Computational protocol: Enterovirus D68 in Hospitalized Children: Sequence Variation, Viral Loads and Clinical Outcomes

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Protocol publication

[…] A single step, EV-D68-specific reverse transcriptase real-time PCR was developed in house during the 2014 outbreak. The primer and probe sequences were designed based on in silico analysis of all the available EV-D68 sequences deposited in NCBI, targeting the 5’ non-translated region of the human enterovirus genome. The NCBI database was accessed on September 15, 2014 and at that time < 20% of the EVD68 2014 outbreak sequences were included. Briefly, total nucleic acid was obtained by extraction using the NucliSENS easyMag platform (bioMerieux, Durham, NC) and 4μL of the eluate was added to a 20 μL total volume reaction mixture [1XTaqMan RT-PCR mix (TaqMan RNA-to-CT 1-Step Kit, Life Technologies, Grand Island, NY), 1XTaqMan RT Enzyme Mix, 0.25μM of each primer: EVD68F (5’-AAAACCATGACGCTAGACATGA-3’) and EVD68R (5’-GGCCGGAGGACTCTAT-3’) and 0.9μM of probe EVD68P (5’-VIC-CAAGGTGTGAAGAGTCTATT- MGB-3’)]. The RT-PCR was carried out using the ABI 7500 thermocycler (Life Technologies, Grand Island, NY) with the following running conditions: 48°C for 15 min, followed by 10 min at 95°C, 40 cycles of 95°C for 15s and 50°C for 1 min. Semiquatitative genomic EV-D68 loads were quantified using Ct values, which reflect the number of amplification cycles that are required for a positive PCR test. Thus the higher the Ct value the lower the amount of genomic EV-D68.For EV-D68 sequencing, partial VP1 genes from EV-D68 strains identified at our hospital during the 2014 outbreak and in previous years, were amplified to yield an 805-basepair (bp) PCR product, as described. [] Cycle sequencing was performed with BigDye Terminator v3.1 cycle sequencing Kit (Applied Biosystems) on the automated sequencer 3130xl Genetic Analyzer (Applied Biosystems) bi-directionally. Multiple sequence alignment was performed and phylogenetic trees were generated using the neighbor-joining method implemented in MEGA version 6.0. [] Support for specific tree topologies was estimated by bootstrap analysis with a value of 1000. Using Geneious software (,[]), the amino acid sequences (including BE and DC loops) of partial VP1 were aligned and compared with NCH strains and selected sequences that were previously published. […]

Pipeline specifications

Software tools MEGA, Geneious
Application Phylogenetics