Impact of coronavirus disease 2019 on respiratory surveillance and explanation of high detection rate of human rhinovirus during the pandemic in the Republic of Korea

Background After the detection of the first case of coronavirus disease 2019 (COVID‐19) in South Korea on January 20, 2019, it has triggered three major outbreaks. To decrease the disease burden of COVID‐19, social distancing and active mask wearing were encouraged, reducing the number of patients with influenza‐like illness and altering the detection rate of influenza and respiratory viruses in the Korea Influenza and Respiratory Viruses Surveillance System (KINRESS). We examined the changes in respiratory viruses due to COVID‐19 in South Korea and virological causes of the high detection rate of human rhinovirus (hRV) in 2020. Methods We collected 52 684 oropharyngeal or nasopharyngeal swab samples from patients with influenza‐like illness in cooperation with KINRESS from 2016 to 2020. Influenza virus and other respiratory viruses were confirmed using real‐time RT‐PCR. The weekly detection rate was used to compare virus detection patterns. Results Non‐enveloped virus (hRV, human bocavirus, and human adenovirus) detection rates during the COVID‐19 pandemic were maintained. The detection rate of hRV significantly increased in 2020 compared with that in 2019 and was negatively correlated with number of COVID‐19‐confirmed cases in 2020. The distribution of strains and genetic characteristics in hRV did not differ between 2019 and 2020. Conclusions The COVID‐19 pandemic impacted the respiratory virus detection rate. The extremely low detection rate of enveloped viruses resulted from efforts to prevent the spread of COVID‐19 in South Korea. The high detection rate of hRV may be related to resistance against environmental conditions as a non‐enveloped virus and the long period of viral shedding from patients.

was first detected on January 2020 in South Korea, the first wave emerged around Deagu City and Gyungbuk province from February to March, the second wave emerged in the Metropolitan in August and September 2020, 2,3 and the third wave began in November 2020. 4 The spread of COVID-19 was slowed by implementation of high-intensity social distancing and isolation measures as well as easily available testing for COVID-19 in screening centers in all cities and provinces. 5,6 These active precautions led to reduction in the number of patients with influenza-like illness (ILI) and lower numbers of respiratory specimens collected by the national surveillance program for respiratory virus (Korea Influenza and Respiratory Viruses Surveillance System, KINRESS). 7 According to our national surveillance report, However, human adenovirus (hAdV) was maintained at a steady low detection rate annually and human rhinovirus (hRV) was highly detected in patients with ILI. The detection rate and patterns of respiratory viruses in KINRESS were altered in 2020. In this study, we evaluated the changes in national surveillance for respiratory viruses during the COVID-19 pandemic in South Korea and the virological causes of these changes. This study was conducted for the first time in South Korea to analyze detection pattern of respiratory viruses that have changed significantly since COVID-19 and to establish an effective quarantine policy for COVID-19 and other viral respiratory diseases.

| Respiratory virus surveillance system
The Korea Disease Control and Prevention Agency has been performing national surveillance for KINRESS since 2000. 8 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 collected from patients with ILI, a measured temperature of ≥38 C, and cough, with an onset within the past 10 days. 9   Amplified PCR products were sequenced by Macrogen (Seoul, Korea).

| Genetic analysis of influenza and other respiratory viruses
We used CLC Workbench v X. X (CLC bio, Aarhus, Denmark) for sequencing and assembly.

| Statistical analysis
We used 52 684 oropharyngeal or nasopharyngeal swab samples, with the weekly detection rate calculated from 55 825 tested including co-infection cases. The weekly detection rate of eight respiratory viruses (hRSV, IFV, hPIV, hCoV, hRV, hAdV, hBoV, and hMPV) from 2016 to 2020 in KINRESS and that of COVID-19-confirmed cases from 2020 was used for statistical analysis (supporting information S1). R software (ver. 4.0.2; The R Project for Statistical Computing, Vienna, Austria) was used for most analyses. 11 The statistical significance of the means between two independent groups was analyzed by Welch's t-test. A P-value < 0.05 was considered to indicate statistically significant results. Pearson correlation coefficient (R) was calculated to determine the correlation between severe acute respiratory syndrome coronavirus 2 and hRV using the R heatmap function.

| Sequencing and analysis
We randomly selected 94 samples from 2019 and 2020 for amplification using primers, generating a 635-base pair fragment containing part of the 5 0 untranslated region and all of VP4 and part of the VP2 regions. 12-14 PCR amplicons were purified using the QIA PCR purification kit (Qiagen, Hilden, Germany) and sequenced with appropriate primers 12 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 sequences in the GenBank database 15 using BLASTN (http://blast.ncbI.nlm.nih. gov/Blast.cgi) tools to differentiate between the hRV A, B, and C species.
The nucleotide sequences were aligned using the MUSCLE program in MEGA7 (ver. 7.0.26). 16 Phylogenetic analysis was performed with the 94 generated sequences and 19 reference sequences using RAxML (ver.8.1.21). 17 Maximum likelihood trees with 1000 bootstraps were constructed based on the general time reversible + gamma distribution + proportion of invariable sites model after testing for the bestfit model of nucleotide substitution in jModelTest (ver. 2.1.10). 18

| Submission to GenBank
Ninety-four sequences were submitted to GenBank with part of the 5 0 untranslated region and all of the VP4 and part of the VP2 regions.
The sequences generated in this study were assigned GenBank accession numbers are given in supporting information S2.

| Ethics statement
The study protocol was reviewed and approved by the institutional  at an annual rate of 11.46%, with IFV type A dominant in early winter and followed by IFV type B in late winter and spring. The annual detection rate of hRV was 16.51%, which was the highest among the respiratory viruses evaluated. hRSV is annually detected in 3.73% of patients with ILI and is typically restricted to infants and toddlers in fall and early winter. hADV was detected at a rate of 6.02% in patients with ILI and showed no distinct seasonality. However, some sporadic hADV cases were reported in small outbreak from swimming pool during summer. hPIV type 1, 2, and 3 showed an annual detection rate of 6.26% and was prevalent in summer. hBoV showed the lowest annual detection rate (1.95%) and was coinfected with other respiratory viruses. hMPV was detected mainly in spring with an annual detection rate of 4.17%. Common hCoVs including NL63, OC43, and 229E circulated during influenza season with an annual detection rate of 4.05%.

| Comparison of hRV detection rate according to age between 2019 and 2020
To compare hRV detection patterns by patient age and year, the detection rate of hRV in each quarter was compared among six age groups between 2019 and 2020. The lowest detection rate was observed in the first quarter of both 2019 and 2020. Although a low detection rate was maintained in the second quarter of 2019, the rate was increased in 2020, particularly in the 0-6 years group. In the third quarter, the mean detection rate of hRV in the 0-6 and 7-12 years groups was 40% in 2020 higher than that in 2019 but slightly decreased in the fourth quarter. In contrast, at 50-64 and over 65 years, the mean detection rate of hRV was around 10% in 2019, which was higher than that in 2020 ( Figure 6).

| Distribution of hRV species by age and year (2019-2020)
We investigated the distribution of hRV species over 2 years (2019-2020) in KINRESS. We randomly selected 52 and 42 respiratory specimens from 2019 and 2020, respectively, and the partial 5 0 untranslated region and all of the VP4 and partial VP2 region (624 base pairs) were sequenced to identify the clinical strains (hRV A, B, and C). All sequences showed >98% similarity to hRV sequences with partial coding sequences available in GenBank. The A species was dominant in all age groups in both 2019 and 2020 at 76.9% and 85.7%, respectively. The hRV C species was detected in 15.4% and 11.9% of samples from 2019 and 2020, respectively, and frequently detected in the 0-6 and over 65 years groups. HRV B showed the lowest detection rates of 7.7% and 2.4% in 2019 and 2020, respectively, mostly in the 0-6 years group. There were no significant differences in the distribution of hRV species between 2019 and 2020 ( Table 1).

| DISCUSSION AND CONCLUSION
COVID-19 outbreak has not only caused public health challenges worldwide but also greatly impacted the economy, society, and normal daily lives. 19,20 Since COVID-19 was first detected in January 20, 2020 in South Korea, it spread as a local epidemic among religious groups, schools, rallies, and even in family groups. [2][3][4] The first wave of COVID-19 occurred on March, 2020 leading to 200-300 patients becoming infected with severe acute respiratory syndrome coronavirus 2 in 1 day. After the first wave, intense social distancing and active mask-wearing measures prevented the spread of COVID-19 and led to reduced respiratory virus infections as well as changes in the detection patterns in KINRESS compared with that in previous years. 7 The total number of specimens collected from the centers in 2020 was 6094, which is 53.2% lower than that in 2019. This may be because patients with ILI visited COVID-19 screening centers rather than KINRESS centers. However, this did not significantly affect the analysis of the weekly detection rate of respiratory viruses. South Korea has four seasons (spring, summer, fall, and winter), and the detection patterns of respiratory viruses differ by season. 21 However, during the COVID-19 pandemic, enveloped viruses (IFV, hCoV, hPIV, hRSV, and hMPV) were rarely detected in patients with ILI in KINRESS. Only non-enveloped viruses (hRV, hAdV, and hBoV) were detected, with the rate of hRV in 2020 higher than those in the previous 4 years (2016-2019). A high detection rate of hRV was also observed in private diagnostic sectors, which diagnose larger numbers of patients than the national surveillance (KINRESS). Although the number of patients with ILI was decreased because of social distancing and quarantine measures (such as wearing masks and washing hands properly), hRV showed a high detection rate, as it is a non-enveloped virus that is resistant to environmental stress and exhibits prolonged viral shedding for an average of 10-14 days from immunocompetent subjects. 22,23 A negative correlation between influenza A virus and hRV has been observed in some studies as well as in KINRESS. 24,25 Since the start of the COVID-19 pandemic, the detection rate of IFV has dramatically decreased, eventually reaching 0%. Rather than IFV, COVID-19 showed a negative correlation with hRV. The hRV A species was detected in KINRESS in all age groups (0-6, 7-12, 13-18, 19-49, 50-64, and over 65 years) in 2019 and 2020. Interestingly, the hRV C species was detected mostly in the 0-6 years age group in 2019 and 2020, as observed previously. 26,27 It is thought that hRV C sensitivity is high in children with relatively low exposure to the virus. those in previous years based on the partial 5 0 untranslated region and VP4-partial VP2 coding regions. This result demonstrates that the high detection rate of hRV in 2020 when COVID-19 was an epidemic did not occur because of changes in the genetic characteristics of hRV but rather because of the prolonged viral shedding period of hRVinfected patients and its environmental resistance. Other study confirmed that clinical manifestations in patients with hRV C were more severe than in those with hRV A and B 28,29 ; however, the difference in clinical manifestations (fever, cough, sore throat, wheezing, chill, headache, muscle ache, nasal discharge, dyspnea, and phlegm) was not significant in this study (data not shown).

ACKNOWLEDGEMENTS
We thank the team members of the regional laboratories (Public Health