two non-neutralizable HEV-C isolates split off from the CV-A13/CV-A18 branch, from which they diverged by 15?7-18?2 %. Three of the six non-neutralizable isolates, W553-130/99, W543-122/99 and W137-126/99, diverged by >24?2 % from the most similar prototype in the compared region. The complete VP1 was therefore sequenced and found to diverge by >29 % from all prototypes and by >28 % from each other. Strains similar to W553-130/99 that have been identified in the USA are tentatively designated EV-74. The two other isolates fulfil the molecular criterion for being new types. Since strains designated EV-75 and EV-76 have been identified in the USA, we have proposed the tentative designations EV-77 and EV-78 for these two new members of HEV-B.
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Section: Rna Extraction and Diagnosticmentioning
confidence: 99%
“…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].…”
Human enteroviruses (HEV) are one of the major causes of central nervous system (CNS) infections in pediatrics. A prospective study was conducted to assess the epidemiological, clinical, and laboratory characteristics of enterovirus (EV) infections of the CNS in children under 15-years-old, suspected of having viral CNS infections and admitted to the Pediatric Department of Monastir University Hospital, Tunisia. Enteroviral RNA was detected by 5 0 NCR nested RT-PCR assay in 33 % (20 out of 60) of cerebrospinal fluid specimens, whereas only six samples (10 %) were EV positive in cell culture. EV-positive patients were clustered according to their clinical manifestations, predominantly diagnosed as aseptic meningitis (65 %) and meningoencephalitis (20 %). Fever, headache, vomiting, and neck stiffness were the most pronounced symptoms. Pleocytosis with the predominance of lymphocytes was observed in 60 % of EV positive specimens. Although patients suffering from EV infections were encountered throughout the year, most occurred during spring and summer months. Using VP1-2A nested RT-PCR and sequence analysis, three of the 20 positive HEV were identified as Echovirus (E)-9. This is the first report of a cluster of aseptic meningitis cases caused by E-9 in Monastir.
“…-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 .…”
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