Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses

Abstract

The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 39-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68?3 % of isolates, while the HEV-A and HEV-C primers accounted for 9?7 and 11?3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6?5 %) were amplified by more than one primer set and eight isolates (4?3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 39-non-translated region sequences. INTRODUCTIONThe 65 human enterovirus serotypes (family Picornaviridae) are classified into four species, Human enterovirus (HEV)-A, HEV-B, HEV-C and HEV-D, largely on the basis of phylogenetic analysis of multiple genome regions (King et al., 2000). Analysis of the complete genome sequences of all the prototype strains has shown that the species remain phylogenetically coherent in all genome regions except in the 59-non-translated region (NTR) (Brown et al., 2003; Oberste et al., 2004a, d). Seven of the 13 simian enterovirus prototype strains are provisional members of two HEV species, with six strains in HEV-A and one in HEV-B (Oberste et al., 2002). The remaining six strains are proposed to define three new species in the genus Enterovirus (Oberste et al., 2002), while the bovine and porcine enteroviruses comprise two additional species (King et al., 2000). Recombination, a major mechanism of enterovirus evolution, has been observed to occur in nature only among members of the same species, except in the 59-NTR (Brown et al., 2003; Lindberg et al., 2003;Lukashev et al., 2003 Lukashev et al., , 2004 Oberste et al., 2004a, d, e;Oprisan et al., 2002). Species identity therefore appears to have some biological significance integral to virus survival.Genus-specific primers are widely used for the amplification of enteroviruses from clinical specimens by RT-PCR (Rotbart & Ro...

Diversity of picornaviruses in rural Bolivia

“…Several other studies, using both classical and molecular methods, have detected EV, HPeV, SAFV and CoSV in faecal samples from AFP cases (Bingjun et al, 2008;Junttila et al, 2007;Kapoor et al, 2001;Oberste et al, 2006;Saeed et al, 2007). Most previous studies, including our own, used specific PCR assays to detect members of one or two genera (usually only Enterovirus), due to specificity, low cost and potential for high throughput, though some investigators are starting to use metagenomics methods to expand the range of viruses that can be detected , with limitations of higher per-sample cost and lower overall throughput.…”

“…Surveillance for AFP is recommended by the World Health Organization (WHO) using the standard approach for poliovirus surveillance for the purposes of Global Polio Eradication (Anonymous, 2006). A wide variety of nonpolio enteroviruses and other picornaviruses have been identified in stool specimens of AFP cases (Bingjun et al, 2008;Kapoor et al, 2008;Oberste et al, 2006;Saeed et al, 2007;Santos et al, 2002;Shoja et al, 2007). Detection of high-prevalence agents from a non-sterile site is not sufficient to conclusively establish an aetiological link of picornavirus infection with disease; however, given the known ability of many picornaviruses to cause central nervous system (CNS) disease, it is likely that these viruses contribute to at least a fraction of AFP cases.…”

Molecular characterization of enterovirus 71 and coxsackievirus A16 using the 5′ untranslated region and VP1 region

“…Published primers 423-426, 480-483 and 486-489 for PCR amplification and/or sequencing of the complete VP1 were obtained (Oberste et al, 2006), as well as PCR amplification and sequencing primers 292 and 222 for analysis of partial VP1 sequences (Oberste et al, 2006).…”

Phylogenetic analysis of complete VP1 sequences of echoviruses 11 and 6: high genetic diversity and circulation of genotypes with a wide geographical and temporal range

“…The mixture was incubated at 42 uC for 45 min, heat inactivated at 95 uC for 5 min and immediately chilled on ice. Two reported primer pairs (490/492 and 491/493) were used to amplify two overlapping fragments of the VP1 region (Oberste et al, 2006). PCR amplification was performed with 5 ml cDNA in a 50 ml reaction mixture containing 3 ml 106 polymerase buffer, 3 ml MgCl 2 , 2.5 U recombinant Taq polymerase, and 50 pmol each forward and reverse primer.…”