Nasal-spraying Bacillus spores as an effective symptomatic treatment for children with acute respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a leading cause of Acute Respiratory Tract Infections (ARTIs) in young children. However, there is currently no vaccine or treatment available for children. Here, we demonstrated that nasal-spraying probiotics containing 5 billion of Bacillus spores (LiveSpo Navax) is an effective symptomatic treatment in a 6-day randomized controlled clinical study for RSV-infected children (n = 40–46/group). Navax treatment resulted in 1-day faster recovery-time and 10–50% better efficacy in relieving ARTI symptoms. At day 3, RSV load and level of pro-inflammatory cytokines in nasopharyngeal samples was reduced by 630 folds and 2.7–12.7 folds respectively. This showed 53-fold and 1.8–3.6-fold more effective than those in the control-standard of care-group. In summary, nasal-spraying Bacillus spores can rapidly and effectively relieve symptoms of RSV-induced ARTIs while exhibit strong impacts in reducing viral load and inflammation. Our nasal-spraying probiotics may provide a basis for simple-to-use, low-cost, and effective treatment against viral infection in general.

and Δ pulse (beats/min) values of both groups fluctuated within safe limits of 2 beats/min on average, ranging from − 5 to + 5 beats/min for Δ breath and from − 14 to + 15 beats/min for Δ pulse for individuals. In the meantime, we recorded that Δ temperature (°C) slightly decreased (within 0.1 °C on average, ranging from − 1 to + 0.8 °C for individuals) and Δ SpO 2 (%) slightly increased (within 1% on average, ranging from − 2 to + 4% for individuals) after nasal spraying in both groups. When sprayed, all participants in both groups did not choke, showed no signs of nasal mucosa irritation, had no symptoms of local bacterial infection, or had any digestive problems such as vomiting, diarrhea. During the observed treatment period, no patient reported abnormality in vital signs. Taken together, the data indicated that nasal-spraying Bacillus spores administration is safe and comfortable for pediatric RSV patients.
To examine the effectiveness of LiveSpo Navax, we evaluated typical clinical features of RSV infection in patients at day 3 and day 6 and observed notable differences between the Navax and the Control groups (Table 1). In comparison to the Control group, the Navax group showed a lower percentage of patients who still had symptoms like moist rales (32.61% in Navax vs. 55.00% in Control; p = 0.04), and fast pulse (8.70% in Navax vs. 30.00% in Control; p = 0.02) at day 3. At day 6, there was no patient with runny nose in Navax group vs. 17.50% in Control (p = 0.04). Because most patients had recovered by the third day of treatment, there was no statistically significant difference in either fever, pulse oxymetry, or fast breath between the two groups. The median days of treatment and changes in percentage (%) of patient's symptoms free over time in the two groups were then analyzed and shown in Fig. 3A1-G1 and A2-G2, respectively. Remarkably, the Navax group recovered from most of the symptoms, including runny nose (day 4 in Navax vs. day 5 in Control; p = 0.0014; Fig. 3A1), difficulty breathing (day 2 in Navax vs. day 3 in Control; p = 0.0042; Fig. 3B1), chest depression (day 2 in Navax vs. day 3 in Control; p = 0.0042; Fig. 3C1), dry and moist rales (day 4 in Navax vs. day 5 in Control; p = 0.0149 and p = 0.012, respectively; Fig. 3D1-E1), fast pulse (day 3 in Navax vs. day 4 in Control; p < 0.0001; Fig. 3F1). Although the Navax group's fast breathing symptom was recovered 1-day earlier than that of the Control group, the difference is not statistically significant (p = 0.3515) (Fig. 3G1). We also discovered that in the Navax group, the Days of Treatment when 50% patients are no longer symptomatic (DT 50 ) for all observed seven symptoms were shorter than in the Control group. In details, pairwise comparison between the Navax group vs. the Control Acute respiratory infection assessed for eligibility n = 197 Consented and randomized n = 100 Excluded round 1 (n = 97) -not meeting inclusion: criteria: negative with RSV (n = 55); newborn (n = 3) -did not consent (n = 39) LiveSpo Navax + routine treatment n = 50 Excluded round 2 (n = 10) withdrawn from trial (n = 4) lost to follow-up (n = 2) discharge before day 3 (n = 3) oxygen therapy (n = 1) Analyzed (n = 40) NaCl 0.9% + routine treatment n = 50

Follow up
Analysis Excluded round 2 (n = 4) withdrawn from trial (n = 1) lost to follow-up (n = 1) discharge before day 3 (n = 1) continuing in trial but missing data (n = 1) Figure 1. Diagram displaying the flow of participants involved in the study. From the clinical database, participants were screened for eligibility. Eligible participants who provided consent were randomized in either Control or Navax group. Measurements took place between baseline (day 0) and day 6 of treatment period. Participants were recruited from August 2020 to July 2021. Clinical and subclinical data collection and analysis were conducted from September 2020 to August 2021.  3G2). This data indicated that Navax helped to increase the efficiency of symptomatic treatment by 10-50% with chest depression being the most improved. Taken all together, we concluded that spraying Bacillus spores alleviates normal RSV-infection symptoms 1-day early and 10-50% more effectively.
Lowering RSV load and co-infection bacterial concentration by nasal-spraying Bacillus spores. Next, to determine how Bacillus spores act to relieve symptoms, we conducted real-time PCR TaqMan probes to measure RSV loads in nasopharyngeal samples. The changes in RSV loads and co-infection bacteria concentrations were semi-quantified using 2 △Ct values where △C t is the difference between threshold cycle (C t ) at day 3 and C t at day 0. Day 3 was chosen since it was noted that the benefit of LiveSpo Navax treatment in improving symptoms in RSV-infected patients was highest at this time point. As shown in Fig. 4A-B, two representative amplification curves taken from two representative nasopharyngeal samples of each group demonstrated that RSV load in both groups was much lower (or not detectable) at day 3 compared to before treatment. Amplification curves with similar results were seen in other samples of both groups (Figs. S1-S2). Moreover, the 2 △Ct values showed that RSV viral load in the Navax group was reduced by more than 630-fold,  Fig. S1-2). In fact, amplification thresholds for B. subtilis and B. clausii in the Navax group were at C t values (median, lower-upper confidence limit) of 29.6 (28.9-30.1) and 33.6 (33.1-33.9), respectively, and non-detectable in the Control group (Fig. 4F). The data confirmed the correct administration of LiveSpo Navax and the control product in our trial. Given that bacterial co-infection in nasal tract is common in patients with RSV infection and is the cause of complication, we developed a multiplex real-time PCR assay to evaluate bacterial co-infection. As shown in Table 3, ten individuals in each group who were tested positive with S. pneumoniae and H. influenzae with C t values of < 30 at day 0 were further tested at day 3. Concentrations below this threshold cycle do not pose a high risk of bacterial co-infection in the respiratory tracts and do not necessitate the use of antibiotics in routine hospital care. Surprisingly, we observed that 9 out of 10 patients in Navax group infected with S. pneumoniae (C t range: [25][26][27][28][29] or/and H. influenzae (C t range: 26-29) at day 0 became negative (C t > 40) at day 3, meaning that the reducing folds (2 △Ct ) in these cases were more than 10 3 -10 4 . The remained one case had unchanged H. influenzae concentration. By contrast, there were only 3 out of 10 patients in the Control group infected with either S. pneumoniae or H. influenzae showed a negative result at day 3. Meanwhile, 3 cases had 3.5-42-fold decrease; 1 case was unchanged; and 2 cases showed 4.0-6.0-fold increase. Comparing the number of negative vs. positive results obtained after 3 days of treatment between Navax and Control, the difference was statistically significant (OR Navax/Control = 21; p = 0.0157).      . Thus, IL-6, IL-8, and TNF-α were lowered by 3.1-fold (p = 0.2219), 1.8-fold (p = 0. 0199), and 3.6-fold (p = 0.0012), respectively, when comparing the levels of those cytokines at day 3 between the Navax and the Control groups (Fig. 5D). The data strongly indicated that nasal-spraying Bacillus spores effectively suppress cytokine overreacted production in immune response to RSV infection.

Associations between improvements in clinical symptoms, reduction in RSV load, and reductions in cytokines levels.
To uncover the associations between days of treatment for the typical symptoms of ARTIs, reduction in RSV load, and reductions in cytokines levels, we performed Spearman's rank correlation coefficients (rho) analysis (Fig. S3). Regarding the symptoms, there was a monotonic correlation between the relief from chest depression and difficulty breathing (rho = + 1.00; p = 0.001), but a weak correlation between dry rales and runny nose (rho = + 0.34; p = 0.019) in the Navax group. In the Control group, there was a moderate association between dry rales relief and runny nose relief (rho = + 0.44; p = 0.007), but difficulty breathing relief was more strongly connected with dry rales relief (rho = + 0.79; p = 0.010) than with chest depression relief (rho = + 0.55; p = 0.155). Relief from symptoms in patients of Navax group was proportional with reductions in RSV load (rho = + 0.30 in breath difficulty and chest depression), IL-6 (rho = + 0.32 in moist rales; rho = + 0.22 in runny nose), IL-8 levels (rho = + 0.23 in dry rales; rho = + 0.17 in runny nose), or in TNF-α levels (rho = + 0.25 in dry rales; rho = + 0.24 in fast pulse). However, these correlations were not statistically significant, possibly due to the size of the experimental groups. On the other hand, patients in the Navax group exhibited a stronger association between RSV load and IL-8 level reduction (rho = + 0.49; p = 0.003) than those in the Control group (rho = + 0.19; p = 0.36). Furthermore, in the Navax group, the IL-8 reduction was found to be linked with the IL-6 reduction (rho = + 0.48; p = 0.02), whereas this proportional connection (rho = + 0.38; p = 0.121) was lower and not statistically significant in the Control group. Overall, there were notable correlations between improve-   www.nature.com/scientificreports/ ments in several major clinical symptoms, reduction in RSV load, and cytokines levels, suggesting a potential mechanism of action for nasal-spraying Bacillus spores in lowering viral load and modulating immune response.

Discussion
Safe Bacillus species like B. subtilis, B. clausii, and B. coagulans can retain their efficacy for a long time because they can form heat-stable spores under harsh conditions such as starvation 24,[34][35][36][37] . Bacillus spore probiotics can be made as a liquid suspension with extremely pure and concentrated spores for direct spraying into the nasal tract. In this clinical trial with a moderate number of 86 patients (40 in Control vs. 46 in Navax), the data showed that LiveSpo Navax was safe for all participants. Correct administration of LiveSpo Navax provided 1-day earlier and 10-50% more effective symptomatic-relief including runny nose (p = 0.0014), chest depression (p = 0.0042), difficulty breathing (p = 0.0042), dry rales (p = 0.0149), moist rales (p = 0.012), fast pulse (p < 0.0001), and possibly fast breath (p = 0.3515). All patients in the Navax group were fully recovered during 6-day follow up, while several patients in the Control group still had runny nose (8/40 cases, 26.67%) and dry rales (3/40 cases, 10%) at day 6. These findings formed a foundation for future clinical trials with a larger sample size to further evaluate the effects of nasal-spraying Bacillus spores in symptomatic treatment for children with RSV and other viral infection. The ability of LiveSpo Navax in shortening one day treatment while improving 10-50% efficacy is beneficial not only to the children's health but also alleviate parents' anxiety and caring time while lowering the cost of therapy and hospitalization. Our data indicated a faster and superior efficacy in supportive treatment of ARTIs using nasal-spray Bacillus spore probiotics when compared to previous results using oral administrative ones. In the clinical study in 80 healthy children aged 6-8 years to assess the effects of oral Bacillus coagulans GBI-30, 6086 probiotics against upper respiratory tract infections (URTIs) after up to three months usage, there is a lower incidence of URTI symptoms such as nasal congestion, bloody nasal mucus, itchy nose, and hoarseness, as well as a shorter duration of URTI-related symptoms such as hoarseness, headache, red eyes, and fatigue 31 . In another study, Slivnik et al. 2020 have conducted a clinical trial in children aged 2-6 years, which looked at the effects of B. subtilis DE111 probiotics on gastrointestinal tract infections and URTIs. Even after long observation period (up to 102 days), despite the gastrointestinal efficacy, there was no change in the incidence of respiratory infection (41.3% probiotic vs. 36.2% placebo, p = 0.60) 38 .
Results from real time PCR assays revealed that nasal-spraying Bacillus spores reduced RSV viral load with a remarkable efficacy of over 53-fold more effective than standard of care treatment. This data also provided a mechanistic insight underlying the benefit of Bacillus spore probiotics in relieving typical symptoms due to RSV infection. These findings are in agreement with prior studies that looked at the impact of nasal-dropping Bacillus spores on viral infection in mice. Song et al. (2012) have found that B. subtilis PY79 spores can non-specifically adsorb H5N1 influenza virus at an estimated 8 virions per one spore, and that the complex virus-spore suspension can function as a nasal mucosal vaccine with 80% protection of mice against H5N1 viral challenge infection at Co-infection with these pathogens worsens the respiratory failure, lengthens the treatment period, and raises the cost of antibiotic treatment 41 . Antibiotic resistance represents a rising problem in patients infected with S. pneumoniae and H. influenzae 5,42 . In our study, bacterial co-infection cases accounted for 50-60% of RSV-infected patients, which is similar to recent reports 41,[43][44][45] . The primary data from semiquantitative real-time PCR of co-infection bacteria in both groups (n = 10/group) showed that 9/10 probiotictreated patients in the Navax group became negative at day 3 of treatment, while unclear reduction in bacterial concentration was observed in 7/10 patients in the Control group. The data suggests that Bacillus spores sprayed directly into the nose can block co-infection bacteria's growth far more efficiently than physiological saline. To the best of our knowledge, this is the first study to demonstrate the efficacy of probiotics in reduction of bacterial concentrations in nasal tract.
We hypothesized that sprayed Bacillus spores can compete with RSV in interacting with nasal epithelium, resulting in a less systemic inflammatory response syndrome. Mechanistically, our data suggested that Bacillus spore spraying could diminish "cytokine storm" in the nasal cavity of RSV-infected patients by regulating the cell mediated immune system. Given the limited material of the nasopharyngeal samples, the most relevant cytokines IL-6, IL-8 and TNF-α in respiratory airway, which were reported as potential biomarkers of severity and prognosis for RSV infection, were chosen in our study 7,8,10 . We found that the IL-8 level detected in nasopharyngeal samples at day 0 was the highest, followed by IL-6 level and the lowest was TNF-α level. The correlation of these three cytokines levels in nasal fluid of RSV-infected children is similar to a recent study by Garcia et al. 46 . After 3 days of treatment, levels of IL-6, IL-8, and TNF-α in nasopharyngeal samples of patients in Navax group were about 2.5-fold more effective than those in the Control group. The IL-6 level (12.0 pg/mL) measured in nasopharyngeal samples of Navax group at day 3 was close to those (14.9-16.6 pg/mL) measured in intravenous blood of healthy children reported recently 47,48 . T-helper responses are critical in stimulating cytotoxic T lymphocyte (CTL) proliferation, as well as the production of cytokines 39 . IL-6, along with IL-1, IL-2, IL-8 and TNF-α, play important role in modulating the host immune response during viral infection 49 . In infants with acute RSV infection, inflammation in the upper and lower airways is dominated by an intense neutrophilia, with overreacted release of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α produced in response to RSV 11 . A number of in-vitro and animal studies have shown that certain probiotic strains can protect against virus infections by triggering or modulating cytokine responses in respiratory epithelial cells or immune cells 22,26 . Therefore, our data support the notion that while cell mediated immunity does not specially prevent viral infection, it can aid the recovery of infection-related-consequences.
Notably, there is statistically significant correlation between reduction of RSV load and decreased levels of IL-8, IL-6 and TNF-α in Navax group. One possible mode of action for Bacillus spores is the ability of ligands on their outer coat layer to adsorb RSV virions while simultaneously competing with the viruses for interaction with nasal epithelium. This results in reducing activation of the innate immune system in the nasal-associated lymphoid tissue, hence modulating the hyper-induction of pro-inflammatory cytokines. Due to ethical concerns, limited nasopharyngeal samples were collected only once for each time point, which is sufficient only for measuring the loads of RSV, co-infection bacteria, Bacillus spores by real-time RT/PCR, and concentrations of the three cytokines by conventional ELISA method. Therefore, other pro-inflammatory cytokines with potential prognostic value in RSV infection, such as IL-3 and IL-12 (Th1-type cytokines), IL-13 and IL-33 (Th2-type cytokines) 10 , were not included in the evaluation. The procedure thus limited the measurement of other immuno-stimulatory parameters reflecting activation of macrophages, maturation of dendritic cells (DC), recruitment of natural killer (NK) cells, and other cytokines relating to Th1/Th2 balance. In the future, by applying Luminex XMAP technology for measuring multiple cytokines in a single test, changes in key cytokines relating to multiple pathways that regulate immune cell proliferation and differentiation following RSV infection will provide us with more insights on mechanisms of action for nasal-spraying Bacillus spores against RSV infection.
Since the mechanism of interaction between Bacillus spores with nasal mucosal immune system, virus and bacteria relies on non-specific interaction, our findings suggest that nasal-spraying Bacillus spores could be useful not only for supportive treatment of RSV infection, but also for other rapidly emerging RTIs virus like influenza virus or coronavirus. People who have been vaccinated via the intramuscular route are at risk of ARTIs due to either low in-situ antibody levels in the nasal tract or appearance of new immunity-escape mutations. Nasal-spraying Bacillus spores can provide an additional barrier of protection to help increase the effectiveness of preventing viral infection in general. This novel probiotic treatment has a distinct advantage of being simple to use, low-cost, and effective against viral infection, making it ideal for developing countries with limited medical resources. Future clinical trials of nasal-spraying Bacillus spores with these viruses, if successful, could pave the way for the prevention and supportive treatment of viral infectious respiratory diseases in children for which there is current no vaccination, vaccination with low protection level, or specific drug therapy.

Conclusion
This study is the first randomized and blind clinical trial in RSV-infected children to demonstrate safety and beneficial effects of a nasal-spraying liquid suspension of Bacillus spore probiotics in relieving clinical respiratory symptoms of RSV infection, thereby shortening the treatment period 1 day and improving symptomatic treatment efficacy by 10-50%. The sprayed Bacillus spores significantly inhibited the multiplications of RSV (630 folds) and co-infection bacteria S. pneumoniae and H. influenzae (10 3 -10 4 folds), thereby reducing the overreacted immune response of epithelium cells to lower IL-6, IL-8, TNF-α pro-inflammatory cytokine levels (2.7-12.7 folds) in nasal tract.  (Table S1), (ii) antibiotics susceptibility (Table S2), (iii) 16S rRNA sequencing analysis (Figs. S4-S6), and (iv) sequence analysis of antibiotic resistance and toxic genes in B. subtilis ANA4 and B. clausii ANA39 whole genomes (Table S3-S5) were shown in Supplemental data, indicating that the two strains are safe in-vitro tests, have high spore formulation efficiency (> 90%), heat-stability (> 65 °C), and survive in both aerobic/anerobic conditions. These advantage properties are suitable for low-cost production of spore suspension in physiological saline NaCl 0.9%, stable quality during storage at room temperature, and being alive to exhibit their effects in nasal tract. Acute and sub-acute toxicity studies in mice and rabbit for LiveSpo Navax conducted in Vietnam National Drug Quality Control Institute have indicated that the product is non-toxic. LiveSpo Navax (intervention product) and 0.9% NaCl physiological saline (control product) were indistinguishable regarding taste and smell. The color and turbidity of LiveSpo Navax suspension is unrecognizable to nurses and patient's parents due to opaque plastic spray bottle. Nasal-spraying 0.9% NaCl physiological saline (control product) was prepared by extracting 5 mL from 0.9% NaCl intravenous infusion 500 mL PP bottle (B. Braun, Germany), and then transferring it into the opaque plastic spray bottle that is used for LiveSpo Navax. The control and intervention products were coded number 1 and 2, respectively, and this coding information was blind to most investigators and nurses (except the PI, the data analyst, and the chief nurse who is responsible for product preparation and coding) and to all parents of children.

Study design and patient collection.
This was a blind, randomized, controlled trial, with the Control group used 0.9% NaCl physiological saline and an experimental group (named the "Navax" group) used the probiotics LiveSpo Navax. The study lasted for 12 months, from August 2020 to August 2021, and included pediatric patients (both male and female sex) having acute respiratory infection symptoms and were diagnosed with positive RSV infection at International Center, Vietnam National Children's Hospital. Sample size was calculated based on a hypothesis is that LiveSpo Navax alleviates RSV-infection symptoms about 25% more effectively, as indicated by 90% of patients in the Navax group are symptom free at day 3-6 of intervention, compared to 65% of patients in the Control group. Estimated required sample size for each group was 43 at the end of intervention (α = 0.05; power level = 0.8). Thus, sample size at the beginning of intervention was decided to be 50 per each group to reduce the risk of patient's drop out during follow-up treatment. Totally 100 eligible RSV-infected participants were randomly assigned by lottery to the Control and the Navax groups. In detail, the chief nurse randomly took paper sheets coding either number 1 or 2 from a box and assigned the coding number to each participant right after his/her parents signed the informed consent form. The numbers 1 and 2 were assigned to the Control and the Navax groups, respectively, and this information was also blind to all parents of children, nurses, and investigators, except the PI and the data analyst. The flow charge of study was shown in Fig. 1 required to provide the following information of their children: full name, sex, age, obstetric history, vaccination history, antibiotic use history, underlying diseases. The patient was given a coded spray in the form of a blind sample to ensure the objectivity of the study. Nurses were instructed and trained to use sprayers with dosages of 3 presses (each press is about 50 µl 0.9% NaCl physiological saline (with/without 2.5 × 10 8 Bacillus spores)) per each nasal cavity/ time × 3 times/ day directly into the nasal cavity continuously for maximal follow-up 6 days of treatment. The nasal spray products were applied in parallel with routine treatment drugs at hospital such as antipyretics, anti-inflammatory corticosteroids, and aerosol therapy as described in Table 2. Antibiotic susceptibility tests and adequate antibiotic medication were assigned for all patients who tested positive for bacterial co-infection. During treatment, patients were monitored daily for typical clinical symptoms of RSV-induced respiratory tract infections, including runny nose, chest depression, difficulty breathing, dry rales, moist rales, body temperature (°C), pulse oxymetry (SpO 2 ) (%), pulse (beats/min), and breath (beats/min) until discharged. The patients' health conditions were observed by doctors and nurses, and their pieces of information were filled in medical records.
Routine diagnostics at hospital. Screening of RSV-infected cases from nasopharyngeal samples at day 0 was firstly conducted by using "BD Veritor System for rapid detection of RSV" kit (Becton Dickinson, NJ, US). Detection of respiratory infection bacteria from nasopharyngeal samples was conducted by microbial culture assays on specific mediums. CRP concentrations and white blood cell counts were measured to access the level of infection. Chest radiography was appointed for visualization of lung hyperinflation, atelectasis... Due to ethical concerns, all these tests were conducted only at day 0 following routine procedures at Vietnam National Children's Hospital. For RSV detection at day 0 and day 3, the following primers (5'→ 3') and probes (5'→ 3') were used as described previously 50  ELISA assays for cytokine levels. Pro-inflammatory cytokines levels (pg/mL) including interleukin (IL-6, IL-8) and TNF-α in nasopharyngeal samples at day 0 and 3 were quantified using an enzyme-linked immunosorbent assay kit (ELISA) according to the manufacturer's instructions. IL-6 and TNF-α were quantified from 100 µL samples by the Human IL-6 DuoSet ELISA and the Human TNF-α ELISA kit, respectively (R&D Systems, MN, US). IL-8 was quantified by 50 µL samples by IL-8 Human ELISA kit (Invitrogen/Thermo Fisher Scientific, MA, US). Samples were measured by the SpectraMax Plus384 Microplate Reader system. The results were analyzed by using SoftMax Pro 6.3 Software (Molecular Devices, CA, US). The number of measurable sam- www.nature.com/scientificreports/ ples for IL-6, IL-8 and TNF-α levels was lower than number of collected patients, both in the Control and the Navax groups was due to (i) lack of sample volume left over from failed and repeated performance in ELISA and real-time PCR assays that occurred at random in each sample; (ii) measured values at day 0 were found below the detection range.

Data collection and statistical analysis.
Individual medical records were collected, and the patient's information was then gathered and systematized in a data set. The efficacy of LiveSpo Navax was evaluated and compared to 0.9% NaCl physiological saline based on following clinical and sub-clinical criteria obtained in the Navax and the Control groups: (i) the symptomatic-relieving day; (ii) the reduction levels (2 △Ct ) of RSV load and co-infection bacteria concentrations. △C t for target genes was calculated as C t (threshold cycle) at day 3-C t at day 0 while C t of internal control were adjusted to be equal among all samples. Negative result or non-detectable signal was assigned to C t of 40; (iii) the reduction levels of IL-6, IL-8, and TNF-α cytokines. Tabular analysis was performed on dichotomous variables using the Fisher's exact test when the expected value of any cell was below five ( Table 3). Distributions of the data were verified using both the normality test and QQ plot. Variables were compared using either the Wilcoxon test or Mann-Whitney test due to the unnormal distribution of data. The Wilcoxon two-tail test was used on paired samples (Fig. 5A-C for IL6, IL8, and TNF-concentrations at day 3 versus day 0 in each group). The Mann-Whitney test was used on independent samples (Figs. 3, 4E for RSV-load reducing folds at day 3 between the two groups; Fig. 5A-C for IL6, IL8, and TNF-concentrations at either day 0 or day 3 between the two groups; Fig. 5D for IL6, IL8, and TNF-reducing folds at day 3 between the two groups). The correlations among the variables were assessed by Spearman's correlation analysis. Statistical and graphical analysis were performed on GraphPad Prism v8.4.3 software (GraphPad Software, CA, USA). The significance level of all analyzes was set at the p < 0.05.