RESUMEN
Molecular methods, based on sequencing the region encoding the VP1 major capsid protein, have recently become the gold standard for enterovirus typing. In the most commonly used scheme, sequences more than 75% identical (>85% amino acid identity) in complete or partial VP1 sequence are considered to represent the same type. However, as sequence data have accumulated, it has become clear that the '75%/85% rule' may not be universally applicable. To address this issue, we have determined nucleotide sequences for the complete P1 capsid region of a collection of 53 isolates from the species Human enterovirus C (HEV-C), comparing them with each other and with those of 20 reference strains. Pairwise identities, similarity plots and phylogenetic reconstructions identified three potential new enterovirus types, EV96, EV99 and EV102. When pairwise sequence comparisons were considered in aggregate, there was overlap in percentage identity between comparisons of homotypic strains and heterotypic strains. In particular, the differences between coxsackievirus (CV) A13 and CVA17, CVA24 and EV99, and CVA20 and EV102 were difficult to discern, largely because of intratypic sequence diversity. Closer inspection revealed the minimum intratypic values and maximum intratypic values varied by type, suggesting that the rules were at least consistent within a type. By plotting VP1 amino acid identity vs nucleotide identity for each sequence pair and considering each type separately, members of each type were fully resolved from those of other types. This study suggests that a more stringent value of 88% VP1 amino acid identity is more appropriate for routine typing and that other criteria may need to be applied, on a case by case basis, where lower values are seen.
Asunto(s)
Proteínas de la Cápside/genética , Enterovirus Humano C/clasificación , Enterovirus Humano C/genética , ARN Viral/genética , Análisis por Conglomerados , Enterovirus Humano C/aislamiento & purificación , Genotipo , Humanos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Molecular methods have enabled the rapid identification of new enterovirus (EV) serotypes that are untypeable using existing neutralizing antisera. As a result, sequencing of the VP1 capsid gene has been developed as a surrogate for antigenic typing to distinguish enterovirus types. In this study, 17 enterovirus isolates from four countries were identified as members of 13 new types within the species Human Enterovirus B (HEV-B) by complete genome sequencing. Members of each of these new types are at least 75% identical to one another (91% amino acid identity) in VP1, but members of different types differ from one another and from other enteroviruses by at least 27% in nucleotide sequence (26% amino acid sequence difference). The complete P1 (capsid) sequences of the new types are at least 17% different from those of all other enterovirus serotypes (14.5% amino acid sequence difference), but they are highly conserved within a type (<8% amino acid sequence difference). For both VP1 and P1, the 17 isolates are monophyletic by type with respect to all other EV serotypes. The P2 and P3 sequences are closely related to those of other HEV-B viruses (>93% amino acid identity), confirming that the 17 new strains belong to HEV-B. We propose that these 17 isolates be classified as members of 13 new human enterovirus types, enteroviruses 79-88, 97, and 100-101.
Asunto(s)
Enterovirus Humano B/clasificación , Enterovirus Humano B/genética , Infecciones por Enterovirus/virología , Animales , Proteínas de la Cápside/genética , Línea Celular , Enterovirus Humano B/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Serotipificación , Especificidad de la EspecieRESUMEN
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 3'-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 3'-non-translated region sequences.
Asunto(s)
Regiones no Traducidas 3'/análisis , Proteínas de la Cápside/genética , Enterovirus/aislamiento & purificación , Genoma Viral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Regiones no Traducidas 3'/genética , Secuencia de Aminoácidos , Cápside/química , Cartilla de ADN , Enterovirus/genética , Humanos , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
Molecular methods have enabled the rapid identification of new enterovirus (EV) serotypes that would have been untypable using existing neutralizing antisera. Nineteen strains of four new EV types termed EV76 (11 isolates), EV89 (two isolates), EV90 (four isolates) and EV91 (two isolates), isolated from clinical specimens from patients in France (one isolate) and Bangladesh (18 isolates), are described. Nucleotide sequences encoding the VP1 capsid protein (882-888 nt) are less than 65 % identical to the homologous sequences of the recognized human EV serotypes, but within each group the sequences are more than 78 % identical. The deduced amino acid sequences of the complete capsid (P1) region are more than 94 % identical within type but less than 76 % identical to those of the recognized serotypes. For both VP1 and P1, the 19 isolates are monophyletic by type with respect to all other EV serotypes. Using the proposed molecular typing scheme, these data support their identification as four new types within the species Human enterovirus A (HEV-A). In almost all cases, the VP1 sequences were more similar to those of some simian EVs than to the human EVs. Partial 3D sequences of all 19 isolates also clustered within HEV-A; they were monophyletic as a group, but not by type, suggesting that recombination has occurred among viruses of these four types. Partial 3D sequences were more closely related to those of simian EVs than to human viruses in HEV-A. These results suggest that the four new types may represent a new subgroup within HEV-A, in addition to the existing human and simian subgroups.
Asunto(s)
Enterovirus Humano A/clasificación , Bangladesh , Proteínas de la Cápside/genética , Enterovirus Humano A/genética , Francia , Genotipo , Humanos , Datos de Secuencia Molecular , FilogeniaRESUMEN
Sequencing of the gene that encodes the capsid protein VP1 has been used as a surrogate for antigenic typing in order to distinguish enterovirus serotypes; three new serotypes were identified recently by this method. In this study, 14 enterovirus isolates from six countries were characterized as members of two new types within the species Human enterovirus B, based on sequencing of the complete capsid-encoding (P1) region. Isolates within each of these two types differed significantly from one another and from all other known enterovirus serotypes on the basis of sequences that encode either VP1 alone or the entire P1 region. Members of each type were > or =77.2 % identical to one another (89.5 % amino acid identity) in VP1, but members of the two different types differed from one another and from other enteroviruses by > or =31 % in nucleotide sequence (25 % amino acid sequence difference), indicating that the two groups represent separate new candidate enterovirus types. The complete P1 sequences differed from those of all other enterovirus serotypes by > or =31 % (26 % amino acid sequence difference), but were highly conserved within a serotype (<8 % amino acid sequence difference). Phylogenetic analyses demonstrated that isolates of the same serotype were monophyletic in both VP1 and the capsid as a whole, as shown previously for other enterovirus serotypes. This paper proposes that these 14 isolates should be classified as members of two new human enterovirus types, enteroviruses 74 and 75 (EV74 and EV75).