Detection and Typing of Human Enteroviruses from Clinical Samples by Entire-Capsid Next Generation Sequencing

Abstract

There are increasing concerns of infections by enteroviruses (EVs) causing severe disease in humans. EV diagnostic laboratory methods show differences in sensitivity and specificity as well as the level of genetic information provided. We examined a detection method for EVs based on next generation sequencing (NGS) analysis of amplicons covering the entire capsid coding region directly synthesized from clinical samples. One hundred and twelve clinical samples from England; previously shown to be positive for EVs, were analyzed. There was high concordance between the results obtained by the new NGS approach and those from the conventional Sanger method used originally with agreement in the serotypes identified in the 83 samples that were typed by both methods. The sensitivity and specificity of the NGS method compared to those of the conventional Sanger sequencing typing assay were 94.74% (95% confidence interval, 73.97% to 99.87%) and 97.85% (92.45% to 99.74%) for Enterovirus A, 93.75% (82.80% to 98.69%) and 89.06% (78.75% to 95.49%) for Enterovirus B, 100% (59.04% to 100%) and 98.10% (93.29% to 99.77%) for Enterovirus C, and 100% (75.29% to 100%) and 100% (96.34% to 100%) for Enterovirus D. The NGS method identified five EVs in previously untyped samples as well as additional viruses in some samples, indicating co-infection. This method can be easily expanded to generate whole-genome EV sequences as we show here for EV-D68. Information from capsid and whole-genome sequences is critical to help identifying the genetic basis for changes in viral properties and establishing accurate spatial-temporal associations between EV strains of public health relevance.

Molecular Characterization and Taxonomic Assignment of Three Phage Isolates from a Collection Infecting Pseudomonas syringae pv. actinidiae and P. syringae pv. phaseolicola from Northern Italy

“…Phage cocktail persistence in the open field could be improved with specific formulations [ 9 ] and their action can be increased in synergy with the addition with other compounds such as carvacrol [ 20 ] and garlic extract [ 24 ]. Additionally, the phages we isolated can be a useful source of endolysins as an alternative Psa and Pph biocontrol method [ 78 ]; the vast number of functionally uncharacterized ORFs they encode could contribute to further control approaches once their role is elucidated, urging a vast effort to assign function to the unprecedented proteomic diversity harbored by phages [ 67 , 79 , 80 ].…”

Surveillance of rhinovirus diversity among a university community identifies multiple types from all three species including an unassigned rhinovirus A genotype

“…Furthermore, though originally designed to amplify complete capsid protein sequence region of poliovirus (an Enterovirus C. species member), 12 coupled with the findings of this study, we and others have now shown that the PanEV RT-PCR assay can amplify the same region from other enterovirus species (particularly enterovirus species A, B, C, D, and G and rhinovirus species A, B, and C) and canine picornavirus genomes. [13][14][15] We are aware that the diversity of rhinovirus types described here might not be a complete catalog of all the types present in the samples screened or that circulated in the university community during the period sampled. Particularly, the fact that we pooled only 20 μl of each resuspended NP swab sample coupled with the stringent (>2000 nt contig length and >50x coverage; Table 1) cutoff used in our assembly and detection pipeline could have prevented detection of low titer rhinovirus (or other enterovirus) types present in the samples.…”

“…species member), 12 coupled with the findings of this study, we and others have now shown that the PanEV RT‐PCR assay can amplify the same region from other enterovirus species (particularly enterovirus species A, B, C, D, and G and rhinovirus species A, B, and C) and canine picornavirus genomes. 13 , 14 , 15 …”

Echovirus 9 genetic diversity detected in whole-capsid genome sequences obtained directly from clinical specimens using next generation sequencing

“…We used an echovirus adapted version of the entire capsidcoding region amplification (ECRA) method originally designed for poliovirus by Arita et al [14] and modified for enteroviruses [13,15,16] (Fig. 1A).…”

“…Routinely generated nucleotide sequencing information about EVs from clinical samples is usually limited to short, partial genomic sequences of VP1 that enable identifying the genotype but have limited use for detailed genetic characterisation. Several approaches to EV NGS have been described previously for sequencing viruses directly from clinical samples and from cell culture isolates with metagenomics or target-specific techniques, still, available E9 virus sequences covering the whole capsid are scarce [9][10][11][12][13].…”