Xuanfei Formula inhibited RSV infection by normalizing the SREBP2-mediated cholesterol synthesis process.

Background and aims: Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children and the elderly, often progressing to pneumonia and severe sequelae. However, there are currently no feasible and cost-effective interventions with proven efficacy for children, making medications with anti-RSV activity urgently needed. Traditional Chinese medicine has shown promising therapeutic efficacy in alleviating viral infection symptoms. Therefore, we aimed to develop effective strategies for RSV treatment based on traditional Chinese medicine. Methods and results: The infection status was assessed in BALB/c mice with or without Xuanfei Formula (XFF) treatment over a one-week period using H&E staining, cytokine assays and RSV titer testing after RSV challenge. Remarkably, on the first day of XFF intervention, both the pro-inflammation cytokine levels in the serum and RSV-N gene copies in the lung of mice were plummeted, compared to the RSV-infected group. This implied that XFF might possess the immune-independent anti-RSV capability. To elucidate the underlying mechanism, we employed transcriptome analysis followed by k-means analysis. The reversal effects of XFF against RSV primarily focused on the processes of innate and adaptive immunity. Additionally, we found that XFF administration corrected the disordered fatty acid and cholesterol metabolism processes during RSV infection. Lipidomics profiling indicated consistent cholesterol abundance with transcriptional changes but not fatty acids. Cholesterol synthesis-related genes mRNA levels and cholesterol synthesis intermediates detection supported XFF's repression upon cholesterol biosynthesis. Aberrantly increased cholesterol production has been reported as necessary for RSV infection. To mimic that, we observed lovastatin treatment inhibited RSV replication and pro-inflammation cytokine expression in vitro. Transcription factor prediction of differentially expressed genes (DEGs) involved in cholesterol synthesis implicated SREBP2. Through network pharmacology, stigmasterol and ß-sitosterol were identified as the effective active ingredients within the XFF, with the help of further molecular docking and mass spectrum detection. In vitro experiments demonstrated ß-sitosterol and stigmasterol reinforced the bonding between SREBP cleavage-activation protein (SCAP) and insulin-induced gene proteins (INSIGs) to inhibit SREBP2 cleavage maturation and consequent RSV infection. Conclusion: Xuanfei Formula (XFF) exhibits excellent anti-RSV efficacy by inhibiting SREBP2-mediated cholesterol synthesis to reduce RSV replication and ameliorate inflammation in the lung of infected mice.

Virus-like particles containing a prefusion-stabilized F protein induce a balanced immune response and confer protection against respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children worldwide. Currently, no licensed RSV vaccines are available. In this study, we explored stable prefusion conformation virus-like particles (Pre-F VLPs) as RSV vaccine candidates. RSV fusion (F) protein mutants were constructed to form stabilized Pre-F or postfusion (Post-F) configurations. VLPs containing Pre-F or Post-F protein were generated using a recombinant baculovirus (rBV)-insect cell expression system. The assembly and immunological properties of Pre-F or Post-F VLPs were investigated. Pre-F and Post-F VLPs contained antigenic sites Ø and I of pre- and postfusion conformations, respectively. Compared with Post-F VLPs, immunization with Pre-F VLPs elicited upregulation of IFN-γ, IL-2 and IL-10 and downregulation of IL-4 and IL-5 cytokine production in mice. A high percentage of CD25+ Foxp3+ cells or a low percentage of IL-17A-producing cells among CD4+ T cells was observed in the lungs of mice vaccinated with Pre-F VLPs. Importantly, immunization with Pre-F VLPs induced a high level of RSV neutralizing antibody and a balanced immune response, which protected mice against RSV infection without evidence of immunopathology. Our results suggested that Pre-F VLPs generated from rBV-insect cells represent promising RSV vaccine candidates.

The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus.

BACKGROUND: Classical swine fever (CSF) virus is the causative agent of an economically important, highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV). BVDV infection in pigs can mimic CSF clinical signs, which cause difficulty in differentiation. Serological test for detection of virus specific antibodies is a valuable tool for diagnosis and surveillance of CSFV and BVDV infections in animals. The aim of this study was to develop the CSFV Erns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV. METHODS: The CSFV Erns and truncated E2 (tE2, residues 690-865) of BVDV were expressed in E. coli and purified by Ni-NTA affinity chromatography, respectively. Employing Erns or tE2 protein as diagnostic antigen, indirect ELISAs were developed to distinguishably test specific antibodies against CSFV and BVDV. The specificity and sensitivity of ELISAs were evaluated using a panel of virus specific sera of pigs, immunized rabbits and immunized mice. A total 150 clinical serum samples from farm pigs were measured by the developed ELISAs and compared with virus neutralizing test (VNT). RESULTS: Indirect ELISA was established based on recombinant CSFV Erns or BVDV tE2 protein, respectively. No serological cross-reaction between antibodies against CSFV and BVDV was observed in sera of immunized rabbits, immunized mice or farm pigs by detections of the Erns and tE2 -based ELISAs. Compared to VNT, the CSFV Erns -based ELISA displayed a high sensitivity (93.3%), specificity (92.0%) and agreement rate (92.7%), and the sensitivity, specificity and agreement rate of BVDV tE2 -based ELISA was 92.3%, 95.2% and 94.7%, respectively. CONCLUSION: The newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.