The expression and antigenicity of a truncated spike-nucleocapsid fusion protein of severe acute respiratory syndrome-associated coronavirus.

BACKGROUND: In the absence of effective drugs, controlling SARS relies on the rapid identification of cases and appropriate management of the close contacts, or effective vaccines for SARS. Therefore, developing specific and sensitive laboratory tests for SARS as well as effective vaccines are necessary for national authorities. RESULTS: Genes encoding truncated nucleocapsid (N) and spike (S) proteins of SARSCoV were cloned into the expression vector pQE30 and fusionally expressed in Escherichia coli M15. The fusion protein was analyzed for reactivity with SARS patients' sera and with anti-sera against the two human coronaviruses HCoV 229E and HCoV OC43 by ELISA, IFA and immunoblot assays. Furthermore, to evaluate the antigen-specific humoral antibody and T-cell responses in mice, the fusion protein was injected into 6-week-old BALB/c mice and a neutralization test as well as a T-cell analysis was performed. To evaluate the antiviral efficacy of immunization, BALB/c mice were challenged intranasally with SARSCoV at day 33 post injection and viral loads were determined by fluorescent quantitative RT-PCR. Serological results showed that the diagnostic sensitivity and specificity of the truncated S-N fusion protein derived the SARS virus were > 99% (457/460) and 100.00% (650/650), respectively. Furthermore there was no cross-reactivity with other two human coronaviruses. High titers of antibodies to SRASCoV appeared in the immunized mice and the neutralization test showed that antibodies to the fusion protein could inhibit SARSCoV. The T cell proliferation showed that the fusion protein could induce an antigen-specific T-cell response. Fluorescent quantitative RT-PCR showed that BALB/c mice challenged intranasally with SARSCoV at day 33 post injection were completely protected from virus replication. CONCLUSION: The truncated S-N fusion protein is a suitable immunodiagnostic antigen and vaccine candidate.

Real-time RT-PCR for H5N1 avian influenza A virus detection.

The recent recurrence of highly pathogenic avian influenza virus A H5N1 was firstly reported in mid-December 2003 and continued through 2005. This study describes a sensitive and specific real-time RT-PCR method for the detection of influenza A subtype H5 and for monitoring virus loads. Using serial dilutions of influenza A H5N1 cultures, this assay reproducibly determined the lowest detection limit to be approximately 5 x 10(-2) 50 % egg infective doses (EID(50)). In contrast, the minimum detection limit was approximately 3 EID(50) in conventional RT-PCR with WHO primers and 10 EID(50) in antigen-capture ELISA. In tests of serial dilutions of in vitro-transcribed influenza A H5 gene RNA, there was linear amplification from 40 copies to 4 x 10(8) copies of target RNA per reaction and approximately six copies, and sometimes even as few as three copies, of target RNA tested positive in our assay. Thirty-five throat swabs from ill birds were tested: 33 samples tested positive using this assay. In comparison, 27, 13 and 19 samples tested positive using conventional RT-PCR, antigen-capture ELISA and virus isolation, respectively. To evaluate further the sensitivity of this real-time RT-PCR, a standard panel and 60 H5N1 isolates that contained different clades of influenza virus A/H5N1 were tested and all tested positive. To evaluate the specificity of the assay, 60 throat swabs from patients infected with influenza virus A H1 were tested; all were negative. Thirteen other viruses were also tested and all tested negative.

Follow-up study on pulmonary function and lung radiographic changes in rehabilitating severe acute respiratory syndrome patients after discharge.

OBJECTIVES: To follow-up on the changes in lung function and lung radiographic pictures of severe acute respiratory syndrome (SARS) patients discharged from Xiaotangshan Hospital in Beijing (by regularly receiving examination), and to analyze retrospectively the treatment strategy in these patients. METHODS: Surviving SARS patients were seen at least twice within 3 months after discharge and underwent SARS-associated coronavirus (SARS-CoV) IgG antibody testing, pulmonary function testing, and chest radiography and/or high-resolution CT (HRCT) examinations at Chinese PLA General Hospital. The treatments received at Xiaotangshan Hospital were analyzed retrospectively and were correlated to later status. RESULTS: Positive SARS-Co virus IgG antibody results were seen in 208 of 258 patients, with 21.3% (55 of 258 patients) still having a pulmonary diffusion abnormality (D(LCO) < 80% of predicted). By comparing the 155 survivors with positive SARS-CoV IgG antibody results and D(LCO) > or = 80% predicted with the 50 patients with negative SARS-CoV IgG results, we found that 53 patients with positive SARS-CoV IgG results and a lung diffusion abnormality had endured a much longer course of fever and received larger doses of glucocorticoid, as well as higher ratios of oxygen inhalation and noninvasive ventilation treatment. For these patients, 51 of 53 patients with positive SARS-CoV IgG results and a lung diffusion abnormality underwent pulmonary function testing after approximately 1 month. D(LCO) improved in 80.4% of patients (41 of 51 patients). Of the patients with a lung diffusion abnormality, 40 of 51 patients showed lung fibrotic changes in the lung image examination and 22 patients (55%) showed improvement in lung fibrotic changes 1 month later. CONCLUSION: These findings suggest that lung fibrotic changes caused by SARS disease occurred mostly in severely sick patients and may be self-rehabilitated. D(LCO) scores might be more sensitive than HRCT when evaluating lung fibrotic changes.

Dynamic changes of serum SARS-coronavirus IgG, pulmonary function and radiography in patients recovering from SARS after hospital discharge.

OBJECTIVE: The intent of this study was to examine the recovery of individuals who had been hospitalized for severe acute respiratory syndrome (SARS) in the year following their discharge from the hospital. Parameters studied included serum levels of SARS coronavirus (SARS-CoV) IgG antibody, tests of lung function, and imaging data to evaluate changes in lung fibrosis. In addition, we explored the incidence of femoral head necrosis in some of the individuals recovering from SARS. METHODS: The subjects of this study were 383 clinically diagnosed SARS patients in Beijing, China. They were tested regularly for serum levels of SARS-CoV IgG antibody and lung function and were given chest X-rays and/or high resolution computerized tomography (HRCT) examinations at the Chinese PLA General Hospital during the 12 months that followed their release from the hospital. Those individuals who were found to have lung diffusion abnormities (transfer coefficient for carbon monoxide [DLCO] < 80% of predicted value [pred]) received regular lung function tests and HRCT examinations in the follow-up phase in order to document the changes in their lung condition. Some patients who complained of joint pain were given magnetic resonance imaging (MRI) examinations of their femoral heads. FINDINGS: Of all the subjects, 81.2% (311 of 383 patients) tested positive for serum SARS-CoV IgG. Of those testing positive, 27.3% (85 of 311 patients) were suffering from lung diffusion abnormities (DLCO < 80% pred) and 21.5% (67 of 311 patients) exhibited lung fibrotic changes. In the 12 month duration of this study, all of the 40 patients with lung diffusion abnormities who were examined exhibited some improvement of lung function and fibrosis detected by radiography. Of the individuals receiving MRI examinations, 23.1% (18 of 78 patients) showed signs of femoral head necrosis. INTERPRETATION: The lack of sero-positive SARS-CoV in some individuals suggests that there may have been some misdiagnosed cases among the subjects included in this study. Of those testing positive, the serum levels of SARS-CoV IgG antibody decreased significantly during the 12 months after hospital discharge. Additionally, we found that the individuals who had lung fibrosis showed some spontaneous recovery. Finally, some of the subjects developed femoral head necrosis.

Antibody response and viraemia during the course of severe acute respiratory syndrome (SARS)-associated coronavirus infection.

To understand the time-course of viraemia and antibody responses to severe acute respiratory syndrome-associated coronavirus (SARS-CoV), RT-PCR and ELISA were used to assay 376 blood samples from 135 SARS patients at various stages of the illness, including samples from patients who were in their early convalescent phase. The results showed that IgM antibodies decreased and became undetectable 11 weeks into the recovery phase. IgG antibodies, however, remained detectable for a period beyond 11 weeks and were found in 100 % of patients in the early convalescent phase. SARS-CoV viraemia mainly appeared 1 week after the onset of illness and then decreased over a period of 1 month, becoming undetectable in the blood samples of the convalescent patients. At the peak of viraemia, viral RNA was detectable in 75 % of blood samples from patients who were clinically diagnosed with SARS 1 or 2 weeks before the test.

[Dynamic changes of immunoglobulin G in convalescents who have suffered from severe acute respiratory syndrome patients].

OBJECTIVE: To investigate the dynamic changes of the antibody specific to severe acute respiratory syndrome (SARS) coronavirus in convalescents who have suffered from SARS. METHODS: Samples of peripheral blood were collected twice during the first 2 weeks after discharge and then once every 2 - 4 weeks from 310 convalescents of SARS, 131 males and 179 females, aged 18 - 74, discharged from hospitals in Beijing April 3 to June 20 2003 with the average discharge date of June 10, to detect the level of immunoglobulin G (IgG) by ELISA. A curve of level of IgG was drawn to describe the change by the specific month in the year. RESULTS: Most of the 310 convalescents underwent successive or non-successive testing for 2 - 4 months and 15 were tested for 5 months, 15 for 6 months, and 2 for 7 months. IgG was detected in each sample with the mean of A value of 0.97 +/- 0.37 (0.197 - 1.849). The mean IgG level peaked in July (1.203), about 35 days after discharge, and then gradually declined to 0.857 in December, a decline by 27.3%. CONCLUSION: All SARS patients generate specific antibody against the coronavirus. However, the antibody level gradually decline with the lapse of time during the convalescence. Long-term surveillance of the change of antibody is necessary.

[Comparative study of clinical characteristics and prognosis of clinically diagnosed SARS patients with positive and negative serum SARS coronavirus-specific antibodies test].

OBJECTIVE: To investigate clinical characteristics and long-term effects of SARS. METHODS: Clinical characteristics of 197 SARS patients in Xiao Tang-shan hospital were analyzed retrospectively, and prognosis of them were analyzed prospectively. RESULTS: Among the 197 patients, 153 patients (77.7%) have positive results of serum SARS coronavirus-specific antibodies test, and 44 patients (22.3%) have negative results. The average age of SARS and non-SARS patients were (40 +/- 12) and (31 +/- 12) years, male/female ratio were 1.0:1.6 and 2.1:1.0, average body temperature were (38.5 degrees C +/- 0.3 degrees C) and (38.1 degrees C +/- 0.4 degrees C), median of fever length were 7.0 d (0.4 approximately 50 d) and 2.3 d (0.3 approximately 37 d) respectively. The occurrence of dyspnea, malaise and gastrointestinal symptom were more often in SARS patient than in non-SARS patients. Some patients have residual symptoms (such as cough, fatigue, dyspnea, abnormality of liver function, hyperglycemia and hyperglyceridemia), and only few patients have lung fibrosis. CONCLUSION: Some patients with other respiratory diseases were misdiagnosed as SARS. There were several obvious differences of clinical characteristics between SARS patient and non-SARS patients. Prognosis of most patients were preciously well, and few still had abnormality of lung function. Residual symptoms of SARS and side effects of drugs used to treat SARS should be discriminated.

[The quantitative detection of anti-coronavirus antibody titer in medical personnel closely contacted with severe acute respiratory syndrome patients].

OBJECTIVE: To study the serum anti-coronavirus antibody titer in medical personnel who had closely contacted with severe acute respiratory syndrome (SARS) patients. METHODS: The serum anti-coronavirus IgG antibody titer in medical personnel who had closely contacted with SARS patients, healthy individuals, patients with community acquired pneumonia and patients recovered from SARS was detected by using an enzyme-linked immunosorbent assay (ELISA) method. The antibody titer was expressed as the value of absorbency (A) with common logarithm conversion. RESULTS: The serum anti-coronavirus IgG antibody titer in patients recovered from SARS was 0.07 +/- 0.13, which was significantly higher as compared with those in other groups. The antibody titer in medical personnel was -1.18 +/- 0.20, which was also significantly higher as compared with those in community acquired pneumonia patients and healthy persons. In the healthy persons, the antibody titer of serum samples obtained from Beijing in May, 2003 was -1.61 +/- 0.13, which was significantly higher than that of samples obtained from Beijing in 2001 when SARS was not found -1.76 +/- 0.25 and that of samples from Shandong province where SARS was not found in May, 2003 -1.95 +/- 0.44. There was no significant difference in the antibody titer between patients of bacterial pneumonia and patients of atypical pneumonia, which was -1.99 +/- 0.31 and -2.05 +/- 0.23 respectively. CONCLUSION: Close contact with SARS patients can cause the serum anti-coronavirus antibody titer to increase significantly in medical personnel, a phenomenon deserves further study.

[Prognostic analysis of lung function and chest X-ray changes of 258 patients with severe acute respiratory syndrome in rehabilitation after discharge].

OBJECTIVE: To analyze the clinical characteristics and prognostic changes of rehabilitating severe acute respiratory syndrome (SARS) patients through regular lung function tests and lung imaging studies after discharge and to retrospectively analyze the treatment data of these patients. METHODS: 258 discharged SARS patients received regular SARS-Co virus IgG test, lung function test and chest X-ray and/or high resolution computerized tomography (HRCT) examination at General Hospital of PLA two months after discharge, and the treatment data of these patients were retrospectively analyzed. RESULTS: 80.6% patients (208 of 258 patients) were positive for SARS-Co virus IgG. 21.3% patients (55 of 258 patients) showed lung diffusion abnormity (D(LCO) < 80%pred). Compared to 155 SARS-Co virus IgG positive patients without lung diffusion abnormity and 50 SARS-Co virus IgG negative patients, the 53 SARS-Co virus IgG positive patients with lung diffusion abnormity had longer fever course, higher dosages of glucocorticoid therapy, higher percentage of oxygen therapy and non-invasive ventilation. 51 of the 53 patients with lung diffusion abnormity received lung function test after one month, and the results of D(LCO) improved in 80.4% patients (41 of 51 patients). 40 of 51 patients with lung diffusion abnormity showed lung fibrosis, and the fibrosis decreased in 55% patients (22 of 40 patients) after one month. CONCLUSIONS: This finding suggests that lung fibrosis caused by SARS mostly occurs in severe patients, and it can resolve spontaneously. D(LCO) may be more sensitive than HRCT in evaluating the fibrotic changes.

[Expression profile of 60 lung cancer related genes in BEP2D cell and R15Hp35T-2 cell].

BACKGROUND: To profile the expression patterns of 60 lung cancer related genes in human bronchial epithelial cell (BEP2D) and alpha-particle induced malignantly transformed cell (R15Hp35T-2). METHODS: Sixty lung cancer related cDNAs were micro-arrayed onto the microscope slides using Cartesian PixSys5500 cDNA Microarray machine. Total RNA from BEP2D cell and R15Hp35T-2 cell was extracted and labeled by fluorescent dye. The labeled probe was then hybridized with the cDNA. RESULTS: Compared with the BEP2D cell, 27 genes up-regulated and 7 down-regulated in the R15Hp35T-2 cell. The expression abundance of most tumor suppressor genes were similar in the two kinds of cells, however, most oncogenes and growth factor genes were overexpressed in R15Hp35T-2 cell. CONCLUSIONS: In malignantly transformed human bronchial epithelial cell model induced by alpha-particle, some oncogenes and growth factor genes may promote the malignant transformation together.

[Combining SSH and cDNA microarray for identification of lung cancer related genes].

BACKGROUND: To screen and identify differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and some other kinds of tumor tissues using suppression subtractive hybridization (SSH) and cDNA Microarray. METHODS: One cDNA chip was made by gathering clones of three differentially expressed cDNA libraries which came from BEP2D cell lines during three different malignant transformed phases. Then the clones were hybridizated with cDNA probes which extracted from 15 cases of lung cancer tissues, 5 cases of paracancerous pulmonary tissues and 24 cases of other 8 kinds of tumor tissues respectively. RESULTS: Twenty-six cDNAs were obtained which expressed higher in lung cancer tissues than that in paracancerous pulmonary tissues. Thirty-one cDNAs expressed remarkably higher in paracancerous tissues than those in cancer tissues. Compared with other 8 kinds of tumors, paracancerous tissues had 63 overexpressed cDNAs and lung cancer tissues had 87 overexpressed cDNAs. CONCLUSIONS: The combination of SSH and cDNA microarray is rapid and effective for screening and identification of differentially expressed genes in different samples. It may be potentially useful for diagnosis of lung cancer to further study the differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and other tumor tissues.

[Study on the dynamics of IgG antibody in 311 patients with severe acute respiratory syndrome].

OBJECTIVE: To detect the level and dynamic change of severe acute respiratory syndrome (SARS)-coronavirus-specific IgG antibody in conavalescent SARS patients, and to provide information for prevention and vaccine development. METHODS: IgG antibody against coronavirus was detected by ELISA in the blood of 311 convalescent SARS patients for every 2 - 4 weeks. Stata 7.0 statistics software was used to analyse the results. RESULTS: IgG antibody was detected positive on each testing of all the convalescent patients and its peak appeared 35 days after recovery. IgG antibody level showed a 35.8% decrease within one year. CONCLUSION: Data showed that all the SARS convalescent patients had generated high level of specific IgG antibody against coronavirus in the early stage of recovery, but the antibody level declined along with the progress of convalescence, suggesting that the detection of the IgG antibody should go on until it disappeared.

Use of the COOH portion of the nucleocapsid protein in an antigen-capturing enzyme-linked immunosorbent assay for specific and sensitive detection of severe acute respiratory syndrome coronavirus.

Antibody detection with a recombinant COOH portion of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (N) protein, N13 (amino acids 221 to 422), was demonstrated to be more specific and sensitive than that with the full-length N protein, and an N13-based antigen-capturing enzyme-linked immunosorbent assay providing a convenient and specific test for serodiagnosis and epidemiological study of SARS was developed.

Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.

A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.

[Malignant transformation of human bronchial epithelial cell (BEAS-2B) induced by depleted uranium].

BACKGROUND & OBJECTIVE: It is clear from works already reported that depleted uranium (DU) affect human health. However, the late effect, especially the carcinogenesis, was not clearly understood. This study was designed to investigate the malignant transformation of human bronchial epithelial cell induced by insoluble DU and lung cancer related gene expression pattern, through imitating the condition that human absorbs depleted uranium aerosol. METHODS: Adenovirus-12/SV40 virus immortalized human bronchial epithelial cells (BEAS-2B) were reacted with insoluble DU oxide (dUO2); the characteristics of malignant transformation of cells were identified through observing the multiplication time of different generation cells, serum resistance, colony formation rate of semi-solid agar, and tumorigenesis in nude mice. Gene expression pattern of transferred BEAS-2B cell induced by DU was determined using 213 lung cancer related gene arrays. RESULTS: The multiplication time of BEAS-2B cell treated with DU was obviously decreased and the serum reistance was significantly increased in 5th generation; the anchorage independent growth (semi-solid agar colony formation) was appeared in 10th generation cell. The 15th generation cell formed tumor in nude mice. DMSO showed overt protection effect on malignant transformation of BEAS-2B cell. The analyzing results of 213 lung cancer related gene arrays showed that the expression level changed in more than 70 genes of transferred cells, including the overt decrease of level of gene expression in more than 10 genes. CONCLUSION: DU has carcinogenesis in vitro.

[Regulation of Smad7 gene by TGF-beta 1 in process of malignant transformation].

BACKGROUND & OBJECTIVE: Escape from transforming growth factor-beta(TGF-beta)-induced inhibition of growth and proliferation may contribute to tumorigenesis. Smad7 is inhibitory Smads of TGF-beta s signal transduction pathway and prevents TGF-beta signaling. The disorder of Smad7 may lead to the perturbation of TGF-beta signal pathway. In this study, The authors analyzed the expression of Smad7 mRNA and the regulation of Smad7 gene by TGF-beta 1 in the process of malignant transformation of BEP2D cells to investigate the mechanism of cells malignant transformation. METHODS: Cells were cultured and stimulated with TGF-beta 1 followed by RNA extraction. Purified total RNA from TGF-beta 1 treated cells and untreated controls and performed an expression analysis with a human Smad7-specific probe applying Northern blot. As a loading control for the Northern experiment, the membrane was hybridized with a human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) probe. Proteins were extracted from BEP2D and BERP35T-2 cells, then perform Western blot to examine the expression level of TGF-beta 1. RESULTS: Before stimulation with TGF-beta 1, the expression level of Smad7 in the BERP35T-2 cells were higher than that in the BEP2D cells. When stimulated with TGF-beta 1, Smad7 expression levels was upregulated evidently in BEP2D cells, but not significant in BERP35T-2 cells. The expression level of endogenetic TGF-beta 1, BERP35T-2 cells was a little higher than BEP2D cells. CONCLUSION: Over expression of Smad7 mRNA and down-regulation of the cells' responsiveness to TGF-beta 1 in human lung cancer cell line which induced by alpha-particles should be one of the mechanism of radiation induced lung cancer.