**Acknowledgements**

This study is part of the project under a research grant (FRGS-59280) funded by the Ministry of Higher Education (KPT). The authors are grateful to the Ministry of Higher Education, Malaysia, for the financial support granted towards the success of the research project.

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*3.5. E*ff*ect of PAE and CTE on the Production of Viral RNA and Protein in HEp2 Cells*

Inhibition of intracellular viral RNA transcription and protein translation by herb extract treatment was evaluated in HEp2 cells. HEp2 cells were infected with RSV-GFP and treated with PAE and CTE (50 μg/mL) at 2 hpi, then cells were harvested at the indicated time points, and viral gene expression at the RNA and protein level was determined by qRT-PCR and immunoblot analysis, respectively. Interestingly, PAE treatment reduced the transcription of RSV-G mRNA level by 39-fold at 36 hpi and 22-fold at 48 hpi. Similarly, CTE treatment reduced the transcription of RSV-G mRNA level by 10-fold at 36 hpi and 11-fold at 48 hpi (Figure 4A). Furthermore, the reduction in viral gene transcription in HEp2 cells treated with herb extracts was associated with reduced RSV-G protein synthesis (Figure 4B,C). Thus, the reduction of viral gene transcription and protein synthesis by PAE and CTE correlated with their antiviral activity in vitro (Figure 2B,C).

**Figure 4.** Reduction of RSV Glycoprotein (RSV-G) gene transcription, protein translation and syncytium formation in-vitro and inhibition of RSV replication *in-vivo* by PAE and CTE. HEp2 cells were seeded in six well cell culture plats and incubate for 12 h. Medium was changed into DMEM containing 1% FBS and RSV-GFP (0.1MOI) was infected for 2 h. Then cells were treated with 50 μg/mL PAE or CTE. (**A**) Cells were harvested at indicated time points, and RSV-G protein mRNA level was measured at 12, 24, 36, 48 hpi by qRT-PCR, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for the normalization. (**B**,**C**) Immunoblot analysis was performed using cell lysates harvested at indicated time points to measure RSV-G protein and β-actin protein expression level time-dependently. The intensity of the RSV-G was quantified. (**D**) Cell and the GFP image were taken at 48 hpi to see the syncytial formation inhibition by PAE and CTE (400× magnification). (**E**,**F**) Five weeks old (16 g/mice) BALB/c mice (*n* = 5) were intranasally infected with RSV-GFP (1 × 106 PFU/mice) in the total volume of 28 μL. PAE or CTE were orally administrated at a dose of 200 μL/mice (0.5 mg/mL) at 6, 12, 18 and 24 hpi. At 3 and 5-day post infection (dpi), lung tissues were collected, and the transcription level of RSV-G protein mRNA was determined by qRT-PCR. The arrow indicates the RSV syncytium formation in HEp2 cells. mRNA expression, band intensity expressed as mean ± SD. Error bars indicate the range of values obtained from three independent experiments. In vivo experiment was performed in duplicate. (\* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 regarded as significant difference).

#### *3.6. PAE and CTE Inhibits RSV Syncytium Formation*

**0 12 24 36 48**

**(hpi)**

Syncytium formation by RSV is a well-known mechanism of cell-to-cell infection that contributes significantly to virus spread in vivo. Therefore, to determine whether PAE and CTE prevent the cell-to-cell spread of the virus after infection, a syncytium formation assay was performed on infected HEp2 cells. Monolayers of HEp2 cells were infected with RSV-GFP and incubated at 37 ◦C for 2 h. Cells were left untreated or treated with PAE or CTE (50 μg/mL) and examined for syncytium formation. Interestingly, in untreated HEp2 cells, large areas of syncytium formation were visible. By contrast, PAE and CTE treated HEp2 cells showed significantly reduced syncytium formation at 48 hpi (Figure 4D).

#### *3.7. Oral Administration of PAE and CTE Enhance Protection against RSV Infection in BALB*/*c Mice*

Next, we designed a mouse model to evaluate the therapeutic effect of PAE and CTE against RSV infection in vivo. BALB/c mice were intranasally infected with RSV-GFP (1 × 106 PFU/mouse) or left uninfected, and PBS, PAE, or CTE were orally administrated at 6, 12, 18, and 24 hpi. The RSV infection titer (1 × 106 PFU/mouse), inoculation time (6, 12, 18, and 24 hpi) and inoculation dose (200 μL (0.5 mg/mL) mice/time were chosen based on preliminary studies. Lungs were collected aseptically at 3 dpi and 5 dpi. RSV-G protein mRNA level in the lungs was determined by qRT-PCR. As shown in Figure 4E, PAE-treated mice showed significantly reduced viral mRNA in the respiratory tract at both 3 dpi and 5 dpi compared to the control (PBS)-treated group. Similarly, the transcription of RSV-G mRNA was reduced in the CTE-treated group compared to the PBS-treated group at 3 dpi and 5 dpi, and was significantly reduced at 5 dpi (Figure 4F). This data demonstrates that both herb extracts have the ability to inhibit viral replication in the mouse respiratory tract and protect against RSV infection in vivo.

#### *3.8. Acteoside Inhibits RSV Replication at Non-Cytotoxic Concentrations in vitro*

It has been previously reported that acteoside is an important phenolic glycoside in PAE [27] and CTE [15,17]. To investigate for the presence of this glycoside in PAE and CTE, we performed reverse-phase high-performance liquid chromatography (HPLC) on extracts from PAE and CTE. Interestingly, we found that both extracts contained acteoside as one of their major active components (Figure 5A). To assess the antiviral effect of acteoside, a monolayer of HEp2 cells was infected with RSV-GFP, and at 2 hpi, cells were treated with 10, 30, or 50 ng/mL acteoside and virus replication were monitored. As reported in Figure 5C,D, acteoside treatment significantly reduced the GFP expression compared to untreated HEp2 cells. Furthermore, virus titers were determined by plaque formation assay. Similar to the results of the GFP expression analysis, acteoside significantly reduced the RSV plaque titer (Figure 5E). Cell death induced by RSV infection was also reduced in acteoside-treated HEp2 cells compared with untreated cells (Figure 5F). Next, the EC50 and CC50 of acteoside were determined in HEp2 cells as 15.64 ± 1.07 ng/mL and 740.34 ± 8.23 ng/mL, respectively (Figure 5G,H). The SI of acteoside against RSV-GFP was 47.33 (Figure 5I). Based on this data, the reduced viral replication caused by actioside (Figure 5C–F) was due to its antiviral properties and not its cytotoxicity. Moreover, the mRNA and protein expression of viral genes in HEp2 cells treated with acteoside was determined, as with the herb extracts. Interestingly, acteoside-treated cells showed significantly reduced RSV-G mRNA compared with untreated cells (1.7-fold reduction at 36 hpi and 4-fold reduction at 48 hpi) (Figure 6A). Similarly, RSV-G protein synthesis was inhibited by acteoside treatment (Figure 6B). Therefore, our results demonstrate that acteoside can inhibit RSV replication in HEp2 cells.