Immunogenicity of the Spike Glycoprotein of Bat SARS-like Coronavirus

A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γand IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.

The Immunity Induced by Recombinant Spike Proteins of SARS Coronavirus in Balb/c Mice

The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and puri- fied by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with com- plete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.

The possible mechanism of lung injury induced by severe acute respiratory syndrome coronavirus spike glycoprotein

Objective To investigate the role of severe acute respiratory syndrome coronavirus (SARS-CoV) in the induction of acute lung injury by promoting the synthesis of chemokine/cytokines in human endothelial cells. Methods Twenty-three SARS patients were enrolled in this study, comprising 15 males and 8 females, aged 27～55 years, mean (36±6) years. They were treated at Guan-

Bioinformatic Analysis of SARS-CoV-2 Genomes to Develop a Universal Coronavirus Vaccine

COVID-19 is caused by the SARS-CoV-2 virus. Current RNA vaccines Pfizer/BioNTech’s BNT162b2 and Moderna’s mRNA-1273 are more than 94% successful in preventing infection. The spike protein of the virus is essential for the interaction and internalization of the virus in the host cell and is considered a prime target for vaccine development against the SARS virus. This study aims to identify highly conserved sequences in spike protein or other sections of the viral genome that can potentially be used to develop a universal coronavirus vaccine. Bioinformatic analysis of 258,269 full-length SARS-CoV-2 genomic sequences in the NCBI database was carried out using a custom Perl Script. All sequences were compared to the spike protein and full-length viral genome reference to find 100 nucleotide-long segments that were at least 99% conserved across SARS-CoV-2 sequences. The analysis resulted in a >99.5% conserved 114-nucleotide segment on the spike protein and a 99.49% conserved 104-nucleotide segment on the non-spike protein section of the viral genome. The conserved sequences from this study may be useful in developing an RNA or protein vaccine that may be effective against future SARS-CoV-2 strains or could act as a universal vaccine if these sequences are present in other coronavirus families.

Comparing the effects of three decoctions for coronavirus disease 2019 on severe acute respiratory syndrome coronavirus 2-related toll-like receptors-mediated inflammations

OBJECTIVE:To compare the anti-inflammatory effects of three decoctions for coronavirus disease 2019(COVID-19)[Qingfei Paidu Tang(清肺排毒汤),QF;Huashi Baidu Fang(化湿败毒方),HS;Xuanfei Baidu Fang(宣肺败毒方),XF]in parallelly experimental models by using severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)-related toll-like receptors(TLRs)ligands and its spike(S)protein as stimulators.METHODS:RAW264.7 macrophages were used to investigate the effects of three decoctions on the inflammations induced by R848,poly(I:C),lipopolysaccharide(LPS),as well as SARS-CoV-2 S protein,in vitro.Mouse endotoxemia model was used for evaluating their anti-inflammatory actions in vivo.The levels of monocyte chemoattractant protein-1(MCP-1),interleukin-6(IL-6),tumor necrosis factorα(TNF-α),and interferon-β(IFN-β)were determined by enzyme-linked immunosorbent assay.RESULTS:Three decoctions could decrease supernatant IL-6,MCP-1,nitric oxide(NO)and TNF-αto varying degrees in activated macrophages.Meanwhile,they did not increase the level of antiviral cytokine IFN-βinduced by TLR3 and TLR4 ligands,but rather suppressed it,suggesting that externally administrated type I interferons(IFN-Is)may be needed for the severe COVID-19 cases characterized by deficient IFN-Is.In mouse endotoxemia model,all three decoctions could suppress serum pro-inflammatory cytokines,but only QF could relieve hypothermia and antagonize diarrhea.CONCLUSIONS:Collectively,our study compared,for the first time,the effects of three decoctions on SARSCoV-2-related TLRs-mediated inflammations.In vitro,three decoctions exert similar suppressive effects on inflammatory cytokines induced by SARS-CoV-2-related TLRs ligands,as well as S protein.In vivo,QF possesses the strongest effects compared with HS and XF.These findings may provide not only experimental basis for the clinical use of three decoctions,but also a rationale for the combination therapy with IFN-Is.

Using the spike protein feature to predict infection risk and monitor the evolutionary dynamic of coronavirus

Background:Coronavirus can cross the species barrier and infect humans with a severe respiratory syndrome.SARS-CoV-2 with potential origin of bat is still circulating in China.In this study,a prediction model is proposed to evaluate the infection risk of non-human-origin coronavirus for early warning.Methods:The spike protein sequences of 2666 coronaviruses were collected from 2019 Novel Coronavirus Resource(2019nCoVR)Database of China National Genomics Data Center on Jan 29,2020.A total of 507 human-origin viruses were regarded as positive samples,whereas 2159 non-human-origin viruses were regarded as negative.To capture the key information of the spike protein,three feature encoding algorithms(amino acid composition,AAC;parallel correlation-based pseudo-amino-acid composition,PC-PseAAC and G-gap dipeptide composition,GGAP)were used to train 41 random forest models.The optimal feature with the best performance was identified by the multidimensional scaling method,which was used to explore the pattern of human coronavirus.Results:The 10-fold cross-validation results showed that well performance was achieved with the use of the GGAP(g=3)feature.The predictive model achieved the maximum ACC of 98.18%coupled with the Matthews correlation coefficient(MCC)of 0.9638.Seven clusters for human coronaviruses(229E,NL63,OC43,HKU1,MERS-CoV,SARS-CoV,and SARS-CoV-2)were found.The cluster for SARS-CoV-2 was very close to that for SARS-CoV,which suggests that both of viruses have the same human receptor(angiotensin converting enzyme II).The big gap in the distance curve suggests that the origin of SARS-CoV-2 is not clear and further surveillance in the field should be made continuously.The smooth distance curve for SARS-CoV suggests that its close relatives still exist in nature and public health is challenged as usual.Conclusions:The optimal feature(GGAP,g=3)performed well in terms of predicting infection risk and could be used to explore the evolutionary dynamic in a simple,fast and large-scale manner.The study may be beneficial for the surveillance of the genome mutation of coronavirus in the field.

Immune response in Balb/c mice induced by recombinant spike protein of severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly aggressive pathogen that caused SARS in 2003 and 2004.^(1-4) Spike protein (S) on the surface of virus particles mediates the attachment of the virus to cell surface receptors and induces the fusion of viral and cellular membranes. According to the epitope analysis and structure of S protein, we used two fragments of S protein S1 (108-488aa) and S2 (723-938aa) expressed in Escherichia coli and immunized Balb/c mice to investigate the immune response to the recombinant proteins in mice.~5

Dissection of SARS Coronavirus Spike Protein into Discrete Folded Fragments

The spike protein of the severe acute respiratory syndrome coronavirus （SARS-CoV） mediates cell fusion by binding to target cell surface receptors. This paper reports a simple method for dissecting the viral protein and for searching for foldable fragments in a random but systematic manner. The method involves digestion by DNase I to generate a pool of short DNA segments, followed by an additional step of reassembly of these segments to produce a library of DNA fragments with random ends but controllable lengths. To rapidly screen for discrete folded polypeptide fragments, the reassembled gene fragments were further cloned into a vector as N-terminal fusions to a folding reporter gene which was a variant of green fluorescent protein. Two foldable fragments were identified for the SARS-CoV spike protein, which coincide with various anti-SARS peptides derived from the hepated repeat （HR） region 2 of the spike protein. The method should be applicable to other viral proteins to isolate antigen or vaccine candidates, thus providing an alternative to the full-length proteins （subunits） or linear short peptides.

Molecular Docking Study of the Binding Interaction of Hydroxychloroquine, Dexamethasone and Other Anti-Inflammatory Drugs with SARS-CoV-2 Protease and SARS-CoV-2 Spikes Glycoprotein

Aims: The outbreak of the novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still accountable for millions of deaths worldwide and declared as a global pandemic by the World Health Organisation. Despite efforts, there is still limited evidence available on a successful potent inhibitor with a low toxicity profile that can aid in the prevention and/or treatment of COVID-19. This study will focus on four main aspects: 1) screening 19 Food Drug and Administration (FDA) approved drugs using computational molecular docking;2) assessing drug toxicity profiles using biological data;3) recommending potential therapies against COVID-19 and 4) supplementing currently used therapies. Methods: 19 FDA approved drugs were investigated against the crystal structure of SARS-CoV-2 protease (6LU7) and SARS-CoV-2 glycoprotein (6VXX) using a computational molecular docking software, Molecular Operating Environment (MOE). Separately, on MOE, 6LU7 and 6VXX were loaded, prepared, and the binding pockets located. The drug’s canonical SMILES were imported, minimised, and docked on the prepared proteins using a search algorithm to establish the highest stability conformation. Drugs were ranked depending on binding properties and biological data to assess safety;steric clashes and voids in the binding site were also analysed. Results and discussion: Out of the nineteen (19) FDA approved drugs, 18 inhibited 6LU7 and 13 inhibited 6VXX. High-ranked drugs based on binding properties for 6LU7 were hydroxychloroquine, dexamethasone, naproxen, etoricoxib, and ibuprofen. For 6VXX were hydroxychloroquine, celecoxib, etoricoxib, meloxicam, and parecoxib. Considering safety profile, the top 3 drugs in descending order for 6LU7 were etoricoxib, naproxen and dexamethasone and for 6VXX were etoricoxib, meloxicam, and parecoxib. Compared to the literature, the results were consistent for dexamethasone which was effective against 6LU7. However, for hydroxychloroquine and ibuprofen, there was conflicting literature regarding safety and efficacy. Conclusion and future work: The findings suggest that against COVID-19 etoricoxib might be effective as a therapeutic and prophylactic measure. Naproxen and dexamethasone would be more effective as treatment only while meloxicam and parecoxib as prophylaxis. However, future studies are needed to validate these findings. Compared to previous literature, the findings in this study also support the use of dexamethasone over hydroxychloroquine and ibuprofen for COVID-19 based on the binding and safety properties. Despite this, future research should explore the impressive binding properties displayed by hydroxychloroquine and ibuprofen to aid in developing a new drug against COVID-19.

Amino acid variation analysis of surface spike glycoprotein at 614 in SARS-CoV-2 strains

As severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)continues to disperse globally with worrisome speed,identifying amino acid variations in the virus could help to understand the characteristics of it.Here,we studied 489 SARS-CoV-2 genomes obtained from 32 countries from the Nextstrain database and performed phylogenetic tree analysis by clade,country,and genotype of the surface spike glycoprotein(S protein)at site 614.We found that virus strains from China's Mainland were mostly distributed in Clade B and Clade undefined in the phylogenetic tree,with very few found in Clade A.In contrast,Clades A2(one case)and A2a(112 cases)predominantly contained strains from European regions.Moreover,Clades A2 and A2a differed significantly from those of China's Mainland in age of infected population(P Z 0.0071,mean age 40.24 to 46.66),although such differences did not exist between the US and China's Mainland.Further analysis demonstrated that the variation of the Sprotein at site 614(QHD43416.1:p.614D>G)was a characteristic of stains in Clades A2 and A2a.Importantly,this variation was predicted to have neutral or benign effects on the function of the S protein.In addition,global quality estimates and 3D protein structures tended to be different between the two S proteins.In summary,we identified different genomic epidemiology among SARS-CoV-2 strains in different clades,especially in an amino acid variation of the S protein at 614,revealing potential viral genome divergence in SARS-CoV-2 strains.

Dual inhibition of COVID-19 spike glycoprotein and main protease 3CLpro by Withanone from Withania somnifera

Objective:To identify the safe and effective natural inhibitors of spike glycoprotein and main protease 3CLpro using potential natural antiviral compounds which are studied under various animal models and viral cell lines.Methods:First,compounds were retrieved from the Pub Chem database and predicted for their druggability using the Mol Soft web server,and compounds having drug-like property were predicted for major adverse drug reactions like cardiotoxicity,hepatotoxicity,arrhythmia,myocardial infarction,and nephrotoxicity using ADVERpred.Docking of nontoxic antiviral compounds with spike glycoprotein and main protease 3CLpro was performed using Auto Dock vina by PyRx 0.8 version.The stability of compoundprotein interactions was checked by molecular dynamic(MD)simulation using Schrodinger Desmond software.Results:Based on the druggable and nontoxic profile,nine compounds were selected.Among them,Withanone from Withania somnifera showed the highest binding affinity and best fit at active sites 1 of spike glycoprotein(glycosylation site)and main protease 3CLpro via interacting with active site amino acid residues before and after MD simulation at 50 ns.Withanone,which may reduce the glycosylation of SARS-CoV-2 via interacting with Asn343 and inhibit viral replication.Conclusion:The current study reports Withanone as a non-toxic antiviral against SARS-CoV-2 and serve as a potential lead hit for further experimental validation.

The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2

Bat coronavirus(CoV)RaTG13 shares the highest genome sequence identity with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)among all known coronaviruses,and also uses human angiotensin converting enzyme 2(hACE2)for virus entry.Thus,SARS-CoV-2 is thought to have originated from bat.However,whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive.Here,we found that Rhinolophus affinis bat ACE2(Ra ACE2)is an entry receptor for both SARSCoV-2 and Ra TG13,although the binding of Ra ACE2 to the receptor-binding domain(RBD)of SARSCoV-2 is markedly weaker than that of h ACE2.We further evaluated the receptor activities of ACE2 s from additional 16 diverse animal species for Ra TG13,SARS-CoV,and SARS-CoV-2 in terms of S protein binding,membrane fusion,and pseudovirus entry.We found that the Ra TG13 spike(S)protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2,and seven out of sixteen different ACE2 s function as entry receptors for all three viruses,indicating that all three viruses might have broad host rages.Of note,Ra TG13 S pseudovirions can use mouse,but not pangolin ACE2,for virus entry,whereas SARS-CoV-2 S pseudovirions can use pangolin,but not mouse,ACE2 enter cells efficiently.Mutagenesis analysis revealed that residues 484 and 498 in Ra TG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2 s.Finally,two polymorphous Rhinolophous sinicus bat ACE2 s showed different susceptibilities to virus entry by Ra TG13 and SARS-CoV-2 S pseudovirions,suggesting possible coevolution.Our results offer better understanding of the mechanism of coronavirus entry,host range,and virushost coevolution.

牛冠状病毒刺突蛋白研究进展

牛冠状病毒(bovine coronavirus, BCoV)是一种能感染牛肠道、呼吸道引起新生犊牛腹泻、成年牛冬季痢疾和各年龄段的牛呼吸道疾病的重要病原体,严重危害着养牛业的发展。刺突(spike, S)蛋白是该病毒主要的结构蛋白和免疫原性蛋白,在病毒初始感染和诱导保护性免疫中发挥重要作用。本文就该病毒刺突蛋白的结构特征、生物学功能及应用研究、免疫原性、变异原因和潜在双受体系统的最新研究进展做一阐述,期望为新型疫苗研制和靶点治疗药物开发提供科学依据,以便制定快速有效的安全防治对策应对预出现的新毒株。

Characterization of the receptor-binding domain(RBD)of 2019 novel coronavirus:implication for development of RBD protein as a viral attachment inhibitor and vaccine

The outbreak of Coronavirus Disease 2019(COVID-19)has posed a serious threat to global public health,calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),also known as 2019 novel coronavirus(2019-nCoV).The CoV spike(S)protein plays the most important roles in viral attachment,fusion and entry,and serves as a target for development of antibodies,entry inhibitors and vaccines.Here,we identified the receptor-binding domain(RBD)in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2(ACE2)receptors.SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and,hence,attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells,thus inhibiting their infection to host cells.SARS-CoV RBD-specific antibodies could crossreact with SARS-CoV-2 RBD protein,and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2,suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

严重急性呼吸综合征冠状病毒Spike蛋白诱发小鼠免疫损伤的实验研究

目的研究严重急性呼吸综合征冠状病毒(SARS-CoV)的Spike蛋白(S蛋白)引起免疫病理损伤的分子机制。方法采用信号转导通路发现者基因芯片筛选相关诱导免疫炎症的信号转导通路基因的表达,通过RT-PCR对基因芯片筛选基因进一步验证并观察信号分子阻断剂干预的影响;将SARS-CoV的S蛋白及信号分子阻断剂分别经气管滴注、尾静脉注射的途径感染BALB/c小鼠,通过免疫组织化学观察肺及免疫器官的病理变化。结果S蛋白诱导了与γ干扰素(IFN-γ)类似的JAK-STAT的信号通路相关基因的表达,RT-PCR证实S蛋白诱导了人支气管上皮细胞的γ干扰素诱导蛋白10(IP-10)基因表达,该作用能够被JAK3抑制剂完全阻断。经气管滴注或尾静脉注射S蛋白均可引起小鼠肺损伤和脾脏损伤。JAK3抑制剂能够抑制S蛋白诱导小鼠肺内IP-10表达,减轻病理损伤。结论JAK-STAT信号转导通路的激活可能在SARS-CoV的S蛋白诱导的以IP-10为代表的炎症级联中起重要作用,该发现可为发展抗SARS病毒诱导的免疫损伤治疗提供新的策略。

Comparison of lentiviruses pseudotyped with S proteins from coronaviruses and cell tropisms of porcine coronaviruses

The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins(S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus(PEDV) and transmissible gastroenteritis virus(TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected VeroCCL-81(monkey kidney), Huh-7(human liver), and PK-15(pig kidney) cells efficiently. CCL94(cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.

基于增量学习的冠状病毒人际感染预测研究

2019年底至今,新型冠状病毒大流行严重影响了公众卫生和社会秩序,而基于机器学习的预测方法可以判别冠状病毒的可感染性表型和大流行风险。目前,已发现6类感染人的冠状病毒,病毒基因组序列差异显著,病毒持续遗传变异导致机器学习模型性能下降并引发潜在的学习遗忘现象。文章基于增量学习的模型框架,使用One-class SVM算法对冠状病毒新类群进行持续鉴别,并进一步使用参数共享和知识蒸馏的联合策略改造BP神经网络,对冠状病毒人际感染表型进行持续学习和预测。结果显示,One-class SVM对6类病毒区分的权衡参数v组合在0.92、0.81、0.24、0.11、0.55、0.20下达到最优的病毒类群分类效果;当隐藏层节点批次增加为6时,预测模型取得最好性能表现,IAC取得最大值0.9035,BT取得最大值-0.0399,有效地抑制了神经网络模型的学习遗忘趋势,模型的预测性能接近联合训练的性能表现(IAC:0.9236),明显优于未使用知识蒸馏的神经网络(IAC:0.7764),进一步与其他增量方法比较,优于基于样本的ESRIL方法(IAC:0.8662)和基于模型参数的CCLL方法(IAC:0.8853),具有重要的公共卫生应用价值。

靶向SARS-CoV-2的纳米抗体研究进展

席卷全球的新型冠状病毒病(corona virus disease 2019,COVID-19)是由严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)引起的,SARS-CoV-2是继严重急性呼吸综合征冠状病毒(severe acute respiratory syndrome coronavirus,SARS-CoV)和中东呼吸综合征冠状病毒(middle east respiratory syndrome coronavirus,MERS-CoV)后人类发现的第三种引起全球大流行的冠状病毒.刺突蛋白(spike protein,S)的受体结合域(receptor binding domain,RBD)可以与细胞表面的血管紧张素转换酶2(angiotensin converting enzyme2,ACE2)结合,进而入侵细胞内部,启动病毒的复制.为了预防和治疗新冠病毒肺炎,研究人员研发了多种疫苗、小分子药物及抗体药物.纳米抗体(nanobodies,Nbs)是可识别抗原的最小结合片段,具有体积小、渗透性高、热稳定性好、结合特异性高、生产成本低、免疫原性小等优势,在治疗SARS-CoV-2中效果显著.对SARS-CoV-2的纳米抗体研究进展进行了综述,这些纳米抗体的发现,对于新冠病毒病的诊断和治疗均有潜在的应用价值.

糖尿病合并新型冠状病毒S蛋白感染小鼠病理生理特征

目的建立糖尿病(DM)合并新型冠状病毒(SARS-CoV-2)感染小鼠模型,研究DM合并SARS-CoV-2感染病程发展中的重要病理生理变化。方法将野生型(WT)小鼠和由细胞角蛋白18基因启动子驱动的人血管紧张素转化酶2受体(K18-hACE2,简称hACE2)的转基因小鼠分别随机分为溶剂对照组、DM组、SARS-CoV-2病毒刺突蛋白感染(S)组以及DM合并S蛋白感染(DM+S)组,每组10~12只。除溶剂对照组及S组外,其余组通过10周高脂饮食后连续3 d ip给予链脲佐菌素(STZ)40 mg·kg^(-1)诱导DM症状,溶剂对照组及S组给予等体积0.1 mol·L^(-1)柠檬酸钠缓冲液。在此基础上,S组及DM+S组小鼠经鼻腔滴入15μg SARS-CoV-2 S蛋白与1 g·L^(-1)聚肌胞苷酸(Poly[I:C])的混合溶液50μL,溶剂对照组滴鼻给予等体积无菌水。在高脂喂养第6周和ip给予STZ 1周后,以口服葡萄糖耐量实验评价小鼠糖耐量水平及胰岛β细胞功能。高脂喂养第6周至ip给予STZ 2周后,每周用血糖仪检测小鼠随机血糖及空腹血糖。DM小鼠S蛋白感染前及感染24,48和120 h后,每组取3只小鼠颌下静脉取血后处死并取肺组织,采用苏木精-伊红染色法观察合并S蛋白感染前后的肺组织病理改变。S组小鼠在感染S蛋白前及感染6,24,48,72和120 h后采血,Luminex液相芯片技术检测小鼠血浆细胞因子白细胞介素1β(IL-1β)、IL-2、IL-6、IL-10、IL-17、干扰素诱导蛋白10(IP-10)、干扰素γ(IFN-γ)、肿瘤坏死因子α(TNF-α)、单核细胞趋化蛋白1(MCP-1)和粒细胞集落刺激因子(G-CSF)水平,ELISA检测血浆硫酸乙酰肝素(HS)水平;将细胞因子水平、HS水平与小鼠感染S蛋白后的肺部病理损伤程度进行Spearman相关性分析。结果STZ合并高脂饮食可诱导小鼠DM样表现,hACE2-DM组随机血糖(P<0.01)和1周后的空腹血糖(P<0.05)均显著高于WT-DM组,且hACE2-DM小鼠胰岛功能损伤程度显著高于WT-DM小鼠(P<0.05)。与DM组相比,DM+S组小鼠均表现出更严重的肺部病理变化,并伴有大量炎症浸润和肺间质增厚。与溶剂对照组相比,S蛋白感染6 h,WT-S组小鼠血浆中促炎细胞因子G-CSF,IL-6和IP-10均显著升高(P<0.01),S蛋白感染24 h,促炎细胞因子IL-17和抗炎细胞因子IL-10均显著升高(P<0.05);S蛋白感染6 h,hACE2-S组血浆中促炎细胞因子IL-1β,IL-6,TNF-α,MCP-1,G-CSF和IP-10均显著升高(P<0.05,P<0.01);WT-DM+S组和hACE2-DM+S组IL-17分别在S蛋白感染24和6 h显著升高(P<0.01,P<0.05),hACE2-DM+S组IFN-γ和IL-1β延迟至48 h显著升高(P<0.05,P<0.01),MCP-1延迟至72 h显著升高(P<0.05)。与溶剂对照组相比,S蛋白感染6和24 h后,WT-S组血浆中HS水平显著升高(P<0.05,P<0.01),48 h开始降低;同时,与WT-S组相比,感染6 h后,WT-DM+S组HS水平略升高,24 h出现降低;hACE2-S组HS水平于24 h显著升高(P<0.01),且在S蛋白感染24,48和120 h后与WT-S组基本持平;S蛋白感染6,24和48 h后,hACE2-DM+S组血浆HS水平均显著升高(P<0.01,P<0.05),升高持续时间较其S组长。小鼠血浆中IL-1β,IL-10,MCP-1,IP-10,G-CSF以及HS水平与DM+S小鼠肺损伤程度呈正相关(P<0.05,P<0.01)。结论本研究建立的DM合并SARS-CoV-2 S蛋白感染小鼠模型能成功模拟临床患者的部分病理生理特征,主要表现为较单纯感染免疫反应钝化,HS水平升高且持续时间较长。IL-1β,IL-10,MCP-1,IP-10,G-CSF以及HS可能有助于及时了解DM合并SARS-CoV-2感染患者的病程。

SARS-CoV-2刺突糖蛋白对神经细胞的损伤效应及机制

目的探讨严重急性呼吸综合征冠状病毒2(SARS-CoV-2)刺突糖蛋白(S蛋白)对人神经母细胞瘤细胞(SH-SY5Y)的损伤效应及其机制。方法用S蛋白0(细胞对照组),25,50,75和100 mg·L^(-1)处理SH-SY5Y 24 h,CCK-8法检测细胞活力;比色法检测乳酸脱氢酶(LDH)释放率;EdU试剂盒检测细胞增殖;荧光素酶发光法检测细胞内ATP含量;JC-1荧光探针法检测细胞线粒体膜电位(MMP);Seahorse XF检测细胞糖酵解及线粒体氧化磷酸化水平。结果与细胞对照组相比,S蛋白25,50,75和100 mg·L^(-1)组细胞活力显著降低(P<0.01),半数抑制浓度(IC_(50))为65.05 mg·L^(-1);LDH释放率显著增加(P<0.01);EdU阳性细胞比例显著降低(P<0.01);S蛋白75和100 mg·L^(-1)组细胞内ATP含量显著降低(P<0.01);S蛋白50和75 mg·L^(-1)组细胞内MMP显著降低(P<0.05,P<0.01);S蛋白50 mg·L^(-1)组基础糖酵解水平和糖酵解能力的最大值显著升高(P<0.05,P<0.01),S蛋白25和50 mg·L^(-1)组呼吸能力最大值显著升高(P<0.05,P<0.01)。SH-SY5Y细胞活力与细胞内ATP含量和MMP均呈正相关(r^(2)=0.9209,P=0.001;r^(2)=0.6170,P=0.0025);与反映细胞糖酵解水平的细胞基础糖酵解水平和糖酵解能力最大值呈负相关(r^(2)=0.5194,P=0.0285;r^(2)=0.6664,P=0.0073),与反映线粒体氧化磷酸化水平的ATP生成能力呈负相关(r^(2)=0.8204,P=0.0008)。结论S蛋白使SH-SY5Y细胞活力下降,抑制细胞增殖,其机制可能与干扰神经细胞内能量代谢密切相关。