Respiratory syncytial virus (RSV) infects the central nervous system, resulting in neurological symptoms. However, the precise underlying pathogenic mechanisms have not been elucidated. In the present study, the infectivity of RSV on N2a neuronal cells and the possible roles of Toll-like receptor 4 (TLR4) and nucleolin (C23) during RSV infection were investigated. We compared two experimental groups (infected and non-infected) and monitored the RSV viral titers in the culture supernatant by a viral plaque assay. We also inspected the morphology of the nucleus in infected N2a cells. We measured the level of RSV F protein and studied its co-localization with TLR4 and nucleolin using immunofluorescence assays and laser confocal microscopy. The potential interaction of RSV F protein with TLR4 and nucleolin was examined by coimmunoprecipitation. The expression changes of TLR4, nucleolin, TLR3 and TLR7 proteins in N2a cells and IL-6 and TNF-α in the culture supernatant were investigated by Western Blot analysis and ELISA assay. Changes in neuronal cell apoptosis status was examined by flow cytometry. The results demonstrated prolific RSV infection of N2a cells, which triggered a decrease of NeuN protein expression, coinciding with an increase of nuclear lesions, F protein expression, RSV viral titers, and late apoptotic levels of N2a cells. RSV infection induced co-localization of RSV F protein with TLR4 and nucleolin, which could potentially lead to a direct interaction. Furthermore, it was found that TLR4 and nucleolin levels increased early after infection and decreased subsequently, whereas TLR3 and TLR7 expression increased throughout RSV infection. The RSV Long strain can prolifically infect N2a neuronal cells, modulating the expression of TLR4 and nucleolin, as well as TLR3, TLR7 and their downstream inflammatory factors, and inducing the co-localization of the RSV F protein with TLR4 and nucleolin.

Toll‑like receptor 3 (TLR3) can react with double stranded RNA and is involved in the inflammatory response to respiratory syncytial virus (RSV) infection. Also, oxidative stress has been reported to be involved in RSV infection. However, the correlation between oxidative stress and TLR3 activation during RSV infection is unclear. Therefore, the present study investigated the association between TLR3 expression and oxidative stress modulation during RSV infection in A549 cells. For comparison, seven treatment groups were established, including RSV‑treated cells, N‑acetyl‑L‑cysteine (NAC)+RSV‑treated cells, oxidant hydrogen peroxide (H2O2)+RSV‑treated cells, normal cell control, inactivated RSV control, NAC control and H2O2 control. The mRNA expression changes of TLR3, interferon regulatory factor‑3 (IRF3), nuclear factor‑κB (NF‑κB) and superoxide dismutase 1 (SOD1) were measured using semi‑quantitative reverse transcription‑polymerase chain reaction, and the protein changes of TLR3 and phospho‑NF‑κB p65 were determined using western blot in A549 cells from the different treatment groups. The present study also evaluated the differences in hydroxyl free radical (·OH), nitric oxide (NO) and total SOD activity in the different treatment groups. The results demonstrated that RSV infection of A549 cells increased the levels of ·OH and NO, while decreasing the activity of total SOD. Pretreatment of A549 cells with H2O2 prior to RSV infection upregulated the mRNA and protein expression of TLR3 and NF‑κB, and downregulated the mRNA expression of IRF3 and SOD1, as well as the total SOD activity. When the infected cells were pretreated with NAC, the mRNA and protein expression of these genes were reversed. These variations in the TLR3‑mediated signaling pathway molecules suggested that oxidative stress may be a key regulator for TLR3 activation during RSV infection. RSV‑induced oxidative stress may potentially activate TLR3 and enhance TLR3‑mediated inflammation. These results may provide better understanding of the RSV‑induced inflammatory and immune pathways, and may also contribute to the drug development and prevention of human RSV diseases.