Apoptosis induced by the SARS-associated coronavirus in Vero cells is replication-dependent and involves caspase.

The pathogenesis of the severe acute respiratory syndrome (SARS), a newly emerging life-threatening disease in humans, remains unknown. It is believed that the modulation of apoptosis is relevant to diseases that are caused by various viruses. To examine potential apoptotic mechanisms related to SARS, we investigated features of apoptosis induced by the SARS-associated coronavirus (SARS-CoV) in host cells. The results indicated that the SARS-CoV-induced apoptosis in Vero cells in a virus replication-dependent manner. Additionally, the downregulation of Bcl-2, the activation of casapse 3, as well as the upregulation of Bax were detected, suggesting the involvement of the caspase family and the activation of the mitochondrial signaling pathway. Although there is a positive correlation between apoptosis and virus replication, the latter is not significantly blocked by treatment with the caspase inhibitor z-DEVD-FMK. These preliminary data provide important information on both the pathogenesis and potential antiviral targets of SARS-CoV.

Profiles of IgG antibodies to nucleocapsid and spike proteins of the SARS-associated coronavirus in SARS patients.

To evaluate humoral immunity against the SARS-associated coronavirus (SARS-CoV), we studied the profiles of IgG antibodies to the nucleocapsid (N) and spike (S) proteins of SARS-CoV. Serum specimens from 10 SARS patients were analyzed by Western blotting and an enzyme-linked immunosorbent assay (ELISA) using purified recombinant N and truncated S (S1, S2, and S3) proteins as antigens. Western blotting results demonstrated that 100% of the SARS patients tested positive for N protein-specific antibodies, 50% for S1 protein-specific antibodies, 30% for S2 protein-specific antibodies, and 70% for S3 protein-specific antibodies. The ELISA results, which showed positive rates of IgG reactivity against recombinant proteins N, S1, S2, and S3, were, respectively, 28.57, 14.29, 14.29, and 14.29% at week 1, 77.78, 55.56, 44.44, and 66.67% at week 2, 100, 75, 75, and 87.5% at week 3, and 100, 77.78, 77.78, and 88.89% after 3 weeks. The average titers of IgG against recombinant proteins N, S1, S2, and S3 were, respectively, 691, 56, 38, and 84 after 3 weeks. These results suggest that the recombinant proteins N and S3 are potentially useful antigens for a serological diagnosis of SARS. In consideration of possible cross-reactivity among N proteins of SARS-CoV and other coronaviruses, immunoassays using recombinant N protein in combination with S3 as antigens might improve the specificity of SARS diagnoses.

[Study on the RNA of severe acute respiratory syndrome (SARS) associated coronavirus in the blood and excretion of convalescent patients with SARS].

OBJECTIVE: To examine the RNA of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in the blood and excretion of convalescent patient with SARS for prevention and treatment of the disease. METHODS: A total of 276 samples, including plasma, urine, feces and sputum, obtained from 23 convalescent patients with SARS were studied at 3 time-points at least 21 days after the onset of symptoms. RNA was extracted and nested reverse transcription-polymerase chain reaction (RT-PCR) was carried out using SARS-CoV specific primers. RESULTS: Among the 276 samples, SARS-CoV RNA was detected in 6 cases (38.8%) by nested RT-PCR. The positive rates of SARS-CoV RNA was 5.8% in feces and 2.9% in sputum samples but SARS-CoV RNA was not detectable in plasma and urine of all the cases. CONCLUSION: The existence of SARS-CoV RNA in the excretion of some convalescent patients with SARS showed that the excretion from these patients should be carefully treated whilthe re-transmission of SARS by which, should be further studied.

An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus.

A new SARS animal model was established by inoculating SARS coronavirus (SARS-CoV) into rhesus macaques (Macaca mulatta) through the nasal cavity. Pathological pulmonary changes were successively detected on days 5-60 after virus inoculation. All eight animals showed a transient fever 2-3 days after inoculation. Immunological, molecular biological, and pathological studies support the establishment of this SARS animal model. Firstly, SARS-CoV-specific IgGs were detected in the sera of macaques from 11 to 60 days after inoculation. Secondly, SARS-CoV RNA could be detected in pharyngeal swab samples using nested RT-PCR in all infected animals from 5 days after virus inoculation. Finally, histopathological changes of interstitial pneumonia were found in the lungs during the 60 days after viral inoculation: these changes were less marked at later time points, indicating that an active healing process together with resolution of an acute inflammatory response was taking place in these animals. This animal model should provide insight into the mechanisms of SARS-CoV-related pulmonary disease and greatly facilitate the development of vaccines and therapeutics against SARS.

The design and application of DNA chips for early detection of SARS-CoV from clinical samples.

BACKGROUND: SARS coronavirus has been identified as the cause of severe acute respiratory syndrome (SARS). Few tests allow confirmation or exclusion of SARS within the first few days of infection. A gene chip is a useful tool for the study of microbial infections mainly for its capability of performing multi-target analysis in a single test. OBJECTIVES: Investigate the possibility of early detection of SARS virus from clinical samples using the gene chip-based method. STUDY DESIGN: We purified RNA from SARS-CoV obtained from routinely collected peripheral blood and sputum samples of 34 patients who had been identified as probable SARS patients by following the interim U.S. case definition. Four segments of the SARS-CoV were amplified using reverse transcription-nested PCR and the products examined using the 70-mer gene chips for SARS-CoV detection. RESULTS: A blind-test of both peripheral blood and sputum specimens lead to the positive detection of SARS-CoV in 31 out of 34 patients. SARS-CoV was not found in peripheral blood or sputum specimens from three patients. Two of the 34 patients were only 3 days post-onset of symptoms and were subsequently confirmed to be SARS positive. Our results indicate that the gene chip-based molecular test is specific for SARS-CoV and allows early detection of patients with SARS with detection rate about 8% higher than the single PCR test when the sputum sample is available.

[Chlamydia-like and coronavirus-like agents found in dead cases of atypical pneumonia by electron microscopy].

OBJECTIVE: To explore the causative agents of the atypical pneumonia (also SARS) occurred recently in some regions of our country. METHOD: Organ samples of 7 dead cases of SARS were collected from Guangdong, Shanxi, Sichuan Provinces and Beijing for electron microscopic examination. 293 cell line was inoculated with the materials derived from the lungs to isolate causative agent(s). The agents in the organs and cell cultures were revealed by immunoassay. RESULTS: Both Chlamydia-like and coronavirus-like particles were found in EM. Inclusion bodies containing elementary bodies, reticulate antibodies and intermediate bodies of Chlamydia-like agent were visualized in multiple organs from the 7 dead cases, including lungs (7 cases), spleens (2 cases), livers (2 cases), kidneys (3 cases) and lymph nodes (1 cases), by ultrathin section electron microscopy (EM). In some few sections, coronavirus-like particles were concurrently seen. A coronavirus RNA- polymerase segment (440 bp) was amplified from the lung tissues of two cases of the SARS. After inoculated with materials from the lung samples, the similar Chlamydia-like particles were also found in the inoculated 293 cells. Since the Chlamydia-like agents visualized in both organs and cell cultures could not react with the genus specific antibodies against Chlamydia and monoclonal antibodies against C. pneumoniae and C. psittaci, the results might well be suggestive of a novel Chlamydia-like agent. CONCLUSION: Since the novel Chlamydia-like agent was found co-existing with a coronavirus-like agent in the dead cases of SARS, it looks most likely that both the agents play some roles in the disease. At the present time, however, one can hardly determining how did these agents interact each other synergetically, or one follows another, need further study.