恒河猴感染SARS病毒致病模型的建立

为建立恒河猴严重急性呼吸道综合征(SARS)的模型并对其致病特点进行观察,采用病毒分离、免疫荧光、光镜及RT-PCR方法对病毒感染组和非感染组恒河猴不同时间、不同组织或分泌物进行检测。结果显示从恒河猴不同组织中分离到病毒,而且在病毒感染后第2d和第5d的血液、第7、9d的鼻咽分泌物、第3d的粪、第5d的粪尿中均检测到SARS-CoV RNA。光镜观察到病毒感染组肺组织肺泡问隔增宽,有大量淋巴细胞、单核细胞浸润,肺泡腔有渗出,甚至形成透明膜样物;多个肺泡形成机化性肺炎的表现。感染组肝组织可见较大的坏死灶,并伴有大量炎性细胞浸润。结论认为已成功建立了恒河猴SARS模型,可用于评价抗SARS药物和疫苗的研究。     

SARS Research in China

SARS Research in China     

Indirect Enzyme-Linked Immunosorbent Assay based on the nucleocapsid protein of SARS-like coronaviruses

The nucleocapsid protein (N) is a major structural protein of coronaviruses. The N protein of bat SARS-like coronavirus (SL-CoV) has a high similarity with that of SARS-CoV. In this study, the SL-CoV N protein was expressed in Escherichia coli, purified and used as antigen. An Indirect Enzyme-Linked Immunosorbent Assay (indirect ELISA) was developed for detection of SARS- or SL-CoV infections in bat populations. The detection of 573 bat sera with this indirect ELISA demonstrated that SL-CoVs consistently circulate in Rhinilophus species, further supporting the proposal that bats are natural reservoirs of SL-CoVs. This method uses 1-2 μl of serum sample and can be used for preliminary screening of infections by SARS- or SL-CoV with a small amount of serum sample.     

Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin

Severe acute respiratory syndrome (SARS) is caused by the SARS-associated coronavirus (SARS-CoV), which uses angiotensin-converting enzyme 2 (ACE2) as its receptor for cell entry. A group of SARS-like CoVs (SL-CoVs) has been identified in horseshoe bats. SL-CoVs and SARS-CoVs share identical genome organizations and high sequence identities, with the main exception of the N terminus of the spike protein (S), known to be responsible for receptor binding in CoVs. In this study, we investigated the receptor usage of the SL-CoV S by combining a human immunodeficiency virus-based pseudovirus system with cell lines expressing the ACE2 molecules of human, civet, or horseshoe bat. In addition to full-length S of SL-CoV and SARS-CoV, a series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S backbone. Several important observations were made from this study. First, the SL-CoV S was unable to use any of the three ACE2 molecules as its receptor. Second, the SARS-CoV S failed to enter cells expressing the bat ACE2. Third, the chimeric S covering the previously defined receptor-binding domain gained its ability to enter cells via human ACE2, albeit with different efficiencies for different constructs. Fourth, a minimal insert region (amino acids 310 to 518) was found to be sufficient to convert the SL-CoV S from non-ACE2 binding to human ACE2 binding, indicating that the SL-CoV S is largely compatible with SARS-CoV S protein both in structure and in function. The significance of these findings in relation to virus origin, virus recombination, and host switching is discussed.     

冠状病毒研究态势分析

2019年底,一种新型冠状病毒在武汉引起发热性呼吸道疾病(COVID-19),并在我国境内及周边国家持续蔓延,对人类流行病的防控提出了新的要求。基于文献计量学方法梳理了冠状病毒的研究进展,从全球视角对主要研发国家/地区、核心研究机构和热点研究方向进行了分析,旨在揭示冠状病毒的诊断、预防和治疗的研究态势,以期为当前科研联合攻关提供参考。     

实验动物和公园动物中SARS病原来源调查初报

目的　调查SARS冠状病毒的动物来源。方法　用冠状病毒通用引物的反转录巢式PCR方法对采集的动物呼吸道分泌物或咽拭子标本进行检测 ,用血清学方法 (EIASA)对动物血清进行检测 ,确定阳性的初筛动物。结果　分别从各实验动物猴、犬饲养场、广州动物园共采集拭子标本 84份 ,包括的动物品种有猴、犬、小灵猫、果子狸、狒狒、巨蜥、滑鼠蛇、巴西龟、孔雀、鸭子、鸽子等 ,PCR方法检测 ,未检出SARS冠状病毒 ;另抽取 16个单位 3 73只小鼠和 9个单位 198只大鼠的血清 ,用ELISA方法检查 ,有 52只鼠检出鼠冠状病毒抗体。实验动物SARS冠状病毒的追溯研究是从根本上防治SARS病的重要环节之一。     

SARS病毒感染动物模型

SARS-CoV感染动物模型的建立可加深对SARS病原学的了解和发病机制的研究,加速实验室诊断技术的建立、抗病毒药物的筛选和疫苗的开发,同时也有助于给该病一个更精确的定义。可以说SARS动物模型的建立,不但是SARS研究的瓶颈问题,其应用更是贯穿SARS研究的整个过程。到目前为止,已经报道有4种非人灵长类动物(恒河猴、食蟹猴、绒猴、非洲绿猴)和6种啮齿类动物(大鼠、小鼠、豚鼠、田鼠、仓鼠、转基因鼠),以及雪貂、家猫等可以作为SARS动物模型用于实验研究,并已经开始利用动物模型进行疫苗和药物的安全性和有效性评价。本文就已报道的各类SARS动物模型进行综述,并根据动物模型和SARS患者的比对,提出动物模型建立的技术要点。     

SARS-CoV的分子生物学研究

SARS-CoV是引起严重急性呼吸道综合症(SARS)的病原体．更多地了解SARS-CoV的基因组、蛋白结构以及它与其它冠状病毒的关系,将有助于SARS疾病的防治．     

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 t...     

SARS冠状病毒M蛋白的生物信息学研究

针对GenBank上发布的来自不同国家地区的39条SARSCoV推测M蛋白,采用生物信息学软件分析其核酸和氨基酸序列,获得其分子生物学特征,确定突变位点,预测功能结构区、Motif及抗原决定簇,比较基因突变对这些功能结构的影响.结果表明:在39个病毒株M蛋白的666 bp中,共有18个病毒株在7个位点上发生了25次变异.在M蛋白序列上预测获得3个跨膜螺旋序列和一个可能的信号肽序列.氨基酸序列的变异主要发生在其跨膜和胞外区域,胞内区域相对较少.预测发现12个Motif和7个抗原决定簇.提示突变对M蛋白的结构功能区的影响不大,也未造成M蛋白的Motif的数量和构成发生改变.对抗原决定簇的影响也主要体现在序列成分构成的改变上,在设计疫苗时,应考虑由其导致的抗原特性改变.     

Expression and Purification of N and E Proteins from Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus: a Comparative Study

Histidine-tagged N (rNH) and E (rEH) proteins of Severe Acute Respiratory Syndrome (SARS)-coronovirus were expressed in the baculovirus/insect cell system and purified by immobilized metal affinity chromatography. rNH and rEH proteins differed markedly with respect to expression levels, cell death kinetics and subcellular localizations that led to different extraction and purification schemes. The features of both proteins are compared and the potential applications of purified rNH and rEH are discussed.     

Molecular mechanisms of severe acute respiratory syndrome (SARS)

Background

The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells).

Methods

Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits.

Results

In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells.

Conclusion

In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells.     

SARS-CoV动物模型研究之中国现状

目的综合对比SARS-CoV感染的恒河猴、布氏田鼠及Lewis大鼠的病理学、免疫学以及病毒的复制与外排情况的变化,来探讨此三种动物在建立SARS模型上的特点。方法SARS病毒感染8只恒河猴、9只Lewis大鼠和20只布氏田鼠,在感染后不同时间安乐死动物,应用光镜对动物的各脏器进行病理观察研究;用病毒分离和RT-PCR方法检测病毒外排与复制的情况;用ELISA法检测动物产生特异性抗体情况。结果在SARS-CoV感染恒河猴、Lewis大鼠和布氏田鼠后,肺组织均出现一定的与人类SARS疾病相似的病理改变,在动物体内均可检测到活病毒或病毒核酸,并可检测到特异性IgG抗体的存在。在病死率上布氏田鼠最高;在病毒的复制与外排方面恒河猴的检出率最高,持续时间最长;在抗体产生情况上恒河猴与Lewis大鼠基本相似;在病理变化上恒河猴病变最重且最为复杂,与人类SARS疾病的病理变化最为接近。结论布氏田鼠,Lewis大鼠,特别是恒河猴动物模型可以用于SARS发病机制、疫苗和药物的研发,恒河猴动物模型是目前研究SARS疾病最理想的动物模型。     

Immunodetection of occult eosinophils in lung tissue biopsies may help predict survival in acute lung injury

Background

Acute lung injury (ALI) is a serious respiratory disorder for which therapy is primarily supportive once infection is excluded. Surgical lung biopsy may rule out other diagnoses, but has not been generally useful for therapy decisions or prognosis in this setting. Importantly, tissue and peripheral blood eosinophilia, the hallmarks of steroid-responsive acute eosinophilic pneumonia, are not commonly linked with ALI. We hypothesized that occult eosinophilic pneumonia may explain better outcomes for some patients with ALI.

Methods

Immunohistochemistry using a novel monoclonal antibody recognizing eosinophil peroxidase (EPX-mAb) was used to assess intrapulmonary eosinophil accumulation/degranulation. Lung biopsies from ALI patients (n = 20) were identified following review of a pathology database; 45% of which (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Controls were obtained from uninvolved tissue in patients undergoing lobectomy for lung cancer (n = 10). Serial biopsy sections were stained with hematoxylin and eosin (H&E) and subjected to EPX-mAb immunohistochemistry.

Results

EPX-mAb immunohistochemistry provided a >40-fold increased sensitivity to detect eosinophils in the lung relative to H&E stained sections. This increased sensitivity led to the identification of higher numbers of eosinophils in ALI patients compared with controls; differences using H&E staining alone were not significant. Clinical assessments showed that lung infiltrating eosinophil numbers were higher in ALI patients that survived hospitalization compared with non-survivors. A similar conclusion was reached quantifying eosinophil degranulation in each biopsy.

Conclusion

The enhanced sensitivity of EPX-mAb immunohistochemistry uniquely identified eosinophil accumulation/degranulation in patients with ALI relative to controls. More importantly, this method was a prognostic indicator of patient survival. These observations suggest that EPX-mAb immunohistochemistry may represent a diagnostic biomarker identifying a subset of ALI patients with improved clinical outcomes.     

Factores de riesgo asociados a fallecimiento por la variante ómicron de COVID-19: análisis retrospectivo con personas mayores de Canarias

Background and aimsSince the beginning of the COVID-19 pandemic, the elderly population has had the highest rates of complications and mortality. This study aimed to determine the influence of different risk factors on deaths due to the Omicron variant in the Canary Islands.Materials and methodsA retrospective observational study of 16,998 cases of COVID-19 over 40 years of age was conducted in the Canary Islands between August 1, 2022, and January 31, 2023. We extracted sociodemographic data (age and sex) and clinical data (death, vaccination history, hospital admission, previous diseases, and treatments).ResultsAmong the deaths, there was a higher proportion of males aged over 70 years, with diabetes, cardiovascular, renal, respiratory, and systemic diseases, and nursing home residents. Significant differences were observed in the number of doses of the vaccine. The multiple regression model showed that male sex (OR [95% CI] = 1.92 [1.42–2.58]), age (70–79 years, 9.11 [4.27–19.43]; 80–89 years, 21.72 [10.40–45.36]; 90–99 years, 66.24 [31.03–141.38]; 100 years or older, 69.22 [12.97–369.33]), being unvaccinated (6.96, [4.01–12.08]), or having the last dose administered at least 12 months before the diagnosis (2.38, [1.48–3.81]) were significantly associated with mortality.ConclusionsMultiple factors may increase the risk of mortality due to COVID-19 in the elderly population. In our study, we found that only three predictors can effectively explain the variability: older age, male sex, and not being vaccinated or last vaccination date prior to one year.     

The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity

In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.     

32株SARS冠状病毒基因组比对在PCR检测中的应用

进行了32株SARS冠状病毒基因组的多序列比对分析.所得无根进化树揭示SARS冠状病毒之间的同源性.同时,验证了所设计的PCR检测引物对全部32株SARS冠状病毒检测的适用性.