ABSTRACT
Background
Rhinovirus (RV) is a common respiratory virus, but asymptomatic RV infections in adults still require further investigation. Nonpharmaceutical interventions (NPIs) for COVID‐19 markedly reduced other respiratory viruses, yet RV circulation appeared to persist. We describe the epidemiology of asymptomatic and symptomatic adult RV infections in Shanghai across periods with and without strict NPIs.
Methods
We performed a retrospective, hospital‐based repeated cross‐sectional study at a large hospital in Shanghai over two epidemic cycles (March 2021 to February 2022 and June 2023 to May 2024). Adults undergoing routine SARS‐CoV‐2 screening (asymptomatic group) and hospitalized adults with respiratory symptoms (symptomatic group) were systematically sampled. Nasopharyngeal swabs were tested by RT‐PCR, and RV‐positive samples underwent genotyping and quantitative viral load assessment. Clinical characteristics, co‐/secondary infections, and age‐specific patterns were compared using χ 2 tests and nonparametric tests, and associations between RV prevalence and climate variables were evaluated using correlation analysis.
Results
Among 89,571 adults, 865 RV infections were identified. Asymptomatic RV detection remained low and similar during and after strict NPIs (0.68% vs. 0.56%). Adults aged 18–59 years had higher asymptomatic infection rates than those ≥ 60 years (0.68% vs. 0.55%, p = 0.022). RV‐A predominated, while RV‐C was associated with higher viral loads and more frequent symptoms. Total monthly rainfall showed the strongest negative correlation with asymptomatic RV prevalence (r = −0.418, p = 0.042). Symptomatic cases had higher co‐infection rates.
Conclusions
Persistent asymptomatic RV infections in young and middle‐aged adults were unaffected by NPIs. Older adults were more often symptomatic and experienced more frequent co‐infections.
Keywords: asymptomatic infection, nonpharmaceutical interventions, rhinovirus, viral load
1. Introduction
Rhinoviruses (RVs) are the most common respiratory pathogens in humans, accounting for approximately 20%–50% of respiratory tract infections (RTIs) annually [1, 2]. RV infections present with a broad spectrum of clinical manifestations, from asymptomatic cases to rare instances of severe lower respiratory illness [3, 4, 5], and are recognized as potent triggers of exacerbations in chronic respiratory illnesses such as chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis [6, 7, 8]. To date, nearly 170 RV types have been identified, categorized into three genetically distinct species: RV‐A, RV‐B, and RV‐C, with RV‐A being the most prevalent [9]. Asymptomatic RV infections are widespread and often undetectable due to the absence of symptoms, yet they play a crucial role in virus transmission. Investigating the prevalence of asymptomatic infections is essential in understanding the full scope of RV epidemics and transmission dynamics.
Advancements in molecular diagnostics have greatly enhanced our understanding of the etiology of respiratory infections. Previous studies have shown that asymptomatic RV infections are common in children, with frequencies ranging from 2% to 32% [10, 11, 12, 13]. However, the incidence of asymptomatic RV in adults remains understudied and poorly understood. Few studies have focused on asymptomatic RV infections in adults during epidemic seasons, and comprehensive annual prevalence data remain unavailable [1, 14]. During the COVID‐19 pandemic, the prevalence of influenza virus infections significantly declined due to NPIs, whereas RV prevalence remained stable in children during and after the pandemic [15, 16, 17]. High detection rates of asymptomatic RV infections in children have raised concern about the etiological role of RV in certain clinical conditions. Additionally, the climatic conditions, particularly temperature, humidity, and precipitation, are important drivers of respiratory virus seasonality and may shape distinct circulation patterns of respiratory viruses in different temperate regions. Understanding RV epidemiology in different climate zones is therefore valuable for local public health.
Investigating asymptomatic RV infections in adults is crucial for comprehensive epidemiological surveillance, informing public health strategies, understanding interactions with other respiratory pathogens, and guiding the development of vaccines and clinical management practices. In this large‐scale, retrospective, hospital‐based repeated cross‐sectional observational study, we systematically observed and analyzed asymptomatic RV infections in adults across two complete year‐round epidemic cycles (2021–2022 and 2023–2024) to estimate RV prevalence in asymptomatic adults undergoing SARS‐CoV‐2 screening in 2021–2022 (strict NPIs) and 2023–2024 (post‐NPI) and compare clinical features, RV species, and viral load (VL) between asymptomatic adults and symptomatic inpatients with RV infection. Additionally, we examine the association between monthly RV detection rates and local climate factors (monthly mean temperature, total rainfall, and mean relative humidity) in Shanghai and describe the frequency and patterns of co‐infection and secondary infection with other respiratory viruses among RV‐positive adults in both surveillance periods.
2. Methods
2.1. Study Design and Subjects
We performed a retrospective, hospital‐based repeated cross‐sectional observational study at Ruijin Hospital, a large general hospital in Shanghai, China. The study covered two 12‐month surveillance periods: March 2021 to February 2022, when strict national COVID‐19 nonpharmaceutical interventions (NPIs) were implemented, and June 2023 to May 2024, after most NPIs had been lifted. In both periods, we systematically sampled 72,811 asymptomatic adults undergoing routine SARS‐CoV‐2 nucleic acid screening and enrolled a comparison group of 16,760 adult inpatients with acute respiratory symptoms who underwent multiplex RT‐PCR testing for respiratory viruses (Figure 1). Hospital identifiers were used to identify repeat individuals; we retained only the first eligible screening encounter and excluded subsequent duplicates from the analysis. All sampled subjects were adults aged ≥ 18 years, and enrolled subjects were classified into four clinical groups based on clinical manifestation, RV detection, and sampling dates (Table S1): RV‐positive asymptomatic/symptomatic subjects in 2021–2022 and RV‐positive asymptomatic/symptomatic inpatients in 2023–2024.
FIGURE 1.
The inclusion process for asymptomatic and symptomatic rhinovirus infected subjects during two study periods.
Individuals were classified as asymptomatic if (1) no respiratory symptoms (cough, sputum production, shortness of breath, wheezing, coryza, fever, and chest pain) were reported at the time of sampling and (2) there was no emergency or outpatient/inpatient visit for respiratory complaints recorded in the hospital information system within the preceding 7 days (2021–2022) or no respiratory symptoms documented in the admission assessment (2023–2024). In 2021–2022, we used systematic sampling to include the first 900 adults undergoing outpatient SARS‐CoV‐2 nucleic acid screening (In response to the Chinese government's mandate for regular SARS‐CoV‐2 testing in healthy individuals to prevent the spread of COVID‐19 [18]) every Wednesday (900 per week). In 2023–2024, we included the first 500 adults undergoing routine pre‐admission SARS‐CoV‐2 screening every Monday and Thursday (1000 per week). For the weekly systematic samples of asymptomatic adults, the sample size was chosen to provide precise estimates of the prevalence of asymptomatic RV infection, which we expected to be low (approximately 1%–5%) based on prior surveillance and research. Using the standard formula for estimating a single proportion with a 95% confidence level (CI) (Z = 1.96), an expected prevalence of 1%–5% and a desired absolute precision of ± 1.5 percentage points, the required sample size ranges from approximately 200 to 800 individuals per week. Our weekly samples of 900 individuals in 2021–2022 and 1000 individuals in 2023–24 therefore exceed this requirement. Assuming prevalences in the 1%–5% range, these sample sizes yield 95% CI half‐widths of approximately 0.6–1.4 percentage points (95% CI, 0.6%–1.4%), which was adequate for the surveillance objectives.
The symptomatic comparison group comprised adult inpatients with respiratory symptoms of acute respiratory infection who underwent multiplex RT‐PCR testing for respiratory viruses.
Nasopharyngeal swab (NPS) samples were collected from all enrolled subjects and stored at −80°C for subsequent analysis. NPS from asymptomatic subjects were collected on the day of screening, whereas NPS from symptomatic inpatients were collected within 1–3 days of symptom onset. This retrospective study was approved by the ethics committee of Ruijin Hospital (Ruijin Hospital Ethics Committee 2018–48), and all experimental infection procedures were performed in biosafety level 2 facilities.
2.2. Clinical Data Collection
Clinical data were recorded by the resident physician in the electronic medical record management system. The presence or absence of cough, sputum production, shortness of breath, wheezing, coryza, fever, chest pain, muscle ache, headache, disturbed sleep, general malaise, interference with normal activities, and confusion or disorientation were documented. For RV‐positive subjects, we collected laboratory test results for other respiratory viruses (SARS‐CoV‐2, influenza virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, seasonal coronavirus, and adenovirus) performed within 1 month after RV detection. Co‐infections were defined as the detection of RV together with at least one of these respiratory viruses within 48 h of hospital admission, whereas secondary infections were defined as those detected more than 48 h after admission.
2.3. RV RNA Detection
The viral laboratory diagnostics were conducted in the Virology Laboratory of Ruijin Hospital. Total viral RNA was extracted using commercial kits from Shanghai Biogerm Medical Technology Co. Ltd. (Shanghai, China), following the manufacturer's instructions. The real‐time RT‐PCR for RV detection was performed using a HiScript II One Step RT‐PCR Kit (Vazyme Biotech, Nanjing, China) and a set of primers. The target gene region was targeting the VP4/VP2 gene as previously described [18].
2.4. Sequencing and Quantification of RV
RV genotyping was conducted by amplifying and sequencing a partial VP4/VP2 viral genome fragment, as described previously [19]. Initial species identification was performed using the BLASTn tool for nucleotide database analysis. RV VL was quantified using a developed real‐time RT‐PCR assay. The quantification primers and probe were designed as previously described [20, 21]. RV VL was quantified using a real‐time RT‐PCR assay with a plasmid standard curve spanning 3 × 101–3 × 108 copies/μL of a conserved RV fragment. The plasmid containing conserved sequences (447–529), which covers most HRV types, was used to construct the standard curve of CT values. Ct values of clinical samples were converted to copies/μL by interpolation within this curve. When an initial Ct value indicated a VL above the highest standard (3 × 108 copies/μL), the RNA extract was diluted 10‐ to 100‐fold and retested; final VL values were back‐calculated by multiplying the interpolated concentration of the diluted sample by the dilution factor, ensuring that all reported VLs were derived from measurements within the validated linear range of the assay.
2.5. Climate Data
Monthly ground‐level climate data for Shanghai (near‐surface air temperature, total rainfall, and relative humidity) were obtained from the official surface observation network operated by the China Meteorological Data Service Centre/National Meteorological Information Center data service platform (data.cma.cn) and Shanghai Water Authority (swj.sh.gov.cn). For each month between March 2021 and February 2022 and between June 2023 and May 2024, we extracted the mean near‐surface (2 m) air temperature (°C), total rainfall (mm), and relative humidity (%). All data had undergone routine quality control by the meteorological authorities before release. These monthly climate variables were then matched to the corresponding monthly RV detection rates in asymptomatic and symptomatic adults.
2.6. Statistical Analysis
Continuous variables were summarized as medians with interquartile ranges (IQRs) or means with 95% CIs and categorical variables as counts and percentages. The primary outcome was the prevalence of RV infection among adults in each clinical group and study period. Prevalence was calculated as the proportion of RV‐positive individuals among all screened subjects, overall and stratified by clinical group (asymptomatic screening subjects vs. symptomatic inpatients), study period (2021–2022 vs. 2023–2024), and age group (18–59 vs. ≥ 60 years). Differences in prevalence and other categorical characteristics between groups were assessed using chi‐squared (χ 2) tests. For continuous variables such as age and VL, two‐group comparisons were performed using the Mann–Whitney U test or independent t‐test and comparisons across more than two groups using the Kruskal–Wallis test or one‐way analysis of variance (ANOVA), as appropriate. For selected 2 × 2 comparisons of categorical outcomes, crude associations were summarized as odds ratios (ORs) with 95% CIs.
VLs were compared between clinical groups (asymptomatic vs. symptomatic), age groups (18–59 vs. ≥ 60 years) and RV species (A–C) using the nonparametric tests described above (Mann–Whitney U test for two‐group comparisons and Kruskal–Wallis test for comparisons across more than two groups), or one‐way ANOVA when the normality assumption was judged to be reasonable. For selected 2 × 2 comparisons of categorical outcomes (e.g., age group vs. clinical presentation), crude associations were summarized as ORs with 95% CIs derived directly from the contingency tables. For analyses of co‐/secondary infections, the proportion of adults with co‐/secondary infection with another respiratory virus was compared between asymptomatic and symptomatic subjects and between study periods (2021–2022 vs. 2023–2024) using χ 2 tests. Where informative, crude ORs with 95% CIs calculated from 2 × 2 tables are reported to describe the strength of these associations.
Exploratory associations between individual respiratory symptoms and VL among symptomatic adults were assessed using Spearman's rank correlation between log₁₀‐transformed VL and each symptom. Spearman's correlation coefficients (R) and p‐values are reported, based on subjects with available VL measurements, without adjustment for multiple comparisons. For the climate analysis, monthly RV detection rates in asymptomatic and symptomatic adults were calculated and correlated with monthly mean ground temperature, total rainfall, and mean relative humidity using Spearman's rank correlation coefficients. Statistical analyses were performed using IBM SPSS Statistics, version 25.0 for Windows (IBM Corp., Armonk, NY, USA), and two‐sided p‐values < 0.05 were considered statistically significant.
Graphical methods were used to present the distribution and temporal patterns of RV infection. Heatmaps and line graphs were used to depict monthly RV detection rates across study periods and age groups; bar plots were used to display prevalence and proportions in different clinical and demographic strata, and boxplots were used to compare distributions of VL and other continuous variables between groups. All figures were generated using GraphPad Prism 8.4.0 software.
3. Results
3.1. The Prevalence of Asymptomatic RV Infections Among Adults Remains Stable Especially in Young‐ and Middle‐Aged Population and Appears Unaffected by NPIs
During the study period, a total of 72,811 asymptomatic adults undergoing SARS‐CoV‐2 screening and 16,760 hospitalized patients with respiratory symptoms were included (Figure 1). In 2021–2022, 40,848 asymptomatic screening subjects and 4253 symptomatic inpatients were enrolled; in 2023–2024; 31,963 asymptomatic screening subjects and 12,507 symptomatic inpatients were enrolled. The RV infection status across the four groups was presented as follows: (1) asymptomatic subjects in 2021–2022 (n = 277, 32.02%), (2) symptomatic inpatients in 2021–2022 (n = 98, 11.33%), (3) asymptomatic subjects in 2023–2024 (n = 180, 20.81%), and (4) symptomatic subjects in 2023–2024 (n = 310, 35.84%). Among the 865 RV‐positive subjects, 408 (47.17%) reported at least one sign/symptom, and 457 (52.83%) were asymptomatic. Among all asymptomatic subjects, 277 (0.68%) were RV‐positive during 2021–2022 and 180 (0.56%) during 2023–2024, indicating a stable prevalence of asymptomatic RV infection in adults. In contrast, RV positivity was higher among inpatients with symptoms of acute upper RTIs, with 2.30% testing positive during 2021–2022 and 2.48% during 2023–2024. The demographics of symptomatic and asymptomatic RV infection population were presented in Table 1.
TABLE 1.
Demographics of symptomatic and asymptomatic RV infection population.
| Total asymptomatic N = 72,811 | Total symptomatic N = 16,760 | RV asymptomatic N = 457 | RV symptomatic N = 408 | p | |
|---|---|---|---|---|---|
| Female (%) | 34,117 (46.86) | 6189 (36.93) | 206 (45.08) | 129 (31.62) | Total: < 0.0001 RV: < 0.0001 |
| Age (median, IQR) | 55 (39–67) | 60 (43–73) | 52 (35–64) | 63.5 (49–75) | Total:< 0.0001 RV:< 0.0001 |
| Study period | |||||
| 2021.3–2022.2 | 40,848 | 4253 | 277 | 98 | RV asymptomatic: 0.0526 a |
| 2023.6–2024.5 | 31,963 | 12,507 | 180 | 310 | RV Symptomatic: 0.5647 a |
| Underling condition (%) | |||||
| Asthma | 1286 (1.77) | 843 (5.03) | 12 (2.63) | 19 (4.65) | Total: < 0.001, RV: < 0.01 |
| COPD | 2763 (3.79) | 1542 (9.20) | 8 (1.75) | 12 (2.94) | Total: < 0.001, RV: 0.068 |
| Hypertension | 12,834 (17.63) | 5492 (32.77) | 53 (11.60) | 93 (22.79) | Total: < 0.001, RV: < 0.01 |
| Diabetes | 4684 (6.43) | 2634 (15.72) | 26 (5.69) | 37 (9.07) | Total: < 0.001, RV: < 0.001 |
Abbreviations: COPD, chronic obstructive pulmonary disease; RV, rhinovirus.
Comparison of the proportion of RV asymptomatic and symptomatic cases between two study periods.
Further age‐based analysis revealed that the overall age of the asymptomatic infection group was significantly lower than that of the symptomatic group. Notably, asymptomatic RV‐A infections showed the most pronounced age difference, while RV‐B and RV‐C infections were also more prevalent in 18–59 young and middle‐aged individuals (Figure 2A–C). The asymptomatic infection rate was significantly higher in the 18–59 age group compared to those over 60 (0.68% vs. 0.55%, χ 2 ≈ 5.3, p = 0.022, OR 1.25, 95% CI 1.03–1.52), with older HRV‐infected adults showing significantly higher odds of respiratory symptom (2.81% vs. 2.04%, χ 2 ≈ 10.6, p = 0.001, OR 1.39, 95% CI 1.14–1.70) (Figure 2D).
FIGURE 2.
The prevalence of rhinovirus in distinct age groups showed significant differences. No matter the total RV infection age distribution (A) or during certain study period such as 2021–2022 (B) or 2023–2024 (C), RV‐A asymptomatic infections showed the most significant age difference. (D) The asymptomatic infection rate in the 18–59 age group was significantly higher than in those over 60. (E) No significant difference in adult asymptomatic RV infection rates between the period of strict nonpharmaceutical interventions (NPIs) (2021–2022) and the period without NPIs (2023–2024).
Notably, during the COVID‐19 pandemic, China implemented strict public health measures to prevent SARS‐CoV‐2 infection, including travel restrictions, social distancing, and personal protection measures. However, our data showed no significant difference in adult asymptomatic RV infection rates between the period of strict NPIs (2021–2022) and the period lifting NPIs (2023–2024) (Figure 2E). Similarly, symptomatic RV infection rates remained unchanged between these periods, suggesting that NPIs had a minimal impact on RV infection prevalence.
3.2. Asymptomatic and Symptomatic RV Infections Exhibit Alternating Peak Periods and Are Correlated With Total Rainfall in the Shanghai Region
The number of RV detection and species distribution across the four clinical groups over the 2‐year study period was presented in Figure 3. RV demonstrated a year‐round prevalence with two peak periods in spring and autumn/winter. During March 2021 to February 2022, peak asymptomatic RV infection rates were observed from March to July during 2021–2022 and from November to December during 2023–2024, while peak symptomatic RV infection rates occurred from May to July and October to December during 2021–2022 and from October to April during 2023–2024 (Figure 3A). The overall seasonal trends for both asymptomatic and symptomatic infections did not differ significantly. RV‐A was prevalent year‐round, whereas RV‐C was mainly concentrated in autumn and winter, particularly among symptomatic individuals. RV‐B predominantly caused asymptomatic infections in autumn and winter (Figure 3B).
FIGURE 3.
The number of RV detection (A) and species distribution (B) among the four clinical groups over the 2‐year study period. (A) During March 2021 to February 2022, peak asymptomatic RV infection rates were observed from March to July, while peak symptomatic RV infection rates occurred from May to July and October to December. During June 2023 to May 2024, peak RV infection rates among asymptomatic and symptomatic subjects occurred in November to December and October, respectively. (B) RV‐A was prevalent year‐round, whereas RV‐C was mainly concentrated in autumn and winter, particularly among symptomatic individuals. RV‐B predominantly caused asymptomatic infections in autumn and winter.
To investigate whether RV prevalence was influenced by climate, as suggested by multiple studies, we collected corresponding climate data in Shanghai, including ground temperature, total rainfall, and relative humidity. We observed that the peak times of both symptomatic and asymptomatic RV infection rates (Figure 4A) did not align with humidity and precipitation levels (Figure 4B). Statistical analysis revealed a significant negative correlation between infection rates and monthly rainfall for asymptomatic infections (r = −0.418, p = 0.042), whereas temperature and relative humidity showed no significant correlation. For symptomatic infections, none of the three climate factors demonstrated a significant correlation. These results suggested that rainfall might be a meteorological factor affecting the prevalence of asymptomatic RV infections in Shanghai. Furthermore, although relative humidity and temperature did not show significant correlation with RV prevalence in our study, rainfall exhibited significant positive correlations with both temperature (r = 0.780, p < 0.0001) and relative humidity (r = 0.789, p < 0.0001).
FIGURE 4.
(A) Seasonality of rhinovirus (RV) infections and meteorological profiles in Shanghai during March 2021 to February 2022 and June 2023 to May 2024. (B) Bar and line charts illustrating the trends between RV incidence and meteorological factors are depicted. Statistical analysis revealed a significant negative correlation between infection rates and monthly rainfall for asymptomatic infections (r = −0.418, p = 0.042), whereas temperature and relative humidity showed no significant correlation. Rainfall exhibited significant positive correlations with both temperature (r = 0.780, p < 0.0001) and relative humidity (r = 0.789, p < 0.0001).
3.3. RV‐A and RV‐C Are, Respectively, Dominant in Asymptomatic and Symptomatic Infections, While RV‐C Associating With a Significantly Higher Frequency of Clinical Symptoms
A total of 865 NST samples from HRV‐positive subjects were collected during the study period, with 90.87% (786/865) successfully genotyped. Sequencing analysis of the VP4/VP2 gene revealed that RV‐A predominated, infecting 65.78% (517/786) of the subjects, followed by RV‐C (25.95%, 204/786) and RV‐B (8.27%, 65/786). In total, 81 distinct types (RV‐A: 50 types, RV‐B: 9 types, and RV‐C: 22 types) were identified through sequencing analysis. The prevalence of RV‐A and RV‐C infections consistently remained higher than that of RV‐B throughout the study period.
Among asymptomatic and symptomatic infections, RV‐B had a higher proportion of asymptomatic cases, while RV‐C was more frequently associated with symptomatic cases. Further analysis indicated that specific subtypes varied seasonally, with A29, A89, and C7 being dominant from 2021 to 2022 and A46, C1, and C7 appearing more frequently from 2023 to 2024. Overall, RV‐A exhibited the highest diversity among both asymptomatic and symptomatic infections (Figure 5).
FIGURE 5.
Rhinovirus (RV) detection rate and species distribution according to month of detection.
Most RV‐infected subjects experienced cough, sputum production, and coryza. In symptomatic patients, RV‐C infections were associated with a significantly higher frequency of clinical symptoms compared to RV‐A, consistent with findings from other studies (Table 2).
TABLE 2.
Age, gender distribution, and infection symptoms for symptomatic RV infection cases according to rhinovirus species.
| Characteristics | RV‐A | RV‐B | RV‐C | p | Correlation with viral load (Spearman R, p) |
|---|---|---|---|---|---|
| No of cases | 215 | 18 | 144 | ||
| Age (median) | 64 | 60 | 64 | ||
| Female (%) | 29.77 | 38.89 | 31.94 | ||
| Infection symptom (%) | |||||
| Cough | 55.81 | 66.67 | 78.47 | RV‐A vs. RV‐C (< 0.0001) | R = 0.19, p < 0.001 |
| Sputum production | 46.05 | 44.44 | 75.00 | RV‐A vs. RV‐C (< 0.0001) | R = 0.08, p = 0.116 |
| Shortness of breath | 8.84 | 11.11 | 65.97 | RV‐A vs. RV‐C (< 0.0001) | R = 0.17, P = 0.001 |
| Wheeze | 27.91 | 16.67 | 57.64 | RV‐A vs. RV‐C (< 0.0001) | R = 0.13, p = 0.015 |
| Coryza | 50.23 | 50.00 | 57.64 | RV‐A vs. RV‐C (0.2) | R = 0.09, p = 0.096 |
| Fever | 43.72 | 44.44 | 62.50 | RV‐A vs. RV‐C (< 0.001) | R = 0.09, p = 0.090 |
| Chest pain | 29.77 | 11.11 | 52.78 | RV‐A vs. RV‐C (< 0.0001) | R = 0.08, p = 0.144 |
| Muscle aching | 53.02 | 11.11 | 49.31 | RV‐A vs. RV‐C (0.56) | R = 0.01, p = 0.918 |
| Headache | 51.63 | 33.33 | 53.47 | RV‐A vs. RV‐C (0.81) | R = 0.11, p = 0.040 |
| Disturbed sleep | 10.23 | 22.22 | 18.06 | RV‐A vs. RV‐C (0.048) | R = 0.07, p = 0.224 |
| Feeling generally unwell | 56.28 | 33.33 | 52.78 | RV‐A vs. RV‐C (0.59) | R = −0.10, p = 0.063 |
| Interference with normal activities | 12.09 | 0.00 | 50.69 | RV‐A vs. RV‐C (< 0.0001) | R = 0.12, p = 0.030 |
| Confusion/disorientation | 5.12 | 0.00 | 44.44 | RV‐A vs. RV‐C (< 0.0001) | R = 0.09, p = 0.107 |
3.4. The Differences in VL Between Asymptomatic and Symptomatic Infections Varied According to the RV Species
We investigated the early VL differences between asymptomatic and symptomatic RV infections using real‐time RT‐PCR. Cough, shortness of breath, wheeze, and headache were positively correlated with higher VLs (Table 2). Symptomatic RV‐C infections exhibited significantly higher VLs compared to asymptomatic cases. In contrast, RV‐A and RV‐B infections showed no significant differences in VLs between symptomatic and asymptomatic individuals (Figure 6A).
FIGURE 6.
(A) Symptomatic RV‐C infections exhibited significantly higher viral loads compared to asymptomatic cases. In contrast, RV‐A and RV‐B infections showed no significant differences in viral loads between symptomatic and asymptomatic individuals. (B) Asymptomatic RV‐A infections generally exhibited higher viral loads among individuals aged 18–59. (C) Symptomatic RV‐C infections in individuals aged over 60 had significantly higher viral loads compared to asymptomatic cases.
Further age stratification revealed that symptomatic RV‐C infections in individuals over 60 had significantly higher VLs compared to asymptomatic cases (Figure 6C). In contrast, asymptomatic RV‐A infections generally had higher VLs in individuals aged 18–59 (Figure 6B). These findings suggested that RV‐C VLs were highly correlated with clinical severity.
3.5. The Interaction Between Symptomatic RV Infections and Subsequent Viral Infections
We further examined whether RV infection status was associated with the occurrence of other common respiratory viruses. According to the predefined criteria for co‐ and secondary infections, symptomatic RV infections were associated with higher rates of co‐infections or secondary infections with other respiratory viruses compared with asymptomatic RV infections (Figure 7). The most frequently observed co‐ or secondary infections involved SARS‐CoV‐2, influenza virus, seasonal coronavirus, and human metapneumovirus. In addition, the incidence of co‐/secondary infections with other respiratory viruses was higher in 2023–2024 than in 2021–2022 (Figure 7). These findings are consistent with higher circulation of other respiratory viruses after relaxation of NPIs; however, given the observational design of our study, no causal inferences about the effect of NPIs on these infections can be drawn.
FIGURE 7.
Symptomatic RV infections were significantly associated with higher rates of co‐infections or secondary infections with other respiratory viruses compared to asymptomatic cases. The incidence of co‐infections or secondary infections was significantly higher during 2023–2024 compared to 2021–2022.
4. Discussion
Previous studies have confirmed the prevalence of asymptomatic RV infections in children, but there remains a lack of comprehensive data on such infections in adults. This includes crucial aspects such as VL, genetic subtypes, and infection rates. A better understanding of asymptomatic infection rates in adults is essential for gaining a fuller picture of RV epidemiology and for assessing the effectiveness of preventive measures. Our study provides a comprehensive analysis of the epidemiological mechanisms and patterns of RV, utilizing sequence analysis and quantitative viral RNA detection in 457 asymptomatic and 408 symptomatic cases of adult RV infections among 89,571 cases across two independent epidemic seasons during and after the COVID‐19 pandemic.
The overall adult asymptomatic infection rate was 0.63%, with subtype proportions ranging from 0.41% (RV‐A), 0.08% (RV‐C), and 0.06% (RV‐B). This rate is lower than the 4% estimated in a previous European study, probably due to the larger population screened in our study [1]. There were no significant differences in overall asymptomatic infection rates between the two epidemics nor in symptomatic infection rates. This finding also provides insight into a common question observed during the pandemic: Why did strict NPIs during the COVID‐19 pandemic significantly reduce infection rates of respiratory viruses like influenza and respiratory syncytial virus in children but not the infection rate of RV? Our study similarly found no significant differences in the incidence of asymptomatic and symptomatic RV infections during and after the pandemic. This suggests that NPIs did not have the same effect on RV prevalence as they did on viruses like SARS‐CoV‐2 and influenza [22, 23, 24]. One possible underlying reason is the widespread presence of asymptomatic RV infections in adults. These asymptomatic individuals are unlikely to increase their vigilance in reducing social contacts and may interact more readily with children and the elderly, leading to the continued circulation of RV. Extensive data from the later stages of the COVID‐19 pandemic have demonstrated that asymptomatic infections are important contributors to viral transmission [25, 26, 27]. Therefore, asymptomatic RV infections in adults may play a significant role in the global prevalence of RV, particularly since individuals aged 18–59 tend to have higher activity levels and greater mobility compared to children and the elderly. Previous studies in children have shown that a considerable proportion of HRV infections are asymptomatic, with estimates ranging from approximately 12%–32% of detections in children < 4 years and up to 25%–60% of HRV infection episodes in some pediatric cohorts [28, 29, 30]. These reports also suggest that the proportion of asymptomatic infections tends to decrease with increasing age. In our adult population, individuals aged 18–59 years were more likely to have asymptomatic RV infection than those aged ≥ 60 years, whereas older adults more often presented with symptoms when RV‐positive. This pattern is consistent with the hypothesis that younger adults, similar to children, may more frequently experience subclinical RV infection, whereas immunosenescence, comorbidities, and differences in symptom perception or health‐seeking behavior in older adults increase the likelihood of symptomatic disease. However, because our study only included adults and grouped ages as 18–59 vs. ≥ 60 years, we cannot fully descript a continuous age gradient from childhood to older age, and our interpretation should be viewed as hypothesis‐generating rather than causal.
RV infection rates varied depending on the RV types. RV‐C had higher infection rates in autumn and winter, while RV‐A was prevalent throughout the year. Asymptomatic RV‐B infections peaked in autumn and winter, similar to RV‐C, whereas RV‐A maintained year‐round activity. The trends in symptomatic infections align with previous studies in Shanghai, likely due to the higher prevalence of RV‐C in autumn and winter [31, 32]. Subtype classification indicates that RV‐A is the most prevalent in both asymptomatic and symptomatic cases, followed by RV‐C, while RV‐B is more common in asymptomatic infections. These observations are consistent with previous findings on RV infections in both children and adults [2, 33, 34]. Our analysis showed that symptomatic RV‐C infections were associated with higher VLs and more severe clinical symptoms. Asymptomatic RV‐A infections generally exhibited higher VLs in individuals aged 18 to 59, while symptomatic RV‐C infections had significantly higher VLs than asymptomatic RV‐C infections. Additionally, symptomatic RV‐C infections in individuals over 60 had higher VLs compared to asymptomatic cases, underscoring the correlation between RV‐C VLs and clinical severity. These findings align with some previous studies [21, 35, 36], although there is no unified understanding of the relationship between RV species and clinical manifestations due to the significant diversity of RV species.
Additionally, while early studies suggested that RV prevalence might be influenced by climatic factors, conclusions among various studies remain inconsistent [37]. Some studies in tropical climates suggest that RV prevalence is associated with local rainfall and increased humidity [21, 38], yet others have found that RV can prevail during the dry season [39]. In this study, we conducted a preliminary exploration using climate data from the Shanghai area and found the incidence of asymptomatic infections was negatively correlated with rainfall. Considering that Shanghai is located in a subtropical monsoon climate region, we speculate that the actual influence of climatic factors on RV prevalence might require a more specific analysis based on local climate types. Notably, studies conducted in other tropical rainforest climates show a negative correlation between local rainfall and temperature. However, in the Shanghai area, rainfall is highly positively correlated with temperature. This difference may contribute to variations in RV prevalence rates, suggesting that the impact of climatic factors on RV prevalence requires further comprehensive exploration.
The interaction between RV and other respiratory viruses has received considerable attention. Some models suggested that co‐infections with influenza virus and RV resulted in more severe clinical manifestations [40], while others propose that RV infections activated innate immunity, thereby providing protection against other viral infections [41]. We found that during the NPIs phase of 2021–2022, the incidence of secondary or co‐infections with other respiratory viruses following RV infection was significantly lower than in 2023–2024 when the NPIs were lifted. However, due to the limit of observational study and the lack of baseline immune status or vaccination data, it was challenging to determine whether the higher co‐infection rates were directly caused by RV infection or due to inherently weaker immune states in symptomatic individuals. In conclusion, our study systematically investigated the prevalence of asymptomatic RV infections in adults, confirming that individuals under 60 were more susceptible to asymptomatic infections. Individuals over 60 were more likely to exhibit respiratory symptoms and had a higher risk of co‐infections or secondary infections. Given the high prevalence of asymptomatic infections, NPIs had limited effectiveness in controlling RV spread among adults but remained beneficial in reducing co‐infections. Developing vaccines for high‐risk populations, particularly individuals over 60, could help prevent transmission from asymptomatic infections. Our findings underscore the prevalent adult RV infections and highlight the need for continued surveillance and targeted preventive strategies.
Author Contributions
Dong Wei: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, resources, writing – original draft, writing – review and editing. Wei Tan: data curation, formal analysis, investigation, methodology, resources, validation, writing – original draft. Chenyue Tang: formal analysis, methodology, resources. Weihui Ma: formal analysis, data curation, resources. Yulong Li: formal analysis, data curation. Zhitao Yang: conceptualization, funding acquisition, project administration, validation, resources, writing – review and editing. Xinxin Zhang: conceptualization, funding acquisition, project administration, validation, writing – original draft, writing – review and editing.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Table S1: Sample information.
Acknowledgments
This project is financially supported by the grants from the Shanghai Municipal Science and Technology Major Project (ZD2021CY001), the National Natural Science Foundation of China (NSFC‐82402586), the Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases (20dz2261100), the Innovative research team of high‐level local universities in Shanghai, National Key Research and Development Program of China (2021YFC2301500), the Shanghai Municipality Strengthening Public Health System Construction Three‐Year Action Plan (GWVI‐11.2‐YQ04), and the Shanghai Targeted Biomedical Emergency Project (23DX1900300).
Contributor Information
Zhitao Yang, Email: yangzhitao@hotmail.fr.
Xinxin Zhang, Email: zhangx@shsmu.edu.cn.
Data Availability Statement
The data that support the findings of this research are available from the corresponding authors on reasonable request. All sequence data had been submitted to NCBI Genbank database (GenBank Accession: PV787262‐PV788051).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1: Sample information.
Data Availability Statement
The data that support the findings of this research are available from the corresponding authors on reasonable request. All sequence data had been submitted to NCBI Genbank database (GenBank Accession: PV787262‐PV788051).