Ethical approval
The study protocol, involving collection of patient residual specimens, was approved by the Institutional Review Board (IRB) of Chuncheon Sacred Heart Hospital, Hallym University (IRB 2022-05-003). Informed consent was obtained from participating patients and/or his legal guardians. Confirms that all experiments were performed in accordance with relevant named guidelines and regulations.
RNA extraction and cDNA synthesis
Serum samples obtained 2 and 6 days after symptom onset from CHIKV infected patients were provided by Hallym Chuncheon Sacred Heart Hospital, Kangwon province, ROK. Total RNA was extracted using the TRIzol LS Reagent (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg total RNA using the High-Capacity RNA-to-cDNA kit (Applied Biosystems; Foster City, CA, USA) according to the manufacturer’s instructions.
Amplicon-based NGS
The cDNA was amplified using CHIKV-specific primer mixtures and the Solg 2X Uh-Taq PCR Smart mix (Solgent; Seoul, Republic of Korea) according to the manufacturer’s instructions. The first PCR cycling conditions were as follows: initial denaturation at 95˚C for 15 min; followed by 40 cycles of 95˚C for 20 s, 50˚C for 40 s, and 72˚C for 1 min; and a final elongation at 72˚C for 3 min. The second PCR assay was conducted in a 25 μL reaction mixture containing 12.5 μL 2X Uh-Taq PCR Smart mix, 1.0 μL of the first PCR product, 10.0 μL of 0.5 μM of each primer mixture (final concentration, 0.2 μM), and 1.5 μL distilled water. The sequencing library was prepared using a Ligation Sequencing Kit (SQK-LSK109) according to the standard protocols (Oxford Nanopores Technologies, UK) and then ligated to sequencing adapters. The purified library was sequenced using a MinION device by loading onto a FLO-MIN106 (R9.4.1; Oxford Nanopores Technologies, UK) flow cell.
NGS data analysis and phylogenetic analysis
Basecalling was performed using Guppy (v3.0.3) embedded in the MinKNOW system (Oxford Nanopore Technologies). The adaptor sequences were trimmed, and the filtered reads were assembled into a FASTQ file using Porechop (v.0.2.4). Viral reads were mapped to the reference genome sequence CHIKV Thail 2019 (Accession ID MN630017.1) and the consensus was extracted using Medaka (v.12.0).
Phylogenetic analysis
The genomic sequences of the CHIKV were aligned using the MUSCLE algorithm in MEGA 11. The best-fit substitution model was determined by its Bayesian Information Criterion (BIC) as GTR+G+I. Phylogenetic trees were generated using the maximum-likelihood method in MEGA 11. Topologies were assessed by bootstrap analysis of 1,000 iterations.
Estimation of the zoonotic prediction potential of CHIKV
The viral genome was assessed and rated for its potential to cause zoonotic infections through the utilization of a machine learning model [10]. This model determines the probability of human infection by analyzing the host range characteristics encoded in the viral genome. The features encompass biases in the arrangement of the viral genome, such as the proportional occurrence of each codon, amino acid, and dinucleotide. This particular method entailed delineating the viral genome in the FASTA format and manually ascertaining the open reading frames (ORFs). The ORFs in viral genome ORFs were predicted using a gene-finding program called Prodigal [11]. Zoonotic potential rankings for the four genotypes of CHIKV were created by inputting files in fasta format, which included genome and metadata files, and specifying output file names. The zoonotic potential of the viruses was ranked using the machine learning model with a cut-off value of 0.293 as reported in a previous study [12]. Zoonotic potential prediction probabilities were categorized by low, entire 95% confidence interval (CI) of predicted probability > cutoff value; medium, mean prediction > cutoff value; although the CI crosses it; high, mean prediction > cutoff value, with the CI crossing it; and very high, entire CI > cutoff value).