Computational protocol: Genetic Diversity of Human Enterovirus 68 Strains Isolated in Kenya Using the Hypervariable 3′- End of VP1 Gene

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[…] Human enterovirus (HEV) isolates used in this study were retrieved from archives of the respiratory virus surveillance program in the Department of Emerging Infectious Diseases (DEID) of the United States Army Medical Research Unit-Kenya (USAMRU-K), at the National Influenza Center, within the Kenya Medical Research Institute (KEMRI). The program surveillance network was selected to include disparate geographic regions and population demographics across the country and comprised Mbagathi, New Nyanza, Malindi, Isiolo, Mombasa, Port Reitz, Alupe, Kisii and Kericho District Hospitals.Inclusion criteria included being an outpatient, >2 months in age and having influenza-like-illness (ILI) symptoms. Demographic information including age, sex, occupation, workplace and residence history were ascertained for all participants. Clinical parameters and symptoms including recent history of ILI, cough, difficulty in breathing, chills, sore throat, muscle aches, retro-orbital pain, malaise, vomiting, neurological symptoms, abdominal pain, nasal stuffiness, runny nose, sputum production, headache, joint pain, fatigue, diarrhea and bleeding were documented.Nasopharyngeal specimens were collected and viruses isolated using RD cells (ATCC® CCL-136) prepared in culture tubes (Nunc, Denmark). Each tube containing the cell line in Dulbecco’s minimum essential medium (Life Technologies, USA) was inoculated with 100 µl of clinical specimen and incubated at 33°C under 5% CO2, and virus growth monitored with reference to cytopathic effects (CPEs) for up to 2 weeks. The presence of HEV in culture was confirmed by indirect fluorescent antibody test using Millipore Light Diagnostics™ Kit (Millipore Corporation, USA) according to the manufacturer’s instructions. [...] VP1 DNA nucleotide sequence fragments were edited and assembled into consensus contigs using DNA baser version 3.2 . Serotype identity of the isolates was determined by pair-wise comparisons, using CLC genomics Workbench v6.5 software (CLC bio, Denmark). An isolate was determined to be EV68 if it shared ≥75% nucleotide identity (85% amino acid similarity) with the prototype strain (Fermon) , . EV68 VP1 sequences were compared to those previously published and deposited in GenBank. Multiple sequence alignment was performed using Muscle v3.8 software . The mean dN/dS (ω) value and selection pressure at individual codon sites were estimated by the single likelihood ancestor counting (SLAC) and fixed effects likelihood (FEL) methods implemented in the Hypothesis testing using the Phylogenies (HYPHY; ) package. The analyses were performed on web-based Datamonkey interface (; ). The mean dN/dS ratio and 95% confidence interval were computed based on Neighbor-Joining (NJ) trees under the HKY85 substitution model . The ratio of the nonsynonymous substitution rate (dN) to the synonymous rate (dS) was interpreted as follows: dN/dS = natural selection; dN/dS >1 = positive selection, while dN/dS <1 = negative selection. The significance level for selection in both analyses was assessed based on the p-value. P-values <0.05 were considered as thresholds for strong evidence of selection. Phylogenetic relationships were inferred using MrBayes software v3.2 and the generated tree visualized using Fig Tree v1.4.0 software . […]

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