RT-PCR coupled with amplicon sequencing successfully identified 49 known enterovirus serotypes, 2 rhinoviruses, and 4 novel picornaviruses among 55 isolates refractory to classic neutralization.
Does RT-PCR and amplicon sequencing improve the identification of untypeable enteroviruses compared to classic neutralization assays?
RT-PCR coupled with amplicon sequencing is a simple and rapid method for typing picornaviruses that are refractory to traditional neutralization assays.
Members of the family Picornaviridae are the most common viruses infecting humans, and species in several genera also infect a wide variety of other mammals. Picornaviruses have traditionally been classified by antigenic type, based on a serum neutralization assay. However, this method is time-consuming and labor-intensive, is sensitive to virus aggregation and antigenic variation, and requires a large number of antisera to identify all serotypes, even when antiserum pools are used. We developed generic reverse transcription (RT)-PCR primers that will amplify all human enterovirus serotypes, as well as many rhinoviruses and other picornaviruses, and used RT-PCR amplification of the VP1 gene and amplicon sequencing to identify enteroviruses that were refractory to typing by neutralization with pooled antisera. Enterovirus serotypes determined by sequencing were confirmed by neutralization with monospecific antisera. Of 55 isolates tested, 49 were of known enterovirus serotypes, two were rhinoviruses, and four were clearly picornaviruses but did not match any known picornavirus sequence. All four untyped picornaviruses were closely related to one another in sequence, suggesting that they are of the same serotype. RT-PCR, coupled with amplicon sequencing, is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes.
Oberste et al. (Wed,) conducted a other in Untypeable Enteroviruses (n=55). RT-PCR and amplicon sequencing vs. Neutralization with pooled antisera was evaluated on Identification of enterovirus serotypes. RT-PCR coupled with amplicon sequencing successfully identified 49 known enterovirus serotypes, 2 rhinoviruses, and 4 novel picornaviruses among 55 isolates refractory to classic neutralization.