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Simple Summary: H9N2 subtype avian influenza virus (AIV), which is mild but widespread, has been a concern due to the potential threat to poultry farming and public safety. AIV-infected poultry exhibit a complex range of various types of disease, which, coupled with the fact that AIV viruses are prone to genetic mutation and remodeling and exhibit a very high degree of variability, further increases the difficulty of immunization and diagnosis of avian influenza. We focused on the PB1 protein, a vital part of the virus's ability to replicate. In this study, a truncated PB1 protein was designed and expressed in the prokaryotic expression system. Four hybridoma cells secreting the antibody specific to PB1 protein were screened using mouse immunization and cell fusion techniques. Two B cell antigenic determinants were identified by gradually truncating protein expression, and were conserved across different flu strains and located on the surface of PB1 protein. These findings could help make better vaccines against the H9N2 virus and might be important for controlling the spread of bird flu and protecting both animals and humans. Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms. [ABSTRACT FROM AUTHOR]

Mucosal immunity is the main defense line against respiratory disease pathogens. Newcastle disease and avian infectious bronchitis are common respiratory diseases in poultry. However, the mucosal immune response is not sufficiently activated and thus fails to achieve the ideal immune protection. Therefore, it is important to develop a suitable mucosal immune adjuvant to enhance the immune response of live vaccines. Here, the bursal-derived peptide BP7, β-glucan, and hyaluronic acid were selected as the adjuvant to be assembled into the composite nanopolypeptide adjuvant (CNPB7) with ultrasonic dispersion technology. The results showed that after optimizing assembly conditions, the optimal average particle size of nanoparticle CNPB7 was 514.9 nm and PDI was 0.298. To evaluate the non-specific immune responses of nanoparticle CNPB7, the chickens were immunized only with nanoparticle CNPB7. It was confirmed that nanoparticle CNPB7 enhanced the expression of CD3, CD4, CD80, and CD86 factors in the spleen lymphocyte from the chicken immunized with nanoparticle CNPB7. To investigate the mucosal immune response of nanoparticle CNPB7, the chickens were orally immunized with Newcastle disease virus (NDV)–infectious bronchitis virus (IBV) dual vaccines and CNPB7. The results proved that the levels of immunoglobulin SIgA, IL-4, IFN-γ, and IL-13 in the mucus samples from the respiratory and digestive tract in chicken immunized with nanoparticle CNPB7 and vaccines were significantly increased, compared to that of vaccine control. Finally, it was observed that nanoparticle CNPB7 promoted specific increased antibody productions against NDV and IBV in the immunized chicken. These results proved that the assembled nanoparticle CNPB7 could enhance the vaccination efficacy in chicken, which provided the experimental basis for the development of new adjuvants, and offered technical support for preventing virus transmission of avian diseases. [ABSTRACT FROM AUTHOR]

Influenza virus infection poses a great threat to human health globally each year. Non-coding RNAs (ncRNAs) in the human genome have been reported to participate in the replication process of the influenza virus, among which there are still many unknowns about Long Intergenic Non-Coding RNAs (LincRNAs) in the cell cycle of viral infections. Here, we observed an increased expression of Linc01615 in A549 cells upon influenza virus PR8 infection, accompanied by the successful activation of the intracellular immune system. The knockdown of Linc01615 using the shRNAs promoted the proliferation of the influenza A virus, and the intracellular immune system was inhibited, in which the expressions of IFN-β, IL-28A, IL-29, ISG-15, MX1, and MX2 were decreased. Predictions from the catRAPID website suggested a potential interaction between Linc01615 and DHX9. Also, knocking down Linc01615 promoted influenza virus proliferation. The subsequent transcriptome sequencing results indicated a decrease in Linc01615 expression after influenza virus infection when DHX9 was knocked down. Further analysis through cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) in HEK293 cells stably expressing DHX9 confirmed the interaction between DHX9 and Linc01615. We speculate that DHX9 may interact with Linc01615 to partake in influenza virus replication and that Linc01615 helps to activate the intracellular immune system. These findings suggest a deeper connection between DHX9 and Linc01615, which highlights the significant role of Linc01615 in the influenza virus replication process. This research provides valuable insights into understanding influenza virus replication and offers new targets for preventing influenza virus infections. [ABSTRACT FROM AUTHOR]

The Bursa of Fabricius, an avian unique humoral immune organ, is instrumental to B cell development. Bursal-derived peptide BP9 fosters B-cell development and formation. Yet, the exact mechanism wherein BP9 impacts B cell differentiation and antigenic presentation remains undefined. In this paper, B cell activation and differentiation in the spleen cells from mice immunized with the AIV vaccine and BP9 were detected following flow cytometry (FCM) analysis. Furthermore, the molecular mechanism of BP9 in B cell differentiation in vivo was investigated with RNA sequencing technology. To verify the potential functional mechanism of BP9 in the antigenic presentation process, the transcriptome molecular basis of chicken macrophages stimulated by BP9 was measured via high-throughput sequencing technology. The results proved that when given in experimental dosages, BP9 notably accelerated total B cells, and enhanced B-cell differentiation and plasma cell production. The gene expression profiles of B cells from mice immunized with 0.01 mg/mL BP9 and AIV vaccine disclosed that 0.01 mg/mL BP9 initiated the enrichment of several biological functions and significantly stimulated key B-cell pathways in immunized mice. Crucially, a total of 4093 differentially expressed genes were identified in B cells with BP9 stimulation, including 943 upregulated genes and 3150 downregulated genes. Additionally, BP9 induced various cytokine productions in the chicken macrophage HD11 cells and activated 9 upregulated and 20 downregulated differential miRNAs, which were involved in various signal and biological processes. Furthermore, BP9 stimulated the activation of multiple transcription factors in HD11 cells, which was related to antigen presentation processes. In summary, these results suggested that BP9 might promote B cell differentiation and induce antigen presentation, which might provide the valuable insights into the mechanism of B cell differentiation upon bursal-derived immunomodulating peptide stimulation and provide a solid experimental groundwork for enhancing vaccine-induced immunity. [ABSTRACT FROM AUTHOR]

Chicken melanoma differentiation-associated gene 5 (MDA5) is a member of the RLRs family that recognizes the viral RNAs invading cells and activates downstream interferon regulatory pathways, thereby inhibiting viral replication. The caspase activation and recruitment domain (CARD) is the most important region in MDA5 protein. However, the antiviral and immune enhancement of MDA5 with the CARD region remains unclear. In this study, two truncated MDA5 genes with different CARD regions, namely MDA5-1 with CARD1 plus partial CARD2 domain and MDA5-2 with CARD1 plus complete CARD2 domain, were cloned via reverse transcription PCR and ligated into plasmid Flag-N vector to be Flag-MDA5-1 and Flag-MDA5-2 plasmids. DF-1 cells were transfected with two plasmids for 24 h and then inoculated with H9N2 virus (0.1 MOI) for 6 h to detect the levels of IFN-β, PKR, MAVS, and viral HA, NA, and NS proteins expression. The results showed that MDA5-1 and MDA5-2 increased the expression of IFN-β and PKR, activated the downstream molecule MAVS production, and inhibited the expression of HA, NA, and NS proteins. The knockdown of MDA5 genes confirmed that MDA5-2 had a stronger antiviral effect than that of MDA5-1. Furthermore, the recombinant proteins MDA5-1 and MDA5-2 were combined with H9N2 inactivated vaccine to immunize SPF chickens subcutaneously injected in the neck three times. The immune response of the immunized chicken was investigated. It was observed that the antibody titers and expressions of immune-related molecules from the chicken immunized with MDA5-1 and MDA5-2 group were increased, in which the inducing function of MDA5-2 groups was the highest among all immunization groups. These results suggested that the truncated MDA5 recombinant proteins with complete CARD2 region could play vital roles in antiviral and immune enhancement. This study provides important material for the further study of the immunoregulatory function and clinical applications of MDA5 protein. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Identification of Unique and Conserved Neutralizing Epitopes of Vestigial Esterase Domain in HA Protein of the H9N2 Subtype of Avian Influenza Virus." The correction addresses an error in Figure 4A of the original publication, where two duplicated images were mistakenly included. The corrected figure has been provided, and the original publication has been updated accordingly. The article is available as an open access article under the Creative Commons Attribution license. [Extracted from the article]

The H9N2 subtype of avian influenza virus (AIV) has been reported to infect not only birds, but also humans. The hemagglutinin (HA) protein is the main surface antigen of AIV and plays an important role in the viral infection. For treatment strategies and vaccine development, HA protein has been an important target for the development of broadly neutralizing antibodies against influenza A virus. To investigate the vital target determinant cluster in HA protein in this work, HA gene was cloned and expressed in the prokaryotic expression vector pET28a. The spleen lymphocytes from BALC/c mice immunized with the purified recombinant HA protein were fused with SP2/0 cells. After Hypoxanthine-Aminopterin-Thymidine (HAT) medium screening and indirect ELISA detection, six hybridoma cell lines producing anti-HA monoclonal antibodies were screened. The gradually truncated HA gene expression and western blotting were used to identify their major locations in epitopes specific to these monoclonal antibodies. It was found that the epitopes were located in three areas: 112NVENLEEL119, 117EELRSLFS124, and 170PIQDAQ175. Epitope 112NVENLEEL119 has a partial amino acid crossover with 117EELRSLFS124, which is located in the vestigial esterase domain "110-helix" of HA, and the monoclonal antibody recognizing these epitopes showed the neutralizing activity, suggesting that the region 112NVENLEELRSLFS124 might be a novel neutralizing epitope. The results of the homology analysis showed that these three epitopes were generally conserved in H9N2 subtype AIV, and will provide valuable insights into H9N2 vaccine design and improvement, as well as antibody-based therapies for treatment of H9N2 AIV infection. [ABSTRACT FROM AUTHOR]

Avian influenza caused by H9N2 subtype avian influenza virus (AIV) poses a great threat to the healthy development of the poultry industry. Vimentin is closely related to intracellular lipid metabolism, which plays an important role during the viral infection process. However, the function of lipid metabolism and vimentin on H9N2 AIV replication is unclear. In this paper, the cholesterol level and 3-hydroxy-3-methylglutaryl coenzyme a reductase (HMGCR) phosphorylation were investigated in vimentin knockout (KO) and human cervical carcinoma cells (HeLa) cell with or without AIV infection. The results showed that compared to the control group without infected with H9N2 subtype AIV, the cholesterol contents were significantly increased, while HMGCR phosphorylation level was reduced in both KO and HeLa cell after virus infection. Furthermore, viral replication was significantly inhibited in the cells treated with the cholesterol inhibitor lovastatin. Compared with the control group, adenylate activated protein kinase (AMPK), a kinase regulating HMGCR enzymatic activity was inhibited in both KO and HeLa cells in the infected virus group, and AMPK phosphorylation levels were significantly lower in KO HeLa cell than that of HeLa cells. Additionally, after MβCD treatment, viral hemagglutinin (HA) gene level was significantly decreased in HeLa cells, while it was significantly increased in KO HeLa cells. In addition, vimentin expression was significantly increased in MβCD-treated HeLa cells with the viral infection and returned to normal levels after exogenous cholesterol to backfill the MβCD-treated cells. Therefore, the disruption of lipid rafts during the binding phase of viral invasion of cells significantly reduced viral infection. These studies indicated that the lipid rafts and cholesterol levels might be critical for H9N2 subtype AIV infection of human-derived cells and that vimentin might play an important role in the regulation of lipids on viral replication, which provided an important antiviral target against influenza virus. [ABSTRACT FROM AUTHOR]

Avian Influenza (AI) caused by the H9N2 subtype of the avian influenza virus (AIV) poses a serious threat to both the poultry industry and to public health safety. NP is one of the major structural proteins in influenza viruses. B-cell determinants located on NP proteins have attracted increasing attention. In this study, based on the NP sequence of the H9N2 (A/chicken/Shandong/LY1/2017) strain, the truncated NP gene (71 AA–243 AA) was cloned and prokaryotically expressed in a pET-28a (+) vector. BALB/c mice were immunized with a purified recombinant of an NP protein to prepare a monoclonal antibody against NP proteins. The prokaryotic expression of four overlapping fragments, NP-N-96, NP-C-103, NP-C-54 and NP-C-49, were used to recognize an antigenic epitope of the NP protein. The results show that, after cell fusion, one hybridoma cell clone secreted the antibody specific to the NP protein, following screening with ELISA and indirect immunofluorescence, which is named the 4F5 monoclonal antibody (mAb). Western blotting on the overlapping fragments showed that the 230FQTAAQRA237 motif was identified as the minimal motif recognized by 4F5mAb, which was represented as the linear B-cell epitope of the NP protein. Homology analysis of this epitope shows that it was highly conserved in 18 AIVs analyzed in this study, and the epitope prediction results indicate that the epitope may be located on the surface of the NP protein. These results provide a strong experimental basis for studying the function of the NP protein of the H9N2 AIV and also strong technical support for the development of a universal assay based on an anti-NP monoclonal antibody. [ABSTRACT FROM AUTHOR]

As the basic spatial unit of urban planning and management, it is necessary to know the distribution status of urban functional areas in time. Due to the complexity of urban land use, it is difficult to identify the urban functional areas using only remote sensing images. Social perception data can provide additional information for the identification of urban functional areas. However, the sources of remote sensing data and social perception data differ, with some differences in data forms. Existing methods cannot comprehensively consider the characteristics of these data for functional area identification. Therefore, in this study, we propose a multimodal deep learning method with an attention mechanism to fully utilize the data features of these two modalities and apply it to the recognition of urban functional areas. First, the pre-processed remote sensing images, points of interest, and building footprint data are divided into block-based target units of features by the road network. Next, the remote sensing image features and social perception data features of the target unit are extracted separately using a two-branch convolutional network. Finally, features are extracted sequentially along two separate dimensions, being channel and spatial, to generate an attention weight map for the identification and classification mapping of urban functional areas. The model framework was finally applied to the Ningbo dataset for testing, and the recognition accuracy was above 93%. The experimental results deduce, as a whole, that the prediction performance of the deep multimodal fusion model framework with an attention mechanism is comparatively superior to other traditional methods. It can provide a reference for the classification of urban land use and provide data support for urban planning and management. [ABSTRACT FROM AUTHOR]

CpG oligodeoxynucleotides (CpG ODN) present adjuvant activities for antigen proteins, which can induce humoral and cellular immune responses to antigens. However, the immunomodulatory functions of CpG ODNs with different sequences are very different. In this paper, six CpG ODNs with different sequences were designed based on CpG2007 as a template. Through the screening of CEF cells in vitro, the stimulating activity of CpG ODNs was determined. Then, two selected CpG ODN sequence backbones were modified by substituting the oxygen with sulfur (S-CpG) and verifying the immune activity. Next, to prove the feasibility of S-CpG as an immune potentiator, two immune models with or without white oil adjuvant were prepared in 20-day-old chicken vaccinations. The screening experiment in vitro showed that the inducing roles of CpG ODN 4 and 5 could strongly stimulate various immune-related molecular expressions. Additionally, CpG ODN 4 and 5 with sulfation modification significantly induced various cytokines' expressions. Furthermore, CpG ODN 4 and 5 induced the strongly humoral and cellular immune responses during vaccination, in which white oil, as an adjuvant, could significantly improve the immune effect of CpG ODN. These results provide an important experimental basis for exploring the structural characteristics and vaccine immunity of CpG ODN. [ABSTRACT FROM AUTHOR]

Diallyl disulfide (DADS) is the main organosulfur ingredient in garlic, with known antioxidant and anti-inflammatory activities. The aim of the present study was to investigate the effect of DADS on reducing the inflammation and redox imbalance in a rat emphysema model that was induced by intraperitoneal injection of cigarette smoke extract (CSE). Briefly, DADS exerted an anti-inflammation effect on emphysema rats through decreasing cell influx in the bronchoalveolar lavage fluid (BALF) and suppressing pro-inflammation cytokine production including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) via inhibiting the NF-κB pathway. In addition, levels of oxidative stress markers including malondialdehyde (MDA) and myeloperoxidase (MPO) were reduced, while the activities of glutathione (GSH), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were markedly enhanced by DADS. Moreover, MMP-9 and TIMP-1 expression were down-regulated by DADS. Furthermore, the regulation effects of DADS on CD4+ and CD8+ T cells were observed. In conclusion, these encouraging findings suggest that DADS could be considered as a promising anti-inflammation and antioxidative agent for the treatment of emphysema. [ABSTRACT FROM AUTHOR]

Soil dilatancy and partial drainage have great influence on the consolidation coefficient assessment of silty soils with clay content of less than 30% in the Yellow River Delta using the CPTu. This paper discussed the effect of soil dilatancy and partial drainage on the pore pressure response and dissipation of piezocone test in shallow silty soil of the Yellow River Delta through variable penetration rate tests in a pressure chamber and a series of supplementary soil element tests. The results show that the pore pressure dissipation curve is affected by soil type and degree of consolidation. The soil dilatancy is the key factor affecting consolidation coefficient inversion of shallow silt and silty clays. The initial pore pressure is negative and the pore pressure increases to umax first, but the umax value is very small in the Yellow River Delta silt. The inversion method used for shear contractile soil cannot be used to invert the mechanical properties of shallow silty soil directly, and a new consolidation curve normalization method is proposed. This paper provides a reference for the consolidation coefficient inversion of CPTu data in the Yellow River Delta area. [ABSTRACT FROM AUTHOR]

Influenced by climate change, extreme weather events occur frequently, and bring huge impacts to urban areas, including urban waterlogging. Conducting risk assessments of urban waterlogging is a critical step to diagnose problems, improve infrastructure and achieve sustainable development facing extreme weathers. This study takes Ningbo, a typical coastal city in the Yangtze River Delta, as an example to conduct a risk assessment of urban waterlogging with high-resolution remote sensing images and high-precision digital elevation models to further analyze the spatial distribution characteristics of waterlogging risk. Results indicate that waterlogging risk in the city proper of Ningbo is mainly low risk, accounting for 36.9%. The higher-risk and medium-risk areas have the same proportions, accounting for 18.7%. They are followed by the lower-risk and high-risk areas, accounting for 15.5% and 9.6%, respectively. In terms of space, waterlogging risk in the city proper of Ningbo is high in the south and low in the north. The high-risk area is mainly located to the west of Jiangdong district and the middle of Haishu district. The low-risk area is mainly distributed in the north of Jiangbei district. These results are consistent with the historical situation of waterlogging in Ningbo, which prove the effectiveness of the risk assessment model and provide an important reference for the government to prevent and mitigate waterlogging. The optimized risk assessment model is also of importance for waterlogging risk assessments in coastal cities. Based on this model, the waterlogging risk of coastal cities can be quickly assessed, combining with local characteristics, which will help improve the city's capability of responding to waterlogging disasters and reduce socio-economic loss. [ABSTRACT FROM AUTHOR]

Studying land use changes and ecological risk assessment in Yongjiang River Basin in Zhejiang Province, China, provides theoretical references for optimal configuration of land resources and maintaining stability of ecosystems. Given impacts of land use changes on landscape patterns in the Yongjiang River Basin, ecological risk assessment indexes were constructed and used to analyze temporal and spatial variation characteristics of ecological risk within different periods. Results show that (1) the construction land area was increased quickly, while the cultivated area decreased sharply. A prominent characteristic of land use changes was manifested by transforming cultivated area and forestland into construction land. The utilized degree of the land increased continuously. Spatially, the land utilized degree in northern regions was higher than that in southern regions and the degree in eastern regions was higher than that in western regions. (2) The ecological risk in the Yongjiang River Basin was intensified and the area of high ecological risk was expanded by 893.96 km2. Regions with low and relatively low ecological risks concentrated in western and southern regions of the Basin, whereas regions with high ecological risks were mainly in northern and eastern regions. Landscapes in cities and towns at a high economic development level are highly sensitive to human activities. (3) Transformation of ecological risk is complicated. Land area with the ecological risk changing from a low level to a high level was 4.15 times that with the ecological risk changing from a high level to a low level. There were 15 transformation directions among different ecological risk regions. [ABSTRACT FROM AUTHOR]