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Establishment of a new reverse genetics system for respiratory syncytial virus under the control of RNA polymerase II RNA聚合酶ⅱ控制下呼吸道合胞病毒反向遗传新体系的建立
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-07-09 DOI: 10.1111/1348-0421.13088
Tatsuki Takahashi, Shiori Ueno, Yoshiro Sugiura, Kenta Shimizu, Wataru Kamitani

A reverse genetics system for the respiratory syncytial virus (RSV), which causes acute respiratory illness, is an effective tool for understanding the pathogenicity of RSV. To date, a method dependent on T7 RNA polymerase is commonly used for RSV. Although this method is well established and recombinant RSV is well rescued from transfected cells, the requirement for artificial supply of T7 RNA polymerase limits its application. To overcome this, we established a reverse genetics system dependent on RNA polymerase II, which is more convenient for the recovery of recombinant viruses from various cell lines. First, we identified human cell lines with high transfection efficiency in which RSV can replicate effectively. Two human cell lines, Huh-7 and 293T, permitted the propagation of recombinant green fluorescent protein–expressing RSV. Our minigenome system revealed that efficient transcription and replication of RSV occurred in both Huh-7 and 293T cells. We then confirmed that recombinant green fluorescent protein–expressing RSV was rescued in both Huh-7 and 293T cells. Furthermore, the growth capability of viruses rescued from Huh-7 and 293T cells was similar to that of recombinant RSV rescued using the conventional method. Thus, we succeeded in establishing a new reverse genetics system for RSV that is dependent on RNA polymerase II.

呼吸道合胞病毒(RSV)是一种引起急性呼吸道疾病的病毒,其反向遗传系统是了解RSV致病性的有效工具。迄今为止,依赖于T7 RNA聚合酶的方法通常用于RSV。虽然这种方法已经很好地建立,重组RSV也很好地从转染的细胞中获救,但人工供应T7 RNA聚合酶的要求限制了其应用。为了克服这个问题,我们建立了一个依赖于RNA聚合酶II的反向遗传系统,该系统更方便从各种细胞系中恢复重组病毒。首先,我们确定了转染效率高的人细胞系,其中RSV可以有效地复制。两种人类细胞系Huh-7和293T允许表达RSV的重组绿色荧光蛋白的繁殖。我们的小基因组系统显示,RSV的高效转录和复制发生在Huh-7和293T细胞中。然后,我们证实了在Huh-7和293T细胞中都能救出表达重组绿色荧光蛋白的RSV。此外,从Huh-7和293T细胞中提取的病毒的生长能力与用常规方法提取的重组RSV相似。因此,我们成功地建立了一个依赖于RNA聚合酶II的RSV新的反向遗传系统。
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引用次数: 0
Knock-out of multidrug efflux pump MexXY-OprM results in increased susceptibility to antimicrobial peptides in Pseudomonas aeruginosa 敲除多药外排泵MexXY-OprM导致铜绿假单胞菌对抗菌肽的敏感性增加
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-07-09 DOI: 10.1111/1348-0421.13089
Anke Neidig, Nikola Strempel, Nadine Bianca Waeber, Waleska Stephanie da Cruz Nizer, Joerg Overhage

Multidrug efflux systems of the resistance-nodulation-cell division family play a crucial role in resistance of Pseudomonas aeruginosa to a large variety of antibiotics. Here, we investigated the role of clinically relevant efflux pumps MexABOprM, MexCDOprJ, and MexXYOprM in resistance against different cationic antimicrobial peptides (AMPs). Our results indicate that a knock-out in efflux pump MexXY-OprM increased susceptibility to some AMPs by two- to eightfold. Our data suggest a contribution of MexXY-OprM in resistance to certain AMPs in P. aeruginosa, which should be considered in the future development of new and highly active antimicrobial peptides to fight multidrug resistant infections.

耐药-结瘤-细胞分裂家族的多药外排系统在铜绿假单胞菌对多种抗生素的耐药中起着至关重要的作用。在这里,我们研究了临床相关的外排泵MexAB−OprM、MexCD−OprJ和MexXY−OprM在对不同阳离子抗菌肽(amp)的耐药性中的作用。我们的结果表明,外排泵MexXY-OprM的敲除使对某些amp的易感性增加了2至8倍。我们的数据表明,MexXY-OprM在铜绿假单胞菌对某些amp的耐药性中发挥了作用,这应该在未来开发新的高活性抗菌肽以对抗多药耐药感染时加以考虑。
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引用次数: 0
Investigating theobromine as a potential anti-human coronaviral agent 研究可可碱作为一种潜在的抗人类冠状病毒药物
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-07-06 DOI: 10.1111/1348-0421.13086
Jiajing Li, Yining Wang, Sajjan Rajpoot, Marla Lavrijsen, Qiuwei Pan, Pengfei Li, Mirza S. Baig

Coronaviruses (CoVs) have long been known to infect humans, mainly alpha-CoV and beta-CoV. The vaccines developed for SARS-CoV-2 are likely not effective against other coronavirus species, whereas the risk of the emergence of new strains that may cause the next epidemic/pandemic is high. The development of antiviral drugs that are effective across different CoVs represents a viable strategy for improving pandemic preparedness. In this study, we aim to identify pan-coronaviral agents by targeting the conserved main protease (Mpro). For drug screening, the catalytic dyad of four human CoVs (HCoVs: SARS-CoV-2, and seasonal CoV NL63, OC43, and 229E) was targeted by molecular docking. The identified leading candidate theobromine, a xanthine derivative, was further tested in cell culture models of coronavirus infection. Theobromine binds strongly with the catalytic dyad (His41 and Cys144/145) of SARS-CoV-2 and HCoV-NL63 Mpro, mildly with HCoV-OC43, but not with HCoV-229E. However, theobromine only shows dose-dependent inhibition in Calu3 cells inoculated with SARS-CoV-2, but not in cells inoculated with seasonal CoVs. Theobromine exerts antiviral activity against coronavirus infections potentially through targeting Mpro. However, the antiviral potency is distinct among different CoVs.

人们早就知道冠状病毒(cov)会感染人类,主要是甲型冠状病毒和乙型冠状病毒。针对SARS-CoV-2开发的疫苗可能对其他冠状病毒物种无效,而出现可能导致下一次流行/大流行的新菌株的风险很高。开发对不同冠状病毒有效的抗病毒药物是改善大流行防范的一项可行战略。在这项研究中,我们的目标是通过靶向保守主蛋白酶(Mpro)来鉴定泛冠状病毒因子。为了进行药物筛选,我们通过分子对接的方法靶向了4种人类冠状病毒(hcov: SARS-CoV-2和季节性冠状病毒NL63、OC43和229E)的催化二联体。确定的主要候选可可碱是一种黄嘌呤衍生物,在冠状病毒感染的细胞培养模型中进行了进一步测试。可可碱与SARS-CoV-2和HCoV-NL63 Mpro的催化二联体(His41和Cys144/145)结合强烈,与HCoV-OC43结合轻微,但与HCoV-229E不结合。然而,可可碱仅在接种了SARS-CoV-2的Calu3细胞中表现出剂量依赖性抑制,而在接种了季节性cov的细胞中没有表现出剂量依赖性抑制。可可碱可能通过靶向Mpro对冠状病毒感染发挥抗病毒活性。然而,不同冠状病毒的抗病毒效力是不同的。
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引用次数: 0
Analysis of multidrug-resistant determinants of clinically isolated Acinetobacter baumannii CYZ via whole genome sequencing 临床分离鲍曼不动杆菌CYZ多药耐药决定因素全基因组测序分析
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-07-04 DOI: 10.1111/1348-0421.13087
Xia Chuai, Yaya Zhou, Junhua Feng, Menghan Yu, Yan Wu, Lujuan Han, Yan Zhao, Hongxiu Qiao, Zhiyun Gao, Jian Li, Lixin Xie, Wenting Zhao, Changle Wang

Acinetobacter baumannii is a multidrug-resistant coccobacillus responsible for severe nosocomial infectious diseases. This study mainly focuses on investigating the antimicrobial resistance features of a clinically isolated strain (A. baumannii CYZ) using the PacBio Sequel II sequencing platform. The chromosomal size of A. baumannii CYZ is 3,960,760 bp, which contains a total of 3803 genes with a G + C content of 39.06%. Functional analysis performed using the Clusters of Orthologous Groups of Proteins (COGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, as well as the Comprehensive Antibiotic Resistance Database (CARD) revealed a complicated set of antimicrobial resistance determinants in the genome of A. baumannii CYZ, which were mainly classified into multidrug efflux pumps and transport systems, β-lactamase relative and penicillin-binding proteins, aminoglycoside modification enzymes, alternation of antibiotic target sites, lipopolysaccharide relative, and other mechanisms. A total of 35 antibiotics were tested for the antimicrobial susceptibility of A. baumannii CYZ, and the organism exhibited a stronger antimicrobial resistance ability. The phylogenetic relationship indicated that A. baumannii CYZ has high homology with A. baumannii ATCC 17978; however, the former also exhibited its specific genome characteristics. Our research results give insight into the genetic antimicrobial-resistant features of A. baumannii CYZ as well as provide a genetic basis for the further study of the phenotype.

鲍曼不动杆菌是一种多重耐药球芽孢杆菌,可导致严重的医院传染病。本研究主要利用PacBio Sequel II测序平台研究临床分离菌株(鲍曼不动杆菌CYZ)的耐药特征。鲍曼不动杆菌CYZ染色体大小为3960760 bp,共包含3803个基因,G + C含量为39.06%。利用同源蛋白群(Clusters of Orthologous Groups of Proteins, COGs)、基因本体(Gene Ontology, GO)、京都基因与基因组百科全书(KEGG)数据库以及抗生素耐药性综合数据库(Comprehensive Antibiotic Resistance Database, CARD)进行的功能分析显示,鲍曼不动杆菌CYZ基因组中存在一组复杂的耐药决定因素,主要分为多药外排泵和转运系统、β-内酰胺酶相关蛋白和青霉素结合蛋白。氨基糖苷修饰酶,抗生素靶位点的改变,脂多糖相关,和其他机制。鲍曼不动杆菌CYZ共对35种抗生素进行了药敏试验,结果表明该菌具有较强的耐药能力。系统发育关系表明,鲍曼不动杆菌CYZ与鲍曼不动杆菌ATCC 17978具有高度同源性;然而,前者也表现出其特定的基因组特征。我们的研究结果揭示了鲍曼不动杆菌CYZ的遗传耐药特征,并为进一步研究其表型提供了遗传基础。
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引用次数: 0
Issue Information – Cover 问题信息-封面
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-07-03 DOI: 10.1111/1348-0421.12999

Cover photograph: Electron microscopy of lectin-treated SARS-CoV-2. SARS-CoV-2 was concentrated by using a porous membrane-filter unit and incubated with saline or 9 μM OAA, ESA-2, or HypninA-2 for 10 min. The sample was negative-stained and observed by transmission electron microscopy. The left panel for each lectin is a 2000x view, and the right panel is a magnified view of the designated region. The area of n viruses on the electron micrographs was measured and divided into groups corresponding to less than 2 viral particles (~2), 2-5 (2~5), and 5 or more (5~), and the percentages were plotted on a pie chart. For HypninA-2, all virus particles made up about 10 huge clumps, and nothing that appeared to be free virus particles was found. Therefore, no pie chart was drawn. Microbiol Immunol: 67:334–344. Article link here

封面照片:凝集素处理的严重急性呼吸系统综合征冠状病毒2型的电子显微镜。使用多孔膜过滤装置浓缩严重急性呼吸系统综合征冠状病毒2型,并与盐水或9μM OAA、ESA-2或HypninA-2孵育10分钟。样品进行阴性染色,并通过透射电子显微镜进行观察。每个凝集素的左侧面板是2000x视图,右侧面板是指定区域的放大视图。测量n种病毒在电子显微照片上的面积,并将其分为小于2个病毒颗粒(~2)、2-5个(2~5)和5个或更多(5~)的组,并将百分比绘制在饼图上。对于HypninA-2,所有的病毒颗粒组成了大约10个巨大的团块,没有发现任何游离的病毒颗粒。因此,没有绘制饼图。微生物免疫学:67:334-344。此处的文章链接
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引用次数: 0
Nicotine promotes Mycobacterium tuberculosis H37Rv growth and overexpression of virulence genes 尼古丁促进结核分枝杆菌H37Rv的生长和毒力基因的过表达
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-06-28 DOI: 10.1111/1348-0421.13085
Bruno Rivas-Santiago, Jeny de Haro-Acosta, Adrian Rodríguez Carlos, Mariana H. Garcia-Hernandez, Carmen J. Serrano, Irma Gonzalez-Curiel, Cesar Rivas-Santiago

Tobacco consumption increases the susceptibility to develop infectious diseases such as tuberculosis (TB). Nicotine (Nc) is the main component of cigarette smoke with immunomodulatory properties, however, its effect on Mycobacterium tuberculosis (Mtb) has been scarcely investigated. The present study evaluated the effect of nicotine on the growth of Mtb and on the induction of virulence-related genes. Mycobacteria were exposed to different concentrations of nicotine then Mtb growth was evaluated. Subsequently, the expression of the virulence-related genes lysX, pirG, fad26, fbpa, ompa, hbhA, esxA, esxB, hspx, katG, lpqh, and caeA was evaluated by RT-qPCR. The effect of nicotine on intracellular Mtb was also evaluated. The results showed that nicotine promotes the growth of Mtb both extracellularly and intracellularly and increases the expression of genes related to virulence. In summary, nicotine promotes the growth of Mtb and the expression of virulence-related genes that could be correlated with the increased the risk of smokers developing TB.

吸烟会增加患结核病等传染病的易感性。尼古丁是香烟烟雾中的主要成分,具有免疫调节作用,但对其对结核分枝杆菌(Mtb)的作用研究较少。本研究评估了尼古丁对结核分枝杆菌生长和毒力相关基因诱导的影响。将分枝杆菌暴露于不同浓度的尼古丁中,然后评估结核分枝杆菌的生长。随后,通过RT-qPCR评估毒力相关基因lysX、pirG、fad26、fbpa、ompa、hbhA、esxA、esxD、hspx、katG、lpqh和caeA的表达。还评估了尼古丁对细胞内Mtb的影响。结果表明,尼古丁在细胞外和细胞内都能促进结核分枝杆菌的生长,并增加与毒力相关的基因的表达。总之,尼古丁促进结核分枝杆菌的生长和毒力相关基因的表达,这些基因可能与吸烟者患结核病的风险增加有关。
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引用次数: 0
Huoxue Jiedu Recipe represses mitochondrial fission to alleviate submandibular gland inflammation in Sjögren's syndrome 活血解毒方抑制线粒体裂变缓解Sjögren综合征颌下腺炎症
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-06-20 DOI: 10.1111/1348-0421.13084
Jiangxi Xu, Zhengdong Shen, Yun Du, Shai Zhang, Yajing Xi, Yuting Zhang, Dan Wang, Yuelan Zhu

Sjögren's syndrome (SS) is the second most common autoimmune rheumatism. Huoxue Jiedu Recipe (HXJDR) is a kind of traditional Chinese medicine with a variety of pharmacological functions; however, its biological function in SS has not been studied yet. Peripheral blood mononuclear cells (PBMCs) and serum samples were isolated from healthy controls and patients with SS. NOD/Ltj mice were used for developing the SS mouse model. The levels of inflammatory cytokines and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome–related markers as well as dynamin-related protein 1 (Drp1) were determined by ELISA, quantitative real-time PCR, and western blot analysis, respectively. Hematoxylin and eosin and TUNEL staining detected the pathological damage. A transmission electron microscope was used to observe the mitochondrial microstructure. Inflammatory cytokines IL-18, IL-1β, B-cell activating factor (BAFF), BAFF-receptor (BAFF-R), IL-6, and TNF-α in serum samples and NLRP3 inflammasome–related makers (NLRP3, cysteinyl aspartate–specific proteinase 1 [caspase-1], apoptosis-associated speck-like protein containing a caspase-1 recruitment domain [ASC], IL-1β) in PBMCs were greatly upregulated in patients with SS. Furthermore, cytoplasmic phosphorylation of Drp1 and mitochondrial Drp1 level were significantly increased in PBMCs, while mitochondrial swelling and fuzzy inner ridge were observed in PBMCs of patients with SS, suggesting increased mitochondrial fission. Compared with control mice, SS mice showed decreased salivary flow rate, increased submandibular gland index, and more severe inflammatory infiltration and damage as well as mitochondrial fission in submandibular gland tissues. After HXJDR administration, these effects were significantly reversed. HXJDR treatment could alleviate the inflammatory infiltration and pathological damage in submandibular glands of SS mice by inhibiting Drp-1–dependent mitochondrial fission.

干燥综合征(SS)是第二常见的自身免疫性风湿病。活血解毒方是一种具有多种药理作用的中药;然而,其在SS中的生物学功能尚未得到研究。从健康对照组和SS患者中分离外周血单核细胞(PBMC)和血清样本。采用NOD/Ltj小鼠建立SS小鼠模型。分别通过ELISA、实时定量PCR和蛋白质印迹分析测定炎性细胞因子和NOD样受体家族pyrin结构域3(NLRP3)炎症小体相关标记物以及动力蛋白相关蛋白1(Drp1)的水平。苏木精、伊红和TUNEL染色检测病理损伤。用透射电镜观察线粒体的微观结构。SS患者PBMC中的炎性细胞因子IL-18、IL-1β、B细胞活化因子(BAFF)、BAFF受体(BAFF-R)、IL-6和TNF-α以及NLRP3炎症小体相关因子(NLRP3、半胱天冬氨酸-特异性蛋白酶1[case-1]、含有半胱氨酸天冬氨酸蛋白酶1募集结构域的凋亡相关斑点样蛋白[ASC]和IL-1β)显著上调。此外,Drp1的细胞质磷酸化和线粒体Drp1水平在PBMC中显著增加,而在SS患者的PBMC中观察到线粒体肿胀和模糊的内嵴,表明线粒体分裂增加。与对照小鼠相比,SS小鼠的唾液流速降低,下颌下腺指数增加,下颌下腺体组织中炎症浸润和损伤以及线粒体分裂更加严重。服用HXJDR后,这些作用显著逆转。HXJDR治疗可通过抑制Drp-1依赖性线粒体分裂来减轻SS小鼠下颌下腺的炎症浸润和病理损伤。
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引用次数: 0
Re-recognition of innate immune memory as an integrated multidimensional concept 重新认识先天免疫记忆作为一个综合的多维概念
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-06-13 DOI: 10.1111/1348-0421.13083
Chang Tang, Shoichiro Kurata, Naoyuki Fuse

In the past decade, the concept of immunological memory, which has long been considered a phenomenon observed in the adaptive immunity of vertebrates, has been extended to the innate immune system of various organisms. This de novo immunological memory is mainly called “innate immune memory”, “immune priming”, or “trained immunity” and has received increased attention because of its potential for clinical and agricultural applications. However, research on different species, especially invertebrates and vertebrates, has caused controversy regarding this concept. Here we discuss the current studies focusing on this immunological memory and summarize several mechanisms underlying it. We propose “innate immune memory” as a multidimensional concept as an integration between the seemingly different immunological phenomena.

在过去的十年中,免疫记忆的概念已经被扩展到各种生物的先天免疫系统中,这一概念一直被认为是脊椎动物适应性免疫中观察到的一种现象。这种从头开始的免疫记忆主要被称为“先天免疫记忆”、“免疫启动”或“训练免疫”,因其在临床和农业应用方面的潜力而受到越来越多的关注。然而,对不同物种,特别是无脊椎动物和脊椎动物的研究,对这一概念引起了争议。在这里,我们讨论了目前的研究集中在这种免疫记忆,并总结了其背后的几种机制。我们提出“先天免疫记忆”作为一个多维的概念之间的整合看似不同的免疫现象。
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引用次数: 0
Issue Information – Cover 问题信息-封面
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-06-05 DOI: 10.1111/1348-0421.12997

Cover photograph: HCMV infection interrupted mitochondrial dynamics in THP-1 cells. THP-1 cells were infected with HCMV (MOI = 5) for the indicated times. Cells were stained and subjected to confocal microscopy. Microbiol Immunol: 67:303–313. Article link here

封面照片:HCMV感染中断了THP-1细胞的线粒体动力学。THP-1细胞用HCMV(MOI=5)感染所指示的时间。对细胞进行染色并进行共聚焦显微镜检查。微生物免疫学:67:303-313。此处的文章链接
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引用次数: 0
Capture and neutralization of SARS-CoV-2 and influenza virus by algae-derived lectins with high-mannose and core fucose specificities 利用具有高甘露糖和核心焦点特异性的藻类衍生凝集素捕获和中和SARS-CoV-2和流感病毒
IF 2.6 4区 医学 Q3 Immunology and Microbiology Pub Date : 2023-05-29 DOI: 10.1111/1348-0421.13082
Tanuza Nazmul, Basirat M. Lawal-Ayinde, Tomoko Morita, Reiko Yoshimoto, Akifumi Higashiura, Akima Yamamoto, Toshihito Nomura, Yukiko Nakano, Makoto Hirayama, Hiroshi Kurokawa, Yasuyuki Kitamura, Kanji Hori, Takemasa Sakaguchi

We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)–type glycan-specific lectins OAA and KAA-1 or core fucose–specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose–type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.

我们首先研究了几种藻类衍生凝集素与严重急性呼吸综合征冠状病毒-2 (SARS-CoV-2)之间的相互作用。我们使用高甘露糖(HM)型聚糖特异性凝集素OAA和KAA-1或核心焦点特异性凝集素hypninA-2构建凝集素柱,并与SARS-CoV-2进行结合实验。结果表明,这些凝集素能够与病毒结合。此外,在检测9种不同凝集素的中和能力时,发现KAA-1、ESA-2和hypninA-2能有效中和SARS-CoV-2。在与糖蛋白的竞争性抑制实验中,证实了中和是通过hm型或核心焦点型聚糖发生的。然而,与其他凝集素(如OAA)没有观察到中和作用。这种KAA-1和ESA-2具有中和能力而OAA不具有中和能力的趋势也与流感病毒相似。电镜观察显示,KAA-1和hypninA-2对SARS-CoV-2颗粒聚集程度较强,而OAA对SARS-CoV-2颗粒聚集程度较低。我们认为,SARS-CoV-2的中和涉及多种因素,如S蛋白上的聚糖附着位点、凝集素的大小及其聚集倾向,这些因素会抑制受体结合或病毒颗粒的聚集。本研究表明,几种藻类来源的凝集素可以中和SARS-CoV-2,凝集素柱可以有效地回收和浓缩病毒。
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引用次数: 0
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Microbiology and Immunology
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