Delayed respiratory syncytial virus outbreak in 2020 in Taiwan was correlated with two novel RSV‐A genotype ON1 variants

Abstract Background Human respiratory syncytial virus (RSV) is a leading pathogen of acute respiratory tract disease among infants and young children. Compared with previous seasons, RSV outbreaks in Taiwan during the 2020–2021 season were delayed because of COVID‐19 mitigation measures. We conducted this study to determine the association of viral factors with clinical characteristics of preschool children with RSV infection. Methods We performed a molecular epidemiology analysis of RSV among inpatient preschool children in Taiwan. In 80 nasopharyngeal samples positive for RSV, we sequenced and analyzed viral genotypes according to patient data. Patients' clinical data were obtained from medical files, and their clinical profiles were compared with those of RSV cases recorded during the 2014–2017 seasons. Results Phylogenetic analysis revealed that among the RSV‐positive samples, all RSV strains identified during the 2020–2021 season belonged to the ON1 genotype. Most of the Taiwan ON1 strains were categorized into two well‐supported clusters with distinct G protein amino acid substitution patterns that had never been demonstrated previously. Furthermore, the proportion of cases among children aged >24 months increased (P < 0.001). Compared with patients infected during the 2014–2017 seasons, patients infected during the 2020–2021 season were hospitalized for shorter days from hospital admission to dereference (P = 0.004) and had a greater need for oxygen supplements (P = 0.021) and systemic steroid therapy (P = 0.026). Conclusion The delayed 2020–2021 RSV outbreak in Taiwan was caused by two novel RSV ON1.1 variants. How the change in RSV epidemiology affects future RSV outbreaks warrants exploration.


| INTRODUCTION
Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in children aged <5 years of age worldwide. Most children have at least one episode of RSV infection by age 2, but repeated infections are common and can lead to severe diseases in the elderly people and high-risk adults. 1,2 RSV is the leading viral pathogen of childhood community acquired pneumonia. 3,4 According to one global surveillance study in 2015, RSV infection was attributed to 33.1 million episodes of lower respiratory tract infection, 3.2 million hospitalizations, and as many as 118,200 deaths. 5 RSV belongs to the Pneumoviridae family, Orthopneumovirus genus, and consists of a single-stranded, negative sense RNA genome packaged in a lipid envelope. The RSV genome is approximately 15.2 kb in length and encodes 11 proteins. The external glycoproteins F and G are two primary antigenic sites and vital elements for viral attachment. 6 The G protein of RSV is highly glycosylated and contributes to immune evasion and antigenicity. 7 The ectodomain of the G protein contains two hypervariable regions (HVR) spanning a length of 13 amino acids in the central conserved cysteine-rich domain, and its size ranges from 282 to 321 amino acids. The G protein, especially second HVR, is highly diverse and under selection pressure. 8 These genetic and antigenic variations in the protein are used for the molecular characterization of RSV strains. RSV strains have been classified into two subgroups, namely, A and B (RSV-A and RSV-B, respectively), and at least 13 HRSV-A genotypes and 20 HRSV-B genotypes have been identified on the basis of G gene sequences. 9 Although disease severity has been reported to be correlated with specific strains or genotypes, [10][11][12][13] no consistent association has been established yet. 14 The extensive use of strict nonpharmaceutical interventions in 2020 to combat the COVID-19 pandemic changed the RSV circulation pattern and engendered a delay in the annual RSV outbreak in several countries. [15][16][17] In general, RSV infection occurs biennially with peaks in spring and fall in Taiwan. 18 The RSV genotypes ON1 and BA9 have co-circulated in Taiwan with alternating predominance since 2011. 19 Amid the threat of the COVID-19 pandemic, an extremely low level of RSV activity was observed in the spring of 2020 in Taiwan, followed by a delayed outbreak at the end of the year. Accordingly, in this study, we explored the clinical features and genotype evolution of this delayed RSV outbreak in Taiwan

| Phylogenetic and amino acid substitution analysis
Nucleotide sequences of the G genes of RSV with determined genotypes were obtained from GenBank for reference. Sequences derived in our study were aligned with reference sequences by using the MUSCLE program implemented in MEGA7 software. Phylogenetic trees were inferred using the neighbor-joining method implemented in MEGA7, and 1000 replications of bootstrap probabilities were used to evaluate confidence estimates.
We compared the complete sequences of the G genes of all RSVpositive samples from our two studies with representative G gene sequences obtained from GenBank for the periods 2015 and 2020.
Deduced amino acid sequences were translated with standard genetic code using MEGA 7 software. Mutations were determined through comparisons of the strains with the corresponding prototype strains.
The mutations of the RSV-ON1 strains in this study were described with respect to their prototype strains: ON67-1210A (accession number JN257693).

| Statistical analysis
Statistical analysis was performed using SPSS (IBM SPSS Statistics version 22). Descriptive data are presented as proportions.
Continuous data are presented as mean AE standard deviation (SD) and analyzed through an independent t test. Group comparisons were performed through either a chi-square test or Fisher's exact test.
Results with a P value of <0.05 were considered significant.

| RSV epidemiological prevalence
The monthly distribution of identified RSV cases throughout the study period is displayed in Figure 1A. Because of the COVID-19 pandemic, RSV activity was extremely low in Taiwan  Moreover, no differences in total white cell count or C-reactive protein levels were noted, but a higher neutrophil/lymphocyte count ratio was observed in the patients during the 2020-2021 season (P < 0.001). We conducted further multivariable analysis by controlling for age and sex. The analysis results revealed that only three parameters differed significantly between the two study seasons: days from admission to dereference (P = 0.004), need for supplementary oxygen (P = 0.021), and systemic steroid therapy (P = 0.026; Table 2).

| Phylogenetic analysis of RSV
The ectodomain of RSV-G gene representative sequences was suc-

| Deduced amino acid sequence analysis
The ectodomain of the G protein amino acid sequences obtained from the 78 available RSV strains, spanning 100 to 320 amino acids, was aligned with the ON1 reference strain (accession number JN257693; Figure 3A). Seventy-five strains could be grouped into two clusters based on the pattern of amino acid substitutions. A total of six shared mutations were noted among these 75 strains: T113I, V131D, N178G, H258Q, H266L, and Y304H. In addition to these amino acid changes, cluster 1 strains specifically harbored an E257K substitution, and cluster 2 strains had a fixed set of genetic alterations of K204R, V225A, T238I, and Y280H. These amino acid substitutions observed in the 2020 strains were distinct from those observed in the older RSV ON1 strains in Taiwan, which comprised E262K, L274P, L298P, and P300S as the prevailing genetic alterations (unpublished data).
We designated the RSV strains responsible for clusters 1 and 2 as ON1.1 genotype variants 1 and 2, respectively; Figure 3B presents the changes in their amino acid sequences and their relative positions on the RSV-G protein sequence.

| Comparison of clinical characters between two groups infected ON1 variants
We observed 44 patients with variant 1 infection and 31 patients with variant 2 infection. Table 3  Of the RSV proteins, the attachment G protein has the highest genetic diversity and exhibits continual changes. 8,9 The highest frequency of amino acid substitutions was observed in the second HVR of the G protein. Along with the gain or loss of glycosylation, these amino acid changes contribute to the evolution of G antigenic sites and presumably facilitate the weakening of preexisting immunity. The RSV genotype ON1 continues to evolve locally and globally. 21   Abbreviations: CRP, C-reactive protein; N/L ratio, neutrophil/lymphocyte count ratio; RSV, respiratory syncytial virus. Multivariable analysis controlled for age and sex, and data are presented as marginal mean AE standard error. Continuous data were analyzed using a general linear model; categorical data were analyzed using logistic regression.
During the 2020-2021 RSV season, we found two RSV genotype Compared with RSV cases observed during the 2014-2017 seasons, the atypical age distribution for patients with RSV infection during the 2020 RSV season was notable. This phenomenon was also observed in the 2020 RSV season in France, which showed an increase in the proportion of cases among children aged 3 months to 5 years. 16 Human humoral and cellular immunity only confers partial and nondurable protection against subsequent RSV infection. 27,28 RSV antigenic changes, in addition to glycosylation alterations, may have also reduced the effectiveness of preexisting immunity. 11,28,29 Whether an RSV genotype or variant affects disease severity warrants exploration. Several studies have determined that certain RSV genotypes are linked to increased disease severity and a higher rate of lower respiratory tract infections compared with other RSV genotypes; nevertheless, the mechanism underlying these findings remains inconclusive. 12 resources. Teh Ying Chou: Supervision.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/irv.12951.

DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of this study are available within the article.