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Newcastle disease virus (NDV) is a contagious agent of Newcastle disease in avian species and seriously affects the poultry industry. The cleavage site of the viral F protein (Fcs) is a key determinant of membrane fusion and viral virulence. In this study, we investigated the precise effect of variable amino acid sequences of the Fcs on fusogenic activity. Based on viral pathogenicity, the Fcs sequences of natural isolates (n = 1572) are classified into eight types of virulent Fcs (VFcs) with the motif \"G/R/K-R-Q/R/K-R/K-R↓F\" and ten types of the avirulent Fcs (AFcs) with the motif \"G/R/E-R/K/Q-Q-G/E-R↓L\". The VFcs is only found in the Class II cluster of viral classification and not in Class I. The AFcs exists in both Class I and II isolates. The VFc and AFc types present an evolutionary relationship with temporal distribution and host species. Using a fusion assay in vitro, VFcs-1 \"RRQKR↓F\" and VFcs-2 \"RRQRR↓F\" show the highest efficiency in triggering membrane fusion. The neutral residue Q at the P3 position of the VFcs plays an enhancing role compared to effect of the basic residues R and K. A single residue K at P3 or P5 is less efficient of the fusogenic activity in the VFcs with all basic residues. Moreover, the cleavage efficiencies of F0 proteins with different types of Fcs motifs do not appear to affect membrane fusion. Our findings offer insight into the effect of amino acid variation of the Fcs on the fusion triggered by NDV.

Newcastle disease virus (NDV), as an avian pathogen, can infect a broad spectrum of cell lines in vitro. However, noncarcinoma cell lines possessing nonsusceptibility to NDV are rare. Here, we isolated primary mouse embryonic fibroblasts (MEFs), which are nonsusceptible to NDV. MEF-derived cells were generated by passaging the cells over fifty times to achieve spontaneous immortalization. Two of the resulting cell lines were named SLM-21 and MEF50. Karyotype analysis revealed that SLM-21 has a near-tetraploid karyotype and that MEF50 shows a near-tetraploid and near-hexaploid chimeric karyotype. NDV exerted a significant cytopathic effect on MEF50, and substantial viral replication was observed. In contrast, NDV did not have a significant effect on SLM-21, indicating that SLM-21 was a nonsusceptible cell line to NDV, while MEF50 was a susceptible cell line. The NDV authentic sialic acid (SA) receptors SA 2,3-Gal and SA 2,6-Gal were expressed in SLM-21. Transcriptomic analysis revealed that the non-susceptibility of SLM-21 may be related to its broadly activated antiviral pathways and fine-tuned regulation of the cell cycle and DNA damage. This study provides a basic cell platform for exploring viral susceptibility and pathogenesis as well as host–virus interactions during NDV infection.

Surgical resection is commonly used for therapeutic management of different solid tumors and is regarded as a primary standard of care procedure, but precise localization of tumor margins is a major intraoperative challenge. Herein, a generalized method by optimizing gold nanoparticles for intraoperative detection and photothermal ablation of tumor margins is introduced. These nanoparticles are detectable by highly sensitive surface‐enhanced Raman scattering imaging. This non‐invasive technique assists in delineating the two surgically challenged tumors in live mice with orthotopic colon or ovarian tumors. Any remaining residual tumors are also ablated by using post‐surgical adjuvant photothermaltherapy (aPTT), which results in microscale heat generation due to interaction of these nanoparticles with near‐infrared laser. Ablation of these post‐operative residual micro‐tumors prolongs the survival of mice significantly and delays tumor recurrence by 15 days. To validate clinical translatability of this method, the pharmacokinetics, biodistribution, Raman contrast, aPTT efficiency, and toxicity of these nanoparticles are also investigated. The nanoparticles have long blood circulation time (≈24 h), high tumor accumulation (4.87 ± 1.73%ID g−1) and no toxicity. This high‐resolution and sensitive intraoperative approach is versatile and can be potentially used for targeted ablation of residual tumor after resection within different organs. The surface‐enhanced Raman scattering nanoparticles provide intraoperative detection and image‐guided cancer surgery. The remaining residual tumors are then ablated by a post‐surgical adjuvant photothermal therapy. Ablation of these residual microtumors, prolongs the survival significantly and reduces tumor recurrence. This high‐resolution and sensitive intraoperative approach is versatile and can be potentially used for targeted ablation of post‐resection residual tumors.

Background Theranostic nanoparticles (NPs) have achieved rapid development owing to their capacity for personalized multimodal diagnostic imaging and antitumor therapy. However, the efficient delivery and bulk accumulation of NPs in tumors are still the decisive factors in improving therapeutic effect. It is urgent to seek other methods to alters tumor microenvironment (like vascular permeability and density) for enhancing the efficiency of nanoparticles delivery and accumulation at the tumor site. Methods Herein, we developed a Raman-tagged hollow gold nanoparticle (termed as HAuNP@DTTC) with surface-enhanced Raman scattering (SERS) property, which could be accumulated efficiently in tumor site with the pre-irradiation of low-dose (3 Gy) X-ray and then exerted highly antitumor effect in breast cancer model. Results The tumor growth inhibition (TGI) of HAuNP@DTTC-induced photothermal therapy (PTT) was increased from 60% for PTT only to 97%, and the lethal distant metastasis of 4T1 breast cancer (such as lung and liver) were effectively inhibited under the X-ray-assisted PTT treatment. Moreover, with the strong absorbance induced by localized surface plasmon resonance in near-infrared (NIR) region, the signals of Raman/photoacoustic (PA) imaging in tumor was also significantly enhanced after the administration of HAuNP@DTTC, indicating it could be used as the Raman/PA imaging and photothermal agent simultaneously under 808 nm laser irradiation. Conclusions Our studied of the as-prepared HAuNP@DTTC integrated the Raman/PA imaging and PTT functions into the single platform, and showed the good prospects for clinical applications especially with the low-dose X-ray irradiation as an adjuvant, which will be a productive strategy for enhancing drug delivery and accumulation in tumor theranostics. Graphic Abstract

The transcription and replication of the Newcastle disease virus (NDV) strictly rely on the viral ribonucleoprotein (RNP) complex, which is composed of viral NP, P, L and RNA. However, it is not known whether other viral non-RNP proteins participate in this process for viral self-regulation. In this study, we used a minigenome (MG) system to identify the regulatory role of the viral non-RNP proteins V, M, W, F and HN. Among them, V significantly reduced MG-encoded reporter activity compared with the other proteins and inhibited the synthesis of viral mRNA and cRNA. Further, V interacted with NP. A mutation in residue W195 of V diminished V–NP interaction and inhibited inclusion body (IB) formation in NP-P-L-cotransfected cells. Furthermore, a reverse-genetics system for the highly virulent strain F48E9 was established. The mutant rF48E9-VW195R increased viral replication and apparently enhanced IB formation. In vivo experiments demonstrated that rF48E9-VW195R decreased virulence and retarded time of death. Overall, the results indicate that the V–NP interaction of the W195 mutant V decreased, which regulated viral RNA synthesis, IB formation, viral replication and pathogenicity. This study provides insight into the self-regulation of non-RNP proteins in paramyxoviruses.

Background The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a major antigen that can induce protective antibodies in poultry. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the linear epitopes of HN, especially neutralizing epitopes, will be useful for revealing its antigenic characterization. Methods In this study, we analyzed B-cell immunodominant epitopes (IDEs) of the HN protein from the vaccine strain LaSota using pepscan technology with LaSota-specific chicken hyperimmune antisera. We constructed IDEs-RFP plasmids and prepared anti-IDEs peptide mouse sera to identify IDEs through immunological tests. At last, the different diluted anti-IDE antisera were used in BHK-21 cells to perform the neutralization test. Results Five IDEs of the HN were screened and further verified by indirect immunofluorescence assays, dot blots and Western blots with NDV- and IDEs-specific antisera. All five IDEs showed good immunogenicity. IDE5 (328–342 aa) could recognize only class II NDV but did not react with the class I strain. Most of the IDEs are highly conserved among the different strains. A neutralization test in vitro showed that the peptide-specific mouse antisera of IDE4 (242–256 aa) and HN341-355, a reported neutralizing linear epitope, could partially neutralize avirulent LaSota as well as virulent strains at similar levels, suggesting that IDE4 might be a potential neutralizing linear epitope. Conclusion The HN protein is a major protective antigen of NDV that can induce neutralizing antibodies in animals. We identified five IDEs of the HN using a pepscan approach with NDV-specific chicken hyperimmune antisera. The five IDEs could elicit specific antibodies in mice. IDE4 (242–256 aa) was identified as a novel potential neutralizing linear epitope. These results will help elucidate the antigenic epitopes of the HN and facilitate the development of NDV vaccines.

Background Newcastle disease virus (NDV) causes severe diseases in avian species. Its fusion protein cleavage site (Fcs) is a major contributor to virulence and membrane fusion. Previous studies showed that a change from phenylalanine (F) to lysine (L) at position 117 of the virulent strain fusion protein, which has the polybasic amino acid Fcs motif “ 112 RRQKR↓F 117 ”, blocked syncytium formation. However, we observed that F proteins of the virulent strain F48E9 and avirulent strain LaSota substituted with an identical cleavage motif, “ 112 RRQRR↓L 117 ”, induced extensive and slight syncytium formation, respectively. Accordingly, we hypothesized that the difference in syncytium formation is caused by other regions of the fusion protein. Results The exchanged regions between the fusion proteins of two strains, F48E9 and LaSota, showed that the region from amino acid 118–499 plays an important role in modulation of fusogenic activity in transfected cells. Further dissection of this region indicated that replacement of two amino acids (N479D, R486S) in heptad repeat 2 (HR2) of the avirulent fusion protein by the virulent counterpart resulted in fusion promotion. Moreover, the role of these two amino acids in fusion is dependent on the unique Fcs sequence “RRQRR↓L”. Conclusions Our results demonstrated that two amino acids (D479, S486) of the virulent strain F protein with this unique Fcs were critical for promoting fusogenic activity, and residue F or L at position 117 did not affect syncytium formation. These findings provide novel insights into fusogenic triggering by the fusion protein and may be useful for designing antiviral peptides.

Despite sufficient studies performed in non-primate animal models, there exists scanty information obtained from pilot trials in non-human primate animal models, severely hindering nanomaterials moving from basic research into clinical practice. We herein present a pioneering demonstration of nanomaterials based optical imaging-guided surgical operation by using macaques as a typical kind of non-human primate-animal models. Typically, taking advantages of strong and stable fluorescence of the small-sized (diameter: ~ 5 nm) silicon-based nanoparticles (SiNPs), lymphatic drainage patterns can be vividly visualized in a real-time manner, and lymph nodes (LN) are able to be sensitively detected and precisely excised from small animal models (e.g., rats and rabbits) to non-human primate animal models (e.g., cynomolgus macaque ( Macaca fascicularis ) and rhesus macaque ( Macaca mulatta )). Compared to clinically used invisible near-infrared (NIR) lymphatic tracers (i.e., indocyanine green (ICG); etc.), we fully indicate that the SiNPs feature unique advantages for naked-eye visible fluorescence-guided surgical operation in long-term manners. Thorough toxicological analysis in macaque models further provides confirming evidence of favorable biocompatibility of the SiNPs probes. We expect that our findings would facilitate the translation of nanomaterials from the laboratory to the clinic, especially in the field of cancer treatment.

Rabies virus (RABV) causes an acute progressive viral encephalitis. Although currently licensed vaccines have an excellent safety and efficacy record, the development of a safer and more cost-effective vaccine is still being sought. An E1-deleted, replication-defective human adenovirus type 5 (HAd5) vector expressing RABV glycoprotein (HAd5-G) is thought to be a promising candidate vaccine for immune prophylaxis against rabies. Salmonella enterica serovar Typhimurium ( S. Typhimurium) flagellin is a well-known immune adjuvant. In this work, we have researched the adjuvant effect of flagellins (FljB and FliC) for HAd5 in mice for the first time. We found that the recombinant HAd5 expressing RABV glycoprotein and FljB (HAd5-GB), if administered intramuscularly, but not orally, could induce stronger immune responses and provide better protection against rabies than HAd5-G or the recombinant HAd5 expressing glycoprotein and FliC (HAd5-GC). These results suggest that the recombinant HAd5-GB has potential for development as a promising rabies vaccine.

Background The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a major antigen that can induce protective antibodies in poultry. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the linear epitopes of HN, especially neutralizing epitopes, will be useful for revealing its antigenic characterization. Methods In this study, we analyzed B-cell immunodominant epitopes (IDEs) of the HN protein from the vaccine strain LaSota using pepscan technology with LaSota-specific chicken hyperimmune antisera. We constructed IDEs-RFP plasmid and prepared anti-IDEs peptide mouse sera to identify IDEs through immunological tests. At last, the different diluted anti-IDE antisera were used in BHK-21 cells to perform the neutralization test. Results Five IDEs of the HN were screened and further verified by indirect immunofluorescence assays, dot blots and Western blots with NDV- and IDEs-specific antisera. All five IDEs showed good immunogenicity. IDE5 (328-342 aa) could recognize only class II NDV but did not react with the class I strain. Most of the IDEs are highly conserved among the different strains. A neutralization test in vitro showed that the peptide-specific mouse antisera of IDE4 (242-256 aa) and HN341-355, a reported neutralizing linear epitope, could partially neutralize avirulent LaSota as well as virulent strains at similar levels, suggesting that IDE4 might be a potential neutralizing linear epitope. Conclusions The HN protein is a major protective antigen of NDV that can induce neutralizing antibodies in animals. We identified five IDEs of the HN using a pepscan approach with NDV-specific chicken hyperimmune antisera. The five IDEs could elicit specific antibodies in mice. IDE4 (242-256 aa) was identified as a novel potential neutralizing linear epitope. These results will help elucidate the antigenic epitopes of the HN and facilitate the development of NDV vaccines.