The H9N2 subtype avian influenza virus (AIV) is common in poultry and poses significant risks to both the poultry industry and public health. Current control strategies for H9N2 AIV predominantly rely on vaccination; however, these approaches are often undermined by the continuous antigenic drift of hemagglutinin under the pressure of antibodies. This study evaluated the antiviral efficacy of Siji Antiviral Mixture (SAM) against H9N2 infection in specific pathogen-free chickens. Compared with the infected control group, SAM significantly reduced viral loads in multiple tissues, including liver, spleen, lung, kidney, brain, and trachea, demonstrating its direct antiviral activity. Further analysis revealed that SAM modulated H9N2-induced inflammation by downregulating excessive innate immune responses. Specifically, SAM significantly enhanced the expression of antiviral effectors (OASL and MX-1) in the lungs during the early stages of viral infection, while attenuating the production of inflammatory cytokines (IL-6, IL-10) in the later stages. Additionally, SAM regulated the expression levels of pattern recognition receptors (MDA5, TLR3, and TLR7), thereby preventing excessive immune responses. These findings indicate that SAM possesses both antiviral and immunoregulatory effects, highlighting its potential as an effective anti-AIV agent for the management of H9N2 AIV infection.

H9N2 avian influenza virus (AIV) is one of the major pathogens causing respiratory disease in chickens and poses a threat to public health by serving as a gene donor for zoonotic avian influenza strains. The DEAD-box RNA helicase DDX21 is known to play a critical role in innate immunity in mammals during influenza virus infection. However, the function role and regulatory mechanisms of its avian ortholog, chicken DDX21 (chDDX21), remain poorly understood. In this study, the chicken DDX21 (chDDX21) gene was cloned and analyzed using bioinformatics tools. Phylogenetic analysis revealed that chDDX21 clustered within the avian lineage and shared a close relationship with DDX21 from other avian species. Notably, a widespread distribution of chDDX21 was observed in various tissues in healthy chickens. Following infection with H9N2 AIV, chDDX21 mRNA expression was significantly upregulated in tissues of chickens compared to the control group. Similarly, in CEF cells, chDDX21 transcript levels increased post-infection, peaking at 24 h, a trend that corresponded with the kinetics of viral replication. Furthermore, overexpression of chDDX21 activated the IFN-β promoter, and its DEXDc and HELICc domains were identified as critical for this activity. Co-transfection of chDDX21 with chTLR3 or chTRIF in CEFs resulted in significant upregulation of immune-related gene expression, suggesting that chDDX21 acted as a modulator of the chicken TLR3-mediated type I interferon (IFN) signaling pathway. Additionally, overexpression of chDDX21 significantly increased the expression of antiviral immune factors and concurrently suppressed H9N2 AIV replication. Consequently, these results demonstrated that chDDX21 inhibited H9N2 AIV replication by positively regulating the TLR3-mediated type I IFN signaling pathway. This study provides mechanistic insights into the interaction between the avian innate immune system and H9N2 AIV, highlighting chDDX21 as a key regulator of antiviral response in chickens.

Avian infectious bronchitis virus (IBV) is widely distributed worldwide and causes substantial economic losses to the poultry industry. Because IBV undergoes frequent mutation, prevention and control of infection remain challenging. Immunization is an important measure for the prevention and control of IB. Therefore, there is an urgent need for a rapid, sensitive, specific, and convenient method for the detection of antibodies against IBV. In this study, we firstly developed an indirect colloidal gold immunochromatographic strip for the rapid detection of antibodies against IBV based on a conserved epitope peptide. The recombinant epitope peptide recognized by N2D5 monoclonal antibody (mAb) against the N protein of IBV was expressed as a GST fusion protein (GST-N2D5) based on the conserved antigenic epitope previously identified in our laboratory. Colloidal gold-labeled GST-N2D5 was used as the detection reagent to generate visual signals. Rabbit anti-chicken IgY and mouse anti-GST mAb were immobilized on the nitrocellulose membrane as the test line (T line) and control line (C line), respectively. The optimal pH and optimal protein concentration for conjugation of gold nanoparticles (AuNPs) with GST-N2D5 were pH 8.5 and 72 µg/mL, respectively. Specificity was evaluated using common avian pathogens, and no cross-reactivity was observed. The detection limit of the strip for IBV-positive serum was 1:180. In addition, the assay showed good reproducibility and stability, and results could be observed within 5 min without any specialized equipment. Clinical chicken serum samples were tested using both the developed strip and an enzyme-linked immunosorbent assay (ELISA), and the strip showed high agreement with the ELISA. In conclusion, the established immunochromatographic strip is rapid, sensitive, specific, and easy to operate, and therefore has potential as an on-site tool for the rapid detection of antibodies against IBV, particularly in resource-limited settings.

The Taiwan-I-type infectious bronchitis virus (IBV) has become one of the most dominant and threatening genotypes circulating in China poultry farms. In this study, we characterized the Taiwan-I-type IBV strain GX-NN200723 and evaluated its pathogenicity and vaccine potential. Phylogenetic and recombination analyses revealed that the strain shared the highest nucleotide similarity with the vaccine strain QXL87 and originated from a recombination event between CK/CH/LSC-99I (major parent) and TW2575/98 (I) (minor parent) within the S1 gene region. Notably, epitope mapping showed significant differences in the number and sequence of antigenic sites compared to commonly used vaccine strains (H120, 4/91, QXL87, LDT3-A), and these differences may largely account for the poor cross-protection and frequent vaccine failures observed in the field. Pathogenicity assessment in 7-day-old SPF chicks demonstrated broad tissue tropism of the strain, causing 30% mortality along with severe reproductive disorders, notably, testicular atrophy in males was observed for the first time in Taiwan-I-type IBV. Furthermore, an oil-emulsified inactivated vaccine (OEIV) developed from the GX-NN200723 strain induced strong humoral and cell-dependent immune responses. Protective efficacy was only evaluated against homologous challenge in this study, the GX-NN200723-OEIV group exhibited 100% survival, the mildest clinical signs, minimal pathological damage, and the lowest viral shedding among all immunized groups. These findings support the use of the GX-NN200723-OEIV as a promising candidate vaccine for controlling Taiwan-I-type IBV infection. This study provides critical insights into the evolution, pathogenesis, and immunization strategies against this economically significant poultry pathogen.

H3N2 subtype avian influenza virus (AIV) is prevalent in poultry and wild birds and typically causes asymptomatic or mild respiratory infections. However, genetic reassortment between H3N2 and other AIV subtypes generates novel strains capable of crossing the species barrier, posing a threat to both poultry and public health. In this study, nine H3N2 AIVs were isolated from ducks in live poultry markets (LPMs) in Guangxi, southern China, during 2022-2024. Phylogenetic analysis revealed that all eight gene segments of the nine isolates were clustered within the Eurasian lineage, with internal genes derived from multiple subtypes, including H1, H2, H3, H4, H5, H6, H7, and H9. These findings indicate complex gene reassortment of H3N2 AIVs in Guangxi. Importantly, the PB2 genes of certain isolates were closely related to those of highly pathogenic H5 subtype viruses, suggesting that H3N2 AIVs may contribute internal genes to H5 viruses. Three representative isolates (LZD44, NND98, and NND100) were assessed for pathogenicity in SPF chickens and mice. All three strains successfully replicated in the respiratory tissues of both species. Notably, the LZD44 virus, which harbored the mammalian-adaptive mutations PB2-MVV and NP-I353V, presented significantly higher virulence in chickens and mice than the other two strains. These results demonstrate that H3N2 subtype AIVs are capable of replicating in certain tissues of chickens and mice without prior adaptation, underscoring a potential risk for cross-species transmission. Consequently, sustained surveillance of H3N2 subtype AIVs is essential to prevent the spillover of novel recombinants into the human population.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), and swine influenza virus (SIV) are important pathogens of significant infectious diseases. They cause similar clinical respiratory symptoms, including fever, cough, runny nose, and respiratory distress, which makes these diseases difficult to distinguish from each other. In this study, three pairs of specific primers and TaqMan probes were designed for the conserved regions of the PRCoV S gene, PRRSV N gene, and SIV M gene, respectively. The annealing temperature, primer and probe concentrations, and reaction cycle were optimized, and a triplex crystal digital PCR (cdPCR) assay was established for the detection of PRCoV, PRRSV, and SIV. According to the test results, the assay was capable of specifically detecting PRCoV, PRRSV, and SIV, and there was no cross-reaction with other control swine viruses. Based on the Poisson distribution analysis, the limits of detection (LODs) for PRCoV, PRRSV, and SIV were 6.00, 5.75 and 6.00 copies/reaction, respectively, and the sensitivity was 26 times higher than those of the corresponding multiplex RT-qPCR. The coefficients of variation (CVs) of the intra-assay and inter-assay ranged from 0.19 to 1.84%. The assay was used to test 1,657 clinical samples, and the positivity rates of PRCoV, PRRSV, and SIV were 1.15, 12.79, and 2.05%, respectively. It showed diagnostic sensitivity and specificity of 100 and 99.82% for PRCoV, 100 and 99.24% for PRRSV, and 100 and 99.69% for SIV, respectively. These results indicated that the triplex cdPCR assay has strong specificity, high sensitivity, and excellent repeatability, which provides a valuable tool for the detection and differentiation of PRCoV, PRRSV, and SIV.

Infectious diseases caused by pathogenic microorganisms have caused serious economic losses to animal husbandry, and the use of appropriate disinfectants is crucial for eliminating these pathogens. Plant essential oils (PEOs), as natural bioproducts, have the characteristics of safety, non-toxicity, and broad spectrum. In this study, the inhibition efficacies against bacteria, viruses, and mycoplasmas of a compound PEO disinfectant (designated as Lei-Huo-Fu) were evaluated through determination of minimum inhibitory concentration (MIC) and bactericidal rate against Escherichia coli, Staphylococcus aureus, and Salmonella spp.; inactivation rate of avian infectious bronchitis virus (IBV); as well as determination of MIC of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The results showed that the MIC values of the PEO disinfectant against Escherichia coli, Staphylococcus, and Salmonella spp. were as low as 0.00375 µg/mL to 0.03 µg/mL. The bactericidal rates against Escherichia coli, Staphylococcus aureus, and Salmonella spp. reached over 95% within 30 min at a concentration of 0.03 µg/mL. For three dominant prevalent genotype strains of LX4-type, Mass-type, and Taiwan-type of IBV, the inactivation rates achieved by the PEO disinfectant at a concentration of 0.015 µg/mL and a disinfection time of 30 min were all above 99.9%. The MIC of the PEO disinfectant against MG and MS was 0.001875 µg/mL and 0.00375 µg/mL, respectively. In conclusion, the compound PEO disinfectant (Lei-Huo-Fu) has significant inhibitory effects on bacteria, viruses, and mycoplasmas, and possesses broad-spectrum antimicrobial activity. However, it is important to note that these findings are based on laboratory assays, and the efficacy in practical settings, along with the exact mechanisms of action, require further investigation. In this study, the compound PEO disinfectant demonstrates promising in vitro efficacy, suggesting its potential as a candidate for development into a safe, efficient, and natural disinfectant, pending further validation.

Infectious bronchitis virus (IBV) is one of the major diseases affecting the poultry industry worldwide. The high mutation rates of viral genome pose serious challenges to the prevention and control of IB. Based on our previous studies on IBV isolates in southern China during 2009-2017, we continued to carry out the genetic evolution analyses of S1 gene from IBV isolates during 2018-2023. Furthermore, we investigated the temporal and geographical origin of 245 IBV strains from southern China during 1985-2023, and reconstructed their transmission dynamics. Our findings revealed that GI-19 (LX4-type) was still the most dominant genotype, with the coexistence of multiple genotypes. Notably, the GI-19 strains were further classified into three subgroups, one of which was a new GI-19 subtype with nucleotide sequence similarities less than 90 % compared to the vaccine strain QXL87, along with five and eight consecutive amino acid substitutions, respectively. GI-28 (LDT3-A-type) strains re-emerged in 2019 and remained predominant thereafter. In addition, there was an increasing isolation rate of GI-7 (Taiwan-I-type). The purification pressure was detected in the S1 protein IBV isolates during 2018-2023, but eight positive selection sites with high entropy values were identified. Five isolates were confirmed to be recombinants, and GI-22 strains involved in all five recombinants as major or minor parents. The estimated times for the most recent common ancestors based on the S1 gene was the years of 1905. Bayesian skyline analysis indicated a rapid increase in genetic diversity of the S1 gene in IBV strains since late 2015. Bayesian geographical analysis demonstrated that the southern China IBVs originated from Nanning and identified four principal transmission pathways. In conclusion, our findings revealed the emergence of a new GI-19 subtype of IBV, the re-emergence of GI-28, and multiple transmission routes in southern China during 2018-2023, underscoring the importance of real-time and continuous surveillance of circulating strains and the urgency of developing safe, effective, and broad-spectrum new vaccines against emerging and re-emerging IBVs.

Avian infectious bronchitis virus (IBV) infection has caused significant economic losses to the poultry industry. Unfortunately, there is currently no effective cure for this disease. Understanding the pathogenic mechanism is crucial for the treatment of the disease. Studying the pathogenic mechanism of IBV based on metabolomics analysis is helpful for identifying antiviral drugs. However, studies on metabolomics analysis of IBV infection have been relatively limited, particularly without metabolomics analysis in sera after IBV infection. In this study, 17-day-old SPF chicks were infected with the IBV GX-YL5 strain, and serum samples were collected 7 days post-infection (DPI) for metabolomics analysis using ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). A total of 143 differential metabolites were identified across 20 metabolic pathways, with the phenylalanine pathway showing the most significant changes. The level of cinnamic acid (CA), an upstream metabolite in the phenylalanine pathway, was notably increased following IBV infection. To investigate the antiviral effects of CA, chicken embryo kidney (CEK) cells and SPF chicks infected with IBV were treated with different concentrations of CA to assess its effect on viral replication. The results demonstrated that CA at 25 μg/mL effectively inhibited IBV replication in vitro; meanwhile, CA at 50 μg/mL and 25 μg/mL effectively inhibited IBV replication in vivo. Molecular docking and molecular dynamics simulation studies showed that CA interacts with the N domains of the IBV nucleocapsid (N) protein. In conclusion, the serum metabolite CA is significantly elevated following IBV infection and demonstrates remarkable antiviral effects both in vitro and in vivo, providing a promising avenue for the development of antiviral therapies to combat IBV infection.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RFRAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39degree celsius within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no crossreaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/mu L for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and realtime quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.

Avian infectious bronchitis virus (IBV) still causes serious economic losses in the poultry industry. Currently, there are multiple prevalent genotypes and serotypes of IBVs. It is imperative to develop a new diagnosis method that is fast, sensitive, specific, simple, and broad-spectrum. A monoclonal hybridoma cell, N2D5, against the IBV N protein was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The N2D5 monoclonal antibody (mAb) and the previously prepared mouse polyclonal antibody against the IBV N protein were used to target IBV as a colloidal gold–mAb conjugate and a captured antibody, respectively, in order to develop an immunochromatographic strip. The optimal pH and minimum antibody concentration in the reaction system for colloidal gold–mAb N2D5 conjugation were pH 6.5 and 30 μg/mL, respectively. Common avian pathogens were tested to evaluate the specificity of the strip and no cross-reaction was observed. The sensitivity of the strip for detecting IBV was 10−1.4522 EID50/mL. The strip showed a broad-spectrum cross-reactive capacity for detecting IBV antigens, including multiple IBV genotypes in China and all of the seven serotypes of IBV that are currently prevalent in southern China. Additionally, the result can be observed within 2 min without any equipment. The throat and cloacal swab samples of chickens that were artificially infected with three IBV strains were tested using the developed strip and the qPCR method; the strip test demonstrated a high consistency in detecting IBV via qPCR gene detection. In conclusion, the immunochromatographic strip that was established is rapid, sensitive, specific, simple, practical, and broad-spectrum; additionally, it has the potential to serve as an on-site rapid detection method of IBV and can facilitate the surveillance and control of the disease, especially in resource-limited areas.

Novel variant infectious bursal disease virus (nvIBDV) is an emerging genotype (A2dB1b) that can cause severe and prolonged immunosuppression in young chickens. Despite current commercial vaccines being proven to lack complete protection against nvIBDV, it remains unclear whether the oil emulsion inactivated vaccines (OEVs) of the homologous and heterologous virus or booster immunization can provide effective protection. In this study, OEVs with two types of nvIBDV isolates QZ191002 (A-nv/B-nv) and YL160304 (A-nv/B-HLJ0504-like) were prepared and evaluated the protective effects of OEVs plus the booster immunizations with different current commercial vaccines against the challenge of nvIBDVs. The results from vaccination-challenge experiments showed that nvIBDV could break through the protection provided by only one immunization dose of the commercial vaccines, with the protection rates ranging from 40% to 60%. Interestingly, even with booster immunization with different commercial vaccines, the protection rates could only be increased to 60%–80%. As expected, only the OEVs of the homologous virus could provide 100% protection against the homologous nvIBDV, which could induce high-level specific antibodies, ameliorate target organ damage, and significantly reduce the viral load of the bursal in the challenged chickens. Notably, YL160304-OEV performed better than QZ191002-OEV, providing 100% protection not only against the challenge of homologous strain but also against that of heterologous QZ191002 strain. Antibody levels of the immunized chickens gradually increased after a short decline and reached the highest level on the age of 28 days. Similarly, the percentages of lymphocytes CD4+, CD8+ T, and B in peripheral blood lymphocytes (PBLs) were significantly increased on 21 d and 28 d. Notably, despite the nvIBDV, OEVs initially induced a delayed responses in the early stages but ultimately reach higher levels of CD4+ and CD8+ T lymphocytes. The results of study suggest that even booster immunization with different commercial vaccines cannot provide complete protection against nvIBDV, while the OEVs made by the nvIBDVs can provide full protection. Moreover, YL160304-OEV exhibits a broader protective spectrum against different nvIBDV strains, making it a potential candidate for the development of new vaccine.

With the emergence of new variant strains resulting from high mutation rates and genome recombination, avian infectious bronchitis virus (IBV) has caused significant economic losses to the poultry industry worldwide. Little is known about the underlying mechanisms of IBV-host interactions, particularly how IBV utilizes host metabolic pathways for efficient viral replication and transmission. In the present study, the effects of the cell membrane, viral envelope membrane, and viperin-mediated cholesterol synthesis on IBV replication were explored. Our results revealed significant increase in cholesterol levels and the expression of viperin after IBV infection. Acute cholesterol depletion in the cell membrane and viral envelope membrane by treating cells with methyl-β-cyclodextrin (MβCD) obviously inhibited IBV replication; thereafter, replenishment of the cell membrane with cholesterol successfully restored viral replication, and direct addition of exogenous cholesterol to the cell membrane significantly promoted IBV infection during the early stages of infection. In addition, overexpression of viperin effectively suppressed cholesterol synthesis, as well as IBV replication, whereas knockdown of viperin (gene silencing with siRNA targeting viperin, siViperin) significantly increased IBV replication and cholesterol levels, whereas supplementation with exogenous cholesterol to viperin-transfected cells markedly restored viral replication. In conclusion, the increase in viperin induced by IBV infection plays an important role in IBV replication by affecting cholesterol production, providing a theoretical basis for understanding the pathogenesis of IBV and discovering new potential antiviral targets.

African swine fever (ASF) is a severe and highly contagious viral disease that affects domestic pigs and wild boars, characterized by a high fever and internal bleeding. The disease is caused by African swine fever virus (ASFV), which is prevalent worldwide and has led to significant economic losses in the global pig industry. In this study, three pairs of specific primers and TaqMan probes were designed for the ASFV B646L, MGF505-2R and I177L genes. After optimizing the reaction conditions of the annealing temperature, primer concentration and probe concentration, triplex crystal digital PCR (cdPCR) and triplex real-time quantitative PCR (qPCR) were developed for the detection and differentiation of the wild-type ASFV strain and the MGF505-2R and/or I177L gene-deleted ASFV strains. The results indicate that both triplex cdPCR and triplex qPCR were highly specific, sensitive and repeatable. The assays could detect only the B646L, MGF505-2R and I177L genes, without cross-reaction with other swine viruses (i.e., PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV). The limit of detection (LOD) of triplex cdPCR was 12 copies/reaction, and the LOD of triplex qPCR was 500 copies/reaction. The intra-assay and inter-assay coefficients of variation (CVs) for repeatability and reproducibility were less than 2.7% for triplex cdPCR and less than 1.8% for triplex qPCR. A total of 1510 clinical tissue samples were tested with both methods, and the positivity rates of ASFV were 14.17% (214/1510) with triplex cdPCR and 12.98% (196/1510) with triplex qPCR, with a coincidence rate of 98.81% between the two methods. The positivity rate for the MGF505-2R gene-deleted ASFV strains was 0.33% (5/1510), and no I177L gene-deleted ASFV strain was found. The results indicate that triplex cdPCR and triplex qPCR developed in this study can provide rapid, sensitive and accurate methods for the detection and differentiation of the ASFV B646L, MGF505-2R and I177L genes.

鸡传染性支气管炎是由鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)感染引起的鸡的一种急性、高度接触性传染性病.IBV基因组非常容易发生突变和重组,给该病防控带来巨大困难,因此高效疫苗和有效药物的研发非常重要.反向遗传技术是研究RNA病毒的重要技术.通过反向遗传技术可以在DNA分子水平定向改造病毒序列,对病毒的基因功能、致病机制以及新型疫苗进行研究.论文对构建IBV操作系统的策略、利用反向遗传技术研究IBV基因结构和功能、研发新型疫苗以及开发病毒载体的最新进展进行综述,为IBV及其他冠状病毒相关研究提供参考.

应用噬菌体随机十二肽库技术对针对IBV GX-YL5株N蛋白的单抗N2D5进行抗原表位的鉴定.将淘选得到的噬菌体随机十二肽库第3轮洗脱液进行二代测序,将测序结果中出现频率最高的3个十二肽序列合成肽后验证是否为模拟表位;根据获得的模拟表位预测相应的天然表位序列并合成多肽和原核表达进行验证,同时对获得的天然表位进行保守性和交叉反应性分析.结果显示,第3轮筛选洗脱液测序出现频率最高的3个序列中有2个为模拟表位,即 VVGRAMAYSTIP和DGVLLGTSGEST;预测天然表位序列为158IPLNRGRGGRST169;预测天然表位的合成肽的ELISA和Dot-blotting分析以及其原核表达蛋白的Western-blotting分析表明,158IPLNRGRGGRST169是IBV的一个天然表位序列;保守性和交叉反应性分析显示该天然表位具有高度保守性.结果表明,本研究成功鉴定出IBV GX-YL5 N蛋白上的一个高度保守的表位158IPLNRGRGGRST169,为IBV N蛋白的抗原结构和功能的研究及诊断试剂和表位疫苗的研制奠定了基础.

[Objective]This paper screened out the amino acid region with good antigenicity of non-structural protein 4(nsp4)of avian infectious bronchitis virus(IBV)and prepared polyclonal antibody,so as to provide a material basis for further research on the function of nsp4 protein in the process of IBV replication and lay a foundation for the research and development of new diagnostic kits and vaccines for IBV.[Method]In this study,nsp4 protein of IBV Beaudette strain was prokaryotically expressed.Rabbit and chicken polyclonal antibodies were prepared by immunizing Japanese white rabbit and healthy negative chicken with the fusion protein nsp4 as immunogen.And the biological function of the prepared polyclonal antibodies were investigated by indirect enzyme linked immunosorbent assay(ELISA),Western blot-ting and indirect immunofluorescence assay(IFA).[Result]The nsp4 protein prokaryotic expression vector pCZN-1-HIS-nsp4 and nsp4 protein eukaryotic expression vector pVAX1-nsp4-HA were successfully constructed by selecting amino acids at positions 408-514 of nsp4 protein as prokaryotic expression sequences.The prokaryotic expression of fusion pro-tein nsp4 was 13.6 kD,which was consistent with its predicted size,and the fusion protein nsp4 was able to react with positive whole virus serum against IBV GX-YL5 and M41 strains.The serum titers of prepared rabbit and chicken poly-clonal antibodies were 1∶128000 and 1∶6400 respectively.Additionally,both polyclonal antibodies could react with the fusion protein nsp4 and IBV whole virus.Moreover,they could react with nsp4 protein expressed in chicken embryonic kidney(CEK)cells infected with IBV GX-YL5,Beaudette and M41 strains,and could react with nsp4 protein expressed in the transfected or IBV infected African green monkey kidney cells(Vero)Beaudette strain.The results of real-time fluo-rescence quantitative PCR showed that the viral loads of Vero cells increased gradually after transfection of nsp4 protein eukaryotic expression vector pVAX1-nsp4-HA.In other words,in vitro overexpressed nsp4 protein promoted IBV replica-tion.[Conclusion]Two polyclonal antibodies against nsp4 of IBV strain with high titer,good reactivity and strong speci-ficity are successfully prepared,which can be used as tools for the study of protein characteristics during cell localization and expression phase analysis.In addition,they can be used to analyze the action mechanism of nsp4 protein in the proli-feration process of IBV,and also provide reference for the study of other coronavirus nsp4 protein.

Context Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still ongoing and currently the most striking epidemic disease. With the rapid global spread of SARS-CoV-2 variants, new antivirals are urgently needed to avert a more serious crisis. Inhibitors from traditional medicines or natural plants have shown promising results to fight COVID-19 with different mechanisms of action.Objectives To provide comprehensive and promising approaches to the medical community in the fight against this epidemic by reviewing potential plant-derived anti-SARS-CoV-2 inhibitors.Methods Structural databases such as TCMSP (http://lsp.nwu.edu.cn/tcmsp.php), TCM Database @ Taiwan (http://tcm.cmu.edu.tw/), BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm/) and TCMID (http://www.megabionet.org/tcmid/), as well as PubMed, Sci Finder, Research Gate, Science Direct, CNKI, Web of Science and Google Scholar were searched for relevant articles on TCMs and natural products against SARS-CoV-2.Results Seven traditional Chinese medicines formulas have unique advantages in regulating the immune system for treating COVID-19. The plant-derived natural compounds as anti-SARS-CoV-2 inhibitors were identified based on 5 SARS-CoV-2 key proteins, namely, angiotensin-converting enzyme 2 (ACE2), 3 C-like protease (3CLpro), papain-like protease (PLpro), spike (S) protein, and nucleocapsid (N) protein.Conclusions A variety of natural products, such as flavonoids, terpenoids, phenols, and alkaloids, were identified, which could be used as potential SASR-Cov-2 inhibitors. These shed new light on the efficient discovery of SASR-Cov-2 inhibitors from natural products.

为了建立一种快速、简便的基于重组酶介导等温扩增技术(RAA)检测滑液囊支原体(MS)的新型方法,本研究针对MS vlhA基因设计了2 对特异性引物.经过优化引物和反应条件,建立了MS的RAA新型检测方法,并对其特异性、敏感性和重复性进行评价,还将其敏感性与常规PCR方法进行比较.应用建立的方法对疑似或确诊的临床样品共 100 份进行检测.结果表明:建立的基础RAA法可在 29℃恒温操作 10～15 min即可完成对MS 核酸模板的检测;RAA法与大肠杆菌、肠炎沙门菌、鸡毒支原体、新城疫病毒、禽流感病毒、禽传染性喉气管炎病毒、禽传染性支气管炎病毒和禽呼肠孤病毒等其他病原均无交叉反应;对MS基因组DNA的最低检出限为 6.53×105 copies/μL,比PCR方法敏感 100倍;应用该方法检测了100 份临床样本,结果 71 份阳性,与PCR检测结果一致.研究表明,建立的MS RAA方法具有特异、敏感、快速、简便的特点,方便基层实验室应用.