Sentinel surveillance system
The KDCA has been performing national surveillance for KINRESS since 2000. The system includes 63 clinics and 18 regional laboratories (Public Health and Environment Research Institute, PHERI). The clinic provides up to eight upper respiratory specimens per week; these are collected from patients with influenza-like illnesses, a measured temperature of ≥ 38 °C, and cough with an onset within the past 10 days [5]. PHERI uses commercial respiratory virus detection real-time reverse transcription (RT)-polymerase chain reaction (PCR) or real-time PCR kits (PowerCheck™ Real-time PCR kit series, Kogenebiotech, Seoul, South Korea) to detect IFV (A/H1N1pdm09, A/H3N2, and B), hRSV (A and B), hPIV (1, 2, and 3), human coronavirus (hCoV-OC43, 229E, and NL63), human metapneumovirus (hMPV), hBoV, hAdV, and hRV. The diagnostic results are then reported to the KDCA, which shares the national respiratory virus surveillance data with the public through the National Influenza Center [6].
Non-sentinel surveillance system
Five private medical diagnostic centers (Seegene Medical Center, Green Cross Laboratory, Eone Laboratory, Seoul Clinical Laboratories, and Samkwang Medical Laboratories) collect respiratory samples from clinics and hospitals across the country. The centers uses commercial respiratory virus detection real-time RT-PCR or real-time PCR kits (Allplex™ Respiratory Panel Assays, Seegene, Seoul, South Korea) to detect IFV (A/H1N1pdm09, A/H3N2, and B), hRSV (A and B), hPIV (1, 2, and 3), hCoV (OC43, 229E, and NL63), hMPV, hBoV, hAdV, and hRV, and report the diagnostic results to the KDCA weekly.
Clinical specimen collection
A total of 56,984 and 257,217 oropharyngeal or nasopharyngeal swabs were collected from patients through sentinel surveillance (between the 1st week of 2016 and the 47th week of 2021) and non-sentinel surveillance (between the 1st week of 2020 and the 47th week of 2021), respectively. The oropharyngeal and nasopharyngeal swabs were stored in Universal Transport Media (Copan Diagnostics, Inc., Murrieta, CA, USA) before analysis.
RNA or DNA extraction
Viral RNA or DNA was extracted from 140 µL of transport medium using the MagNA Pure 96 automated extractor (Roche Life Science, Basel, Switzerland) with the DNA and Viral Nucleic Acid Small Volume kit according to the manufacturer’s instructions.
Genetic analysis of influenza and other respiratory viruses
Commercially available respiratory virus detection real-time PCR kits were used to detect eight respiratory viruses and their subtypes of, including hRSV (types A and B), IFV (type A/H1N1pdm09, A/H3N2, and B), hPIV (type 1, 2, and 3), hCoV (type OC43, 229E, and NL63), hRV, hAdV, hBoV, and hMPV [7]. Viral cDNAs, except those from hAdV and hBoV, were synthesized from 5 μL of extracted nucleic acids using a reverse transcriptase; the reaction mixture was incubated at 50 °C for 30 min, followed by inactivation of the reverse transcriptase at 95 °C for 10 min. PCR amplification was performed with 40 cycles at 95 °C for 15 s and 60 °C for 1 min using an ABI 7500 Fast instrument (Applied Biosystems, Foster City, CA, USA) or a CFX96 real-time cycler (Bio-Rad, Hercules, CA, USA). The amplified PCR products were sequenced by Macrogen (Seoul, South Korea). We used CLC Workbench v X.X (CLC Bio, Aarhus, Denmark) for sequencing and assembly.
Statistical analysis
The test results of 56,984 sentinel specimens and 257,217 non-sentinel specimens were analyzed; weekly detection rates of eight respiratory viruses (hPIV, IFV, hRSV, hCoV, hRV, hAdV, hBoV, and hMPV) were used for the statistical analysis (Additional file 1). R software (ver. 4.0.2; The R Project for Statistical Computing, Vienna, Austria) was used for most analyses. [8] Statistical significance of the means between two independent groups was analyzed using Welch’s t-test. Statistical significance was set at P < 0.05.
Sequencing and analysis
Five hPIV 3-positive samples were randomly selected each year from 2016 and 2021 for amplification using the primers HPIV3-Seq_6714_sense (5′-AAAGTTACGCAATCCAACTC-3′), HPIV3_Seq_7400_antisense (5′-GAGGTATAAGCATAAATCAGA-3′), HPIV3_Seq_7257_ sense (5′-CACGTCTGGTCTTCCATCT-3′), HPIV3_Seq_7929_ antisense (5′-AACAATGATGGAGTTGACCAT-3′), HPIV3_Seq_7785_ sense (5′-TGGGTATGGAGGTCTTGAAC-3′), and HPIV2_Seq_8577_ antisense (5′-TGTCTATTGTCTGATTGCTGATTA-3′) to generate a 1719-base pair fragment containing the full region of the hemagglutinin-neuraminidase (HN) gene [9]. The amplicons were purified using a QIA PCR purification kit (Qiagen, Hilden, Germany) and sequenced using each sense and antisense primers in both directions on an ABI‐3100 Prism Genetic Analyzer using the BigDye Terminator version 3.1 sequencing kit (Applied Biosystems). The sequences were compared with all available HN sequences of hPIV3 in the National Center for Biotechnology Information (NCBI) Virus annotated specimen collection, taking the date and country name into account. In total, 621 sequences were used to construct a phylogenetic tree, including 25 sequences isolated from the Republic of Korea. Each sequence was aligned using Geneious Prime software (https://www.geneious.com/) and manually trimmed to equal lengths. Temporal phylogenies were inferred in BEAST v1.10.4 [10] using a strict clock, Hasegawa–Kishono–Yano substitution model, and constant coalescent tree prior. Markov chain Monte Carlo chains were run for 100 million steps, with sampling every 10,000 generations. Run convergence was confirmed using Tracer v1.7.1, after 10% burn-in removal [11]. Maximum clade credibility trees were generated using Tree Annotator v1.8 [10] Trees were visualized using FigTree v1.4.4.
Submission to GenBank
Twenty-five sequences were submitted to GenBank with the full HN region of hPIV3 generated from the samples obtained during the sentinel surveillance. The GenBank accession numbers of the sequences generated in this study are provided in Additional file 2.
