SARS动物模型的研究

利用分离的SARS CoV毒株BJ 0 1,经滴鼻等途径感染大鼠、豚鼠、黑线仓鼠、白化仓鼠和雏鸡等 5个种属的动物 ,筛选对SARS易感的小动物。在此基础上 ,选择食蟹猴和恒河猴进行SARS的人工感染实验 ,评价其作为SARS动物模型的可能性。结果表明 ,大鼠、豚鼠、黑线仓鼠、白化仓鼠和雏鸡等动物对SARS均不易感 ,感染后未观察到任何的临床及病理学改变 ,不过从感染 2周后的大鼠和豚鼠的肺和咽等组织样本中检测到了的特异的核酸 ,提示SARS CoV能够在这两种动物的体内复制。从感染猴子的分泌物和脏器中分离出了病毒 ,证明SARS CoV也能够在猴子体内复制。临床和病理组织学检查结果显示 ,SARS病毒接种食蟹猴和恒河猴后 ,可以引起所有实验猴发生间质性肺炎 ,其病理学改变与人类感染SARS病毒后肺部病变近似 ,但病变的严重程度比较人类的轻得多 ,除此之外无任何其它的明显的临床表现及组织病理学改变 ,按照动物模型的指标判断食蟹猴和恒河猴并不是SARS的理想动物模型 ,不过在目前尚没有更理想的动物模型情况下 ,以间质性肺炎为病理学检查指标 ,恒河猴和食蟹猴可以作为评价抗SARS药物和疫苗的模型动物     

非典-SARS-冠状病毒

2002年11月,一种神秘不明的疾病出现在我国广东省继页肆虐全球,世界上有27个国家和地区相继报道出现这种疫情.     

Proteasome Inhibition In Vivo Promotes Survival in a Lethal Murine Model of Severe Acute Respiratory Syndrome

Ubiquitination is a critical regulator of the host immune response to viral infection, and many viruses, including coronaviruses, encode proteins that target the ubiquitination system. To explore the link between coronavirus infection and the ubiquitin system, we asked whether protein degradation by the 26S proteasome plays a role in severe coronavirus infections using a murine model of SARS-like pneumonitis induced by murine hepatitis virus strain 1 (MHV-1). In vitro, the pretreatment of peritoneal macrophages with inhibitors of the proteasome (pyrrolidine dithiocarbamate [PDTC], MG132, and PS-341) markedly inhibited MHV-1 replication at an early step in its replication cycle, as evidenced by inhibition of viral RNA production. Proteasome inhibition also blocked viral cytotoxicity in macrophages, as well as the induction of inflammatory mediators such as IP-10, gamma interferon (IFN-γ), and monocyte chemoattractant protein 1 (MCP-1). In vivo, intranasal inoculation of MHV-1 results in a lethal pneumonitis in A/J mice. Treatment of A/J mice with the proteasome inhibitor PDTC, MG132, or PS-341 led to 40% survival (P < 0.01), with a concomitant improvement of lung histology, reduced pulmonary viral replication, decreased pulmonary STAT phosphorylation, and reduced pulmonary inflammatory cytokine expression. These data demonstrate that inhibition of the cellular proteasome attenuates pneumonitis and cytokine gene expression in vivo by reducing MHV-1 replication and the resulting inflammatory response. The results further suggest that targeting the proteasome may be an effective new treatment for severe coronavirus infections.Severe acute respiratory syndrome (SARS) was first introduced into the human population in the Guangdong Province in China and rapidly spread to several other countries (31). SARS is caused by infection with the SARS coronavirus (SARS-CoV) and is characterized by an atypical pneumonia and lymphopenia. Two-thirds of the SARS-infected patients developed a viral pneumonitis, of which 10% developed acute respiratory distress syndrome. During the outbreak in 2002 to 2003, 8,000 people were infected and 774 people died from respiratory failure (36; WHO, Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003 [http://www.who.int]). At present there are no effective treatments for SARS as well as other coronavirus infections. Finding an effective treatment for coronavirus infections could be protective in the event of a reemergent coronavirus outbreak (7).We have recently reported that a rodent model of SARS mimics many of the features of severe clinical SARS pathology (11, 12). Intranasal infection of A/J mice with strain 1 of murine hepatitis virus (MHV-1) causes a lethal form of pneumonitis, characterized by marked innate immune inflammatory cytokine production and replication of the virus in pulmonary macrophages (11, 12). MHV-1 infection is uniformly fatal in infected A/J mice; the resultant disease serves as a model to understand the pathology of the most severe SARS cases. In mice, the pulmonary damage is histologically similar to that seen in human SARS and is similarly associated with a marked upregulation of inflammatory mediators, including monocyte chemoattractant protein 1 (MCP-1), IP-10, MIG, gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-6 (11, 12, 25). These innate immune mediators are likely to play roles in human SARS and MHV-1 SARS-like pathogenesis.A critical aspect of the host innate immune response to viral illness is the upregulation of the antiviral type 1 IFN response. With respect to SARS, type 1 IFN responses have been reported to be suppressed by SARS-CoV in several models and in clinical cases (11, 39, 45, 52). In our model, MHV-1-infected A/J mice produce less type 1 IFN than resistant strains of mice and they respond poorly to IFN-β therapy (11). Type I IFN has been used clinically in the treatment of established SARS infections but has shown only limited efficacy (25). In the absence of an effective antiviral treatment, the innate immune pathways present a potential target for therapeutic intervention (7).Ubiquitination, the process by which cellular proteins are conjugated to the 7.5-kDa ubiquitin (Ub) protein, is a critical regulator of innate and adaptive immune pathways (40). There are several possible fates for ubiquitinated proteins: degradation by the 26S proteasome, trafficking to various subcellular sites, altered interactions with other proteins, and altered signal transduction functions (28). The fates of the ubiquitinated proteins, many of which overlap, can play a role in innate immunity. Since the first discovery that papillomavirus encodes an E3 ubiquitin ligase that targets p53, it has become widely appreciated that many viruses encode proteins that target or exploit ubiquitination pathways (37, 43). For example, Epstein-Barr virus and herpes simplex virus proteins interact with the host deubiquitinating (DUB) protein USP7 (13, 17). Ubiquitination of IRF3 has been implicated in the viral control of the innate immune system (22, 48, 49). DUB may also be important for viral functions, such as the assembly of viral replicase proteins with double-membrane vesicles at the site of replication, a process that parasitizes autophagy (39).All coronaviruses, including MHV (A59 and JHM), infectious bronchitis virus, and human CoV229E SARS coronavirus, encode one or more papain-like proteases (PLpros) (PL1pro and PL2pro) (3, 5, 19, 23, 50). One role for the PL2pro proteases is to cleave the coronavirus polyprotein into its component parts. This enzyme, isolated from the SARS-CoV, has also been shown to have DUB activity both in vitro and in HeLa cells (23), suggesting that it might also play a role in modulating the host ubiquitination pathways. PLpro proteases harbor an N-terminal Ub-like domain reported to mediate interactions between PLpro DUB activity and the cellular proteasome (35). Although there is no direct link between the proteasome and SARS-CoV DUB activity, the presence of the Ub1 domain and of SARS-CoV DUB activity suggests that the proteasome may be being exploited by the virus either to evade the immune response or to promote viral replication. These interactions also suggest that the ubiquitination system might be a target for antiviral therapeutic intervention.We explored the role of the cellular proteasome in MHV-1 replication and in the innate immune response to the virus by testing the effects of small-molecule proteasome inhibitors in both cell-based and murine models of SARS pneumonitis. We compared the results in the SARS model to a well-described model of lymphocytic choriomeningitis virus (LCMV) hepatitis in order to test for virus-specific effects. To control for nonspecific effects of the inhibitors, we used three different agents: pyrrolidine dithiocarbamate (PDTC), MG132, and PS-341 (bortezomib, Velcade). PDTC is a chelating agent that reversibly inhibits the proteasome complex, MG132 is a peptide aldehyde protease inhibitor, and PS-341 is a peptide boronic acid inhibitor (1, 20, 38). PS-341 is a clinically approved drug currently being used in the treatment of multiple myeloma.     

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间隔增宽,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.     

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。     

Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.     

SARS冠状病毒的分子生物学研究

严重急性呼吸系统综合征(SARS)是2002年底爆发于中国广东,后蔓延全球的传染性疾病。其病原体为一种未知的新型冠状病毒,章从SARS冠状病毒的蛋白构成和功能研究、SARS冠状病毒感染机制(表型变化,受体)、SARS冠状病毒分子进化这几个方面对现有研究进展做一综述。     

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.     

Comparative Epidemiology of Human Infections with Middle East Respiratory Syndrome and Severe Acute Respiratory Syndrome Coronaviruses among Healthcare Personnel

The largest nosocomial outbreak of Middle East respiratory syndrome (MERS) occurred in South Korea in 2015. Health Care Personnel (HCP) are at high risk of acquiring MERS-Coronavirus (MERS-CoV) infections, similar to the severe acute respiratory syndrome (SARS)-Coronavirus (SARS-CoV) infections first identified in 2003. This study described the similarities and differences in epidemiological and clinical characteristics of 183 confirmed global MERS cases and 98 SARS cases in Taiwan associated with HCP. The epidemiological findings showed that the mean age of MERS-HCP and total MERS cases were 40 (24~74) and 49 (2~90) years, respectively, much older than those in SARS [SARS-HCP: 35 (21~68) years, p = 0.006; total SARS: 42 (0~94) years, p = 0.0002]. The case fatality rates (CFR) was much lower in MERS-HCP [7.03% (9/128)] or SARS-HCP [12.24% (12/98)] than the MERS-non-HCP [36.96% (34/92), p<0.001] or SARS-non-HCP [24.50% (61/249), p<0.001], however, no difference was found between MERS-HCP and SARS-HCP [p = 0.181]. In terms of clinical period, the days from onset to death [13 (4~17) vs 14.5 (0~52), p = 0.045] and to discharge [11 (5~24) vs 24 (0~74), p = 0.010] and be hospitalized days [9.5 (3~22) vs 22 (0~69), p = 0.040] were much shorter in MERS-HCP than SARS-HCP. Similarly, days from onset to confirmation were shorter in MERS-HCP than MERS-non-HCP [6 (1~14) vs 10 (1~21), p = 0.044]. In conclusion, the severity of MERS-HCP and SARS-HCP was lower than that of MERS-non-HCP and SARS-non-HCP due to younger age and early confirmation in HCP groups. However, no statistical difference was found in MERS-HCP and SARS-HCP. Thus, prevention of nosocomial infections involving both novel Coronavirus is crucially important to protect HCP.     

严重急性呼吸综合征动物模型中血清因子的检测

严重急性呼吸综合征是一种新近出现的严重传染性疾病,病原体为一种新的冠状病毒。但其免疫病理的发病机制尚未阐明,我们用SARS病毒感染了恒河猴和leiws大鼠,经PCR和抗体检测,证明病毒在动物体内有复制。用酶连免疫吸附试验测量动物血清中白介素(IL)-6,白介素(IL)-10,γ-干扰素(INF),肿瘤坏死因子(TNF)-α的含量。结果显示,血清中IL-10和INF-γ的含量在感染前后无显著性差异。感染后动物体内IL-6显著升高,其含量与肺部病变程度呈正相关。TNF-α的含量降低。动物模型中血清免疫因子的测定避免了临床病人由于使用抗病毒药物和激素造成的干扰,对于我们了解免疫因子在SARS免疫病理发病机制的作用有一定帮助。