DNA vaccine of SARS-Cov S gene induces antibody response in mice

The spike (S) protein, a main surface antigen of SARS-coronavirus (SARS-CoV), is one of themost important antigen candidates for vaccine design. In the present study, three fragments of the truncated S protein were expressed in E. coli, and analyzed with pooled sera of convalescence phase of SARS patients. The full length S gene DNA vaccine was constructed and used to immunize BALB/c mice. The mouse serum IgG antibody against SARS-CoV was measured by ELISA with E. coli expressed truncated S protein or SARS-CoV lysate as diagnostic antigen. The results showed that all the three fragments of S protein expressed by E. coli was able to react with sera of SARS patients and the S gene DNA candidate vaccine could induce the production of specific IgG antibody against SARS-CoV efficiently in mice with seroconversion ratio of 75% after 3 times of immunization. These findings lay some foundations for further understanding the immunology of SARS-CoV and developing SARS vaccines.     

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.     

Preliminary study on the detection of the SARS-CoV specific target cDNA fragments by multiplex PCR

The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The re~sults indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully.     

SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway

While severe acute respiratory syndrome coronavirus （SARS-CoV）~as initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that yirus entry may also involve endocytosis. We have found that SARS-CoV enters cells viapH- and receptor-dependent endocytosis. Treatment of cells with either SARS-COV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 （ACE2）, the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic path- way inhibitors and dominant-negative Epsl5 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.     

Early Pattern of Epstein-Barr Virus Infection in Gastric Epithelial Cells by “Cell-in-cell”

Epstein-Barr virus(EBV)is an important human dsDNA virus,which has been shown to be associated with several malignancies including about 10%of gastric carcinomas.How EBV enters an epithelial cell has been an interesting project for investigation."Cell-in-cell"infection was recently reported an efficient way for the entry of EBV into nasopharynx epithelial cells.The present approach was to explore the feasibility of this mode for EBV infection in gastric epithelial cells and the dynamic change of host inflammatory reaction.The EBV-positive lymphoblastic cells of Akata containing a GFP tag in the viral genome were co-cultured with the gastric epithelial cells(GES-1).The infection situation was observed under fluorescence and electron microscopies.Real-time quantitative PCR and Western-blotting assay were employed to detect the expression of a few specific cytokines and inflammatory factors.The results demonstrated that EBV could get into gastric epithelial cells by"cell-in-cell"infection but not fully successful due to the host fighting.IL-1β,IL-6 and IL-8 played prominent roles in the cellular response to the infection.The activation of NF-κB and HSP70 was also required for the host antiviral response.The results imply that the gastric epithelial cells could powerfully resist the virus invader via cell-in-cell at the early stage through inflammatory and innate immune responses.     

The inhibitory effect of Chinese herb on SARS virus infection

[Subject]Severe acute respiratory syndrome(SARS)is a contagious atypical pneumonia with a high mortality rate.SARS coronavirus(SARS-CoV)is the pathogenof SARS.We established SARS-CoVS/HIVpseudotyped(SHP)virussystemandthe cell fusion assay systemto screeninhibitors for entry of SARS-CoV.[Materials and methods]SHPor VSV-Gpseudotype(VHP)virus was made bytransfecting pCMVΔR8·2,pHR’CMV-Luc and pCMV/R-SARS-S or pMDGplasmids into293Tcells.5ng p24of SHPor VHPvirus was …     

我国一株鹦鹉病毒的鉴定及其生化特性

A strain of new virus was isolated from fledgling budgerigars by authors in 1995. Cytopathic effect became visible after the isolate was passaged 4 generations on budgerigar embryo fibroblast and 5 generations on Chicken embryo fibroblast. The SDS-PAGE proved that viral capsid was composed of eight polypeptides with molecular weight from 60 000 to 14 500 D. The protein was rich in Asp, Glu and Leu, but comparatively poorer in Arg and Met. The ratio of acidic amino acid to basic amino acid was 1∶2.51. The nucleic acid was dsDNA and existed as supercoiled circular, relaxed circular and linear molecules. G+C of viral DNA was 45%. The molecular weight of DNA was 3.3×10⁶D by restriction endonuclease analysis. According to standards of virus classification, it is proved that the isolate was budgerigar fledgling disease virus which had placed into the Polyomavirus genus of Papovaviridae.     

Active expression of Gγ globin gene on chromosome 11 with Yunnanese (Ayγδβ)~0-thalasseinia deletion in MEL cells

A permanent lymphocyte cell line of a heterozygote with Yunnanese (Aγδβ)0-thalassemia deletion, associated with an increased production of Cry globin in adult, was founded using Epstein-Barr virus transformation. The hybrids of the lymphocyte cell and mouse erythroleukemia cell (MEL) were achieved and the hybrids containing human chromosome 11 were selected with the monoclonal antibody 53/6. The subclones containing only either the normal or the abnormal human chromosome 11 were separated and the expression of the human globin genes was studied. Expression of the β-globin gene, but not the Cγ and Aγ, was observed in the hybrids containing only the normal human chromosome 11, while active expression of the Cγ globin gene was observed in the hybrids containing only the abnormal human chromosome 11. These results have confirmed that the DNA deletion in the β-globin gene cluster is the cause of persistent active expression of the Cγ globin gene in the Yunnanese mutant.     

Cotton Leaf Curl Virus： Ionic Status of Leaves and Symptom Development

In the present study, the relationship between the nutritional status of leaves and the development of symptoms of cotton leaf curl virus （CLCuV） in two cotton （Gossypium hirsutum L.） cuItlvars （I.e. CIM-240 and S-12） was Investigated. The incidence of disease attack was found to be 100% In the S-12 cuItlvar and 16% in the CIM-240 cuItivar. Geminivirus particles in infected leaves were confirmed by transmission electron microscope examination of highly specific geminivirus coat protein antlsera-treated cell sap. The CLCuV Impaired the accumulation of different nutrients in both cuItivars. A marked decrease in the accumulation of Ca^2＋ and K^＋ was observed in infected leaves. However, the disease had no effect on leaf concentrations of Na^＋, N, and P. It was observed that the curling of leaf margins in CLCuV-Infected plants was associated with the leaf Ca^2＋ content; leaf curling was severe in plants with a significant reduction In Ca^2＋ content. Moreover, leaf K＆＋ content was found to be associated with resistance/susceptibility to CLCuV infection.     

SARS冠状病毒感染Lewis大鼠模型的建立

目的通过观察SARS-CoV感染Lewis大鼠后,病理学、免疫学以及病毒的复制与外排情况变化,探讨其能否作为有效的SARS动物模型。方法SARS病毒感染9只Lewis大鼠,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Lewis大鼠后,肺组织出现一定的与人类SARS疾病相似的病理改变,在动物体内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。结论Lewis大鼠出现了特异的免疫反应及特征性病理改变,可做为灵长类SARS动物模型的有益补充用于SARS发病机理及疫苗的研发等。     

老龄ICR小鼠对SARS-CoV的易感性

目的为探讨SARS的发病机制并提供易感的SARS动物模型。方法利用RT-PCR和病毒分离后免疫荧光技术检测成龄鼠和老龄鼠接种SARS-CoV后肺组织内病毒复制情况,同时观察两组动物的肺脏和肺外组织器官的病理变化,对肺组织进行免疫组化分析,观察SARS-CoV在肺内复制的主要部位。结果老龄鼠的感染率明显高于成龄鼠;老龄鼠肺脏出现更为严重的弥漫性肺泡损伤,其中两只老龄鼠的肺外器官出现了变性、灶状坏死以及血管广泛的扩张充血等全身中毒性变化;肺脏内病毒抗原主要存在于肺泡上皮细胞和间质的血管内皮细胞。结论老龄ICR小鼠对SARS-CoV的易感性明显高于成龄鼠,有可能作为研究SARS发病机制以及药物评价的动物模型。     

Persistent replication of severe acute respiratory syndrome coronavirus in human tubular kidney cells selects for adaptive mutations in the membrane protein

Severe acute respiratory syndrome (SARS) is a systemic disease characterized by both lung pathology and widespread extrapulmonary virus dissemination causing multiple organ injuries. In this regard, renal dysfunction is an ominous sign in patients with SARS. Indeed, clusters of SARS coronavirus (SARS-CoV) particles have been detected in the cytoplasm of renal tubular epithelial cells in postmortem studies, explaining the presence of infectious virus in the urine of SARS patients. In order to investigate the potential SARS-CoV kidney tropism, we have evaluated the susceptibility of human renal cells of tubular and glomerular origin to in vitro SARS-CoV infection. Immortalized cultures of differentiated proximal tubular epithelial cells (PTEC), glomerular mesangial cells (MC), and glomerular epithelial cells (podocytes) were found to express the SARS-CoV receptor angiotensin-converting enzyme 2 on their surface. Productive infection, however, occurred only in PTEC but not in glomerular cells. A transient infection with poor virus production was observed in MC, whereas podocytes were not permissive to SARS-CoV infection. In contrast to the cytopathic infection of the Vero E6 cell line, SARS-CoV did not cause overt cytopathic effects in PTEC or MC. Of interest, PTEC, but not MC, maintained stable levels of SARS-CoV production in serial subcultures, suggesting a persistent state of infection. In this regard, a SARS-CoV variant with increased replication capacity in PTEC was selected after four serial subculture passages. This SARS-CoV variant acquired a single nonconservative amino acid change from glutamic acid (E) to alanine (A) at position 11 in the viral membrane (M) protein. The E11A point mutation was sufficient for enhanced SARS-CoV replication and persistence in PTEC when introduced in a SARS-CoV recombinant infectious clone. These findings indicate that human PTEC may represent a site of SARS-CoV productive and persistent replication favoring the emergence of viral variants with increased replication capacity, at least in these kidney cells.     

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。     

S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approximately 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T- and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replication-competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.     

Participation of both host and virus factors in induction of severe acute respiratory syndrome (SARS) in F344 rats infected with SARS coronavirus

To understand the pathogenesis and develop an animal model of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), the Frankfurt 1 SARS-CoV isolate was passaged serially in young F344 rats. Young rats were susceptible to SARS-CoV but cleared the virus rapidly within 3 to 5 days of intranasal inoculation. After 10 serial passages, replication and virulence of SARS-CoV were increased in the respiratory tract of young rats without clinical signs. By contrast, adult rats infected with the passaged virus showed respiratory symptoms and severe pathological lesions in the lung. Levels of inflammatory cytokines in sera and lung tissues were significantly higher in adult F344 rats than in young rats. During in vivo passage of SARS-CoV, a single amino acid substitution was introduced within the binding domain of the viral spike protein recognizing angiotensin-converting enzyme 2 (ACE2), which is known as a SARS-CoV receptor. The rat-passaged virus more efficiently infected CHO cells expressing rat ACE2 than did the original isolate. These results strongly indicate that host and virus factors such as advanced age and virus adaptation are critical for the development of SARS in rats.     

Interactions between M protein and other structural proteins of severe,acute respiratory syndrome-associated coronavirus

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) structural proteins (S, E, M, and NC) localize in different subcellular positions when expressed individually. However, SARS-CoV M protein is co-localized almost entirely with S, E, or NC protein when co-expressed in the cells. On the other hand, only partial co-localization was observed when S and E, S and NC, or E and NC were co-expressed in the cells. Interactions between SARS-CoV M and other structural proteins but not interactions between S and E, S and NC, or E and NC were further demonstrated by co-immunoprecipitation assay. These results indicate that SARS-CoV M protein, similar to the M proteins of other coronaviruses, plays a pivotal role in virus assembly. The cytoplasmic C-terminus domain of SARS-CoV M protein was responsible for binding to NC protein. Multiple regions of M protein interacted with E and S proteins. A model for the interactions between SARS-CoV M protein and other structural proteins is proposed. This study helps us better understand protein-protein interactions during viral assembly of SARS-CoV. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.