Development of a virus-based affinity ultrafiltration method for screening virus-surface-protein-targeted compounds from complex matrixes: Herbal medicines as a case study.

Herbal medicines (HMs) are one of the main sources for the development of lead antiviral compounds. However, due to the complex composition of HMs, the screening of active compounds within these is inefficient and requires a significant time investment. We report a novel and efficient virus-based screening method for antiviral active compounds in HMs. This method involves the centrifugal ultrafiltration of viruses, known as the virus-based affinity ultrafiltration method (VAUM). This method is suitable to identify virus specific active compounds from complex matrices such as HMs. The effectiveness of the VAUM was evaluated using influenza A virus (IAV) H1N1. Using this method, four compounds that bind to the surface protein of H1N1 were identified from dried fruits of Terminalia chebula (TC). Through competitive inhibition assays, the influenza surface protein, neuraminidase (NA), was identified as the target protein of these four TC-derived compounds. Three compounds were identified by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS), and their anti-H1N1 activities were verified by examining the cytopathic effect (CPE) and by performing a virus yield reduction assay. Further mechanistic studies demonstrated that these three compounds directly bind to NA and inhibit its activity. In summary, we describe here a VAUM that we designed, one that can be used to accurately screen antiviral active compounds in HMs and also help improve the efficiency of screening antiviral drugs found in natural products.

Qingjin Huatan decoction protects mice against influenza a virus pneumonia via the chemokine signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingjin Huatan Decoction (QJHTT) consists of 11 herbal medicines: Scutellaria baicalensis Georgi, Gardenia jasminoides J.Ellis, Platycodon grandiflorus (Jacq.) A.DC., Ophiopogon japonicus (Thunb.) Ker Gawl., Morus alba L., Fritillaria thunbergii Miq., Anemarrhena asphodeloides Bunge, Trichosanthes kirilowii Maxim., Citrus reticulata Blanco, Poria cocos (Schw.) Wolf, and Glycyrrhiza uralensis Fisch. As a traditional compound Chinese medicinal formula, QJHTT has been used for more than 400 years in China. Historically, it was used to treat respiratory diseases and had shown beneficial clinical results for diseases related to lung inflammation. AIM OF THE STUDY: To investigate the therapeutic effect of QJHTT on influenza A virus (IAV) pneumonia in mice and explore its possible mechanism of action. MATERIALS AND METHODS: The components in QJHTT were analyzed by UPLC-Q-TOF-MS and some antiviral active components reported in the literature were determined and quantified by HPLC. The protective effects of QJHTT were investigated using lethal and sublethal doses (2 LD50 or 0.8 LD50 viral suspension, separately) of H1N1-infected mice. Mortality and lung lesions in H1N1-infected mice were used to evaluate the efficacy of QJHTT. The potential mechanism of QJHTT in the treatment of viral pneumonia was determined at the gene level by RNA sequencing and validated by qRT-PCR. Following this, the changes in protein levels of JAK2/STAT3 were analyzed since it is a key downstream target of the chemokine signaling pathways. Preliminary elucidation of the mechanism of QJHTT to protect mice against IAV pneumonia through this pathway was conducted. RESULTS: In this study, 12 antiviral active constituents including baicalin, geniposide, and mangiferin were identified from QJHTT. In vivo treatment of QJHTT reduced the virus titers of lung tissue significantly and improved the survival rate, lung index, and pulmonary histopathological changes; additionally, a reduction in the serum levels of TNF-α, IL-1ß, IL-6, and IFN-γ inflammatory factors in H1N1-infected mice was observed. RNA-seq analysis and qRT-PCR showed that QJHTT primarily reversed the activities CCL2, CCL7, CCR1, and other chemokines and their reception-related genes, suggesting that QJHTT may produce disease-resistant pneumonia by inhibiting the downstream JAK2/STAT3 pathway. Western blot analysis confirmed that QJHTT effectively reduced the protein levels of JAK2, STAT3, and related phosphorylated products in the lung tissue of H1N1-infected mice. CONCLUSIONS: Our results indicated that QJHTT alleviated IAV pneumonia in mice by regulating related chemokines and their receptor-related genes in lung tissue, thereby inhibiting JAK2/STAT3 pathway. This could pave way for the design of novel therapeutic strategies to treat viral pneumonia.

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.