Recomendaciones sobre el manejo clínico de la infección por el «nuevo coronavirus» SARS-CoV2. Grupo de trabajo de la Asociación Española de Pediatría (AEP)

On 31 December 2019, the Wuhan Municipal Committee of Health and Healthcare (Hubei Province, China) reported that there were 27 cases of pneumonia of unknown origin with symptoms starting on the 8 December. There were 7 serious cases with common exposure in market with shellfish, fish, and live animals, in the city of Wuhan. On 7 January 2020, the Chinese authorities identified that the agent causing the outbreak was a new type of virus of the Coronaviridae family, temporarily called «new coronavirus», 2019-nCoV. On January 30th, 2020, the World Health Organisation (WHO) declared the outbreak an International Emergency. On 11 February 2020 the WHO assigned it the name of SARS-CoV2 and COVID-19 (SARS-CoV2 and COVID-19).The Ministry of Health summoned the Specialties Societies to prepare a clinical protocol for the management of COVID-19. The Spanish Paediatric Association appointed a Working Group of the Societies of Paediatric Infectious Diseases and Paediatric Intensive Care to prepare the present recommendations with the evidence available at the time of preparing them.     

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.     

Serum neopterin for early assessment of severity of severe acute respiratory syndrome

Neopterin and C-reactive protein (CRP) concentrations were determined in serum samples from 129 severe acute respiratory syndrome (SARS) patients and 156 healthy blood donors. In the patients with confirmed SARS, an early neopterin elevation was detected already at the day of onset of symptoms and rose to a maximum level of 45.0 nmol/L 3 days after the onset. All SARS patients had elevated neopterin concentrations (>10 nmol/L) within 9 days after the onset. The mean neopterin concentrations were 34.2 nmol/L in acute sera of SARS patients, 5.1 nmol/L in convalescent sera, and 6.7 nmol/L in healthy controls. In contrast, the mean CRP concentrations in both acute and convalescent sera of SARS patients were in the normal range (<10 mg/L). Serum neopterin level in SARS patients was associated with fever period and thus the clinical progression of the disease, while there was no significant correlation between the CRP level and the fever period. Serum neopterin may allow early assessment of the severity of SARS. The decrease of neopterin level was found after steroid treatment, which indicates that blood samples should be collected before steroid treatment for the neopterin measurement.     

SARS冠状病毒棘突蛋白的S1蛋白诱导小鼠产生中和抗体的研究

目的 研究SARS冠状病毒棘突蛋白受体结合部位S1的免疫原性，为SARS的实验诊断和新型疫苗的研究提供依据。方法 用克隆有哺乳动物细胞密码子优化的SARS-CoV S1基因的质粒pcDNA3．1／S1或P-S1Ig转染293T细胞，用细胞的上清液纯化S1蛋白。以pcDNA3．1／S1质粒对BALB／c小鼠进行2次基因免疫，以纯化的S1蛋白进行加强免疫。用ELISA法检测小鼠抗SARS-CoV的特异性IgG抗体，并在Vero E6细胞上做体外中和实验，检测中和抗体。结果 S1蛋白诱导小鼠产生抗SARS-CoV的特异性抗体；1：1499．68稀释的S1蛋白免疫的小鼠血清可保护50％的细胞对1000TCID50的病毒攻击，而阴性对照血清不能保护细胞对病毒的感染。结论 SAPS冠状病毒棘突蛋白受体结合部位S1能有效诱导机体产生具有高效保护作用的中和抗体免疫反应，可望发展成为理想的SARS棘突蛋白亚单位疫苗。     

SARS-CoV、MERS-CoV、SARS-CoV-2的基因结构特点与致病机制

高致病性冠状病毒具有高发病率、高病死率以及全球大流行等特点,严重威胁人类健康,给社会经济带来巨大挑战。目前关于高致病冠状病毒的致病机制,主要从直接细胞病变效应和间接免疫病理损伤两方面进行讨论,但具体作用基因的研究尚需进一步深入。本文从高致病性冠状病毒的基因结构特点与致病机制的相关性进行综述,为预防和治疗高致病性冠状病毒感染提供参考。     

SARS疫苗的研究简述

严重急性呼吸系统综合症曾一度流行,造成巨大的经济损失,引起全球的关注。而引起SARS的SARS—CoV是冠状病毒的一个变种,目前对它的致病机理了解的还不够清楚。尚没有有效治疗SARS的抗病毒药物,研制有效的疫苗可防止SARS的再度流行。在疫苗的研制方面,虽然在一些动物模型上得到了一些进展,但是在人体上的应用受到了限制。本文综述了SARS疫苗的研究进展和存在的一些问题。     

SARS冠状病毒S2基因原核表达及免疫学特性

目的研究严重急性呼吸道综合征（SARS）冠状病毒S2蛋白表达并对其免疫学特性。方法克隆SARS冠状病毒S2基因。并在大肠埃希菌中表达谷胱甘肽硫转移酶（GST-S2）融合蛋白。通过免疫印迹（Western blot）和酶联免疫吸附试验（ELISA）方法鉴定表达GST—S2融合蛋白的活性。结果通过原核系统表达的GST-S2融合蛋白可与谷胱甘肽硫转移酶（GST）多克隆抗体结合，并在85千道尔顿（KD）处出现特异性结合条带。GST-S2蛋白能与SARS患者恢复期血清反应，而不与正常人血清反应。结论本项研究获得了SARS冠状病毒GST-82融合蛋白，它可与GST多克隆抗体特异性结合，并与SARS患者恢复期血清发生特异性反应。     

SARS冠状病毒受体结合域在大肠杆菌中的融合表达和鉴定

目的为了研究SARS冠状病毒感染与免疫和跨种属传播机制,从来源于人和果子狸的SARS冠状病毒spike蛋白基因中,克隆病毒受体结合域（receptor binding domain,RBD）基因片段,在大肠杆菌中进行表达。方法用PCR方法扩增RBD基因,先经T-A连接,转化大肠杆菌DH5α,挑选阳性克隆测序鉴定,双酶切后进一步亚克隆至质粒pGEX-6p-3,构建原核表达重组质粒,通过IPTG诱导在大肠杆菌BL21中表达,并用SDS-PAGE和Western-blotting方法检测表达情况。结果扩增了RBD的编码基因并成功构建了其原核表达载体pGEX-6p-3-hsRBD和pGEX-6p-3-csRBD,RBD能够在大肠杆菌中获得良好的表达,表达的融合蛋白相对分子质量约为47000。结论本工作利用基因工程技术表达了两物种来源的SARS冠状病毒的RBD,两者具有高度的同源性,因此我们可通过配基受体结合实验,为进一步研究SARS冠状病毒感染与免疫及跨种属传播机制奠定了基础。     

SARS冠状病毒N蛋白的原核表达及活性测定

目的克隆编码严重急性呼吸综合征冠状病毒（sARSCoV）N蛋白的DNA,构建原核表达质粒pGEX-2T／N,并诱导表达。方法采用RT-PCR方法从病毒培养液中获得N基因片段。将N蛋白基因序列定向插入原核表达载体pGEX-2T中,在大肠杆菌中表达融合蛋白。用表达产物与抗SARS-CoV抗体阳性血清做Western blot。结果获得N基因片段;GST-N融合蛋白以可溶形式表达;Western blot检测表明,其与抗SARS-CoV抗体阳性血清的反应呈阳性。结论成功地构建原核表达质粒pGEX-2T／N,并表达GST-N融合蛋白,为下一步的研究奠定了基础。